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User:Marios F. Sardis
From OpenWetWare
Revision as of 07:24, 3 November 2007 by Marios F. Sardis (Talk | contribs)
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current status:Studying, MS Protein Biotechnology
Sardis Marios Frantzeskos
Department of Biology
University of Crete
Herakleion, Crete
Greece
hartigan.sc@gmail.com
Education
2006-........MS in Protein Biotechnology, Department of Biology,University of Crete
2002-2006 BS in Biology (Biomolecular science and Biotechnology), University of Crete.
2001-2002 Certificate of Higher Education (in Biology), University of Cardiff, Wales, U.K.
Research experience
Past research interests
MHC class II molecules
Current research interests
Molecular motors Protein translocation mechanisms in E.coli through the SecA-SecYEG system-Institute of Molecular Biology and Biotechnology-Foundation of Research and Technology Hellas (FORTH), Herakleion, Greece
SecA translocase
Ecolab
Events/News
Past Great Events
1st Joint Meeting of European National Societies of Immunology 6-9 September, 2006, Paris
Summer school in Protein Biotechnology, 23-26 May 2007, Fodele Crete, Greece
Journals
General
Nature Publishing Group
Cell
Science
Annual Reviews
New Scientist
Open Access
PLoS
PLoS
[* BMC journals here!!!
Links
University of Crete
Department of Biology Department of Biology, University of Crete, Greece
M.Sc. Protein Biotechnology, University of Crete M.Sc. Protein Biotechnology of the Department of Biology, University of Crete, Home Page
University of Cardiff University of Cardiff, Wales, U.K.
University of Newport University of Newport, Wales, U.K.
BiocompareGuide for life sciences products
BIO 502Bio 502 Economou & Tokatlidis
How to collect and see bacteria
Atlas of Protein Side-Chain Interactions
This introduction to experimental biosciences will introduce and reinforce practical skills in the context of three different laboratory investigations..
Superb presentation explaining how PAGE works
The Escherichia coli database
Todar's Online Textbook of Bacteriology
Ken Todar's Microbial World
Quorum sensing-University of Nottingham
Fotis Kafatos: the model mentor
Introduction to Molecular Orbital Theory
What are van der Waals forces?
Internet tools
PubMed-Major source of Bibliography and useful tools
PAGE (SDS/Native) calculator
Molarity calculator
The Lipid Library
Protein interactions in Yeast
Recombinant Protein Solubility Prediction
Serine proteases mechanism
Proteomics
ExPASy Proteomics Server
SWISS-MODEL is a fully automated protein structure homology-modeling server, accessible via the ExPASy web server, or from the program DeepView
EBI Tools: ClustalW
[ ]
[ ]
Interests
eBay UKThe best place for shopping!!!
PaypalThe best way to pay what you buy!!!
Myspace Profile
Richard Dawkins net
Richard Dawkins foundation
Visits
Personal tools
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User:Mobeen Ashraf/Notebook/Chem-581/2012/10/19
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Experimental Chemistry Main project page
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Objective
Description
• 3 PVOH films were prepared: 0.5 g MW of 22,000, 1 ml glutaraldehyde, and 1 ml of porphrine dihydrogen chloride.
• AAS studies for all the Cu standards: NaMT- concentration of 1.25 g/500 ml, for 1min, 5 min and 10 mins standards, 50 % CEC exchange-concentration of 1.25 g/500 ml, for 1 min, 5 min and 10 mins standards, 100% CEC exchange, for 1 min, 5 min and 10 mins standards, and Fe3O4-concentration of 1.25 g/500 ml, for 1 min, 5 min and 10 mins standards.
Data
• Add data and results here...
Notes
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Use categories like tags. Change the "Course" category to the one corresponding to your course. The "Miscellaneous" tag can be used for particular experiments, as instructed by your professor. Please be sure to change or delete this tag as required so that the categories remain well organized.
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Quotation added by staff
Why not add this quote to your bookmarks?
Talk is cheap, except when Congress does it. Hightower, Cullen
This quote is about speakers and speaking · Search on Google Books to find all references and sources for this quotation.
A bit about Hightower, Cullen ...
After serving in the U.S. Army during World War II Cullen Tower began a career as a salesman and sales trainer. He retired and moved to Florida where he began writing the quips and advice he had been compiling throughout his career. His work was published in a newspaper feature called Salty Sally and was often found in other popular magazines including Forbes and Reader's Digest. He eventually published a collection of quotations in a book entitled Cullen Hightower's Wit Kit.
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The best chess-player in Christendom may be little more than the best player of chess; but proficiency in whist implies capacity for success in all these more important undertakings where mind struggles with mind. Poe, Edgar Allan
This quote is about cards · Search on Google Books to find all references and sources for this quotation.
A bit about Poe, Edgar Allan ...
Edgar Allan Poe (January 19, 1809 October 7, 1849) was an American poet, short story writer, editor and critic and one of the leaders of the American Romantics. He is best known for his tales of the macabre and his poems, as well as being one of the early practitioners of the short story and a progenitor of detective fiction, as well as crime fiction in the United States. Poe died at the age of 40, the cause of his death a final mystery. His exact burial location is also a source of controversy.
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It's easy! Just pick the product you like and click-through to buy it from trusted partners of Quotations Book. We hope you like these personalized gifts as much as we do.
Make and then buy your OWN fantastic personalized gift from this quote
When a person is down in the world, an ounce of help is better than a pound of preaching. Bulwer-Lytton, Edward G.
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212 - The Extra Degree
The one extra degree makes the difference. This simple analogy reflects the ultimate definition of excellence. Because it's the one extra degree of effort, in business and life, that can separate the good from the great. This powerful book by S.L. Parker and Mac Anderson gives great examples, great quotes and great stories to illustrate the 212° concept. A warning - once you read it, it will be hard to forget. Your company will have a target for everything you do ... 212°
Click here to buy this »
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Tuesday, July 19, 2005
Zionist behind Iraqi insurgency - Iran's Supreme Leader
Tehran Times: Today, Iran's Supreme Leader spoke out about the Iraqi insurgency:
"There is a will that seeks to obstruct the establishment of stability in Iraq through bitter incidents and blind terrorist acts.
"The Zionists are probably involved in planning these events but the Iraqi nation and government can reconstruct Iraq as deems fit in view of their natural and human resources and through a strong will."
What is amazing about the remark is that it has been Iranian intelligence operatives who have been captured and confessed to organizing the Iraqi insurgency. For instance, WND reported, January 22, 2005:
An agent of Iran's prestigious Jerusalem Force was arrested in Iraq carrying money and planning attacks against U.S. troops.
There is even video proof that Iran has been funding Iraqi Insurgents
Commander of Saddam Hussein's "The Army of Muhammad" Confesses: We Received Aid in Money and Arms from Syria and Iran and transcript.
But an Iraqi blogger, Iraq The Model said it well when he challenged Iran's statements that Iran is not supporting the Iraqi insurgency, saying:
Now, he's trying to say that Iran was NOT supporting the insurgency, hmmm, then what about that meeting between Asad and Khatemi last year in Tehran when they both vowed to support the Iraqi "resistance"?
And what about the recruitment centers where idiots signup under the Mullahs' noses (and with their blessings) to fight and become "martyrs" in Iraq? Not to mention Iran's involvement in providing Muqtada with money and weapons and training his gangs in Iranian camps.
Spiritual leaders should speak the truth, even if you are the Supreme Leader.
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Ceanothus Crassifolius
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Title: Ceanothus Crassifolius
Description: Steel Engravings. Series of plates: Botany of the Boundary.
Citation: Ceanothus Crassifolius. Illustrations. 1859. From Rice University http://hdl.handle.net/1911/35576.
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Australian Bureau of Statistics
Celebrating the International Year of Statistics 2013
ABS Home > Statistics > By Release Date
8752.3 - Building Activity, Queensland, Mar 1996
Previous ISSUE Released at 11:30 AM (CANBERRA TIME) 17/07/1996
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8752.3 - Building Activity, Queensland
© Commonwealth of Australia 2013
Unless otherwise noted, content on this website is licensed under a Creative Commons Attribution 2.5 Australia Licence together with any terms, conditions and exclusions as set out in the website Copyright notice. For permission to do anything beyond the scope of this licence and copyright terms contact us.
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Australian Bureau of Statistics
Celebrating the International Year of Statistics 2013
ABS Home > Statistics > By Release Date
9314.0 - Sales of New Motor Vehicles, Australia, Dec 2012 Quality Declaration
Previous ISSUE Released at 11:30 AM (CANBERRA TIME) 16/01/2013
© Commonwealth of Australia 2013
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Research article
Type II diabetes patients in primary care: profiles of healthcare utilization obtained from observational data
Christel E van Dijk1*, Trynke Hoekstra2,3, Robert A Verheij1, Jos WR Twisk2,3, Peter P Groenewegen1,4, François G Schellevis1,5 and Dinny H de Bakker1,6
Author Affiliations
1 NIVEL, Netherlands Institute for Health Services Research, P.O.Box 1568, Utrecht, 3500 BN, The Netherlands
2 VU University, Faculty of Earth- and Life Sciences, Department of Health Sciences and the EMGO Institute for Health and Care Research, Boelelaan 1085, Amsterdam, 1081 HV, The Netherlands
3 VU University medical center, Department of Epidemiology and Biostatistics and the EMGO Institute for Health and Care Research, Boelelaan 1085, Amsterdam, 1081 HV, The Netherlands
4 Utrecht University, Department of Sociology, Department of Human Geography, P.O.Box 80140, Utrecht, 3508 TC, The Netherlands
5 VU University medical center, Department of General Practice and the EMGO Institute for Health and Care Research, Van der Boechorstraat 7, Amsterdam, 1081 BT, The Netherlands
6 Tilburg University, Scientific Centre for Transformation in Care and Welfare (TRANZO), Tilburg 90153, 5037 AB, The Netherlands
For all author emails, please log on.
BMC Health Services Research 2013, 13:7 doi:10.1186/1472-6963-13-7
The electronic version of this article is the complete one and can be found online at: http://www.biomedcentral.com/1472-6963/13/7
Received:21 May 2012
Accepted:28 December 2012
Published:4 January 2013
© 2013 van Dijk et al.; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background
The high burden of diabetes for healthcare costs and their impact on quality of life and management of the disease have triggered the design and introduction of disease management programmes (DMPs) in many countries. The extent to which diabetes patients vary with regard to their healthcare utilisation and costs is largely unknown and could impact on the design of DMPs. The objectives of this study are to develop profiles based on both the diabetes-related healthcare utilisation and total healthcare utilisation in primary care, to investigate which patient and disease characteristics determine ‘membership’ of each profile, and to investigate the association between these profiles.
Methods
Data were used from electronic medical records of 6721 known type II diabetes patients listed in 48 Dutch general practices. Latent Class Analyses were conducted to identify profiles of healthcare and regression analyses were used to analyse the characteristics of the profiles.
Results
For both diabetes-related healthcare utilisation and total healthcare utilisation three profiles could be distinguished: for the diabetes-related healthcare utilisation these were characterised as ‘high utilisation and frequent home visits’ (n=393), ‘low utilisation, GP only’ (n=3231) and ‘high utilisation, GP and nurse’ (n=3097). Profiles differed with respect to the patients’ age and type of medication; the oldest patients using insulin were dominant in the ‘high utilisation, GP and nurse’ profile. High total healthcare utilisation was not associated with high diabetes-related healthcare utilisation.
Conclusions
Healthcare utilisation of diabetes patients is heterogeneous. This challenges the development of distinguishable DMPs.
Keywords:
Type II diabetes mellitus; Healthcare utilisation profiles; Primary care; Latent Class Analyses
Background
The number of people with type II diabetes mellitus is increasing [1]. Due to the high burden of diabetes in particular and chronic diseases in general for healthcare costs and their impact on quality of life, management of these diseases has become an important issue in health policy in many countries [2]. This has triggered the design and introduction of disease management programmes (DMPs) for type II diabetes mellitus in particular. According to the Disease Management Association of America (DMAA) disease management is defined as a system of coordinated healthcare interventions and communications for populations with conditions in which patients’ self-care efforts are significant. Disease management supports the physician or practitioner/patient relationship and plan of care, emphasizes prevention of exacerbations and complications through the use of evidence-based practice guidelines and patient empowerment, and evaluates clinical, humanistic, and economic outcomes on an ongoing basis with the goal of improving overall health [3]. DMPs are expected to be a solution for the inadequate coordination of care between health services, variation in quality of care and increasing costs for chronic illnesses [4]. However, literature on the design and effects on health and disease outcomes is inconclusive and research currently focuses mainly on refining these issues, such as defining the optimal patient group per programme [5,6].
Remuneration of DMPs differs between European countries: a yearly price for total care for a chronic disease (e.g. Denmark, UK, the Netherlands), a financial bonus for general practitioners (GPs) per patient that is included in a DMP (e.g. France), dedication of one percent of the total health care budget and refunding additional services for DMP-patients (e.g. Germany) [7]. A position paper showed that providing financial incentives to relevant stakeholders is important for facilitating successful implementation of DMPs [4]. Stakeholders may be reluctant to invest in better chronic care if their investments are not accompanied by better payment, or at least equal compensation [8]. Setting the incentives correctly will encourage healthcare providers to efficiently provide healthcare services. In the case of DMPs, varying healthcare demands between and within patients over time might make it problematic to design a good financial compensation system for DMPs. If physicians are paid an equal amount per patient, they might be reluctant to include patients with a high healthcare demand and consequently risk of financial losses or risk of under-treatment [9]. On the other hand, a fee-for-service remuneration system might provide the incentive for physicians to increase the number of services, with a risk for increased costs and over-treatment [9,10]. Incorporating possible heterogeneity of patients’ healthcare demands in the design of DMPs and remuneration of professionals might be a solution to this problem. However, to what extent diabetes patients vary with regard to their healthcare utilisation and costs is largely unknown. El Fakiri et al. distinguished healthcare utilisation patterns for diabetes patients in the Netherlands [11]. However, these patterns were based on the total healthcare utilisation of diabetes patients and did not distinguish between type I and type II diabetes patients, although it is known that healthcare utilisation of type I and type II diabetes patients is different [11-13]. Moreover, DMPs have been focusing mainly on just one disease and not on multiple diseases. Most diabetes patients, however, also suffer from other chronic diseases. Sixty percent of diabetes type II patients have at least one other chronic disease and twelve percent of the patients even have three or more other chronic conditions [14]. Although it might be more realistic to incorporate all utilised healthcare instead of diabetes-related healthcare utilisation only in DMPs, we do not know whether diabetes patients with a high healthcare demand related to diabetes also have a higher demand for healthcare in general and, if so, whether this is equally true for all diabetes patients.
The objective of this study was therefore to empirically develop profiles of healthcare use of type II diabetes patients based on both the total healthcare utilisation and diabetes-related healthcare utilisation in primary care; to determine which patient and disease characteristics determine the ‘membership’ of each profile; and to assess the association between profiles of total healthcare utilisation and diabetes-related utilisation. Such empirically derived profiles may be useful when designing DMPs and planning remuneration of professionals.
Methods
Study design
For the purpose of this retrospective study, primary care utilisation of known diabetes patients was assessed in 2008 by developing profiles of diabetes-related healthcare utilisation and of total health-care utilisation separately. Data from the Netherlands Information Network of General Practice (LINH) were used, which is a representative sample of GP-practices in the Netherlands that provide routinely recorded data from their electronic medical records (EMRs) of all patients listed in their practice. The Dutch healthcare system is very useful for analysing longitudinal data. All Dutch inhabitants are obligatory listed in a general practice and the GP acts as gatekeeper for specialized health care. Therefore, the EMR kept by the GP is the most complete record. The LINH-database holds longitudinal data on morbidity, drug prescriptions and referrals of approximately 90 GP-practices and 350,000 listed patients [15]. The network is a dynamic pool of practices, with each year some minor changes in the composition of practices. Diagnoses are coded by the GPs using the International Classification of Primary Care (ICPC) [16]. LINH is registered with the Dutch Data Protection Authority; data are handled according to the data protection guidelines of the authority. According to the Dutch legislation, ethical approval is not required for observational studies.
For our analyses, we used data from practices that a) participated in both 2007 (for determining known diabetes patients) and 2008 and b) provided recorded year-round data for consultations, prescriptions, and morbidity and referral records in 2008.
Patients were selected for this study if 1) they had consulted their GP for type II diabetes at least once in 2007 and 2) were registered with the practice during the whole year in 2008 and 3) were 18 or older. Type II diabetes patients were selected on the basis of a recorded ICPC-code T90. GPs participating in LINH do generally not record on ICPC sub codes (T90.1 or T90.2), and therefore we could not distinguish between type I and type II diabetes patients on the basis of ICPC-codes. For the purpose of this study, type I diabetes patients were excluded on the basis of having received a prescription of insulin (ATC-code A10A), but not any oral anti-diabetic medication (ATC-code A10B) [14,17]. In total, data of 48 GP-practices and 6,721 type II diabetes patients were included. Reasons for exclusion were 1) incomplete data on consultations (16% of practices), 2) incomplete data on prescriptions (28%) and/or 3) incomplete data on referrals (44%).Overall, these GP-practices were representative of the Dutch GP-practices with respect to degree of urbanisation and region, but not with respect to practice type (overrepresentation of group practices or health centres, underrepresentation of single handed practices).
Healthcare utilisation in primary care
Healthcare utilisation of subjects consisted of contacts with general practice, drug prescriptions and referrals to allied healthcare: primary healthcare and medication. Healthcare utilisation was regarded as diabetes-related, if the care provided was mentioned in the multidisciplinary healthcare standard of the Dutch Diabetes Federation (NDF: “Nederlandse Diabetes Federatie”) [18]. We only took into account healthcare utilisation for which an ICPC code was recorded by the GP. Additional file 1 shows the definition of diabetes related healthcare for known type II diabetes patients with the corresponding ICPC-codes.
Additional file 1. Healthcare utilisation for known type II diabetes patients based on Dutch Diabetes Federation type II diabetes guideline.
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Contacts in general practice were derived from claims data in the EMR in which a distinction was made between consultations in the practice, home visits and telephone consultations with GPs and primary care nurses. The numbers of consultations, home visits and telephone consultations with both GPs and primary care nurses separately, were included in the analyses.
Drug prescriptions were coded using the ATC classification system. Prescriptions mentioned in de NDF-guideline (see Additional file 1) as well as prescriptions related to the diabetes-related health problems were included in the analyses of diabetes-related profiles. A list of included drug prescriptions is provided in Additional file 2. The number of different drug prescriptions based on ATC codes at the 4 digit level was included in the analyses. If a prescription could be captured with an ATC code at the 5 digit level, the ATC code at the 5 digit level was included.
Additional file 2. Prescriptions related to diabetes care.
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Healthcare utilisation of patients with allied healthcare providers was estimated on the basis of GP referrals. Referrals for diabetes-related health problems to a physiotherapist or exercise therapist, dietician and podiatrist were included in the analyses.
Patient and disease characteristics
Patient and disease characteristics included in this study were age (categorised), gender, urbanisation (categorised), diabetes medication type (no treatment, oral treatment or oral treatment and insulin) and comorbidity; all data were derived from the EMR. Comorbidity was divided into several categories of related and unrelated comorbidity according to Struijs et al. (2006) [19]. The following comorbid diseases were regarded as diabetes-related: (with ICPC-codes): heart diseases (K74-K76), stroke (K90), retinopathy (F83), nephropathy (U99) and diabetic foot (K99.06 and N94; deviated from Struijs et al.). Non-related comorbidity included depression (P76), lung diseases (R91, R95, and R96), musculoskeletal diseases (L01-L03, L08, L13, L15, L84, L86, and L89-91), neurological diseases (N86-N88) and cancer (B74, D74, D75, D77, R84, S77, X76, Y77).
Statistical analyses
To analyse the different profiles, Latent Class Analyses (LCA) were performed to identify distinct classes of patients with specific combinations of healthcare utilisation. LCA is a type of cluster analysis used to group patients into k number of unique (otherwise unobserved) categories, where, within each category patients are most similar to each other regarding their healthcare utilisation, and between the categories patients are most different [20-22]. To find the optimal number of categories, a 2–6 class solution was modelled and output was assessed and compared according to a stepwise approach described elsewhere [20,22]. To determine the final solution several model fit indicators were used [23]. The Bayesian Information Criterion (BIC) (where a lower BIC indicates a better fit) and posterior probabilities (where probabilities close to 1 indicates a better classification and posterior probabilities at least 0.8 are advised [24,25]) were used as model fit indicators. Also, we assessed the usefulness and clinical interpretation of each solution. The usefulness was assessed by considering the solutions based on the number of people in each class (hereby rejecting solutions with small groups: minimum N = 200). Mplus was used to perform LCA because within Mplus, LCA can adequately cluster a combination of both categorical (also binary variables) and count data [26]. LCA was conducted for both diabetes-related healthcare utilisation and total healthcare utilisation separately. Each profile was given a label resembling their healthcare utilization. Subsequently, a predictive model was made using multilevel multinomial regression analyses (patients nested in practices) for the diabetes-related healthcare utilisation profiles. In this analysis, it was assessed whether patient and disease characteristics were associated to profile membership. Analyses were performed using STATA, Mplus and MLwiN.
Results
For both the diabetes-related primary healthcare and total primary healthcare, a three-class solution was found to be the best model according to the criteria described in the statistical analyses section above (BIC: 102255, posterior probabilities 0.969, 0.941 and 0.963 respectively and BIC: 148819 and posterior probabilities 0.913, 0.949 and 0.917 respectively). Table 1 presents the descriptive information of the profiles regarding healthcare utilisation. Table 2 describes the diabetes-related healthcare-profiles in terms of patient and disease characteristics.
Table 1. Description of profiles of diabetes-related and total primary healthcare utilisation of diabetes type II patients
Table 2. Patient and disease characteristics of profiles of diabetes-related primary healthcare of diabetes type II patients
Profiles of diabetes-related primary healthcare utilisation
In the first three columns of Table 1 the descriptive information of the profiles regarding diabetes-related healthcare is presented. The first profile ‘high utilisation and frequent home visits’ was mainly characterised by a high number of home visits and telephone consultations by both GPs and primary care nurses. Consultations in the practice were less common for diabetes patients in this profile. The second profile ‘low utilisation, GP only’ was characterised by a relatively low number of consultations. Patients in profile 3 ‘high utilisation, GP and nurse’ were characterised by a high number of consultations in the practice, especially frequent consultations with primary care nurses. Diabetes patients in profile ‘high utilisation and frequent home visits’ had on average 4.7 face-to-face contacts related to diabetes with GPs and/or primary care nurses, patients in profile ‘low utilisation, GP only’ 1.9 and diabetes patients in profile ‘high utilisation, GP and nurse’ had 5.8 contacts.
Table 2 shows the comparison of the three profiles with regard to patient and disease characteristics; Table 3 shows the results of the multinomial logistic regression analysis. Clearly, diabetes patients in profile 1 were significantly older, mostly female, more of them had had a stroke, and had a significantly higher prescription rate of both oral medication only and of the combination of oral and insulin medication. Patients in profile 2 and profile 3 were difficult to distinguish; the only clear difference was the age range and medication usage, with the youngest patients and patients with no diabetes medication classified in profile 2.
Table 3. Results of multilevel multinomial logistic regression analysis predicting cluster membership
Although corrected for patient and disease characteristics, large practice variance still existed with regards to profile membership. For example, for 13 practices all patients were assigned to profile 2, and for one practice all patients were assigned to profile 3. General practices with patients assigned in profile 2 had less often a primary care nurse working in the practice, were more often single handed practices and less often group practices compared to general practices with more variation in patients’ profiles.
Profiles of total primary healthcare utilisation
The last three columns of Table 1 show the descriptive information of the profiles including total primary healthcare. The first profile ‘low utilisation, GP only’ was characterised by GP-consultations in the practice only in combination with a relatively low number of different drug prescriptions and referrals, although 4.2% of the patients were referred to a podiatrist. The second profile ‘medium-high utilisation, GP and nurse’ was also characterised by a high number of GP-consultations in the practice, and by a higher number of consultations with a primary care nurse in the practice. Patients in profile 3 ‘high utilisation’ were particularly characterised by contacts with a GP in practice and home visits by both GP and nurse. Moreover, patients in this profile were characterised by a high prescription rate and referrals to physiotherapists. Diabetes patients in profile ‘low utilisation, GP only’ had on average 4.6 face-to-face contacts with GPs and/or primary care nurses, patients in profile ‘consultation by GP and nurse’ 8.2 and diabetes patient in the profile ‘high utilisation’ 12.0 contacts.
Comparing membership of profiles of diabetes-related primary care with membership of profiles of total primary healthcare utilisation
Table 4 shows the cross tabulation between the diabetes-related primary care profiles and total primary healthcare profiles. Low healthcare utilisation for total primary healthcare (profile 1) was associated with a low diabetes-related healthcare utilisation (profile 2), whereas a high total healthcare utilisation (profile 3) did not necessarily imply a high healthcare utilisation profile for diabetes-related primary healthcare. Comparing the two profiles with high diabetes-related primary healthcare utilisation (profile 1 and 3) for diabetes patients with the total primary healthcare profile ‘high utilisation’ showed that diabetes patients in the ‘high utilisation and frequent home visits’ profile (n=386) were more often women and aged 75 year or older, and diabetes patients in the ‘high utilisation, GP and nurse’ (n=297) more often had diabetes related comorbidity (heart disease and stroke) and unrelated comorbidity (lung- and musculoskeletal diseases).
Table 4. Cross tabulation of profiles of diabetes-related and total primary healthcare utilisation of diabetes type II patients
Discussion
The purpose of this study was to develop profiles based on both total and diabetes-related primary healthcare utilisation and to investigate the association between profiles of total healthcare utilisation and diabetes-related utilisation. For both diabetes and total primary healthcare utilisation, three clearly distinct profiles were found with regard to the type of contacts and type of healthcare provider (GP or primary care nurse). Patient and disease characteristics were, however, not always associated with the membership of each profile. Age and type of medication – no medication, oral medication or oral medication and insulin – were the strongest indicators for diabetes-related primary healthcare profiles. Diabetes patients with a high total healthcare utilisation (profile ‘high utilisation’), were not always patients with a high utilisation pattern for diabetes (‘high utilisation, GP and nurse’), whereas having a low total healthcare utilisation profiles was associated with a low contact rate for diabetes.
Profiles of diabetes-related primary care utilisation
According to the guidelines, type II diabetes patients under supervision of GPs should have four regular check-ups within the practice per year [18]. Of our three diabetes-related primary healthcare utilisation profiles, only the patients in profile ‘low utilisation, GP only’ had on average less than the recommended four contacts for diabetes-related issues. Interestingly, this profile represented almost half of the type II diabetes population in general practice. Principal treatment in secondary care (by an internist – in the Netherlands internists are not seen as primary care specialists) could explain the low number of contacts in primary care for part of this subgroup, but we do not have information available in our dataset to confirm this. However, from a report published by the National Institute for Public Health and the Environment (RIVM), it is known that only a small number of type II diabetes patients is under treatment solely by an internist [27], thereby possibly not providing a full explanation for our findings. Thus, part of the type II diabetes patients did not have the recommended four contacts annually for diabetes-related health problems. In general, the patients in the profile with low frequency of contacts are the youngest in the sample, and also show the lowest prevalence of co-morbidity. This might coincide with well-controlled diabetes, indicating a less-frequent need for primary care consultations [28]. In this respect, our findings showing that patients from a quarter of the practices were all assigned to the 'low utilisation, GP only' profile, are notable. It might be that these practices do not provide adequate care to type II diabetes patients, which may be explained by unavailability of primary care nurses.
We showed that diabetes-related primary healthcare utilisation is heterogeneous. Only one previous study has researched this heterogeneous presentation also [11]. This study, conducted in a much smaller sample of Dutch diabetes patients (around 400 patients), included both type I and type II diabetes patients and total care utilised in both primary and secondary care, found four distinct profiles of healthcare utilisation. Although difficult to compare due to methodological issues, our profiles point to a fairly similar picture; for example, we also find a large subgroup of patients with low healthcare utilisation.
Determinants of diabetes-related primary healthcare profile membership
Diabetes-related primary healthcare profiles could only partly be explained by patient and disease characteristics. Age and use of oral medication and insulin were the strongest predictors for membership in a diabetes-related primary healthcare profile with high utilisation. In agreement with our study, El Fakiri et al. also found, except for the type of diabetes, little effect of possible predictors for the different healthcare profiles. However, this study investigated other predictors than we did [11]. They did show that the patients classified into the profile with the highest number of contacts in general practice more often had comorbidity, which was not consistently found in our study. However, this difference can be explained by the fact that our membership of the profiles was determined on diabetes-related primary healthcare utilisation only. These results illustrate the difficulty of predicting healthcare utilisation for diabetes patients. With the consequence that it is also problematic to develop different DMP for diabetes type II based on patient and disease characteristics, since it does not resemble the healthcare utilisation and therefore costs. In conclusion, these results do not assist health planners in allocating diabetes type II patients in different DMPs. However, differentiations in the remuneration system for patients with differing healthcare demands might also lead to an unnecessary complexity in the design of such DMPs, coinciding with an increase in administrative costs.
Association between diabetes-related and total primary healthcare utilisation
Our study showed that a high total primary healthcare utilisation profile was not generally associated with a high diabetes-related primary healthcare utilisation profile. Both age and the existence of related and unrelated comorbidity were determinants for having both a higher total and diabetes-related primary healthcare utilisation profile. This is in accordance with previous research that showed that both diabetes-related and diabetes-unrelated comorbidity increased the use of medical care in diabetes patients [19]. A recent review also showed a positive association between multiple chronic conditions and healthcare utilisation and expenditure [29]. These results indicate that total primary healthcare utilisation is not a good indicator for disease specific healthcare utilisation. When incorporating total utilised healthcare instead of diabetes-related healthcare utilisation only in a DMP, specific attention should be paid to the role of age and comorbidity, in particular as only these patient characteristics predict high healthcare utilisation for both total and diabetes-related care.
Strengths and limitations
The empirical derivation of profiles of healthcare utilisation of type II diabetes patients as opposed to self-defined profiles provides new insights in healthcare utilisation and demands of type II diabetes patients. A number of points should be considered in our study. First, not all GPs’ actions were recorded in a structured way in their EMR and could for that reason not be incorporated in our analyses. We chose to include only the information that was recorded in a concise and structured way by all GPs. This meant that we unfortunately were unable to include information about the exact content of the consultations in general practice and do not know whether for example lifestyle advice was given, nor were structured clinical outcome data available (e.g. glycated haemoglobin level or blood pressure). This then makes it impossible to make inferences about the effect of the different primary healthcare utilisation profiles on patient outcomes. This should be addressed in future research. Second, no referral to a physiotherapist is needed since 2006 and therefore the number of patients visiting a physiotherapist was underestimated. Research shows that mostly patients with acute problems (instead of chronic problems) visit a physical therapist on their own initiative, which is not often the case with diabetes patients [30]. In some practices no primary care nurse was working in the practice, and therefore these patients may not be assigned to profiles which are largely described by contacts with primary care nurses. Additional analyses (available upon request by the first authors) limited to practices with a primary care nurse showed similar effects of determinants of diabetes-related primary healthcare profile membership, although the profile ‘high utilisation, GP and nurse’ in comparison to the profile ‘low utilisation, GP only’ showed slightly underestimated effects of the main medication type for diabetes compared to the model with all practices. In addition, healthcare utilisation as presented in this study does not reflect the ideal or needed level of healthcare.
Implications of the findings
DMPs are expected to be the solution for the inadequate coordination of care, variation in quality of care and increasing costs for illnesses [4]. The design of such standardised programmes ultimately requires a homogeneous patient population or in case of a heterogeneous population at least one that is easily explained by clear patient and disease characteristics. Unexplained heterogeneity in healthcare demands of these patients, therefore, means that a standardised programme might be insufficient or inadequate for some patients. Moreover, a heterogeneous patient population with diverse healthcare demands might cause physicians to be reluctant in the inclusion of patients with high healthcare demands. The issue of multimorbidity is also predominant in type II diabetes patients. This results in the fact that a large part of the healthcare utilisation of these patients might not be included in diabetes DMPs if these DMPs would focus exclusively on diabetes neglecting other existing health problems [14]. With a non-explained heterogeneous diabetes type II population and large non-diabetes-related primary healthcare utilisation, health planners might consider putting more emphasis on case management instead of disease management.
Conclusions
In conclusion, we have shown that primary healthcare utilisation of diabetes patients is heterogeneous, which could be captured in three distinct profiles of diabetes-related and total healthcare utilisation. The diabetes-related profiles were only partly explained by patient and disease characteristics, posing difficulties for the future development of distinguishable disease management programmes. Further, we have shown that total primary healthcare utilisation is not a good indicator for diabetes-related primary healthcare utilisation for diabetes patients. This fact should also be taken into account in the remuneration system of DMPs.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
CD and TH were involved in the conception of the research question and in analysing the data. All authors had full access to all the data and contributed to the interpretation of the data. CD and TH drafted the manuscript, which was reviewed by all authors. All authors read and approved the final manuscript.
Acknowledgements
We would like to thank all staff at the practices of the Netherlands Information Network of General Practice for their cooperation.
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Gray wolves under the Endangered Species Act: Distinct population segments and experimental populations
Gray wolves under the Endangered Species Act: Distinct population segments and experimental populations
This article has been reviewed by the following Topic Editor: Peter Saundry
The wolf was among the first animals protected under the Endangered Species Preservation Act, a predecessor to the current Endangered Species Act (ESA). In 1978 the gray wolf was listed as endangered in all of the conterminous 48 states except Minnesota, where it was listed as threatened. With the exception of experimental populations established in the 1990s, the protections for the gray wolf have been diminishing since that date, as wolf populations have increased in some areas. The use of distinct population segments (DPSs), a term created in the 1978 ESA amendments, has played a role in that reduced protection. DPSs allow vertebrate species to be divided into distinct groups, based on geography and genetic distinctions.
ESA protection for DPSs has changed back-and-forth since the first DPSs—Western and Eastern—were proposed in 2003. In 2004, the U.S. Fish and Wildlife Service (FWS) determined that those DPSs no longer needed the protection of the ESA and so were delisted. The Western and Eastern DPS designations and delistings were nullified by courts. In 2007, FWS designated and delisted the Western Great Lakes DPS, and in early 2008, FWS designated and delisted the Northern Rocky Mountains DPS. However, courts found both delistings flawed and vacated the rulemaking. In December 2008 FWS returned the wolves in the Western Great Lakes and parts of the Northern Rocky Mountains areas to their former protected status, eliminating the DPSs. That same rulemaking redesignated wolves in south Montana, southern Idaho and all of Wyoming as “nonessential experimental populations,” which they were prior to the DPS efforts. The rulemaking took effect on December 11, 2008, meaning it appears to be outside the scope of White House Chief of Staff Rahm Emanuel’s memorandum of January 20, 2009, halting the effect of all non-final regulations pending further review. An additional rulemaking to delist Western Great Lakes and Northern Rocky Mountain wolves (except within Wyoming) was announced on January 14, 2009, but was not published.
This report analyzes the DPS designation process as it is applied to the gray wolf. It also examines experimental populations of wolves under the ESA and their protections. As part of its oversight responsibilities, Congress has conducted hearings on the Fish and Wildlife Service’s application of science to endangered species.
Background and regulatory history
The history of gray wolf protection is interconnected with the history of the Endangered Species Act (ESA) (16 U.S.C. §§ 1531- 1543). Gray wolf protection began at the nascency of the ESA, when it was one of the first species covered under the Endangered Species Protection Act of 1966.[1] As the ESA has been amended, so has gray wolf protection. The act provides the basis for determining which species are threatened and endangered, and how those listed species will be protected. Amendments allow consideration of distinct groups within species for protection. The act allows introduction of experimental populations to areas where the species no longer exists, and provides regulatory protections for that introduction. Each of these elements will be discussed in this report generally, and more specifically in the context of gray wolf protection.
For centuries, wolf populations have been under attack by humans. The effort to reduce or eliminate the species was designed to protect humans from a perceived direct threat to humans or to protect livestock or favored game species. Wolves were eventually eliminated in most states in an effort supported by the science community at the time. But coinciding roughly with the forester Aldo Leopold’s essay, “Killing the Wolf,” in A Sand County Almanac in 1948, this view began to change. Leopold wrote:
I was young then, and full of trigger-itch; I thought that because fewer wolves meant more deer, that no wolves would mean hunters’ paradise. But after seeing the green fire die, I sensed that neither the wolf nor the mountain agreed with such a view.... Since then I have lived to see state after state extirpate its wolves. I have watched the face of many a newly wolfless mountain, and seen the south-facing slopes wrinkle with a maze of new deer trails. I have seen every edible bush and seedling browsed, first to anaemic desuetude, and then to death.
In 1967 when the gray wolf was listed under the first version of the Endangered Species Act, it was listed in two subspecies, the eastern timber wolf, and the northern Rocky Mountain wolf.[2] In 1978 the gray wolf was relisted as endangered at the species level throughout the lower 48 states, with the exception of Minnesota, where it was listed as threatened.[3] In the 1990s actions were taken to reintroduce the wolf into areas where it had been eradicated. Experimental populations were introduced into the Yellowstone area and central Idaho,[4] and in Arizona, New Mexico and Texas.[5] Efforts to protect the wolf have always been controversial, however. The Fish and Wildlife Service (FWS) reports receiving, and denying, “several petitions” to delist the wolf in all or part of the 48 states.[6] Additionally, in 1987 legislation was introduced to remove the gray wolf from the ESA protected list.[7] The amendment failed.
Wolf populations: A taxonomic view
Like many large mammals, such as bears (Ursus arctos), mountain lions (Felis concolor), and white-tailed deer (Odocoileus virginianus), gray wolves (Canis lupus) have a complicated, even convoluted, taxonomic history. Variations in color, size, and bone structure have led some mammalogists to name wolves in various areas as different subspecies or populations, where other credible experts would see only a single species with variability. Here are scientific definitions of a few key terms:
• A population is a group of “organisms of the same species that inhabit a specific area.”
• A species is a “naturally [occurring] population or a group of potentially interbreeding populations that is reproductively isolated (i.e.,cannot exchange genetic material) from other such populations or groups.”
• A subspecies is a “taxonomic category that subdivides species into morphologically distinct groups of individuals representing a step toward the production of a new species, although they are still fully capable of interbreeding. Subspecies are usually geographically isolated.”
• Taxon, or the plural taxa, is defined as: “a grouping of organisms given a formal taxonomic name at any rank: species, genus, family, order, class, division, phylum, or kingdom.”[8]
These terms may appear clear; however, there are no simple measures to draw unequivocal distinctions. Biologists commonly divide their colleagues into “lumpers” and “splitters,” based on their inclinations in classifying organisms. As the names suggest, lumpers are those who tend to minimize differences, and see one or a few species, perhaps with some variations, while splitters would tend to emphasize those differences, dividing a species into many subspecies, or populations. For wolves, which are (or were) found in temperate and polar areas throughout the Northern Hemisphere, some observers (splitters) would argue that there are as many as 24 subspecies in North America and eight in Europe and Asia.[9] More recently, lumpers have had the upper hand, and FWS recognizes two species (gray and red wolves), and divides the gray wolf into six “distinct population segments,” based in part on administrative and procedural criteria.[10]
While confusing to the non-scientist, this muddled state of taxonomic affairs is entirely predictable for several reasons. First, wolves are extremely wide-ranging, both as a species and as individuals, so interbreeding among them could certainly muddy the picture. Second, the consistency of variations over time is hard to determine, since long-range studies of long-lived species are rare. Third, evolutionary change does not stop, and wolves are an adaptable species, as shown by their behavior and by their presence in a tremendous variety of ecosystems.[11] If FWS scientists’ choice of state boundaries to delineate wolf populations is criticized as arbitrary, the debate among academic scientists also has an air of informed judgment — and there is no reason to predict that either debate will end any time soon.
Experimental populations
In 1982 Congress added the concept of experimental populations to the ESA as a way of reintroducing species without severe restrictions on the use of private and public land in the area.[12] Experimental population designations are sometimes referred to as Section 10(j) rules. The practice allows introduction of a species outside its current range to restore it to its historic range.
Two criteria must be met for an experimental population to comply with the law. First, the Department of Interior (DOI) must have authorized the release of the population. Second, the population must be wholly separate geographically from other animals of that species.[13] Congress required the separation so that the introduced population could be clearly distinguished.
Members of an experimental population are considered to be threatened under the act, and thus, can have special rules written for them.[14] In fact, Congress referred to special rules for experimental populations as a way to reduce public opposition to the release of certain species, using the red wolf as an example.[15] Congress suggested in a report that the special regulations could allow killing members of the species:
The committee fully expects that there will be instances where the regulations allow for the incidental take of experimental populations .... The committee also expects that, where appropriate, the regulations could allow for the directed taking of experimental populations. For example, the release of experimental populations of predators, such as red wolves, could allow for the taking of these animals if depredations occur or if the release of these populations will continue to be frustrated by public opposition.[16]
Unlike distinct population segments (DPSs), experimental populations may not necessarily have the same protections under the ESA. Section 10 requires FWS to determine whether the experimental population is of a species that is in imminent danger of extinction. That decision is based on whether the loss of the population would appreciably diminish the species’ prospect for survival. If so, the experimental population is deemed essential and is treated as an endangered species. Currently, there are no essential experimental populations. If it is deemed nonessential, the experimental population is treated as a species that is proposed for listing as threatened or endangered. There is no critical habitat designation for an experimental population if it is nonessential. Also, federal actions that may take a member of the population do not require a Section 7(a)(2) consultation under the ESA, unless the species is in a national wildlife refuge or a national park, although agencies are required to confer under Section 7(a)(4).
Examples of species with nonessential experimental populations are the Colorado pikeminnow (or squawfish), the southern sea otter, the gray wolf in the Southwest and in the Yellowstone area, theblack-footed ferret, and the whooping crane.
Experimental populations of gray wolves
Figure 1. Reintroduction Zone of the Central Idaho Experimental Population Source: 59 Fed. Reg. 60281 (November 22, 1994), as modified by CRS.
Figure 2. Reintroduction Zone of the Yellowstone Experimental Population Source: 59 Fed. Reg. 60281 (November 22, 1994), as modified by CRS.
Despite near-eradication of the wolf in the lower 48 states, some of the wolf’s old territories survived, though in a highly modified form. At the end of the 20th century, FWS planned to reintroduce the wolf to parts of its historic range, using the experimental population provisions of the ESA. No reintroduction was more controversial than that in the greater Yellowstone ecosystem, where all other large vertebrates were still present, and where many scientists agreed that elk populations—a favorite wolf prey—had reached harmful levels. In 1995 and 1996 FWS released 66 gray wolves from Canada in Yellowstone and central Idaho. (See Figure 1 and Figure 2).
When wolves were returned, the science community was nearly giddy anticipating the potential effects from a first-ever return of a major predator to a nearly intact ecosystem. The excitement was intense partly because the Yellowstone area was already well studied, with long-term data on many species, including both competitors (e.g., coyotes and, to some extent, grizzlies) and potential prey (e.g., elk, moose, and bison). As scientists had expected, wolves had a profound effect on elk, but there is also evidence of effects that were less predictable — on aspens, cottonwoods, beavers, beetles, mice, red foxes, ravens, and voles, among others.[17] However, the road to the reintroduction was, and still is, fraught with litigation and controversy.
Wolves also had been exterminated in the southwest. FWS recognized a separate subspecies, the Mexican wolf (Canis lupus baileyi), which was probably gone from the United States but found in very low numbers in Mexico. After a cooperative and successful captive breeding program of wolves obtained from Mexico, reintroduction was begun in 1998, in an area centered in the Apache National Forest in Arizona and the Gila National Forest in New Mexico. In both the Yellowstone and the Southwest cases, litigation was a major factor in the reintroduction effort.
Yellowstone litigation
While the recovery plan for the Northern Rockies gray wolf acknowledged that the species may have to be reintroduced into the area around Yellowstone National Park, that decision was controversial. Suit was filed to compel FWS to bring some wolves to Yellowstone. However, the court ruled the action was moot as it could not compel FWS to act,[18] and an appropriations rider in 1992 blocked any funding for bringing the wolf to the area.
In 1995 and 1996 FWS released 66 gray wolves from Canada in Yellowstone and central Idaho. A man accused of violating the ESA for killing one member of the Yellowstone experimental population argued that he had killed a Canada wolf, which was not an endangered species. This argument failed. The Ninth Circuit upheld the regulations for the experimental population, holding that once the wolves from Canada were introduced into the park, they became protected under the ESA.[19]
Another lawsuit argued that because the Yellowstone experimental population may interact and breed with the few lone wolves in the area the experimental population designation violated Section 10(j) of the ESA. (The lone wolves may have been remaining wolves that somehow survived extermination, feral wolves, or wolves naturally dispersing from farther north.) Section 10(j) requires that experimental populations must be “wholly separate geographically from nonexperimental populations of the same species.”[20] The district court ruled that the Yellowstone population would have to be removed. However, the Tenth Circuit overruled the decision.[21] The court rejected the argument that the legislative history of experimental populations (as discussed earlier in this report) meant that the experimental population must be separate from every naturally occurring individual animal. The court deferred to the DOI management plan for the reintroduction, finding it did not conflict with the statute.
A more recent claim disputed FWS management of the wolves. A rancher argued the agency failed to control wolves that were preying on livestock. After FWS killed three wolves, including the lead male wolf of the offending pack, no more depredations were found. The court dismissed the claims on procedural grounds.[22]
After courts ruled that FWS had not satisfied the requirements of the ESA in making distinct population segments in the Northern Rockies area, FWS re-established the non-essential experimental populations of the Yellowstone and Central Idaho areas, now spread to all of
Wyoming, the southern half of Montana, and all but the northern panhandle of Idaho.
Southwest litigation
The reintroduction of the wolf to the Southwest was no less controversial. Ranchers sued, claiming the action violated the National Environmental Policy Act (NEPA), as well as the ESA. The court found for FWS, even though livestock owners and FWS had different estimates as to the impact of the wolves on domesticated stock.[23]
Once the wolf was reintroduced to the Southwest, environmentalists sued FWS for not acting to modify the reintroduction regulations.[24] The action was dismissed as moot. The area remains a center of intense public controversy about wolves.
Species and distinct population segments
If the scientific community is somewhat inconsistent in identifying species, the law has fared no better. The ESA definition of species has changed since the early days of the act. In 1973 the definition included “any subspecies of fish or wildlife or plants and any other group of fish or wildlife of the same species or smaller taxa in common spatial arrangement that interbreed when mature.”[25] In 1978, Congress amended that definition to include the term distinct population segment (DPS) and was limited to vertebrate DPS's only.[26] The change was controversial.
The General Accounting Office (GAO) (now the Government Accountability Office) recommended limiting the definition of species to higher taxonomic categories than populations, and exclude all distinct populations, including geographically separated populations.[27] GAO proposed the following definition: “The term ‘species’ includes any subspecies of fish, wildlife, or plants.”[28] GAO found the 1973 definition to be overly broad:
We found that Interior’s Fish and Wildlife Service is listing populations of species in limited geographical areas as endangered or threatened instead of listing the entire species. This has occurred because the Service has interpreted the definition of “species” to include populations, regardless of their size, location, or total numbers. Using the Service’s interpretation of the term, squirrels in a specific city park could be listed as endangered even though there is an abundance of squirrels in other parks in the same city and elsewhere. Such listings had increased the number of potential conflicts between endangered and threatened species and federal, state, and private projects and programs.[29]
Congress did not follow the GAO recommendation. It agreed with FWS that the service needed to be able to adopt different management practices for different populations, based on their need. A Senate committee report discussing populations said “the committee agrees that there may be instances in which FWS should provide for different levels of protection for populations of the same species,” although it advised the practice be used “sparingly and only when the biological evidence indicates that such action is warranted.”[30]
Thus, Congress revised and limited the definition of species in 1978 by eliminating taxonomic categories below subspecies from the definition, except for vertebrates.[31] The revised, and still current, definition is: “any subspecies of fish or wildlife or plants, and any distinct population segment of any species of vertebrate fish or wildlife which interbreeds when mature.”[32] However, the phrase distinct population segment had no meaning in the scientific community outside of the ESA, and was not used in endangered species listings for nearly two decades.
Regulatory history of distinct population segments
A DPS generally refers to a portion of a listed species, separated from the rest of the species by genetic distinction and range. The legislative history offers two examples of when different protection is appropriate within a species: 1) when a U.S. population of an animal is near extinction even though another population outside the United States is more abundant; and 2) where conclusive data have been available only for certain populations of a species and not for the species as a whole.[33]
In 1996 a policy regarding DPS was introduced by FWS (hereinafter referred to as “the Policy”).[34] The Policy contains the criteria that must be met for protection of a species at the population level. First, the population segment must be discrete. Factors considered to determine discreteness are whether the segment is “markedly separated from other populations of the same taxon as a consequence of physical, physiological, ecological, or behavioral factors.”[35] Discreteness can also be found if the population is delimited by international governmental boundaries. Although state boundaries are frequently used to describe a DPS, they cannot be used under the Policy to determine discreteness.
Next, the population segment must be found to be significant, meaning its demise would be an important loss of genetic diversity.[36] Four factors are listed in the policy for determining a species’ significance: 1) persistence of the segment in an ecological setting unusual or unique for the taxon; 2) evidence that loss of the DPS would result in a significant gap in the range of the taxon; 3) evidence that the DPS represents the only surviving natural occurrence of a taxon within its historic range; or 4) evidence that the DPS differs markedly from other populations of the species in its genetic characteristics. Genetic evidence is allowed to be considered but is not required. The policy indicates that “available scientific evidence of the discrete population segment’s importance” will be considered in finding significance, but does not specify the best available scientific evidence.
If a species is found to be both discrete and significant, then its status is reviewed to see whether it is endangered or threatened. A DPS species is reviewed to determine whether it should be listed under exactly the same procedures as any other listing. The listing determination is to be based solely on the “best scientific and commercial data available.”[37]
Pros and cons of distinct population segments
Agency efficiency and focus were two intended benefits of DPSs, according to the Policy. The Policy said determining DPSs will “concentrate ... efforts toward the conservation of biological resources at risk of extinction.”[38] The Policy suggested the practice of using DPSs could help endangered species by focusing on smaller groups:
This may allow protection and recovery of declining organisms in a more timely and less costly manner, and on a smaller scale than the more costly and extensive efforts that might be needed to recover an entire species or subspecies. The Services’ [FWS & the National Marine Fisheries Service’s] ability to address local issues (without the need to list, recover, and consult rangewide) will result in a more effective program.[39]
The FWS has followed Congress’s admonition to apply the practice “sparingly.” According to FWS, only 39 of the 374 vertebrates listed under the ESA are DPSs.
Some have criticized DPSs as being used to remove ESA protections from certain segments of a listed species. In three cases the listing classification of DPSs appears to be used solely to remove animals from protected status. The DPS designation and the delisting occurred on the same day in the same Federal Register notice. Those three cases are:
• Columbian white-tailed deer, Douglas Co. DPS — July 24, 2003;
• Gray wolf, Western Great Lakes DPS — February 8, 2007; [40]
• Grizzly bear, Yellowstone DPS — March 29, 2007. [41]
• Gray wolf, Northern Rocky Mountains DPS—February 28, 2008.[42]
One district court suggested that this practice was contrary to the ESA, which was brought before it on a challenge to the Western Great Lakes DPS designation and delisting.[43] The court said the ESA did not unambiguously allow a species to be designated as a DPS at the same time it was delisted, noting that a goal of the act was to protect species. The court vacated the designation and the delisting, and remanded the matter to the FWS.
In other examples, the species has become downlisted (having its status dropped from endangered to threatened) the same day as being designated a DPS:
• Gray wolf, Western DPS — downlisted April 1, 2003;
• Gray wolf, Eastern DPS — downlisted April 1, 2003.
However, for many more species, the designation of a DPS improved its protection status. Here are some examples:
However, for many more species, the designation of a DPS increased its protection status, by protecting a group, even though the species as a whole was not covered by the act.[44] Here are some examples:
• California Bighorn Sheep, Sierra Nevada DPS — listed as endangered January 3, 2000;
• Canada Lynx, contiguous U.S. DPS — listed as threatened March 24, 2000;
• Atlantic Salmon, Gulf of Maine DPS — listed as endangered November 17, 2000;
• Dusky Gopher Frog, Mississippi DPS — listed as endangered December 4, 2001;
• Pygmy Rabbit, Columbia Basin DPS — listed as endangered March 5, 2003;
• California Tiger Salamander, Sonoma County DPS — listed as endangered March 19, 2003;
• Northern Sea Otter, Southwest Alaska DPS — listed as threatened August 9, 2005.
Gray wolf distinct population segments
Since the issuance of the Policy, FWS has pursued dividing the gray wolf into more DPSs.[45] These DPSs are considered separately from the experimental populations. All designations were challenged in federal court (the lawsuits will be discussed later in this report), and each court rejected the FWS’s action. In 2003 FWS divided wolves into three DPSs: Western, Eastern and Southwestern.[46] This rulemaking then downlisted the Eastern and Western DPSs from endangered to threatened under the ESA. At the same time, gray wolves were removed from protection in 14 southern and eastern states where they have not occurred in recent times. This rulemaking was vacated by two federal courts.
Figure 3. Northern Rocky Mountain Gray Wolf DPS Area Showing Individual Wolf Pack Territories. Source: 73 Fed. Reg. 10517 (February 27, 2008).
After the 2003 DPS rulemaking was nullified, two other DPSs of the gray wolf were proposed: Northern Rocky Mountain, and Western Great Lakes.[47] The Western Great Lakes population was declared a distinct population segment in 2007, and delisted at the same time.[48] That final rule was vacated by the District Court for the District of Columbia.[49] On the same date as the Western Great Lakes rule, FWS proposed designating the Northern Rockies DPS and delisting the population (See Figure 3), except for the population in Wyoming because Wyoming’s state laws were found not to provide enough protection for the wolf.[50] Wyoming’s population was subsequently delisted.[51] Following litigation, in September 2008, FWS voluntarily vacated the determination that both designated the Northern Rockies DPS and delisted it, returning wolves in that area to the list of endangered and threatened species.[52]
As a result, from 1978 to 2008 wolves in the United States were listed in all of the available categories for a vertebrate species: (a) never listed (Alaska); (b) delisted (the DPSs described above); (c) experimental (Southwest; Yellowstone and Central Idaho); (d) threatened (Minnesota); and (e) endangered (every wolf that was not a DPS, experimental population, or in Minnesota).
As noted above, wolf taxonomy is complex, but the choices made in distinguishing DPSs have significant effects. In particular, if FWS agrees with taxonomic lumpers, and recognizes only a few DPSs, then the recovery task becomes simpler than it would be if many DPSs are recognized. Some observers would argue that because wolf sightings in Northern New York and New England are very rare, those animals should represent a portion of a DPS which extends into Canada, and therefore entitle them to recovery in their own plan.[53] In addition, whether a few lone wolves inhabit the area or simply visit it occasionally, reintroduction proponents would further argue that an abundance of apparently suitable habitat and high prey populations make the region suitable for recovery efforts.
Regardless of the merits or demerits of this argument and the surrounding facts, Northeast wolf designation hinges on a taxonomic assessment that the language of the ESA elevates far beyond an academic debate between lumpers and splitters. FWS considers the wolves (if any) in that area to be part of the same DPS of much larger area.[54] And if a portion of the DPS reaches its recovery goals, FWS — arguing that it does not have legal responsibility to recover a species throughout its historic range — would be relieved of the burden of recovering the species in the remainder of the DPS’s range. Thus, the decision of whether to mount an effort to recover relatively rare wolves in the remote Northeast (and certain other areas) depends on two questions, one legal and one scientific:
• Does ESA require that a species be recovered in all or most of the remaining areas of suitable habitat?
• Do (or did) the wolves of the Northeast constitute a DPS, and if none remain, should presumably genetically similar wolves in nearby parts of Canada be used to repopulate the area?
Litigation regarding western and eastern DPSs.
The rule designating three DPSs of the gray wolf (Western, Eastern, and Southwestern) and downlisting the Western and Eastern DPSs in 2003 was challenged in two federal district courts. The plaintiff environmental groups before the District Court for the District of Oregon disputed how the DPS ranges were designated. They argued that FWS considered only where the wolves were currently located when determining their viability. This allowed FWS to count wolves only in the areas they occupied. However, areas outside of the wolves’ current range were suitable habitat, according to FWS, although no wolves were present, but FWS did not include those areas in defining the DPS ranges. The plaintiffs argued that this method was contrary to the ESA and prior caselaw, because the act requires that a species is endangered if it is at risk of extinction in “all or a significant portion of its range.” The court agreed that FWS had violated the ESA by equating the wolves’ current range with a “significant portion of its range.”[55] The court vacated the rule, effectively eliminating the three DPSs.
The other suit was before the District Court for the District of Vermont, which issued a decision eight months after the Oregon court. The plaintiffs in Vermont challenged the final rule’s designation of an Eastern DPS, which changed the proposed rule’s two DPSs for that area: a Northeast DPS and a Western Great Lakes DPS. The court found procedural flaws and also that the FWS failed to consider the “significant portion of its range” in a way consistent with the ESA.[56] The court criticized FWS’s method before vacating the rule: “The FWS simply cannot downlist or delist an area that it previously determined warrants an endangered listing because it ‘lumps together’ a core population with a low to non¬existent population outside of the core area.”[57]
Litigation Regarding the Northern Rocky Mountains DPS
In July 2008 the District Court for the District of Montana issued a preliminary injunction halting the effectiveness of the FWS delisting of the Northern Rockies DPS.[58] The delisting had added Wyoming to Idaho and Montana as states that had adequate wildlife management programs to support populations above recovery levels.[59] The July order rejected FWS’s contention that there was genetic exchange between the Yellowstone experimental population and the Northern Rockies animals. (Figure 3 shows the distribution of wolf packs in the two areas.) Without sufficient genetic exchange, the isolated wolf populations would not be genetically diverse enough to avoid inbreeding, and therefore could not be termed “recovered.” The court also found that the states’ management plans did not seem adequate to support wolf recovery levels. The order reinstated the wolf as endangered until final disposition. In September 2008, FWS voluntarily moved to withdraw the final rule that both designated the Northern Rockies DPS and declared it recovered.[60] Wolves in that area returned to being nonessential experimental populations with special regulations allowing takes.[61] In January 2009, FWS announced that it planned to delist the Northern Rockies population, with the exception of the Wyoming population.[62] (See below at “Section 4(d) Rules for Yellowstone and Idaho Experimental Populations.”) It appears that FWS will not issue the rule, however, in light of the January 20, 2009 memorandum by White House Chief of Staff Rahm Emanuel halting publication of rules that were not reviewed by an official appointed by President Obama.[63]
Litigation Regarding the Western Great Lakes DPS
In September 2008 the District Court for the District of Columbia vacated the final rule that designated the Western Great Lakes gray wolf as a DPS and delisted that DPS.[64] Unlike the holding in the Northern Rockies DPS case, this decision focused on the procedure, not the science, behind the designation and delisting rule. The plaintiffs claimed that FWS had violated the act by issuing the designation and delisting simultaneously. FWS argued that the ESA “unambiguously” supported its rulemaking. The court found the ESA was not unambiguous, in light of the act’s purpose in conserving species. The action was remanded to the agency to find a “reasonable explanation” for its interpretation that the ESA supports its designation/delisting rule.[65]
FWS reinstated the Western Great Lakes DPS as an endangered species in December 2008.[66] In January 2009, FWS announced it was delisting the DPS,[67] but the rulemaking was halted.[68] Those wolves are considered endangered except for in Minnesota where they are listed as threatened.
Section 4(d) Rules
Special rules may be issued for both distinct population segments and experimental populations. When a species is designated as threatened, rather than endangered, FWS has discretion to issue special rules for that species. (Endangered species have protections that are expressly stated in the act.) Under Section 4(d) of the ESA, FWS may decide how the protections of the act related to taking, or harming of the threatened species, are applied. These regulations are called Section 4(d) rules or special rules. A DPS is treated like a species under the act; therefore, the special regulation provision also applies to threatened DPSs. Under Section 10(j)(3)(C), experimental populations are treated as threatened species, and so are also covered under this provision. Special rules provide customized protection that FWS deems necessary and advisable for the species’ conservation. FWS is not limited in determining the protections and can allow the full range of protections in the act to threatened species. The special rules are promulgated in Title 50 (Part 17) of the Code of Federal Regulations.
Section 4(d) Rules for Gray Wolves.
According to FWS, Section 4(d) rules are intended to reduce conflicts between the provisions of the act and needs of people near the areas occupied by the species. This type of special rule has been in effect for the threatened gray wolves in Minnesota for many years, and was extended to gray wolves in other states, when and where the wolf was downlisted. Under the rule for Minnesota, individual wolves that have preyed on domestic animals can be killed by designated government agents. FWS asserts that this rule avoids even larger numbers of wolves being killed by private citizens who otherwise might take wolf control into their own hands.[69]
In 2003 as part of the rulemaking that was vacated, FWS issued Section 4(d) rules for two DPSs: Eastern and Western. The special rules would have allowed individuals to kill Western DPS wolves in the act of attacking livestock on private land, and to harass wolves near livestock. Permits to kill wolves could also be issued to landowners who showed wolves routinely were present and formed a significant risk to livestock. FWS said that, as in Minnesota, the rule would “increase human tolerance of wolves in order to enhance the survival and recovery of the wolf population.”[70] Michigan and Wisconsin citizens would be able to kill any wolf within one mile of killed livestock, and in other Eastern states beside Minnesota, any lethal measures could be used within four miles of such a site. [71] This rule was vacated, as discussed earlier in Litigation Regarding Western and Eastern DPSs.
Section 4(d) rules for Yellowstone and Idaho experimental populations
In 2005, after the FWS found that the wolf population had exceeded its minimum goals of 30 breeding pairs for Yellowstone and Central Idaho, it issued a rule to manage wolves where they had an unacceptable impact on ungulate populations.[72] This 2005 Rule modified the provisions put in effect when the wolves were first introduced, which stated that “wolves could not be deliberately killed solely to resolve predation conflicts with big game.”[73] The 2005 Rule allowed States and Tribes in the area to kill wolves where it was shown they were adversely affecting the populations of deer, antelope, elk, big horn sheep, mountain goats, bison, or moose in the area. Before the states and tribes could act, they were required to submit the plan for peer review, public comment, and FWS approval. Data at the time, from many sources cited by FWS, showed that wolf predation was “unlikely to be the primary cause of a reduction of any ungulate herd or population in Idaho, Wyoming, or Montana.”[74] FWS reported that more wolves were killed in 2007 and 2008 than were cattle.[75] In 2008, the wolf population in the area was estimated at 1,463.[76]
In 2008 FWS changed the special rule.[77] FWS determined that the definition of unacceptable impact had to be altered, as wolves were not the primary cause in ungulate population decreases. Accordingly, the definition was modified to mean: “Impact to a wild ungulate population or herd where a State or Tribe has determined that wolves are one of the major causes of the population or herd not meeting established State or Tribal population or herd management goals.”[78] Public and peer reviews are still required. The plan allows a state to kill wolves, provided the experimental population does not go below 20 breeding pairs in the state.[79]
The 2008 Rule also expands the provision for killing wolves when they are in the act of attacking livestock or dogs. The 2005 Rule allowed an individual to “take” a wolf that was in the act of attacking stock animals or dogs on private property. The 2008 Rule allows individuals to take wolves that are in the act of attacking livestock or dogs on public lands as well, except for National Environmental Policy Act (NEPA) property.[80]
References
1. ^ P.L. 89-669, 80 Stat. 926 (October 15, 1966).
2. ^ The first list of endangered species included the timber wolf (Canis lupus lycaon) and the red wolf (Canis niger, now called Canis rufus). 32 Fed. Reg. 4001 (March 11, 1967).
3. ^ 43 Fed. Reg. 9607 (March 9, 1978).
4. ^ 59 Fed. Reg. 60252 (November 22, 1994).
5. ^ 63 Fed. Reg. 1752 (January 12, 1998).
6. ^ 68 Fed. Reg. 15803, 15807 (April 1, 2003).
7. ^ The proposed legislation would have amended an authorizations act to fund the ESA. H.Amdt. 576 (100th Congress): “(b) Exception to Listing. — Upon enactment of this subsection of this Act, the gray wolf, Canis Lupus, shall not be considered an endangered or threatened species under the Endangered Species Act of 1973.”
8. ^ All the definitions are from Henry W. Art (ed.), The Dictionary of Ecology and Environmental Science (New York: Henry Holt and Co. 1993).
9. ^ See discussion, citing various authors, in L. David Mech, The Wolf: The Ecology and Behavior of an Endangered Species, pp. 29-31 ( Garden City, NY: Natural History Press 1970).
10. ^ The wolves of Alaska, which have never been listed under the ESA, would constitute a seventh population, with equally uncertain boundaries.
11. ^ For example, should global warming proceed and arctic snow cover diminish, will the genes for white coats diminish in the arctic wolves? That may be likely, since more brightly colored wolves would be at a disadvantage in much of the year and over a growing area. Natural selection would then tend to disfavor these animals and their offspring.
12. ^ P.L. 97-304 §6(6), 96 Stat. 1424; 16 U.S.C. § 1539(j).
13. ^ ESA § 10(j); 16 U.S.C. § 1539(j).
14. ^ ESA § 10(j)(2)(C); 16 U.S.C. § 1539(j)(2)(C). See discussion of Section 4(d) Rules, below.
15. ^ 1982 U.S. Code Cong. and Admin. News, p. 2807.
16. ^ 1982 U.S. Code Cong. and Admin. News, at 2834.
17. ^ For an account of some of the changes, and a sense of the excitement in the scientific community, see Jim Robbins, “Lessons from the Wolf,” Scientific American (June 2004).
18. ^ Defenders of Wildlife v. Lujan, 792 F. Supp. 834 (D.D.C. 1992) (referring to P.L. 102¬154, 105 Stat. 970, 993-94 (1991)).
19. ^ United States v. McKittrick, 142 F.3d 1170 (9th Cir. 1998).
20. ^ ESA § 10(j)(1); 16 U.S.C. § 1539(j)(1).
21. ^ Wyoming Farm Bureau Federation v. Babbitt, 199 F.3d 1224 (10th Cir. 2000).
22. ^ Gordon v. Norton, 322 F.3d 1213 (10th Cir. 2003).
23. ^ New Mexico Cattle Growers v. U.S. Fish and Wildlife Service, 1999 WL 34797509
(D.N.M. 1999).
24. ^ Center for Biological Diversity v. Kempthorne, 498 F. Supp. 2d 293 (D.D.C. 2007).
25. ^ P.L. 93-205, § 3(11), 87 Stat. 886.
26. ^ P.L. 95-632; 16 U.S.C. § 1532(16).
27. ^ GAO Testimony before the Subcommittee on Resource Protection, No. 108960, p. 5 (April 3, 1979).
28. ^ GAO Testimony before the Subcommittee on Resource Protection, No. 108960, Attach. 1 (April 3, 1979).
29. ^ GAO Testimony before the Subcommittee on Resource Protection, No. 108960, p. 3-4 (April 3, 1979).
30. ^ S.Rept. 96-151, p. 7 (May 15, 1979). The discussion occurs after the amendment, because, according to the Senate report, “some clarification would be useful.”
31. ^ H.Rept. 95-1625 at 25 (September 25, 1978). Restriction to vertebrates is a severe limitation, in terms of numbers of species able to enjoy this level of protection. Insects alone outnumber all other animals — including vertebrates — by three to one. Donald J. Borror, et al., An Introduction to the Study of Insects, p. 1. (Saunders College Publishing: New York, 5th ed. 1981).
32. ^ ESA § 3; 16 U.S.C. § 1532(16).
33. ^ S.Rept. 96-151, p. 7 (May 15, 1979).
34. ^ 61 Fed. Reg. 4722 (February 7, 1996).
35. ^ 61 Fed. Reg. at 4725.
36. ^ 61 Fed. Reg. at 4724.
37. ^ ESA § 4(b); 16 U.S.C. § 1533(b).
38. ^ 61 Fed. Reg. at 4725.
39. ^ 61 Fed. Reg. at 4725.
40. ^See Humane Society of the United States v. Kempthorne, No. 07-0677 PLF (D.D.C. Sept. 29, 2008) (vacating final rule that designated and delisted the Great Lakes DPS).
41. ^Two pending lawsuits challenge this final rule. See Western Watersheds Project v. Servheen, No. 07-CV-243-EJL
(D. Idaho), and Aland v. Kempthorne, No. CV08-24-S-EJL (D. Idaho).
42. ^The Northern Rockies DPS was later vacated by FWS. See Defenders of Wildlife v. Hall, 08-cv-56-M-DWM (D. Mont. filed Sept. 22, 2008).
43. ^Humane Society of the United States v. Kempthorne, No. 07-0677 PLF (D.D.C. Sept. 29, 2008).
44. ^In the case of the Sonoran Desert bald eagle, a petition to recognize the DPS was filed at the time the entire bald eagle species was being removed from the ESA. The DPS designation would have kept ESA protections in place for the Sonoran Desert bald eagle. However, FWS found the Sonoran population did not meet the criteria for a DPS. 72 Fed. Reg. 37345, 37357 (July 7, 2007). The court found that decision arbitrary and capricious and ordered the population to be listed as threatened. Center for Biological Diversity v. Kempthorne, CV-07-0038-PHX-MHM, 2008 WL 659822 (D. Ariz. March 5, 2008). In May, FWS listed the “potential Sonoran Desert Bald Eagle Distinct Population Segment” as threatened (73 Fed. Reg. 23966 (May 1, 2008)), and also initiated a status review of the listing. 73 Fed. Reg. 29096 (May 20, 2008). It is not designated a DPS, although it has distinct protection due to the court order. For an analysis of the Sonoran Desert bald eagle listing, see CRS Report RL34174, What Happens to the Bald Eagle Now That It Is Not Protected Under the Endangered Species Act (ESA)?, by Kristina Alexander.
45. ^Efforts to name the wolves of the Alexander Archipelago in Alaska as threatened or endangered have not succeeded. See Biodiversity Legal Foundation v. Babbitt, 943 F. Supp. 23 (D.D.C. 1996) (remanding the decision not to list the Alexander Archipelago gray wolf to DOI, as its decision was not based solely on the best scientific and commercial data); 62 Fed. Reg. 46709 (Sept. 4, 1997) (upon remand, no finding that the wolf was threatened).
46. ^ 68 Fed. Reg. 15803 (April 1, 2003). This rule was vacated by court order. See Defenders of Wildlife v. U.S. Dept. of the Interior, 354 F. Supp. 2d 1156 (D. Or. 2005). A discussion of that case is presented later in this report.
47. ^ The Northern Rocky Mountain DPS includes Washington, Oregon, Utah, Montana, Idaho, and Wyoming; the Western Great Lakes DPS includes North Dakota, South Dakota, Minnesota, Wisconsin, and Michigan.
48. ^ 72 Fed. Reg. 6052 (February 8, 2007).
49. ^Humane Society of the United States v. Kempthorne, No. 07-0677 (PLF) (D.D.C. Sept. 29, 2008).
50. ^ 72 Fed. Reg. 6106 (February 8, 2007). In December 2007, FWS approved Wyoming’s wolf management plan and scheduled the delisting of the gray wolf in Wyoming for February 29, 2008. See 72 Fed. Reg. 36969 (July 6, 2007) for the proposed rule to delist.
51. ^73 Fed. Reg. 10513 (Feb. 27, 2008).
52. ^Defenders of Wildlife v. Hall, cv-08-56-M-DWM (D. Mont. Sept. 22, 2008) (FWS motion for voluntary remand and vacatur); 73 Fed. Reg. 75356 (Dec. 11, 2008) (reinstating gray wolves as endangered, except in Minnesota (threatened), and in the Northern Rocky Mountains (nonessential experimental populations)).
53. ^ See 72 Fed. Reg. 6051-103 (February 8, 2007) for a discussion of wolf recovery goals, including goals for establishing additional populations of the Western Great Lakes DPS.
54. ^ Essentially, one DPS of the lower 48 states is created from all regions not in three named areas. Thus, according to FWS, the lower 48 states has one population of wolves, except:
1. Where listed as an experimental population;
2. Minnesota, Wisconsin, Michigan, eastern North Dakota (that portion north and east of the Missouri River upstream to Lake Sakakawea and east of the centerline of Highway 83 from Lake Sakakawea to the Canadian border), eastern South Dakota (that portion north and east of the Missouri River), northern Iowa, northern Illinois, and northern Indiana (those portions of IA, IL, and IN north of the centerline of Interstate Highway 80), and northwestern Ohio (that portion north of the centerline of Interstate Highway 80 and west of the Maumee River at Toledo); and
3. Mexico.
See FWS website
55. ^ Defenders of Wildlife v. U.S. Dept. of the Interior, 354 F. Supp. 2d 1156 (D. Ore. 2005).
56. ^ National Wildlife Federation v. Norton, 386 F. Supp. 2d 553 (D. Vt. 2005). FWS has since changed its interpretation of a “significant portion of its range.” See DOI Solicitor’s Opinion, M-37013 (March 16, 2007), available at: http://www.doi.gov/solicitor/M37013.pdf.
57. ^ National Wildlife Federation v. Norton, 386 F. Supp. 2d at 556.
58. ^Defenders of Wildlife v. Hall, 08-cv-56-M-DWM, 2008 U.S. Dist. LEXIS 55071 (D. Mont. July 18, 2008).
59. ^73 Fed. Reg. 10513 (Feb. 27, 2008).
60. ^Defenders of Wildlife v. Hall, 08-cv-56-M-DWM (D. Mont. filed Sept. 22, 2008).
61. ^73 Fed. Reg. 75356 (Dec. 11, 2008).
62. ^See FWS Press Release, Service Removes Western Great Lakes, Portion of Northern Rocky Mountain Gray Wolf Populations from Endangered Species List (Jan. 14, 2009), available online at http://www.fws.gov/news/NewsReleases.
63. ^See Rahm Emanuel, Memorandum for the Heads of Executive Departments and Agencies (Jan. 20, 2009) http://ombwatch.org/regs/midnightregfreezememo.pdf.
64. ^Humane Society of the United States v. Kempthorne, No. 07-0677 PLF (D.D.C. September 29, 2008). As a result of this ruling, wolves in that area were returned to the endangered species list.
65. ^Humane Society of the United States v. Kempthorne, No. 07-0677 PLF, *24 (D.D.C. Sept. 29, 2008).
66. ^73 Fed. Reg. 75356 (Dec. 11, 2008).
67. ^See FWS Press Release, Service Removes Western Great Lakes, Portion of Northern Rocky Mountain Gray Wolf Populations from Endangered Species List (Jan. 14, 2009), available online at http://www.fws.gov/news/NewsReleases.
68. ^See Rahm Emanuel, Memorandum for the Heads of Executive Departments and Agencies (Jan. 20, 2009) http://ombwatch.org/regs/midnightregfreezememo.pdf.
69. ^ 50 C.F.R. § 17.40(d).
70. ^ 68 Fed. Reg. at 15864.
71. ^68 Fed. Reg. at 15868.
72. ^ 70 Fed. Reg. 1285 (January 6, 2005) (hereinafter the “2005 Rule”). Unacceptable impact was defined as: “State or Tribally-determined decline in a wild ungulate population or herd, primarily caused by wolf predation, so that the population or herd is not meeting established State or Tribal management goals.” Id. at 1307.
73. ^ 59 Fed. Reg. 60252, 60255 (November 22, 1994) (Yellowstone); 59 Fed. Reg. 60266, 60272 (November 22, 1994) (Idaho) (“wolves will not be deliberately killed solely to address ungulate-wolf conflicts”).
74. ^ See 2008 Rule, p. 6-7, citing Bangs, et al. 2004, pp. 89-100; National Research Council 1997, pp. 185-186; Mech and Peterson 2003, p. 159; Pletscher et al. 1991, pp. 545-548.
75. ^73 Fed. Reg. at 63928 (Oct. 28, 2008) (reporting that in 2007, 112 cattle were killed by wolves, and 135 depredating wolves were killed; in 2008, 170 cattle killed by wolves and 172 depredating wolves killed).
76. ^73 Fed. Reg. at 63923 (Oct. 28, 2008) (down from 1,544 wolves in 2007).
77. ^Revision of Special Regulation for the Central Idaho and Yellowstone Area Nonessential Experimental Populations of Gray Wolves in the Northern Rocky Mountains, (hereinafter the “2008 Rule”). 73 Fed. Reg. 4720 (Jan. 28, 2008). See also 73 Fed. Reg. 75356 (Dec. 11, 2008)(reinstating the special rules).
78. ^ 2008 Rule, p. 8.
79. ^ According to FWS, at the time of the 2008 Rule, Montana had 394 wolves, including 37 breeding pairs; Idaho had 788 wolves, including 41 breeding pairs; and Wyoming had 362 wolves, including 27 breeding pairs. 2008 Rule, p. 11.
80. ^ 2008 Rule, pp. 15-16.
Disclaimer: This article is taken wholly from, or contains information that was originally published by, the Congressional Research Service. Topic editors and authors for the Encyclopedia of Earth may have edited its content or added new information. The use of information from the Congressional Research Service should not be construed as support for or endorsement by that organization for any new information added by EoE personnel, or for any editing of the original content.
Note: The first version of this article was drawn from RL34238 Gray Wolves Under the Endangered Species Act: Distinct Population Segments and Experimental Populations by Kristina Alexander and M. Lynne Corn, Congressional Research Service on February 13, 2009.
View/Download Attached File: RL34238.pdf
Citation
Crs (Content Source);Peter Saundry (Topic Editor) "Gray wolves under the Endangered Species Act: Distinct population segments and experimental populations". In: Encyclopedia of Earth. Eds. Cutler J. Cleveland (Washington, D.C.: Environmental Information Coalition, National Council for Science and the Environment). [First published in the Encyclopedia of Earth October 6, 2009; Last revised Date October 6, 2009; Retrieved May 18, 2013 <http://www.eoearth.org/article/Gray_wolves_under_the_Endangered_Species_Act:_Distinct_population_segments_and_experimental_populations>
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Cambridgeshire, England, Baptism Index 1801-1837Edit This Page
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Monterey County, CaliforniaEdit This Page
From FamilySearch Wiki
Revision as of 22:18, 3 January 2013 by Lsgc (Talk | contribs)
United States California Monterey County
California
Online Records
Contents
County Courthouse
Monterey County Courthouse
240 Church Street
PO Box 29
Salinas, CA 93092
Phone 831.755.5041
County Recorder has birth and death records and marriage records
from 1893 Clerk Superior Court has probate and land rececords
County Court has divorce and court records[1]
Historical Facts
Wikipedia has more about this subject: Monterey County, California
Parent County
18 February 1850: Monterey County was created as an original county. County seat: Salinas [2]
Boundary Changes
Record Loss
Places/Localities
Populated Places
Aromas
Big Sur
Boronda
Bradley
Carmel
Carmel Highlands
Carmel Valley Village
Castroville
Chualar
Del Rey Oaks
Fort Hunter Liggett
Fort Ord
Gilroy
Gonzales
Greenfield
Jolon
Lockwood
Lucia
King City
Gorda
Marina
Monterey
Pacific Grove
Pajaro
Parkfield
Pebble Beach
Prunedale
Salinas
San Ardo
San Benancio
San Lucas
Sand City
Seaside
Soledad
Spreckels
Neighboring Counties
Resources
Bible Records
Biography
Cemeteries
CASTROVILLE
Castroville Public Cemetery District
8442 Moss Landing Road, Moss Landing, CA 95039
List of Burials - Contributed by Jodi Brewster (belukha6@yahoo.com) 5 Aug 2003 Updated 10 Jun 2003
http://files.usgwarchives.net/ca/monterey/cemeteries/castroville-dates.txt
EL CARMETO
Pacific Grove
BillionGraves
FORT ORD
Whitcher Family Cemetery
Monterey County Herald, CA June 6, 1999
Final resting place far from today’s uproars by Mary Barker
GONZALES
Gonzales Cemetery District
Highway 1 & S. Alto St., Gonzales, CA 93926
KING CITY
King City District Cemetery
1010 Broadway Street, King City, CA 93930
Place to document burials
http://cagenweb.com/montereybbs/viewforum.php?f=34
MONTEREY
Cementerio El Encinal and Monterey City Cemetery
Fremont at Camino Aguajito, Del Rey Oaks, CA 93940
http://www.monterey.org/cemetery/
list of burials http://freepages.history.rootsweb.ancestry.com/~westroot/cemetery/encinal.html
Mission Memorial Park
1915 Ord Grove Avenue, Sand City, CA 93955
Presidio of Monterey Cemetery, Presidio of Monterey
The Presidio of Monterey is located in the City of Monterey. It is an “Active Post” and it is closed to the public. The cemetery is located within the Presidio and is classified as “Inactive”, that is, all of the gravesites have been used or reserved prior to 1 May 1975 per Army Regulation 210-190. ACCESS TO THE PRESIDIO OF MONTEREY IS LIMITED TO ACTIVE MILITARY, RETIRED MILITARY, EMPLOYEES, AND SPONSORED GUESTS.
Information page http://freepages.history.rootsweb.ancestry.com/~westroot/cemetery/rpc.html
References:
1. Presidio of Monterey, Monterey, Monterey County, CA by David Johnson, 4 Aug, 2005 (Part of California Tombstone Project)
http://www.rootsweb.com/~cemetery/california/californ.html
2. Monterey County Herald, CA May 30, 2010
Event re-dedicates Presidio of Monterey Cemetery
Royal Presidio Chapel de San Carlos de Borromeo, Monterey, California
Information page
http://freepages.history.rootsweb.ancestry.com/~westroot/cemetery/rpc.html
San Carlos Catholic Cemetery
792 Fremont Street, Del Rey Oaks, CA 93940
Place to document burials
http://cagenweb.com/montereybbs/viewforum.php?f=32
PACIFIC GROVE
El Carmelo Cemetery and the Little Chapel by-the-Sea
Asilomar Blvd, Pacific Grove, CA 93950
List of Burials - Contributed by: Kristina Magill <kam1242@attbi.com> 24 Sep 2002
http://files.usgwarchives.net/ca/monterey/cemeteries/elcarmelo-ac.txt
Old list of burials, Updated 24 Sep. 2004
http://freepages.history.rootsweb.ancestry.com/~westroot/cemetery/elcaf.html
Full obits of burials – compiled by Mary S. Taylor, ongoing
http://cagenweb.com/montereybbs/viewforum.php?f=9
List of burials compiled from diverse sources – compiled by Mary S. Taylor
http://cagenweb.com/montereybbs/viewforum.php?f=33
References:
1 Carmel Pine Cone, CA Dec. 16, 2010
P.G. Cemetery costs skyrocket
2. Cedar Street Times, Pacific Grove, CA Aug. 28, 2010
Darlene Billstrom is back in charge
3. Cedar Street Times, Pacific Grove, CA Aug. 28, 2010
El Carmelo Cemetery: Keeping it neat for the ages
4. Pacific Grove Hometown Bulletin, CA Jan. 18, 2006 p12
Board and Batten, Pacific Grove Heritage Society
El Carmelo Cemetery
(From the Dec 89/Jan 1990 Newsletter of the Pacific Grove Heritage Society.)
5. Carmel Pine Cone, CA March 9, 2005
Last Hometown adds 800 eternal bunk beds
By KIRSTIE WILDE
6. Monterey Peninsula Herald, CA Jan. 5, 1963
New Partner in P.G. Mortuary
7. Monterey Peninsula Herald, CA May 29, 1947 p2
El Carmelo Cemetery Entrance Changed
8. Pacific Grove Tribune, CA June 1, 1945
El Carmelo Cemetery Transferred
Mutual Agreement Made By Cemetery Board And City
PRUNEDALE
Queen of Heaven Cemetery & Mausoleum
18200 Damian Way, Prunedale, CA 93907
SALINAS
Garden of Memories Memorial Park (I.O.O.F.)
768 Abbot St., Salinas, CA 93901
List of Burials - Submitted by Val Hoover 26 Jul 2005
http://files.usgwarchives.net/ca/monterey/cemeteries/garden-of-memories.txt
Place to document burials
http://cagenweb.com/montereybbs/viewforum.php?f=31
Yamat Cemetery
1165 Abbott Street, Salinas, CA 93905
Healey Mortuary and Crematory
405 N. Sanborn Road, Salinas, CA 93905
Old Calvary Catholic Cemetery
Salinas, Monterey County, CA
Index of Burials - Submitted by Val Hoover 26 Jul 2005
http://files.usgwarchives.net/ca/monterey/cemeteries/old-cavalry-catholic.txt
SAN LUCAS
San Lucas Cemetery
Monterey County, CA
List of Burials - Submitted by Betsy Wood 15 Jan 2007
http://files.usgwarchives.net/ca/monterey/cemeteries/san-lucas.txt
SOLEDAD
Soledad Cemetery District
1711 Metz Road, Soledad, CA 93960
MONTEREY COUNTY
Sands Cemetery, Monterey County, CA
All Markers in the Sands Cemetery were wooden and burned one summer when a fire swept through the area. There are some more recent stones in the Sands Cemetery but for the “old wooden markers” we only have word of mouth as to who is buried there & their approximate dates.
List of Burials - Submitted by Betsy Wood 13 Jan 2007
http://files.usgwarchives.net/ca/monterey/cemeteries/sands.txt
Pleyto (Pleito) Cemetery, Carmel Valley, Monterey County, CA
Pleyto or Pleito Cemetery was moved when the San Antonio Dam was built in 1967 and today it is called Cemetery Cove. (Part of CA Tombstone Project)
List of burials Submitted by Betsy Wood 13 Jan 2007
http://ftp.rootsweb.com/pub/usgenweb/ca/monterey/cemeteries/pleyto.txt
http://cagenweb.com/monterey/cempleyto.html
Census
For tips on accessing Monterey County, California census records online, see: California Census.
Church History and Records
LDS Ward and Branch Records
• Monterey
• Pacific Grove
• Salinas
Court Records
Superior Court of California – County of Monterey
Case Lookup – Index
https://www.justicepartners.monterey.courts.ca.gov/Public/JPPublicIndex.aspx
Probate Notes
http://www.monterey.courts.ca.gov/Probate/ProbateNote.aspx
Probate/Guardianship. Conservatorship Case Fillings
http://www.monterey.courts.ca.gov/Probate/
http://www.monterey.courts.ca.gov/Documents/Administration/Access-to-Administrative-and-Court-Records.pdf
Requesting Court Case File and Adjudicative Records (divorce, criminal, complaints, judgments, traffic tickets, case information) Mostly available at the clerk’s office at the court location where record was originally filed. Requests to inspect and copy case file and other adjudicative records prepared for or filed or used in a court proceeding may be made at the courthouse where they are filed. There is a copying fee of $0.50 per page, which must be paid in advance. Please direct requests for access to administrative records maintained by the Monterey County Superior Court to:
PUBLIC INFORMATION OFFICE Superior Court of California
County of Monterey 240 Church Street
Salinas, CA 93901
Phone: (831) 775-5400 x3020
Fax: (831) 775-5499
Mediainfo@monterey.courts.ca.gov
Crime and Criminals
Directories
Ethnic, Political, or Religious Groups
Germans
Gazetteers
Genealogy
History
Land and Property
Maps
Migration
Military History and Records
Naturalization and Citizenship
Newspapers
(note: some of these dates may be off due to incomplete holdings lists and variations in titles of the newspapers)
BIG SUR
Big Sur Gazette (1978-1981)
CARMEL and CARMEL VALLEY
Carmel Californian (1936-1937)
Carmel Cottager (1934)
Carmel Cymbal (1926-1941)
Carmel Cymbal and Masten’s Gazette (1941-1942)
Carmel, Pebble Beach, Carmel Valley Spectator (1949)
Carmel Pine Cone (1915-present)
Carmel Pine Cone and Carmel Valley Outlook (1981-1993)
Carmel Pine Cone Cymbal (1942-1962)
Carmel Spectator (1949-1955)
Carmel Sun (1992-1994)
Carmel Valley News (1951-1955)
Carmel Valley Sun (1983-1990)
Carmel Valley Outlook (1979-1981)
Carmel Valley Weekly Sun (1990-1993)
Carmel Village Daily (1933)
Masten’s Gazette (1941)
Paisano (1948)
Peninsula Spectator (1959)
Spectator (1949-1950)
CASTROVILLE and MOSS LANDING
Castroville News (1936-1942)
Castroville Times (1950-1952)
Castroville Times Journal (1950)
Castroville Times and Moss Landing Harbor News (1952-1975)
North County News (1975-1982)
FORT ORD
Fort Ord Panorama (1980-1982)
GONZALES
Gonzales Tribune (1956-1991)
GREENFIELD
Greenfield News (1952-1991)
KING CITY
King City Rustler (1901-1997)
Rustler and King City Herald (1961-1964)
Semi Weekly Rustler (1936-1937)
MARINA
Marina Tribune (1981-1982)
Marina Weekly Tribune (1979)
MONTEREY
Army News (1904)
At Ease (1990)
Monterey American (1913-1916)
Monterey Argus (1887-1888)
Monterey Bay Labor News 1955-1985
Monterey Bay News (1947-1950)
Monterey Bay and Pacific Grove/Pebble Beach Tribune (1985)
Monterey Bay Tribune (1985-1987)
Monterey County Herald (1992-present)
Monterey County Journal (1864)
Monterey County Post (1929-1933)
Monterey Daily Cypress 1889-1922)
Monterey Democrat (1868-1889)
Monterey Enterprise (?)
Monterey Evening Herald (1909-1910)
Monterey Gazette (1863-1868)
Monterey Herald (1986-1992)
Monterey Labor News (1937)
Monterey New Era (1890-1909)
Monterey News Daily and Monterey Trader (1940)
Monterey Peninsula Herald (1929-1992)
Monterey Sentinel (1855-1856)
Monterey Trader (1933-1951)
Monterey Union (1862)
Montereyan (1951-1955)
Peninsula Advocate (1910-1911)
Peninsula Daily Herald (1927-1929)
Peninsula Daily Herald and Monterey Daily Cypress and Monterey American (1923-1927)
Peninsula Merchants’ Semi-Monthly Bulletin (1915-1916)
PACIFIC GROVE
Pacific Grove Beacon (1994-1995)
Pacific Grove Daily Review (1892-1926)
Pacific Grove Monarch (1987-1992)
Pacific Grove News (1916)
Pacific Grove/Pebble Beach Tribune (1976-1984)
Pacific Grove Progress (1916)
Pacific Grove Review (1888-1912)
Pacific Grove Tide (1936-1943)
Pacific Grove Tribune (1943-1951, 1984-1985)
Pacific Grove Weekly Tribune (1978-1979)
Peninsula Pelican (1925)
Peninsular Review and Pacific Grove Daily Review (1926-1932)
SALINAS
Monterey County Labor News (1942-1954)
Monterey County Post (1929-1933)
North Monterey County Fortnighter (1983-1989)
Owl (1894)
Salinas Californian (1942-present)
Salinas Daily Index (1894-1928)
Salinas Daily Journal (1889-1897)
Salinas Daily Post and Monterey County Post (1933-1942)
Salinas Democrat (1889-1896)
Salinas Index (1895-1904)
Salinas Index Journal (1928-1942)
Salinas Index Weekly (1873-1925)
Salinas Labor News (1937-1942)
Salinas Morning Post (1936-1942)
Salinas Standard (1869-1872)
Salinas Valley Rustler (1906-1923)
Salinas Valley Settler (1889)
Salinas Village Crier (1942)
Salinas Weekly Index (1883-1895)
Salinas Weekly Journal (1867-1915
SEASIDE
Free Weekly Sentinel (1987)
Right-On Post (1970)
Seaside News Sentinel (1950-1970)
Seaside Post (1969-1970)
Seaside Post News Sentinel (1970-1988)
Seaside Post Sentinel (1970)
Seaside Sentinel (1988-1996)
Seaside Weekly Sentinel (1987-1988)
SPRECKELS
Spreckles Courier (1909-1914)
WATSONVILLE
Watsonville Rustler (1888-1897)
References:
1. “Californian.” In the California Weekly, vol. 1, August 27, 1909, p629.
2. “Californian.” In the Grizzly Bear, vol. 2, Feb. 1908 p23
3. Denny, Melcena Burns. “California’s First newspaper Printed without a Letter ‘W’ on Flimsy Cigarette Paper.” In Montana, the Magazine of Western History, vol. 15, no. 4, October 1965 p29-41.
4. Dethlefs, Helen. “First California Newspaper Founded 100 Years Ago, August 15, 1846.” In California Publisher, August 1946, p11.
5. “First newspaper in California.” In The Pony Express, September 1946 p2.
6. Harding, Geroge L. “The Origin of California’s First printing Press.” In Book Club of California, Quarterly news-letter 2:5-8, June 1934.
7. “The Salinas Californian; Newspaper’s Growth Tied to a Dream.” In California Publisher, Vol. 1, Nos 2 & 3, Nov-Dec 1971, p34-35.
8. “Newspaper Holdings of the California State Library” compiled by Marianne Leach
On-line
• Carmel Pine Cone, Carmel http://www.pineconearchive.com/
• Monterey County Herald, Monterey http://www.montereyherald.com/news
• Monterey County Weekly, Monterey http://www.montereycountyweekly.com/
• Monterey County News Topix http://www.topix.com/county/monterey-ca
• Cedar Street Times, Pacific Grove http://www.cedarstreettimes.com/
• Pacific Grove Hometown Bulletin http://www.pgbulletin.com/archives.php
• King City Rustler http://www.kingcityrustler.com/v2_main_page.php
• Salinas Californian http://www.thecalifornian.com/apps/pbcs.dll/frontpage
• South County News – Gonzales, Greenfield, King City, Soledad http://southcounty.kionrightnow.com/
Obituaries
Periodicals
Probate Records
Repositories
Archives, Libraries and Museums
County Courthouse
Monterey Courthouse, 1200 Aguajito Rd, Monterey, CA 93940; (831) 647-5800
History - Monterey County Courthouse Completed 1878
http://www.monterey.courts.ca.gov/General_Information/History.aspx
The military and social capital of Alta California during Spanish and Mexican rule, the town of Monterey naturally became the county seat when the 27 original counties were formed in, 1850. But when it was learned that the railroad was to go through the valley and not along the coast, the government center was moved to Salinas. After the house used as a courthouse burned, this much larger brick Victorian building was commissioned. The courthouse remained in use while the current one was being constructed around it, and then was demolished. Today a courtyard, lily pond, and commemorative sculpture occupy the site.
Marina Courthouse, 3180 Del Monte Blvd, Marina, CA 93933; (831) 883-5300
King City Courthouse, 250 Franciscan Way, King City, CA 93930; (831) 386-5200
Salinas Courthouse, 240 Church St, Salinas, CA 93901; (831) 775-5400
Family History Centers
Societies
Taxation
Vital Records
Birth
Marriage
Divorce
Death
Voters Registers
The following is a list of original voter registration records recently transferred from Monterey County Elections Department to the Monterey County Historical Society archival facility in Salinas. To access records, see: http://www.mchsmuseum.com/
Great Register of Voters:
Ledger Books:
1866-1870 1 book
1870 1 book
1881-1883,1886,1888,1890 1 book
1892 (Vol. 1, Vol. 2) 2 books
1896 (Vol. 1, Vol. 2) 2 books
1898 (Vol. 3) 2 books
1900 (Vol. 1,Vol. 2) 2 books
1902 (Vol. 1, Vol. 2) 2 books
1904 (Vol. 1, Vol.2) 2 books
1906-1907 (Vol. 1, Vol. 2) 2 books
1908-1909 (A-L, M-Z) 2 books
1910-1911 (A-L, M-Z) 2 books
1912-1913 (A-L, M-Z) 4 books
1914-1915 (A-L, M-Z) 2 books
Scattered records:
Voter affidavits*:
1922-23 (Affidavits, Pleyto precinct)
1930-31 (Affidavits, Blanco precinct)
Indexes (no voter affidavits):
1938, August primary (Greenfield precincts No.1 and No. 2)
Description of collection:
The ledger books1 titled Great Register of Voters are the original handwritten manuscripts kept by the County Clerk’s (Registrar of Voters) office of the County of Monterey beginning in 1866.2 The entries are made in alphabetical sections (with alpha sub-sections) and show chronological appearance of voters as they were sworn and registered to vote. The entries include place of nativity (birthplace); age of voter; place of residence (post office or precinct); date and place of naturalization, if applicable; whether citizen by virtue of Treaty of Hildago or citizen by virtue of father’s naturalization (and in later years a woman’s citizenship status by marriage); occupation; date of appearance to be sworn; and date of registration. Later registration data includes a physical description of the voter and party affiliation. The Registrar of Voters also appended notes to the entries to indicate reasons for cancellation whether due to removal to another county or state, transfer to another county due to change of boundary lines (i.e. to San Benito County in 1874), death, incarceration or “Section 1106” (failure to vote). These notations usually contained dates if known. Copies of the list of voters not thus cancelled, were made every two years and sometimes more frequently if a special election occurred (polling lists). These copies of uncancelled voters were sent to the state and other repositories on a routine basis. Microfilm of these typeset lists is available at the California State Library in Sacramento, California, and other repositories.3
These original handwritten Great Register ledger books have not been microfilmed or transcribed. There was a partial transcription done of the 1890 Great Register as part of a census substitute project4 but the transcription does not include cancellation information.
Note: The Great Register books contain information for years other than marked on the book spines. For example, the 1866-1870 Great Register contains several entries made after 1870 when cancellations were noted and the 1890 Great Register shows voter registration dates which occurred years earlier.
• Voter affidavits are registration forms signed by the voters and contain birth date, birth place, residence address, occupation, party affiliation and sometimes cancellation information.
• List of records
1 Some years are missing.
2 Beginning in the year 1866, California state law required a list of voters to be kept.
3 Monterey County (California) County Clerk. Great Register of Voters 1867, 1869, 1872,
1875-1876, 1879-1880, 1884, 1886, 1888, 1890, 1892, 1894, 1896, 1898.
(Salt Lake City: Filmed by the Genealogical Society of Utah, 1975).
4 Bettyann Lockwood Hedegard, ed., 1890 great register of Monterey County, California.
(Salinas, CA: Monterey County Genealogy Society, c1993).
Courtesy of Junel Davidsen
Websites
References
1. Handybook for Genealogists: United States of America, 10th ed. (Draper, Utah: Everton Pub., 2002), Monterey County, California. Page 86 At various libraries (WorldCat); FHL Book 973 D27e 2002.
2. The Handybook for Genealogists: United States of America,10th ed. (Draper, UT:Everton Publishers, 2002).
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Norway OccupationsEdit This Page
From FamilySearch Wiki
Revision as of 03:42, 7 October 2011 by SteuartRC (Talk | contribs)
Occupations were a measure of social status. Some trades were viewed as more prestigious than others. For example, goldsmiths had more prestige than shoemakers. Many trades, such as butchers, tanners, shoemakers, tailors, and others were organized into guilds, which were in charge of training apprentices and regulating a trade's practice in an area.
Guilds were usually established in each city. Guild records include lists of members, information on journeymen practicing in the town, marriages of journeymen, and advancements from the rank of apprentice to journeyman and from journeyman to master craftsman. In addition, contracts between masters and parents of apprentices may be included.
There are records not often used that can be of great help and interest for the researcher, and they are:
Borgerskap (citizenship) records in Norway
A new idea, that resulted in little used record show up in Oslo during the 1300’s.
A man could apply for “borgerskap” in a specific city, and if granted would be given certain rights, as well as certain responsibilities. As a “borger” or citizen he would be able to set up shop in town; as a merchant, skipper, master craftsman with apprentices etc.
He would also be expected to pay taxes and accept different city assignments from the government.
The word “borgerskap” can have many different meanings. In modern times we talk about having citizenship in a country, or the word can refer to a specific social group in society; “borgerskap” versus the working class or nobility.
In earlier years the word “borgerskap” describes the judicial relationship a person had with the city he lived in, (“borgerskap” was not made available to women in earlier times).
In the 1600’s it became required that the new “borger” swore allegiance to the King and city, and paid a licensing fee. He was then recorded in the city’s “borgerbok”, and given a “borgerbrev” as a proof that he was a “borger”.
The eldest known “borgerbok” today is from mid 1551-1751, and is found in Bergen city.
The “borgerbøker” can be very helpful to the genealogist, especially during the time period before the parish registers were kept.
Information about a person’s name, birth place, address, and farm name if connected to a farm is given. His particular job is also listed. Used with other genealogical records it can be very helpful to researchers, adding information not usually found in church records.
Norway Occupations
Norwegian occupations: '
Contents
A
A/S Abb. for aksjeselskap or share company
Aar year
Adel nobility
Adelsmann nobleman
Adjunct secondary school teacher
Advokat lawyer, solicitor
Agent representative, commercial agent
Agronom graduate of an agricultural college
Aksjonær shareholder
Almisse charity or alms
Almisselem charity recipient, poor person
Almueskolen public school
Amanuens assistant
Amme nurse
Amt administrative district / county
Amtmann county governor, county administrator
Apotek pharmacy
Apoteker pharmacist
Abr abb. for “arbeider” or laborer
Arbeidskarl laborer
Artillerist soldier
Assessor assistant judge
B
Br. Abb. for “bruker” or farmer
B.F. abb. for “barnefader” or the child’s father
Baadsmand boats man
Bager baker
Bager dreng baker apprentice
Bager/baker mester master baker
Bagwe/baker svend baker journeyman
Bager/baker baker
Bager/baker lærling baker assistant
Baneformann foreman at the railroad
Banevokter length man
Bankbogholder bank accountant
Bergarbeider mine worker
Bergverksarbeider mine worker
Bestyrer manager
Besøkende visitor
Betjent assistant, servant
Betler beggar
Bibliotekar librarian
Biskop bishop
Blikkenslager tinsmith
Bog/bok trykker book printer
Bog/bok binder book binder
Bokhandler book seller
Bokholder bookkeeper
Bonde farmer
Borgerkrig soldat soldier in the civil war
Borgemester mayor
Brannmann fireman
Brennevinsbrenner distiller of spirits
Brendevins handler liquor dealer
Bruger/bruker farmer/ user of a farm
Brygge arbeider dock worker
Brygger brewer
Bryggeri arbeider brewery worker
Budeie milkmaid
Bundtmager/maker furrier
Bureiser settler
Butikk dame sales woman in a store
Butikk sjef store manager
Bygde folk country people, villagers
Byggmester master builder, contractor
Bygnings arbeider construction worker
Bygsel mann leaseholder
Bødkemester master cooper, barrel maker
Bøksel mand lease holder
Bønder farmers
Børsemager/maker gunsmith
Båd/båtbygger ship builder
C
Cammerrrad chamber councilor
Cancelliraad same as above
Cand. Juris Bachelor of Laws
Cautionister witnesses, sponsors
Contorist secretary, office worker
Copist transcriber
D
Dagsarbeider day laborer
Danne kvinde gentlewoman
Dannemann gentleman
Degn pariah clerk
Diakon deacon, male nurse
Dosent associate professor
Dommer judge
Domprost cathedral dean
Dragon light cavalryman
Dreng boy, farm hand
Dug/duk mager/maker sail maker
Dyrlege veterinarian
E
Enroullert enrolled
Eier owner
Ekspeditør shopkeeper
Elektriker electrician
Embedsmand civil servant
Emigrant emigrant
Emissær travelling lay preacher
F
Faarehyrde shepherd
Fabrikk arbeider factory worker
Farger dyer
Fattig folk poor folk
Fattiglem pauper
Fehandler person in the cattle business
Felemaker fiddle maker
Felespiller fiddle player
Fell maker rug maker
Fenrik ensign, second lieutenant
Fergemann ferryman
Fisker fisherman
Fiskehandler fish merchant
Fjøsstell dairy management
Flaademand log driver
Flaadmand ferry man
Flødnings arbeider log floating worker
Fmd. “føderaadsmand” or pensioner
Fogd bailiff, sheriff
Forbryter criminal
Forettningsmand businessman
Forfatter writer, author
Forhen. previous
Forhenværende previous
Forlover best man, sponsor
Forlovede fiancé
Forløftningsmænd guarantors
Forman foreman, supervisor
Formynder guardian
Forpakter leaseholder
Forvalter manager
Fotograf photographer
Fraktmand cargo handler, transporter
Frisør dame hairdresser
Fullmektig head clerk, agent
Fylkesmand county governor
Fylkesrådmand county commissioner
Fyrbøter stoker, lighthouse keeper
Fyrmester lighthouse boss
Færdesmand traveler
Føderaad pensioner
Føderaadsmand, føderaadskone
Førnemde the former, previously mentioned
Fører pilot, guide, operator
Fårehyrde shepherd
G
Grdbr., selveier farmer, owner
Gaar i dagleie working as a daylaborer
Gaar paa skole in school
Gaardbruger/bruker farmer
Gaard far head of household
Gardsfolk farmers
Gartner gardener
Garver tanner
Garver svend tanner apprentice
General løyntnant lieutenant general
Gevorben enlisted
Gjestgiver innkeeper
Gjeter shepherd
Gjetergutt shepherd boy
Gjørtler brazier, brass smith
Godseier estate owner
Guldsmed Goldsmith
Graver sexton, grave digger
Grenader grenadier, infantryman
Grenselos border pilot
Grossist wholesaler
Grovsmed blacksmith
Grube arbeider miner
Grunnlegger founder
Gråsten arbeider stonecutter
Gårdsarbeider farmer, field hand
Gårdsbestyrer farm manager
Gårdsfolk farm people, owners or tenants
Gårds gutt farm boy
Gårdsbruker farmer
Gårdmann farm owner
H
Haandværker tradesman
Hammersmed hammer smith
Handelsbestyrer shop manager
Handelsbetjent salesman, shop assistant
Handelsfuldmægtig business agent
Handelsmand merchant, trader
Hanskemaker glove maker
Hattemaker hat maker
Heradsskriver district taxation registrar
Hjelpepleier enrolled nurse
Hjulmaker wheelwright
Husbestyrinde housekeeper
Huseier homeowner
Hushjelp housemaid
Husholderske housekeeper
Husmand cottager, tenant farmer, crofter
Husmand med jord cottager who rented a small plot of land
Husfader head of household
Husmoder house wife
Høker small grocer/ shopkeeper, huckster
Høvedsmand fishing boat master, shack master
Håndlanger assistant, hodman
Håndverker tradesman
Håndsverksvend journeyman
I
Inderst renter, lodger on a farm
Indehaver owner
Indsitter renter, lodger
Ingeniør engineer
Innvandrer immigrant
Isskjauer dockhand
Isskjærer ice cutter
J
Jaegere elite troops of the army
Jeger hunter, trapper
Jeger light infantry soldier
Jernbane arbeider railroad worker
Jernbane fuldmektig managing clerk at the railroad
Jernverks arbeider mine worker
Jord arbeider farm worker, field worker
Jordbruker farmer
Jordbruksarbeider farm hand, farm worker
Jordeier landowner, landed proprietor
Jordløs husmand cottager without land
Jordmor midwife
Jordrotten landowner
Journalist journalist
Jungmand ordinary seaman
Jurist lawyer
Justis justice, administration of justice
Jæger hunter
Jætergut shepherd boy
Jøkel glacier
K
Kaarmand retired man living on his own farm with accommodations and support
Kadet cadet (army)
Kapelan curate, cleric who assists the priest, deacon
Kaptein captain
Karetmaker coach builder
Kasserer treasurer
Kaveringsmand best man
Kemner town treasurer
Kgl. Fuldmægtig royal attorney
Kongelig fuldmægtig royal attorney
Kirkesanger church singer
Kirkeverge church warden
Kirurg surgeon
Kjelearbeider boiler worker
Kjæreste lover, sweetheart, friend
Kjøpmand merchant
Kjørekarl driver of a horse
Klokker bell ringer, parish clerk, sexton
Kommune arbeider local government worker
Konditor confectioner
Konduktør railway conductor
Konsulent consultant
Kontor betjent office clerk
Kontorfuldmægtig head clerk
Kontormand office clerk
Kontrollør inspector, supervisor
Kranfører crane operator
Krigsfange prisoner of war
Krigsminister minister of war
Kræmmer shopkeeper
Krøpling cripple
Kuldbrenner charcoal burner
Kuldhugger coal miner
Kuldhandler coal dealer
Kurvmaker basket maker
Kusk coachman, teamster
Kvartermester chief petty officer
Kystvakt coastguard
Kæmner town treasurer
Kårfolk retired, elderly couple living on their farm and receiving support
L
Lagerarbeider warehouse laborer
Lagrettsmand court member
Landhandler rural store owner
Landsfogd county police commissioner
Lasaron tramp, bum
Laugverge widow’s guardian
Legdekjerring woman on welfare
Legdekall man on welfare
Legdslem welfare recipient
Leieboer tenant
Leilending leaseholder, tenant farmer
Lekmand lay preacher
Lensmand sheriff
Likrøver grave robber
Livsarving heir, issue
Livsfange life prisoner
Logerende lodger
Losoldermand pilot master (ship)
Lutbrukar using a part of a farm
Læregutt apprentice
Lærer teacher
Lærerinde female teacher
Lærling apprentice, assistant
Løitnant lieutenant
Løsarbeider day laborer
Løskarl free hand, takes any job that comes along
Låsesmed locksmith
M
Magistrate town administrator, magistrate
Major major, squadron leader
Maler painter
Malersvend painter apprentice
Malmlaster ore loader
Mandskap crew
Marine navy
Maskinist engineer
Masovn arbeider blast furnace worker
Matros sailor
Medeier part owner
Medhjelper assistant, helper
Megler mediator, negotiator
Mekaniker mechanic
Menig soldier with rank of private
Messing smed brazier, brass smith
Mester master of a trade
Mestermand executioner
Militær military
Mine arbeider mine worker
Minister priest
Misjonær missionary
Montør fitter
Murarbeider brick layer
Murer brick layer
Musiker musician
Mægler mediator, negotiator
Møbel snekker furniture maker/carpenter
Møller miller
Møllearbeider worker at a mill
N
Nabo neighbor
Nyder almisse receives charity
Nyder vildkaar person receiving pension paid by new owner of the farm
O
Odelsberettiget freehold right
Odelsbonde freeholder, allodialist
Odelsgutt oldest son with right to inherit the farm
Odelsjente oldest daughter with inheritance right
Ombudsmand trade-union representative
Omflakkende handelsmand traveling salesman
Omgangs skolelærer circuit teacher
Omreisende handelskarl traveling salesman
Omvandrende persons moving from place to place
Oppsitter leaseholder, tenant farmer, occupant
Oppsynsmand supervisor, inspector, warden
Optiker optician
Ordfører mayor
P
Pakkhus arbeider ware house worker
Parykk maker wig maker
Pastor vicar, minister
Pensjonær boarder
Pensionist retired person
Permitert be on leave
Pige/Pike girl, maid, unmarried female
Plate arbeider sheet metal worker
Pleieassistant nursing assistant
Politiker politician
Postbud postman, mail carrier
Pottemaker potter
Praest priest
Preses president, chairman
Privat lærer private teacher
Prost/provst senior rector, dean
Pølsemaker sausage maker
Q
Quinde female person
Quindemenneske female person
R
Radiorelegrafist radio operator
Redactør chief editor
Rector head teacher
Reparatør repairman, mender
Representant representative
Repslager ropemaker
Revisor auditor
Ridder knight
Ritmester cavalry captain
Roarskarl oarsman
Rydningsmand pioneer, original clearer of land
Rytter cavalryman
Rørlegger plummer
Rådmand councilman
S
Sadelmagerlærling saddle maker’s apprentice
Sagarbeider miller
Sagbruksarbeider sawmills worker
Sagmester master saw miller
Sakfører attorney
Salmaker saddle maker
Seildukmaker canvas maker
Seilmaker sail maker
Sekretær secretary
Selveier owner, freeholder
Sersjant sergeant
Sildearbeider herring processing
Sjauer dockhand, long shore man
Sjef chief, manager
Sjøfarende seafaring, sailor
Sjøfolk seaman
Sjøkadett naval cadet
Sjøkaptein captain on a vessel
Sjåfør chauffer, driver
Skarpretter hangman, executioner
Skarpskytter sharpshooter
Skattefut tax collector
Skipsfører ship’s captain
Skieløber skier, infantry soldier on skies
Skifterettsdommer probate judge
Skipper skipper
Skipsbygger shipbuilder
Skipstømmermand carpenter onboard a ship, carpenter building ship
Skoemager shoemaker
Skogsarbeider lumberjack, logger, forest worker
Skogfut forest bailiff
Skogridder forest manager
Skogvokter forester
Skoleholder schoolmaster, teacher
Skolemester schoolmaster
Skomager shoemaker
Skomagerdreng shoemaker apprentice
Skomagersvend shoemaker journeyman
Skrabhandler scrap dealer
Skraeder tailor
Skreppekar peddler
Skriver writer, scribe
Skrædder tailor
Skræddermester master tailor
Skurekone cleaning lady
Slagter butcher
Slakter butcher
Smedlærling blacksmith apprentice
Smedmester master blacksmith
Småbruker a farmer of a small farm
Snedker carpenter
Sogneprest parish priest
Soldat soldier, usually an infantryman
Sorenskriver magistrate, probate judge
Spillemand fiddler, musician
Spinderi arbeider spinning mill worker
Spinderske female spinner
Staldkarl stall groom
Stamfar progenitor
Stasjonsmester station master at the railroad
Statsminister prime minister
Stattholder governor, vice regent
Sten arbeider stone worker
Stenhugger stone cutter
Straffange prisoner
Strandsitjar person renting a cottage near or at the coastline
Strandsitter person renting a cottage near or at the coastline
Strikkerske knitter
Strygerske ironer, laundry maid
Stubbebryter stump puller
Student student
Stuepige chambermaid
Stuert steward
Styrer ruler, director
Styrmand ships mate
Støber/støper foundry worker
Svend/svenn journeyman
Svensk Swedish
Sygepleierske nurse
Sykepleier nurse
Sypige/sypike seamstress
Søfarende sailor
Sølvverksarbeider silver smelter worker
Sømand seaman
T
Tambur drummer (soldier)
Tannlege dentist
Tegelbrænder brick/tile burner
Tekstil arbeider textile worker
Telegrafist telegraph operator
Tiener servant
Tieneste dreng servant boy, farm hand
Tieneste pige servant girl
Tigger beggar
Tilsynsmand supervisor
Timmermand lumberman
Tjener servant, farm hand
Tjeneste dreng servant boy, farm hand
Tjeneste folk servants, domestic or hired hands
Tjeneste gutt servant boy, farm hand
Tjeneste karl male employee
Tjeneste kone female servant
Tjeneste jente servant girl
Tjeneste pige servant girl
Tjeneste quinde female servant
Tollbetjent costums officer
Tomte arbeider working at a building site
Trædreier wood turner
Træsliper worker at a pulp mill
Tuskhandel barter
Tvangsfange prisoner at hard labor
Typograf typographer
Tyskertøs German tart
Tyv thief
Tømmer arbeider lumber jack, lumber man
Tømmerflaader log driver (down the river)
Tømmerfløter log driver (down the river)
Tømmerhugger logger
Tømmerfut saw mill owner
U
Ufør crippled
Uhrmager/urmaker watchmaker
Underskrevne the undersigned
Ungkarl bachelor
Utkommandert called, mobilized
Utvandrer emigrant
Utyske monster
V
Vaegter public watcher
Vanfør crippled, disabled
Vanskapt deformed, handicapped
Vaskekone cleaning woman
Vegarbeider road worker
Veiarbeider road worker
Vejarbeider road worker
Vekter public watchman
Verge guardian
Verksarbeider factory worker
Verkseier factory owner
Veterinær veterinarian
Vever weaver
Veveriarbeider worker in a weaving factory
Vesjemaker weave spool maker
Vikar substitute
Visegutt errand boy
Vitne witness
Vognmaker wagon maker
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About this Journal Submit a Manuscript Table of Contents
Journal of Biomedicine and Biotechnology
Volume 2012 (2012), Article ID 186982, 7 pages
doi:10.1155/2012/186982
Research Article
Production of Ethanol from Sugars and Lignocellulosic Biomass by Thermoanaerobacter J1 Isolated from a Hot Spring in Iceland
Faculty of Natural Resource Sciences, University of Akureyri, Borgir, Nordurslod 2, 600 Akureyri, Iceland
Received 8 June 2012; Revised 16 August 2012; Accepted 4 September 2012
Academic Editor: Anuj K. Chandel
Copyright © 2012 Jan Eric Jessen and Johann Orlygsson. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Thermophilic bacteria have gained increased attention as candidates for bioethanol production from lignocellulosic biomass. This study investigated ethanol production by Thermoanaerobacter strain J1 from hydrolysates made from lignocellulosic biomass in batch cultures. The effect of increased initial glucose concentration and the partial pressure of hydrogen on end product formation were examined. The strain showed a broad substrate spectrum, and high ethanol yields were observed on glucose (1.70 mol/mol) and xylose (1.25 mol/mol). Ethanol yields were, however, dramatically lowered by adding thiosulfate or by cocultivating strain J1 with a hydrogenotrophic methanogen with acetate becoming the major end product. Ethanol production from 4.5 g/L of lignocellulosic biomass hydrolysates (grass, hemp stem, wheat straw, newspaper, and cellulose) pretreated with acid or alkali and the enzymes Celluclast and Novozymes 188 was investigated. The highest ethanol yields were obtained on cellulose (7.5 mM·g−1) but the lowest on straw (0.8 mM·g−1). Chemical pretreatment increased ethanol yields substantially from lignocellulosic biomass but not from cellulose. The largest increase was on straw hydrolysates where ethanol production increased from 0.8 mM·g−1 to 3.3 mM·g−1 using alkali-pretreated biomass. The highest ethanol yields on lignocellulosic hydrolysates were observed with hemp hydrolysates pretreated with acid, 4.2 mM·g−1.
1. Background
More than 95% of the ethanol produced today is from simple biomass like mono- and disaccharides and starch [1]. The use of this type of biomass has been increasingly debated due to its impact on food and feed prices as well as for environmental reasons [2]. Therefore, complex (lignocellulosic) biomass has been put forward as a feasible alternative due to its abundance in nature and the large quantities generated as waste from agricultural activities [2, 3]. Lignocellulosic biomass is primarily composed of cellulose, hemicellulose, and lignin. Cellulose and hemicellulose are the main substrates used for ethanol production, but lignin is composed of aromatic lignols that need to be separated and removed before enzymatic hydrolysis. Today, expensive pretreatments are the main reason for unsuccessful implementation of complex lignocellulosic biomasses as a starting material for ethanol production [2].
The best-known microorganisms used for ethanol production today are the yeast Saccharomyces cerevisiae and the bacterium Zymomonas mobilis. Both organisms have very high yields of ethanol (1.9 mol ethanol/mol hexose) but very narrow substrate spectra and thus are not suitable for ethanol production from complex substrates. Therefore, the use of thermophilic bacteria with broad substrate range and high yields may be a better option for ethanol production from complex biomasses. It has been known for some time now that many thermophilic bacteria are highly efficient ethanol producers [4]. After the oil crisis in the 1980s, there was a peak in investigations on thermophilic ethanol-producing bacteria; bacteria within the genera of Thermoanaerobacterium, Thermoanaerobacter, and Clostridium have demonstrated good ethanol yields and fast growth rates [58]. There are several advantages in using these thermophilic bacteria: the increased temperature deters contamination from mesophilic bacteria and fungi, possible self-distillation of ethanol avoiding the generally low ethanol tolerance problem with those bacteria, and broad substrate spectrum [9, 10]. Some of these strains produce more than 1.5 mol ethanol/mol hexose [1116], whereas the theoretical maximum yield is 2.0 mol ethanol/mol hexose degraded. The main reasons for low yields are the formation of other end products such as acetate, butyrate, and CO2 [1116].
The present study focuses on a recently isolated thermophilic bacterium, strain J1, which is most closely related to species within the genus Thermoanaerobacter. Bacteria within this genus seem to be among the most efficient ethanol producers known and show very high yields from simple sugar fermentations [1214, 16] as well as from complex lignocellulosic biomass [10, 13, 1719]. These bacteria are Gram-variable rods with broad substrate spectrum (mostly sugars) and produces ethanol, acetate, lactate, hydrogen, and carbon dioxide during anaerobic fermentation [20, 21]. The physiological characteristics of Thermoanaerobacter strain J1, isolated from Icelandic hot spring, were investigated in detail with the main aim of exploring the ethanol production capacity both from simple sugars as well as from various lignocellulosic biomass.
2. Methods
2.1. Medium
The composition and preparation of the medium used has been described earlier [12]. This medium, referred to as basal medium (BM) hereafter, contains yeast extract (2 g/L) in addition to glucose or other carbon sources. All experiments were performed at 65°C at pH 7.0 without agitation with the exception of the temperature and pH optimum experiments. The inoculum volume was 2% (v/v) in all experiments which were always performed in duplicates.
2.2. Isolation of Strain J1
The strain was isolated in BM with glucose (20 mM) from a hot spring (69°C, pH 7.5) in Grensdalur in Southwest of Iceland. Samples were enriched on glucose, and positive samples (increase in growth and production of hydrogen) were reinoculated five times. From the final enrichment series, end point dilutions were performed by using BM containing agar (30 gL−1). Colonies were picked from final positive dilution and reinoculated to liquid BM with glucose. Isolation of the hydrogenotrophic methane producing strain has been described elsewhere [22].
2.3. Optimum pH and Temperature Growth Experiments
To determine the strain’s growth characteristics at various pHs and temperatures, the strain was cultivated on glucose (20 mM), and cell concentration was measured by increase in absorbance at 600 nm by a Perkin-Elmer Lambda 25 UV-Vis spectrophotometer. Maximum (specific) growth rate () for each experiment was derived from absorbance data. For pH optimum experiments, the initial pH was set to various levels in the range from 3.0 to 9.0 with increments of 1.0 pH unit. The experimental bottles were supplemented with acid (HCl) and alkali (NaOH) to set the pH accordingly. To determine the optimum temperature for growth, the incubation temperature varied from 35°C to 80°C. For the pH optimum experiments, the strain was cultivated at 65°C, and for the temperature optimum experiments, the pH was 7.0. Optimal pH and temperature were used in all experiments performed. Experiments were done in 117.5 mL serum bottles with 50 mL liquid medium.
2.4. Phylogenetic Analysis
Full 16S rRNA analysis of 1479-nucleotide long sequence was done according to Orlygsson and Baldursson [23] and references therein. Sequences from 16S rRNA analysis were compared to sequences in the NCBI database using the nucleotide-nucleotide BLAST (BLAST-N) tool. The most similar sequences were aligned with the sequencing results in the programs BioEdit [24] and CLUSTAL_X [25]. Finally, the trees were displayed with the program TreeView. Caloramator viterbiensis was used as an outgroup.
2.5. Effect of Initial Glucose Concentration on End Product Formation
The effect of initial glucose concentration on strain J1, by varying the concentration from 5 to 200 mM, was tested. Control samples contained only yeast extract. Glucose, hydrogen, acetate, and ethanol concentrations were measured at the beginning and at the end of incubation time (7 days). Experiments were done in 117.5 mL serum bottles with 60 mL liquid medium, and the pH was measured at the end of incubation time.
2.6. Substrate Utilization Spectrum
The ability of strain J1 to utilize different substrates was tested using the BM medium supplemented with various carbon substrates (xylose, arabinose, glucose, mannose, galactose, fructose, rhamnose, maltose, cellobiose, sucrose, lactose, trehalose, raffinose, starch, cellulose, CMC, avicel, xylan (from oat spelt), glycerol, pyruvate, serine, and threonine). All substrates were added from filter-sterilized (0.45 m) substrates except for xylan, starch, CMC, cellulose, and avicel which were autoclaved with the medium. In all cases, the concentration of substrates was 20 mM except for xylan, starch, CMC, cellulose, and avicel when 2 gL−1 was used. Hydrogen, acetate, and ethanol concentrations were analysed after one week of incubation. Experiments were performed in 24.5 mL serum bottles with 10 mL liquid medium.
2.7. Pretreatment of Biomass and Hydrolysates Preparation
Hydrolysates (HLs) were made from different biomasses: Whatman no. 1 filter paper, newspaper, hemp stem (Cannabis sativa), barley straw (Hordeum vulgare), and grass (Phleum pratense). Hydrolysates were prepared according to Sveinsdottir et al. [12], and the final concentration of each biomass type was 22.5 gL−1. Biomass was pretreated chemically by using 0.50% (v/v) of acid (H2SO4) or alkali (NaOH) (control was without chemical pretreatment) before heating (121°C, 60 min). Two commercial enzyme solutions, Celluclast (Novozyme, 750 Ug−1) and Novozyme 188 (Sigma C6105, 200 Ug−1), were added to each bottle after chemical pretreatment; the bottles were cooled down to room temperature and the pH adjusted to 5.0 before enzymes were added. The hydrolysates were incubated in water bath at 45°C for 68 h. After the enzyme treatment, the pH was adjusted with NaOH or HCl to pH 7.0 which is the pH optimum of the strain. The hydrolysates were then filtered (Whatman-WeiBrand; 0.45 m) into sterile bottles.
2.8. Fermentation during External Electron-Scavenging Systems
In one set of experiments, strain J1 was incubated on glucose (20 mM) in the presence of sodium thiosulfate (40 mM) and in coculture with a hydrogenotrophic methanogen. The methanogen was precultivated in BM medium with a gas phase consisting of 80% of H2 and 20% of CO2 for one week. Then the experimental culture bottles were flushed with nitrogen prior to the addition of glucose (20 mM) and strain J1. The coculture was incubated at 65°C for one week.
2.9. Fermentation of Hydrolysates
Fermentation of carbohydrates present in the hydrolysates after chemical and enzymatic pretreatment was performed in 24.5 mL serum bottles. The BM medium and inoculum (8.0 mL) were supplemented with different hydrolysates (2.0 mL, total liquid volume of 10 mL) giving a final hydrolysate concentration of 4.5 gL−1. Control samples did not contain hydrolysate; the only carbon source was yeast extract.
2.10. Analytical Methods
Hydrogen, ethanol, and volatile fatty acids were measured by gas chromatography as previously described [23]. Glucose was determined by slight modification of the method from Laurentin and Edwards [26]; supernatant broth (400 L) was mixed with 2 mL of anthrone solution (0.2% (w/v) of anthrone in 72% (v/v) of sulphuric acid). The sample was boiled for 11 minutes and then cooled down on ice. Absorbance was then measured at 600 nm by using Perkin-Elmer Lambda 25 UV-Vis spectrophotometer.
3. Results and Discussion
3.1. Phylogeny
Figure 1 shows that strain J1 belongs to the genus Thermoanaerobacter with its closest neighbours being T. uzonensis (97.7% homology) and T. sulfurigenes (95.5%). The genus Thermoanaerobacter falls into clusters V in the phylogenetic interrelationship of Clostridium according to Collins and coworkers [27]. All species within the genus are obligate anaerobes and ferment various carbohydrates to ethanol, acetate, lactate, hydrogen, and carbon dioxide [20], while some species can degrade amino acids [28]. Most strains can reduce thiosulfate to hydrogen sulphide [20, 28]. Today, the genus consists of 18 species according to the Euzeby list of prokaryotes.
Figure 1: Phylogeny of strain J1 based on partial 16S rRNA sequence analysis. The phylogenetic tree was generated by using distance matrix and neighbor-joining algorithms. Caloramator viterbensis was selected as outgroup. The bar indicates 0.01 substitutions per nucleotide position.
3.2. Optimum Growth Conditions
The strain was able to grow between 55.0°C and 75.0°C with optimal temperature being 65.0°C (; 0.23 h). The pH optimum was 7.0 (; 0.19 h). No growth was observed below pH 4.0 and above pH 9.0.
3.3. End Product Production from Sugars and Other Substrates
One of the main reasons for increased interest in using thermophilic bacteria for second-generation ethanol production is because of their broad substrate spectrum. Therefore, it was decided to cultivate the strain on the most common sugars present in lignocellulosic biomass as well as pyruvate, glycerol, serine, and threonine (Figure 2). Clearly, the strain is a very powerful ethanol producer; it produces 1.70 mol ethanol/mol glucose and 1.25 mol ethanol/mol xylose (control values subtracted) or 85.0 and 75.0% of theoretical yields, respectively. The following stoichiometry from glucose and xylose was observed:
Figure 2: End product formation from various substrates by strain J1. Data represents average of two replicate experiments. Standard deviation are shown as error bars. From left to right; ethanol, acetate and hydrogen.
Lactate was not analysed in the present paper, but high carbon recoveries from analysed end products from glucose and xylose (92.5 and 87.4%, resp.) indicate that if it was produced, its significance is very little. The substrate spectrum of the strain shows a broad capacity in degrading pentoses (xylose, arabinose), hexoses (glucose, mannose, galactose, fructose, and rhamnose), disaccharides (maltose, cellobiose, lactose, trehalose, and sucrose) the trisaccharide raffinose, and starch, pyruvate, and serine. In all the cases, the major end product is ethanol except for serine and pyruvate in which acetate is the primary end product. The highest ethanol concentrations were produced from the trisaccharide raffinose (75.2 mM). As earlier mentioned, the strain is most closely related to T. uzonensis (strain JW/IW010) which also produces ethanol and acetate as the only volatile end products, but the ratio between ethanol and acetate is 1.35 in that strain [28]. However, T. uzonensis has a more narrow sugar degradation spectrum as compared to strain J1; it cannot degrade arabinose and rhamnose. Other well-known ethanol producers within the genus are T. ethanolicus, T. thermohydrosulfuricus, and T. finnii with yields between 1.5 and 1.9 mol ethanol/mol glucose [11, 13, 14, 29].
During growth on serine and pyruvate, the carbon flow was shifted away from ethanol to acetate and hydrogen. This can be explained by the oxidation state of these substrates as compared to sugars; the oxidation state of the carbon in glucose is zero, and during its oxidation to pyruvate, the electrons are transferred to NAD+ leading to the formation of NADH. Reoxidation of NADH to NAD+ by the strain occurs most likely through acetaldehyde dehydrogenase and alcohol dehydrogenase rendering ethanol as the main product. However, both pyruvate and serine are more oxidized substrates as compared to sugars (glucose), and there is no need to reoxidize NADH. Instead, the strain deaminates serine directly to pyruvate which is decarboxylated to acetyl phosphate (by phosphotransacetylase) and further to acetate (by acetate kinase) resulting in ATP formation. However, since hydrogen production is less as compared to acetate, it is likely that the strain is also producing formate (not analyzed) instead of hydrogen from these substrates.
3.4. Effect of Initial Glucose Loadings on Ethanol Production
High initial substrate concentrations may inhibit substrate utilization and/or decrease end product yields [5, 10, 30]. In closed systems, such as batch cultures, the limited buffer capacity of the medium may be overloaded by the accumulation of organic acids resulting in a pH drop and the inhibition of substrate fermentation utilization [30]. To investigate the influence of initial substrate concentration on end product formation, changes in pH, and substrate degradation, strain J1 was cultivated with different concentration of glucose (0 to 200 mM). The strain completely degraded glucose in all experiments, except for the highest (200 mM) initial glucose loadings, and ethanol yields were between 1.2 and 1.7 mol ethanol/mol glucose (Figure 3). Acetate formation increased from 2.7 mM in control bottles (without glucose) to 9.5 mM at 100 mM glucose concentrations which was directly linked to a decrease from pH 7.0 (control) to 5.2 (100 mM glucose). At 200 mM glucose concentrations, acetate was only slightly higher as compared to 100 mM glucose concentrations, the pH dropped from 5.2 to 4.8, and only 110 mM of glucose was degraded. Thus, the limit of glucose seems to be pH related, because of the formation of acetate, rather than substrate inhibition. The strain seems to be more tolerant for initial substrate concentrations as compared to many other thermophilic bacteria where often a concentration between 20 and 30 mM is too high for a complete degradation [7, 8]. In those cases, however, more acetate was produced as compared to ethanol and may be crucial for lowering the pH at lower substrate concentrations.
Figure 3: End product formation from different initial glucose concentrations. Also shown are percent of glucose degraded. Values represent means of two replicates and standard deviation are shown as error bars. Columns from left to right; ethanol, acetate, hydrogen. pH measered after fermentation ().
3.5. Effect of Hydrogen-Scavenging Systems on End Product Formation
It is well known that Thermoanaerobacter species are highly flexible concerning end product formation depending on the culture conditions. Fardeau et al. [31] showed a dramatic shift in end product formation by Thermoanaerobacter finnii when grown on glucose in the presence and absence of thiosulfate. In that case, both ethanol and lactate decreased during thiosulfate reduction to hydrogen sulphide, whereas the acetate concentration increased. The influence of using biological hydrogen-scavenging systems has also been investigated throughout Thermoanaerobacter brockii during amino acid degradation [27]. Both thiosulfate and the presence of a hydrogen-scavenging methanogen were crucial for the oxidative deamination of the branched chain amino acids by this strain. However, degradation of a substrate that is thermodynamically easier to degrade, for example, the amino acid serine, was completely degraded in the presence and absence of thiosulfate and Methanobacterium sp. although a shift occurred between ethanol and acetate formation [27]. To investigate the influence of low partial pressure (pH2) on end product formation, strain J1 was cultivated in the presence of thiosulfate and in coculture with a hydrogenotrophic methanogen. As observed earlier, strain J1 produced ethanol as the main end product during glucose fermentation only (Table 1). The addition of thiosulfate to glucose fermentations resulted in a shift towards acetate from ethanol where the ratio between ethanol and acetate changed from 6.90 to 1.29. Cocultivating strain J1 with a hydrogenotrophic methanogen led even to more dramatic shift towards acetate (and methane), and the ratio of ethanol and acetate was 0.14. This difference in end product formation by using thiosulfate or a hydrogenotrophic methanogen is surprisingly big considering that the concentration of hydrogen is very low at the end of experimental time (0.3 to 0.5 mmolL−1) in both cases. This difference could be caused by more rapid uptake of hydrogen in the coculture experiment, but end products were only analysed at the end of the experimental time.
Table 1: Utilization of glucose by strain J1 in the presence of thiosulfate or a hydrogenotrophic methanogen. Data represents average of two replicate experiments ± standard deviation.
3.6. Fermentation of Hydrolysates from Lignocellulosic Biomass
The strain is producing maximally 33.9 mM (1.56 g/L) of ethanol from 4.5 g/L of hydrolysates made from cellulose (Figure 4). The yields on cellulose pretreated only with enzymes and heat are 7.5 mMg−1 dry weight (dw) which is considered lower as compared to glucose degradation alone (1.70 mol ethanol/mol glucose; 9.4 mMg−1 glucose). No glucose was analysed in the cellulose hydrolysate after fermentation. Thus, the lower ethanol yields on cellulose as compared to glucose indicate that the cellulose was not completely degraded during enzymatic hydrolysis. Chemical pretreatment of cellulose by the addition of acid or alkali did not increase the end product formation yields on cellulose. The highest ethanol yields on the more complex biomass types (without chemical pretreatment) were observed on hemp (11.6 mM; 2.6 mMg−1 dw) but lowest on straw (3.5 mM; 0.8 mMg−1 dw). Chemical pretreatment by adding either acid or alkali increased yields substantially on most of the lignocellulosic biomasses tested. The increase was most profound on hydrolysates from straw pretreated with alkali where ethanol production was increased from 3.5 to 14.8 mM (controls subtracted). The highest ethanol yields were however observed on hemp, 4.3 mMg−1 dw (19.0 mM). The highest ethanol yields by Thermoanaerobacter species have been reported by continuous cultures of Thermoanaerobacter strain BG1L1 on wheat straw [17] and corn stover [18], or 8.5–9.2 mMg−1 sugar consumed. Thermoanaerobacter ethanolicus has been reported to produce 4.5 and 4.8 mM ethanolg−1 hexose equivalent degraded from wood hydrolysate and beet molasses, respectively [13, 32]. Thermoanaerobacter mathranii, isolated from the same geographical area in Iceland [33] as strain J1 produced 5.3 mMg−1 sugar from wheat straw hydrolysate [34]. Recently, a new Thermoanaerobacter strain, AK5 closely related to T. thermohydrosulfuricus and T. ethanolicus, was isolated from a hot spring in Iceland and has similar yields on cellulose (7.7 mMg−1), hemp (3.1 mMg−1), and grass (4.1 mMg−1) hydrolysates [22].
Figure 4: Production of end products from hydrolysates (4.5 gL−1) from different biomasses. Values represent mean of two replicates (standard deviation). From left to right: ethanol, acetate, and hydrogen.
4. Conclusion
Ethanol production was studied by Thermoanaerobacter J1 isolated from hot spring in Iceland. The main aim of the study was to investigate the importance of various factors on ethanol production from both sugars and complex lignocellulosic biomass. The strain produces 1.70 mol ethanol/mol glucose and 1.25 mol ethanol/mol xylose and shows a broad substrate spectrum, degrading various sugars and starch but not cellulosic substrates. High ethanol yields were observed at initial glucose concentrations up to 100 mM. During growth under hydrogen removal, a shift from ethanol to acetate formation occurs. The strain produces up to 7.5 mM ethanolg−1 cellulose and 4.2 mMg−1 hemp hydrolysate.
Authors’ Contribution
J. E. Jessen carried out all experimental procedures. J. Orlygsson planned the experimental procedure and drafted the paper. Both authors read and approved the final paper.
Conflict of Interests
The authors declare that there is no conflict of interests.
Acknowledgments
This work was sponsored by RANNÍS, Technology Development Fund, projects 081303408 (BioEthanol) and RAN091016-2376 (BioFuel), and the Research Fund of the University of Akureyri. Special thanks are due to Margret Audur Sigurbjornsdottir for aligning the 16S rRNA sequences and building the phylogenetic tree and to Sean M. Scully for proofreading the paper.
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32. J. Wiegel, L. H. Carreira, C. P. Mothershed, and J. Puls, “Production of ethanol from biopolymers by anaerobic, thermophilic, and extreme thermophilic bacteria. II. Thermoanaerobacter ethanolicus JW200 and its mutants in batch cultures and resting cell experiments,” Biotechnology Bioengineering Symposium, vol. 13, no. 13, pp. 193–205, 1983. View at Scopus
33. L. Larsen, P. Nielsen, and B. K. Ahring, “Thermoanaerobacter mathranii sp. nov., an ethanol-producing, extremely thermophilic anaerobic bacterium from a hot spring in Iceland,” Archives of Microbiology, vol. 168, no. 2, pp. 114–119, 1997. View at Publisher · View at Google Scholar · View at Scopus
34. B. K. Ahring, D. Licht, A. S. Schmidt, P. Sommer, and A. B. Thomsen, “Production of ethanol from wet oxidised wheat straw by Thermoanaerobacter mathranii,” Bioresource Technology, vol. 68, no. 1, pp. 3–9, 1999. View at Publisher · View at Google Scholar · View at Scopus
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The free office suite
Download LibreOffice
LibreOffice Linux - deb (x86_64), version 3.5.7, Tsonga. Not the version you wanted? Change System, Version or Language
You need to download and install these files in order:
• Source code
LibreOffice is an open source project and you can therefore download the source code to build your own installer.
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Mar. Drugs 2011, 9(7), 1210-1219; doi:10.3390/md9071210
Article
A New Hydroxylated Nonaprenylhydroquinone from the Mediterranean Marine Sponge Sarcotragus spinosulus
1 Chemistry Laboratory of Biomolecules and Aroma, Nice Institute of Chemistry, UFR of Sciences, University of Nice-Sophia Antipolis, UMR-CNRS 6001, Parc Valrose, F-06108 Nice Cedex 02, France 2 Marseille Oceanology Center, University of Aix-Marseille, CNRS UMR 6540 DIMAR, Endoume Marine Station, Batterie des Lions Street, 13007 Marseille, France 3 INSERM UMR 895, Team 2, Cell Death Differentiation and Cancer, Batiment ARCHIMED, 151 Saint-Antoine de Ginestiere Road, BP2 3194, 06204 Nice Cedex 3, France 4 Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles, CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France
* Author to whom correspondence should be addressed.
Received: 23 May 2011; in revised form: 16 June 2011 / Accepted: 23 June 2011 / Published: 7 July 2011
(This article belongs to the Special Issue Bioactive Compounds from Marine Sponges)
Download PDF Full-Text [212 KB, Updated Version, uploaded 8 July 2011 11:55 CEST]
The original version is still available [213 KB, uploaded 7 July 2011 12:25 CEST]
Abstract: Chemical investigation of the Mediterranean sponge Sarcotragus spinosulus led to the isolation of a new hydroxylated nonaprenylhydroquinone, along with two known metabolites, hepta- and octaprenylhydroquinones. The structure of the new metabolite was assigned by extensive 1D and 2D NMR analyses and MS studies. The antileukemic effect of the three compounds towards the chronic myelogenous leukemia (CML) cells line K562 was also evaluated.
Keywords: ponges; Sarcotragus spinosulus; marine natural products; hydroxylated polyprenylhydroquinone; bioactivity
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Cite This Article
MDPI and ACS Style
Abed, C.; Legrave, N.; Dufies, M.; Robert, G.; Guérineau, V.; Vacelet, J.; Auberger, P.; Amade, P.; Mehiri, M. A New Hydroxylated Nonaprenylhydroquinone from the Mediterranean Marine Sponge Sarcotragus spinosulus. Mar. Drugs 2011, 9, 1210-1219.
AMA Style
Abed C, Legrave N, Dufies M, Robert G, Guérineau V, Vacelet J, Auberger P, Amade P, Mehiri M. A New Hydroxylated Nonaprenylhydroquinone from the Mediterranean Marine Sponge Sarcotragus spinosulus. Marine Drugs. 2011; 9(7):1210-1219.
Chicago/Turabian Style
Abed, Charline; Legrave, Nathalie; Dufies, Maeva; Robert, Guillaume; Guérineau, Vincent; Vacelet, Jean; Auberger, Patrick; Amade, Philippe; Mehiri, Mohamed. 2011. "A New Hydroxylated Nonaprenylhydroquinone from the Mediterranean Marine Sponge Sarcotragus spinosulus." Mar. Drugs 9, no. 7: 1210-1219.
Mar. Drugs EISSN 1660-3397 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert
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Nano Express
Atomic layer deposition of high-density Pt nanodots on Al2O3 film using (MeCp)Pt(Me)3 and O2 precursors for nonvolatile memory applications
Shi-Jin Ding, Hong-Bing Chen, Xing-Mei Cui, Sun Chen, Qing-Qing Sun, Peng Zhou, Hong-Liang Lu, David Wei Zhang and Chen Shen
For all author emails, please log on.
Nanoscale Research Letters 2013, 8:80 doi:10.1186/1556-276X-8-80
Published: 15 February 2013
Abstract (provisional)
Pt nanodots have been grown on Al2O3 film via atomic layer deposition (ALD) using (MeCp)Pt(Me)3 and O2 precursors. Influence of the substrate temperature, pulse time of (MeCp)Pt(Me)3, and deposition cycles on ALD Pt has been studied comprehensively by scanning electron microscopy, transmission electron microscopy, and X-ray photoelectron spectroscopy. Therefore, Pt nanodots with a high density of approximately 2 x 1012 cm-2 have been achieved under optimized conditions: 300[degree sign]C substrate temperature, 1 s pulse time of (MeCp)Pt(Me)3, and 70 deposition cycles. Further, metal-oxide-semiconductor capacitors with Pt nanodots embedded in ALD Al2O3 dielectric have been fabricated and characterized electrically, indicating noticeable electron trapping capacity, efficient programmable and erasable characteristics, and good charge retention.
The complete article is available as a provisional PDF. The fully formatted PDF and HTML versions are in production.
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[6] For the flattened piece of bread had the force of a perforated, or negative, ballot. And if one such is found in the bowl, the candidate is not admitted to the mess, because they wish all its members to be congenial. The candidate thus rejected is said to have been ‘caddished,’ for ‘caddichus’ 1 is the name of the bowl into which they cast the pieces of bread. Of their dishes, the black broth is held in the highest esteem, so that the elderly men do not even ask for a bit of meat, but leave it for the young men, while they themselves have the broth poured out for their meals.
1 Or ‘caddos,’ from which the verb in the Greek text is formed.
This work is licensed under a Creative Commons Attribution-ShareAlike 3.0 United States License.
An XML version of this text is available for download, with the additional restriction that you offer Perseus any modifications you make. Perseus provides credit for all accepted changes, storing new additions in a versioning system.
load focus Greek (Bernadotte Perrin, 1914)
hideData/Identifiers
Citation URN: urn:cts:greekLit:tlg0007.tlg004.perseus-eng1:12.6
Document URN: urn:cts:greekLit:tlg0007.tlg004.perseus-eng1
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Neusoft Mobile Solutions receives Silver Award from GSMA joyn Innovation Challenge
Printer-friendly versionPDF version
SHENYANG, China, March 13, 2013 -- Neusoft Mobile Solutions, a fully owned subsidiary of Neusoft Corporation (Neusoft), announced that its innovation, 'Family joyn,' received the Silver Award from GSMA joyn Innovation Challenge. The joyn Innovation Challenge was an initiative to encourage innovations based on RCS. The challenge stimulates mobile and web app developers and technology innovators to build on the core capabilities of joyn and explore ideas for new and attractive services. Using the joyn 'innovation accelerator,' a developer can use APIs on a live GSMA Rich Communication Services (RCS) network. As these APIs have already been standardized, it will be easy for any new services to be transferred to a live operator commercial network as they become available.
A competition was held and 17 ideas from 12 companies were short listed. The companies were then asked to present their ideas to the judging panel. The best new ideas were showcased at the GSMA Pavilion at the Mobile World Congress 2013.
Neusoft's 'Family joyn' client is expanding the communication capability within the daily life. The application is mainly targeted at family members who are the most vulnerable and at the same time most valuable – children and elders.
The application offers a simplified and locked RCS user-interface, which ensures the right functions are continuously available. The most important contacts are available under one-click for immediate Voice and Video Share connection. With the RCS real-live location information pull, parents can have continuous information about their children's location and safety.
"The approach for the innovation was set to be very practical and the ground-rule was to create something that can be actually used within real-life" explained Customer Services Manager, Antti Miikkulainen. "Actually the whole idea then started from the daily need at home," he laughs.
Neusoft Mobile Solutions' 'Family joyn' offers excellent enhancement to RCS. The client enables:
• re-use of 2-3 years old returned subscription phones to turn them into "Family joyn" phones;
• "Lifetime SIM" to decrease churn;
• improved RCS knowledge and penetration.
News Source : Neusoft Mobile Solutions receives Silver Award from GSMA joyn Innovation Challenge
Copy this html code to your website/blog and link to this press release.
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Weekly Search Buzz RoundUp - 4/6/07
Apr 6, 2007 • 12:40 pm | (2) by | Filed Under Search Buzz Video Round Recap
Ah, the month of April. We opened the month with a few fun tricks all around and then Barry and I went off to celebrate the first days of Passover. What did you (and I) miss in the week of search? Let's take a look.
April 1st: Funny?
Barry found some great Google Maps April Fools Day finds. He then created some funny maps with screenshots including an earthquake in NYC, an alien bug just north of Central Park, and a sinkhole over Manhattan zipcode 10025 (where I used to live! Hmph!).
(By the way, Google's pre-Katrina maps blunder was not a joke, and they addressed it pretty quickly, as Andy noticed.)
The Search Community also participated in April Fools Day. Search Engine Roundtable (yes, that's us) had a silly red background, ad-free WebmasterWorld put up AdSense for a day, and Matt Cutts had only 5% of us fooled with his hacked blog. I covered the other Google jokes (Google TiSP and Gmail Paper) in this week's Digg Digest.
Cuttscon was a joke too, guys. Don't get too excited. Matt may originally be from Kentucky, but I'm pretty sure he doesn't live there anymore.
Click, click: Microsoft and Yell
Is Microsoft buying Doubleclick? The cost is apparently two billion dollars too steep, so Microsoft will probably pass for now.
Yell appears to have launched its own PPC advertising solution. They're a little late, but evilgreenmonkey thinks that they have a lot of potential.
Google's Forever-Changing Algorithm
Chris Boggs wrote an interesting post about freshness of Google results and says that updated content does play a role in the results. I followed up a day later with some other similar findings on the Google algorithm, this time related to relevancy of Google results. That makes me think: is Google putting more emphasis on more updated content instead of on relevancy? I guess we'll have to wait this one out.
Ads? What ads?
Google made its way into radio, mobile phones, and now ... it's Google TV. This week, Google announced that it forged a partnership with Echostar and Astound Cable to offer targeted television ads, since yes, people (like Danny) still do seem to watch TV.
Ask.com is under fire this week for its very questionable tactics in the Information Revolution marketing campaign. Should Ask really be trying to hijack small companies' traffic onto its search engine? Where would you draw the line?
Google Announces My Maps
Speaking of drawing lines, I had great fun this morning when I illustrated the nifty little Google Personalized Maps tool. Great fun! Check out the coverage on Google My Maps at Search Engine Land for coverage into this new tool, including comparisons from other search engines.
Yahoo Wants Elevator Pitches
Yahoo has asked advertisers to shorten their Yahoo Sponsored Listing descriptions by May/June of this year. The new character requirement has been shortened from 190 characters to 75. That's even shorter than Twitter! As many of you know, I like to microblog, so this hurts. (By the way, Matt Cutts, after all I wrote about you, you hurt me too. Sigh.)
Contextual Ads For the Win, Banner Ads -- Not So Much
Okay, so I made a booboo. Ben blogged about the banner blindness study in my absence, which indicated that people are starting to ignore ads that seem too obvious. I, confused from a real delayed flight, wrote about the same thing a day later. Ben then called me out on it. The joke's on me. Regardless, the findings are pretty important, especially if you're an advertiser, so maybe it does deserve two posts after all.
Search Engine Optimization
Barry, Ben Pfeiffer, and Chris Boggs are just three of over thirty well-respected industry experts who were asked to submit feedback for SEOmoz's updated Search Engine Optimization ranking factors. This chart is pretty cool and very flashy. Great design, guys!
The question has arisen yet again. Should you outsource your link development in SEO? No! The discussion goes on to defining what a quality link is and what to look out for. Ben's post is a great read.
Chris also writes about how to cajole developers into creating search-engine friendly URLs. He references a thread that tells you to buy them a beer. I suppose that is a good way to lead me into next week's events...
Search Engine Strategies NY
Remember, Search Engine Strategies NY is next week. As we wrote last week, will have complete coverage of the sessions, but new this week is the SES NY 07 party schedule. SES is a great place to network, so be sure to check out the events after the daily sessions.
Next week in search
Barry and I are off on Monday and Tuesday once again for the last days of the Passover holiday, but we'll be joining the rest of you at SES on Wednesday. We have a few posts lined up for next week already, but you can expect for the SES coverage to be massive. I am truly looking forward to meeting you all!
Previous story: Ask.com Markets on Google to Lure Visitors to its Own Search Engine
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CMD sent two reporters to track ALEC in Oklahoma
Click here to help support our future investigations.
Alexander Shakow
From SourceWatch
Jump to: navigation, search
Alexander Shakow "joined the board of NetSol Technologies on June 4, 2007. Mr. Shakow had a distinguished career with the World Bank where he held various high level positions from 1981-2002. Since 2002, he has been an independent consultant for various international organizations. From 1968-1981, Mr. Shakow held many senior positions at the United States Agency for International Development, including Assistant Administrator for Program and Policy; Director -Office of Development and Planning, Bureau for Asia; and, Director - Indonesia, Malaysia and Singapore affairs. Mr. Shakow was also a staff member of the United States Peace Corps from 1963-1968, including director in Indonesia. Mr. Shakow received his PhD from the London School of Economics and Political Science in 1962. He earned his undergraduate degree with honors from Swarthmore College in 1958. Mr. Shakow is listed in Who’s Who in America and Who’s Who in the World; and currently is a member of the Board of Trustees of EnterpriseWorks/VITA. Mr. Shakow is a member of the Audit, Compensation and Nominating and Corporate Governance Committees." [1]
Resources and articles
Related Sourcewatch articles
References
1. Alexander Shakow, Reuters, accessed August 31, 2008.
2. Staff, Cultural Change Institute, accessed December 12, 2010.
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User:Parsa/George Banks
Watchers
Contents
George Lovell Banks Research
Research page for George L. Banks.
Personal Facts
• Birth: 13 Oct 1839 — Find A Grave, Connelley, Duncan, Chapman
• Medal of Honor awarded: 28 Sep 1897 — Civil War MOH recipients
• Death: 20 Aug 1924 — Find A Grave
Family Facts
Employment Facts
Service and Fraternal Facts
Residences
• Lake County, Ohio
• Laporte Counties, Indiana
• Lake County, Indiana
• St. Anthony, Minnesota
• Northern Michigan, 1857
• Lake County, Indiana
• (Civil War)
• Lake County, Indiana
• Fawn Creek Township, Montgomery County, Kansas, 1871
• Angola, Indiana, Fall 1886
• Hillsdale County, Michigan, Spring 1887
• Montgomery County, Kansas, (farm) c 1893
• Independence, Montgomery, Kansas
• Montgomery County, Kansas, (farm) 1896
• 417 North Fifth Street, Independence, Montgomery, Kansas, 1903
• concurrently owned his farm near Bolton, KS, Dearing, KS, and Chambers County, Texas.
Source codes
• Connelley = A Standard History of Kansas and Kansans, written and compiled by William E. Connelley, Secretary of the Kansas State Historical Society, Topeka. Chicago: Lewis Publishing Company, copyright 1918
• Duncan = History of Montgomery County, Kansas, By Its Own People, published by L. Wallace Duncan, Iola, Kansas, 1903, pgs. 468-470
• Chapman = Portrait and biographical album of Hillsdale County, Mich: containing full page portraits and biographical sketches of prominent and representative citizens of the county, together with portraits and biographies of all the governors of the state, and of the presidents of the United States. (Chicago: Chapman Bros., 1888)
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Tell me more ×
Answers OnStartups is a question and answer site for entrepreneurs looking to start or run a new business. It's 100% free, no registration required.
I just wondering what is the reason for some companies to not obfuscate their product that written in Java/.NET ?
I've worked with expensive enterprise software, and I helped since the software don't apply obfuscation on their web app client, so I can made understand how it work and write customization easily. Could this is one of the reason ?
share|improve this question
2 Answers
For me the important things to consider are:
-What could the client do with my source code if they had it? Could they cost me money etc.?
-What kind of client am I dealing with, are they trustworthy?
I am in the USA. If I have a signed agreement with a major USA company and they agree not to reverse engineer or "steal" the software then I am generally not worried. If it turns out that they violate the agreement and it causes me to loose money I take them to court and collect big bucks in damages. Major companies just don't like to take such risks, and thus generally don't ripoff software on a major scale.
If on the other hand I had to send my software to some third-world country with no effective intellectual property protection and a track record of bootlegging like crazy it would behoove me to obfuscation as much as possible and understand that no matter what I do they are probably still going to steal it if they want to.
Now to the other point, what can they do with it to hurt me if they steal it? Most people think their software is worth far more than it probably is. Think about the major companies; Microsoft Windows is pirated all the time, but Microsoft is still making lots of money.
Then there is the question of if the costs associated with obfuscation outweigh the benefits. Perhaps there is a parallel to copy protection; entertainment companies spend big dollars on copy protection and yet they get ripped off anyway while inconveniencing legitimate users. Consider how Apple decided to drop copy protection from the music they sell.
Finally there is some software that perhaps is so sensitive that it could affect national defense etc. In the USA there are laws and export controls on such things, but this is not your average software package.
share|improve this answer
If it's a webapp then the important part is on the server where you can't get to it.
share|improve this answer
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My fiancée and I, this weekend, attended what for reasons of space will be described as "professional development for engaged couples."
There were threats all throughout the literature: if you left early, brought a cell phone, left the grounds at all, or committed any number of minor-league infractions, you would not receive a certificate of completion.
Nope. Don't — no, y'know — just stop begging. Stop it. You were seven-and-a-half minutes late to our noon session. No certificate.
Thing was, me and my girl didn't much care for a certificate. And after it became evident our presenters were going to read from a script all day, we left.
The relevance of this anecdote to classroom management is left as an exercise to the reader.
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RandomFields (2.0.66)
0 users
Simulation and Analysis of Random Fields.
http://ms.math.uni-mannheim.de
http://cran.r-project.org/web/packages/RandomFields
Simulation of Gaussian and extreme value random fields; conditional simulation; kriging
Maintainer: Martin Schlather
Author(s): Martin Schlather [aut, cre], Peter Menck [aut], Richard Singleton [ctb], Ben Pfaff [ctb], R Core team [ctb]
License: GPL (>= 2)
Uses: Does not use any package
Reverse depends: CircSpatial, DSpat, Geneland, geoR, glmmBUGS, lgcp, MarkedPointProcess, ProbForecastGOP, SoPhy, SpatialExtremes, spatstat, WeedMap
Reverse suggests: CompRandFld, fractaldim, geoR, rpanel, spatstat
Released 3 months ago.
22 previous versions
Ratings
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Log in to vote.
Reviews
No one has written a review of RandomFields yet. Want to be the first? Write one now.
Related packages: DCluster, FieldSim, Geneland, GeoXp, MarkedPointProcess, PBSmapping, PBSmodelling, RArcInfo, RColorBrewer, RPyGeo, ade4, adehabitat, ads, akima, ash, aspace, classInt, clustTool, deldir, ecespa(20 best matches, based on common tags.)
Search for RandomFields on google, google scholar, r-help, r-devel.
Visit RandomFields on R Graphical Manual.
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Error!
Success!
Dean Hume - HTTP HEAD Request - Benefits / Usages
0
kicks
Dean Hume - HTTP HEAD Request - Benefits / Usages (Unpublished)
Until recently, I had heard of the HTTP HEAD method but never actually used it, or quite understood it's usages. I'm sure most of us have used some of the common HTTP Verbs before such as GET or POST. Let me explain a little bit about the HTTP HEAD request method.
Kicked By:
Drop Kicked By:
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Squash FS Howto
From eLinux.org
Revision as of 19:04, 12 December 2006 by Wmat (Talk | contribs)
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Contents
SquashFS HOWTO
Artemiy I. Pavlov
Artemio.net
<artemio (at) artemio (dot) net>
2004-06-07 Revision History Revision 1.5 2004-06-07 Changes according to SquashFS release 2.0 alpha. Lots of description improvements and clarifications. Split instructions for Linux kernels of 2.6.x (new) and 2.4.x series. Revision 1.1 2004-05-22 Changes according to SquashFS release 1.3r3. Revision 1.0 2004-02-19 Initial Release, reviewed by LDP. Revision 0.2 2003-12-08 Text corrections, license added. Revision 0.1 2003-11-24 Initial version. Instructions for SquashFS release 1.3r2.
Abstract
This HOWTO describes the usage of SquashFS - a highly-compressed read-only file system for Linux, which is intended for use in tiny-sized and embedded systems, and anywhere else you'd want to use a compressed file system. With this document, you'll learn how to prepare a SquashFS-ready Linux kernel, create a sqaushed file system and happily use it.
Home of this HOWTO
The SquashFS HOWTO lives at http://artemio.net/projects/linuxdoc/squashfs. There you will always find the latest version of the document, and will be able to send your feedback.
What is SquashFS
Introduction
When creating tiny-sized and embedded Linux systems, every byte of the storage device (floppy, flash disk, etc.) is very important, so compression is used everywhere possible. Also, compressed file systems are frequently needed for archiving purposes. For huge public archives, as well as for personal media archives, this is essential.
SquashFS brings all this to a new level. It is a read-only file system that lets you compress whole file systems or single directories, write them to other devices/partitions or to ordinary files, and then mount them directly (if a device) or using a loopback device (if it is a file). The modular, compact system design of SquashFS is bliss. For archiving purposes, SquashFS gives you a lot more flexibility and performance speed than a .tar.gz archive.
SquashFS is distributed as a Linux kernel source patch (which enables SquashFS read support in your kernel), and the mksquashfs tool, which creates squashed file systems (in a file or on a block device).
The latest SquashFS release tree is 2.x, the former one was 1.x. This document describes both these releases with proper notes given. For example, if some feature or parameter is different in these release trees, it will be written as follows: new value (2.x) or old value (1.x)
Overview of SquashFS
• Data, inodes and directories are compressed
• SquashFS stores full uid/gids (32 bits), and file creation time
• Files up to 2^32 bytes are supported; file systems can be up to 2^32 bytes
• Inode and directory data are highly compacted, and packed on byte boundaries; each compressed inode is on average 8 bytes in length (the exact length varies on file type, i.e. regular file, directory, symbolic link, and block/character device inodes have different sizes)
• SquashFS can use block sizes up to 32 Kb (1.x) and 64Kb (2.x), which achieves greater compression ratios than the normal 4K block size
• SquashFS 2.x inroduced the concept of fragment blocks: an ability to join multiple files smaller than block size into a single block, achieving greater compression ratios
• File duplicates are detected and removed
• Both big and little endian architectures are supported; SquashFS can mount file systems created on different byte-order machines
Making it clear
Now let's make sure any further discussions will be clearer for you to understand. The procedure of getting SquashFS working, basically, consists of the following steps:
1. Patching and recompiling the target Linux kernel to enable SquashFS support
2. Compiling the mksquashfs tool
3. Creating a compressed file system with mksquashfs
4. Testing: mounting a squashed file system to a temporary location
5. Modifying the /etc/fstab or startup scripts of your target Linux system to mount the new squashed file system when needed
Getting ready for SquashFS
Acquiring SquashFS
The SquashFS home site is located at http://squashfs.sourceforge.net/ - it contains news for the latest release and it's changelog, as well as general information about SquashFS. You can grab the latest version at the SqaushFS project page at SourceForge.
Preparing a SquashFS-capable kernel
In order to read SquashFS, you need it supported in your kernel - just as if it was a reiserfs or ext3 file system. You have to make sure there is an appropriate patch for your kernel version - it should be located in linux-2.x.y subdirectory of the SquashFS source tree. Also, remember that in most cases you will need a clean (original) Linux kernel source from kernel.org. If your kernel source is from a distro vendor, it may be already pre-patched with custom vendor patches, and patching with a SquashFS patch will almost surely not work, as SquashFS patches are made against original Linux kernels.
Patching the kernel source
With a kernel source and a proper SquashFS patch present, all you have to do is (we'll assume that you have your Linux kernel source in /usr/src/linux and that you have the SquashFS source in /usr/src/squashfs):
Change to the SquashFS source directory and copy the kernel patch (we'll assume it's named squashfs-patch) to /usr/src/linux.
bash# cd /usr/src/squashfs
bash# cp linux-2.x.y/squashfs-patch /usr/src/linux
Go to the linux kernel source directory /usr/src/linux:
bash# cd /usr/src/linux
Note: please remember that we will not be leaving this directory during all further kernel-related procedures, and all paths will be given relative to /usr/src/linux.
Now patch the source with the SquashFS patch:
bash# patch -p1 < squashfs-patch
Compiling a 2.6.x kernel
Cleanup and prepare the kernel source:
bash# make distclean
bash# make mrproper
Configure the kernel using your favourite method (config/menuconfig/xconfig/gconfig): bash# make menuconfig
1. In the "File systems" section, "Miscellaneous file systems" subsection, enable the "Squashed filesystem" option, whether as module or bundled with the kernel. It is only obligatory to compile SquashFS inside the kernel if you plan using squashed initial RAM disks (initrd).
2. If you would like to use a squashed initial RAM disk, enable the "Initial RAM disk support" in the "Device drivets" section, "Block devices" subsection. If you want to be able to mount the squashed file system via a loopback device in future, you should enable
3. "Loopback device support" in the "Device drivers" section,
4. "Block devices" subsection.
Now you may compile the kernel and modules:
bash# make
Compiling a 2.4.x kernel
Configure the kernel:
bash# make menuconfig
1. In the "File systems" section, enable the "Squashed filesystem" option, whether as module or bundled with the kernel. It is only obligatory to compile SquashFS inside
the kernel if you plan using squashed initial RAM disks (initrd).
1. If you would like to use a squashed initial RAM disk, enable the "Initial RAM disk support" in the "Block devices" section.
2. If you want to be able to mount the squashed file system via a loopback device in future, you should enable "Loopback device support" in the "Block devices" section.
Now you may compile the kernel and modules:
bash# make dep
bash# make bzImage
bash# make modules
Installing and testing the kernel
It's time to install your new SquashFS-enabled kernel. The instructions below are for installing and booting the kernel on the host machine. You may want to install and test it on the target system.
We assume that the kernel was compiled for a x86 architecture, and the compressed kernel image is located in the arch/i386/boot/ subdirectory of the kernel tree. Now copy the kernel to the /boot directory (and name it bzImage-sqsh for convenience, if you like):
bash# cp arch/i386/boot/bzImage /boot/bzImage-sqsh
Don't forget to install the kernel modules if you have any:
bash# make modules_install
Modify your boot loader's configuration file to include your new kernel and install (update) the boot loader. Now you may reboot with your new kernel. When it boots, check that everything went fine:
bash# cat /proc/filesystems
Or, if you built SquashFS support as a kernel module:
bash# insmod squashfs
bash# cat /proc/filesystems
If you see the squashfs line among other file systems, this means you have successfully enabled SquashFS in your kernel.
Compiling the mksquashfs tool
Now you need to compile mksquashfs - the tool for creating squashed file systems.
bash# cd /usr/src/squashfs/squashfs-tools
Compile and install mksquashfs:
bash# make
bash# cp mksquashfs /usr/sbin
If everything went fine, typing mksquashfs at the shell prompt should print it's "usage" message.
The mksquashfs tool, exposed
Using mksquashfs
mksquashfs is the tool for creating new squashed file systems, and for appending new data to existing squashed file systems. The general command-line format for mksquashfs is:
bash# mksquashfs source1 source2 ... destination [options]
• source1, source2, etc.: files and directories to be added to the resulting filke system, given with relative and/or absolute paths
• destination: a regular file (filesystem image file), or a block device (such as /dev/fd0 or /dev/hda3) where you want to have your squashed file system
Notes for default mksquashfs behavior:
• When the new files are added to the new file system or appended to an existing one, mksquashfs will automatically rename files with duplicate names: if two or more files named text will appear in the same resulting directory, the second file will be renamed to text_1, third one to text_2 and so on.
• Duplicate files will be removed, so there will be only one physical instance (with SquashFS 2.x, you can disable the detection/rtemoval of the duplicates with the -no-duplicates option).
• If destination has a pre-existing SquashFS file system on it, by default, the new source items will be appended to the existing root directory. Examine the options table below to force mksquashfs to overwrite the whole destination and/or change the way new source items are added. Please note that it is not possible to append to a file system created with mksquashfs 1.x using mksquashfs 2.x. You will need to mount the SquashFS-1.x file system and copy the files to some location, and then join them with other needed files to create a SquashFS-2.x file system.
• If a single source file or directory is given, it becomes the root in a newly created file system. If two or more source files and/or directories are given, they will all become sub-items in the root of the new file system.
• The resulting filesystem will be padded to a multiple of 4Kb: this is required for filesystems to be used on block devices. If you are very sure you don't ned this, use the -nopad option to disable this operation.
See the next section for more details about all possible options.
Command-line options
All possible options for mksquashfs are shown in the table below.
Table 1. Command-line options of the mksquashfs tool
Option Description
-always-use-fragments divide all files greater than block size
into fragments (2.x only, will result in greater compression ratios)
-b [block size] use [block size] filesystem block size (32
Kbytes default) - this can be either 512, 1024, 2048, 4096, 8192, 16384 or 32768
-be or -le force a big or little endian file system,
respectively
-check-data enable additional file system checks
-e [file1] ( [file2] ... ) specify which files and/or
directories to omit from the new file system that is to be created
-ef [file] specify a file which contains the list of
files/directories to exclude
-info print files, their original size and compression ratio,
as they are added to the file system
-keep-as-directory if the source is a single directory, force
this directory to be a subdirectory of the root in the created file system
-noappend if the destination file/device already contains a
squashed file system, overwrite it, rather than append the new data to an existing file system
-no-duplicates do not detect/remove duplicate file names
-noD or -noDataCompression do not compress the data
-noF or -noFragmentCompression do not compress the fragments
(2.x only)
-no-fragments do not generate fragment blocks (2.x only, this
will produce almost the same filesystem as 1.x did)
-noI or -noInodeCompression do not compress the inode table
-nopad do not pad the resulting file system to a multiple of 4
KBytes
-root-becomes [name] can be used while appending to a
pre-existing squashed file system: it will make a new root, and [name] directory will contain all pre-existing files/directories
-version print the version, copyright and license message
In most cases, you should leave all compression/block options by default, as they allow mksquashfs to achieve the best possible compression ratios.
Creating and using squashed file systems
Basic steps
In order to create a squashed file system out of a single directory (say, /some/dir), and output it to a regular file (thus, producing a file system image), you need to say only one magic phrase:
bash# mksquashfs /some/dir dir.sqsh
mksquashfs will perform the squashing and print the resulting number of inodes and size of data written, as well as the average compression ratio. Now you have your /some/dir directory image in the dir.sqsh file. You can now use the mount command to mount it using a loopback device:
bash# mkdir /mnt/dir
bash# mount dir.sqsh /mnt/dir -t squashfs -o loop
To check if you have what's expected:
bash# ls /mnt/dir
If you want to output the file system directly into a device (say, your floppy at /dev/fd0):
bash# mksquashfs /some/dir /dev/fd0
Then just mount the device:
bash# mount /dev/fd0 /mnt/floppy -t squashfs
And check if it's okay:
bash# ls /mnt/floppy
Squashing file systems
Operations described here correspond to most cases where a read-only compressed file system can be used, whether you want it to be on a block device or in a file. This could be anything from large FTP/HTTP-served archives that don't change often, to having a squashed /usr partition and anything alike with these.
Example 1
Let's suppose you have a /var/arch directory with lots of files and that you want to turn it into a squashed file system and keep it on your root partition as a file (it will be a file system image that you will mount via a loopback device). The operations needed to perform are as follows.
Squash the directory, then mount it via loopback to test it:
bash# mksquashfs /var/arch /var/arch.sqsh
bash# mkdir /mnt/tmp
bash# mount /var/arch.sqsh /mnt/tmp -t squashfs -o loop
bash# ls /mnt/tmp
If everything is as expected, make this file system mount automatically at boot time by adding this line to your /etc/fstab:
/var/arch.sqsh /var/arch squashfs ro,defaults 0 0
Unmount the file system from the temporary mount point, and mount using it's fstab entry:
bash# umount /mnt/tmp
bash# mount /var/arch
Now just ensure that everything works fine:
bash# ls /var/arch
Example 2
Say you have two hard disk partitions, /dev/hda6 (which is empty) and /dev/hda7 (which is bigger than /dev/hda6, mounted at /var/arch, contains some data and is full). Now, say you want to squash the /dev/hda7 file system and move it to /dev/hda6, then use /dev/hda7 for some other purposes. We will suppose you have the following line in /etc/fstab (reiserfs is just an example file system used on /dev/hda7):
/dev/hda7 /var/arch reiserfs defaults 0 0
In the same fashion as with the previous example:
bash# mksquashfs /var/arch /var/arch.sqsh
bash# mkdir /mnt/tmp
bash# mount /var/arch.sqsh /mnt/tmp -t squashfs -o loop
bash# ls /mnt/tmp
If everything went fine, unmount /dev/hda7 and use dd to copy
/var/arch.sqsh to /dev/hda6:
bash# umount /dev/hda7
bash# dd if=/var/arch.sqsh of=/dev/hda7
Now change the line in /etc/fstab for /dev/hda7 to:
/dev/hda6 /var/arch squashfs ro,defaults 0 0
Mount the new file system and check to see if all went fine:
bash# mount /var/arch
bash# ls /var/arch
Don't forget to erase the unneeded file system image:
bash# rm /var/arch.sqsh
Creating tiny/embedded systems
By saying "tiny/embedded", I mean Linux systems that are being built for booting from floppy disks, IDE/USB flash disks, iso9660 CD-ROMs, small-sized hard drives and the like. Whether you want to have your whole root file system on a single media (a single partition, a single floppy), or have a modular system (several floppies or disk partitions), the procedure is almost identical. Creating such Linux systems themselves is out of scope of this HOWTO - there are dedicated HOWTOs and guides for this (like the Bootdisk HOWTO and Linux From Scratch - visit www.tldp.org to retrieve these documents).
Squashed file systems on floppy/flash/hard disks
In order to use SquashFS for creating Linux systems on small disks, you just have to follow the usual steps for creating a minimal system, performing the following operations at respective points:
1. When developing a kernel for your system, make sure you enable SquashFS support so it can mount squashed file systems
2. Use mksquashfs for creating read-only initial ram disks and/or root and/or other file systems
3. Don't forget to set file system types to squashfs in /etc/fstab and/or the startup scripts of your system for mounting squashed file systems
Floppy example. Let's say you have your floppy system tree at /home/user/floppylinux and you want to place the root file system on one floppy and /usr on another. What you should do is:
bash# cd /home/user
bash# mksquashfs floppylinux root.sqsh -e usr
bash# mksquashfs floppylinux/usr usr.sqsh
Note 1: you can see here how we use the -e option to exclude the /usr directory for root file system's image.
Note 2: don't forget to specify squashfs in your root disk's /etc/fstab or startup scripts when mounting the /usr file system.
Insert a root disk in your 3.5" floppy drive (I assume you have a lilo or grub on it, and, thus, a file system exists on this floppy, and the root file system will reside under the /boot directory of this file system):
bash# mount /mnt/floppy
bash# cp root.sqsh /mnt/floppy/boot
When done, unmount the root floppy, change the floppy to a /usr disk and use dd to transfer the usr file system:
bash# dd if=usr.sqsh of=/dev/fd0
Squashed file systems on CD-ROMs
With SquashFS, you can compress large file systems that will be used in live CDs (just as an example).
1. Enable SquashFS in the linux kernel of the target system
2. Create a squashed root file system
3. Modify the /etc/fstab or startup scripts of the target system to mount the squashd file system when you need it
If you create a root file system out of a running Linux system, use the -e option for mksquashfs to exclude all pseudo-filesystems such as /proc, /sys (on linux kernels after 2.5.x) and /dev (when using DevFS). Also, don't forget to add the file system image itself that is being created with mksquashfs (I think you know the reasons for these exclusions).
Acknowledgements
I would like to express my sincere thanks and immeasurable respect to:
• Phillip Lougher - for his brilliant work under squashfs, for creating an exculsive patch for linux-2.4.18, for his help with polishing this howto and answers to my mails
• Tabatha Marshall at TLDP for helping me with bringing this HOWTO to the final 1.0 release
• Everybody at The Linux Documentation Project for their great work under all the HOWTOs and guides that helped me a lot with exploring and hacking Linux
• All those at the TLDP mailing lists who helped me with getting started
• Endless thanks and respect to everybody who develops open-source software
License
This document may be used and distributed under the terms and conditions set forth in the Open Content licence. In short, this means that you can freely modify and re-distribute the HOWTO under the main condition that you keep the author and copyright the article along. The full text of the licence is available at http://www.opencontent.org/opl.shtml
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Crow IndiansEdit This Page
From FamilySearch Wiki
Indians of Montana > Crow Indians
To get started in American Indian Research
Crow
Population
1990 8,491
1980 3,953
1944 abt. 2,500
1923 1,777 [1]
1904 1,826 [2]
1871 abt. 4,100 [3]
1804 abt. 3,500 [4]
Regions with significant populations
Ancestral Homelands:
Upper plains on or near the Missouri River and its tributaries, especially the Bighorn and Yellowstone Rivers
Descendants:
Crow Reservation in Bighorn County, Montana.
Status
Federally recognized as Crow Tribe of Montana
Linguistic Group
Siouan
Other Related Ethnic Groups
Mountain Crow, River Crow, Hidatsa
Bands and Groups of Crow -- Achepabecha, Ahaharopirnopa, Ahachik, Ashinadea, Ashbochiah, Ashkanena, Booadasha, Ehartsar, Esachkabuk, Esekepkabuk, Hokarutcha, Noota Oosabotsee, Pareescar, Petchaleruhpaka, Shiptetza, and others.
Alternate Names and Spellings: Crow Tribe of Montana[5], Absaroka[6], Apsáalooke[7]
Contents
Tribal Headquarters
Crow Tribe of Montana
P. O. Box 159
Crow Agency, MT 59022
Phone: 1.406.638.3708
Fax: 1.406.638.7301
Website: www.crowtribe.com
History
The first recorded contact between the Crows and non-Indians was with the Lewis and Clark Expedition in 1806. The tribe was later involved with trading and interacting with mountain men during rendezvous.
In 1825 strong tribal leaders initiated a division of the tribe, and the Mountain Crow and River Crow tribes were formed. This same year some Crow warriors assisted the United States military in fighting other Indian tribes.
A treaty was signed in 1851 at Fort Laramie which included the Crow Tribe, but it was the 1868 Treaty which established a reservation for the Crows in Montana.
During the 1870s and the Indian Wars for the West, the Crow warriors served as scouts fighting against the Sioux and Nez Perce. In the historic Battle of the Little Big Horn, General Custer had Crows serving as scouts.
The 1880 treaty specified that the "Crow Indians shall consent to permit cattle to be driven across their reservation or grazed on the same, the Secretary of the Interior shall fix the amount to be paid by parties desiring to so drive or graze cattle; all moneys arising from this source to be paid to the Indians..."
Even though they had served the U.S. military, the tribe was removed to the Crow Reservation in Big Horn and Yellowstone Counties, Montana.
The Crow Tribe adopted their Constitution and By-Laws in 2001.
Brief Timeline
• 1806: The Lewis and Clark expedition encountered the Tribe
• 1821: The tribe interacted with mountain men during fur-trading rendezvous
• 1825: divide into Mountain Crow and River Crow
• 1825: joined the United States soldiers in fighting other Indian tribes
• 1851: Treaty signed at Fort Laramie, Wyoming (38.5 million acres in Montana)
• 1868: Treaty at Fort Laramie established a reservation in Montana, south to the Yellowstone River
• 1870: During the wars for the West the Crow were allies of the U.S. Army, serving as scouts, and fought against the Sioux and the Nez Perce.
• 1876: Crow warriors acted as scouts for General Custer; Custer defeated at Little Bighorn in July 1876
• 1880: Treaty at Washington D.C.,
• 1887: Crow Indian outbreak led by Deaf Bull near Crow Agency, Montana.
• 1888: ceded most of their land; removed to Crow Reservation in Big Horn and Yellowstone Counties, Montana
Additional References to the History of the Tribe
Records
Agencies
The following agencies of the Bureau of Indian Affairs had jurisdiction over the Crow for the time periods indicated. BIA agencies were responsible to keep such records as census rolls, allotment (land) records, annuity rolls, school records, correspondence, and other records of individual Indians under their jurisdiction. For details, see the page for the respective agency.
The agencies which had jurisdiction over a major portion of the Crow in the United States were:
Census
The Bureau of Indian Affairs compiled annual Indian Census Rolls on many of the reservations from 1885 to 1940. They list the names of individuals, their age, and other details about each person enumerated. For more information about these records, click here.
The following table lists the census rolls for the Crow Indians:
Agency Location of Original Records
Post- 1885 Census
M595 RG 75 -- 692 Rolls
Roll Number
FHL
Film Number
Crow Agency, 1891-1940 National Archives in Washington D.C. Rolls 79-86 FHL 575771-575778
Correspondence
There are several sets of correspondence between the supervising offices of the Bureau of Indian Affairs and the local offices -- agencies, subagencies, etc. The correspondence is often historical in nature, including reports of the conditions among local groups of Indians, hostilities, plans for building facilities, activities of traders or missionaries, etc. Occasionally, there will be names of individuals but little detail about them. For more information about American Indian correspondence, click here.
The following table lists some correspondence relating to the Crow Indians:
BIA Field Office Location of Original Records
Pre-1880 Correspondence
M234 RG 75 --
962 Rolls
Roll Number
FHL film number
Upper Missouri Agency, 1824-66 Washington D.C. Rolls 883-886 FHL 1661613-1661616
Fort Berthold Agency, 1867-70 Washington D.C. Roll 292 FHL 1661022
Treaties
During the latter part of the 18th Century and most of the 19th Century, treaties were negotiated between the federal government and individual Indian tribes. The treaties provide helpful information about the history of the tribe, but usually only include the names of those persons who signed the treaty. For more information about treaties, click here.
Treaties to which the Crow Indians were a part were:
The year link (year of the treaty) will connect to an online copy of the treaty.
• 1825 August 4, at Mandan Village
• 1851 September 17, at Fort Laramie
• 1868 May 7, at Fort Laramie
• 1880 May 14, at Washington - unratified
Tribal Office Records
The Tribal Office is responsible for enrollment records, vital records, tribal police records, tribal court records, employment records and many others. They are an entirely different set of records from those kept by the Bureau of Indian Affairs. Most of them remain in the Tribal Office. For details, contact that office at the address for the Tribal Headquarters listed above.
Vital Records
Prior to the Indian Reorganization Act, the Bureau of Indian Affairs, through their agencies, may have recorded some vital events. Some were recorded on health forms, such as the "Sanitary Record of Sick, Injured, Births, Deaths, etc." Others were recorded as supplements to the "Indian Census Rolls." Some were included in the unindexed reports and other correspondence of the Bureau of Indian Affairs.
Some vital records for the Crow Indians include:
• Crow Agency, M595, births and deaths 1925-1932, FHL 575776 (Supplements to the Indian Census Rolls)
Important Web Sites
References
1. John Swanton. The Indian Tribes of North America. Smithsonian Institution, Bureau of American Ethnology, Bulletin #145.
2. Frederick Webb Hodge. Handbook of American Indians North of Mexico. Washington, DC: Smithsonian Institution, 1906.
3. Indian Affairs Report, 1871 as quoted in Frederick Webb Hodge. Handbook of American Indians North of Mexico. Washington, DC: Smithsonian Institution, 1906.
4. John Swanton. The Indian Tribes of North America. Smithsonian Institution, Bureau of American Ethnology, Bulletin #145.
5. Federally recognized name, as recorded in Federal Register, Vol. 72, No. 55, Thursday, March 22, 2007
6. Frederick Webb Hodge. Handbook of American Indians North of Mexico. Washington, DC: Smithsonian Institution, 1906.
7. Official Tribal website of the Crow Indians of Montana.
Bibliography
Crow Tribe
• Carlson, Paul H. The Plains Indians. College Station, Texas: Texas A.M. University Press, c1998. FHL Book 970.1 C197p
• Denig, Edwin Thompson. Five Indian Tribes of the Upper Missouri: Sioux, Arickaras, Assiniboines, Crees, Crows. Norman, Oklahoma: University of Oklahoma Press, c1981. The Civilization of American Indian Series:059. FHL Book 970.1 D415f
• Hoxie, Frederick E. Parading Through History: The Making of the Crow Nation in America, 1805-1935. New York, New York: Cambridge University Press, c1995. FHL Book 970.3 C885h
• Lowie, Robert H. The Crow Indians. New York, New York: Farrar & Rinehart, c1935. FHL Book 970.3 C885L
General
• Guide to Federal Records in the National Archives; Record Group 75, Records of the Bureau of Indian Affairs.
• Hodge, Frederick Webb. Handbook of American Indians North of Mexico. Washington, DC: Smithsonian Institution, 1906 Available online.
• Klein, Barry T., ed. Reference Encyclopedia of the American Indian. Nyack, New York: Todd Publications, 2009. 10th ed. WorldCat 317923332; FHL 970.1 R259e; WorldCat 37475188; FHL book 970.1 R259e.
• Malinowski, Sharon and Sheets, Anna, eds. The Gale Encyclopedia of Native American Tribes. Detroit: Gale Publishing, 1998. 4 volumes. Includes: Lists of Federally Recognized Tribes for U.S., Alaska, and Canada – pp. 513-529 Alphabetical Listing of Tribes, with reference to volume and page in this series Map of “Historic Locations of U.S. Native Groups” Map of “Historic Locations of Canadian Native Groups” Map of “Historic Locations of Mexican, Hawaiian and Caribbean Native Groups” Maps of “State and Federally Recognized U.S. Indian Reservations. WorldCat 37475188; FHL book 970.1 G131g.
Vol. 1 -- Northeast, Southeast, Caribbean
Vol. 2 -- Great Basin, Southwest, Middle America
Vol. 3 -- Arctic, Subarctic, Great Plains, Plateau
Vol. 4 -- California, Pacific Northwest, Pacific Islands
• Sturtevant, William C. Handbook of North American Indians. 20 vols., some not yet published. Washington, DC: Smithsonian Institution, 1978– .
Volume 1 -- Not yet published
Volume 2 -- Indians in Contemporary Society (pub. 2008) -- WorldCat 234303751
Volume 3 -- Environment, Origins, and Population (pub. 2006) -- WorldCat 255572371
Volume 4 -- History of Indian-White Relations (pub. 1988) -- WorldCat 19331914; FHL book 970.1 H191h v.4.
Volume 5 -- Arctic (pub. 1984) -- WorldCat 299653808; FHL book 970.1 H191h v.5.
Volume 6 -- Subarctic (pub. 1981) -- WorldCat 247493742; FHL book 970.1 H191h v.6.
Volume 7 -- Northwest Coast (pub. 1990) -- WorldCat 247493311
Volume 8 -- California (pub. 1978) -- WorldCat 13240086; FHL book 970.1 H191h v.8.
Volume 9 -- Southwest (pub. 1979) -- WorldCat 26140053; FHL book 970.1 H191h v.9.
Volume 10 -- Southwest (pub. 1983) -- WorldCat 301504096; FHL book 970.1 H191h v.10.
Volume 11 -- Great Basin (pub. 1986) -- WorldCat 256516416; FHL book 970.1 H191h v.11.
Volume 12 -- Plateau (pub. 1998) -- WorldCat 39401371; FHL book 970.1 H191h v.12.
Volume 13 -- Plains, 2 vols. (pub. 2001) -- WorldCat 48209643
Volume 14 -- Southeast (pub. 2004) -- WorldCat 254277176
Volume 15 -- Northwest (pub. 1978) -- WorldCat 356517503; FHL book 970.1 H191h v.15.
Volume 16 -- Not yet published
Volume 17 -- Languages (pub. 1996) -- WorldCat 43957746
Volume 18 -- Not yet published
Volume 19 -- Not yet published
Volume 20 -- Not yet published
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• This page was last modified on 7 May 2012, at 04:25.
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For the half-year to 30 June 2013, the IPKat's regular team is supplemented by contributions from guest bloggers Stefano Barazza, Matthias Lamping and Jeff John Roberts.
Two of our regular Kats are currently on blogging sabbaticals. They are Birgit Clark and Catherine Lee.
Tuesday, 16 June 2009
"But you never asked ..."
An interesting little decision on the mechanics of proof of use of trade mark cropped up in the Court of First Instance of the European Communities last Friday in Case T‑450/07, Harwin International LLC v Office for Harmonisation in the Internal Market, Cuadrado, SA. Harwin registered the figurative Pickwick mark (right) as a Community trade mark for ‘clothing, footwear, headgear’ in Class 25. Cuadrado sought cancellation, citing its earlier word mark PICK OUIC and figurative mark (below left), both of which were registered for various items in Class 25, and maintaining that on account of the similarity of marks and overlap of goods there was a likelihood of confusion.
The Cancellation Division annulled the registration in a decision which was upheld by the Board of Appeal. According to the Board, Harwin's appeal was based on the complaint that the Board had not considered whether Cuadrado had demonstrated use of its earlier marks, but the Cancellation Division didn't need to do so since Harwin hadn't expressly asked it to. Having failed to ask the Cancellation Division to assess whether there was use of the earlier marks, Harwin was too late to do so before the Board of Appeal.
The Court of First Instance allowed Harwin's appeal. When Cuadrado submitted evidence of use of its earlier marks before the Cancellation Division, Harwin commented that that evidence was insufficient to prove genuine use. In response, Cuadrado produced additional documents on proof of use. The CFI continued:
"23 For OHIM, the effect of the absence of an express request for proof of use, which should, for example, have been made by [Harwin] when OHIM invited it to submit its observations on the application for a declaration of invalidity, was to deprive [Harwin] of any opportunity to dispute the genuine use of the earlier mark relied on in support of the application for a declaration of invalidity. In the present case, OHIM refused to treat the observations submitted on that issue by [Harwin] in its response to the application for a declaration of invalidity as an express request ....
24 Such an approach does not take into account the relevant evidence submitted on that issue in due time by the parties to the Cancellation Division and, subsequently, to the Board of Appeal.
25 So far as the principles underlying it are concerned, the system provided for by Article 56 of Regulation No 40/94 ... must be described as follows:
– the proprietor of an earlier trade mark is obliged to prove the use of its mark only if that use is challenged by the proprietor of the trade mark which is the subject-matter of an application for a declaration of invalidity;
– in the absence of such a challenge, OHIM may confine itself to considering whether a likelihood of confusion exists, without considering proof of use;
– where there is such a challenge of the use, whether by means of a request for proof of use submitted by the proprietor of the trade mark which is the subject-matter of an application for a declaration of invalidity or by means of that party challenging the evidence submitted by the proprietor of the earlier mark to prove use, OHIM is required to examine the issue of proof of use prior to that of the existence of a likelihood of confusion.
26 Concerning the literal interpretation of the words ‘[i]f the proprietor of the Community trade mark so requests’ used in Article 56(2) of Regulation No 40/94, it must be observed that they may be understood as referring to a request such as that set out in the applicant’s observations on the application for a declaration of invalidity. That request was furthermore fully understood by the applicant for a declaration of invalidity, since it produced additional evidence in order to reply to the applicant (see paragraph 21 above)....
29 ... the request for proof of use was made by [Harwin] expressly and timeously. In any event, it was fully understood by [Cuadrado], which answered it in its response to the observations submitted by [Harwin] on that subject. ...
32 The situation here is not comparable to that which gave rise to the judgment in MUNDICOR .... In the present case, the issue of the use of the earlier mark was the subject of an exchange of arguments between the parties. That exchange, which was commenced in the application for a declaration of invalidity, was expressly instigated by[Harwin] both in its observations relating to that application for a declaration of invalidity and again following those observations, without [Cuadrado] being mistaken as to the nature of that exchange.
33 Consequently, in the light of the observations submitted by [Harwin] concerning the genuine use of the earlier mark in response to the observations made on that issue by [Cuadrado] and following which the latter submitted new evidence in that regard, the Board of Appeal infringed Article 56(2) and (3) by holding in the contested decision that the issue of the genuine use of the earlier mark did not have to be examined by the Cancellation Division".
The IPKat thinks this is correct, but notes the contrast between Harwin -- which didn't expressly say what it meant even once -- and the CFI which tends to say the same thing over and over again. Merpel laments the annoying fact that both parties are 'applicants', Cuadrado being the applicant for invalidity and Harwin being the applicant for annulment of the Board's decision; these appellations change in accordance with the nature of the proceedings as they wend their way from Cancellation Division to Board of Appeal to CFI to Court of Justice, which can confuse for the sake of achieving clarity. For the benefit of readers of the decision and users of the Community trade mark system it would be much easier if the owner of the challenged mark were simply referred to as the 'proprietor' throughout.
Pickwick Papers here
Picnic Papers here
Picnic Cat here
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26 February 2010
Schneier Nails it on CCTV Folly
Another brilliant essay on security from Bruce Schneier. It's all well-worth reading, but here's the nub:
If universal surveillance were the answer, lots of us would have moved to the former East Germany. If surveillance cameras were the answer, camera-happy London, with something like 500,000 of them at a cost of $700 million, would be the safest city on the planet.
We didn't, and it isn't, because surveillance and surveillance cameras don't make us safer. The money spent on cameras in London, and in cities across America, could be much better spent on actual policing.
When will the politicians face up to the facts on CCTV? (Via Boing Boing.)
Follow me @glynmoody on Twitter or identi.ca.
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Protein Purification - Salting Out
From OpenWetWare
(Difference between revisions)
Jump to: navigation, search
Current revision (23:12, 14 April 2010) (view source)
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[[Category:protocol]]
[[Category:protocol]]
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[[Category:in vitro]]
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[[Category:In vitro]]
[[Category:protein]]
[[Category:protein]]
Current revision
Different proteins salt out at different concentrations of ammonium sulfate. Here are some references on salting out and tools for calculating the amounts needed.
Current Protocols in Protein Science, appendix 3F contains information on salting out. DOI:10.1002/0471140864.psa03fs13
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User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/11/20
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Experimental Biological Chemistry I Main project page
Previous entry Next entry
Purpose
• ADA protein were transferred from dialysis tubing into 15mL falcon tubes
• Make Au/ADA samples with the following mole ratios: 60 - 70 - 80 - 90 - 100 - 110 - 120 - 130 - 140 - 150, with ADA fraction 2 after dialysis.
• Un-dialyzed Au/ADA samples and Au/HRP made on 2012/11/14 were run on UV-vis spectrometer and Atomic Absorption Spectrometer in order to compare spectra results for Au/ADA, Au/HRP, Au/Lysozyme, and Au/BSA.
• Resuspend Au/HRP samples in different concentration and pH of Tris buffer to test ionic strength.
Procedure for Dialysis
• Dialysis beaker containing dialysis tubing enclosed with ADA protein fractions were taken from the 4°C cold room into room temperature lab room.
• The dialysis clips were taken off from dialysis tubing. ADA protein fractions were poured into a sterile 15mL falcon tubes.
• This process was repeated for all three protein fractions: ADA fraction 1&3 made on 2012/11/06, ADA fraction 2 made on 2012/11/06, and ADA fraction purified on 2012/10/03.
• ADA fractions in 15mL falcon tubes were stored in 4°C refrigerator.
Procedure for making dialyzed Au/ADA samples
• Au/ADA samples were made with the following mole ratios:
60 - 70 - 80 - 90 - 100 - 110 - 120 - 130 - 140 - 150
• Stock solution of HAuCl4 was made on 2012/09/05 with a concentration of 10.5uM.
• Stock solution for ADA was the ADA protein fraction made in 2012/10/03 after dialysis. The ADA stock solution has a concentration of 58.36μM.
• Volume of ADA protein was set at 137.1uL and a range of HAuCl4 was used from 45.71μL to 114.3μL. Water was added to the sample to increase the volume of sample to 8mL. Volumes of each reactants are shown in table below:
Au/ADA ratio ADA added[uL] HAuCl4 Added [uL] Water Added[uL] [ADA]final[uM] [HAuCl4]final[uM]
60137.145.717817.2160
70137.153.37809.6170
80137.160.97802180
90137.168.67794.4190
100137.176.27786.81100
110137.183.87779.11110
120137.191.47771.51120
130137.1997763.91130
140137.1106.77756.31140
150137.1114.37748.71150
• The samples were made in 15mL non-sterile falcon tubes. After all reactants were added, samples are capped and wrapped around with aluminum foil.
• Samples were placed in incubator at 85°C for 4 hours then cooled down to room temperature.
Procedure for Running UV-vis Spectrometer on Au/ADA and Au/HRP samples
• UV-vis spectrometer were run in spectrum method. A range of 200nm to 800nm were ran on all Au/ADA and Au/HRP samples made on 2012/11/14.
• 3mL of distilled water was placed in quartz cuvette with 1cm pathlength and set as base line solution. 3mL taken from supernatant of Au/ADA and Au/HRP samples were placed in an identical quartz cuvette with 1cm pathlength for spectra measurement.
Results for Running UV-vis Spectrometer on Au/ADA and Au/HRP samples
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He who is false to present duty breaks a thread in the loom, and will find the flaw when he may have forgotten its cause. Beecher, Henry Ward
Source: Life Thoughts · This quote is about duty · Search on Google Books to find all references and sources for this quotation.
A bit about Beecher, Henry Ward ...
Henry Ward Beecher (June 24, 1813 - March 8, 1887) was a theologically liberal American Congregationalist clergyman and reformer, and author who was born in Litchfield, Connecticut, the eighth of nine children of Lyman Beecher by his first wife (and the eighth of thirteen children in all). One of his elder sisters was Harriet Beecher Stowe, author of Uncle Tom's Cabin.
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Truth alone wounds. Bonaparte, Napoleon
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A bit about Bonaparte, Napoleon ...
Napoleon is the French leader famed for his military successes and for not quite conquering Europe. Starting as a second lieutenant in the French artillery, he rose quickly through the ranks until he became First Consul of France. (Later he crowned himself Emperor.) He led his armies to victory after victory, and by 1807 he ruled territory that stretched from Portugal to Italy and north to the river Elbe. But his attempts to conquer the rest of Europe failed; a defeat in Moscow in 1812 nearly destroyed his empire, and his 1815 loss to the Duke of Wellington at Waterloo finished the job. He was sent into exile on the island of St. Helena, where he died in 1821.
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The great gift of conversation lies less in displaying it ourselves than in drawing it out of others. He who leaves your company pleased with himself and his own cleverness is perfectly well pleased with you. Bruyere, Jean De La
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Quotes by Shakespeare, William
Born ca. 1564 and died ca. 1616 during the Renaissance period (1450-1599). One of the greatest writers of all time, Shakespeare, the peerless poet of the Sonnets and the creator of such dramatic masterpieces as Romeo and Juliet, Hamlet, and King Lear, is a playwright of paradigmatic originality. In his discussion of the Western literary canon, critic Harold Bloom declared: "Shakespeare and Dante are the center of the Canon because they excel all other Western writer in cognitive acuity, linguistic energy, and power of invention." However, one could go a step further and suggest that Shakespeare defines the Western canon because he transcends it. If Shakespeare, as Ben Jonson declared, "was not of an age, but for all time," the great dramatist, one could argue, spoke to the ultimate concerns of humankind, regardless of period or cultural tradition..
"A politician is one that would circumvent God."
Shakespeare, William on politics
3 fans of this quote
"There have been many great men that have flattered the people who never loved them."
Shakespeare, William on politics
"I durst not laugh for fear of opening my lips and receiving the bad air."
Shakespeare, William on pollution
"My bounty is as boundless as the sea, my love as deep. The more I give thee, the more I have, For both are infinite"
Shakespeare, William on possibilities
7 fans of this quote
"For he was likely, had he been put on, to have proved most royally."
Shakespeare, William on potential
"Lord we may know what we are, but know not what we may be."
Shakespeare, William on potential
7 fans of this quote
"O world, how apt the poor are to be proud!"
Shakespeare, William on poverty and the poor
"Madness in great ones must not unwatched go."
Shakespeare, William on power
"There's not one wise man among twenty will praise himself."
Shakespeare, William on praise
"Bow, stubborn knees!"
Shakespeare, William on prayer
"But, good my brother, do not, as some ungracious pastors do. Show me the steep and thorny way to heaven whilst like a puffed and reckless libertine himself the primrose path of dalliance treads and recks not his own rede."
Shakespeare, William on preachers and preaching
"Man, proud man, drest in a little brief authority, most ignorant of what he's most assur d, glassy essence, like an angry ape, plays such fantastic tricks before high heaven, as make the angels weep."
Shakespeare, William on pride
"Defer no time, delays have dangerous ends."
Shakespeare, William on procrastination
4 fans of this quote
"In delay there lies no plenty."
Shakespeare, William on procrastination
3 fans of this quote
"He plough'd her, and she cropp'd."
Shakespeare, William on creation
"Beware of the ides of March."
Shakespeare, William on prophecy
"The proverb is something musty."
Shakespeare, William on proverbs
"Canst thou not minister to a mind diseased, pluck from the memory a rooted sorrow, raze out the written troubles of the brain, and with some sweet oblivious antidote cleanse the fraught bosom of that perilous stuff which weighs upon the heart?"
Shakespeare, William on psychiatry
"I have bought golden opinions from all sorts of people."
Shakespeare, William on publicity
"Better three hours too soon than a minute too late."
Shakespeare, William on punctuality
4 fans of this quote
"And where the offence is, let the great axe fall."
Shakespeare, William on punishment
"Every why has a wherefore."
Shakespeare, William on purpose
6 fans of this quote
This quotation can be viewed in the context of a book
"What we determine we often break. Purpose is but the slave to memory."
Shakespeare, William on purpose
"The course of true love never did run smooth."
Shakespeare, William on quarrels
9 fans of this quote
"To be or not to be that is the question. Whether it is nobler in the mind to suffer the stings and arrows of outrageous fortune, or take up arms against a sea of troubles, and by opposing them, end them. [Hamlet]"
Shakespeare, William on questions
3 fans of this quote
"What is a man, If his chief good and market of his time Be but to sleep and feed? a beast, no more. Sure, he that made us with such large discourse, Looking before and after, gave us not That capability and god-like reason To fust in us unused."
Shakespeare, William on reason
"Strong reasons make strong actions."
Shakespeare, William on reason
"Let's not burden our remembrance with a heaviness that's gone."
Shakespeare, William on regret
4 fans of this quote
"Reputation, reputation, reputation! O, I ha lost my reputation, I ha lost the immortal part of myself, and what remains is bestial!"
Shakespeare, William on reputation
"For I am full of spirit and resolve to meet all perils very constantly."
Shakespeare, William on resolution
"Who is so firm that can't be seduced?"
Shakespeare, William on resolution
"Nothing will come of nothing."
Shakespeare, William on results
"Fear no more the heat o the sun, nor the furious winter's rages. Thou thy worldly task hast done, home art gone and taken thy wages."
Shakespeare, William on retirement
"Our life, exempt from public haunt, finds tongues in trees, books in the running brooks, sermons in stones, and good in everything."
Shakespeare, William on retirement
"Heat not a furnace for your foe so hot that it do singe yourself."
Shakespeare, William on revenge
"If you prick us do we not bleed? If you tickle us do we not laugh? If you poison us do we not die? And if you wrong us shall we not revenge?"
Shakespeare, William on revenge
10 fans of this quote
"O, what a world of vile ill-favored faults, looks handsome in three hundred pounds a year!"
Shakespeare, William on riches
"The path is smooth that leadeth on to danger."
Shakespeare, William on risk
4 fans of this quote
This quotation can be viewed in the context of a book
"Virtue is bold and goodness never fearful."
Shakespeare, William on risk
"Uneasy lies the head that wears a crown."
Shakespeare, William on royalty
But wait... There are more: prev 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 next
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Showing results 1 to 3 of 3 @ 0.00 seconds. Search: Posts Made By: ahagman
Forum: Applications 05-21-2011, 06:50 AM
Replies: 10
Views: 3,224
Posted By ahagman
Re: What's the deal with us not having Pixel Pipe anymore?
It seems you can still download it from this link, the .install file will be downloaded if you click the download from ovi store button.
...
Forum: General 03-28-2011, 03:58 PM
Replies: 13
Views: 2,750
Posted By ahagman
Re: Can't access tweetgo.net
The site seems to be down, i's not reachable with ping.
Here is the end of a trace
17 183 ms 187 ms 186 ms xen-tarzan.nodesdirect.com [204.74.211.210]
18 xen-tarzan.nodesdirect.com...
Forum: General 01-24-2011, 07:51 PM
Replies: 6
Views: 2,226
Posted By ahagman
Re: (Maemo 5) Lets compare our Megabytes.....
My last backup was 72 MB, it has grown 30MB in a month.
Showing results 1 to 3 of 3
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Integration of Geographic Information System and 2D Imaging to investigate the effects of subsurface conditions on flood occurrence
AbdulNafiu Kola Adiat, Mohd Nordin M Nawawi, Khirudin Abdullah
Abstract
Availability of accurate flood maps and adequate understanding of the subsurface conditions can enhance the effective management of flood disasters. In this study, the GIS and the 2D resistivity imaging had been employed to carry out the preparedness phase of flood management. The objectives of the study include developing flood risk map and establishing link between the subsurface conditions and flood occurrence. The results showed that about 93% of the area is vulnerable to flood occurrence. The subsurface investigations revealed that the area is characterized by the presence of thick column of impermeable clayey overburden material. Correlation of 78.57% was established between the thickness of clayey overburden and the flood vulnerability. This suggests that the flood occurrence in the area is largely dependent on geologic factor. The study is useful in reducing flood damages and planning mitigation measures.
Full Text: PDF DOI: 10.5539/mas.v6n3p11
This work is licensed under a Creative Commons Attribution 3.0 License.
Modern Applied Science ISSN 1913-1844 (Print) ISSN 1913-1852 (Online)
Copyright © Canadian Center of Science and Education
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Recipe for Disaster and Other Stories (1998 series) (Fantagraphics)
Highlighted fields represent added, changed, and deleted data.
Field Previous Change / Next
Name Recipe for Disaster Recipe for Disaster and Other Stories
Leading Article No No
Format Black & White; Softcover; One-Shot Black & White; 8" x 10.87" (20.32 cm x 27.6 cm); Softcover; One-Shot
Color
Dimensions
Paper Stock
Binding
Publishing Format
Year Began 1999 1998
Year Began Uncertain No No
Year Ended 1999 1998
Year Ended Uncertain No No
Current? No No
Country United States United States
Language English English
Has Barcode Yes Yes
Has Indicia Frequency Yes No
Has Isbn Yes Yes
Has Issue Title No No
Has Volume Yes No
Is Comics Publication Yes Yes
Tracking Notes
Notes
Keywords
Comments
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You are here: Home / Data and maps / Maps and graphs / Distance-to-target-path for EU-15 Member States in 2005
Distance-to-target-path for EU-15 Member States in 2005
Created : Nov 12, 2009 Published : Dec 04, 2007 Last modified : Nov 29, 2012 11:39 AM
The distance-to-target-path indicator (DTPI) measures the deviation in percentage points of actual emissions in 2005 from a (hypothetical) linear path between base-year emissions and the burden-sharing target for 2010
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A positive value suggests an underachievement and a negative value an overachievement by 2005. The DTPI is used as an early indication of progress towards the Kyoto and Member States burden-sharing targets.
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Phone: +45 3336 7100
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advanced search
Category: Organizations > Wildlife > Wildlife Sanctuaries
STAR ECO Station
STAR ECO Station is an 18,000 square-foot environmental science and wildlife rescue center that provides educational field trips, assemblies, classroom instruction, and special events that reach 1,000,000 students of all ages on a yearly basis.
Ratings/Review of this resource:
Address:
10101 W. Jefferson Blvd.
Culver City , CA 90232
USA
Phone: 310-842-8060
Fax: 310-842-8280
E-Mail: ecostation@starinc.org
Website: http://www.ecostation.org
Detailed Information:
Through a partnership with the U.S. Fish and Wildlife Service, STAR ECO Station rescues and houses "at-risk" exotic wildlife.
Visitors study ecology and environmentalism on a wild and fun encounter with the STAR ECO Station's rescued tropical birds, exotic reptiles, wildcats and ocean life. Designed by real Hollywood set designers to transport visitors to a tropical jungle setting, the STAR ECO Station offers interdisciplinary, hands-on lessons of compassion and empathy for the natural world and inspires visitors to a deep understanding of their role in protecting the Earth.
With a mission of conservation through education, STAR ECO Station is for adults and children alike; it is open to the public on weekends for hands-on eco-educational tours. Admission is $5 for children, $6 for seniors, and $7 for adults.
The STAR ECO Station is also available to host a variety of special events throughout the year, including workshops, conferences and birthday parties.
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Adams Township, OhioEdit This Page
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Perry Township, Ohio may refer to:
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• This page was last modified on 26 September 2011, at 13:29.
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Changes related to "Haiti"
From FamilySearch Wiki
Haiti
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Please share your favorite website
Newbie Member
18Feb2007,04:55 #1
Yo! Great way to find full software and download it , I would appriciate you if share some links with me. Thank you in advance for you help ! Bye, Bye
Last edited by shabbir; 18Feb2007 at 09:30.. Reason: Confine links to signature only
Go4Expert Founder
18Feb2007,09:30 #2
We both are on my fav site.
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Henry Wadsworth Longfellow - Summary Bibliography
You are not logged in. If you create a free account and sign in, you will be able to customize what is displayed.
Other views: Awards Alphabetical Chronological
Poem Series Poems Essays
Copyright (c) 1995-2011 Al von Ruff.
ISFDB Engine - Version 4.00 (04/24/06)
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Bibliography: A Conversation with David B. Silva
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Interview Title: A Conversation with David B. Silva
Interviewees: David B. Silva
Author: Ivy Fehervari
Year: 1998
Type: INTERVIEW
ISFDB Record Number: 1196369
User Rating: This title has fewer than 5 votes. VOTE
Current Tags: None Add Tags
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Copyright (c) 1995-2011 Al von Ruff.
ISFDB Engine - Version 4.00 (04/24/06)
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Argentinean Couple Shoot Kids, Kill Themselves Over Global Warming
March 1, 2010
By
Infowars reports:
A seven month old baby has miraculously survived being shot after the parents killed themselves and their two year old son, citing fears over a lack of government action on global warming.
Francisco Lotero, 56, and Miriam Coletti, 23, are said to have shot their young son in the back, killing the toddler instantly.
Neighbours called the police, after complaining of a stench coming from the house.
Clearly this couple were either already very much deranged or were so terrified that they ended their own lives believing that they were contributing to the destruction of the planet.
In a written press release, Al Gore released this statement:
I applaud the family for taking the steps necessary to reduce global warming. I have been working toward a “final solution” to the problem that involves the US military moving to reduce greenhouse gas emissions of the US population. Its important to remember people should not kill themselves by using bullets, which take CO2 emissions to create, they should instead suffocate themselves using biodegradable rope that they cultivate from plant fibers themselves.
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The free office suite
Download LibreOffice
LibreOffice Linux - deb (x86), version 4.0.1, Oriya. Not the version you wanted? Change System, Version or Language
This version of LibreOffice is prepared with care and presented with pride by the LibreOffice community. PLEASE NOTE that, since this is the very second version in the series, make sure to read the release notes (under "Handy resources").
You need to download and install these files in order:
• Source code
LibreOffice is an open source project and you can therefore download the source code to build your own installer.
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BISC110/F12
From OpenWetWare
Revision as of 13:42, 8 May 2012 by Tucker Crum (Talk | contribs)
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Wellesley College
BISC110/112- Introduction to Cell Biology Lab- Fall 2012
Introduction to Cell Biology Lab Calendar/Assignments Resources OWW Basics Appendices
Series 1: Scientific Investigation Series 2: Genetics Series 3: Photosynthesis
Fall 2012
Lecture Sections:
BISC110 Section 1 M/Th 8:30-9:50 TBA
BISC110 Section 2 M/Th 11:10-12:20 Michelle LaBonte
BISC110 Section 3 T/F 9:50-11:00 Gary Harris
BISC112 M 11:10-12:20 Yui Suzuki
BISC112FY Th 11:10-12:20 Yui Suzuki
Laboratory Instructors: Michelle LaBonte, Martina Koniger, Jocelyne Dolce, Melissa Beers, Kaye Peterman, TBA
Lab Prep:Sherly Veeraragavan and Mbaira Maorongarti
Labs: M 1:30-5 Koniger; M 6:30-10 TBA; T 12:30-4 LaBonte; W 2:15-5:45 TBA ; Th (112FY seminar students only) 1-4:30 Peterman; Th 6:30-10 TBA;
; F 1:00-4:30 TBA
Welcome to BISC 110/112 LAB
Please complete a brief entry survey found at add link . Click on the link to take this survey. The survey will be open through ????.
The labs for BISC 110/112 are designed to familiarize you with how experimental science is designed, performed and how it is communicated. Over the course of the semester, you will be designing and performing experiments that reinforce concepts covered in the lecture portion of the class. Your job will be to think like scientists when designing experiments to answer hypothesize driven questions about basic cellular processes. You will learn to perform the experiments properly, to keep good records of your results, and to communicate the results and conclusions of your work, both orally and in written reports.
These are ambitious goals for an introductory class such as BISC 110/112. Many of you will be starting this class with a strong background in biology and in lab work, but just as many of you will have had little previous training in biology and in performing experiments. The labs for this class are designed to bring everyone in the class to a working level of expertise and then to build from there.
The laboratory part of the course can be divided into sections of several labs each.
Schedule of Experiments
Section Lab # Activities
1 1-5 Boot Camp: Metric Measurement; Using Basic Lab Equipment; Making Solutions and Dilutions; Microscopy; Scientific Investigation; Designing & Executing Experiments; Data Analysis & Display; Statistical parameters; Scientific Writing
2 6-8 Genetics: DNA extraction, Separation of DNA fragments & Visualization in agarose gel electrophoresis; Genotype/phenotype via Taster SNP data collection and analysis
3 8-11 Photosynthesis: Spectrophotometry: Beer-Lambert Law, Linear regression analysis; Hill Reaction: Self-designed investigation of factors affecting photosystems
Lab Practical 12 Hands-on skills assessment:
Lab attendance is MANDATORY. Some experiments take more than one lab to complete; therefore, it is very disruptive to miss lab, particularly for your partner.Severe illness or serious family crisis, are the only permissible excuses for missing lab. Your lab instructor must be notified IN ADVANCE if you must miss lab. There are NO lab "make-ups". Do not schedule interviews, doctor appointments or other activities during lab time. For all wet lab work, you will be working with a partner. Your instructor may rotate partners during the semester to give you an opportunity to work with several people in the class.
There will be assignments for you to complete after most lab periods. Assignments are to be turned in at the beginning of the lab on the due date. It is your responsibility to turn in your assignment on time. “I forgot to give it to you” is not a valid excuse for handing in an assignment late. Assignments will be graded by your lab instructor, and will be penalized at least 5% a day for each day they are late. Work that is one week late or later will not be given any point credit.
To pass BISC110/112 you will have to pass both the lecture and the lab component of the class. You are strongly encouraged to turn in all your lab assignments.
You are expected to keep a lab notebook in which you keep track of your experimental procedures, record all your data, and perform calculations. These notebooks will be collected twice and graded once at the end of the semester.
We sincerely hope that you will find your introduction to cell biology an interesting and rewarding experience. As always, we would appreciate your comments and suggestions as to how to improve the program.
Welcome to the lab!
Your BISC 110/112 Instructors
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BISC 219/F10: RNAi Lab 6
From OpenWetWare
Revision as of 10:45, 15 October 2010 by Tucker Crum (Talk | contribs)
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Contents
Lab 6: Series 3: Reverse Genetics- Cloning Your Gene of Interest
To see this process as an animation created by the DOLAN DNA center, go to | http://www.dnalc.org/view/15476-Genetic-engineering-inserting-new-DNA-into-a-plasmid-vector-3D-animation-with-with-basic-narration.html
Plasmids are circular pieces of DNA that can replicate in bacteria but are not part of the bacterial chromosome. Plasmids are generally circular molecules with fewer base pairs of DNA than the chromosome and with certain sequence elements (called the origin or ori) that allow the plasmid to replicate within the bacterial cytoplasm. Many naturally occurring plasmids have been modified for the purposes of using them as research tools. For example, a gene encoding resistance to an antibiotic can be added to a plasmid so that bacteria carrying the plasmid will become antibiotic resistant. This modification allows for selection of cells that carry plasmid DNA. A simplified map of the C. elegans RNAi plasmid is below:
Our goal is to insert our gene of interest into the pL4440 plasmid and transform bacteria with the newly created plasmid.
Restriction enzyme digest of PCR product
To see an animation of this concept and process go to the Dolan DNA center at | http://www.dnalc.org/resources/animations/restriction.html
Once you have analyzed the agarose gel from last week and determined if the PCR amplification of your gene of interest was successful, you are ready to proceed to the next step: Restriction Enzyme Digestion of the amplified DNA.
What do restriction enzymes/endonucleases do? They are enzymes have been isolated from bacteria and cut single or double stranded DNA at specific recognition sequences. These recognition sequences are often palindromic (read the same forwards and backwards). The purpose of these enzymes in bacteria is to eliminate foreign DNA that enters the cells (ie bacteriaphage genomes) to protect the host genome. Companies now purify these enzymes from the bacteria and we can use them to manipulate DNA to link different DNA strands together.
There are two kinds of "cuts" a restriction enzyme can make, either blunt ended or overhangs called "sticky" ends. The blunt ended cuts cause the DNA to have no single stranded "overhangs" that can facilitate base pairing with another strand of complimentary DNA. This makes joining two pieces together harder, but it can be done. The "sticky" ends make joining two pieces together with complimentary base pair overhangs much easier to do and it is much more likely to happen during a ligation reaction.
For more information on restriction enzymes you can read the information on Wikipedia or my favorite site for restriction endonuclease information New England Biolabs. At NEB you can click on the different enzymes and look at the information they have available about the recognition sequence for cutting, the conditions for effectiveness and lots more.
Protocol for RE digest of bli-1
1. Obtain your frozen PCR product from last week's C. elegans gene amplification from your instructor.
2. Pipette 10 μL of your PCR into a clean 1.5 ml microfuge tube.
3. Add 10 μL of Master Mix that contains:
1. 2 μL of 10X NEB restriction buffer 2
2. 1 μL of 100xBSA (bovine serum albumin)
3. 1 μL of enzyme 1 (for bli-1 that enzyme is called BglII)
4. 1 μL of enzyme 2 (for bli-1 it's SpeI)
5. 5 μL of dH2O
4. The total volume of your reaction should be 20 μL
Incubate the reaction at 37°C for 45 minutes to allow for proper digestion.
Your instructor will have already cut and purified the pL4440 vector for you. You will need the vector for the ligation reaction.
Purification of samples (remove enzymes)
In order for the ligation of your new construct to be successful you MUST remove the restriction enzymes from the reaction. There are a few ways of doing this. One is to denature the enzymes with heat, although not all enzymes can be "heat killed". Thus the better option is to purify the DNA away from the enzymes (proteins) and there are wonderful kits out there to do this in a few easy steps. We will use the QIAquick PCR Purification Kit from Qiagen.
We will follow the manufacturer's instructions:
1. Obtain a collection tube and spin column (contains a silica matrix) from your instructor.
2. Make sure the spin column is seated properly in the collection tube.
3. Add 250 μL of buffer PB to the spin column.
4. Add your entire 20 μL restriction enzyme digest sample to the spin column. Close the lid tightly
5. Invert a few times to mix.
6. Centrifuge your sample for 1 minute at 13,200g (highest speed in your microcentrifuge).
7. Separate the spin column from the collection tube; Discard the flow through in the collection tube; put your spin column back into the emptied collection tube.
8. Add 700 ul of PE (Wash Buffer contains EtOH) to the spin column.
9. Centrifuge for 1 minute at 13,200g (highest speed).
10. Discard the flow through in the collection tube; put the spin column back into the emptied collection tube.
11. Centrifuge for 1 minute at 13,200g again. This centrifugation removes any residual wash buffer that might contaminate your final elution.
12. Discard the collection tube(throw it into your autoclave bag) but DO NOT discard the spin column!
13. Put the spin column into a clean 1.5 ml microcentrifuge tube.
14. Add 25 μL of Elution buffer to the center of the silica matrix in the spin column without touching the matrix.
15. Centrifuge for 1.5 minutes at highest speed to elute your purified DNA into the microfuge tube.
16. Discard the spin column in your autoclave bag. Label your elutate with your initials and the name of the C. elegans gene. This DNA will be your "insert" in the next ligation step.
17. Store it in your ice bucket
Ligation into the pL4440 vector
To see an animation of this process go to the Dolan DNA center at | http://www.dnalc.org/view/15541-DNA-ligase-joining-two-lengths-of-DNA-at-their-sticky-ends.html
You have now "cut" the ends of your PCR product to make them amenable to being joined or ligated back together with other DNA molecules cut with the same restriction enzyme to make complementary base pairing possible. This is a very common practice in molecular biology to "clone" or insert genes or pieces of genes into plasmids. We will be using an enzyme called T4 DNA ligase from bacteriophage T4. For more information about ligases see Wikipedia or our exact enzyme from NEB.
Ligation Protocol:
For an effective ligation you want an excess of stick end inserts - typically the smaller piece of DNA to the plasmid, the larger piece of DNA.
We will do a 20 μL reaction.
Add 10 μL of the master mix that contains:
1. 2 μL of 10X T4 DNA Ligase Reaction Buffer
2. 3 μL of pL4440 vector that your instructor digested
3. 5 μL of dH2O
To that you will add:
9 μL of insert (stored in your ice bucket after digestion and purification).
Bring your final mixture to your instructor for addition of:
1 μL of T4 DNA ligase
Incubate the reaction at room temperature for 30 minutes.
Proceed to the transformation when the incubation is complete.
Transformation into competent cloning cells
Here are two animations from the Dolan DNA center that describe the history and the process of interspecies incorporation and expression of DNA (transformation): | http://www.dnalc.org/resources/animations/transformation1.html AND | http://www.dnalc.org/resources/animations/transformation2.html
During “transformation,” a single plasmid enters a single bacterium and, once inside, replicates and expresses the genes it encodes. In this case, the relevant genes expressed are for ampicillin resistance and for the piece of the C. elegans gene of interest. The transformation mixes were given a short time to express these gene products and then were spread on an agar plate that contained nutrients and the antibiotics tetracyclin (encoded by the bacteria) and ampicillin (encoded by the plasmid). Only the cells that incorporated the plasmid DNA and expressed the plasmid genes grew to form colonies of bacteria in the presence of ampicillin. The untransformed bacteria failed to form visible colonies on the ampicillin containing agar surface.
Most bacteria do not usually exist in a “transformation ready” state, but the bacteria can be made permeable to the plasmid DNA by exposing them to calcium chloride. Cells that have been treated with calcium chloride or are otherwise capable of transformation are referred to as “competent.” Competent cells are extremely fragile and must be handled gently, i.e. kept cold, not vortexed, etc. The transformation procedure is efficient enough for most lab purposes; with efficiencies as high as 107 transformed cells per microgram of DNA, but it is important to realize that only 1 cell in about 10,000 is successfully transformed.
This is especially true for "cloning competent" bacteria. The newly ligated plasmids are few in number compared to the number that can be isolated during a miniprep (you will do this more efficient isolation in a future step) so the cells are made "ultra-competent" and usually purchased from a company for A LOT of money - around $300 for 20 transformations! The newly ligated plasmid DNA is not as tightly wound as one isolated from a cell so the plasmids themselves are fragile and can be sheared and rendered untransformable. Please be gentle with your cells and your newly formed plasmids!
Transformation of newly ligated plasmid DNA into Invitrogen TOP10 cells
The Invitrogen TOP10 Chemically Competent bacterial cells are on the instructor’s bench You will transform some of your plasmid DNA into this strain. The cells are very fragile, so treat them gently.
1. Label the top or the side of the tube the cells came in with TOP10, pL4440+your gene name, and your initials or team color.
2. Add 10 μL of your ligated plasmid DNA to the tube. Swirl gently to mix the DNA and the cells.
3. Close the cap and let the transformation mixture sit on ice for 30 minutes.
4. Heat shock by incubating the transformation mix in the heat block at 42°C for 60 seconds, exactly. This step must be timed exactly!!! Remove the tubes at the end of 60 seconds directly to your ice bucket for about 2 minutes.
5. Pour 3 mls of Luria-Bertoni broth (LB) from the stock bottle into a clean and sterile 15ml conical tube. By doing this, you will minimize the amount of LB that will be contaminated if you accidentally touch the media with something that is not sterile. Contaminated media looks cloudy, so be sure to swirl and examine the stock bottle of LB to make sure it is not contaminated.
6. Add 250 microliters of the LB in your conical tube to the transformation mix. When pipetting the media, remember to release your thumb on your micropipet slowly, to avoid splashing the liquid on the end of the barrel. The barrel is not sterile and if you see the liquid touch it, then discard the media in the waste beaker and try again with a new tip.
7. Once you have added the media, close the cap and invert the tube once or twice to mix the contents.
8. Incubate at 37°C for 30 minutes.
9. While the plasmid DNA is being taken up by the competent cells and the new genes provided by the plasmid are being expressed by the bacteria, label two LB + amp agar plates. Label the bottom of the plates with the bacterial strain name(TOP10), the plasmid used (pL4440+bli-1), the date, your initials and team color. Label one 200 μL. You must label the bottom of the plate since the tops are easily switched.
10. Put these plates in the hood with the blower on and with the lid ajar to dry the surface of the agar for about 10 minutes or until the surface looks dry but is not badly dehydrated.
11. Once the transformation mix has incubated at 37°C for ~30 minutes, invert it to mix the contents and pipet 200 μL of transformed cells onto the center of one plate.
12. Pipet 50 μL of transformed cells onto the second plate.
13. Pour 5-10 sterile glass beads (found in a flask at your bench) onto the plates.
14. Put the lid back on and gently swirl the beads all over the plates to spread the transformed bacteria around. When you are done - pour the beads into the disinfectant beaker on the instructor's bench. Do not discard them!
15. Leave the agar plates undisturbed for a few minutes.
16. Once they have dried enough that the surface doesn’t appear wet, invert the plates and incubate at 37°C on the shelf labeled with your lab day. Incubate for 24-48 hours. The plates should be incubated with the agar side up so that condensation will not drip onto the surface of the agar and smear the colonies that will be growing there. The 37°C incubator is by the door to the lab.
17. Save the remaining transformation mix for 24 hours or until we are sure that there is at least one colony growing on each of your plates.Give it to your instructor to refrigerate for you.
What would it mean if you had no colonies on your plate? Normally, you would expect to have around 10-100 pale color colonies on each plate. After a 24-48 hour growth period the plate to allow formation of medium size but well isolated colonies, the plate should be stored in the rack in the refrigerator labeled with your lab day. If you have no colonies on one or more of your plates, please notify your instructor right away.
Before leaving lab today, give the rest of your ligated plasmid DNA to your instructor in a labeled microfuge tube. Make sure your tube is labeled with your name, lab day, plasmid name and color coded with a piece of tape in your team color.
Outline of Experimental Design for REVERSE Genetics Project
Where are you now in this process?(What have you done so far; What's next?)
Make the feeder strain of bacteria
1. Amplify gene of interest by pcr ;
2. Restriction Enzyme digestion of amplified DNA to create "sticky ends" for ligation;
3. Clean up DNA (remove enzymes);
4. Cloning: ligate gene into vector plasmid with amp resistance gene ;
5. Transform competent bacterial cells of a strain genetically modified to be tetracycline resistant;
6. Select for transformants on media with tetracycline and ampicillin;
7. Perform colony pcr on several transformants to be sure to find one colony containing a vector plasmid with the gene of interst
8. Culture the selected colony from colony pcr to create a lot of copies of these bacteria
9. Isolate the cloned plasmid DNA from that cultured colony by miniprep;
10. Retransform isolated plasmids (with gene interest) into HT115 (DE3)cells genetically modified to have impaired ability to degrade RNA;
11. Select for transformants on media with tetracycline and ampicillin
12. Choose an isolated colony to culture and make lots of feeder strain bacteria;
13. Induce expression of C. elegans gene dsRNA from the pL4440 vector in the bacteria by IPTG induction.
14. Seed NM lite worm growth media plates with feeder strain produced as described
Plate wild type C. elegans worms (N2 and rrf-3 strains) on feeder plates made as described (containing bacteria expressing dsRNA of our gene of interest).
Observe phenotype change in progeny caused by RNAi silencing or knockdown of the gene of interest compared to control worms of same strains that were NOT fed feeder strain bacteria.
Isolate RNA from RNAi worms and control worms of same strains.
Perform RT-PCR (Reverse Transcriptase) using the mRNA of the gene of interest as template, isolated from the RNAi worms.
Visualize cDNA in the pcr product by agarose gel electrophoresis and compare size of amplified fragment to known size of coding regions of gene of interest.
Assignment
Remember to check the Assignment section of the wiki for instructions about the graded assignment due in the next lab and check the Weekly Calendar for other work to accomplish before the next lab.
Links to Labs& Project Info
Series1:
Worm Info
Lab 1: Worm Boot Camp & Sex-Linked or Autosomal Start
Lab 2: Sex-Linked or Autosomal Finale
Series2:
Background: Classical Forward Genetics and Gene Mapping
Lab 2: Mutant Hunt
Lab 3: Linkage Test Part 1
Lab 4: Linkage Test Part 2, Mapping and Complementation
Lab 5: Finish Complementation; Mapping Con't
Lab 6: DNA sequence analysis; Mapping Con't
Lab 7: Complete Mapping: Score
Series3:
Schedule of Reverse Genetics Project
RNAi General Information
Media Recipes
Lab 5: Picking your gene to RNAi
Lab 6: Cloning your gene of interest
Lab 7: Picking your transformant
Lab 8: Plasmid purification and transformation
Lab 9: Induction of bacteria for RNAi
Lab 10: Scoring your worms and RNA purification
Lab 11: RT PCR reactions
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RPA rep. says emigration rooted in Armenian psychology
PanARMENIAN.Net - The ruling Republican Party of Armenia (RPA) parliamentary group member Manvel Badeyan has voiced concern over the continued emigration.
“There are a number of factors that affect the migration rates, with the recorded injustices among them. Justice reforms are currently in progress, with the results to be revealed in the near future,” he said, noting the economic conditions as the second factor.
“Measures are being taken to expand Armenia’s market, with the RPA program focusing on creation of jobs,” the MP said, deeming the psychological factor as the third element affecting migration flows.
“Emigration has been deeply rooted in Armenian psychology for centuries,” Mr. Badeyan said.
Partner news
Top stories
Dwelling on the hike in gas prices, Iskandaryan said the change also affected other countries importing Russian gas.
Armen Martirosyan noted that the price rise will further deteriorate the situation in the country, fostering emigration.
In this connection, Foreign Minister Edward Nalbandian left for Strasbourg to attend CoE foreign ministers meeting.
Orinats Yerkir party held a political council meeting on May 15, chaired by party leader Artur Baghdasaryan.
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ALMA to host first of its new series "In Conversation"
PanARMENIAN.Net - On Wednesday, Feb 20, the Armenian Museum and Library of America (ALMA) will host the first of its new series "In Conversation", beginning with poet and singer/songwriter Arto Vaun reading from his work. The conversation will be moderated by Prof. Taline Voskeritchian.
The event will center on the question of literature's relevance in diasporic culture and space. This ongoing series seeks to be a forum where intellectuals and artists, along with the audience, engage in an open conversation.
Born in Cambridge, MA, Arto Vaun has attended Harvard and Glasgow University where he is currently finishing a PhD in English Literature. Vaun was the co-founder of Aspora Literary Journal in Los Angeles, a co-editor of The Armenian Weekly, and is the current poetry editor of Glimpse Journal. His next book of poems, "Isinglass", is forthcoming from Carcanet Press and his new recording, "The Cynthia Sessions", is being released in February 2013.
Professor Taline Voskeritchian teaches writing and literature at Boston University. Her work has appeared in Agni Review, London Review of Books, Bookforum, The Nation, Jadaliyya, among others.
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Possible ways to advance the peaceful settlement of Nagorno Karabakh conflict were in the focus of the discussion.
A meeting between Edward Nalbandian and Azerbaijani FM Elmar Mammadyarov will be held on the sidelines of the event.
Bergen began by recounting his 1997 meeting with Bin Laden in Afghanistan after a long process of negotiations.
Arman Kirakosyan urged Azerbaijan to accept suggestions of Minsk Group to achieve a breakthrough in Karabakh settlement.
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What Are The Most Overrated SEO Tips?
Mar 17, 2011 • 9:13 am | (43) by | Filed Under SEO - Search Engine Optimization
Matt McGee posted a nice discussion at Sphinn asking what are the most overrated SEO techniques you know of.
This comes after the most underused SEO techniques.
There are a lot of people in the SEO space that may obsess of specific techniques, when those techniques have little or no value in the search results. What are they?
The Sphinn members shared what they felt are the most overrated SEO techniques:
• XML Sitemaps
• META keywords tag
• 301 redirecting bought domains
• HTML standards compliant
• Keyword density
• Toolbar PageRank
• Directory Submission
What would you add or delete from this list?
Forum discussion at Sphinn.
Previous story: Confirmed: Google AdSense Ads User Interface Tests
blog comments powered by Disqus
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Glutamic acid
(Redirected from Glutamate)
Jump to: navigation, search
Glutamic acid
Glutamic acid
General
Systematic name ?
Other names ?
Molecular formula ?
SMILES ?
Molar mass ?.?? g/mol
Appearance ?
CAS number [?-?-?]
Properties
Density and phase ? g/cm³, ?
Solubility in water ? g/100 ml (?°C)
Melting point ?°C (? K)
Boiling point ?°C (? K)
Acidity (pKa) ?
Basicity (pKb) ?
Chiral rotation [α]D ?°
Viscosity ? cP at ?°C
Structure
Molecular shape ?
Coordination
geometry
?
Crystal structure ?
Dipole moment ? D
Hazards
MSDS External MSDS
Main hazards ?
NFPA 704
Flash point ?°C
R/S statement R: ?
S: ?
RTECS number ?
Supplementary data page
Structure and
properties
n, εr, etc.
Thermodynamic
data
Phase behaviour
Solid, liquid, gas
Spectral data UV, IR, NMR, MS
Related compounds
Other anions ?
Other cations ?
Related ? ?
Related compounds ?
Except where noted otherwise, data are given for
materials in their standard state (at 25 °C, 100 kPa)
Infobox disclaimer and references
Glutamic acid (abbreviated as Glu or E) is one of the 20 proteinogenic amino acids, and its codons are GAA and GAG. It is a non-essential amino acid. The carboxylate anions and salts of glutamic acid are known as glutamates. In neuroscience, glutamate is an important neurotransmitter which plays a key role in long term potentiation and is important for learning and memory.[1]
Contents
Chemistry
The side chain carboxylic acid functional group has pKa of 4.1 and exists in its negatively charged deprotonated carboxylate form at physiological pH.
History
Although they occur naturally in many foods, the flavor contributions made by glutamic acid and other amino acids were only scientifically identified early in the twentieth century. The substance was discovered and identified in the year 1866, by the German chemist Karl Heinrich Leopold Ritthausen. In 1907 Japanese researcher Kikunae Ikeda of the Tokyo Imperial University identified brown crystals left behind after the evaporation of a large amount of kombu broth as glutamic acid. These crystals, when tasted, reproduced the ineffable but undeniable flavor he detected in many foods, most especially in seaweed. Professor Ikeda termed this flavor umami. He then patented a method of mass-producing a crystalline salt of glutamic acid, monosodium glutamate.[2][3]
Biosynthesis
Reactants Products Enzymes
Glutamine + H2O Glu + NH3 GLS, GLS2
NAcGlu + H2O Glu + Acetate (unknown)
α-ketoglutarate + NADPH + NH4+ Glu + NADP+ + H2O GLUD1, GLUD2
α-ketoglutarate + α-amino acid Glu + α-oxo acid transaminase
1-Pyrroline-5-carboxylate + NAD+ + H2O Glu + NADH ALDH4A1
N-formimino-L-glutamate + FH4 Glu + 5-formimino-FH4 FTCD
Function and uses
Metabolism
Glutamate is a key molecule in cellular metabolism. In humans, dietary proteins are broken down by digestion into amino acids, which serve as metabolic fuel for other functional roles in the body. A key process in amino acid degradation is transamination, in which the amino group of an amino acid is transferred to an α-ketoacid, typically catalysed by a transaminase. The reaction can be generalised as such:
R1-amino acid + R2-α-ketoacid R1-α-ketoacid + R2-amino acid
A very common α-keto acid is α-ketoglutarate, an intermediate in the citric acid cycle. Transamination of α-ketoglutarate gives glutamate. The resulting α-ketoacid product is often a useful one as well, which can contribute as fuel or as a substrate for further metabolism processes. Examples are as follows:
Alanine + α-ketoglutarate pyruvate + glutamate
Aspartate + α-ketoglutarate oxaloacetate + glutamate
Both pyruvate and oxaloacetate are key components of cellular metabolism, contributing as substrates or intermediates in fundamental processes such as glycolysis, gluconeogenesis, and the citric acid cycle.
Glutamate also plays an important role in the body's disposal of excess or waste nitrogen. Glutamate undergoes deamination, an oxidative reaction catalysed by glutamate dehydrogenase, as follows:
glutamate + H2O + NADP+ → α-ketoglutarate + NADPH + NH3 + H+
Ammonia (as ammonium) is then excreted predominantly as urea, synthesised in the liver. Transamination can, thus, be linked to deamination, effectively allowing nitrogen from the amine groups of amino acids to be removed, via glutamate as an intermediate, and finally excreted from the body in the form of urea.
Neurotransmitter
Glutamate is the most abundant excitatory neurotransmitter in the vertebrate nervous system. At chemical synapses, glutamate is stored in vesicles. Nerve impulses trigger release of glutamate from the pre-synaptic cell. In the opposing post-synaptic cell, glutamate receptors, such as the NMDA receptor, bind glutamate and are activated. Because of its role in synaptic plasticity, glutamate is involved in cognitive functions like learning and memory in the brain.[4] The form of plasticity known as long-term potentiation takes place at glutamatergic synapses in the hippocampus, neocortex, and other parts of the brain. Glutamate works not only as a point-to-point transmitter but also through spill-over synaptic crosstalk between synapses in which summation of glutamate released from a neighboring synapse creates extrasynaptic signaling/volume transmission.[5]
Glutamate transporters[6] are found in neuronal and glial membranes. They rapidly remove glutamate from the extracellular space. In brain injury or disease, they can work in reverse, and excess glutamate can accumulate outside cells. This process causes calcium ions to enter cells via NMDA receptor channels, leading to neuronal damage and eventual cell death, and is called excitotoxicity. The mechanisms of cell death include
• Glu/Ca2+-mediated promotion of transcription factors for pro-apoptotic genes, or downregulation of transcription factors for anti-apoptotic genes
Excitotoxicity due to glutamate occurs as part of the ischemic cascade and is associated with stroke[1] and diseases like amyotrophic lateral sclerosis, lathyrism, autism, some forms of mental retardation, and Alzheimer's disease.[8][1]
Glutamic acid has been implicated in epileptic seizures. Microinjection of glutamic acid into neurons produces spontaneous depolarisations around one second apart, and this firing pattern is similar to what is known as paroxysmal depolarizing shift in epileptic attacks. This change in the resting membrane potential at seizure foci could cause spontaneous opening of voltage-activated calcium channels, leading to glutamic acid release and further depolarization.
Experimental techniques to detect glutamate in intact cells include using a genetically-engineered nanosensor.[9] The sensor is a fusion of a glutamate-binding protein and two fluorescent proteins. When glutamate binds, the fluorescence of the sensor under ultraviolet light changes by resonance between the two fluorophores. Introduction of the nanosensor into cells enables optical detection of the glutamate concentration. Synthetic analogs of glutamic acid that can be activated by ultraviolet light and two-photon excitation microscopy have also been described.[10] This method of rapidly uncaging by photostimulation is useful for mapping the connections between neurons, and understanding synapse function.
Evolution of glutamate receptors is entirely the opposite in invertebrates, in particular, arthropods and nematodes, where glutamate stimulates glutamate-gated chloride channels. The beta subunits of the receptor respond with very high affinity to glutamate and glycine.[11] Targeting these receptors has been the therapeutic goal of anthelmintic therapy using avermectins. Avermectins target the alpha-subunit of glutamate-gated chloride channels with high affinity.[12] These receptors have also been described in arthropods, such as Drosophila melanogaster[13] and Lepeophtheirus salmonis.[14] Irreversible activation of these receptors with avermectins results in hyperpolarization at synapses and neuromuscular junctions resulting in flaccid paralysis and death of nematodes and arthropods.
File:L-Glutamate Structural Formulae.png
L-Glutamate at physiological conditions
Brain nonsynaptic glutamatergic signaling circuits
Extracellular glutamate in Drosophila brains has been found to regulate postsynaptic glutamate receptor clustering, via a process involving receptor desensitization.[15] A gene expressed in glial cells actively transports glutamate into the extracellular space,[15] while in the nucleus accumbens stimulating group II metabotropic glutamate receptors, this gene was found to reduce extracellular glutamate levels.[16] This raises the possibility that this extracellular glutamate plays an "endocrine-like" role as part of a larger homeostatic system.
GABA precursor
Glutamate also serves as the precursor for the synthesis of the inhibitory GABA in GABA-ergic neurons. This reaction is catalyzed by glutamate decarboxylase (GAD), which is most abundant in the cerebellum and pancreas.
Stiff-man syndrome is a neurologic disorder caused by anti-GAD antibodies, leading to a decrease in GABA synthesis and, therefore, impaired motor function such as muscle stiffness and spasm. Since the pancreas is also abundant for the enzyme GAD, a direct immunological destruction occurs in the pancreas and the patients will have diabetes mellitus.
Flavor enhancer
Free glutamic acid is present in a wide variety of foods, including cheese and soy sauce, and is responsible for umami, one of the five basic tastes of the human sense of taste. Glutamic acid is often used as a food additive and flavour enhancer in the form of its sodium salt monosodium glutamate (MSG).
Nutrient
All meats, poultry, fish, eggs, dairy products, as well as kombu are excellent sources of glutamic acid. Some protein-rich plant foods also serve as sources. Thirty to 35% of the protein in wheat is glutamic acid. Ninety-five percent of the dietary glutamate is metabolized by intestinal cells in a first pass.[17]
Plant growth
Auxigro is a plant growth preparation that contains 30% glutamic acid.
Production
China-based Fufeng Group Limited is the largest producer of glutamic acid in the world, with capacity increasing to 300,000 tons at the end of 2006 from 180,000 tons during 2006, putting them at 25%–30% of the Chinese market. Meihua is the second-largest Chinese producer. Together, the top-five producers have roughly 50% share in China. Chinese demand is roughly 1.1 million tons per year, while global demand, including China, is 1.7 million tons per year.
Pharmacology
The drug phencyclidine (more commonly known as PCP) antagonizes glutamic acid non-competitively at the NMDA receptor. For the same reasons, sub-anaesthetic doses of ketamine have strong dissociative and hallucinogenic effects. Glutamate does not easily pass the blood brain barrier, but, instead, is transported by a high-affinity transport system.[18] It can also be converted into glutamine.
See also
References
Template:Commons category
1. 1.0 1.1 1.2 Robert Sapolsky. "Biology and Human Behavior: The Neurological Origins of Individuality, 2nd edition", The Teaching Company. Retrieved on 2010-11-10. “see pages 19 and 20 of Guide Book”
2. Renton, Alex. "If MSG is so bad for you, why doesn't everyone in Asia have a headache?", The Guardian, 2005-07-10. Retrieved on 2008-11-21.
3. Kikunae Ikeda Sodium Glutamate. Japan Patent Office (2002-10-07). Retrieved on 2008-11-21.
4. McEntee, W. & Crook, T (1993). "Glutamate: its role in learning, memory, and the aging brain.". Psychopharmacology 111 (4): 391–401. doi:10.1007/BF02253527. PMID 7870979.
5. Okubo Y, Sekiya H, Namiki S, Sakamoto H, Iinuma S, Yamasaki M, Watanabe M, Hirose K, Iino M. (2010). Imaging extrasynaptic glutamate dynamics in the brain. Proc Natl Acad Sci U S A. 107:6526–6531. doi:10.1073/pnas.0913154107 PMID 20308566
6. Shigeri Y, Seal RP, Shimamoto K (July 2004). "Molecular pharmacology of glutamate transporters, EAATs and VGLUTs". Brain Res. Brain Res. Rev. 45 (3): 250–65. doi:10.1016/j.brainresrev.2004.04.004. PMID 15210307.
7. Manev H, Favaron M, Guidotti A, Costa E (July 1989). "Delayed increase of Ca2+ influx elicited by glutamate: role in neuronal death". Mol. Pharmacol. 36 (1): 106–12. PMID 2568579.
8. Hynd MR, Scott HL, Dodd PR. (October 2004). "Glutamate-mediated excitotoxicity and neurodegeneration in Alzheimer's disease.". Neurochem Int. 45 (5): 583–95. doi:10.1016/j.neuint.2004.03.007. PMID 15234100.
9. Okumoto, S., et al. (2005). "Detection of glutamate release from neurons by genetically encoded surface-displayed FRET nanosensors". Proceedings of the National Academy of Sciences U.S.A 102 (24): 8740–8745. doi:10.1073/pnas.0503274102. PMID 15939876.
10. Ellis-Davies, G.C.R., et al. (2007). "4- Carboxymethoxy-5,7-dinitroindolinyl-Glu: an improved caged glutamate for expeditious ultra-violet and 2-photon photolysis in brain slices". Journal of Neuroscience 27 (June): 6601–6604. doi:10.1523/JNEUROSCI.1519-07.2007. PMID 17581946.
11. Laughton, D.L., Wheeler, S.V., Lunt, G.G. and Wolstenholme, A.J. 1995. "The beta-subunit of Caenorhabditis elegans avermectin receptor responds to glycine and is encoded by chromosome 1". J. Neurochem. 64, 2354-2357
12. Cully, D.F., Vassilatis, D.K., Liu, K.K., Paress, P.S., Van der Ploeg, L.H.T., Schaeffer, J.M. and Arena, J.P. 1994. "Cloning of an avermectin-sensitive glutamate gated choride channels from Caenorhabditis elegans". Nature 371, 707-711
13. Cully, D.F., Paress, P.S., Liu, K.K., Schaeffer, J.M. and Arena, J.P. 1996. "Identification of a Drosophila melanogaster glutamate-gated chloride channel sensitive to the antiparasitic agent avermectin". J. Biol. Chem. '271, 20187-20191'
14. Tribble, N.D., Burka, J.F. and Kibenge, F.S.B. 2007. "Identification of the genes encoding for putative gamma aminobutyric acid (GABA) and glutamate-gated chloride channel (GluCl) alpha receptor subunits in sea lice (Lepeophtheirus salmonis)". J. Vet. Pharmacol. Ther. 30, 163-167
15. 15.0 15.1 Augustin H, Grosjean Y, Chen K, Sheng Q, Featherstone DE (2007). "Nonvesicular release of glutamate by glial xCT transporters suppresses glutamate receptor clustering in vivo". Journal of Neuroscience 27 (1): 111–123. doi:10.1523/JNEUROSCI.4770-06.2007. PMID 17202478.
16. Zheng Xi, Baker DA, Shen H, Carson DS, Kalivas PW (2002). "Group II metabotropic glutamate receptors modulate extracellular glutamate in the nucleus accumbens". Journal of Pharmacology and Experimental Therapeutics 300 (1): 162–171. doi:10.1124/jpet.300.1.162. PMID 11752112.
17. Reeds, P.J., et al. (1 April 2000). "Intestinal glutamate metabolism". Journal of Nutrition 130 (4s): 978S–982S. PMID 10736365.
18. Smith QR (April 2000). "Transport of glutamate and other amino acids at the blood-brain barrier". J. Nutr. 130 (4S Suppl): 1016S–22S. PMID 10736373.
Further reading
<tr bgcolor="#ccccff"><td colspan="3" align="center">
v d e
Major families of biochemicals</td></tr><tr><td colspan="3" style="text-align: center;">Peptides | Amino acids | Nucleic acids | Carbohydrates | Nucleotide sugars | Lipids | Terpenes | Carotenoids | Tetrapyrroles | Enzyme cofactors | Steroids | Flavonoids | Alkaloids | Polyketides | Glycosides</td></tr><tr bgcolor="pink"><td style="white-space: nowrap; width: 10%; color: pink;">Analogues of nucleic acids:</td><td align="center">The 20 Common Amino Acids ("dp" = data page)</td><td style="white-space: nowrap; width: 10%; color: pink;">Analogues of nucleic acids:</td></tr>
Alanine (dp) | Arginine (dp) | Asparagine (dp) | Aspartic acid (dp) | Cysteine (dp) | Glutamic acid (dp) | Glutamine (dp) | Glycine (dp) | Histidine (dp) | Isoleucine (dp) | Leucine (dp) | Lysine (dp) | Methionine (dp) | Phenylalanine (dp) | Proline (dp) | Serine (dp) | Threonine (dp) | Tryptophan (dp) | Tyrosine (dp) | Valine (dp)
Template:Glutamatergics
Template:Neurotransmitters Template:Neurotoxinsbn:গ্লুটামিক অ্যাসিড zh-min-nan:Glutamine sng ca:Àcid glutàmic cs:Kyselina glutamová da:Glutaminsyre de:Glutaminsäureeo:Glutama acido eu:Azido glutamiko fa:اسید گلوتامیکko:글루탐산 id:Asam glutamat it:Acido glutammico he:חומצה גלוטמית lv:Glutamīnskābe lb:Glutamat lt:Glutamo rūgštis hu:Glutaminsav nl:Glutaminezuurno:Glutaminsyre oc:Acid glutamicsk:Kyselina glutámová sl:Glutaminska kislina sr:Glutaminska kiselina fi:Glutamiinihappo sv:Glutaminsyra ta:குளூட்டாமிக் காடி th:กลูตาเมตuk:Глутамінова кислота
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Pransu
Created by Jijith Nadumuri at 24 Jul 2011 17:22 and updated at 24 Jul 2011 17:22
VISHNU PURANA NOUN
vp.4.1 Before the evolution of the mundane egg, existed Brahma, who was Hiranyagarbha, the form of that supreme Brahma which consists of Vishnu as identical with the Rig, Yajur, and Sama Vedas; the primeval, uncreated cause of all worlds. From the right thumb of Brahma was born the patriarch Daksha 3; his daughter was Aditi, who was the mother of the sun. The Manu Vaivaswata was the son of the celestial luminary; and his sons were Ikshwaku, Nriga, Dhrishta, saryati, Narishyanta, Pransu, Nabhaga, Nedishta, Karusha, and Prishadhra 4.
vp.4.1 a Vaisya 10: his son was Bhalandana 11; whose son was the celebrated Vatsapri 12: his son was Pransu; whose son was Prajani 13; whose son was Khanitra 14; whose son was the very valiant Chakshupa 15; whose son was Vinsa 16; whose son was Vivinsati 17; whose son was Khaninetra; whose son was the powerful, wealthy, and valiant Karandhama 18; whose son was Avikshi (or Avikshit 19); whose son was the mighty Marutta, of whom this well known verse is recited; "There never was beheld on earth a sacrifice equal to the sacrifice of Marutta: all the implements
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Source link: http://archive.mises.org/4716/study-guide-classic-do-we-ever-really-get-out-of-anarchy/
Study Guide Classic: Do We Ever Really Get Out of Anarchy?
February 21, 2006 by
Alfred G. Cuzán contributed “Do We Ever Really Get Out of Anarchy?” (pdf) to the Summer 1979 issue of the Journal of Libertarian Studies, an article which Roderick Long describes as “invaluable”, (also see Stephan Kinsella). This short article, only 7 pages of text, is one of those little pieces of conceptual dynamite that blows apart the way you’ve seen the world.
Cuzán challenges the notion that we are ever really under “Government” if what Government means is a “third party” external to all relations in society. He points out that “such a ‘third party’ arrangement for society is non-existent among those who exercise the power of government themselves.” (p. 152) Therefore “whereas without government it was market or natural anarchy, it is now a political anarchy, an anarchy inside power.” (pp. 152-3) Cuzán goes on to explain that not all these political anarchies are created equal. Some are more violent than others. The least violent political anarchies begin to look more and more like market anarchy… So wouldn’t outright market anarchy be the least violent of all? Read the whole thing.
For more on this topic see the Anarchy subject. This is part of the new Libertarian Studies category of the Study Guide which I have been creating to categorize the large amount of non-economic libertarian literature in the Study Guide.
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Wikimedia blog
News from the Wikimedia Foundation and about the Wikimedia movement
Wiki Loves Monuments breaks Guinness World Record for largest photo competition
One of the finalist photos from Wiki Loves Monuments 2011
Only days after Wiki Loves Monuments 2012 wrapped up this September, organizers got the word that Guinness World Records had officially certified last year’s 2011 contest as the largest photo competition in the world.
Wiki Loves Monuments 2011 attracted over 5,000 participants from 18 countries, who uploaded 168,208 photos under a free license for use on Wikipedia and other freely licensed projects. The 2011 contest surpassed the previous Guinness record of just over 120,000 photos.
“The Guinness World Record for largest photography competition is a recognition of the effort of the hundreds of volunteers that have helped pull off this contest in the past years” said Lodewijk Gelauff, one of the international coordinators of Wiki Loves Monuments.
Of course, those of you following the current contest know that Wiki Loves Monuments 2012 was an even bigger success. It was expanded to 35 countries, including first-time participants such as the United States, Argentina, South Africa and India. While the 2012 competition is still ongoing in Israel, and the results are not yet final, it’s safe to say the Wikimedia community is about to break its own new world record.
More than 15,000 photographers have already submitted more than 350,000 photos in 2012, doubling the total certified by Guinness World Records for 2011. The most photos came from Poland, with over 50,000 uploads by nearly 700 participants. India had the greatest number of participants, with over 2,200 photographers submitting more than 16,000 photos. And amazingly, Catalan user Pere prlpz uploaded nearly 9,000 photos individually!
For many of the organizers of the contest, the most gratifying number is the following: of the uploaders, more than 11,000 had never contributed to Wikimedia projects before.
At the Wikimedia Foundation, we spend a lot of effort bringing new contributors to the Wikimedia projects and keeping active contributors happy with their experience, so we’re very excited that a contest like Wiki Loves Monuments — which was started by volunteers in the Netherlands in 2010 and was organized by volunteers in every participating country — can prove itself such a demonstrable success.
Best of all, participants discovered that sharing photos is an easy way to help Wikipedia expand its base of free knowledge. “Thanks to the contest thousands of people have made their first steps towards contributing to Wikipedia by submitting a photo,” Gelauff said. “I find it heartwarming that so many people wanted to share their photos freely with the whole world.”
To coincide with the 2012 contest, the Wikimedia Foundation developed a Wiki Loves Monuments app, which enabled Wikipedians for the first time to officially upload photos to Wikipedia projects from a mobile device. There were over 3,000 uploads via mobile worldwide.
Photos uploaded this September as part of the contest are being judged first at the national level, where juries in every country will select their favorite 10 photos based on photographic merit and encyclopedic value. The winning photos from national contests are then considered by an international jury in November, which will announce the top-12 photos and the overall best picture winner in early December. The grand prize winner will be flown to Hong Kong for a photo tour as part of the Wikimania 2013 conference.
Of course, this being Wikimedia, organizers are already planning Wiki Loves Monuments 2013, which they hope will expand to more countries to bring in many more contributors. If you would like to see your country participate next year, go to the international planning site on Wikimedia Commons and take part.
Check back here in early December to find out the results of this year’s contest. For now, feel free to upload your photos to Commons and contribute to Wikipedia’s growing image database year round.
Matthew Roth, Global Communications Manager
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Commentary
During critical illness the gut does not pass the acid test
John C Alverdy
Author Affiliations
Center for Surgical Infection Research and Therapeutics, University of Chicago Medical Center, 5841 S. Maryland MC 6090, Chicago, IL 60637, USA
Critical Care 2012, 16:150 doi:10.1186/cc11474
See related research by Osuka et al., http://ccforum.com/content/16/4/R119
Published: 12 September 2012
Abstract
The composition and function of intestinal microflora are emerging as integral to both health and disease. During critical illness the normal microbiota are rapidly replaced by pathogenic species as a result of both the physiologic stress itself and the use of antibiotics. In this report, the authors use fecal pH as a surrogate marker to determine the predictive value of the functional output of the intestinal microflora during critical illness. Fecal pH appears to be highly predictive of outcome from critical illness, and may reflect the output of key organic acids such as the short-chain fatty acids, lactic acid, and other important products of the gut microflora.
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NOTE: If you are a developer, please use a private wiki based on foswiki/trunk on a daily base ...or use trunk.foswiki.org to view this page for some minimal testing.
Use Item9693 for docu changes for 1.2 and 2.0.
Item9837: TemplateLogin fails with PasswordManager set to none
Priority: CurrentState: AppliesTo: Component: WaitingFor:
Urgent Closed Engine TemplateLogin
With PasswordManager set to None, the initially registered user is left logged in. However if you log out, TemplateLogin prompts for a password during login, and it doesn't matter what is entered, the user is rejected with Oops: we could not recognize you. Try again or reset your password.
I went back into late August with git bisect and didn't find a trunk rev level where this works.
-- GeorgeClark - 18 Oct 2010
I think that this is ready for release. My only concern is did I place the check in the correct module. I changed TopicUserMapping.pm - checkPassword is implemented in:
lib/Foswiki/Users/ApacheHtpasswdUser.pm
lib/Foswiki/Users/BaseUserMapping.pm
lib/Foswiki/Users/Password.pm
lib/Foswiki/Users/HtPasswdUser.pm
lib/Foswiki/Users/TopicUserMapping.pm
lib/Foswiki/Users.pm
lib/Foswiki/UserMapping.pm
-- GeorgeClark - 21 Oct 2010
Everything seems to work like in 1.0.10 now. The combination is a bit silly and totally unsafe but for those that are in a small team and trust each other behind a firewall it will work. Waiting For Release. Thanks George.
-- KennethLavrsen - 25 Oct 2010
Topic revision: r6 - 25 Oct 2010, KennethLavrsen
The copyright of the content on this website is held by the contributing authors, except where stated elsewhere. see CopyrightStatement.
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source: josm/trunk/data/cs.lang @ 2952
Last change on this file since 2952 was 2952, checked in by bastiK, 3 years ago
Updated i18n
• Property svn:mime-type set to application/octet-stream
File size: 113.0 KB
HTML preview not available, since no preview renderer could handle it. Try downloading the file instead.
Note: See TracBrowser for help on using the repository browser.
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o6vdcgnrpqsbs3dit427g7aoaj3gyzpb
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Modify
Opened 10 months ago
Closed 8 weeks ago
#7895 closed enhancement (fixed)
possibility to select several "member of" entries in properties toggle dialogue
Reported by: skyper Owned by: team
Priority: normal Component: Core
Version: Keywords: properties dialog relation
Cc:
Description
Please make it possible to select several entries in the "member of" section in the properties toogle dialogue.
Perfect would be if you are able to select properties and memberships at once.
Thanks
r5355
Attachments (0)
Change History (1)
comment:1 Changed 8 weeks ago by akks
• Resolution set to fixed
• Status changed from new to closed
Modify Ticket
Change Properties
<Author field>
Action
as closed .
as The resolution will be set. Next status will be 'closed'.
The resolution will be deleted. Next status will be 'reopened'.
Author
E-mail address and user name can be saved in the Preferences.
Note: See TracTickets for help on using tickets.
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{
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"warc_date": "2013-11-22T19:23:47.000Z",
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"warc_url": "http://openwetware.org/index.php?title=BioMicroCenter:Sequencing&diff=604650&oldid=604641"
}
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BioMicroCenter:Sequencing
From OpenWetWare
(Difference between revisions)
Jump to: navigation, search
(Additional Information)
(Additional Information)
Line 123: Line 123:
=== Data Analysis ===
=== Data Analysis ===
+
Illumina sequencing at the BioMicro Center includes basic informatic analysis of the data. These steps include:
+
* Image analysis to locate clusters
+
* Basecalling
+
* Demultiplexing of lanes
+
* Alignment of sequences to a reference genome
+
* Quality control
+
* Delivery of the data to a user accessible folder
+
All of these steps are run by our [http://bmc-pipeline.mit.edu/flowcell_data_guide.html automated analysis pipeline]currently in active development. For users requiring further analysis, we have a staff of [[BioMicroCenter:BioInformaticsStaff||bioinformaticists]] that can assist you in analyzing your data.
<BR>
<BR>
Revision as of 14:16, 29 May 2012
The MIT BioMicro Center has five high-throughput Illumina sequencers, including a HiSeq 2000, three Genome Analyzers and one MiSeq. We support a wide variety of applications, such as ChIP-Seq, miRNA sequencing and RNA-seq. Each lane can potentially accomodate dozens of barcoded samples (depending on sequence complexity and desired coverage). Read lengths vary, depending on users, between 36nt and 150nt per end.
All questions about Illumina Sequencing can be directed to Kevin Thai at kthai@mit.edu.
Contents
Illumina Massively Parallel Sequencing
Illumina sequencing works by binding randomly fragmented DNA to an optical flowcell. Fragments are sequenced by sequentially incorporating and imaging fluorescently labeled nucleotides in a “Sequencing-By-Synthesis” reaction. Illumina recently rolled out its TruSeq v3 reagent kits, improving read quality and reducing GC bias at high cluster densities. For an in-depth overview of the Illumina sequencing chemistry, please refer to Kirchner et al 2009.
Platforms
HiSeq 2000
The Illumina HiSeq 2000 is the workhorse of BMC's Illumina fleet and is optimized for maximum yield and the lowest price per basepair. Each lane on the HiSeq is typically produces between 160 and 220 million reads passing our quality filter (for high quality libraries). HiSeq flowcells have 8 lanes, one of which is committed to a control sample that is used for base normalization (lane 1). Read lengths on the HiSeq very between 40 and 100nt per side and nearly all flowcells use barcoding to run multiple samples in each lane.
In order to optimize work flow and keep costs under control, only full flowcells are run. Since all 8 lanes of the flowcell must be run at equal lengths, submissions of single lanes must be grouped with other similar read lengths. This means that some read lengths move through our queue faster then others because more samples of that length are submitted to the BioMicro Center for sequencing. 40nt single end (SE) samples are by far the most common and move through the queue rapidly followed by short paired end (40+40) runs. Many lengths are very unusual (eg. 100nt single end) and can wait months for sequencing. We strongly recommend moving samples with unusual read requirements to one of the other platforms. If you have questions about this (or any other aspect of sequencing) please do not hesitate to contact us.
The HiSeq2000 is ideal for:
• High numbers of multiplexed samples
• De novo sequencing
• SNP detection
• ChIPseq
• Bisulfite sequencing
• RNAseq
• Exome sequencing
The HiSeq2000 was donated to the BioMicro Center by Drs. Penny Chisholm and Chris Burge.
MiSeq
The MiSeq is the newest sequencer in the BioMicro Center. Unlike the HiSeq, the MiSeq is optimized for speed. The MiSeq has a single lane that can produce approximately 5 million reads passing filter. The MiSeq does *not* have a control lane so having good base balance is critical for runs on this sequencer. Amplicons, such as 16S, can be run on the sequencer but should be constructed to have phased sequencing. Highly unbalanced libraries, such as RRBS, should not be run on the MiSeq.
The strength of the MiSeq is its speed and read length. The MiSeq is able to sequence 12nt/hour which allows it to complete a 150+150nt paired end read, from cluster to fastq files, in a little over a day. This compares to 2-3 weeks of sequencing on the HiSeq. Because the chemistry is on the flowcell for less time, error rates are much lower for the MiSeq then the HiSeq. New kits should push read length even longer, with 250+250PE kits coming soon and the Broad Institute having reported 400+400PE runs.
The MiSeq is ideal for:
• Small genome resequencing
• Targeted resequencing
• Metagenomics
• smRNA
• barcode sequencing
The HiSeq2000 was donated to the BioMicro Center by Dr. Chris Love.
Genome Analyzer IIx
The Genome Analyzer II (GAII) are the oldest sequencers in the BioMicro Center and remain the most flexible. The newer generations of Illumina sequencers have been designed with increasing focus on clinical applications and have removed some of the "hands on" aspects of the older GAIIs. The GAIIs remain the only sequencers where the actual images of the flowcell can be reprocessed for example. The GAII/IIx can produce 20-40m reads per lane passing filter and typically runs read lengths of 36-150nt per side.
With the addition of the MiSeq, we have reworked how we are processing GAII flowcells. We have been able to create partial flowcells on the GAII by altering recipes. This has allowed us to move from a model like the HiSeq where we need a full flowcell before we run to a model where we can run as soon as the samples pass quality control, more like the MiSeq. However, unlike the MiSeq, we can run multiple lanes at once. Some critical caveats: First, these methods are not supported by Illumina so we cannot offer to replace failed runs. Second, unlike the HiSeq, the PhiX lane is *not* included. You must choose to sequence a lane of PhiX if you want to do control normalization. Finally, this service is completely "a la carte" so the pricing schema is quite different.
# of Lanes cycles per day cycles per kit
8 24 45
4 36 72
2 48 120
1 72 180
Using fewer lanes on each flowcell has allowed us to decrease the cycle time by not imaging all the lanes. In a typical 8 lane run, 20 minutes is spent doing chemistry followed by 40 minutes of imaging (each lane takes ~5 minutes to image). Therefore, a 2 lane flowcell runs twice as fast as an 8 lane flowcell. Also, since the chemistry is not running in to all of the lanes, each sequencing kit can go to a longer read length. The relationships are summarized in the chart on the left. Pricing is set on the number of lanes you are using, the number of days you are running the GAII, and the number of sequencing kits you are using. For example, if you wanted to run a 96+96 PE flowcell using 2 lanes, the cost would be the initial cost for the 2 lane PE flowcell plus an additional 3 days (one day is included in the original price) plus a second sequencing kit. That kit would not be completely used up (you would have an extra 48nt left that would be thrown away).
The GAII/GAIIx is ideal for:
• Unusual read lengths
• Protocol Prototyping
• Non-standard assays such as HITS-FLIP
This GAII pricing model is an experimental model and is subject to change
The Genome Analyzer IIs were donated to the BioMicro Center by Drs. Penny Chisholm, Chris Burge, Ernest Fraenkel and the Dept of Biology with contributions from many others
Platform Comparison
SPEC HiSeq2000 GAII/IIx MiSeq
Machine Names FonZie Ryland
Boris
Preston
MiAmore
# reads / lane 160-220m 20-40m 4-6m
# lanes coprocessed 7+PhiX 1 to 8 1
nt / day 24 24-72 288
Additional Information
Pricing and Priority
Full pricing information is available on our price list.
Priority for Illumina sequencing is given to labs associated with the BioMicro Center Core departments on a first-come first-served basis. Users requiring expedited services should contact Stuart Levine. We are able offer our services to other MIT and non-MIT users as space allows.
Library Preparation
Illumina sequencing requires the input of libraries with inserts between 10 and 1000bp in length and have specific adapters attached to the 5' and 3' ends. The BioMicro Center accepts custom samples of all types provided the user also submits sequencing primers (though we do not assume responsibility if the samples fail). Samples submitted for Illumina sequencing should be at ~10ng/ul and the user should provide at least 10λ of samples. This is an ideal situation but we do have protocols available to help users with much less concentrated samples. Please submit your sample along with a completed Illumina sequencing form.
In addition to accepting finished libraries, the Biomicro Center supports a number of different sample preparation methodologies for different applications including RNAseq, ChIPseq and genome sequencing. All samples prepped in the BioMicro Center are barcoded for multiplexing.
Quality Control
The BioMicro Center undertakes a number of quality assurance methods to ensure that we produce high quality data for you. All samples submitted for Illumina sequencing are checked for size distribution, presence of proper 5' and 3' adapters, and actual concentration using the Agilent Bioanalyzer and qPCR. For more information on library quality can be found on the Sequencing Quality Control page.
Additional quality metrics are done during all sequencing runs as part of the standard Illumina process. All samples are spiked with ~0.5% of the bacteriophage ΦX174. The ΦX library is primed off the standard Illumina sequencing primers and is used to both ensure the quality of the reagents used in the run and to measure the background sequencing error rates. ΦX reads will not be detected on non-standard libraries using custom priming.
Finally, several additional quality metrics are included in the automated analysis pipeline currently under active development in the Center. These include standard metrics of base composition, GC%, library complexity and overrepresented reads that are in the TagCount and Fastqc files. In addition, we now evaluate libraries for contamination from common laboratory species (human, mouse, yeast and E.coli). More information can be found on the Flowcell data guide page.
Data Analysis
Illumina sequencing at the BioMicro Center includes basic informatic analysis of the data. These steps include:
• Image analysis to locate clusters
• Basecalling
• Demultiplexing of lanes
• Alignment of sequences to a reference genome
• Quality control
• Delivery of the data to a user accessible folder
All of these steps are run by our automated analysis pipelinecurrently in active development. For users requiring further analysis, we have a staff of |bioinformaticists that can assist you in analyzing your data.
MIT Core Collaboration
Because of the layout of Illumina flowcells, samples must be run in batches of 7 lanes (a pool of multiplexed samples counts as one lane). In order to ensure quick throughput, we have established a collaboration that allows us to move partial flowcells between the various centers at MIT. For users with less then 4 samples, their samples may be moved between the BioMicro Center, the Whitehead Institute Center for Genome Technologies and the Koch Institute Biopolymer Center. Samples will be moved only to fill out runs or to expedite processing. The Centers are committed to working together to maintain consistent quality between the different cores, so you should see no difference whether your samples are run in BioMicro or at one of our sister centers. Transfers are only available for members of the MIT community.
View current samples queuing for Illumina
OLDE LINKS
Personal tools
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{
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"provenance": "cccc-CC-MAIN-2013-20-0000.json.gz:56774",
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Chan Yul Yoo
From OpenWetWare
Jump to: navigation, search
Contents
Contact Info
Chan Yul Yoo, Ph.D. (an artistic interpretation)
Chan Yul Yoo, Ph.D.
Purdue University
625 Agriculture Mall Dr. West Lafayette, IN, USA
yoo6@purdue.edu, chanyulyoo@hotmail.com
Email me through OpenWetWare
Education
• 2011, PhD, Purdue University, USA
• 2004, BS, Yonsei University, South Korea
Manuscript in Preparation or in Submission
1. Yoo Chan Yul, Sufang Weng, Hasegawa PM, Mickelbart MV. Environmental Control of Adaptation and Acclimation to Regulate Stomatal Density and Plant Water Use. Trends Plant Sci
2. Yoo Chan Yul, Aliza Finkler, Hua Weng, A.S.N. Reddy, B.W. Poovaiah, Hillel Fromm, Mickelbart MV, Hasegawa PM. Ca2+/calmodulin allosterically inhibits GTL1 transcriptional repressor activity. (Under Review).
Publications
(IF denotes 5 years Journal Impact Factor assigned by ISI, Thomson Reuters)
1. Weng H, Yoo CY, Gosney MJ, Hasegawa PM, Mickelbart MV (2012) Poplar GTL1 is a Ca2+/calmodulin-binding transcription factor that functions in plant water use efficiency and drought tolerance. PLoS One e32925 IF = 4.54
2. Yoo CY, Hasegawa PM, Mickelbart MV (2011) Regulation of stomatal density by the GTL1 transcription factor for improving water use efficiency. Plant Signaling & Behavior 6:1069-1071 IF=2.0. predicted
3. Miura K, Lee J, Gong Q, Ma S, Jin JB, Yoo CY, Miura T, Sato A, Bohnert HJ, Hasegawa PM (2011) SIZ1 regulation of phosphate starvation-induced root architecture remodeling involves the control of auxin accumulation. Plant Physiol. 155:1000-1012 (Cited by 14) IF=7.05
4. Yoo CY, Pence HE, Jin JB, Miura K, Gosney M, Hasegawa PM, Mickelbart MV (2010) The Arabidopsis GTL1 transcription factor regulates water use efficiency and drought tolerance by modulating via transrepression of SDD1. Plant Cell 22:4128-4141 (Cited by 9) IF=10.22
5. Yoo CY, Pence HE, Hasegawa PM, Mickelbart MV (2009) Regulation of transpiration to improve crop water use. Crit. Rev. Plant Sci. 28:410-431 (Cited by 26) IF=6.46
6. Miura K, Lee J, Jin JB, Yoo CY, Miura T, Hasegawa PM (2009) Sumoylation of ABI5 by the Arabidopsis SUMO E3 ligase SIZ1 negatively regulates abscisic acid signaling. Proc. Natl. Acad. Sci. USA. 106(13): 5418-5423 (Cited by 65) IF=10.47
7. Jin JB, Jin YH, Lee J, Miura K, Yoo CY, Kim WY, Van Oosten M, Hyun Y, Somers DE, Lee I, Yun DJ, Bressan RA, Hasegawa PM (2008) The SUMO E3 ligase, AtSIZ1, regulates flowering by controlling a salicylic promotion pathway and through affects on FLC chromatin structure. Plant J. 53:530-540 (Cited by 67) IF=7.12
8. Miura K, Jin JB, Lee J, Yoo CY, Stirm V, Miura T, Ashworth EN, Bressan RA, Yun DJ, Hasegawa PM (2007) SIZ1-mediated sumoylation of ICE1 controls CBF3/DREB1A expression and freezing tolerance in Arabidopsis. Plant Cell 19:1403-1414 (Cited by 169) IF=10.22
9. Lee J, Nam J, Park HC, Na G, Miura K, Jin JB, Yoo CY, Baek D, Kim DH, Jeong JC, Kim D, Lee SY, Salt DE, Mengiste T, Gong Q, Ma S, Bohnert HJ, Kwak SS, Bressan RA, Hasegawa PM, Yun DJ (2007) Salicylic acid-mediated innate immunity in Arabidopsis is regulated by SIZ1 SUMO E3 ligase. Plant J. 49:79-90 (Cited by 86) IF=7.12
10. Yoo CY, Miura K, Jin JB, Lee J, Park HC, Salt DE, Yun DJ, Bressan RA, Hasegawa PM (2006) SIZ1 small ubiquitin-like modifier E3 ligase facilitates basal thermotolerance in Arabidopsis independent of salicylic acid. Plant Physiol. 142:1548-1558 (Cited by 65) IF=7.05
Fellowships, Awards, and Grants
• 2012, 1st place of Post-doc Poster Presentation in HLA Research Retreat, West Lafayette, IN
• 2012, Keystone Symposia Future of Science Fund Scholarship
• 2011, Bilsland Fellowship
• 2011, 2nd place of Graduate Student Oral Presentation in HLA (Department of Horticulture and Landscape Architectures) Research Retreat, West Lafayette, IN
• 2010, 1st place of Outstanding Poster Presentation in American Society of Plant Biologist (ASPB) Midwestern Section Annual Meeting
• 2009, Graduate Student Award for Outstanding Teaching, Purdue University Teaching Academy (HORT301, Plant Physiology)
• 2009, Purdue Research Foundation (PRF) Research Grant, “Characterization of Calcium/Calmodulin-Regulated Transcription Factors involved in Drought Tolerance and Water Use Efficiency”
• 2008, US Departments of Energy and Agriculture-sponsered: United States Registration Award for 19th International Conference on Arabidopsis Research, Montreal, Canada
• 2007, Outstanding Poster Presentation - HLA (Department of Horticulture and Landscape architecture) Research Retreat, West Lafayette, IN
Academic Presentation (Invited Talk)
• 2012, Keystone Symposia Nuclear Events in Plant Gene Expression and Signaling, Taos, NM Mar 6-11 (short talk in plenary session). Title: Calcium/calmodulin regulates SDD1 expression by controlling DNA-binding activity of GTL1 transcriptional repressor.
• 2011, Tel Aviv University, Israel, Title: Integration of drought stress into stomatal development for efficient water use.
• 2011, Purdue University, Research Retreat of Horticulture Department, Title: Calcium/calmodulin regulates the chromatin accessibility of GTL1 from SDD1 promoter.
• 2010, Yonsei University, South Korea, Title: AtGTL1 regulates drought tolerance and water-use efficiency by modulating stomatal density via transrepression of SDD1.
• 2010, Purdue Plant Biology Student/Postdic seminar, West Lafayette, IN, Title: AtGTL1 regulates transpiration and water use efficiency by controlling stomatal number through transcriptional repression of SDD1.
• 2008, 19th International Conference on Arabidopsis Research, Montreal, Canada July 23 – 27 (presented in concurrent session) Title: AtGTL1 transcription factor regulates water use efficiency and drought adaptation through Ca2+/Calmodulin signaling.
• 2007, American Society of Plant Biologist (ASPB) meeting, Chicago, IL, July 7-13, (presented in minisymposium) Title: AtGTL1 transcription factor regulates drought adaptation through Ca2+/Calmodulin signaling.
Grant Proposals
• 2011 National Science Foundation (NSF) - Division of Molecular and Cellular Biosciences (MCB): Investigator-initiated research projects - Program: Networks and regulation (11-545)
Title: Calcium and calmodulin teracts with the GTL1 transcription factor to regulate SDD1 expression and stomatal development. Status: Pending
• 2011 United States Department of Agriculture (USDA) - National Institute of Food and Agriculture (NIFA) - Agriculture and Food Research Initiative (AFRI) Foundational Program - Biology of Agricultural Plants (A1101)
Title: Improvement of water-use efficiency through regulation of transpiration by GTL transcription factors. Status: Not funded
• 2009 USDA-NIFA-AFRI-Plant Biology: Environmental Stress
Title: Improvement of drought tolerance and water-use efficiency through regulation of transpiration by the GTL1 transcription factor. Status: Not funded, ranked as high priority
• 2008 Binational Agricultural Research and Development (BARD)
Title: Calcium-regulated transcription factors mediating carbon metabolism in response to drought. Status: Funded
• 2008 USDA-NIFA-AFRI
Title: Determination of GTL1 transcription factor functions in regulating drought adaptation and WUE through calcium/calmodulin signaling. Status: Not funded, ranked as low priority
• 2007 BARD
Title: Calcium-regulated transcription factor mediating carbon metabolism and partitioning in response to drought. Status: Not funded
Professional Activities
• 2011 Reviewer for Planta
• 2008 Teaching Assistant – Fall, HORT301 Plant Physiology, 3 credits
Membership
American Society of Plant Biologists
Useful links
Personal tools
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{
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brutec1980's bookmarks
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simon shepherd's quote collection
I'm male and made my book on 2nd March 2007.
My book as a pdf
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"uncompressed_offset": 181420256,
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Quotation added by staff
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No man is wise enough by himself. Plautus, Titus Maccius
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Dunfermline
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Dunfermline [1] is a historic town in the Kingdom of Fife, Scotland. It is the ancient capital of Scotland, and the birthplace of philanthropist Andrew Carnegie - at one time the richest man in the world. Dunfermline Abbey is the burial place of Robert The Bruce, while Pittencrieff Park (known locally as The Glen) is one of the most attractive parks in Scotland .
Dunfermline Abbey from Pittencrieff Park
Get in
By Air
The nearest airport to Dunfermline is Edinburgh International Airport (EDI), situated to the south of the Firth of Forth, 14 miles/22km from Dunfermline. The airport offers a wide range of domestic and international flights to Europe and North America. It costs in the region of £30 for a each way by taxi. (See the Edinburgh article for more details)
By Train
Dunfermline is served by 2 railway stations. Dunfermline Town, which is located in the centre of the town, and Dunfermline Queen Margaret, which is located in the northeast of the town, near Queen Margaret Hospital. There is a half hourly (hourly after 19:00) Fife Circle service to Edinburgh Waverley. (See the First ScotRail website for timetable information)
By Car
Dunfermline is located near the M90 motorway which runs from the Forth Road Bridge to Perth. Access to the south and centre is via M90 junction 2, and the west is accessed from via junction 3.
By Bus
There are frequent bus services to Edinburgh, Glasgow, Dundee, Perth, and surrounding towns. The Bus Station has been temporarily relocated to the East Port whilst renovation work goes on, including the demolition of the old bus station to allow for the building of a Kingsgate extension.
Get around
• Dunfermline's town centre is fairly well equipped with amenities for the 21st century shopper. Most sites are within walking distance and a bus station is provided for journeys to surrounding areas.
"PlanaJourney" is a free integrated public transport journey planner that covers Dunfermline and includes much of the Scottish, Northern Ireland and UK public transport network. It includes bus, rail, subway, Scottish ferries and internal UK flights. It can assist with planning journeys into and out of Dunfermline from anywhere in the Dunfermline or Fife area or more widely from anywhere in Scotland and the UK. Outside of Scotland and Northern Ireland the bus information is limited.
See
Do
• Take a stroll around Pittencreiff Park this park was purchased by Andrew Carnegie and donated to the people of the town. There are signed walks of various lengths. The park contains a small museum, gardens and hothouses. In the north eastern section of the park is the remains of King Malcolm's tower. This tower appears on the town crest and dates back to around 1000 AD. Snack vendors in the park also sell bags of nuts with which to feed the squirrels.
Buy
Eat
If you are just stopping off as part of a longer journey there are several restaurants and fast food places at the Fife Leisure Park. (Exit the M90 at junction 3 and take the Duloch Park exit on the roundabout at the top of the slip road).
• Frankie and Benny's, Whimbrel Place, Fife Leisure Place, Dunfermline, KY11 8EX (01383 622 477), [2].
• Brewer's Fayre, Crooked Glen, Fife Leisure Park, Whimbrel Place, Dunfermline, KY11 8EX (0870 600 1486), [3].
• Pizza Hut Fife Leisure Park, Halbeath, Dunfermline KY11 8EX.
If you are staying in Dunfermline, there are a number of good local restaurants.
• The China Ruby, Dalgety Bay. Thoroughly recommended.
• The hideaway, North-east of the town centre on the road to Kingseat.
• There is a very good cafe between the Abbey and the library, with good views of the Abbey from the terrace - traditional home made cooking.
• The Old Inn Carnock, West of the town on the road to Alloa. This bar and restaurant has a fabulous well priced menu and very friendly staff.
• Domino's Pizza, 106-114 Hospital Hill, Dunfermline, KY11 3AU (01383 722 422), [4].
Drink
Sleep
• Pitbauchlie House Hotel, Aberdour Road, +44 (0)1383 722282 (, fax: +44 (0)1383 620738), [5]. Located near the center of town.
• King Malcolm Hotel, Queensferry Road, +44 (0)1383 722611 (fax: +44 (0)1383 730865), [6]. Situated in the south of the town near the Pittrievie business park.
• Express By Holiday Inn Dunfermline, Halbeath Road, Halbeath, +44-1383-748220 (fax: +44-1383-748221), [7]. Located to the east of the town.
• Premier Travel Inn Dunfermline, 4-12 Whimbrel Place, Fife Leisure Park, +44 (0)870 600 1486 (fax: +44 (0)870 600 1487), [8]. Located to the east of the town. GBP 50-55.
• Travel Lodge Dunfermline, Halbeath Junction, Halbeath, +44 (0)870 191 1787 (fax: +44 (0)1383 844649), [9]. Located to the east of the town.
Get out
• Aberdour - Described as "The Jewel of Fife", Aberdour is a historic and stunningly attractive coastal village. Aberdour Castle is a must-see, as well as the Blue-Flag awarded beach the Silver Sands. There are also several pubs, restaurants, and boutique shops. 15 minutes drive East of Dunfermline, or hop on the regular train/bus services.
• Culross - Some 10 miles to the west - the village that time forgot. A quaint selection of dwellings, all restored, dating from the 17th and 18th century. Abbey, Town House, Study and Palace all worth a visit.
• Dollar - It takes about 40 minutes by car. Take the A823 in the direction of Crieff and follow the signs for Dollar en route. There are great walks around Dollar Glen and there is the partial ruin of Castle Campbell to explore.
• Falkland - A small village with historic palace, about 40 minutes drive away.
• Kirkcaldy - 15 minutes in car via the A92 dual carriageway or easily by train or bus. Excellent shopping and theatre, parks, museum and art gallery.
This is a usable article. It has information for getting in as well as some complete entries for restaurants and hotels. An adventurous person could use this article, but please plunge forward and help it grow!
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Revision history of "Lizard"
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Winter Park (Colorado)
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Winter Park, Colorado, is a ski resort in the Front Range. It has many pastimes for both summer and winter visitors. There are several places that close during the spring and fall, however.
Get in
Winter Park is a little over 65 miles from Denver, and is one of the closest ski resorts from Denver. The route is I-70 West most of the way, with the remainder on U.S. Route 40 West, which goes over Berthoud Pass. Be sure to check [1] for current conditions on Berthoud Pass, as it's common for it to be snowing (sometimes heavily) on the pass with no snow in Denver or on the way up I-70.
There are shuttles to Winter Park from Denver International Airport, and the town has an adequate free shuttle between Winter Park Resort, the town of Winter Park, and the town of Fraser.
Get around
Powderhound Transportation [2] provides shuttle service from Denver International Airport to Winter Park Colorado.
See
Do
• Winter Park Resort, 239 Winter Park Drive, 303-892-0961, [3]. Ski Resort. Check out the Alpine Slide in the Summer!
• Hot Sulphur Springs Resort. Hot Sulpher Springs is about 45 minutes north of Winter Park. They have a fantastic set of hot springs pools, fun in both the winter and summer. They also have lodging here, but there's a level train crossing and 3 to 4 trains come through in the middle of the night blowing their whistles within half a mile of the rooms.
• Grand Adventures, Snowmobiling, Sleigh Rides, ATV Rentals, and Horseback Riding. Physical Address: 81699 US Highway 40, Winter Park, CO 80482. Mailing Address: P.O Box 1329, Winter Park, CO 80482. Toll Free:(800)726-9247 [4] Email: info@grandadventures.com
• Pole Creek Golf Course. 27 holes of Colorado Mountain Golf on 3 distinct courses - The Meadow, The Ranch and The Ridge. Course is at 8,600 feet, and plays to 7,107 yards from the back tees. Voted Best New Public Course in North America in 1985. 970-887-9195. Located at 6827 County rd 51, Tabernash, Colorado 80478.
Buy
Eat
• Hernando's, Pizza and Pasta. (Local, $7 per person)
• Mad Munchies (30 miles north in Grandby), fantastic sandwiches. (Local, $6 per person)
• Pizza Shop in Frasier. Good pizza and Italian food. (Local, $6 per person)
• De Antonio's, Pizza and Pasta (2 miles north in Frazier). In the Alco strip-mall, across the street from Safeway. Some of the best pizza by the slice in the area. (Local, $7 per person)
• Mountain Rose, breakfast in downtown Winter Park. It's fairly small, and almost always seems to have a wait. This seems mostly to be due to slow table turnover and a small dining-room. They're fairly heavy on attitude, and the sign out front says "Hours Iffy". Good food. (Local, $8 per person)
• Fontenot's Seafood Restaurant, 78336 US Hwy 40, 970-726-4021, [5]. This restaurant has been around since 1990 and is favored by the locals. Seafood is their specialty with or without a Cajun slant, but they also serve steaks, chicken, and pasta. A great selection of wines by the glass as well as some micro brews. Lunch is great value and they also have great happy hour deals on appetizers. Located in King’s Crossing Shopping Center, downtown Winter Park.
• Gasthaus Eichler Restaurant, 78786 US Hwy 40 (on Main Street, across the street from Hideaway Park and the Winter Park Chamber, central downtown), 970-726-5133, [6]. Back under management by the original owners, Hans & Hanne Eichler. Using family recipes and everything hand-made, it serves up European and Continental cuisine. Their traditional German dishes have a huge local following, and includes such favorites as Paprika Goulasch, Jagerschnitzel, and Wiener Schnitzel. However, do not overlook the Chateaubriand, Steak Diane and the Rocky Mountain Trout. Serving lunch and dinner daily, with take-out available.
• Pearl Dragon II, 45 County Road 804, Fraser, CO 80442 (in the strip mall next to Safeway), (970) 726-5252. Great Chinese food made fresh to order. Generous portions, reasonable prices and a great atmosphere. The family that runs Pearl Dragon is frendly and welcoming. A must for locals and out of town guest!
Drink
• Hernando's Pizza Pub, 78199 Us Hwy 40,(970) 726-5409
Good variety of pizza and beer.
Sleep
• Alpine Resort Properties, 970.726.5701, [7]. Is an all-season Winter Park accommodation specialist, which includes our surrounding towns of Fraser, Tabernash and SolVista/Granby Ranch.
• Iron Horse Resort, 101 Iron Horse Way, 800-621-8190, [8]. Ski-In/Ski-Out, Full-Service resort offering condominiums in hotel style atmosphere.
• Moose Mountain Inn, Tel: (970) 726-8255, Toll Free: 888-999-1616, [9]. Moose Mountain Inn offers luxurious lodging in Winter Park, Colorado.
• Best Western Alpenglo Lodge, 78665 US Highway 40, +1 970 726-8088, Toll-free: +1 888 726-8088, Fax: +1 970 726-0331, [10].
• Winter Park Real Estate, [11]. Coyote Creek mountain homes and real estate.
• The Rocky Mountain Inn and Hostel, Frasier, [12]. Very cheap rooms and communal rooms. Has a computer with dial-up net access, but also has very good CDMA coverage.
• About 10 miles north of Frasier is a golf resort that has many very nice condos as well as hotel rooms, for around $140 per night. The condos all seem to have hot tubs in them. No CDMA coverage though.
• Beaver Village Condominiums (Teverbaugh Heaton Management), 50 Village Drive Winter Park, Co 80482 (From Denver- Exit 232 Highway 40 west, 26 miles on the left, marked by a large oval green sign.), 970-726-8813, [13]. checkin: 4PM; checkout: 10AM. Condos range in size from 1-4 bedrooms. Located only 2 blocks from downtown and 1.5 miles to the base of the ski resort, easily accessible to all of Winter Park. Free town ski shuttle will transport to the ski area directly from all of the condos. Centrally located Clubhouse has a heated indoor pool, hot tubs, sauna, guest laundry and 1200 square feet of meeting space. The Clubhouse and condos are also equipped with free high speed wireless internet services. from $95.
• Vacations Inc, 78415 US Highway 40, (970)726-9421, [14]. checkin: 4PM; checkout: 10AM. Vacations Inc is the longest established rental management company in Winter Park. Has studios, condos, town houses, cabins and private homes throughout the Winter Park & Fraser Valley.
• Wild Horse Inn, 1536 County Road 83, Fraser, Colorado, +1-970-726-0456, [15]. Winter Park Colorado Lodging with intimate lodge rooms and 3 private cabins. Breakfast included at Wild Horse Inn! Great for all seasons. $135 - $235 for individual rooms, $195 - $285 individual cabins.
• Winter Park Lodging Company (WPLCO), 78260 US Highway 40 (Winter Park, CO), 877.329.1383, [16]. checkin: 4PM; checkout: 10AM. Reserve Winter Park lodging accommodations. The Winter Park Lodging Company provides new, clean condominiums, cabins, homes & town homes in Winter Park, CO plus lift ticket and ski rental deals. With over 100 properties WPLCo has the perfect accommodations for you. $175.
Contact
Winter Park has great Sprint CDMA coverage. This coverage extends north to Frasier, but not much further. The hostel has dial-in on a public terminal.
Wireless Internet
• Rocky Mountain Roastery and Coffee Company, with branches in both Fraser and Winter Park, have just recently (2004-08) added pay-per-use wireless internet connectivity.
• Winter Park Coffee Company [17] has free wireless with a purchase. Located next to Deno's restaurant, in the main street Real Estate building.
Get out
This article is an outline and needs more content. It has a template, but there is not enough information present. Please plunge forward and help it grow!
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Research article
Proteomic analysis of lamellar bodies isolated from rat lungs
Pengcheng Wang1, Narendranath R Chintagari1, Jeyaparthasarathy Narayanaperumal1, Sahlu Ayalew2, Steven Hartson3 and Lin Liu1*
Author Affiliations
1 Department of Physiological Sciences, Oklahoma State University, Stillwater, OK 74078, USA
2 Department of Pathobiology, Oklahoma State University, Stillwater, OK 74078, USA
3 Department of Biochemistry & Molecular Biology, Oklahoma State University, Stillwater, OK 74078, USA
For all author emails, please log on.
BMC Cell Biology 2008, 9:34 doi:10.1186/1471-2121-9-34
The electronic version of this article is the complete one and can be found online at: http://www.biomedcentral.com/1471-2121/9/34
Received:12 January 2008
Accepted:24 June 2008
Published:24 June 2008
© 2008 Wang et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background
Lamellar bodies are lysosome-related secretory granules and store lung surfactant in alveolar type II cells. To better understand the mechanisms of surfactant secretion, we carried out proteomic analyses of lamellar bodies isolated from rat lungs.
Results
With peptide mass fingerprinting by Matrix Assisted Laser Desorption/Ionization – Time of Flight mass spectrometry, 44 proteins were identified with high confidence. These proteins fell into diverse functional categories: surfactant-related, membrane trafficking, calcium binding, signal transduction, cell structure, ion channels, protein processing and miscellaneous. Selected proteins were verified by Western blot and immunohistochemistry.
Conclusion
This proteomic profiling of lamellar bodies provides a basis for further investigations of functional roles of the identified proteins in lamellar body biogenesis and surfactant secretion.
Background
Lung surfactant is a surface active material. It reduces the surface tension of the air-liquid interface in alveoli, thus preventing alveoli from collapsing. Deficiency of surfactant at the alveolar surface results in respiratory distress syndromes (RDS) in both newborns and adults [1]. Lung surfactant is synthesized and secreted by alveolar type II cells. It is mainly composed of phospholipids and surfactant proteins A, B and C (SP-A, SP-B and SP-C). Major components of surfactant are synthesized in the endoplasmic reticulum and stored in specialized organelles called lamellar bodies [2].
Lamellar bodies are lysosome-related, large secretory organelles that are 1 to 2 micrometers in size [3]. Similar to lysosomes, lamellar bodies contain soluble lysosomal enzymes, such as acid phosphatase and lysosome associated membrane proteins [3]. Lamellar bodies have an acidic interior with a pH of about 6.1 or below [4]. However, lamellar bodies are different from lysosomes in that they are specialized for storage and secretion of surfactant rather than for degradation processes. The principal components of lamellar bodies, phospholipids, are tightly packed as concentric arrangements of bi-layer membranes. Secretion of surfactant involves the translocation, docking and fusion of lamellar bodies with the apical plasma membrane [5,6].
The molecular mechanisms that control the exocytosis of lamellar bodies are still poorly understood [5]. The recent emergence of powerful proteomic techniques has made it possible to profile the protein components in a specific tissue or subcellular organelle [7-9]. To better understand the regulation of lamellar body biogenesis and exocytosis, we performed proteomic analysis of lamellar bodies isolated from rat lungs. We carried out both one-dimensional and two-dimensional gel electrophoresis, followed by Matrix Assisted Laser Desorption/Ionization – Time Of Flight mass spectrometry (MALDI-TOF) and immunohistochemistry. Here, we report the first proteomic profiling of lamellar bodies, which will aid in defining the mechanisms of lamellar body exocytosis.
Results
The method used to isolate lamellar bodies was based on the work of Chander et al. [10] and is in routine use in our laboratory [11-13]. The isolated lamellar body fraction consists of intact lamellar bodies and large amounts of concentric multilamellated membrane structures and contains no other organelles except for very low amounts of microsomes.
One-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) staining with colloidal Coomassie Brilliant Blue revealed more than 50 protein bands. Due to the complexity of protein components of lamellar bodies, we also performed two-dimensional PAGE to get better separation of the proteins. The two-dimensional gel electophoresis revealed more than 100 spots. Well-resolved protein bands or spots were harvested for peptide mass fingerprint analysis. Figs. 1 and 2 show some of the identified proteins from representive gels.
Figure 1. One-dimensional SDS-PAGE of lamellar body proteins. Lamellar bodies were isolated from perfused rat lungs as described in materials and methods. Approximately 100 μg of total protein were loaded on 10% Bis-Tris polyacrylamide gels. Protein bands were visualized by staining with Coomassie Brilliant Blue G-250.
Figure 2. Two-dimensional SDS-PAGE of lamellar body proteins. Isolated lamellar bodies (500 μg of protein) were analyzed by 2-D polyacrylamide gel electrophoresis. Protein spots were visualized by staining with Coomassie Brilliant Blue G-250. Isoelectric points (pI) and molecular masses are shown on the top and the left of the panel, respectively.
To identify protein components in lamellar bodies, we utilized trypsinolytic fingerprinting, MALDI-TOF mass spectrometry and statistically scored database searching (Mascot®). Table 1 lists the 44 proteins identified from the highly purified lamellar bodies. Listed for each protein include the National Center for Biotechnology Information (NCBI) accession number, the number of peptides matched and percent of sequence covered, the Mascot® Probability Based Mowse (PBM) score and the molecular mass. By applying each of these proteomics search criteria, the proteins were identified with great confidence.
Table 1. Proteins identified in lamellar bodies with MALDI-TOF MS
The functional classification was performed by a literature search in the Pubmed database. The functional categories include calcium-binding proteins, structural proteins, surfactant-related proteins, ion channels, membrane traffic, protein processing, signal transduction and miscellaneous proteins (Fig. 3).
Figure 3. Pie chart showing the functional classifications. The 44 identified proteins were categorized into 8 different functional categories. The pie chart represents the percentage of identified proteins under each category. The percentages are shown within the pie chart.
Based on the availability of antibodies, we selected several important proteins identified by trypsinolytic fingerprinting from each functional category and verified their identities by Western blotting and immunohistochemistry. Actin, Annexin A2, calreticulin, EH domain-containing 1 protein (EHD1), Rho-GDP dissociation inhibitor alpha (GDI-alpha) and vimentin were confirmed to be present in lung tissue and lamellar bodies as seen by Western blot (Fig. 4). Lamellar bodies contain more EHD1 than freshly isolated type II cell lysate. Only a small portion of GDI-alpha and actin were present in lamellar bodies, indicating these proteins were also localized elsewhere in type II cells. Annexin A2 and calreticulin existed in lamellar bodies and freshly isolated type II cell lysate in a similar amount. Vimentin was abundantly present in lamellar bodies, however undetectable in freshly isolated type II cells. The reason is unclear but could be due to the enrichment of this protein in lamellar bodies and the detection sensitivity. EHD1 along with the carbonic anhydrase IV (CAIV) protein were further investigated for their cellular localization in overnight-cultured type II cells because of their suitability for double-immunostaining. As seen in Fig. 5, both EHD1 and CAIV partially colocalized with the lamellar body marker, LB-180. The localization of these proteins at lamellar bodies further suggests their roles in lamellar body function.
Figure 4. Western blotting of selected proteins identified in lamellar bodies. Lung tissue homogenate (LH), freshly isolated type II cells (T2), and lamellar body fraction (LB) were lysed and 20 μg of total protein were separated by 10% SDS-PAGE and probed with corresponding antibodies.
Figure 5. Immunolocalization of selected proteins identified in lamellar bodies. Type II cells were cultured overnight on glass cover slips. The cells were fixed and subjected to double-labeling with antibodies against EHD1 or CAIV and LB-180 proteins. Shown are the representative images indicating the co-localization of EHD1 or CAIV with the LB-180 protein.
Since some of the proteins are developmentally regulated, we also determined the developmental profiles of two of the identified proteins (Fig. 6). The protein expression levels of EHD1 peaked postnatally. Calreticulin was expressed constantly throughout lung development.
Figure 6. Developmental profiles of EHD1 and calreticulin in the lung. Tissues were collected from fetal lungs with gestational days 18, 19, and 21 (E18, E19, E21) and the lungs from newborn (NB), postnatal day 14 (P14) and adult (AD) rats. The protein levels of EHD1 and calreticulin were determined by Western blot. The β-actin was used as a loading control.
Discussion and conclusion
Although proteomic analyses have been performed on several secretory granules [7-9], proteomic prolifes of lamellar bodies of alveolar type II cells have remained a mystery. Here, we have identified 44 proteins present in lamellar bodies by using one- and two dimensional electrophoresis and trypsinolytic fingerprinting. The identification of these proteins provides a basis for further studying their functions in lamellar body exocytosis and biogenesis.
The proteins in lamellar bodies fell into diversified functional categories. Several calcium-binding proteins were identified, including the annexin family and calreticulin. There are some controversies as to which annexins are expressed in alveolar type II cells in literature. We previously showed that annexin A1, A2, A3, A6, but not A4 and A5 were present in rat alveolar type II cells as determined by immunoblotting [14]. However, Sohma et al. were able to detect annexin A4 in type II cell lysate [15]. Mayran et al. observed the presence of annexin A1, A4 and A6 in type II cells by using immunohistochemistry [16]. The inconsistency is likely due to differences among the antibodies used in the respective experiments. Using the proteomic approach, we identified seven members of the annexin family, annexin A1-7, in lamellar bodies.
Consistent with its representation in the lamellar body proteome, annexin A2 is important in various aspects of membrane trafficking [17]. A series of studies from our laboratory support a role of annexin A2 in lung surfactant secretion. It mediates the fusion between lamellar bodies and the plasma membrane [11,14]. The silencing of annexin A2 by RNA interference inhibits regulated lung surfactant secretion [18]. Annexin A2 functions in lung surfactant secretion via its interaction with SNAP-23 [13]. Disruption of lipid rafts, a platform organizing exocytotic proteins on a membrane, reduced annexin A2-mediated fusion of lamellar bodies with the plasma membrane [12]. In addition to annexin A2, annexin A7 has also been proposed to be involved in regulating lung surfactant secretion [19].
We find that annexin A1, A5 and A6 are the components of the lamellar body proteome. Annexin A1 and A6 have been reported to play roles in endocytosis [20,21]. Annexin A5 was reported to be secreted from type II cells. Because its secretion was stimulated by phorbol-12-myristate-13-acetate (PMA) and inhibited by SP-A, showing the same pattern as that of surfactant secretion [22], annexin A5 may be released through lamellar bodies. Annexins play important roles in governing lamellar body exocytosis and endocytosis, membrane organization, membrane cytoskeleton linkage and ion conductance.
Soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs) are a conserved mechanism for membrane targeting, docking and fusion [23]. We have previously shown that the plasma membrane SNAREs, syntaxin 2 and SNAP-23 and their co-factor, α-SNAP are essential for regulated surfactant secretion [24,25]. We have also detected the vesicle SNARE, VAMP-2 in lamellar bodies of type II cells using immunofluoresence [26]. However, our proteomic analysis of lamellar bodies did not show the presence of VAMP-2. The possible reasons include its low abundance or loss during sample preparation and separation.
We note that calreticulin is a component of the lamellar body proteome. Calreticulin is a calcium-binding protein for calcium storage in the endoplasmic reticulum (ER) [27]. It has also been reported to act as an important modulator of the regulation of gene transcription by nuclear hormone receptors [28]. Consistent with its representation in our demonstration of the lamellar body proteome, lamellar bodies contain a high level of calcium, especially those in the apical area. It was reported that the exocytotic lamellar bodies contain significantly higher calcium compared to those in the perinuclear area [29]. This suggests that this high content of calcium in lamellar bodies may be a supply for the increase in local calcium concentration during fusion events. Thus, our results indicate that calreticulin may be the main calcium-binding protein in lamellar bodies and may control the release of calcium ions during exocytosis.
In agreement with the nature of lamellar bodies as discrete cytological structures, we identified several cytoskeletal proteins in lamellar bodies by proteomic analysis. These proteins include β-actin, myosin-9, tropomysin 1, moesin, cytokeratin 19 and vimentin. Cytoskeleton is composed of 3 filamentous structures: actin filaments, intermediate filaments and microtubes of myosin. It has been reported that actin [30] and microtubules [31] play a role in the transportation of lamellar bodies. The cortical cytoskeleton in type II cells undergoes disassembly and assembly after being stimulated with lung surfactant secretagogues [32]. This process is regulated by annexin A2 and is essential for the lamellar body's access to the apical plasma membrane. Cytokertain 19 and vimentin are components of the intermediate filaments [33]. Tropomysin 1 is a ~40 kDa ubiquitous protein that is associated with the actin filaments and regulates the functions of actin cytoskeleton [34]. Moesin is a member of the ezrin/adixin/moesin family and plays a role in the linking of the plasma membrane and cytoskeleton [35]. These proteins may also be involved in the movement of lamellar bodies.
Among the major surfactant-related proteins described here, SP-A and SP-B are known components of lung surfactant. However, we did not detect SP-C. This is likely due to its loss during sample preparation and gel separation since SP-C is a hydrophobic and low molecular mass peptide. We also observed that several other proteins previously implicated in lamellar body biogenesis are in fact part of the lamellar body proteome. ATP-binding cassette transporter A3 (ABCA3) is a unique type II cell marker that was originally identified by a monoclonal antibody screen using lamellar body limited membrane [36]. It transports lipids into lamellar bodies [37]. ABCA3 mutations result in fatal neonatal lung diseases [38]. Peroxiredoxin-6, which possesses both phospholipase A2 and peroxidase activity, is responsible for the degradation of the main recycled surfactant phospholipid, Dipalmitoylphosphatidylcholine (DPPC), in type II cells [39]. Apolipoprotein A is the major apoprotein of high-density-lipoprotein (HDL) [40]. The identification of apolipoproteins in lamellar bodies suggests that the cholesterol of surfactant may originate from HDL. Our findings suggest that these proteins are important components of lamellar bodies, thus demanding closer examination of their roles in lamellar body structure, function and regulation.
Our results support the idea that lamellar bodies are lysosome-related organelles that retain some lysosome-like features. We note that several lysosome-like proteins are the components of lamellar bodies. Along with lysosome membrane protein II [41], some proteases (Dipeptidyl peptidase IV) [42] and protease inhibitors (Serpinh1 and alpha-1-antitrypsin precursor) [43,44] were identified, which suggests that lamellar bodies may not only store surfactant, but they may also play a role in surfactant processing. We also identified the vacuolar proton transporting ATPase (V-ATPase) subunit (H+-transporting two-sector ATPase alpha chain precursor). V-ATPases may pump protons into lamellar bodies to maintain the low pH inside lamellar bodies [4,45]., which is essential for surfactant processing [46,47] and calcium uptake [48].
Our analyses of the lamellar bodies also implicate novel proteins that may regulate lamellar body function. One is the EH domain-containing 1 protein (EHD1). The EH domain includes an EF-calcium-binding motif, a highly conserved ATP/GTP-binding domain and a central coiled-coil structure [49]. It has been reported that EHD1 interacts with SNAP-29 and plays a role in the endocytosis of Insulin-like Growth Factor 1 (IGF1) receptors [50]. Its role in the regulation of endocytic recycling has been further confirmed in other systems [51]. While EHD1 has not been previously implicated in endocytosis of surfactant, our findings imply that EHD1 may have a role in lamellar body function.
Our indentificatoin of GDI alpha as a major component of lamellar bodies suggest that it may be involved in the docking of lamellar bodies at the plasma membrane. Rab proteins are small GTP-binding proteins that play roles in vesicular trafficking of molecules between cellular organelles [52]. They serve as functional switches for the GTP-GDP exchange reaction. Rab GDP dissociation inhibitors (GDIs) can reduce the rate of GDP dissociation from Rab proteins [53]. Rab GDI alpha has been reported to bind Rab3A and modulates their activity and vesicle-mediated transport [54]. Small GTP-binding proteins are associated with lamellar bodies [55]. Rab3D only exists in subpopulations of lamellar bodies (~25% close to the apical membrane) [56].
Carbonic anhydrase (CA) is a zinc metalloenzyme, which reversibly catalyses the conversion of CO2 to HCO3 and a proton [57]. They are predominantly involved in maintaining acid-base balance. CAII was present in both lung type I and type II cells [58,59]. CAIV were detected in alveolar capillary endothelium but not on large blood vessels [60]. Our results indicated that CAIV was localized on the lamellar bodies of type II cells. Lamellar bodies maintain acidic interiors, which favor not only processing of SP-B and SP-C but also aggregation of surfactant lipids [46,47,61]. Thus, the presence of CAIV on lamellar bodies might be one of the additional mechanisms in maintaining and regulating the acidic interiors.
Our results document the first comprehensive proteome of the isolated rat lung lamellar bodies. However, lamellar bodies likely contain proteins that were not detected in our study due to their low abundance or due to individual molecular masses and isoelectric point values that hinder analysis by gel-based techniques. There may also be more proteins that we could not identify due to the incomplete nature of the Rattus sequence database. It is also noted that a few identified proteins have lower molecular masses than the calculated values. This is probably due to degradation or processing. Our initial characterization of the protein constituents of lamellar bodies provides a platform for further studying lamellar body biogenesis and surfactant secretion.
Methods
Reagents and chemicals
The ReadyPrep 2-D cleanup kit, ReadyPrep reduction-alkylation kit, ReadyPrep protein extraction kit, non-linear pH 3-10 ReadyStrip IPG strips and horse-radish peroxidase (HPR)-conjugated goat anti-rabbit and anti-mouse antibodies were from Bio-Rad Laboratories (Hercules, CA). Rabbit polyclonal anti-β-actin antibodies were from Sigma (St Louis, MO). Monoclonal anti-annexin A2 and rabbit polyclonal anti-Rho-GDP dissociation inhibitor (GDI)-alpha antibodies were from Invitrogen (Carlsbad, CA). Rabbit polyclonal anti-vimentin, anti-dipeptidyl peptidase IV (CD26) and anti-calreticulin antibodies were from Abcam (Cambridge, MA). Polyclonal rabbit anti-EH domain-containing 1 protein (EHD1) and anti-carbonic anhydrase IV (CAIV) antibodies were from Santz Cruz (Santz Cruz, CA). Mouse anti-LB-180 antibody was from Covance Research Products (Richmond, VA).
Isolation of lamellar bodies from rat lung
Lamellar bodies were isolated from rat lungs by upward flotation on a discontinuous sucrose gradient, as described by Chander et al. [10] and Chattopadhyay et al. [11]. The Oklahoma State University Animal Use and Care Committee approved all animal procedures used in this study. A perfused rat lung was homogenized in 1 M sucrose and loaded at the bottom of a sucrose gradient (0.2, 0.3, 0.4, 0.5, 0.6, 0.7, and 0.8 M). After centrifugation at 80,000 × g for 3 hours, the lamellar body fraction was collected at the 0.4 and 0.5 M interface, and diluted to 0.24 M with cold water. Lamellar bodies were then spun down at 20,000 × g and resuspended in 0.24 M sucrose containing 10 mM Tris and 50 mM Hepes (pH 7.0). The protein concentration of lamellar bodies was determined by the RC-DC protein assay (Bio-Rad). We typically used 6 rats for the isolation of lamellar bodies, which yielded ~1 mg protein of lamellar bodies.
One-dimensional SDS-PAGE
Lamellar bodies (~100 μg protein) were directly lysed in 1 × SDS sample buffer and fractionated on 10% SDS-PAGE under reducing conditions. The gel was subjected to colloidal coomassie brilliant blue staining to visualize protein bands as described below.
Two-dimensional gel electrophoresis
Lamellar bodies (~500 μg protein) were processed by the ReadyPrep 2-D cleanup kit according to the manufacturer's protocol. The pellet was air-dried at room temperature and resuspended in an appropriate volume of 2-D rehydration/sample buffer (8 M Urea, 2% CHAPS, 50 mM DTT, 0.2% Bio-Lyte 3/10 ampolyte, and 0.002% bromophenol blue). The non-solubilized particulate matter was removed by brief centrifugation and the supernatant was stored at -80°C or used to passively hydrate 17 cm non-linear, pH 3–10, immobilized pH gradient strips (IPG) and isoelectric focusing was performed on a Protean IEF Cell (Bio-Rad). The IPG strips were equilibrated in equilibration buffers I (6 M urera, 0.375 M Tris, pH 8.8, 2% SDS, 20% glycerol. 2% (w/v) DTT) and II (6 M urera, 0.375 M Tris, pH 8.8, 2% SDS, 20% glycerol, 2.5% (w/v) iodoacetaminde) and loaded onto 8 – 16% gradient gel where the proteins were resolved in a secocd dimension gel electrophoresis. After SDS-PAGE, the gels were fixed with 40% ethanol and 10% acetic acid overnight, followed by washing with water twice for 10 min. The colloidal coomassie brilliant staining working solution was made by mixing 80% stock solution (0.1% coomassie brilliant blue G-250, 2% ortho-phosphoric acid, 10% ammonium sulfate) with 20% methanol. The gels were stained overnight and destained with 1% acetic acid until satisfactory destaining was achieved.
MALDI-TOF mass spectrometry
Acrylamide bands or spots were harvested, washed with ammonium bicarbonate and digested for 8 h with 8 μg/ml trypsin. Trypsonolysis products were extracted and analyzed by reflectron MALDI-TOF mass spectrometry (Applied Biosystems DE-Pro) using alpha-cyano-4-hydroxy cinnamic acid as a matrix. Spectra were calibrated to 100 ppm mass accuracy using a mix of 5 known peptides (m/z 904 to 2465) spotted proximal to each sample on the MALDI probes.
Protein identification analysis
Protein identification was carried out by searching the peptide spectra against the Mass Spectrometry protein sequence database (MSDB) using the Mascot web based search engine. The search parameters used were: taxonomy, Rattus; allow up to 1 missed cleavage; variable modifications, carbamidomethyl (C), oxidation (M), propionamide (C), and pyro-glu (N-term Q); mass value, MH+; allowed error, 100 ppm.
Alveolar type II cell isolation
Type II cells were isolated from male Sprague-Dawley rats (180–200 g) as previously described [14]. Briefly, the perfused and lavaged lungs were digested with elastase (3 U/ml) at 37°C. The lung tissues were chopped and filtered through 160-, 37- and 15-μm mesh size nylon gauze. The cells were centrifuged, resuspended in culture medium and panned in a rat IgG-coated bacteriological plastic dish to remove alveolar macrophages. The unattached cells were collected. The resultant type II cells had a purity of >90% and a viability of >95%.
Western blotting
Lung tissue, freshly isolated type II cells and lamellar bodies (20 μg protein each) were fractionated on 10% SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked with 5% fat-free milk in Tris-buffered-saline with Tween 20 (TTBS, 20 mM Tris-HCl, pH 7.6, 150 mM NaCl and 0.1% Tween 20). The membrane was then incubated with appropriate primary antibodies (β-Actin, 1:2000; Annexin A2, 1:1000; Calreticulin, 1:1000; EHD1, 1:200; GDP-1, 1 μg/ml; Vimentin, 1:500) at 4°C overnight, and then with secondary antibody (1:2500) at room temperature for 1 h. Finally, the signal was developed with ECL reagents and exposed to X-ray film.
Immunocytochemistry
Type II cells were suspended in DMEM (supplemented with 10% FBS, non-essential amino acids and antibiotics) and cultured on glass cover slips overnight. Following culture, the cells were washed three times with ice-cold 50 mM phosphate buffered saline (PBS) and fixed in 4% paraformaldehyde at room temperature for 20 min. The cells were then permeablized with 0.5% Triton X-100 for 20 min at room temperature and then incubated with 10% fetal bovine serum to prevent non-specific binding of antibodies. The cells were subsequently incubated with polyclonal rabbit anti-EHD1 or anti-CAIV at 1:50 and mouse anti-LB-180 at 1:500 overnight at 4°C. After washing 3 times with PBS for 5 min each, the cells were incubated with alexa 488-conjugated goat anti-rabbit and Cy3-conjugated goat anti-mouse antibodies at a 1:250 dilution for 1 hour at room temperature. After washing 3 times with PBS, the cover slips were mounted onto glass slides. Anti-fade solution [60% (v/v) glycerol; 1.5% (w/v) n-propyl gallate in 50 mM PBS] was added to prevent fading before sealing the cover slips. Pictures were taken with a Nikon Eclipse E600 microscope (Nikon, Lewisville, TX). Negative controls were incubated with secondary antibodies only. Additional controls were included to eliminate the possibility of cross-reactivity among the antibodies (data not shown).
Authors' contributions
PW performed proteomic analysis and Western blot and drafted the manuscript. NRC performed immunostaining. JN performed part of Western blot analysis. NRC and SA participated in 2-D gel. SH participated in MALDI-TOF MS. LL conceived of the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.
Acknowledgements
This work was supported by NIH R01 HL-052146, R01 HL071628 and R01 HL083188 (LL) and a seed grant from CVHS, OSU (WP). We thank Dr. Heidi Stricker for editorial assistance.
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20 Ways To Get More Website Traffic In 2013
Posted by tcamba under Online Marketing
From http://www.killerstartups.com 136 days ago
Made Hot by: y-dolphin on January 5, 2013 3:18 am
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New Brunswick NewspapersEdit This Page
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Canada New Brunswick Newspapers
Contents
Resources for New Brunswick Newspapers
Many early newspapers mention marriages, deaths, and a few births. J. Russell Harper's Historical Directory of New Brunswick Newspapers and Periodicals lists the different newspapers in New Brunswick and tells where they can be found.
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How to analyze negative external link?
Go4Expert Member
16Jun2010,13:34 #1
My client has asked me to analyze his site. Now, I need the tool for analysis of negative aspects of external links. He wants to know what kind of links is pointing towards his site. He also wants to know if the site has any negative links.
The problem is that I know how to look for links with Google’s back link search. Now the concern is that how do I know that these links have negative impact. The site has over five hundred external links. He wants to be warned of links from web spam sites/pages and known link brokers/sellers.
Kindly share.
Go4Expert Founder
16Jun2010,20:08 #2
Use Google Webmaster tools and see the links to the site.
Then see if the site is related and if not then it is not adding any value as link
Go4Expert Member
5Sep2010,08:01 #3
If the link is not adding any value it does not mean it is negative, just leave it and don't waste your time! Inbound links cannot be negative because google knows you cannot control inbound links, if they would be able to hurt you site, people would load link farms with their competitors!!!
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User talk:KhanWiz
From Grand Theft Wiki
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Welcome KhanWiz
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Hi Khan White, welcome to Grand Theft Wiki! I hope you like the place and decide to stay.
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-- gboyers talk 15:27, 28 October 2011 (BST)
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Bibliography: Jack of Shadows
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Title: Jack of Shadows
Author: Roger Zelazny
Year: 1971
Type: NOVEL
Select 2 publications to diff:
Copyright (c) 1995-2011 Al von Ruff.
ISFDB Engine - Version 4.00 (04/24/06)
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Nano Express
Noninjection Synthesis of CdS and Alloyed CdSxSe1−xNanocrystals Without Nucleation Initiators
Yu Zou, Dongsheng Li* and Deren Yang
Author Affiliations
State Key Laboratory of Silicon Materials and Department of Materials Science and Engineering, Zhejiang University, 310027, Hangzhou, People’s Republic of China
For all author emails, please log on.
Nanoscale Research Letters 2010, 5:966-971 doi:10.1007/s11671-010-9593-2
The electronic version of this article is the complete one and can be found online at:
Received:11 March 2010
Accepted:27 March 2010
Published:11 April 2010
© 2010 The Author(s)
Abstract
CdS and alloyed CdSxSe1−x nanocrystals were prepared by a simple noninjection method without nucleation initiators. Oleic acid (OA) was used to stabilize the growth of the CdS nanocrystals. The size of the CdS nanocrystals can be tuned by changing the OA/Cd molar ratios. On the basis of the successful synthesis of CdS nanocrystals, alloyed CdSxSe1−x nanocrystals can also be prepared by simply replacing certain amount of S precursor with equal amount of Se precursor, verified by TEM, XRD, EDX as well as UV–Vis absorption analysis. The optical properties of the alloyed CdSxSe1−x nanocrystals can be tuned by adjusting the S/Se feed molar ratios. This synthetic approach developed is highly reproducible and can be readily scaled up for potential industrial production.
Keywords:
Nanocrystals; CdS; CdSxSe1−x; Noninjection synthesis; Oleic acid
Introduction
Colloidal semiconductor nanocrystals have been intensively studied due to their unique physical properties. They exhibit quantum confinement effect such as size-dependent optical and electric properties for applications in optoelectronic devices [1-3] and biological fluorescence labeling [4,5]. CdS, with a bulk band gap of ~2.42 eV (~512 nm) and exciton Bohr radius of ~3 nm, is a candidate for quantum-dot blue light-emitting diodes [6] and can be used in photovoltaic devices [7]. In order to tune the optical properties of CdS quantum dots for specific applications, two schemes have been put forward. One is to change the size of the quantum dots, based on the quantum confinement effect. The other is to alloy CdS with other materials, such as CdSe, to form alloyed quantum dots whose band gap energy can vary with composition without changing size. In the past decades, significant advancement has been made in the synthesis of CdS [8-10] and CdSxSe1−x[11-13] quantum dots based on the classic hot-injection method developed by Bawendi’s group [14], the key part of which is the injection of room-temperature organometallic precursors into well-stirred hot organic solvents. However, these injection-based methods are not suitable for large-scale industrial preparation due to the difficulty of large mass transfer in the process of the injection.
In 2004, Cao et al. [15] reported a novel synthetic method for producing high-quality CdS quantum dots by simply heating the reaction mixture containing Cadmium myristate, Sulfur (S) and 1-octadecene without injection. They added two nucleation initiators (tetraethylthiuram and 2,2′-dithiobisbenzothiazole) into the reaction medium to increase the reactivity of S and found that the nucleation initiators were the key to successfully prepare the high-quality quantum dots. Ouyang et al. [16] extended this noninjection method to synthesize alloyed CdSxSe1−x quantum dots. Nucleation initiator 2,2′-dithiobisbenzothiazole is still applied in their work. Moreover, CdSe quantum dots have been prepared without precursor injection and nucleation initiators simultaneously by Cao’s group [17]. Oleic acid, a natural surfactant, was chosen to stabilize the growth of the quantum dots. From this viewpoint, CdS and alloyed CdSxSe1−x quantum dots may be synthesized without nucleation initiators if oleic acid is added into the reaction mixture, which could make the synthesis greener. Recently, Li et al. [18] have prepared CdS magic-sized nanocrystals using OA as stabilizer and highly reactive bis(trimethylsilyl)sulfide as S source at low temperatures (90–140°C).
Herein, we report the successful synthesis of CdS and alloyed CdSxSe1−x quantum dots without injection and nucleation initiators by simply heating the reaction medium. All the source chemicals used including cadmium acetate, sulfur, selenium, oleic acid and 1-octadecene are air-stable and inexpensive relatively. The optical properties of the obtained quantum dots can be tuned by changing the OA/Cd or the S/Se feed molar ratio. The former and latter changing of feed molar ratio results in the change of the size and the composition, respectively. This synthetic approach developed is very simple and highly reproducible and thus suitable for large-scale preparation of CdS and CdSxSe1−x nanocrystals.
Experiment
Materials
All of the chemicals are commercially available and were used as received. Cadmium acetate dihydrate (Cd(OAc)2·2H2O, 99.5%), sulfur (S, 99.5%) and Selenium (Se, 99.95%) were purchased from Shanghai Chemical Reagent Ltd.. Oleic acid (OA, tech. 90%) and 1-octadecene (ODE, tech. 90%) were purchased from Aldrich.
Synthesis
In a typical synthesis of CdS nanocrystals, a 100-ml three-neck flask containing 1 mmol Cd(OAc)2, 6 mmol OA and 15 ml ODE was heated to 120°C and was degassed under vacuum for 30 min. The flask then was filled with argon gas, and its temperature was lowed to about 30°C. A total of 0.5 mmol S was added into the flask, and then the mixture was heated to 240°C at a rate of ~20°C/min and reacted at this temperature for 60 min under a flow of argon gas. To monitor the growth of nanocrystals, small aliquots (~0.1 ml) of the reaction mixture were taken out from the flask and quenched in cold hexane (25°C) in order to stop further growth. The time was counted as 0 when the temperature reached to 240°C. At last, the quantum dots were isolated by precipitation with ethanol, then centrifuged and the resulted nanocrystals were redispersed in hexane. This process was repeated several times to wash the samples. To investigate the effect of OA, only the amount of OA was changed. For the synthesis of CdSxSe1−x nanocrystals, certain amount of S was replaced with equal amount of Se while the amount of OA was fixed at 6 mmol. The other experimental conditions were the same as those to the preparation and purification of CdS nanocrystals.
Characterization
Transmission electron microscopy (TEM) and High-resolution transmission electron microscopy (HRTEM) images were collected using a Philips CM200 transmission electron microscope operating at 160 kV. A sample for TEM analysis was prepared by drying a drop of nanocrystal hexane solution on a carbon-coated copper grid and letting it dry in air. X-ray powder diffraction (XRD) was conducted on a Rigaku D/max-ga X-ray diffractometer with graphite monochromatized Cu Kα radiation (λ = 1.5418 Å). Energy-dispersive X-ray (EDX) analysis was performed using an EDAX X-ray detector attached to a Hitachi S-4800 scanning electron microscope. Ultraviolet–Visible absorption spectra were recorded on a U-4100 UV–Vis spectrophotometer. Photoluminescence (PL) spectra were taken with a He–Cd laser as the excitation source and an excitation wavelength of 325 nm.
Results and Discussions
Figure 1 shows the temporal evolution of absorption and PL spectra of the CdS nanocrystals prepared with the OA/Cd molar ratio of 6. It can be seen that small nanocrystals did appear when the reaction temperature reached 220°C, indicated by the appearance of the absorption and PL peak. The nanocrystals grown fast, and the size distribution decreased evidently with the temperature increasing to 240°C, confirmed by the narrowing of the PL peaks. After the temperature reached 240°C, the growth was slow relatively, and no additional nucleation occurred. The narrow size distribution was maintained in the first 10 min and increased slightly after a 60-min growth. The PL spectra of the CdS quantum dots contain a higher energy and narrow peak assigned to band gap emission, and a low-energy and wide peak assigned to trap emission, which is the same as the PL of the as-prepared CdS nanocrystals obtained in the presence of nucleation initiators [15]. In general, the growth kinetics is similar to that in the presence of nucleation initiators, and the quality of the CdS nanocrystals is comparable, indicating the noninjection synthesis of CdS nanocrystals without nucleation initiators in the presence of OA is feasible undoubtedly.
Figure 1. Temporal UV–Vis absorption and PL spectra of the CdS nanocrystals prepared with the OA/Cd molar ratio of 6
Compared with the synthesis in the presence of nucleation initiators, OA must play an important role here. To investigate the effect of OA, we changed the molar ratio of OA to Cd in the reaction mixture (the amount of Cd precursors is fixed at 1 mmol). Figure 2a2c show the TEM images of the CdS nanocrystals after 60-min growth. Highly monodispersed nanocrystals with narrow size distribution are obtained for all molar ratios of OA to Cd, and these nanocrystals can readily form array even superlattice on the carbon films. The HRTEM image shown in Fig. 2d confirms the obtained nanocrystals are crystalline. The mean size of the CdS nanocrystals increases with the increase in the molar ratio of OA to Cd, which are 2.5, 3.6 and 4 nm, respectively, when the molar ratio is 2, 6 and 10. The change in size is attributed to the tunable reactivity of Cd monomers in the reaction medium [8,19]. The reactivity of Cd monomers decreases with the increasing of the ratio of OA to Cd, leading to the decrease in the number of nuclei formed in the nucleation stage and the increase in the monomer concentration remained in the reaction medium for further growth. As a result, nanocrystals with larger size are obtained when the molar ratio of OA to Cd is higher. The absorption and PL spectra of the CdS nanocrystals prepared with different OA/Cd feed molar ratios presented in Fig. 3 also confirm that the more OA added, the larger CdS nanocrystals obtained, indicated by the systematic red-shift of the spectra with the increase in the amount of OA added. The distinct absorption peaks of each spectrum suggest that the high-quality CdS nanocrystals could be obtained for all the OA/Cd molar ratios. Therefore, we can conclude that it is not the OA/Cd molar ratio but the OA itself that results in the successful synthesis of high-quality CdS nanocrystals in the absence of nucleation initiators.
Figure 2. TEM images of the CdS nanocrystals prepared at 240°C with different OA/Cd molar ratios: a 2, b 6 and c 10. d HRTEM image of the CdS nanocrystals shown in b
Figure 3. UV–Vis absorption (up) and PL spectra (down) of the CdS nanocrystals prepared at 240°C with different OA/Cd molar ratios. The amount of Cd is fixed at 1 mmol
OA has been noticed as a special ligand due to its unique configuration of hydrocarbon chains. OA is more bending than saturated fatty acid, such as myristic acid, due to the existence of a cis-double bond in the middle of the hydrocarbon chains, which makes the Cd–Oleate complex more reactive [19]. Therefore, OA should play two roles here. First, OA accelerates the nucleation just as the nucleation initiators do. In contrast to the nucleation initiators that increase the reactivity of S [15], OA increases the reactivity of Cd precursors relative to myristic acid. Secondly, OA stabilizes the further growth as myristic acid does. As a result, the presence of OA separates and balances the nucleation and growth of CdS nanocrystals, leading to the successful synthesis of high-quality CdS nanocrystals without nucleation initiators.
On the basis of the synthesis of the CdS nanocrystals, we extended this simple scheme to prepare alloyed CdSxSe1−x nanocrystals. By substituting certain amount (25, 50 and 75%) of S with equal amount of Se, we obtained CdSxSe1−x nanocrystals. EDX analysis was used to determine the composition of the obtained nanocrystals, which are, CdS0.78Se0.22, CdS0.44Se0.56 and CdS0.28Se0.72, close to the feed molar ratios. Although the EDX results are not very accurate, they confirm the increase in Se content in the nanocrystals with the increase in Se/S feed molar ratio qualitatively at least. Figure 4a4c shows the TEM images of the CdSxSe1−x nanocrystals after 60-min growth while the OA/Cd feed molar ratio is fixed at 6. Highly monodispersed nanocrystals with narrow size distribution are obtained for all the molar ratios of S to Se. The mean size of the CdSxSe1−x nanocrystals is quite similar to that of the CdS nanocrystals prepared under the same experiment conditions, i.e. 3.6 nm. Fig. 4d shows a typical HRTEM image of CdS0.44Se0.56, which indicates the obtained nanocrystals are crystalline. UV–Vis absorption spectra of the CdSxSe1−x nanocrystals are shown in Fig. 5. The spectra shift to large wavelength with the increase in Se/S molar ratio, suggesting the nanocrystals are alloyed, because the size of the nanocrystals with different Se/S molar ratio is quite close and the change of the band gap energy attributed to the quantum confinement effect should be small. The result is consistent with that from the injection approach [12] or the noninjection approach in the presence of nucleation initiators [16]. However, the optical bowing effect is not observed distinctly in this work, and the band gap energy of the CdSxSe1−x nanocrystals shifts almost linearly with composition. Compared with CdSexTe1−x and CdSxTe1−x nanocrystals [20,21], the optical bowing effect observed in CdSxSe1−x nanocrystals is minor [12]. The bowing constant of CdSxSe1−x nanocrystals is only 0.3, while that of CdSexTe1−x and CdSxTe1−x nanocrystals is 1.19 and 3.17, respectively. The error of the composition of CdSxSe1−x nanocrystals obtained by EDX analysis may also influence the observation of the bowing effect. Regardless of the optical bowing effect, the results confirm that the optical properties of the nanocrystals can be tuned by adjusting the composition without changing the size.
Figure 4. TEM images of the CdSSe nanocrystals prepared at 240°C with different Se/S feed molar ratios: a 1/3, b 1/1 and c 3/1. The total amount of Se and S is fixed at 0.5 mmol. d HRTEM image of the CdSSe nanocrystals shown in b
Figure 5. UV–Vis absorption spectra of the CdSSe nanocrystals prepared at 240°C with different Se/S feed molar ratios
The XRD patterns of the CdS and CdSxSe1−x nanocrystals as shown in Fig. 6 confirm a cubic structure (zinc-blende) throughout the range of compositions, as indicated by the absence of the characteristic diffraction peaks of (102) and (103) planes of the hexagonal structure. Moreover, the spectra shift to smaller 2θ with the increase in Se/S molar ratio, indicating a linear increase in lattice spacing due to the increase in Se content in the nanocrystals, which is another proof of formation of alloy nanocrystals [12,16].
Figure 6. XRD patterns of CdS nanocrystals and CdSSe nanocrystals prepared at 240°C with different Se/S feed molar ratios. The spectra characterize a cubic structure and shift to small 2θ with the increasing of Se/S feed molar ratios
Conclusions
We report the successful synthesis of CdS and CdSSe nanocrystals without injection and nucleation initiators. Highly monodispersed nanocrystals with narrow size distribution are obtained by simply heating a mixture containing Cd(OAc)2, S, Se, OA and ODE. This method is very economical and suitable for industrial production. OA plays an important role in the reaction, which increases the reactivity of Cd precursors and stabilizes the growth of nanocrystals. The effects of the OA/Cd or Se/S feed molar ratios on the optical properties of the nanocrystals have been studied. High OA/Cd molar ratios and high Se/S feed molar ratios lead to nanocrystals with small band gap.
Acknowledgments
The authors express their appreciations to the National Basic Research Program of China (973 Program) (Grant No.2007CB613403), 2008DFR50250 of MOST and PCSIRT project for the financial support.
Open Access
This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
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14. Murray CB, Norris DJ, Bawendi MG:
J. Am. Chem. Soc.. 1993, 115:8706.
COI number [1:CAS:528:DyaK3sXlsV2ltL8%3D]
Publisher Full Text
15. Cao YC, Wang JH:
J. Am. Chem. Soc.. 2004, 126:14336.
COI number [1:CAS:528:DC%2BD2cXos1Wjs7s%3D]
PubMed Abstract | Publisher Full Text
16. Ouyang JY, Vincent M, Kingston D, Descours P, Boivineau T, Zaman MdB, Wu XH, Yu K:
J. Phys. Chem. C. 2009, 113:5193.
COI number [1:CAS:528:DC%2BD1MXisFamtLc%3D]
Publisher Full Text
17. Yang YA, Wu HM, Williams KR, Cao YC:
Angew. Chem. Int. Ed.. 2005, 44:6712.
COI number [1:CAS:528:DC%2BD2MXht1SnsLfO]
Publisher Full Text
18. Li MJ, Ouyang JY, Ratcliffe CI, Pietri L, Wu XH, Leek DM, Moudrakovski I, Lin Q, Yang B, Yu K:
ACS Nano. 2009, 12:3832. Publisher Full Text
19. Ouyang JY, Kuijper J, Brot S, Kingston D, Wu XH, Leek DM, Hu MZ, Ripmeester JA, Yu K:
J. Phys. Chem. C. 2009, 113:7579.
COI number [1:CAS:528:DC%2BD1MXktlSlsLg%3D]
Publisher Full Text
20. Bailey RE, Nie SM:
J. Am. Chem. Soc.. 2003, 125:7100.
COI number [1:CAS:528:DC%2BD3sXjsleit7c%3D]
PubMed Abstract | Publisher Full Text
21. Gurusinghe NP, Hewa-Kasakarage NN, Zamkov M:
J. Phys. Chem. C. 2008, 112:12795.
COI number [1:CAS:528:DC%2BD1cXptVCqu7c%3D]
Publisher Full Text
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Inactive
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Isolation of murine splenocytes
From OpenWetWare
Revision as of 14:39, 9 May 2007 by Reshma P. Shetty (Talk | contribs)
Jump to: navigation, search
Contents
Overview
In order to study spleen cells (e.g. lymphocytes, granulocytes, other immune cells), it helps to make single-cell suspensions so that the cells can be manipulated ex vivo easily. This protocol suggests ways in which you can do this without a lot of equipment or expensive supplies. This protocol can also be used to make cell suspensions from other lymphoid organs, such as the thymus or lymph nodes (see Current Protocols in Immunology, Unit 1.9 [1]).
Materials
All materials listed are for use with one mouse.
Supplies
• 15ml conical tube
• 60mm petri dish
• 5ml pipet
• 100μm cell strainer (can substitute autoclaved fine nylon mesh for Protocol B)
• 3mL sterile disposable syringe, no needle attached (Protocol A only)
• frosted-end glass slides (x 2) (Protocol B only)
• 50ml conical tube (Protocol B only)
Reagents
• DMEM-10 (about 20mL)
• 1L DMEM (with 4.5g/L glucose, L-glutamine, sodium pyruvate; from Mediatech, catalog# 10-013-CM)
• 100mL fetal bovine/calf serum
• 10mL 100X PSG (penicillin G sodium, streptomycin sulfate, L-glutamine; from Gibco, catalog# 10378-016)
• sterilize using 0.2μm filter; store at 4°
• ACK lysis buffer (1mL)
• 1L deionized water
• 8.29g NH4Cl
• 1g KHCO3
• 37.2mg Na2-EDTA
• pH solution to 7.2-7.4; sterilize using 0.2μm filter; store at 4°
• 70% ethanol
• trypan blue solution
Equipment
• scissors
• forceps
• small plastic or glass beaker
• dissection stage (can be styrofoam shipping box lid wrapped in aluminum foil)
• P1000 pipette
• hemacytometer
• phase microscope
• centrifuge
Procedure
Protocol A
Set-Up
1. Clean dissection stage with 70% ethanol.
2. Add ethanol to the beaker and place ends of scissors and forceps into the beaker to sterilize.
3. Add 8-10mL of DMEM-10 to the petri dish.
4. Place the cell strainer into the dish with the DMEM-10.
Procedure
1. Wet fur on left side of sacrificed mouse using 70% ethanol.
2. Cut out the spleen.
1. Cut away the fur along the left side of the mouse, about half-way between the front and back legs.
2. Cut open the body cavity.
3. Remove the spleen using the forceps (the spleen is the color of a kidney bean; it is longer and flatter than the kidney).
3. Place the spleen into the cell strainer. Using the plunger end of the syringe, mash the spleen through the cell strainer into the petri dish.
4. Rinse the cell strainer with 5mL DMEM-10. Discard the strainer.
5. Transfer the suspended cells to a 15mL conical.
6. Spin cells at 800xg for 3 minutes.
7. Discard supernatant and resuspend pellet in 1mL ACK lysis buffer. Incubate at RT for 5-10 minutes. Add 9mL DMEM-10 and spin as before.
8. Discard supernatant and resuspend pellet in 3mL DMEM-10, discarding any dead cell mass.
9. Count cells (dilute 10μL cell suspension in trypan blue, and count with hemcytometer).
Protocol B
Set-Up
• same as Procedure B, except replace step 4 with:
1. Sterilize the frosted end of the glass slides by dipping in or spraying with ethanol. Take care to only touch the non-frosted ends with your gloves.
Procedure
• same as Procedure B, except replace steps 3 with:
1. Place the spleen directly into the DMEM in the petri dish.
2. Homogenize the spleen between the frosted ends of the slides.
3. Pass the homogenized spleen through the cell strainer (or nylon mesh) mounted on a 50mL conical.
4. Continue with step 4 of Procedure A.
Notes
• Keep cells on ice or at 4° if you do not plan to use them right away.
• If sterility is desired, perform all steps in a laminar flow culture hood.
References
1. Current Protocols in Immunology, Unit 1.9: Removal of Lymphoid Organs link (subscription required) [CP1]
2. Current Protocols in Immunology, Unit 3.1: Isolation of Mouse Mononuclear Cells link (subscription required)
[CP3]
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OSDDMalaria:GSK Arylpyrrole Series
From OpenWetWare
Revision as of 19:38, 4 April 2012 by Paul M. Ylioja (Talk | contribs)
Jump to: navigation, search
OSDD Home Malaria Home The Story So Far GSK Arylpyrrole Series GSK Triazolourea Singleton GSK Amino-thienopyrimidine Series Other GSK Series Under Consideration Links
Contents
The Arylpyrrole Series
These compounds are currently under study by the Todd lab at the University of Sydney and the Medicines for Malaria Venture, Geneva. As an open source project, anyone may participate:
Coordination/Discussion site
Electronic Lab Notebooks
Undergraduate Project Report
The initial two subseries of leads have the general structure:
• TCMDC Aryl pyrrole core InChi Key: LNROIXNEIZSESG-UHFFFAOYSA-N
• Near-Neighbour core InChi Key: JZQOEGKHCXONAV-ZROIWOOFSA-N
GSK Tres Cantos Arylpyrrole Generic Structure
Near Neighbour Generic Structure
A number of these compounds and intermediates are available for biological testing from Todd group by request.
The two known TCMDC series starting points
Compounds are commercially-available
Series was listed as one of the most promising leads from TCAMS set (though lacking aryl F)
TCMDC 123812
TCMDC123812
InChI=1/C15H15FN2O3/c1-9-7-13(15(20)21-8-14(17)19)10(2)18(9)12-5-3-11(16)4-6-12/h3-7H,8H2,1-2H3,(H2,17,19)
SMILES: O=C(N)COC(=O)c2c(n(c1ccc(F)cc1)c(c2)C)C
CAS Registry Number: 733026-12-5
Chemspider page
ChEMBL Page
Resynthesis:
TCMDC 123794
TCMDC123794
InChI=1/C26H25FN4O4/c1-16-14-22(17(2)30(16)20-12-10-19(27)11-13-20)26(34)35-15-23(32)28-24-18(3)29(4)31(25(24)33)21-8-6-5-7-9-21/h5-14H,15H2,1-4H3,(H,28,32)
SMILES: Fc1ccc(cc1)n2c(cc(c2C)C(=O)OCC(=O)NC=4C(=O)N(c3ccccc3)N(C=4C)C)C
Chemspider page
ChEMBL Page
Resynthesis:
The proposed resynthesis strategy for these two compounds:
Proposed Synthesis Strategy
The Near-Neighbour Sub-series
Known "Near Neighbours" contained in the Tres Cantos set
The original idea for investigation of the near-neighbour set came from Paul Willis and was posted on the synaptic leap.
Known GSK Arylpyrrole Near Neighbours
Data/links for these compounds:
TCMDC-123563, CHEMBL546966, CHEMBL page: 637010 Cc1ccc(cc1)n2c(cc(c2C)C(=O)CN3C(=O)C(NC3=O)Cc4ccccc4)C
TCMDC-125698, CHEMBL587989, CHEMBL: 627784 Cc1cc(c(n1c2ccc(cc2)Cl)C)C=C3C(=O)N(C(=Nc4ccccc4)S3)C5CCCC5
TCMDC-125697, CHEMBL581336, CHEMBL: 640978 CCOC(=O)c1ccc(cc1)n2c(cc(c2C)C=C3C(=O)N(C(=Nc4ccccc4)S3)C5CCCC5)C
TCMDC-125659, CHEMBL528140, CHEMBL: 626220 Cc1ccnc(c1)n2c(cc(c2C)C=C3C(=O)N(C(=Nc4ccccc4)S3)Cc5ccco5)C
TCMDC-124103, CHEMBL588465, CHEMBL: 643107 Cc1cc(cc(c1)n2c(cc(c2C)C=C3C(=O)NC(=Nc4ccc(cc4)Cl)S3)C)C
TCMDC-124456, CHEMBL548395, CHEMBL: 640006 CCn1c(cc(c1C)C=C2C(=O)NC(=Nc3ccccc3)S2)C
Initial synthesis strategy toward near-neighbours
Initial synthesis of near neighbours
Experimental information available from PMY 13-1, PMY 14-1 and PMY 16-1, PMY 14-3.
Current synthesis strategy toward near-neighbours
Current synthesis of near neighbours
Synthesis method taken from this paper.
Alternative side-chains
Some alternative heterocyclic side-chain ideas have been proposed here at the Synaptic Leap.
Oxazoles
The [1] side chain would target ester isosteres (see TCMDC-123812) but with greater biological stability.
Other known incidences of these molecules/this series
Related compounds are known to inhibit the proteasome, according to this Nature paper.
According to this paper, similar compounds are agonists of the sphingosine-1-phosphate receptor subtypes 1-5, which have a role in immune system function.
A new class of antimalarial was recently published by Novartis, Imidazolopiperazines.
Misc papers to digest/assimilate regarding antimycobacterial/tuberculosis activity: Antimycobacterial 1,5-diphenyl pyrroles, 10.1016/j.ejmech.2009.06.005, 10.1016/j.bmc.2010.09.006, 10.1002/cmdc.201000526
Scifinder Search: 2,4,5-alkyl-1-aryl-pyrrole: 77552 hits, 905 biological studies:
WO 2011127333 (A2)
CXCR4 active compound
US 2011251184 (A1)
HDAC6 inhibitor
Obesity/diabetes GIP receptor inhibitor JP 2011184298 (A)
GIP inhibitor
Proteasome WO 2011094545 (A2)
Proteasome agonist
WO2006076202 Steroid nuclear receptor ligands
WO 2011075684 (A1) Inhibitors of Plasma Kallikrein/Protease
WO 2009137133 (A2) 5-Substituted-2-Imino-Thiazolidinone Compounds And Their Use As Inhibitors Of Bacterial Infection
Bacterial inhibitor
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Drummond:Akashi's Test
From OpenWetWare
Jump to: navigation, search
the drummond lab
home people research publications news jobs protocols contact
Contents
Introduction
In 1994, Hiroshi Akashi developed an elegant test for translational accuracy selection on coding sequences [1]. Some codons, particularly those corresponding to abundant tRNAs, are translated more accurately than others. Under selection for translational accuracy, usage of those more-accurate synonymous codons will be favored at important (e.g., evolutionarily conserved) amino-acid sites, where translation errors could disrupt protein folding or function. At less-important (e.g., evolutionarily variable) amino-acid sites, errors are presumably more tolerable, and therefore more-accurate codons are less likely to be favored. Akashi's test asks:
1. How strong is the association between preferred codons and conserved amino acids, controlling for differences between amino acids and between genes?
2. How likely is that association to have occurred by chance?
Akashi's test on a single gene
Akashi's test is technically very simple to carry out. The hard part is just tabulating data. Suppose you have two aligned codon sequences (a target sequence and an orthologous sequence) and a list of preferred codons. From the aligned codon sequences, build a 2x2 contingency table with entries a, b, c, and d like this:
AA=SerConservedVariable
Preferredab
Unpreferredcd
for each amino acid. You'll usually have 18 tables; W and M have no synonymous codon alternatives and therefore don't contribute to Akashi's test.
• a = the number of codons in your target sequence that encode amino acid AA, are PREFERRED, and encode an AA which is unchanged (CONSERVED) in the orthologous sequence
• b = the number of codons in your target sequence that encode amino acid AA, are PREFERRED and encode an AAwhich is different (VARIABLE) in the orthologous sequence
• c = the number of codons in your target sequence that encode amino acid AA, are UNPREFERRED and encode an AA which is unchanged (CONSERVED) in the orthologous sequence
• d = the number of codons in your target sequence that encode amino acid AA, are UNPREFERRED and encode an AA which is different (VARIABLE) in the orthologous sequence
Now the statistics. Assuming no association -- that is, assuming that the probability of a codon being preferred (which we designate p) is independent of the probability that it encodes a conserved amino acid (which we designate q) -- we can write down estimates for the expected value and variance of a, E(a) and V(a):
With the mean and variance, we can write down a Z-score for one table:
And because a Z-score only gives us a measure of statistical significance (question #2 above), we also want an effect size -- the magnitude of the association between preferred codons and conserved sites -- which we can compute as an odds ratio, the odds of finding a preferred/conserved association divided by the odds of finding a nonpreferred/variable association. The odds ratio answers question #1 above. An unbiased estimate of the odds ratio ψ is given by:
.
Akashi's test on multiple genes
Estimating Z and ψ for a single amino acid in a single gene is perhaps of limited interest. How do we combine tables so that we can ask questions like, "What is the overall association between preferred codons and conserved sites across the genome?" or, "How statistically significant is the preferred/conserved association for alanine compared to glycine?"
To combine tables, we use the Mantel-Haenszel procedure. The basic principle is that tables are independent. Expectations add, variances add, and observed values add. That is, indexing tables by i, with table i given by
AA=XConservedVariable
Preferredaibi
Unpreferredcidi
and and computed as before for each table, we have the combined Z-score
and the Mantel-Haenszel estimator for the common odds ratio (i.e., the single odds ratio ψ assumed to underlie all tables being analyzed),
With enough tables, we assume that follows the standard normal distribution Φ, so that a P-value can be computed as .
Akashi's test equates statistical significance of a preferred-codon/conserved-site association with the influence of translational accuracy selection. One simply asks how statistically significant is.
Implementation
The open-source statistical package R includes an implementation of the Mantel-Haenszel test (mantelhaen.test), which is sufficient to carry out Akashi's test.
Examples
Coming...
References
1. Akashi H. . pmid:8005445. PubMed HubMed [Akashi-Genetics-1994]
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Google Forcing AdWords Advertisers To Set Up SSL & Privacy/Opt Out Policies
May 5, 2011 • 8:42 am | (1) by | Filed Under Google AdWords
Google announced they will begin requiring many of their advertisers to ensure their web site practices and technology is in accordance with rules to make the AdWords marketplace "safe, fair, and trusted marketplace" for their searches.
The new rules will require a (1) valid SSL connection when collecting payment and certain financial and personal information, (2) a method to to discontinue direct communications (opt out) when collecting information that can be used for communication and (3) a policy that is available to their web site users for disclosure before visitors submit personal information (privacy policy).
These three new requirements go into effect May 17, 2011.
For more information on the privacy policy and disclosure information, see this help document and for more on the SSL requirements, see this help document.
A WebmasterWorld thread is very skeptical that these new policies will prevent shady advertisers from advertising on Google and scamming Google's searchers and users. One advertiser said:
Either way, requiring SSL means nothing at this point, SSL certificates can be acquired for peanuts or even free. They should have been more specific about the type of the SSL certificate.
Forum discussion at WebmasterWorld.
Previous story: Google's Panda Update Hopes To Curate An Apple-Like Web
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Australian Bureau of Statistics
Celebrating the International Year of Statistics 2013
ABS Home > Statistics > By Catalogue Number
4704.0 - The Health and Welfare of Australia's Aboriginal and Torres Strait Islander Peoples, Oct 2010
Latest ISSUE Released at 11:30 AM (CANBERRA TIME) 29/10/2010 Final
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Publications
4704.0 - Indigenous Regions 2006
Data Cubes
Demographic, social and economic characteristics
Education
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Disability Released 17/02/2011
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Unless otherwise noted, content on this website is licensed under a Creative Commons Attribution 2.5 Australia Licence together with any terms, conditions and exclusions as set out in the website Copyright notice. For permission to do anything beyond the scope of this licence and copyright terms contact us.
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Australian Bureau of Statistics
Celebrating the International Year of Statistics 2013
ABS Home > Statistics > By Catalogue Number
5228.0 - Australian National Accounts: Quarterly Data on Floppy Disk, Dec 1996
Latest ISSUE Released at 11:30 AM (CANBERRA TIME) 05/03/1997 Ceased
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• About this Release
ABOUT THIS RELEASE
Floppy disk(s) containing the full-time series of quarterly national accounts data published in 5206.0 in spreadsheet format. Most series are provided quarterly from the September quarter 1959.
Note: This data continues to be available on a consultancy basis.
© Commonwealth of Australia 2013
Unless otherwise noted, content on this website is licensed under a Creative Commons Attribution 2.5 Australia Licence together with any terms, conditions and exclusions as set out in the website Copyright notice. For permission to do anything beyond the scope of this licence and copyright terms contact us.
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Source link: http://archive.mises.org/10740/we-call-upon-the-hedge-funds/
We Call Upon the Hedge Funds
September 30, 2009 by
You, within the hedge-fund community, are amongst the few in American society who remain relatively free from the shackles of government. This is your gift. We are not asking you to share your earnings, but merely to use your talent, capital, and influence in order to protect your own industry and help strive towards a more free-market, capitalist system. FULL ARTICLE by Dan O’Connor
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Ask Your Question
2
Remove "alright" from standard.dic?
asked 2012-05-22 10:04:22 +0200
housiemousie2
53 1 4 6
updated 2012-05-22 15:23:55 +0200
cloph
2792 4 17 43
The word "alright" is not all right, in point of fact, is it all wrong. It is not a real word, but yet the standard English dictionary used to check spelling passes over it without highlighting it as a misspelled word.
I know how to remove entries I have added to the standard and custom dictionaries, but not how to edit the entries the dictionary came with.
Anyone know how to do this?
Libra Office 3.5.3.2 Ubuntu 12.04
delete close flag offensive retag edit
1 Answer
Sort by » oldest newest most voted
3
answered 2012-05-22 10:44:56 +0200
manj_k
5622 4 31 48
updated 2012-05-22 10:53:51 +0200
Create a user-defined exception dictionary.
See also:
(1) → http://ask.libreoffice.org/question/1699/e-possivel-editar-o-db-do-corretor-ortografico-can?answer=1705#answer-container-1705
(2) LibreOffice Help (F1) → Index: exceptions;user-defined dictionaries
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Asked: 2012-05-22 10:04:22 +0200
Seen: 281 times
Last updated: May 22 '12
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You are here: Home » Content
The content in Connexions comes in two formats: modules, which are like small "knowledge chunks," and collections, groups of modules structured into books or course notes, or for other uses. Our open license allows for free use and reuse of all our content.
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Saturday, March 25, 2006
Searching Questions
As I have mentioned before, the SiteMeter reports for my blog include information about internet searches that led people here. Sometimes these tend towards the silly, and believe me, I have some more of those coming up soon. But more often than not, the searches reflect information that people want to find. So in this post I will attempt to answer some of them.
How did wood warblers get their name?
This question is difficult to answer, because I am not sure if this person wanted information about the name of the entire family, or names of individual species. I will assume the former, since the latter would take too long to answer. "Warbler" is used to refer to two types of birds, Old World Warblers (Family Sylviidae) and New World, or Wood, Warblers (Family Parulidae). Very few Old World Warblers breed in the Americas; the only representative is the Arctic Warbler. Wood Warblers, on the other hand, are widespread throughout North and South America. There are 116 species overall, with 53 occurring in North America. Wood warblers do not really warble, which means singing with clear trills and sustained notes; their calls tend to be more buzzy and repetitive. The name "warbler" was most likely attached to the American species due to superficial resemblance to their Old World cousins, and "wood" is attached as a modifier to distinguish the two distinct families.
What types of animals are in Jamaica Bay?
There are, in fact, many types of animals in Jamaica Bay, New York. One place to start would be the Jamaica Bay Unit of the Gateway National Recreation Area. There you can find a list of the bird species recorded in Jamaica Bay (link opens pdf). That does not answer for all animals, but it is a start.
How dangerous is a hurricane?
It depends on the strength of the storm. See the NOAA website for an explanation of the Saffir-Simpson Hurricane Scale and what sort of damage can be expected. Hurricane Katrina (pdf) was a Category 3 storm when it made landfall on the Louisiana-Mississippi border in 2005.
Does wind effect woodcock display?
The USFWS seems to think that wind may suppress woodcock activity, since it schedules woodcock counts on days without wind or other adverse weather conditions.
Why do my pigeons lose their feathers?
All birds molt every feather at least once a year. Feathers wear down from exposure to the weather and use in flight. Molting keeps them fresh and suitable for flight and insulation. Some captive birds engage in feather-plucking, in which the bird deliberately chews or plucks its feathers, much like we bite our fingernails. Sometimes this is the result of disease and other times it may be the result of boredom or frustration.
When do Washington, D.C., robins migrate?
Many robins have become nonmigratory in recent years due to two reasons. One is that winters have become warmer, making it easier to find worms and other invertebrates for food. Robins have also benefited from the surge in backyard bird feeding, which provides an alternate source of food during subfreezing snaps. There are, however, migratory movements of robins. Northward migration in this area starts in March, and southward migration peaks in September and October. For more information, see bootstrap analysis and the DC Audubon webpage.
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Error!
Success!
WSE Settings 3.0 tool
0
kicks
WSE Settings 3.0 tool (Unpublished)
The WSE features can be enabled by using WSE 3.0 tool which is a graphical user interface in visual studio 2005. You have to download the WSE 3.0 for .NET to configure the WSE 3.0 tool. To open the WSE Settings 3.0 tool from Visual Studio 2005. 1. Open the Solution or project that you want to use the WSE with.
Kicked By:
Drop Kicked By:
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Wikia
SRD:Antimagic Armor
Talk0
9,503pages on
this wiki
Revision as of 01:58, April 23, 2009 by Dmilewski (Talk)
(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)
This material is published under the OGL
Antimagic Armor
This +1 negating full plate armor of invulnerability is crafted of adamantine (and thus has damage reduction 3/–). The armor provides a –5 penalty on dispel checks made against it or its wearer.
Caster Level: 21st; Prerequisites: Craft Magic Arms and Armor, Craft Epic Magic Arms and Armor, greater dispel magic, stoneskin, wish or miracle; Market Price: 871,500 gp; Cost to Create: 436,500 gp + 18,700 XP.
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RPi Noob Guide for Wheezy and vsftpd
From eLinux.org
Revision as of 08:31, 6 August 2012 by JimJKla (Talk | contribs)
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Page in Development
Contents
FTP File Transfer Protocol
During this bit of noob enlightenment you will be hacking around at the innards of Wheezy always remember you can always re-image your SD card and re-start.
And the Win32Image software will allow you to make a backup of your SD card at any stage .
In setting up ftp you make your RPi open to external change but until you set up your router to port forward (something your not doing here) it’s only visible to machines connected to your router.
You should have your router secured with a good password for administration and for wi-fi if it has it.
As this is noob territory I should start with a basic explanation of ftp.
ftp is a way by which you can copy files from one machine to another using the internet or a local switch/router.
Most browsers have some ftp download ability built in, but it’s going to be useful to use one of the free tools available.
I use “core ftp le” as this version of core is free for the home user and will probably be all you will ever need.
Core FTP Lite
and “filezilla” which comes from the same family as firefox.
Filezilla
I find core easier.
There are others like ”cute ftp” which is a 30 day trial I think it goes to annoy ware after that, but it’s good fairly cheap well supported and stable
Cute FTP
In reality it does not mater which you choose.
ftp connects using port 21 something you don’t need to worry about for now it’s just something you may need if you decide to make your RPi visible across the internet.
Sot hat is your client end of the deal but to play you need to set up the server end and for that you need the ftp server software.
Debian Linux and Fedora and their derivatives (Ubuntu, Raspibian, Wheezy etc.) come with a package available via the apt-get route.
The package is vsftpd (Very Secure FTP Daemon) to load it into your Wheezy image you are going to need to run some command line stuff If you are running on a TV using HDMI it may be worth changing the default font or at least till you get familiar with what is going on or run Blind Login both of these set up’s have instructions for the noob on the wiki and will make your life easier and eyesight last longer.
Note neither are essential they just may make your life easier.
OK It looks like neither the Beta or the latest version of Wheezy that is 2012-06-18-wheezy-beta or 2012-07-15-wheezy-raspbian have vsftpd pre installed.
Either way they don’t have the config file in place so the apt-get install will sort that for you.
Editing the vsftpd.conf
Heads up either at the start of the session type
sudo su
run as super user or type sudo before every line it is your choice. When you come to reboot this revert’s to a normal login so “sudo su” can save you typing
I recommend you run
apt-get update
This just insures you are running the latest list of installs for the package you are using.
apt-get install vsftpd
You may get asked about using the extra space it is sort of pointless to say no.
I have to assume you have at least read the stuff with the Blind Login because that opens with a section that tells you about your RPi’s IP address and your going to need that.
reboot
at this point you just need your RPi running you don’t need to login the ftp does its own login.
If you fire up core now you will get a connection using the ip address of your RPi and user of anonymous but you will not be able to do much because the default settings of vsftpd are fairly restrictive.
But in the Local window you have a tree of your PC and in the remote window you should have a folder icon followed by two dots.
This indicates you have a connection but if you try to do anything useful it will probably fail.
OK you need to get yourself to a position where you are logged into your RPi either directly on a TV using Composite or HDMI or remotely using Putty or the like and an ssh connection.
Either way you should be able to see the RPi Wheezy command line prompt
pi@raspberrypi ~ $
enter sudo su
sudo su
the prompt should change to technically this is the bash shell but do not worry about this here
root@raspberrypi:/home/pi#
This indicates you are logged in as root with all that this implies and your working directory is
/home/pi
If you were on a windows PC that would be like C:\home\pi
Note the slashes point the opposite way in Linux (Unix etc) the leading slash says root
If you are planning on playing in the deep endof command line Linux it may be worth picking up the Linux Pocket Guide published by O’Reilly it’s not always the easiest to understand but it’s cheap and it will tell you what a command does.
There’s plenty of online help but I find you can all to easily be diverted it won’t do you any harm to get familiar with the following
cd = change directory
pwd = print working directory
ls = list files
the dos equivalents are
cd = cd
pwd = there is no equivalent you are expected either know or read the prompt.
ls = dir
It’s probably a good time to have an explore of the file space if you have run the sudo su then you are logged as root the super user and typing cd on its own will change your working directory to root.
cd/ will take you to the root of the RPi drive and an ls at this point will show you the root structure and it’s worth knowing that the whole of the structure of the operating system is visible.
Everything (And I mean Everything) in Unix/Linux is a file so the hardware is here as well as the software so do not go deleting stuff unless you want to cultivate familiarity with the re-image process :D
However the back side to this warning is that anything you do can be undone by re-imaging your SD card.
So we need to navigate to the etc folder and to edit the configuration file for vsftpd you can edit without the navigation but it will be worth your while as a noob to understand what is going on.
So
cd /etc
ls
note there is a space between the cd and the /etc so you are looking at the long list of stuff in the etc folder you are going to edit one of the core files of Wheezy that is vsftpd.conf.
Now I have a preference for nano as an editor of choice there are others feel free to explore note pico is the same as nano and if I explain using nano at least for now you will know what is happening so
nano vsftpd.conf
Now the vsftpd.conf file looks daunting but the commenting is excellent any line that starts with the hash # is a comment and is ignored.
To activate the line remove the hash.
Example of making changes in vsftpd.conf
Lets look at one small section so you can follow this. Find the following part
#
# Run standalone with IPv6
# Like the listen parameter, except vsftpd will listen on a IPv6 socket
# instead of an IPv4 one. This parameter and the listen parameter are mutually
# exclusive
# listen_ipv6=YES
#
For the time being you don’t want to worry about what IPv6 (Internet Protocol version 6) is for now nuff said it’s the future of IP and for now it is not that widely supported.
Ok hash (#) on it’s own is a blank line (white space to make the file easier to read).
The next three lines tell you what the command does.
It also tells you that this command and the listen command (it is earlier in the file) are “mutually exclusive”
This means you can either listen (listen happens to imply listen using IPv4) or listen_ipv6 but not both so if you remove the hash here you need to put on onto the start of the listen=YES line.
By default IPv4 uses port 21 for ftp transfers IPv6 doesn’t for now that’s probably enough about that
So if you want to BREAK vsftpd so it does not work then remove the hash from the
#listen_ipv6=Yes
line but the suggestion is leave it alone this is just an example.
Note these two following links are not uploaded yet...
Here is a link to my un altered original vsftpd.conf
Here is a link to my modified one
What follows is some detail of the changes.
The first change I would suggest you do is enable (remove the hash and the space from the start of) the line write_enable= YES so that the line starts hard left (note no leading spaces)
So that it looks like
write _enable= YES
now change the line #anon_upload_enable=YES by removing its hash and change the Yes to a NO
so it reads
anon_upload_enable=NO
Here are the remaining changes
local_enable=YES
local_unmask=022
anon_upload_enable
ascii_upload_enable=YES
ascii_download_enable=YES
How to exit the editor saving the changes
Ok we need to write these changes to the saved file so [Ctrl][x] (that’s if you are using nano or pico)
Notice the highlighted line at the bottom of the page.
You need to hit the “y “ key just to get to the next stage.
Enter accepts the given name and drops you back to the prompt now you need to reboot so type reboot and wait till your RPi has restarted.
Run yor FTP Client Example here uses Core FTP Lite
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"url": "ficly.com/tags/nentar",
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Stories tagged “nentar”
• Nentar, Quadblade
A golden day, afternoon bringing a bit of light to what looks like a field of pure gold. In a path through this gold, a single man walks through. His hands are at his side, his gait is a relaxed one. He looks somewhat fatigued, yet he looks pretty ener...
• Author: Clotifoth
• Posted over 3 years ago.
• 5 out of 5
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"provenance": "cccc-CC-MAIN-2013-20-0000.json.gz:57014",
"uncompressed_offset": 95787014,
"url": "genomebiology.com/2001/2/10/research/0043/abstract",
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"warc_filename": "<urn:uuid:9346a902-b0f9-4e38-b652-c189ce0ff1e8>",
"warc_url": "http://genomebiology.com/2001/2/10/research/0043/abstract"
}
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Research
Characterization of alternatively spliced products and tissue-specific isoforms of USP28 and USP25
Rebeca Valero1, Mònica Bayés2, M Francisca Sánchez-Font1, Olga González-Angulo1, Roser Gonzàlez-Duarte1 and Gemma Marfany1*
Author Affiliations
1 Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, Av. Diagonal 645, 08028 Barcelona, Spain
2 Unitat de Genètica, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, Dr Aiguader 80, 08003 Barcelona, Spain
For all author emails, please log on.
Genome Biology 2001, 2:research0043-research0043.10 doi:10.1186/gb-2001-2-10-research0043
Published: 13 September 2001
Abstract
Background
The ubiquitin-dependent protein degradation pathway is essential for the proteolysis of intracellular proteins and peptides. Deubiquitinating enzymes constitute a complex protein family involved in a multitude of cellular processes. The ubiquitin-specific proteases (UBP) are a group of enzymes whose predicted function is to reverse the ubiquitinating reaction by removing ubiquitin from a large variety of substrates. We have lately reported the characterization of human USP25, a specific-ubiquitin protease gene at 21q11.2, with a specific pattern of expression in murine fetal brains and adult testis.
Results
Database homology searches at the DNA and protein levels and cDNA library screenings led to the identification of a new UBP member in the human genome, named USP28, at 11q23. This novel gene showed preferential expression in heart and muscle. Moreover, cDNA, expressed sequence tag and RT-PCR analyses provided evidence for alternatively spliced products and tissue-specific isoforms. Concerning function, USP25 overexpression in Down syndrome fetal brains was shown by real-time PCR.
Conclusions
On the basis of the genomic and protein sequence as well as the functional data, USP28 and USP25 establish a new subfamily of deubiquitinating enzymes. Both genes have alternatively spliced exons that could generate protein isoforms with distinct tissue-specific activity. The overexpression of USP25 in Down syndrome fetal brains supports the gene-dosage effects suggested for other UBP members related to aneuploidy syndromes.
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"provenance": "cccc-CC-MAIN-2013-20-0000.json.gz:57080",
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It's easy! Just pick the product you like and click-through to buy it from trusted partners of Quotations Book. We hope you like these personalized gifts as much as we do.
Make and then buy your OWN fantastic personalized gift from this quote
With a new familiarity and a flesh-creeping homeliness entirely of this unreal, materialistic world, where all sentiment is coarsely manufactured and advertised in colossal sickly captions, disguised for the sweet tooth of a monstrous baby called the Public, the family as it is, broken up on all hands by the agency of feminist and economic propaganda, reconstitutes itself in the image of the state. Lewis, Wyndham
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212 - The Extra Degree
The one extra degree makes the difference. This simple analogy reflects the ultimate definition of excellence. Because it's the one extra degree of effort, in business and life, that can separate the good from the great. This powerful book by S.L. Parker and Mac Anderson gives great examples, great quotes and great stories to illustrate the 212° concept. A warning - once you read it, it will be hard to forget. Your company will have a target for everything you do ... 212°
Click here to buy this »
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{
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"provenance": "cccc-CC-MAIN-2013-20-0000.json.gz:57081",
"uncompressed_offset": 182684278,
"url": "quotationsbook.com/quote/gift/3306/",
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"warc_filename": "<urn:uuid:9346a902-b0f9-4e38-b652-c189ce0ff1e8>",
"warc_url": "http://quotationsbook.com/quote/gift/3306/"
}
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It's easy! Just pick the product you like and click-through to buy it from trusted partners of Quotations Book. We hope you like these personalized gifts as much as we do.
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Ask with urgency and passion. Balfour, Arthur James
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212 - The Extra Degree
The one extra degree makes the difference. This simple analogy reflects the ultimate definition of excellence. Because it's the one extra degree of effort, in business and life, that can separate the good from the great. This powerful book by S.L. Parker and Mac Anderson gives great examples, great quotes and great stories to illustrate the 212° concept. A warning - once you read it, it will be hard to forget. Your company will have a target for everything you do ... 212°
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2024-06-03T21:29:49.458Z
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2013-05-18T08:51:45.000Z
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Quotes by Majeed, Muhammad Tariq
Dr Muhammad Tariq Majeed is Assistant professor of Economics at Quaid-i-Azam University, Islamabad, Pakistan. He did his PhD in Economic from University of Glasgow in 2012..
"In the longrun, flexible minds win."
Majeed, Muhammad Tariq on success
"Intelligence is a relative term. It is hard work that matters more. "
Majeed, Muhammad Tariq on success
"Competition brings efficiency. True! Competition with yourself, rather than others, brings more efficiency."
Majeed, Muhammad Tariq on success
"It is not difficult to convert a negative into a positive, given that a high level of tolerance remains persistent."
Majeed, Muhammad Tariq on tolerance
"If there is no shock, life remains in a lock."
Majeed, Muhammad Tariq on life
"When I waste minutes, hours take revenge."
Majeed, Muhammad Tariq on time
"To contribute means add a positive or take away own negative."
Majeed, Muhammad Tariq on character
"Laziness is a luxury."
Majeed, Muhammad Tariq on luxury
"If you cannot improve and diversify your skills then get ready to assist your own juniors."
Majeed, Muhammad Tariq on struggle
"In academic research, efforts wasted in wrong direction are not wasted as they contribute latently."
Majeed, Muhammad Tariq on research
Take a look at recent activity on QB!
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2024-06-03T21:29:49.458Z
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2013-05-18T08:47:07.000Z
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Rondo has to cash the check Perk’s mouth wrote
RedsArmyAdmin November 1, 2009 Uncategorized 8 Comments
Chris Paul is pretty much regarded as the best point guard in the league. Rajon Rondo, however, doesn't seem to be impressed. At least… if you believe SI's Chris Mannix
Kendrick
Perkins just told me that Rondo said "Chris Paul has the stats that he
has because he has the ball in his hands all game." Yikes
Whoa… since when is Kendrick Perkins the "mouth off to the reporter" guy? And we're tweaking one of the best players in the league? HornetsHype sums it up nicely:
This is terrifyingly like poking a dragon with a stick. OK, the Hornets
look like a massive work in progress still, while the Celtics already
seem to be clicking the best out of the potential title contenders in
these early days of the season. So it’s not like we’re expecting a huge
victory Sunday, or anything. But the fact remains that you don’t poke a
dragon with a stick. It breathes motherfreakin’ fire. It’s not
safe.
I’m not saying the Hornets are going to come out and win this game,
bulletin board material or not. I’m just saying if they do, the Celtics
will know who is to blame
What else can I say but… bingo.
The Celtics defense is good. Damn good. But you don't need to give one of the best any added motivation.
But then again… it's gonna set up one hell of a battle between Rondo and Paul. We might have one of those "neither guy can stop the other" type battles brewing… which will at least make for an exciting game.
Oh, and Chris Mannix? I'd tread lightly the next time you come across Kendrick Perkins… unless you're absolutely sure he was cool with you tweeting that. Yeah, maybe Perk needs a refresher on what "off the record" means… but that sounds so far out of Perk's character, I find it hard to believe he was telling you that in a "go ahead and print it" kind of way.
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