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Fatty acids taken up by enterocytes are repackaged into complex lipids at the endoplasmic reticulum and are subsequently stored in enterocyte lipid droplets or directed to lipoprotein synthesis for export. Lipid droplets are composed primarily of TGs and CE in the interior, and bounded by a PL monolayer with associated proteins such as perilipins (70). Though the mechanisms by which lipid droplets grow and shrink are well characterized, the regulation of lipid droplet size and number in various tissues is not as well understood, and most current research efforts focus on adipose and hepatic lipid droplets (71). As the intestine is not a site of long-term lipid storage in vertebrates including larval zebrafish, enterocyte lipid droplets are highly dynamic, temporary structures that respond with high sensitivity to the nutritive state of the animal. This property combined with the relative ease of live imaging in the larval zebrafish intestine compared with other animal models makes for an ideal system for the study of lipid droplet dynamics and regulation. When 5 dpf larvae are fed a high-fat/high-cholesterol meal of chicken egg yolk, both the average lipid droplet number per enterocyte and total area of the cell covered by lipid droplets increase significantly by 1 h post-feeding. Lipid droplet number peaks at 1 h and then gradually decreases, while total lipid droplet area is maintained up to 3 h following the meal, suggesting that smaller lipid droplets fuse as they mature (72). The gut microbiota also influence enterocyte lipid droplet number and size. Intestinal lipid droplets are both larger and more numerous in conventionally raised larvae after feeding than in germ-free larvae. Furthermore, conditioned media from a Firmicutes bacterial strain found to promote dietary fatty acid uptake and export to the liver was sufficient to increase enterocyte lipid droplet number but not the average lipid droplet size (69). These results have begun to reveal the diverse mechanisms by which different members of the gut microbiota influence lipid droplet dynamics and dietary lipid metabolism.
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study
| 99.7 |
Lipoproteins are essential for the export of the products of dietary lipid from enterocytes into the circulation. Expression and function of apolipoproteins in the zebrafish is similar to that observed in mammals; at least one paralog from each of the ApoA-I, ApoB, ApoE, and ApoA-IV families is expressed in the larval zebrafish intestine (13). There is evidence that division of apolipoprotein function among organs is regulated by different mechanisms that achieve the same end in zebrafish and mammals: while different variants of ApoB are produced in the mammalian intestine and liver via RNA editing, larval zebrafish produce mRNA for the ApoB paralog b.1 in the intestine and liver and ApoBb.2 in the liver only. Similar compartmentalization of paralog expression between the liver and intestine is observed in the other apolipoprotein families as well (13) (Figure 3). Intestinal lipid accumulation in animals treated with an MTP inhibitor shows that as in the larval zebrafish yolk, availability of functional ApoB in necessary for normal rates of lipid export from the intestine, and that enterocyte lipid droplets are the destination of excess dietary fatty acids when export is slowed (73–75). The MTP inhibitor lomitapide is effective in larval zebrafish (72). It has also been observed that in mammals as the dietary fat content increases, chylomicron number reaches a plateau but average chylomicron size continues to increase, suggesting that apolipoprotein expression is the limiting factor in the rate of lipid export from the intestine (76).
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study
| 100.0 |
Zebrafish apolipoprotein genes are expressed in the larval digestive system. In situ hybridization reveals expression of 10 of the 11 zebrafish apolipoprotein genes in the apoB, apoA-IV, apoE, and apoA-I families in the liver and/or intestine of the 6 dpf larva. Dissected intestines probed for apoA-Ia are shown, and the gut of a larva probed for apoA-1b is magnified below the image of the whole larva. L, liver, I, intestine. Adapted and reprinted from Ref. (13), Figures 2–5, under a CC-BY license.
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study
| 99.9 |
The larval zebrafish intestine is not only an excellent model for the study of lipid droplet and lipoprotein packaging, but also a site of differential channeling of dietary fatty acids depending on their chemical properties. The amenability of this model to biochemistry due to the ease of obtaining large numbers of embryos and larvae and performing lipid extractions from them, combined with the transparency of the larva, provides an opportunity unique among vertebrates to perform live imaging and metabolic labeling experiments in parallel using the same fluorescent lipid reagents (28, 77–79). Additionally, the whole-body lipid composition of the larval zebrafish is highly sensitive to changes in diet: the TG content of the 6 dpf larva increases 10-fold 24 h after a single high-fat meal (compared with a standard low-fat diet, and allowing time for the intestinal lumen to clear) (29). (In these experiments, the high-fat meal was chicken egg yolk; ~50% lipid dry weight, and the low-fat meal was SERA Micron larval growth food; 7% lipid. The lipid content of “standard chow” for zebrafish larvae is typically 5–15%.) Working at developmental stages before adipose tissue appears (~14 dpf) avoids signal to noise problems that may occur when the neutral lipid stored in adipose is included in the whole-body lipid profile. Also, at these early developmental stages examination of dietary lipid processing in the intestine can be isolated from potential regulatory influences from adipose tissue.
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study
| 99.94 |
Though it was beyond the scope of our recent metabolic labeling study (29), the biochemical techniques described therein could be applied to later-stage larvae in order to examine potential crosstalk between adipose tissue and the enterocytes that could influence dietary lipid partitioning. We have also developed methods for using fluorescent fatty acids as metabolic labels in the context of standard and lipid-enriched diets in larval zebrafish (Table 2). In addition to exploring the metabolic labeling potential of fluorescent lipids whose product profiles were not previously described, we have also applied HPLC with charged aerosol (total lipid detection) and fluorescence detection to obtain a greater depth of information than previous studies using fluorescent TLC (28). Initial findings indicate that the partitioning of saturated fluorescent fatty acids among complex lipid classes varies with carbon chain length, the total fat and cholesterol content of the diet, and the type of fluorescent tag (29). Metabolic labeling with fluorescent fatty acids in the context of lipid metabolism by the larval zebrafish is summarized in Figure 4.
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study
| 99.94 |
Metabolic labeling with fluorescent fatty acids is performed in the context of zebrafish development, yolk absorption, and dietary lipid metabolism. Fluorescent fatty acids (BODIPY-FL-C12 depicted) are trafficked and metabolized along with native yolk or dietary lipids when delivered to the developing zebrafish by yolk injection or feeding. LD, cytoplasmic lipid droplet, LP, lipoprotein, VLDL, very low-density lipoprotein. Embryo and larva illustrations adapted from Ref. (18).
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| 99.7 |
Potential mechanisms regulating the rate of lipid export from the intestine beyond lipoprotein levels, the regulation and physiological effects of the size of enterocyte lipid droplets, and the channeling of newly absorbed dietary fatty acids into the different classes of complex lipids are currently largely uncharacterized. The optically clear and genetically tractable larval zebrafish model presents an ideal system in which to investigate these questions relating to energy homeostasis with a combined live imaging and biochemical approach.
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study
| 99.94 |
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer JM declared a past collaboration with one of the authors SF to the handling editor.
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| 99.94 |
The recent and rapid increase in the number of vehicles has led to growing pollution, especially in large cities. This is one of the main causes of global warming, which is why many governments have begun to take steps to try to reduce it. The most common measures are the promotion of public transport and the limitation in the use of vehicles in city centres through the prohibition of cars with a single occupant or with even or odd number plates depending on the day of the week, etc. However, it could be said that so far none of these solutions has proven to be the most convenient and/or affordable solution for everybody.
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| 99.9 |
Another practical solution would consist of avoiding the low occupancy of most vehicles in cities, through car sharing, so that empty seats are used in most trips. This modality is known by the term carpooling and has been proposed as an effective way to reduce pollution and cut expenses. A related but different approach, known as carsharing, is based on collective fleets of cars that can be temporally rented by multiple users, but such a solution does not solve the aforementioned problem because it does not imply that users share the vehicle at the same moment. There is another term, ridesharing, which is generally used to refer to different solutions to share the use of a car with other people in order to travel to a particular destination. In addition to carsharing and carpooling (also known as real-time or instant or dynamic or ad hoc ridesharing), ridesharing also includes other versions known as slugging, lift sharing and covoiturage. This work does not address ridesharing solutions different from carpooling.
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| 99.9 |
Both types of collaborative solutions have been increasingly used since the beginning of the economic crisis, thanks to technology 2.0. They are applicable in many different cases, but are especially useful in environments and situations like universities, holidays, long journeys and urban centres. This is because, in these cases, both owners and passengers of vehicles have the same motivations to consider the carpooling solution. Their main goal is usually to share the cost of fuel, but they might have other reasons, such as trying to avoid parking problems, wanting to talk, meet new people or make a contribution to environmental protection.
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other
| 99.94 |
One of the main problems of carpooling is reliability, due to the fact that the service requires users to be confident that the driver will take them to their destination, and that drivers be sure that the accompanying people will behave properly during the trip. A practical improvement applicable to existing carpooling systems is described here, based on the use of the latest technological advances in smartphones and social networks to increase reliability. The proposed solution first allows the establishment of trust between drivers and passengers through reputation accounting, and, second, protects the privacy of all users. This is the main aim of this work, but there are others related to carpooling such as route optimization or enhancement of cooperation, which are not addressed here.
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| 99.8 |
This paper is organized as follows. Section 2 mentions several related works, and Section 3 introduces the general design of the proposal, which is mainly based on the reputation algorithm sketched in Section 4. In Section 5, the developed Android application is described. Section 6 briefly analyses the security of the proposal. Finally, some conclusions and open problems can be found in Section 7.
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| 99.94 |
Carpooling has a long history as it had a boom in the United States during World War II, and later, in the mid-1970s, it was once again used as a solution during the oil and energy crises . However, in those days, without the technology available today, carpooling had to face many difficult obstacles, such as the need to develop a network of users and find convenient forms of communication. Gradually, the means used to organize the trips were changing from the telephone to other more flexible technologies like Internet, email and smartphones. Nowadays, there are many different carpooling platforms and services, but, even today, they can be considered in their early stages because none of them has reached a large mass of users.
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| 99.94 |
Several features of various existing carpooling systems are shown in Table 1, including the most relevant security-related ones. In particular, this comparative analysis includes as representative systems: Amovens , Blablacar , Compartir.org , and ZimRide .
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| 99.9 |
For instance, BlaBlaCar is the largest carpooling network in Europe. It is a service focused on long distance trips and uses social networks for registration and feedback and as an enabler of real connections between users. The biggest carpooling service in the United States is ZimRide , where payments are made through credit card account and PayPal. The main system to build trust in all of these platforms is based on user-given points. However, ignoring this security system is quite easy because users who get a negative score, can create a new profile with new credentials and no points.
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| 99.94 |
Apart from the aforementioned practical platforms, which are already in operation, there are several papers that propose different solutions for carpooling. Some of them are centred on route optimization, and others focus on cooperation enforcement or passenger matching, and others are centred in multi-agent platforms. First, the work shows an integrated system with an optimization module, which applies heuristic methods to solve the organization of the routing problem in carpooling services by using different technologies such as Web, Geographic Information System (GIS) and Short Message Service (SMS). Second, the paper defines a push service to promote carpooling through instant processing. The paper presents a carpooling architecture that uses a credit mechanism to encourage cooperation among users, by defining a business model where such a credit can be used in parking facilities and can be bought with real money. A more recent work is , where an algorithm to encourage carpooling is proposed based on assigning priority to users with positive feedback through a fuzzy logic scheme. Thirdly, an automatic service to match commuting trips is presented in . In addition, passenger matching is the topic of the paper , where a system is proposed to help users in choosing a transport solution according to its ecological footprint, matching their needs, preferences, and actual location. Finally, another interesting proposal is , based on a multi-agent platform that focuses on security services that allow the mutual authentication of the users and of the application components with the system.
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| 99.9 |
This work differs from all the aforementioned because its main goal is trust, obtained through a combination of reputation measurements and privacy protection that can be used in existing carpooling services. Additionally, the proposed system has been tested through the implementation of an open-source carpooling platform where any driver can insert the availability of empty seats on his/her car for a planned trip and potential passengers can search for trips and contact him/her.
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other
| 99.9 |
Although the main objective of the proposed design is to increase trust in users, there are also other factors that have been taken into account, such as privacy protection, user-friendliness and usability. Thus, one of the main features of the proposal is that users who publish their trips have their privacy fully protected. Unlike other carpooling platforms, in the described system, no user is allowed to access data such as email, phone, full name or other data of another user, unless it is authenticated on the platform, and the algorithm for checking mutual trust returns a valid permission for it. In this case, the interested user will be able to see the relevant data. Otherwise, it can only submit a request to another user (driver) so that this can decide whether the applicant is reliable enough to have access to those data.
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other
| 99.94 |
The proposal is based on trust relationships in such a way that those who want to use it first have to authenticate it in the platform through some social network such as Facebook, Twitter or Google+. In this way, the proposal checks the existence of some chain of trust between the applicant and other users, based on the so-called rule of six degrees of separation . This rule is the theory that everyone is six or fewer steps away from each other person in the world, so that a chain of ’a friend of a friend’ statements can be made to connect any two people in up to six steps.
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| 99.94 |
In addition, the reputation gained through the use of the application is an influential factor considered in the decision on whether to share a car with some person. To do this, at the end of every shared travel, the application asks both driver and passengers to score the other users in the trip. Such scores are used in future trips so that seats offered by drivers with good scores are more likely to be selected than others with lower scores. Furthermore, well-scored passengers are more likely to have access to details of other users when selecting a new trip.
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other
| 99.94 |
The general architecture used in the proposed application model is known as client–server (see Figure 1). Its different elements are the following:Client: Mobile device used for the system.Server: Hosted in the cloud, and divided into two parts. The first part is the Google Cloud Messaging (GCM) server that handles all of the notifications and is responsible for sending the notification when the receiving clients are alive. The second part is the DataBase (DB) dedicated server, which stores all data related to users and the system in a database. It also serves as a gateway for sending notifications between the client and the GCM server.
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| 99.94 |
Server: Hosted in the cloud, and divided into two parts. The first part is the Google Cloud Messaging (GCM) server that handles all of the notifications and is responsible for sending the notification when the receiving clients are alive. The second part is the DataBase (DB) dedicated server, which stores all data related to users and the system in a database. It also serves as a gateway for sending notifications between the client and the GCM server.
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| 99.94 |
The proposal protects user privacy through limited and controlled access to user data, according to the trust level stated for the relationship between each pair of users. This trust level is obtained through the combination of direct scores and information obtained from trust networks so that, by combining both sources, the system is provided with enough data to deduce whether people can trust each other or not. In this way, privacy is dealt with accordingly as one of the most important aspects of the proposed carpooling system.
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| 99.94 |
The PageRank algorithm used by Google Search to rank websites in their search engine results is used here as a first approach for the development of a trust measurement algorithm that provides a value to each pair of users. This algorithm is used here to predict whether two people can trust each other. However, since it does not fully fit the morphology of the specific problem, a second approach is also used here to complement it, based on Bayesian networks to know whether people can trust each other. The refined algorithm for trust measurement contained in the developed Android application is explained below.
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| 99.9 |
To the best of our knowledge, no existing carpooling proposal offers a quantitative method based on the theory of six degrees of separation to decide if two users should trust each other to share a car or not. Some of the proposals do not even allow drivers to decide who can or cannot apply their route. There are some proposals that use a quantitative method based on the similarities among users. Their main problem is that they have to collect information about the attributes and characteristics of each user. The proposal aims to be simple for the user, so that he/she does not have to fill in any information about his/her attributes. With a simple click to enable social login on the network after a previous registration, the user can log into the system and start using the platform.
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other
| 99.9 |
The reputation algorithm is the main feature of the proposal because, thanks to it, the user can use a quantitative measure to decide whether to trust another user or not. The algorithm is based on the theory of six degrees of separation and individual scores within our platform. This number of steps may be reduced significantly by introducing the concept of social networks. Our application uses social networks when logging into the application to create a network of user, which is used to interconnect users and provide a reliable measure of confidence among them. Through the use of social networks, the six degrees of separation can be reduced on average to less than four. In particular, according to several studies on Facebook , the average distance is 3.9, corresponding to intermediaries or ’degrees of separation’, which shows that the world is even smaller than expected.
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| 99.9 |
The trust rate is a value between 0 and 1, shown to the user as a character (A, B, C, E, F) and computed for each pair of users to inform about the trust between them. This measure is calculated taking into account the two parameters explained below: degree of friendship and ratings of users in the platform.
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| 99.94 |
Let F=[dfij] denote the weight matrix of G defined by Equation (1):(1)dfij=∑(FAij·wAij)MFCi,eij∈E,0,otherwise, where FAij is called Friendship Action, and may be a Comment, a Like, or any other action from user i on user j on a social network;wAij is the weight of the Action FAij and represents its importance over other actions. Its value is between 0 and 1. For example, for Facebook, a Comment is considered more important than a Like in the proposal. In particular, in the proposal, the weight of a positive Comment is 0.727, while the weight of a Like is 0.273, according to the values obtained from the data shown in the survey . The system may detect positive, negative and neutral Comments using machine learning algorithms ;MFCi is called the Maximal Friendship Coefficient, and represents the maximum sum of weighted actions performed by a user i. Its value is: maxj(∑(FAij·wAij)).
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study
| 66.56 |
wAij is the weight of the Action FAij and represents its importance over other actions. Its value is between 0 and 1. For example, for Facebook, a Comment is considered more important than a Like in the proposal. In particular, in the proposal, the weight of a positive Comment is 0.727, while the weight of a Like is 0.273, according to the values obtained from the data shown in the survey . The system may detect positive, negative and neutral Comments using machine learning algorithms ;
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other
| 99.9 |
The remaining scores are obtained from the assessments of users after sharing cars. When a route is completed, the users who participated in it can vote between 1 and 5 stars. Each passenger individually assesses the driver, and the driver individually assesses each passenger.
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other
| 99.94 |
The methodology used for determining the rating estimation for drivers and passengers is based on the risk assumed by each one. This risk has been established taking into account the number of verifiable data, the elements that can be damaged, and the greater importance attached to the supply rather than the demand. With this in mind, it is assumed that a driver has more verifiable data and can suffer more damage as he/she provides the vehicle, with all its features and an up-to-date insurance. Thus, at least initially, the system assigns greater value to users who offer empty seats in their cars than to passengers who are looking for empty seats because, without an offer, it is impossible to meet a demand. Therefore, the scores of Table 2 have been adjusted according to that argument and taking into account that a parameter greater than three stars is considered a positive rating, less than three is negative, and equal to three is neutral. In addition, the system gives the driver added impact due to the fact that he/she provides more verifiable information.
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| 99.9 |
The final metric rating of the assessments given to a user i by other users in the proposal is computed through ari, the average of all the ratings received by i measured in points. A user who has been using the system for a while normally would have more ratings than a recent user, unless the behaviour of the first user has been incorrect. Thus, the way the proposed system can compare the performance of both users is by averaging their ratings because, otherwise, if the simple sum of ratings were used, the user who has been using the system longer would always have more points than a recent one, although the recent one has had a better behaviour or performance.
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other
| 99.9 |
Finally, the metric TRij (Trust Rate) indicating the degree of trust in user i from user j, is given by Equation (2): (2)TRij=DTij,if users i and j are direct friends ,ITij,if some friendship chain exists from user i to user j,NTij,if no friendship chain exists from user i to user j, where If users i and j are direct friends, Direct Trust (DT) is obtained using Equation (3) (3)DTij=dfij·wF+ari·wR.If some chain of friends from user i to user j exists, Indirect Trust (IT) is given by Equation (4) (4)ITij=F(i→j)·wF+ari·wR.If there is no friendship chain from user i to user j, Equation (5) is applied (5)NTij=ari·wR, where wF is called weight of Friendship, and its value (0.625) is obtained by the average happiness of people with their friends in the social network of Facebook ;wR is the weight of Rating, whose value is obtained using 1−wF, so it is 0.375;F(i→j) gives the degree of friendship from user i to user j obtained through the friendship chain {i,i1,i2,....,ic,j} between both. This value is obtained considering the probability of independent events. F(i→j)=dfii1·dfi1i2···dficj. This value is always between 0 and 1, and decreases along with the length of the friendship chain.
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| 56.34 |
F(i→j) gives the degree of friendship from user i to user j obtained through the friendship chain {i,i1,i2,....,ic,j} between both. This value is obtained considering the probability of independent events. F(i→j)=dfii1·dfi1i2···dficj. This value is always between 0 and 1, and decreases along with the length of the friendship chain.
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other
| 99.9 |
The maximum trust rate that a user can get is 1 (represented as A score), which corresponds to the situation when the user direct friends have rated him/her with the highest scores. On the other hand, a user has F valuation if he/she does not have any degree of friendship or is starting to be known, and/or has had mostly negative reviews.
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| 99.94 |
This valuation is dynamically calculated as a function of the friendship degree that a user has in each moment, and the ratings he/she has received by his/her use of the platform till then. Thus, the system helps users to have an up-to-date reliability measure about whether to trust another user or not. In addition, only users who have a valuation higher than B and/or users who have been accepted by the driver to make the route can see certain driver data, such as the phone number or other specific data. The system threshold for reliability has been set to a value higher than C, that is A or B, because this is the minimum value that represents direct friendship.
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| 99.9 |
The reputation algorithm to compute trust rates is run in parallel using different threads to optimize efficiency. For the calculation of IT value, where two users are not direct friends, but they share a common chain of friendship, and each partial degree of friendship between direct friends is calculated in a different thread.
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other
| 99.94 |
In order to discern whether an action taken on social network is positive or negative, the scheme uses AlchemyAPI based on Machine Learning algorithms to provide the social sentiment of a text. In addition, the proposal only takes into account the latest 100 actions of interactions between users on social networks. Thus, each TRij value is computed dynamically for each pair of users i and j as it depends on the degree of friendship between the pair of users and their last interactions in social networks.
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| 99.9 |
For example, in order to compute the trust rate TRAB in user A regarding his relationship with user B, the required calculations are as shown in Equation (6). (6)DTAB=(78×0.273)+(19×0.727)40.822×0.625+0.75+1+0.5+0.5+0.75+0.56×0.375=35.10740.822×0.625+46×0.375=0.537+0.25=0.787.
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other
| 99.9 |
To compute the trust rate TRDA in user D regarding his relationship with user A, the required calculations are as shown in Equation (7) (7)ITDA=(42×0.273)+(7×0.727)40.876×(4×0.273)+(2×0.727)41.738×(82×0.273)+(12×0.727)40.880×0.625+0.15+0.25+0.153×0.375=(0.405×0.061×0.761)×0.625+0.553×0.375=0.019×0.625+0.183×0.375=0.012+0.069=0.081.
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other
| 99.9 |
The proposed system is fully dynamic and depends on the relationships between each pair of users. Figure 3 shows a comparison between the trust rates of each pair of users and the classical simple rating used in other proposals that are based only on ratings received from users.
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other
| 99.94 |
The Android application is developed for the versions 3.0 or higher of the operating system. APIs like Google Maps v3.0, Google Places, Google Cloud Messaging, etc., and Facebook SDKs 3.0 and libraries like Action Bar Sherlock are used for the functionality of the new versions of Android on older versions. Autocomplete in address searches, Google Maps 3D Technology, design based on the latest versions of Android, push notifications with requests or responses of passengers or drivers, etc. are among the features of the Android Application.
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other
| 99.94 |
The use model of the implemented application is shown in Figure 5. Each user can see the routes he/she proposes as driver, and whether potential passengers exist for those routes. In addition, with colour codes, he/she can know the routes that each user has already made and the routes that have been confirmed by users. For the assessment of users participating in a route, after finishing it, each one can give a score. In order to deploy the carpooling platform, a server was developed using JavaScript technologies by frameworks like ‘node.js’ and ‘express.js’. As a database for all the centralized data on this server, a No SQL database, such as MongoDB , was adopted. The server was deployed on a micro instance of Amazon Web Services, specifically under an Ubuntu machine with Amazon EC2 account.
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other
| 99.94 |
Although the Carpoolap application that includes the proposed trust algorithm has been published in the Google Play Store, the lack of marketing campaign and consequent lack of users means that data from the real application are not enough to extract useful conclusions.
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other
| 99.94 |
Regarding the security of the platform, Sybil attack is the most notorious attack in traditional carpooling systems. This type of attack is a hacking attack on peer-to-peer networks where a malicious device illegitimately takes multiple identities by forging them. Due to the privacy-preserving environment of carpooling schemes, Sybil vulnerability is generally hard to defend against.
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other
| 99.94 |
In a Sybil attack, the attacker subverts the reputation system of a peer-to-peer network by creating a large number of pseudonymous identities, using them to gain a disproportionately large influence. The vulnerability of a reputation system to a Sybil attack depends on how cheaply identities can be generated, the degree to which the reputation system accepts inputs from entities that do not have a chain of trust linking them to a trusted entity, and whether the reputation system deals with all entities in the same way.
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other
| 99.94 |
An entity of the analysed peer-to-peer network is a piece of software that has access to local resources. It advertises itself on the peer-to-peer network by presenting an identity. However, more than one identity could correspond to a single entity. In other words, the mapping of identities to entities could be many to one. Entities in peer-to-peer networks could use multiple identities for purposes of redundancy, resource sharing, reliability and integrity because, in peer-to-peer networks, the identity is used as an abstraction so a remote entity could be aware of other identities without necessarily knowing the correspondence of identities to local entities. By default, each different identity is usually assumed to correspond to a different local entity. However, actually, many identities may correspond to the same local entity.
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other
| 99.9 |
A dishonest member or an adversary node may present multiple identities to a peer-to-peer network in order to appear and work as multiple distinct nodes. After becoming part of the peer-to-peer network, the adversary may then overhear communications or act maliciously. By masquerading and presenting multiple identities, the adversary can control the network substantially.
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other
| 99.94 |
The proposed reputation algorithm reduces significantly the vulnerability to the Sybil attack described above because most of the score of the reputation algorithm is preceded by confidence in the degrees of friendship that binds each user to another user. Thus, if a user does not know (at all) another user, very high ratings of the latter in the system are not enough for guaranteeing reliability for the first user because, in the proposed system, the weight of the average rating is 37.5% of the total trust rate, while the weight of the degree of friendship is 62.5%.
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other
| 99.9 |
This paper proposes a new system to increase the safety and reliability of existing carpooling systems, in order to overcome the psychological barrier that slows down the use of many potential users of carpooling. In order to achieve this objective, the system includes, on the one hand, a strong social component that fosters trust among users, and, on the other hand, progressive access to user data based on the estimation of the confidence levels provided by the system. The design of the proposal has been tested for its implementation in a smartphone application that supports the Android platform, is shared as an Open Source Project, and has been published in the Google Play Store. This work is in progress, so there are still several open issues, such as the development of the application on other platforms, the creation of an API for use in third-party applications or the study of existing agent-based models to compare the performance of the proposed system with the implementation of such mechanisms.
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other
| 99.9 |
Clonorchiasis is a major endemic disease affecting over 35 million people in Asian countries including Korea, China, Thailand and Vietnam [1–3]. Clonorchis sinensis infections are caused by the ingestion of raw or undercooked freshwater fish that harbor the metacercariae . Complications associated with the infection increase with an increase in the intensity and duration of the infection. Clonorchis sinensis has also been recognized by the World Health Organization as a biological carcinogen that can induce cholangiocarcinoma in humans .
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review
| 99.8 |
C. sinensis migrates into the bile duct of the host and lives there. However, the bile duct can be regarded as an extreme environment, since accumulation of bile juice can be toxic to the worm’s tissues and cells . Among the various bile juice, lithocholic acid (LCA) has been proven to have a toxic effect on the survival of juvenile C. sinensis . Therefore, it is important that the influx and efflux of bile acids should be balanced to prevent bile intoxication in the worm’s body. In humans, there are many importers and exporters of bile juice circulation , such as apical sodium-dependent bile acid transporter (ASBT), Na+ taurocholate co-transporting polypeptide (NTCP), Multidrug resistance protein (MRP), bile salts export pump (BSEP), and organic solute transporter (OST). Therefore, we believe that C. sinensis needs these bile transporters to reduce accumulation of bile acids within its body.
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| 99.94 |
MRPs belong to a subfamily of the ATP binding cassette (ABC) transporter family [9, 10]. In higher animals, MRP4 is a unidirectional and distinctive bilaterally localized transporter in polarized cells, such as baso-lateral membrane of hepatocytes and choroid plexus epithelial cells [12, 13]. Such tissue-specific distribution suggests that MRP4 has multiple functions. MRP4 is responsible for pumping out a broad range of substrates, including bile acids, as well as for physiological regulation via transport of cyclic nucleotides out of cells .
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Therefore in this study, we identified and characterized C. sinensis MRP4 (CsMRP4), the first MRP4 in trematodes, at the in silico, molecular, and biochemical levels. The structure of CsMRP4 was built using homology modeling, and the structural features and bile acid-binding affinities were investigated. CsMRP4 was found to be localized mainly in the mesenchymal tissues and oral suckers of C. sinensis adults and metacercariae.
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Pungtungia herzi (Jinju, Korea), the second intermediate host of C. sinensis, was ground and digested as described by Dai et al. . Metacercariae were then collected from the saline-rinsed digestive leavings under a dissecting microscope. Next, New Zealand white rabbits (2.3 kg; Koatech, Seoul, Korea) were infected with 200 metacercariae per rabbit twice in 1 week. Adult C. sinensis were then recovered from the rabbit livers after 2 months and stored in a -80 °C freezer until use. Female 7 week-old BALB/c mice (Orient Bio Inc., Gyeonggi-do, Korea) were immunized with a bacterially-produced recombinant protein.
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A putative MRP4 polypeptide sequence (GenBank ID: GAA49862.1) of C. sinensis was retrieved from the National Center for Biotechnology Information (NCBI) database. The coding DNA sequence (CDS) was obtained from the C. sinensis DNA scaffold (GenBank ID: DF142991.1) to which it belonged. DNA-walking was performed twice for CsMRP4-I and CsMRP4-II due to the long size (approximately 4 kb) of the products. Two sets of PCR primers were designed according to CDS and synthesized (Bioneer, Daejeon, Korea) (Additional file 1: Table S1). Total cDNA of C. sinensis was prepared as described previously , and 50 ng per reaction was used as the template for DNA-walking. PCR amplification was performed under the following conditions: pre-denaturation (94 °C for 5 min), amplification phase with 35 cycles (94 °C for 30 s, 55 °C for 30 s, 72 °C for 2 min 15 s), and final extension (72 °C for 10 min). The PCR products were then purified using the QIAquick PCR purification kit (Qiagen, Hilden, Germany) and sequenced (Macrogen Inc., Seoul, Korea). The CsMRP4-I and CsMRP4-II sequences were used for assembling the putative CDS and translated into amino acid (aa) sequences. However, CsMRP4 was assumed to be incomplete at the 5′-end upon comparison with MRP4 of other species. Therefore, 5′-rapid amplification of the cDNA ends (5′-RACE) was carried out to obtain the entire CDS. Total cDNA of C. sinensis was synthesized using the SMARTer™ RACE cDNA amplification kit (Clontech, Mountain View, CA, USA) according to the manufacturer’s instructions. The missing 5′-end of CsMRP4 was amplified by RACE-PCR run using the 5′-RACE universal primer mix (UPM) and gene specific reverse primer (GSP). The PCR product was then confirmed using nested PCR, purified, and subjected to TOPO TA cloning (Invitrogen, Carlsbad, CA, USA). Through blue-white screening, the positive white colony was selected and reconfirmed by rapid colony PCR. Its plasmid DNA was extracted using the Plasmid Miniprep kit (Qiagen, Seoul, Korea) and sequenced (Macrogen Inc., Seoul, Korea). The primers used for RACE-PCR, DNA-walking, and multiple sequencing are listed in Additional file 1: Table S1.
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For CsMRP4, the isoelectric point (pI) and molecular weight (Mr) was estimated using the ExPASy ProtParam Tool (http://web.expasy.org/protparam/). CsMRP4 was blasted against UniProtKB/Swiss-Prot v. 2017_07 . Domain organization and residue annotation were conducted using the Conserved Domain Database (CDD) and InterProScan v. 64 .
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In order to confirm that CsMRP4 belongs to a MRP subfamily and to infer its phylogenetic relationship with the ABCC and ABCB subfamilies, 12 canonical ABCC proteins and 11 canonical ABCB proteins were retrieved from UniProtKB/Swiss-Prot v. 2017_07 . Multiple sequence alignment was performed using the L-INS-i method of MAFFT v. 7.299 . An evolutionary history was inferred by employing the neighbor-joining (NJ) method using MEGA v. 6.06 . All the positions containing gaps and missing data were eliminated.
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The standard protocol of YASARA Structure v. 17.6.5 was used to build the homology models of CsMRP4. To obtain these models, PSI-BLAST was carried out against PDB entries (updated August, 2017) . After building the homology models for each template, the models were submitted to high-resolution energy minimization using a YASARA force field . The result was then validated to ensure that the refinement did not move the model in the wrong direction. Finally, a hybrid homology model was obtained by combining the best scoring parts of the four models. In addition, potential errors in the 3D models were evaluated using a Ramachandran plot and ERRAT .
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Structural conservation was calculated and visualized using ENDscript/ESPript v. 3.0 with the PDBAA95 database, E-value of 1e-12, and contact range of 2.7 Å. COACH was used to predict ligand-binding sites in CsMRP4. SDF files for bile acids were retrieved from the PubChem database as of August 2017 and transformed into the MOL2 format using OpenBabel . Bile acids were docked into CsMRP4 using PyRx v. 0.8 , which includes AutoGrid and AutoDock Vina . A grid box extended to all membrane-spanning domains (MSDs) of CsMRP4; no information regarding the exact location of the binding sites of the various bile acids was available. Active site dimensions were set as the grid size of center X: 25.6 Å, center Y: -13.5 Å, and center Z: -6.5 Å, and 8 maximum exhaustiveness was calculated for each bile acid. All structure visualizations were carried out using UCSF CHIMERA v. 1.10.2 and PyMOL Molecular Graphics System v. 1.7.4.5 (Schrödinger, LLC., New York, NY, USA).
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To evaluate the mRNA expression level in different developmental stages, quantitative real time PCR (Q-rt.-PCR) was performed. Primers were designed using Oligo-primer analysis software v. 6.71 (Molecular Biology Insights, Cascade, WA, USA) (Additional file 1: Table S1). Calcyphosine (CAP) and phosphoglycerate kinase (PGK) were employed as the reference genes . Q-rt.-PCR reaction mixtures were prepared in triplicate using LightCycler FastStart DNA Master SYBR Green I Kit (Roche, Mannheim, Germany), with each reaction containing 50 ng of total cDNA of the adults or metacercariae. Q-rt.-PCR was performed on LightCycler 2.0 (Roche, Penzberg, Germany) with the following thermal cycle parameters: pre-heating (95 °C for 15 min), 40 cycles of 95 °C for 10 s, 48 °C for 10 s, and 72 °C for 30 s. The relative transcription ratio was calculated according to the 2-ΔΔCt method .
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A cDNA fragment encoding CsMRP4-NBD1 was amplified using PCR (Additional file 1: Table S1). The purified PCR product was then subcloned into pET23b and confirmed by colony PCR and restriction enzyme digestion. Plasmid DNA of the positive clone was extracted using the QIAprep Spin Miniprep kit (Qiagen, Hilden, Germany) and sequenced (Macrogen, Seoul, Korea). The correct construct was then transformed in Escherichia coli BL21[DE3]pLysS (Novagen, San Diego, CA, USA) by heat-shock at 42 °C for 30 s and spread on LB/ampicillin/chloramphenicol agar. After overnight incubation at 37 °C, a single colony was picked from the LB plate and grown in LB/ampicillin liquid medium by shaking vigorously at 37 °C. The recombinant(r) CsMRP4-NBD1 was then induced with 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) (TaKaRa, Shiga, Japan) for 5 h. The bacteria were then harvested, and recombinant protein was purified as described previously .
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The rCsMRP4-NBD1 was separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and cut off alone to obtain specific antigens for mouse immunization. The gel slice was equilibrated and homogenized in pre-cooled 1× PBS by complete grinding. The liquid homogenate containing rCsMRP4-NBD1 was then injected into BALB/c mice according to an immunization method . Blood was drawn from the eye and stored at room temperature for 1 to 2 h. The immune serum was obtained by centrifugation at 4000× rpm for 20 min. In order to examine the antibody titer in the immune serum against rCsMRP4-NBD1 and native CsMRP4, C. sinensis crude extracts were prepared using the Mem-PER Plus Membrane Protein Extraction Kit (Thermo scientific, Rockford, USA) following the manufacturer’s instructions. The crude antigen was then examined to determine its concentration using the Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA) and then stored as aliquots at -70 °C until use.
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The reactivity of the antibody was checked by western blot against recombinant protein and immuno-enhanced chemiluminescence (ECL) against native CsMRP4 in crude extracts. The rCsMRP4-NBD1 and C. sinensis crude antigens were loaded onto SDS gels for electrophoresis and then transferred to nitrocellulose membranes (GE Healthcare Life Sciences, Seoul, Korea). The membranes were then blocked using 5% skim milk in PBS/0.05% Tween20, followed by incubation with mouse immune serum at 1:400 at 4 °C overnight and then with goat-anti-mouse-IgG alkaline phosphatase-conjugated antibody (Sigma-Aldrich, St. Louis, MO, USA) at 1:5000 for western blotting or with peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG antibody (Jackson ImmunoResearch Inc., West Grove, PA, USA) at 1:10,000 for ECL at room temperature for 2 h. Normal mouse serum was used as the negative control. The recombinant protein was visualized by color developing in BCIP/NBT (Sigma-Aldrich, St. Louis, MO, USA). The native CsMRP4 was detected using a ECL solution kit (Bio Sesang, Seoul, Korea) and visualized using ImageQuant LAS 4000 (GE Healthcare Bio-Sciences, Amersham, UK).
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Paraffin block preparation and immunohistochemical staining were performed using our previously described methods . Mouse anti-NBD1 immune serum diluted at 1:200 served as the primary antibody. Normal mouse serum was used as the negative control. Dako EnVision + System-horseradish peroxidase-labeled polymer anti-mouse IgG (Dako Cytomation, Glostrup, Denmark) diluted at 1:400 was used as the secondary antibody.
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The complete coding cDNA sequence (4410 nt) of CsMRP4 was obtained through DNA-walking and 5′-RACE on the C. sinensis total cDNA. Its open reading frame was 1469 aa in length (Additional file 2: Figure S1 and Additional file 3: Figure S2). The Mr. of CsMRP4 was about 165.5 kDa and its pI was estimated to be 6.5. BLASTP was performed against the UniProtKB/Swiss-Prot database , which is a high-quality, manually annotated, and non-redundant protein database. Annotation information from NCBI non-redundant and UniProtKB/TrEMBL databases need to be further reviewed, since those contents were generated using in silico annotation or large-scale functional prediction. CsMRP4 is the closest to human MRP4 (HsMRP4) (UniProt ID: O15439) with an E-value of 5.3e-147 and identity of 44.2%, followed by HsMRP6 (UniProt ID: O95255) of 8.8e-89 and 38.4%, Mus musculus MRP3 (MmMRP3) (UniProt ID: B2RX12) of 5.1e-104 and 38.2%, and MmMRP5 (UniProt ID: Q9R1X5) of 2.9e-115 and 38.0%. CsMRP4 was significantly matched to multiple MRPs since the highest-scoring pairwise alignment was found predominantly in the NBD2 region, which is particularly conserved among ABC family transporters . As a subfamily of the ABC family of transporters, the MRP subfamily contains 12 members including MRP1-9, cystic fibrosis transmembrane conductance regulator (CFTR), sulfonylurea receptor 1 (SUR1), and SUR2 [9, 10]. The highest identity (39.0%) was observed between CsMRP4 and HsMRP4 in comparison with canonical human MRP/SUR/CFTR subfamily (Additional file 4: Table S2). Thus, this clone was designated as CsMRP4.
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Functional domains in CsMRP4 revealed diverse characteristics of ABC transporters. There were “ABC transporter type 1, transmembrane domain” (InterProScan ID: IPR011527) in two regions, aa64–378 and aa831–1162. Two domains, MSD1 and MSD2, consisted of six transmembrane α-helices each. As intracellular NBDs that linked with the MSDs, “ABCC_MRP_domain1” (CDD ID: cd03250) for NBD1 and “ABCC_MRP_domain2” (CDD ID: cd03244) for NBD2 were found in the aa407–603 and aa1174–1394 regions, respectively. CsMRP4 had a single four-domain organization of MSD1-NBD1-MSD2-NBD2, which is common to all short forms of the ABCC subfamily, such as MRP4, 5, 8, 9, and CFTR .
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Like in typical MRPs, there were several conserved motifs in the NBD1 of CsMRP4, such as an ATP-binding site (441GCxKSSx26Qx78DDx31N585), ABC transporter signature motif (528LSGGQKARIG537), Walker A/P-loop (438GPVGCGKS445), Walker B (548FLLLDD553), D-loop (556AAVD559), Q-loop/lid (470YMPQ473), and H-loop/switch region (581LLVTNQL587). These motifs play pivotal roles in transporting substrates via conformational changes between outward-facing and inward-facing forms. Dimerization of two NBDs forms the nucleotide-binding site between the Walker A/P-loop, Walker B, and ABC signature motif. The bound ATP is hydrolyzed to provide energy in order to efflux endogenous and xenobiotic substrates from cells to the extracellular milieu .
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An NJ method-based phylogenetic inference verified that CsMRP4 belongs to the MRP/SUR/CFTR subfamily by comparing it with the 12 members of subfamily C and 11 members of subfamily B of the ABC family (Fig. 1). Members of the ABCB subfamily were clearly out-grouped as one cluster despite the close similarity in terms of both sequence and structure between MRP and P-glycoprotein (P-gp) of ABCB members . Moreover, CsMRP7 (GenBank ID: AOE23877.1), which was annotated using the same approach, was grouped with HsMRP7.Fig. 1Phylogenetic relationship between CsMRP4 and members of the MRP/SUR/CFTR subfamily. Bootstrap values (1000 replicates) are shown next to the branches. Scale bar represents amino acid substitutions. UniProtKB/Swiss-Prot IDs for the ABCC subfamily are shown in parentheses. IDs for the ABCB subfamily are P-gp1 (P08183), ATP1 (Q03518), ATP2 (Q03519), P-gp3 (P21439), ABCB5 (Q2M3G0), MT-ABC3 (Q9NP58), ABC7 (O75027), M-ABC1 (Q9NUT2), TAPL (Q9NP78), M-ABC2 (Q9NRK6), and BSEP (O95342)
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Phylogenetic relationship between CsMRP4 and members of the MRP/SUR/CFTR subfamily. Bootstrap values (1000 replicates) are shown next to the branches. Scale bar represents amino acid substitutions. UniProtKB/Swiss-Prot IDs for the ABCC subfamily are shown in parentheses. IDs for the ABCB subfamily are P-gp1 (P08183), ATP1 (Q03518), ATP2 (Q03519), P-gp3 (P21439), ABCB5 (Q2M3G0), MT-ABC3 (Q9NP58), ABC7 (O75027), M-ABC1 (Q9NUT2), TAPL (Q9NP78), M-ABC2 (Q9NRK6), and BSEP (O95342)
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Several MRP4-related PDB structures were found from the PSI-BLAST search with experimentally characterized PDB structures, although there is no solved PDB structure of MRP4 elucidated so far. YASARA Structure selected the templates, such as Bos taurus MRP1 (PDB ID: 5UJA) with 35.2% identity, Caenorhabditis elegans P-gp (PDB ID: 4F4C) with 19.1% identity, MmMRP1 (PDB ID: 4M1M) with 18.9% identity, and MmMRP1 (PDB ID: 4Q9H) with 18.3% identity. Each homology model, built with each template, was refined by unrestrained high-resolution energy minimization using the latest knowledge-based YASARA force field . As a final model, a hybrid homology model was assembled by combining the best scoring parts of the four models and then refined with energy minimization. Out of 1469 residues, 1425 residues were modeled, omitting 44 C-terminal residues, since YASARA Structure does not perform ab initio or threading modeling (Fig. 2).Fig. 2Structural characterization and conservation of CsMRP4. a A 3D homology model was built based on the solved structure templates. The α-helix and β-strand are depicted as ribbon diagrams, and coiled-coil is depicted as a line. TM α-helices are colored and numbered from the N-terminus (blue) to the C-terminus (red). The red rectangles indicate the entrance of inner cavity, and spheres correspond to residues coordinating ATP-binding sites. b TM α-helices, as viewed perpendicular to the horizontal plane marked with red arrowheads. c The degree of sequence conservation is colored using a color gradient from white (divergent) to red (conserved). Structural conservation corresponds to the radii of the backbone sausage representation, which is proportional to the root-mean-squared deviation at each position between structure alignments. PDB IDs of the identified homologs are as follows: 5W81_A, 5UAK_A, 5UJ9_A, 4C3Z_A, 2PZG_A, 3GD7_A, 4Q4J_B, 1R0Z_A, 2HYD_A, 4Q7M_B, 4Q4J_A, 5DGX_A, 5IDV_A, 4MYC_A, 3WMF_A, 5MKK_A, 1MV5_A, 4MRN_A, 3NH6_A, 2FFB_A, 5EUM_A, 4F4C_A, 2GHI_A, 5MKK_B, 4Q9H_A, 4AYW_A, 3VX4_A, 5U1D_B, 5U1D_A, 4PL0_A, 5L22_B, 4U00_A, 4K8O_A, 4MKI_B, 4HUQ_B, 3TUJ_C, 5NIK_J, and 5JSZ_A. Structural alignment and image rendering were carried out using ENDscript and PyMOL (See details in the Methods section)
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Structural characterization and conservation of CsMRP4. a A 3D homology model was built based on the solved structure templates. The α-helix and β-strand are depicted as ribbon diagrams, and coiled-coil is depicted as a line. TM α-helices are colored and numbered from the N-terminus (blue) to the C-terminus (red). The red rectangles indicate the entrance of inner cavity, and spheres correspond to residues coordinating ATP-binding sites. b TM α-helices, as viewed perpendicular to the horizontal plane marked with red arrowheads. c The degree of sequence conservation is colored using a color gradient from white (divergent) to red (conserved). Structural conservation corresponds to the radii of the backbone sausage representation, which is proportional to the root-mean-squared deviation at each position between structure alignments. PDB IDs of the identified homologs are as follows: 5W81_A, 5UAK_A, 5UJ9_A, 4C3Z_A, 2PZG_A, 3GD7_A, 4Q4J_B, 1R0Z_A, 2HYD_A, 4Q7M_B, 4Q4J_A, 5DGX_A, 5IDV_A, 4MYC_A, 3WMF_A, 5MKK_A, 1MV5_A, 4MRN_A, 3NH6_A, 2FFB_A, 5EUM_A, 4F4C_A, 2GHI_A, 5MKK_B, 4Q9H_A, 4AYW_A, 3VX4_A, 5U1D_B, 5U1D_A, 4PL0_A, 5L22_B, 4U00_A, 4K8O_A, 4MKI_B, 4HUQ_B, 3TUJ_C, 5NIK_J, and 5JSZ_A. Structural alignment and image rendering were carried out using ENDscript and PyMOL (See details in the Methods section)
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The final model proved highly accurate based on the following validation. The overall Z-score of the resulting hybrid model was -1.4 using internal quality evaluation of YASARA Structure. A Z-score indicates the number of standard deviations the model quality is away from the average high-resolution X-ray structure. Moreover, a Ramachandran plot of the final model showed that 90.7% of all the residues were found in the most favored regions, 8.4% in additional allowed regions, and only 0.3% in disallowed regions (Additional file 5: Figure S3). These results indicated that the backbone dihedral angles were highly accurate. The ERRAT value, as an overall quality score, was 98.4% (Additional file 6: Figure S4). The final model of the CsMRP4 in PDB format can be found in Additional file 7.
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The 3D structure formed MSD1-NBD1-MSD2-NBD2 as a common structural fold of ABC transporters (Fig. 2a). MSD1 and MSD2 were made up of TM1–6 and TM7–12, respectively (Fig. 2b). When CsMRP4 was compared structurally with the 38 homologs from PDB entries using ENDscript with strict parameters, two NBDs were found to be highly conserved and two MSDs were less conserved (Fig. 2c). Furthermore, NBD2 of CsMRP4 was significantly more conserved than NBD1. At the sequence level, even though CsMRP4 was compared with five short forms of the ABCC subfamily, NBD2 contained 56 identical residues but NBD1 had only 41 identical residues (Fig. 3a, b). These results corroborate previous findings showing that NBD2 of CsMRP7 is more conserved than NBD1 at the structural level .Fig. 3Comparison of the amino acid sequence of CsMRP4 with short forms of the ABCC subfamily. CsMRP4 was aligned with HsMRP4, 5, 8, 9, and CFTR using MAFFT and rendered using ESPript. Out of the alignment profile, the NBD1 region (a) and NBD2 region (b) were selected for visualization. Red bold and red letters indicate identical and similar amino acid residues, respectively. Conserved sequences are indicated by a box if more than 70% of the residues are similar
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Comparison of the amino acid sequence of CsMRP4 with short forms of the ABCC subfamily. CsMRP4 was aligned with HsMRP4, 5, 8, 9, and CFTR using MAFFT and rendered using ESPript. Out of the alignment profile, the NBD1 region (a) and NBD2 region (b) were selected for visualization. Red bold and red letters indicate identical and similar amino acid residues, respectively. Conserved sequences are indicated by a box if more than 70% of the residues are similar
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Various substrates were moved out via binding with MRP4 transporters. The export of inhibitors and bile acids can confer drug resistance and bile recirculation, respectively. Three MRP1 proteins and a P-gp, used as the four PDB templates, showed an open inward-facing conformation with inner cavity, which appears to be suitable for substrate uptake. These templates provided a possible structural foundation to perform in silico prediction of ligand binding by docking simulation. Thus, we investigated ligands and their binding sites for CsMRP4 using two methods. First, probable ligands were analyzed based on the identification of analogs with similar binding sites as the solved structures using COACH . Then, Mg2+ and ATP were predicted to bind to NBD1 and NBD2 of CsMRP4 based on data for HsMRP1 (Table 1 and Fig. 2a). Mg2+ is necessary for ATP hydrolysis and results in the formation of Mg-ATP dimers . Moreover, the cyclic peptide inhibitor, QZ59-SSS (a.k.a. OZ-VAL or 2 J8), was predicted to bind to CsMRP4 according to the ligand-bound pockets of three experimentally characterized P-glycoproteins [43–45] (Table 1). Among them, Ile at the position 1093 was commonly involved in coordinating the inhibitor.Table 1Ligand and ligand-binding residues predicted using COACHLigandRegionConsensus binding residuesPDB templateATPNBD1W412, T420, V440, G441, C442, G443, K444, S445, S446, Q4732CBZ_AMg2+ NBD1S445, Q4732CBZ_AAMP-PNPa NBD2Y1185, A1192, T1212, G1213, A1214, G1215, K1216, S1217, S1218, V1227, Q1258, E1338, H13692ONJ_AMg2+ NBD2S1217, Q1258, D1337, E1338, V13674FWI_B2J8CavityL94, P98, M101, S348, L864, I1093, V10974M2T_AQZ59-VALCavityP98, I10934Q9J_A0JZCavityY349, L353, I1093, I1118, V11223G61_B aAMP-PNP is an ATP analogue
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Secondly, docking simulations were performed using AutoDock Vina to evaluate the binding energies of CsMRP4 with nine bile acids (Table 2 and Fig. 4). All the bile acids tested bound favorably to the inner cavity of CsMRP4 (Fig. 4a). Taurolithocholic acid (TLCA) (Fig. 4b) and LCA (Fig. 4c) showed the highest affinities with CsMRP4, whereas deoxycholic acid (DCA) (Fig. 4i) and cholic acid (CA) (Fig. 4j) revealed moderate affinities. Interestingly, our docking results are in line with previous transport assay data, which indicated that TLCA bound favorably to MRP4 at a low concentration, but other bile acids needed much higher concentrations . TLCA had the highest affinity for MRP4 overexpressed in HEK cells, followed by taurochenodeoxycholic acid (TCDCA), taurodeoxycholic acid (TDCA), taurocholic acid (TCA), glycocholic acid (GCA) and cholic acid (CA). We also then added more primary and secondary bile acids such as LCA, chenodeoxycholic acid (CDCA), and DCA. Among them, LCA at 2–4 μM concentration was reported to have a significant adverse effect on the survival of juvenile C. sinensis . Thus, the high affinity of LCA could be required for removing LCA from the worm’s body for survival. However, these findings remain to be established at the biochemical level, which needs to be studied in the future.Table 2Docking results between CsMRP4 and bile acids using AutoDock VinaBile acidsPubChem IDBinding energy (kcal/mol)No. of configurationsTaurolithocholic acid (TLCA)a SID 103579026-13.43Lithocholic acid (LCA)SID 103542513-12.23Taurochenodeoxycholic acid (TCDCA)a SID 312642451-10.15Chenodeoxycholic acid (CDCA)SID 24875071-9.97Taurodeoxycholic acid (TDCA)a CID 2733768-9.75Taurocholic acid (TCA)a SID 828139-9.36Glycocholic acid (GCA)a SID 177011773-9.27Deoxycholic acid (DCA)CID 222528-8.45Cholic acid (CA)a SID 223730521-8.15 aBinding affinities of bile acids to MRP4 in HEK cells Fig. 4Best docking conformation of bile acids with CsMRP4. The 3D model of CsMRP4 is visualized as a white ribbon diagram, which is depicted in transparency (90%) to show the bile acid docked deep into the inner cavity (a). The red circle indicates the inner cavity for bile acid binding. The results are arranged according to the order of calculated binding affinities of complex bile acid-CsMRP4, as listed in Table 2: b TLCA, c LCA, d TCDCA, e CDCA, f TDCA, g TCA, h GCA, i DCA, j CA
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Best docking conformation of bile acids with CsMRP4. The 3D model of CsMRP4 is visualized as a white ribbon diagram, which is depicted in transparency (90%) to show the bile acid docked deep into the inner cavity (a). The red circle indicates the inner cavity for bile acid binding. The results are arranged according to the order of calculated binding affinities of complex bile acid-CsMRP4, as listed in Table 2: b TLCA, c LCA, d TCDCA, e CDCA, f TDCA, g TCA, h GCA, i DCA, j CA
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CsMRP4 mRNA was expressed at both developmental stages, in the metacercariae and in the adults, but the expression in the metacercariae was 1.91 times higher (Fig. 5a). This result suggested that the metacercariae might need the transcript for the efflux of bile acids from the fluke’s body during their survival in the bile duct of the final host. In the metacercarial stage of C. sinensis, diverse genes have been reported to be highly expressed in response to environmentally induced changes, such as sodium/bile acid cotransporter and several heat-shock proteins . Recently, the mRNA level of CsMRP7 was also reported to be elevated in the metacercariae .Fig. 5Differential expression of the CsMRP4 and reactivity of mouse anti-NBD1 immune serum. a Relative mRNA level of CsMRP4 gene in the adults and metacercariae of C. sinensis, measured using Q-rt.-PCR. b Reaction detected by western blot and immuno-ECL. The mouse immune serum reacted well with rCsMRP4-NBD1 (left) and specifically detected native CsMRP4 (right) in the crude extracts of C. sinensis. Lane NBD1: mouse anti-NBD1 immune serum; Lane Normal: normal mouse serum; Abbreviations: R, recombinant protein; N, native CsMRP4
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Differential expression of the CsMRP4 and reactivity of mouse anti-NBD1 immune serum. a Relative mRNA level of CsMRP4 gene in the adults and metacercariae of C. sinensis, measured using Q-rt.-PCR. b Reaction detected by western blot and immuno-ECL. The mouse immune serum reacted well with rCsMRP4-NBD1 (left) and specifically detected native CsMRP4 (right) in the crude extracts of C. sinensis. Lane NBD1: mouse anti-NBD1 immune serum; Lane Normal: normal mouse serum; Abbreviations: R, recombinant protein; N, native CsMRP4
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| 100.0 |
The tissue distribution of CsMRP4 in both adults and metacercariae was investigated via immunohistochemistry. The NBD1 region was chosen as the immune antigen for mice immunization, since it was more specific than NBD2 and the non-membrane-spanning region. With this strategy, we successfully produced and purified NBD1 of ABCC subfamily transporters . The 6× histidine-tagged rCsMRP4-NBD1 was then purified using Ni-NTA agarose (Additional file 8: Figure S5). The mouse anti-CsMRP4-NBD1 immune serum reacted well with rCsMRP4-NBD1 (24.9 kDa) by apparently detecting the native CsMRP4 in the crude extract of the adult worm (Fig. 5b). It was, therefore, applied to immunohistochemical staining. CsMRP4 was distributed mainly in the oral sucker and mesenchymal tissues of the adults (Fig. 6a, b) and metacercariae (Fig. 6c). Moreover, the ventral sucker of the metacercariae showed strong localization of CsMRP4.Fig. 6Localization of CsMRP4 in C. sinensis adults (a, b, d, e) and metacercariae (c, f) detected by immunohistochemistry. Top panels (a - c) were stained with mouse anti-NBD1 immune serum and bottom panels (d - f) were stained with normal mouse serum. Abbreviations: EB, excretory bladder; MT, mesenchymal tissue; OS, oral sucker; T, testis; VS, ventral sucker
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study
| 100.0 |
Localization of CsMRP4 in C. sinensis adults (a, b, d, e) and metacercariae (c, f) detected by immunohistochemistry. Top panels (a - c) were stained with mouse anti-NBD1 immune serum and bottom panels (d - f) were stained with normal mouse serum. Abbreviations: EB, excretory bladder; MT, mesenchymal tissue; OS, oral sucker; T, testis; VS, ventral sucker
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study
| 99.94 |
CsMRP4 was mainly localized in the mesenchymal tissues in our study. In trematodes, several transporters have been found to be expressed in mesenchymal tissues. BSEP and MRP1 of adult Fasciola hepatica have been shown to be localized in not only mesenchymal tissues but also the tegumental cell layer, implying that the two transporters facilitate the diffusion of bile salts and chemicals in flukes . CsMRP7, which might be involved in drug resistance, has also been found to be expressed in the mesenchymal tissues . It is therefore speculated that mesenchymal tissues may be regions of strategic importance for transporter functions throughout the body of flukes.
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study
| 100.0 |
The function of CsMRP4 seems to be to export bile acids from the worm’s body, which is similar to the function of typical MRP4. C. sinensis adults and metacercariae immerse themselves in bile juice, which can exert toxic effects and impair the tissues and cells of the worm’ bodies. Bile acids have also been reported to decrease the locomotive cycles of juvenile F. hepatica and to provoke parasite death . Together, these data suggest that C. sinensis needs to dilute the high concentrations of bile acids in the interior of the body by pumping them out and that CsMRP4 plays a role in transporting bile acids in coordination with other bile acid exporters.
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study
| 100.0 |
In summary, we cloned and characterized CsMRP4 using computational, molecular, and biochemical approaches in this study. In addition to structural similarities, sequence similarities were also found between CsMRP4 and human MRP4 (39% identity), and CsMRP4 was confirmed as belonging to the ABCC family. A reliable tertiary structure of CsMRP4 was also modeled and shown to have a common structural fold, MSD1-NBD1-MSD2-NBD2. When binding affinities of CsMRP4 with nine bile acids were tested through virtual docking simulation, the results indicated that CsMRP4 could be regarded as a bile transporter. The NBD2 of CsMRP4 was conserved more than NBD1, which was therefore used as a CsMRP4-specific antigen for subsequent immunohistochemistry experiments. In the metacercariae and adults of C. sinensis, CsMRP4 was found to be mainly distributed in mesenchymal tissues, which suggested that these tissues are regions of strategic importance for transporter functions throughout the fluke’s body. These findings suggest that CsMRP4 plays a role in exporting bile acids and inhibitors. The results from this study will also serve as a platform for further research on other bile transporters and homologues in flukes.
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study
| 100.0 |
Additional file 1: Table S1.Primer sets used to amplify CsMRP4 cDNA fragments by PCR. (PDF 20 kb) Additional file 2: Figure S1.Strategy for obtaining the entire coding cDNA sequence of the CsMRP4 gene. a Whole cDNA (4410 bp) was confirmed by combining 5′-RACE PCR and DNA-walking. The used primers are listed in Additional file 1: Table S1. UPM, GSP1, GSP2, and NGSP were primers used for 5′-RACE PCR. CsMRP4-I-F/R and CsMRP4-II-F/R primers were designed for amplification of CsMRP4-I and II fragments through DNA-walking. SF1, SR1, SF2, SR2, and F3 were used for sequencing. b Amplification of the missing 5′-end using RACE-PCR. c RACE-PCR products were confirmed using nested-PCR with UPM and NGSP primers. d The putative CsMRP4 (GenBank ID: GAA49862.1) was confirmed by DNA-walking. (TIFF 3873 kb) Additional file 3: Figure S2.The full cDNA coding sequence and deduced polypeptide sequence of CsMRP4. Through 5′-RACE and DNA-walking, the whole cDNA of 4410 bp was verified to encode a polypeptide of 1496 aa. (TIFF 5203 kb) Additional file 4: Table S2.Identities calculated between CsMRP4 and MRP/SUR/CFTR subfamily members. (PDF 29 kb) Additional file 5: Figure S3.The residue-by-residue stereochemical quality of the CsMRP4 3D model. Ramachandran plot showed the residues in the most favored regions (90.7%), additional allowed regions (8.4%), generously allowed regions (0.6%), and disallowed regions (0.3%). Red (A, B, L), yellow (a, b, l, p), and light yellow (~a, ~b, ~l, ~p) indicate the most favored regions, allowed regions, and generously allowed regions, respectively. White indicates disallowed regions. All the non-glycine and non-proline residues are shown as closed black squares, while glycines (non-end) are shown as closed black triangles. Disallowed residues are colored in red. (TIFF 993 kb) Additional file 6: Figure S4.Accuracy of the non-bonded atomic contacts of the CsMRP4 3D model. The ERRAT plot shows the overall quality factor of 98.45%. (TIFF 1786 kb) Additional file 7:CsMRP4.pdb. The model of CsMRP4 was built using YASARA. (PDB 1780 kb) Additional file 8: Figure S5.Amplification of CsMRP4-NBD1 (a) and purification of the recombinant protein (b). Abbreviations: M, molecular marker (kDa); U, uninduced total lysate; I, induced total lysate; S, urea-treated clear supernatant; PT, Ni-NTA pass-through fraction; W, last washing; Elute 1–6, 1st to 6th fraction eluted from an Ni-NTA column. (TIFF 2938 kb)
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study
| 100.0 |
Strategy for obtaining the entire coding cDNA sequence of the CsMRP4 gene. a Whole cDNA (4410 bp) was confirmed by combining 5′-RACE PCR and DNA-walking. The used primers are listed in Additional file 1: Table S1. UPM, GSP1, GSP2, and NGSP were primers used for 5′-RACE PCR. CsMRP4-I-F/R and CsMRP4-II-F/R primers were designed for amplification of CsMRP4-I and II fragments through DNA-walking. SF1, SR1, SF2, SR2, and F3 were used for sequencing. b Amplification of the missing 5′-end using RACE-PCR. c RACE-PCR products were confirmed using nested-PCR with UPM and NGSP primers. d The putative CsMRP4 (GenBank ID: GAA49862.1) was confirmed by DNA-walking. (TIFF 3873 kb)
|
study
| 99.94 |
The residue-by-residue stereochemical quality of the CsMRP4 3D model. Ramachandran plot showed the residues in the most favored regions (90.7%), additional allowed regions (8.4%), generously allowed regions (0.6%), and disallowed regions (0.3%). Red (A, B, L), yellow (a, b, l, p), and light yellow (~a, ~b, ~l, ~p) indicate the most favored regions, allowed regions, and generously allowed regions, respectively. White indicates disallowed regions. All the non-glycine and non-proline residues are shown as closed black squares, while glycines (non-end) are shown as closed black triangles. Disallowed residues are colored in red. (TIFF 993 kb)
|
study
| 99.94 |
Amplification of CsMRP4-NBD1 (a) and purification of the recombinant protein (b). Abbreviations: M, molecular marker (kDa); U, uninduced total lysate; I, induced total lysate; S, urea-treated clear supernatant; PT, Ni-NTA pass-through fraction; W, last washing; Elute 1–6, 1st to 6th fraction eluted from an Ni-NTA column. (TIFF 2938 kb)
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other
| 99.75 |
Limitless replicative potential is considered a hallmark of cancer, which is achieved by an inappropriate reactivation of the essential enzyme telomerase [1, 2]. Telomerase maintains telomere integrity by adding the six-nucleotide repeat sequence, 5′-TTAGGG, to the ends of the chromosomes using its internal template RNA component (TERC) and reverse transcriptase protein component (TERT), thus counteracting the telomere shortening that naturally occurs during DNA replication [3–6]. Telomerase is not expressed in most adult somatic cells and the telomeres are therefore progressively shortened with each round of replication, which ultimately leads to replicative senescence and thus a finite replicative potential. In contrast, stem cells and cancer cells have high telomerase activity. In fact, TERT has been shown to be overexpressed in almost 90% of all human malignancies, but although there is evidence that mutations in the TERT promoter lead to enhanced TERT expression, the mechanisms by which telomerase is reactivated is still poorly understood [7–11].
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study
| 81.7 |
microRNAs (miRs) are a class of small (∼22 nucleotide) non-coding RNAs, which are loaded onto Argonaute (Ago) proteins forming the miR-induced silencing complex (miRISC) and function as post-transcriptional regulators of gene expression by inducing mRNA instability or translational repression . More than 60% of all protein-coding genes are believed to be subjects of miR regulation and alterations in miR expression can therefore have dire consequences and contribute to the development of a wide variety of human diseases, including cancer [12–17]. Depending on their role in carcinogenesis, miRs can generally be divided into oncogenic miRs (oncomiRs) or tumor suppressor miRs that promote or inhibit tumor development and progression, respectively [17–19]. miRs are suspected to be implicated in telomerase reactivation and a subset has been shown to affect telomerase activity directly, e.g. by inducing TERT expression. For instance, miR-138 has been reported to function as a direct regulator of TERT expression in thyroid carcinoma and at least 5 additional tumor suppressor miRs (let-7g, miR-133a, miR-342, miR-491 and miR-541) have been shown to be capable of regulating TERT expression through direct interaction with TERT mRNA [20, 21].
|
review
| 99.75 |
Aberrant expression of miR-128 is a frequent observation in human malignancies, but depending on the tumor type, it has been shown to be capable of acting both as an oncomiR and a tumor suppressor miR [22, 23]. Most studies have, however, found it to act as a tumor suppressor and downregulation has been documented in a long list of human malignancies, including glioma, prostate, head and neck, lung and colorectal cancer, where it has been shown to function as an inhibitor of cancer cell growth and metastasis [22, 24–28]. We recently demonstrated that miR-128 regulates another cellular reverse transcriptase, namely the Long-Interspersed Element-1 (LINE-1 or L1) by directly interacting with ORF2 L1 RNA, which encodes L1 RT and indirectly by regulating a required host factor (Transportin-1 or TNPO1) needed for nuclear import of L1-RNP complexes [29, 30]. De-repression of L1 elements have been demonstrated to function as driver mutations during tumor initiation, as well as during tumor progression [31–37].
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study
| 99.94 |
In this study, we identified miR-128 as a regulator of telomerase activity in an anti-miR library screen, demonstrating that endogenously expressed miR-128 inhibits telomerase activity in HeLa cells. Furthermore, we found that overexpression of miR-128 decreased TERT mRNA and protein levels and miR-128 depletion enhanced the levels of TERT mRNA and protein, relative to controls, in a panel of cell lines. Finally, we demonstrate that miR-128 regulates telomerase activity by directly targeting two sites in the coding region of TERT mRNA. These findings show that tumor suppressor miR-128 also effect the oncogenic phenotype of cancer cells by regulating telomerase.
|
study
| 100.0 |
We have recently established that miRs (miR-128) can repress the activity of key enzymes in our cells, such as reverse transcriptase (RT) encoded by transposable elements (long-Interspersed element-1, LINE-1 or L1) . With this in mind we turned our attention to the most famous RT in human cells – telomerase – an enzyme which plays a crucial role in cancer, stem cells and aging [7–9].
|
study
| 99.94 |
We developed a lentiviral anti-miR screen as a way to identify miRs that play a regulatory role of telomerase in HeLa cells. In brief, we transduced HeLa cells with an anti-miR library encoding conserved and well-characterized miRs that neutralize the corresponding endogenously expressed miR in the transduced HeLa cells. The lentivirus also encodes copGFP (green) and puromycin for positive selection of transduced cells. This approach favors a physiologically relevant response by avoiding potential artifacts resulting from ectopic overexpression in cells, which does not normally express a specific miR. Following transduction of the anti-miR library or control miRs, we performed single cell dilutions into 96-well plates and performed a qPCR based functional assay of telomerase activity, using the telomeric repeat amplification protocol (q-TRAP) . The q-TRAP assay involves extension of an oligonucleotide through telomerase-mediated enzymatic addition of telomeric DNA repeats and subsequent PCR amplification of the extension products and serves as a great high throughput functional assay for telomerase activity in cells (Figure 1A). Shown in Figure 1B is a panel of miR controls (Controls) and one positive control, tested in parallel with samples, which had been transduced with the anti-miR library (Samples) and plated in single cell dilution and analyzed for telomerase activity.
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study
| 100.0 |
(A) Schematic of the anti-miR library screen to identify miRs that regulate telomerase activity. HeLa cells were transduced using a lentiviral-based, miR-neutralizing shRNA library, selected for puromycin resistance and clonally expanded. Each well represents the neutralization of a single endogenously expressed miR. Cells were then subjected to quantitative PCR-based TRAP assay (q-TRAP) analysis using the quantitative telomeric repeat amplification protocol. (B) Relative telomerase activity of HeLa cells after transduction with lentiviral miR-neutralizing anti-miR library, selection, and clonal expansion as measured by quantitative telomeric repeat amplification protocol (q-TRAP) as shown. Shown is a panel of miR controls (Controls), relative to samples transduced with the anti-miR library (Samples). (C) Secondary measurement of relative telomerase activity in select samples of anti-miR library-expressing HeLa cells, as described for Figure 1B. (D) Single high-titer miR-control, miR-128 and anti-miR-128 lentiviruses were generated and stable miR-modulated HeLa cells were assayed for relative telomerase activity measured by q-TRAP analysis. Results shown as percent change ± SEM (n = 3, independent biological replicates, *p < 0.05, ****p < 0.0001).
|
study
| 100.0 |
We identified anti-miR-128 as an anti-miR, which significantly de-repress telomeric repeat amplification, as determined by q-TRAP analysis, (Figure 1B). Of note, miR-128 was cloned out from 4 independent clones. We repeated the assays with a subset of miR-modulated HeLa samples and verified that anti-miR-128 enhances telomerase activity (Figure 1C). To test the specificity of the anti-miR-128 effect on telomerase activity, we generated high titer miR-128, anti–miR-128 and control miR lentiviruses, transduced HeLa cells and selected for puromycin resistance. The miR-128 cell line panel was then evaluated for telomerase activity as described using the q-TRAP assay. As expected anti-miR-128 significantly increased HeLa cell telomerase activity, compared to cells expressing endogenous miR-128 (Control) (Figure 1D). In contrast, miR-128 significantly reduced the level of telomeric repeat amplification, relative to miR controls (Figure 1D). These experiments suggest that miR-128 regulates telomerase activity in HeLa cells.
|
study
| 100.0 |
We next wished to characterize the mechanism by which miR-128 regulates telomerase activity. First, we performed RT-qPCR analysis of HeLa cells in order to determine if miR-128 acts by regulating the amount of TERT mRNA. Induced miR-128 expression significantly decreased TERT mRNA levels, relative to miR controls, whereas miR-128 neutralization by anti-miR-128 resulted in an enhanced amount of TERT mRNA, compared to HeLa miR control samples (Figure 2A). In addition, miR-128 overexpression resulted in significant degreased TERT mRNA levels in a teratoma cell line (Tera-1 or Tera) and in two iPSC cell lines, relative to miR controls (Supplementary Figure 1). anti-miR-128 showed a similar tendency as seen in HeLa cells, though less substantial in the iPS cell lines (Supplementary Figure 1).
|
study
| 100.0 |
(A) miR-modulated HeLa cell lines were generated (over-expressing miR-128, anti-miR-128 or control miR). Relative expression levels of Tert RNA were determined and normalized to beta-2-microglobulin (B2M) expression levels. (B) Stably miR-128, anti-miR-128 or control miR control HeLa cells lines were analyzed by western blot analysis using antibodies against Tert and α-tubulin. One representative example of three is shown. Quantification of results (n = 3) normalized to α-tubulin is shown (right panel). (C) Stable miR-128, anti-miR-128 and control miR HeLa cell lines were analyzed by immunofluorescence for Tert expression and co-localization with DAPI. Quantification of results is shown (bottom panel) (n = 3). (D) Stable A549, SW620 and PANC1 miR-modulated (miR-128, anti-miR-128 and control miR) cell lines were generated and Western blot analysis were performed using Tert and α-tubulin antibodies. Quantification of results (n = 3) normalized to α-tubulin is shown (bottom panels). All results are shown as mean ± SEM, n = 3, independent biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001, by two-tailed Student’s t test.
|
study
| 100.0 |
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