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The resuscitation of VBNC cells can be triggered by a variety of stimuli factors, such as an increase in the nutrient concentration, increases or decreases in temperature, the presence of chemical stimuli and even co-cultivation with host cells. In 1984, rich medium was first used to resuscitate VBNC cells in S. enteritidis by Roszak et al. (1984). Since then, to identify the factors that stimulate the recovery of VBNC cells, a number of researchers have successively performed resuscitation experiments under different conditions.
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study
| 99.94 |
An increase in temperature is a common physical stimulus to resuscitate most VBNC cells induced by low temperature, such as E. coli O157:H7, A. hydrophila, S. typhimurium, S. dysenteriae, Vibrio spp., E. faecalis, and S. aureus (Table 1). Resuscitation can also be mediated by different kinds of chemical stimuli, including sodium pyruvate (Lleo et al., 2001; Pinto et al., 2011; Morishige et al., 2013; Pasquaroli et al., 2013), amino acids (Pinto et al., 2011), and Tween 80 (Trinh et al., 2015). It is worth mentioning that researchers have different views on the resuscitation role of pyruvate. On the one hand, it is deliberated whether pyruvate cannot recover VBNC cells. Li et al. (2014) reported that the VBNC cells of S. typhimurium could not be recovered by supplementation with antioxidants such as pyruvate, catalase or oxyrase, but could be resuscitated by an autoinducer. On the other hand, some people insist that pyruvate has a significant effect on the resuscitation of VBNC strains. For example, Pinto et al. (2011) found that starving cells of E. coli could easily enter the VBNC state after the addition of pyruvate. Morishige et al. (2013) also discovered that the VBNC cells of S. enteritidis caused by H2O2 stress could regain culturability by the addition of sodium pyruvate but not pyruvate analogs (like phenyl pyruvate or bromoacetone), thus confirming that pyruvate was one of the key molecules in the process of resuscitation by triggering the synthesis of macromolecules such as DNA and protein.
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study
| 99.6 |
In addition, VBNC cells can also be resuscitated by biological stimuli such as eukaryotic cells. Senoh et al. (2010) found that the VBNC cells of V. cholerae could be converted into a culturable state after co-cultivating with eukaryotic cells. However, another study showed that VBNC C. jejuni cells could form colonies on agar plates after co-cultivation with Caco-2 cells, but most VBNC cells could not be resuscitated (Chaisowwong et al., 2012), which indicated that the presence of host cells was a biological stimulus factor that can trigger the resuscitation of a fraction of VBNC cells. Additionally, Imamura et al. (2015) discovered a phenomenon in which VBNC cells of V. cholerae were initially converted into a culturable state by treatment with HT-29 cell extract or catalase but subsequently entered a state from which they could not be resuscitated. These non-resuscitated cells were verified to be viable by fluorescence microscopy and could be resuscitated by co-cultivation with HT-29 human colon adenocarcinoma cells. However, all cells entered a state from which they could not be resuscitated, even by co-cultivation with HT-29. Thus, the VBNC cells that were resuscitated by biological factors could not be maintained for a long time because the requirements for resuscitated VBNC cells changed over time such that the HT-29 cells could not always maintain resuscitation of the VBNC cells.
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study
| 99.94 |
Although the resuscitation mechanism of VBNC cells remains largely unknown, studies examining resuscitation from the VBNC state have provided many promising results, and people have gradually acquired a greater understanding of the resuscitation mechanism with the development of molecular biology. Resuscitation promoting factor (Rpf), a highly conserved protein composed of 220 amino acids that is directly related to the resuscitation of VBNC cells, has been demonstrated to restore the growth and reproductive ability of VBNC cells (Mukamolova et al., 1998). Rpf proteins have been shown to act as cytokines that, when secreted into the medium by growing cells, bind to the surface receptors of dormant cells and trigger resuscitation (Pinto et al., 2015). Panutdaporn et al. (2006) also showed that the growth of S. enterica serovar Oranienburg cells could be enhanced by a certain concentration of rRpf protein. Moreover, Pinto et al. (2011) observed that Rpf supernatant fluid treated with proteinase K could resuscitate the VBNC cells of E. coli, suggesting that the breakdown products of Rpf could also restore VBNC cells. In addition, Pinto et al. (2013) validated the hypothesis that two Rpfs of L. monocytogenes, Lmo0186 and Lmo2522, could promote resuscitation via a mechanism analogous to actinobacteria Rpf proteins. Although the mechanism of Rpf in VBNC cell resuscitation is still not well understood, most researchers believe that the mechanism of Rpf is similar to that of lysozyme, both of which play a role in hydrolysis to divide the peptidoglycan in the cell wall (Keep et al., 2006a). There are two viewpoints regarding the mechanism of Rpf (Figure 2): one is that the breakdown product(s) of peptidoglycan by Rpf may interact with other factors and function as ‘second messengers’ to stimulate the resuscitation and growth of VBNC cells; the other is that Rpf is required to cleave peptidoglycans with inhibitory properties that are distributed in specific areas of the dormant cell wall and thus promote cell division and growth resumption (Keep et al., 2006b). To provide insights into the regulatory mechanism of Rpf protein, Aydin et al. (2011) obtained high-level expression of recombinant V. parahaemolyticus YeaZ in E. coli to determine the atomic structure and elucidate the three-dimensional structural conservation in YeaZ homologs, which may broaden perspectives regarding the mechanism of Rpf. However, the mechanism of Rpf and its breakdown products are still not clear and require further study.
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review
| 99.6 |
Two viewpoints about the mechanism of Rpfs. (1) Rpfs are required to cleave the peptidoglycans with inhibitory properties distributed in specific area of dormant cell wall and thus promote cell division and growth again. (2) The breakdown product(s) of peptidoglycan divided by Rpfs may interact with other factors and function as “second messengers” to stimulate the resuscitation and growth of VBNC cells.
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study
| 99.94 |
In contrast, Moorhead and Griffiths (2011) found that C. jejuni could respond to quorum-sensing (QS) signaling molecules (such as C4-HSL, 3OH-C4-HSL, C12-HSL, and HSL), which indicated that biofilm formation was blocked and the entry of cells into the VBNC state was delayed. It was speculated that QS signaling molecules were related to formation of the VBNC state. This conjecture was further confirmed by Ayrapetyan et al. (2014), who only found that the QS signaling molecule autoinducer-2 (AI-2) could directly awaken the VBNC cells of V. vulnificus but also that the AI-2 deletion mutant lost resuscitation ability from the VBNC state. Bari et al. (2013) also demonstrated that the resuscitation of dormant V. cholera was dramatically improved by the addition to the enrichment medium of biologically synthesized AIs, suggesting that these molecules might signal to dormant cells and then improve conditions for better growth. Furthermore, rpoS deletion mutant strains could not be resuscitated even with the addition of exogenous AI-2. This result shows that RpoS is not only a significant protein for VBNC formation but also an important participant in the resuscitation process mediated by AI-2.
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study
| 100.0 |
It is worth discussing whether VBNC pathogens maintain their pathogenicity if they are unable to be resuscitated. Although there is no relevant information to confirm that pathogenic bacteria that remain in the VBNC state can cause human disease, it has been reported that some VBNC pathogens retain pathogenic effects. For example, Oliver and Bockian (1995) described mice that were in a lethal state after inoculation with VBNC cells of Vibrio vulnificus. Amel et al. (2008) observed a fluid accumulation in the rabbit ileal loop assay (RICA) in response to VBNC V. cholerae O1. VBNC Legionella pneumophila that retained the capacity to infect the amoeba, which is its natural host (Al-Bana et al., 2014). Furthermore, one study demonstrated that VBNC enteropathogenic E. coli showed pathogenicity due to the continual production of enterotoxin (Pommepuy et al., 1996), and another study showed that uropathogenic E. coli that remained in the VBNC state may be the major causative agent of recurrent urinary tract infections in many individuals (Anderson et al., 2004). However, some VBNC pathogens have been confirmed to be non-pathogenic. Compared with culturable cells, the VBNC cells of L. monocytogenes were avirulent because of a failure to colonize the spleen of mice or adhere to HT-29 cells (Cappelier et al., 2007), VBNC C. jejuni cells were unable to colonize the caecum of newly hatched leghorn chicks (Ziprin et al., 2003) and VBNC Salmonella typhimurium could not infect all mice in the experiment (Habimana et al., 2014). In a word, when VBNC cells are unable to resuscitate in animals, some of them are still pathogenic and others are avirulent. The pathogenicity of VBNC cells in animals may differ due to different strains or the type of animals. To better understand the pathogenicity of VBNC cells, we propose to expand the research to include assessments of the pathogenicity of different VBNC cells and to attempt to distinguish the VBNC cells that can directly lead to disease.
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review
| 99.25 |
Interestingly, Amel and Amina (2008) found that VBNC S. typhimurium cells were only recovered into culturable cells by oral administration but not by intraperitoneal injection in mice, indicating that the intestinal environment might be an essential condition for resuscitation. However, Habimana et al. (2014) demonstrated that S. typhimurium failed to resuscitate during passage through the gastrointestinal tract. We speculate that there may be several reasons for the different pathogenic characteristics of the same strain in mice. First, the strains of S. typhimurium were not the same in the two experiments; the former was LT4 and the latter was ATCC 14028. Second, the differences between the experimental animals in the two experiments may have affected the results of the experiment, such as the different genders and ages. Third, the pressure that caused the bacteria to enter the VBNC state and the induction method may determine the resuscitation ability of VBNC cells. As shown in Table 1, most of the VBNC cell resuscitation methods are based on the method of induction, mainly stress relief, such as E. coli O157:H7 induced by low temperature and the corresponding increase in temperature for resuscitation. A similar hypothesis was also proposed by Habimana, who proposed that the phenomenon was dependent on how the pathogens were originally induced into a non-culturable state (Habimana et al., 2014). The specific cause of this phenomenon is still uncertain, requiring further experiments to reach a definitive conclusion.
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study
| 99.94 |
In the previous section (Introduction), we have described the role of VBNC pathogens in public health and food safety, and even their involvement in many foodborne outbreaks; however, there is no evidence to show that VBNC pathogens directly caused the outbreak. We propose that one of the most possible reasons is the undetectability of VBNC cells. Recent findings showed that 20% of illnesses can be linked to known pathogens, but the remaining 80% are due to unspecified or unidentified agents (Nicolò and Guglielmino, 2012), indicating that VBNC pathogens may be ignored during most outbreaks due to undetectability. For example, in 2011 in Germany, there was a large outbreak caused by an E. coli O104:H4 strain expressing genes characteristic of enterohemorrhagic (EHEC) and enteroaggregative E. coli (EAEC), involving more than 3000 cases of bloody diarrhea and haemolytic uremic syndrome. Unfortunately, the local detection department failed to detect or isolate the E. coli O104:H4 strain from the source of contamination until a small amount of the pathogens were isolated from the patients (Aurass et al., 2011; Scheutz et al., 2011). We suggest that the main reason for the failure to isolate the E. coli O104:H4 outbreak strain is that this outbreak strain entered a non-culturable state. Aurass et al. (2011) also confirmed that the E. coli O104:H4 outbreak strain induced by copper ions or tap water of outbreak area entered the VBNC state and was resuscitated to become a potentially pathogenic bacterium by stress relief. This finding implies that VBNC E. coli O104:H4 may invade the human body through contaminated food, undergo resuscitation and thus lead to disease.
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review
| 94.1 |
The presence of VBNC cells in food is widely documented (Rowan et al., 2015). Food is frequently exposed to a complex environmental system, in which physiochemical characteristics (pH, aw, disinfectant and chemical composition) and environmental factors (high pressure CO2, elevated temperatures, storage temperature and time, decontamination treatments, pasteurization and packaging under modified atmosphere) act simultaneously on contaminating bacteria leading to the VBNC state. This alone poses a significant risk to the public health and food safety, as these bacteria cannot be detected by commonly used techniques (Fakruddin et al., 2013). This risk is made even greater by the fact that VBNC cells can resuscitate within the human host (Ayrapetyan and Oliver, 2016). Furthermore, studies have proven that VBNC cells of foodborne pathogens, continue to produce virulence factors in food (Dinu and Bach, 2011). These studies indicate that more effective methods for detection of foodborne pathogen must be employed, to tackle the threat posed by VBNC bacteria with regard to public health and food safety.
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review
| 99.7 |
After decades of research, the VBNC concept has attracted great attention for a variety of foodborne pathogens and the corresponding adaptation mechanisms. It seems clear that the conditions, factors and regulators during the induction and resuscitation of the VBNC state play prominent roles in some strains. However, the formation and resuscitation mechanism of the VBNC state remain unclear and thus require further study. The abilities of VBNC cells to evade detection by conventional plate counting techniques, to tolerate stressful environments including food pasteurization processes and antibiotics, and to resuscitate with virulence and cause disease could pose a great threat to food safety and infectious disease prevention. Therefore, the development of rapid, sensitive, cost-effective, and easy-to-operate methods for detection of the VBNC state is an urgent need. In conclusion, the potential application of fundamental research examining the VBNC state is very important to prevent foodborne infections, protect food safety and identify new treatments to reduce the risk of disease caused by foodborne pathogens.
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review
| 99.75 |
To understand the genetic variability of domesticated or wild plants and animals by 2020 is one of the goals of the United Nations . The aim is to improve biodiversity by safeguarding ecosystems, species, and genetic variability. To achieve this goal, it is necessary to study the largest possible number of species from the different biomes of the world with an emphasis on the understudied biomes. In the Atlantic Forest biome, despite its importance in maintaining the planet’s biodiversity, very few species have been focused in such studies .
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other
| 99.9 |
Among the different types of vegetation that exist in the Atlantic Forest, the ancient grassy fields, known as "Campos de Altitude", form a forest–grassland mosaic in the southern portion of this biome, and are one of the less studied and most threatened vegetation in Brazil . Relative to the species from forests, species from grassy ecosystems are poorly studied and conserved . Furthermore, it is predicted that the medicinal species of these regions and ecosystems are highly vulnerable. Two factors have led to this situation: the lack of study of these species and the increased worldwide demand for medicinal plants resulting in direct removal of these species from their natural habitat . The vulnerability of these species increases because often, the most commonly used medicinal plant species in certain regions of the world are unknown to genetic conservation researchers and therefore, are not considered for conservation.
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other
| 85.56 |
Achyrocline flaccida (Weinm) DC. (Asteraceae) is a medicinal plant species found in the original formation of grassy ecosystems, at forest edges, and in regions of vegetation regeneration in the southern portion of the Atlantic Forest. The species is herbaceous, popularly known as "Macela" and is the most common Achyrocline species in south and southeast Brazil. It is one of the most commonly used native plant species in folk medicine in South America . The infructescence of the species is used in tea preparations or infusions, and in pillows and blankets .
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other
| 99.94 |
The harvest of A. flaccida is closely linked with popular culture in Brazil. For example, every year on Good Friday, before sunrise, families go to the fields to harvest A. flaccida infructescence. It is believed that the plant is most effective in the treatment of diseases when the material is harvested in this manner. Achyrocline flaccida is mainly used in the treatment of digestive system disorders [9–12] and for relaxation when used inside of the pillows and blankets . The plant contains several secondary compounds such as flavonoids, flavones, and coffee acids, as well as essential oils and pigments used in the cosmetic and food industry . The species exhibits biological activities such as antioxidant and cytoprotective activities, inhibitory effects on tumors, and mitogenic activity in spleen and antiviral cells. Despite the ecological, medicinal, and cultural importance of the species, population genetic data for A. flaccida is not available in the literature, which makes it difficult to reach conclusions about its real conservation status.
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study
| 99.8 |
Information on the distribution of genetic variability within and between natural A. flaccida populations can help in conservation and effective management of overexploited plant species . DNA-based molecular markers are very efficient and most preferable in the analysis of the variability and genetic structure in plant species because the information is accessed directly from the genome of the target species, allowing safe analysis without interference of environmental factors . Microsatellite molecular markers are the most suitable for these types of studies; however, these markers are not currently available for A. flaccida. The transferability of microsatellite markers between related species has been successfully achieved [17–19] and can be an alternative to A. flaccida. Additionally, when there is no previous knowledge of microsatellite regions for the genome of the species, ISSR (inter-simple sequence repeat) markers can also be used as an alternative [20–23]. The ISSR markers have the advantage of analyzing multiple loci in a single polymerase chain reaction (PCR); in addition, they are very informative, reliable, and produce a high level of polymorphism .
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study
| 99.25 |
Furthermore, when data on genetic variability and population structure is aligned with the geological and paleoclimatic history of the habitat of a given species, the results can help define the patterns of distribution of a species throughout the historical and present timeline. For this understanding, the individual study of each species is necessary, because the extrapolation of data from one species to another can often lead to inaccurate conclusions owing to physical or biological barriers that influence one species but not the other. Mäder et al. concluded that Calibrachoa heterophylla populations differed because they were influenced by local geomorphological events along the coast of the state of Rio Grande do Sul and northern Uruguay, which could not be identified if only long distance models of isolation and/or other species were used.
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study
| 99.9 |
Aiming to contribute to the reduction of the scientific gap regarding the genetic data of A. flaccida, this work sought to answer the following questions: 1—The transferability of microsatellite markers is an efficient strategy for obtaining genetic data from A. flaccida?; 2—ISSR markers are efficient for obtaining population genetic data in A. flaccida?; 3—The populations of A. flaccida of the Atlantic Forest are structured? and 4—Can high utilization of this species in folk medicine to influence the patterns of genetic variability in the natural populations. The answers to these questions will help determine the real state of A. flaccida conservation in the grassy ecosystems of southern Brazil and, if necessary, contribute to the elaboration of conservation strategies and rational exploitation of the species. Additionally, the data obtained here may help us understand the patterns of maintenance and expansion of the plant populations of the grassy ecosystems in the Atlantic Forest of southern Brazil from a historical perspective.
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study
| 99.94 |
In this study, seven populations of A. flaccida were collected throughout their distribution in the grassy ecosystems of the Atlantic Forest biome in southern Brazil and were named according to the county of origin (Table 1). For all populations, vouchers were made and identified using the key for the genus Achyrocline (Less.) DC. in Brazil proposed by Deble . The number of plants sampled from each population and the characterization of the sampling sites according to original and actual ecosystem are shown in Table 1. From each population, leaves of plants that were spaced at least 20 m from one another were collected. The collected leaves were stored in silica gel until DNA extraction. DNA extraction was performed following the protocol of Doyle and Doyle . The concentration and quality of each DNA sample was checked on a 0.8% agarose gel using standard concentrations of the λ phage DNA (50, 100, and 200 ng).
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study
| 100.0 |
To identify microsatellite primer pairs to be evaluated for transferability to A. flaccida, a literature review was first carried out to identify primer transferability studies among Asteraceae species. Of the primer pairs identified, 15 were selected based on the phylogenetic proximity of A. flaccida to the species for which the primers were initially developed (Table 2). Preference was given to primers pairs that amplified a higher number of alleles and showed transferability to other Asteraceae species as reported in previous studies (Table 2). All of these primers pairs were designed to amplify regions of genomic DNA.
|
review
| 99.9 |
For the transferability evaluation, the 15 primer pairs were first evaluated by PCR in 10 A. flaccida individuals. The amplification was performed in a final volume of 10 μL containing 1X PCR buffer, 3 mM MgCl2, 0.2 mM dNTP, 0.8 mM each of forward and reverse primers, 0.4 U Taq DNA Polymerase), 20 ng DNA, and ultrapure water (to complete the volume).
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study
| 99.94 |
For each primer pair, the annealing temperature described in the literature was initially used (Table 2). When non-specific amplification occurred at these initial temperatures, the annealing temperature was increased by 2°C until specific product amplification was obtained or 60°C was reached. When no amplification occurred, the temperature was decreased by 2°C until amplification was obtained or 45°C was reached. Among the 15 primer pairs, Amb82, Sg-2, Sg-6, Sg-8, and ER-HAJZC, did not require a change in the initial annealing temperature.
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study
| 99.94 |
The amplified products were separated by electrophoresis in a 3% agarose gel at a constant 110 V current for 4 h and visualized under UV light by staining with ethidium bromide (0.5 μg/mL-1). To determine the size of the amplified fragments, a DNA ladder 100–bp marker was used.
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study
| 99.75 |
The primers that showed amplification of fragments of expected sizes were considered positive amplification, but only those that presented a polymorphism were considered transferable to A. flaccida. However, the primers that presented non-specific amplification or did not amplify a product were considered inefficient for genetic studies in A. flaccida.
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study
| 99.94 |
The evaluation of A. flaccida populations using ISSR markers was performed in two steps: Step 1: First, the best ISSR primers for genetic studies in A. flaccida were selected. The DNA of 20 genotypes of the population from Guarapuava, Brazil, was amplified using 42 ISSR primers from the UBC series (University of British Columbia) (Table 3). Step 2: Population genetic data were obtained from the seven populations of A. flaccida. After the analysis of the data obtained in Step 1, the best ISSR primers were identified (Table 3) and used to amplify DNA from 131 plants from the seven populations. In both steps, PCR amplification reactions were conducted in a final volume of 12.5 μL that contained 20 ng DNA, 0.2 μM each primer, 200 μM dNTPs, 1.5 mM MgCl2, 1 U Taq DNA Polymerase, 1X PCR buffer, and water to complete the volume. The thermocycler program consisted of an initial denaturation step at 94°C for 5 min, followed by 35 cycles of 90°C for 45 s, annealing temperature of the primer (Table 3) for 45 s, and 72°C for 60 s. At the end of the cycles, a final step of 72°C for 7 min was added for the complete extension of the fragments. The amplification products were separated by electrophoresis on 1.8% agarose gel at a constant voltage of 110 V for 2 h. The gels were stained with ethidium bromide and visualized under UV light. To determine the size of the amplified fragments, a 100-bp DNA ladder was used.
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study
| 100.0 |
To select the best ISSR primers for A. flaccida in Step 1, each amplified locus was evaluated according to the presence (1) or absence (0) of the DNA fragments (band). Based on the binary matrix obtained, the discriminatory power of ISSR primers was evaluated using three parameters: (i) polymorphism information content (PIC), (ii) marker index (MI), and (iii) resolving power (RP).
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study
| 100.0 |
The RP was calculated according Prevost and Wilkinson : RP = ∑Ib, where, Ib represents how informative the locus is of the polymorphism. The Ib can be translated into a 0–1 scale using the formula: Ib = 1 − (2 × |0.5 –p|), where, p is the proportion of the genotypes showing the locus.
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study
| 55.47 |
The selection of the 10 best ISSR primers for genetic studies in A. flaccida was performed as follows: first, the 10 primers that showed the highest values for each attribute (PIC, MI, and RP) were identified separately. Then, 10 ISSR primers were identified according the following criteria in the following order: 1. primers that showed the highest values for PIC, MI, and RP, 2. primers that showed the highest values for PIC and PR, 3. primers that showed the highest values for MI and RP. Whenever a primer was selected, it was excluded before applying the next criteria.
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study
| 100.0 |
For the analysis of the distribution pattern of each locus in the A. flaccida populations, the exclusive presence and exclusive absence of each locus in each population was determined. Then, the loci were grouped to construct a graphical distribution of the genetic variability with respect to population and sampling sites. For this, the loci were grouped according to the pattern of its presence or absence in the populations. After obtaining the loci groups for each population, the total of possible occurrences (possible bands) for all groups of loci and the number of occurrences observed in each group were computed. The number of occurrences observed for a group of loci was divided by the total possible occurrences in all groups of loci in a population to generate the frequency for that loci group within the population. The frequency values obtained for each group of loci were used to construct the graphs for the distribution and frequency of the loci groups in the populations.
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study
| 100.0 |
The percentage of polymorphism (P), the Shannon diversity index (I), the Nei's genetic diversity (h), the Nei’s genetic distance (D), the coefficient of genetic differentiation (Gst), and gene flow (Nm) among the populations were calculated using the POPGENE software version 1.32 . A dendrogram was constructed based on the Nei’s genetic distance with UPGMA (unweighted pair group method using arithmetic averages).
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study
| 99.94 |
Analysis of molecular variance (AMOVA) and the genetic differentiation index (FST) were performed using the Arlequin software version 3.11 . Mantel test was used to verify the correlation between of Nei’s genetic distance and geographical distance with 1,000 permutations using the NTSYS 2.01 software. The principal coordinates analysis (PCoA) for the observation of the graphical distribution of the samples was performed according to the genetic correlation with the NTSYS 2.01 software.
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study
| 100.0 |
To detect the genetic structure at the population level, Bayesian analysis was performed using Structure software . In order to determine the ideal number of clusters (K), simulations were performed assuming that any number of clusters could be obtained between 1 and 10, with each simulation repeated 10 times. For this analysis, the no admixture ancestry model was used and the allelic frequencies were correlated by 10,000 burn-in and 100,000 MCMC (Markov chain Monte Carlo) repeats after burn-in. The Structure Harvester program was used for the definition of K (number of clusters—genetic groups), which is more probable than those proposed by the analysis the criteria suggested by EVANO et al. .
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study
| 100.0 |
Of the 15 microsatellite primer pairs evaluated, 10 (SS20E, SS24F, Eari4-5, Eari4-6, CO189, Sg-2, Sg-8, ER-HAJZC, LHO35, and HSALZ-12) amplified fragments of expected sizes after optimizing the annealing temperatures. However, when these 10 loci were evaluated in the A. flaccida populations, over 80% of the individuals showed null alleles for all the loci in all the populations, indicating that these loci were not suitable for obtaining the genetic data of A. flaccida.
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study
| 100.0 |
Of the 42 ISSR primers evaluated in A. flaccida, 35 primers (83.3%) showed DNA amplification (Table 3). The total number of amplified fragments was 375 with a mean of 10.7 fragments per primer. The PIC, MI, and RP values for each primer are shown in Table 3. Based on the PIC, MI, and RP data, the primers identified as more informative for genetic studies in A. flaccida were UBC 890, UBC 834, UBC 866, UBC 808, UBC 827, UBC 807, UBC 857, UBC 848, UBC 855, and UBC 835 (Table 3).
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study
| 100.0 |
The 10 ISSR primers identified in the first stage of this study amplified 221 loci when evaluated in the seven populations of A. flaccida. Of these, 190 (85.97%) were polymorphic in all populations. The degree of polymorphism did not show a high variation among populations, with the lowest and highest values of 55.2% and 63.8% in the Porto Barreiro and Campos Novos populations, respectively.
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study
| 100.0 |
Of the 190 loci evaluated, 99 formed 11 groups with at least four loci per group. The largest group comprised 44 loci present in all populations and the smallest group comprised four loci. The remaining 91 loci had no similar pattern of distribution in the sampled populations and are referred to here as “others”. The analysis of the loci pattern allowed us to graphically visualize the distribution of genetic variability in the populations (Fig 1). The highest number of exclusive absence and exclusive presence of loci were observed in the of Marialva and Coxilha populations, respectively (Fig 2).
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study
| 100.0 |
The Nei’s genetic variability of the populations ranged from 0.14 (Marialva) to 0.20 (Panambi), with all populations average of 0.23. The Shannon genetic variability index (I) ranged from 0.23 (Marialva) to 0.31 (Panambi) and all populations average of 0.37 (Table 4).
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study
| 100.0 |
The AMOVA showed that the greatest variation was within the populations (66.71%) compared to the variation among populations (33.29%). GST, the index of the genetic differentiation that considers how the genetic diversity is distributed among the populations was 0.33. The correlation between the Nei’s genetic distances and the geographical distances (in km) between populations (Table 5) was positive and significant (r = 0.77; p < 0.01), as estimated by the Mantel test.
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study
| 100.0 |
Gene flow (Nm) among all A. flaccida populations was 1.39. In pairwise comparisons, the populations showed variable Nm values (Table 6). The greatest gene flow was observed between the Guarapuava and Porto Barreiro populations (5.20) and the least was between the Marialva and Coxilha populations (1.68). It was also observed that the higher the Nm, the lower the Gst values (Table 6).
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study
| 100.0 |
The PCoA allowed for the visualization of the spatial structure of the population distribution. Thus, the populations Coxilha, Panambi, and Campos Novos were isolated in quadrants two and three, although Panambi and Campos Novos contained individuals that were far from the center of the population cluster (Fig 3). The Guarapuava and Porto Barreiro, and Xanxerê and Marialva populations overlapped by more than 85% of their occupied areas in the quadrants (Fig 3).
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study
| 100.0 |
In the Bayesian simulations of the Structure program, the number K (clusters, genetic groups) was defined as six. The distribution of individuals in the six genetic groups obtained (Fig 4) showed that the Porto Barreiro and Guarapuava populations belong to the same genetic group, whereas the other five populations are distinct genetic groups.
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study
| 100.0 |
In Myrtaceae and Rosaceae species, the transferability of microsatellite markers has been extensively explored and proven effective [19,50–53]. This approach has facilitated and accelerated the availability of molecular genetic data for species in absence of their own microsatellite markers. Therefore, it was predicted that the Asteraceae microsatellite primers could be useful in characterizing the A. flaccida populations; however, although Asteraceae primers were selected from the literature based on phylogenetic proximity, reports of transferability, and numbers of amplified alleles, they were not transferrable to A. flaccida. It is interesting that when these microsatellite primers were tested in 10 samples, 66.66% amplified fragments of expected size; however, when they were evaluated in whole populations, these primers were monomorphic and/or had a high number of null alleles. Notably, in the majority of studies that developed microsatellite primers, the authors tested these primers on a small number of samples from other species, which may falsely confirm their transferability. As with A. flaccida, these primers may have an excess of null alleles and/or be monomorphic when evaluated in populations with higher numbers of individuals and therefore, are not useful in population genetic studies. The results obtained in this study reinforce the need for the development of microsatellite primers specific to A. flaccida. As the development of microsatellites is expensive, and our group has been developing works with ISSR markers, we used these markers to get faster and more economical population genetic data of A. flaccida.
|
study
| 99.94 |
The choice of the best ISSR primers is an essential step in obtaining robust data for genetic studies and for this selection, the use of statistical indices should be prioritized by visual evaluation. In this study, the evaluation of PIC, MI, and RP data allowed the selection of the 10 most informative primers for genetic variability studies in A. flaccida (Table 3). These 10 primers amplified 190 polymorphic loci in the seven A. flaccida populations. This number is considered adequate because according to Colombo et al. , seven to 30 primers that amplify between 50 and 200 polymorphic fragments are sufficient for genetic diversity studies within a species.
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| 100.0 |
The percentage of polymorphisms has been used as a measure of variability. In Asteraceae, variable values of polymorphism were found: 98.78% in Aster spathulifolius , 70.0% in Carthamus tinctorious , and 65.05% in Nouelia insignis . Thus, the values of A. flaccida (85.97%) indicates high genetic variability in the species.
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study
| 99.94 |
The frequency of ISSR loci in populations of A. flaccida was variable, with the populations of Marialva PR and Porto Barreiro PR indicating higher deviations in the number of loci and consequently, lower variability (Fig 1). This graphical representation of the distribution of genetic variability is presented for the first time in this study, and when we evaluated the numerical indices that represent the genetic variability and differentiation between the populations (Tables 4–6), we observed that the results were the same (high variability and differentiation between populations). This new form of presentation genetic variability results is an efficient and more visually friendly way to demonstrate genetic differentiation between populations.
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| 100.0 |
The occurrence of exclusive loci in each population (Fig 2) had little variation and did not appear to contribute to the differentiation and distribution of variability among A. flaccida populations. The high exclusive absence of loci (11) in the Marialva population (Fig 2) seems to corroborate with data from the Shannon and Nei’s genetic variability analyses (low indices) of the population. Our previous observations in the last 25 years had not detected the presence of A. flaccida in the Marialva region therefore, it is possible that the species colonized this site in the last five years. The results for this population obtained in this study and absence of the species in the region for a long period, led to the hypothesis that this population is recent and suffered a possible founding effect.
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| 100.0 |
The parameters that quantify the genetic diversity, Nei’s genetic diversity (h), and Shannon diversity index (I) were detected at high levels in A. flaccida (h = 0.23; I = 0.37). These results are in agreement with the proposed by Hamrick and Godt , that generally geographically widespread species tend to maintain higher genetic diversity.
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| 100.0 |
The distribution of the genetic diversity obtained by AMOVA showed higher variation within populations (66.71%) than among populations (33.29%), following the pattern observed in the literature for allogamous and/or pioneer species [59–61]. In addition, with AMOVA it was possible to observe that the genetic differentiation is high among populations (FST 0.33).
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study
| 100.0 |
The comparison of gene flow and Gst (Table 6), and the correlation between the Nei’s genetic and geographical distances (r = 0.77, p < 0.01) among populations shows that A. flaccida populations follow the isolation-by-distance model. The estimated gene flow for a species may describe a migratory pattern of individuals occurring from the distant past to the present , and the data observed here should not be interpreted by considering only the current state of isolation in A. flaccida populations.
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| 100.0 |
The Guarapuava and Porto Barreiro populations presented low genetic distance (Table 5), high gene flow (Table 6), they occupied the same area in the PCoA quadrants (Fig 3), and formed a single genetic group in the Bayesian analysis (Fig 4). This behavior leads to the hypothesis that these populations may assume a metapopulation model. The Marialva, Guarapuava, Porto Barreiro, Xanxerê, and Campos Novos populations were more similar in all analyzed variables, suggesting that they are more related than when compared to the Coxilha and Panambi populations. This finding is even more evident in the dendrogram (Fig 5). The populations of Xanxerê and Campos Novos are genetically closer and physically distant to Marialva population, while genetically distant and physically closer to Coxilha and Panambi. This observation indicates a possible geographic barrier to gene flow between the populations of Xanxerê/Campos Novos and Coxilha/Panambi.
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| 100.0 |
The diversity and genetic structure of natural populations are affected by several factors including migratory capacity, mating systems, ecological characteristics of their habitats, altitude, and recent and historical events . In A. flaccida the altitude was not considered because the populations of Xanxerê (777 m) and Marialva (700 m) are at similar altitude to Coxilha (750 m) and were still genetically distant. The analysis of the current ecosystems over the last 100 years and the history of these regions since the last glacial maximum led us to two complementary hypotheses for the diversity and genetic structure observed in A. flaccida populations.
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study
| 100.0 |
The first hypothesis is based on the historical dynamics of the vegetation in the southern fields of the Atlantic Forest and suggests an answer to why more closely related populations are more genetically distant. In order to understand the dynamics of the vegetation in the glacial and current periods, it is important to observe the topography of the regions where A. flaccida populations were collected. The Guarapuava, Xanxerê, and Campos Novos populations are in a high altitude continuum separated from the Coxilha and Panambi populations by the Uruguay River valley (Fig 6). This valley may have limited the gene flow between these populations.
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study
| 99.94 |
The vegetation of the southern fields in the Atlantic Forest underwent several changes during the ice ages of the Lower and Middle Holocene period . The decrease in temperature and humidity during these periods favored the expansion of grassy vegetation areas, which formed large continuous areas in the southern Brazilian states . This condition favored the wide distribution of typical plant species of grassy vegetation areas and, according to Mayle et al. , among these species, the most abundant in these regions were those from the Poaceae and Asteraceae families; the latter being the family of A. flaccida. Behling proposed that in the glacial periods during the Lower and Middle Holocene, the Araucaria Forest was isolated in refuges located in the river valleys. It is not difficult to conclude that these valleys may have also acted as a barrier to gene flow between populations of the species from grassy vegetation fields. Considering this context, it is possible that the genetic proximity observed between the A. flaccida from Guarapuava, Xanxerê, and Campos Novos populations is partly due to them having been somewhat continuous in the past. When analyzing the topography of the southern states of Brazil (Fig 6), it is possible to observe that such a situation was probable, because among these populations there are no river valleys that could have maintained forests acting as a barrier for the populations during cold and dry periods in the past. Additionally, the Uruguay River valley (Fig 6) may have acted as an important barrier for gene flow among the populations of Paraná/Santa Catarina and Rio Grande do Sul.
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| 100.0 |
Achyrocline. flaccida populations from each region bounded by the Uruguay River valley experienced periods of physical continuity in the last maximum glacial period. The increase on temperature and humidity in this region during the upper Holocene between 5000 and 930 years ago, led to expansion of the Araucaria Forest and isolation of populations due to grassy vegetation retraction. This may be one of the factors that contributed to the actual genetic differentiation observed in A. flaccida populations within each region. This hypothesis need to be tested further by adding data from other populations and using other species typical of the southern Atlantic Forest grassy vegetation field.
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| 99.94 |
The second hypothesis that can also help to understand the pattern of genetic diversity and structure in the A. flaccida populations are human activities. In the last 100 years the isolation of the grassy vegetation has completely changed by deforestation and the establishment of pasture or agriculture, allowing the expansion of species such as A. flaccida. The populations of Porto Barreiro and Marialva are located in regions that were previously dense forests, and the diversity and structure data are consistent with the recent establishment of these populations in these areas.
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study
| 98.56 |
Another factor that may influence the genetic patterns in A. flaccida is the dispersion of species by humans. The seed-bearing A. flaccida infructescence are widely used as medicine and in the manufacturing of pillows and blankets. There are companies that harvest and sell the infructescences throughout southern Brazil. The commercially available infructescences do not undergo processing that hinders germination. Seed germination tests of Achyrocline satureioides, a species in the same genus of A. flaccida, indicated that 25% of the seeds were still viable after 10 months of storage . Then, the movement of seeds by marketing as well as long term seed viability could explain the establishment of a population where the species did not occur, as might be suggested for the Marialva and Porto Barreiro populations. Furthermore, this kind of seed flow may also increase gene flow which can contribute to the genetic similarity between distant populations, like Xanxerê and Marialva.
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study
| 99.94 |
In conclusion, the results provided in this work show that: 1—the transferability of microsatellite markers is not an efficient strategy on A. flaccida; 2—the ISSR markers are a powerful tool to genetic studies on this species; 3—the populations of A. flaccida of grassy vegetation of southern Atlantic Forest have high genetic variability and are structured; 4—human activities (deforestation and folk medicine use) seems to influence the patterns of genetic variability in the natural populations of the species. The lack of previous genetic data of A. flaccida does not allow drawing any inferences about genetic drift on the species, but our results give support to the conclusion that the species is not at risk of extinction in the short term in the studied regions.
|
study
| 99.94 |
Recently, biodegradable polymer scaffolds have been utilized in tissue engineering to regenerate or substitute for a human organs [1, 2]. So numerous researchers had studied and utilized the 3-D scaffolds via various methods and aspects such as using various pore size . The results in many papers are emphasized and come out with numerous characteristics of the 3-D scaffold such as the pore size, porosity, and the mechanical property for tissue engineering [2–4].
|
review
| 99.9 |
Thus, the characteristics of the 3-D scaffold such as mechanical properties, pore size, surface property, and degradability were very important as a culture media for the tissue engineering. The characteristics of the scaffold is also depends on the cell or tissue types. For example, the pore size was also employed differently such as 380~405 μm pore size for chondrocyte and osteoblast growth, and 186~200 μm pore size for fibroblast growth . Besides, there are also many researchers had studied mechanical properties and degradation behaviors for the various biodegradable polymer scaffolds [1, 4–6]. The mechanical properties and degradation behaviors are among the important factors because they are related to the structural and mechanical stability for the tissue regeneration [1, 5]. Thus, the physical properties of 3-D scaffolds must be taken into consideration by using innovative approaches. One of the most important factors for cell proliferation is mechanical stimuli to the cell. There are various methods for mechanical stimulation that will affect the cell behaviors such as compressive loading that is divided into hydrostatic pressure and platen displacement in a 3-D specimen, longitudinal stretch, substrate bending, plane substrate distention, and fluid shear system .
|
review
| 99.5 |
Based on Wolff’s law, the bone cell tends to proliferate by the compressive stimulation because bone composition has piezoelectric properties and bone remodeling is caused by various stimulations . In addition, Compressive stimulation can lead to osteogenic gene expression for proliferation and differentiation [9, 10]. It would depends on the Poisson’s ratio in order to determine the compressive stimulation acted on the bone cells in 3-D scaffolds. The conventional 3-D scaffolds or matrices including all common materials have a positive Poisson’s ratio .
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study
| 100.0 |
The Poisson’s ratio is defined as the negative strain ratio of the longitudinal strain divided by the transverse strain [11, 12]. If the material has a negative Poisson’s ratio, it will conduct high compressibility in multi axial directions, and the material is known as auxetic material . Therefore, the 3-D scaffold with negative Poisson’s ratio will be expected undergoing the bone cell proliferation effectively.
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study
| 99.94 |
Lakes proposed the methods to form a negative Poisson’s ratio to the foam structures, that is, a permanent volumetric compression in three orthogonal directions and heat processing . Lakes and Choi formed the polyurethane foam with a negative Poisson’s ratio via volumetric compression ratio and heat processing . The cell size of foam, different heating temperature and heating time are affected to the value of negative Poisson’s ratios . There are several important conditions such as pore size, compression ratio, heating temperature and heating time needed to form a negative Poisson’s ratio. Kim formed the polyurethane foam with a negative Poisson’s ratio and utilized it as the scaffolds for cartilage regeneration via mechanical stimulation .
|
study
| 99.94 |
poly((D,L-lactic-co-glycolic acid) (PLGA, Lakeshore Biomaterials, Essen, Germany) was used for scaffold fabrication. The composition ratio of lactic and glycolic acid (LA : GA molar ratio) is 50:50. Molecular weight (Mw, Mn) was 124 kDa and 77 kDa and the glass transition temperature (Tg) was 47.7 °C. For scaffold fabrication, chloroform was employed as a solvent and sodium chloride particles were used as porogen in the scaffold.
|
study
| 99.94 |
Solvent casting/salt leaching methods were used to fabricate PLGA porous scaffold. Sodium chloride particulates in the range of 355~400 μm particle size were used for control group and 500~600 μm for experimental group. Both groups were prepared by using a Teflon mold. Control specimens were prepared as 1.5 × 1.5 × 1.5 (cm3) in size and experimental specimens were prepared as 2 × 2 × 2 (cm3) and 2.2 × 2.2 × 2.2 (cm3) in size. In order to make same pore size after applying compression to the experimental specimens, the pore size was bigger for experimental specimen.
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study
| 100.0 |
PLGA copolymer was dissolved in chloroform and then mixed with sodium chloride particles. The mixture solution was placed in the mold and then dried at room temperature for 1 day. After drying, the mixture was removed from the mold and washed with distilled water for 2 days in order to remove sodium chloride particles. Finally, specimens were freeze-dried for 24 h.
|
study
| 99.9 |
Permanent volumetric compression with heat treatment (PVCT) method was employed in order to create a negative Poisson’s ratio to the scaffolds . For the experimental specimen, heat treatment was applied to the temperature above its Tg in a drying oven (DMC-122, DJDAEIL, Daejeon, Korea). The specimen was then compressed in three orthogonal directions at the same time. Firstly, heat treatment was applied to the specimens at 50 °C/5 min, 50 °C/10 min, 60 °C/5 min and 60 °C/10 min. Secondly, tri-axial compression was applied with 2.4:1 and 3.1:1 in volumetric ratio. The experimental specimens were compressed in three orthogonal directions by two Teflon molds up to 1.5 × 1.5 × 1.5 (cm3) in size and became the same size as that of control (see Fig. 1). The specimens were then cooled at room temperature and removed from the mold.Fig. 1Make same pore size to control specimens via permanent volumetric compression. Volumetric compression ratios are 2.4 and 3.1. 2 × 2 × 2 (cm3) to 1.5 × 1.5 × 1.5 (cm3) size is 2.4 : 1 and 2.2 × 2.2 × 2.2 (cm3) to 1.5 × 1.5 × 1.5 (cm3) size 3.1 : 1
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study
| 100.0 |
The Poisson’s ratio for all specimens was analyzed by image processing with Image J program v2.0.0. The Material Testing System (LRX-PLUS, Lloyd instruments, West Sussex, UK) was utilized to test the specimens. A digital microscope (BX51, Olympus Corporation, Tokyo, Japan) was used to capture images in order to measure the displacement of the specimen (see Fig. 2).1\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {\varepsilon}_{\mathrm{x}}=\frac{\left|A-B\right|-\left|A0-B0\right|}{\left|A0-B0\right|} $$\end{document}εx=A−B−A0−B0A0−B0 2\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {\varepsilon}_{\mathrm{y}}=\frac{\left|C-D\right|-\left|C0-D0\right|}{\left|C0-D0\right|} $$\end{document}εy=C−D−C0−D0C0−D0 3\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \nu = \frac{\upvarepsilon \mathrm{x}}{\ \upvarepsilon \mathrm{y}} $$\end{document}ν=εxεywhere:εxstrain of x-axis.εystrain of y-axis.νmaterial Poisson’s ratio.A0~D0initial point (load = 0).A~Dmoved point under load. Fig. 2Points were tracked to estimate Poisson’s ratio of the specimen by image processing. (a). (b) to (c) is changing image when the specimen was compressed
|
study
| 99.94 |
All specimens were compressed from 0 to 25 % strain and the image was captured with 5 % strain intervals. The points A to D were utilized to calculate the Poisson’s ratio by tracking the center of point as shown in Fig. 2. The changes of x-axis and y-axis distance were calculated by using Eqs. (1) and (2), thereby Poisson’s ratio was calculated by Eq. (3).
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study
| 100.0 |
The compressive strengths of the control and experimental groups were analyzed under the conditions shown in Table 1 by using a Material Testing System (MTS) at 10 % compressive strain. The cross head speed of MTS was 1 mm/min.Table 1Conditions of compressive strength measurement for the scaffolds in dry and wet stateTypesConditionsTemperatureAdry/25 °vC (experimental)25 °CBdry/25 °C (control)25 °CCwet/EtOH/PBS (experimental)25 °CDwet/EtOH/PBS (control)25 °CEwet/EtOH/PBS/DMEM (experimental)37 °CFwet/EtOH/PBS/DMEM (control)37 °C
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study
| 100.0 |
In order to analyze the degradation characteristics of the scaffold, we prepared a disc-shaped scaffold. (Diameter = 12 mm, height = 2 mm) The fabricated scaffold was pre-wetted in 70 % ethanol, and then placed in each of 4 ml phosphate buffered saline solution (PBS, pH 7.4). The change in weight of the scaffold in a shaking incubator at 37 °C for 7 weeks was measured. The degradation rate of the scaffold was calculated by Eq. (4).4\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \mathrm{Degradation}\kern0.5em \mathrm{rate}\left(\%\right)=\frac{Initial\kern0.5em weight- loss\kern0.5em weight}{Initial\kern0.5em weight}\times 100 $$\end{document}Degradationrate%=Initialweight−lossweightInitialweight×100
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study
| 100.0 |
Human MG-63 osteoblast-like cells (KCLB, Seoul, Korea) were used for cell attachment and proliferation test. They were cultured at 37 °C in a humidified 5 % CO2 and cultured in DMEM containing 10 % fetal bovine serum and 1 % penicillin/streptomycin. Cells were sub-cultured every 3 days at 90 % confluence in 75 cm2 and 25 cm2 culture flask. 4~6 passage cells were used for this study. The cellular experiments were performed in 24-well plate and 12-well plate where each well was immersed by the culture medium.
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study
| 100.0 |
In cell attachment test, the MG-63 osteoblast-like cells were seeded in density of 3.6 × 105 cells/40 μl for each specimen. After cell seeding, they were incubated for 2 h and then added in immersed culture medium. At each time point (4, 8, 12 and 24 h), specimens were moved to new 24-well plates and washed two times with PBS solution. The attachment rate was measured by using cell counting kit-8 (CCK-8, Sigma-Aldrich) by absorbing read on a micro-plate reader at 450 nm.
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study
| 99.94 |
We prepared our own compressive stimulation device as shown in Fig. 3. It can control applying load with 10 % strain elongation to the scaffold. The applied load was 19.6 Newton. The cells (1.8 × 105 cells/80 μl) were seeded onto the control and experimental specimen groups. The compressive stimulation was applied after 1 day from cell seeding to prevent the loss of cells from the specimens. The cells were seeded into the inner pore of specimens to prevent the damage of cells by compression.Fig. 3An apparatus for cell culture with compressive stimulation. The apparatus was manufactured to apply 10 % compressive strain to the scaffold in 12-well plate
|
study
| 100.0 |
Cell proliferation test were divided into three types as shown in Table 2. The cells were seeded onto the specimens at a density of 1.8 × 105 cells/80 μl. After the cells’ attaching to the specimens, the compressive stimulation was applied to the cells by using the prepared apparatus. At each time of 1-, 3- and 5-day culture, the specimens were moved to new 12-well plates and added with fresh culture medium. Ten percent (v/v) CCK-8 reagent was added to immerse well and incubated at 37 °C in a humidified 5 % CO2 for 4 h. One-hundred microlitre of the reactant were moved into a 96-well plate and the absorbance read was obtained on a micro-plate reader at 450 nm. The cell number was calculated by initial absorbance value.Table 2Conditions of cell proliferation experiment in accordance with the stimulationScaffold typesCompressive stimulationConditionsControl groupX37 °C/5 % CO2 Control groupO37 °C/5 % CO2 Experimental groupO37 °C/5 % CO2
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study
| 100.0 |
Structure of negative Poisson’s ratio was obtained successfully from the process of heat treatment with 3-D compression for the scaffold. Concave porous structure was created in the scaffold specimens. The cross-sectional microstructure of the PLGA scaffold is shown in Fig. 4. The porous shape of control specimen was observed as a cubical and convex type as shown in Fig. 4(a). In contrast, experimental specimen showed partially concave and dented shapes as shown in Fig. 4(b) and (c). This transformation of porous shape is resulted from changing micro porous structure of the scaffold by tri-axial compression. The porous structure of these recessed and dented shapes enable the scaffold have negative Poisson ratio.Fig. 4Cross-sectional SEM images of PLGA scaffold. a control specimen before compression. b experimental specimen after tri-axial compression (2.4 : 1), (c) experimental specimen after tri-axial compression (3.1 : 1). The arrows indicate difference of pore structure
|
study
| 100.0 |
Cross-sectional SEM images of PLGA scaffold. a control specimen before compression. b experimental specimen after tri-axial compression (2.4 : 1), (c) experimental specimen after tri-axial compression (3.1 : 1). The arrows indicate difference of pore structure
|
study
| 57.66 |
The Poisson’s ratio of a control specimen was shown as about 0.11~0.13 when compressed at 5 to 25 % strain (see Fig. 5). For the experimental specimen, the Poisson’s ratios in accordance with the permanent volumetric compression with heat treatment (PVCT) ratio of 2.4 and 3.1 were shown as −0.07 and −0.05, respectively. The specimen of PVCT ratio of 2.4 showed more negative Poisson’s ratio than that of PVCT ratio of 3.1. Thus, extremely high compression ratio of the PVCT ratio of 3.1 can be considered as a factor to cause the shape of the pore structure to collapse. Therefore, PVCT ratio of 2.4 is considered as more effective ratio to form negative Poisson’s ratio.Fig. 5Poisson’s ratio variations for control and experimental specimens. The volumetric compression ratio was 2.4 and 3.1 (n = 4, *p < 0.05)
|
study
| 100.0 |
The heat treatment temperatures (50, 60 °C) and heating time (5, 10 min) with a PVCT ratio of 2.4 were applied during the forming process of a negative Poisson’s ratio. Poisson’s ratios of experimental group were 0.05 at 50 °C/5 min, −0.03 at 50 °C/10 min, −0.04 at 60 °C/5 min and −0.07 at 60 °C/10 min as shown in Fig. 6, respectively. The result showed that PLGA50:50 scaffolds specimen depended on heating temperature and heating time to reveal negative Poisson’s ratio. The lowest negative Poisson’s ratio was −0.07 with 60 °C/10 min at 10 % strain (see Fig. 6). These results are important because the different heating temperature and heating time for the PVCT ratio would affect the negative Poisson’s ratio.Fig. 6Poisson’s ratio variations for control and experimental specimens. Processing was done at 50, 60 °C and for 5, 10 min with a volumetric compression ratio of 2.4 (n = 4, *p < 0.05)
|
study
| 100.0 |
Compressive strengths of the scaffolds in dry and wet state were shown at Fig. 7. Compressive strength of experimental group was about 3.12 times higher than the control group in dry state at 25 °C. The compressive strengths of all groups were tended to be decreased dramatically in wet state than those in dry state. However, compressive strengths of experimental group were higher 3.08 times and 1.88 times at EtOH/PBS (25 °C) and EtOH/PBS/DMEM (37 °C) solution than the control group, respectively. Therefore, the experimental specimens are considered to be able to compensate for decrement of the mechanical strength of the control group in wet state. NPR scaffolds were improve the strength in wet environment compared with control scaffolds.Fig. 7Compressive strengths for the scaffold in dry and wet state
|
study
| 100.0 |
Degradation rates and morphology changes of the PLGA scaffolds in PBS solution during 7 weeks were shown in Fig. 8. As time passed, the weight of the scaffold had been reduced (see Fig. 8(a)). The degradation rate of the scaffold is shown about 16 % weight loss after 5 weeks, but morphology of the scaffold was maintained. The collapse of the scaffold occurred after 6 weeks passed (see Fig. 8(b)).Fig. 8Degradation of control specimens in PBS solution during 7 weeks (a) Degradation rate and (b) morphology change
|
study
| 100.0 |
In cell attachment test, the cell attachment at time points of 4, 8, 12 and 24 h were 12.5, 50.0, 86.1 and 106.0 %, respectively, as shown in Fig. 9. This result means that MG-63 cells needed at least 24 h in order to attach inside of the PLGA scaffold. Because of applying compressive stimulation on scaffold, the seeded cells should be placed inside of the scaffolds as well as enough number of cells should be attached in/on scaffolds.Fig. 9MG-63 cell attachment rate for 1 day for control and experimental specimens. Optical density at 450 nm (a) and cell number (b) of cell attachment at each time point (n = 4, *p < 0.05)
|
study
| 100.0 |
The results of cell proliferation with and without stimulation on control and experimental scaffold specimens are shown in Fig. 10. At 1-day and 3-day culture, there is significant difference between with and without compressive stimulation. Additionally, the experimental group showed 1.46 times higher cell proliferation than control group at day-1 with compressive stimulation, and the experimental group showed 2.09 times higher cell proliferation than control group at 1-day without compressive stimulation. In case of 3-day culture, the experimental group showed 1.32 times higher than control group with no compressive stimulation. However, when applying a compressive stimulation, there was no significant difference between experimental group and control group at 3-day culture. At 5-day culture, there is no significant difference between all groups.Fig. 10MG-63 cell proliferation rate on PLGA scaffolds at 1, 3 and 5 day culture with static stimulation (n = 4, *p < 0.05, **p < 0.01)
|
study
| 100.0 |
As a result of cell proliferation test, the effectiveness of negative Poisson’s ratio was revealed at 1-day and 3-day culture. However, there was no significant difference in cell proliferation due to diminish of mechanical strength of the scaffold as well as disappear the negative Poisson’s ratio in cultivation at 5-day. The diminishment is presumably caused by viscoelastic behavior of the PLGA in the liquid culture media. Hence, static compressive stimulation is no more effective in 5-day culture period due to mechanical strength reduction and stress relaxation in the scaffold. This might be mechanical stimulation to the scaffold could not be sufficiently transmitted to cells.
|
study
| 100.0 |
A negative Poisson’s ratio was moderately formed for the PLGA scaffolds. As previously mentioned, there were various factors to form a negative Poisson’s ratio for foam structure such as tri-axial compression, heat treatment temperatures and time. In this study, negative Poisson’s ratio were successfully embodied, and auxetic PLGA scaffold could be fabricated with the permanent volumetric compression ratio of 2.4 and 60 °C/10 min conditions. The MG-63 osteoblast-like cells were entirely attached after 24 h on the scaffold. The bone cells were grown well via the compressive stimulation. From 1-day to 3-day culture, the result showed the effectiveness of negative Poisson’s ratio of the scaffold for bone cell proliferation. It is considered that the auxetic PLGA scaffolds would affect initial period for bone cell proliferation effectively. Hence, we conclude that mechanical stimulation for bone cell cultivation through auxetic scaffolds should have had beneficial aspects and might be play an important role in bone tissue regeneration.
|
study
| 100.0 |
Women are suffering high levels of morbidity as a result of a prolonged labor [1–5]. The most common solution for a dystocic delivery is a caesarean section. This method of delivery raises concerns not only regarding risks of complications for the current delivery, but also for future deliveries [6–8]. A more restrictive approach is therefore desirable, especially in deliveries of nulliparous women.
|
review
| 93.3 |
For labor to end successfully, the uterus needs to produce strong, coordinated and effective contractions. Under exertion, lactic acid is produced by glycolysis in all human cells. Glycolysis mainly occurs under hypoxic conditions; but the uterus is highly glycolytic and produces lactate even under normoxic conditions [9–11]. Repeated transient hypoxia is a normal feature of labor as the uterine vessels are occluded during each contraction [9–11]. Lactate will be produced and the levels of pH in the tissue will decrease. Decreasing pH leads to intracellular acidification and an inhibition of the Ca2+ channels in the myometrial cells . A decreased inflow of Ca2+ into the muscle means that the contraction will be weaker and consequently less effective [9, 10]. New studies show a significant correlation between lactate production, hypoxia and the effect of oxytocin [13–15]. This is an important interaction if labor is to end normally. The level of lactate has been recognized as a factor in uterine activity and pathophysiological processes during labor [13–15].
|
review
| 96.06 |
A partogram should be used to facilitate early detection of poor progress of labor [16–20]. If poor progress is confirmed, oxytocin is suggested for augmentation. Indeed, responses to the administration of oxytocin are variable, and many labors diagnosed as dystocic will be long, painful and unsafe, as the use of oxytocin does not guarantee vaginal delivery, i.e. labor dystocia occurs despite adequate stimulation with oxytocin. A reasonable assumption is that increased knowledge of uterine activity and pathophysiological processes will be the key to the ability to improve the treatment of dysfunctional labors [13–15].
|
review
| 95.5 |
A close correlation has been described between lactate produced by the uterine unit and the level of lactate in amniotic fluid (AFL) . High levels of AFL are shown to be over-represented in dystocic deliveries compared to deliveries with a normal progress of labor [21–25]. A new, non-invasive method has been developed that makes it possible to detect the AFL level in a small sample of amniotic fluid collected from the vaginal pouch and analysed at the bedside in the delivery room. Levels of AFL can be measured immediately and an answer provided within 15 seconds [21–25].
|
study
| 99.94 |
Nulliparous women in active labor at the selected study locations were invited to take part in the study. Inclusion criteria were: a singleton pregnancy, gestational age between 37–42 weeks, no maternal /fetal chronic and/or pregnancy-related conditions. A spontaneous onset of labor, regular contractions and cervical dilation of at least 3 cm were required before the women were invited to take part in the study. A group of midwives and medical doctors at each clinic assisted with data collection and with enrolling women into the study. For all included deliveries a partogram was recorded to monitor the progress of labor. Included clinics used different alert lines of the partogram, but two criteria for labor dystocia were defined: if cervical dilation crossed the action line in the partogram or if labor progress was arrested for two hours or more. Oxytocin was administered according to local clinical guidelines, but the amount of oxytocin added to the infusion did not differ between clinics. A flowchart of the study design is presented in Fig 3.
|
study
| 100.0 |
Obstetric background data, delivery data and AFL values from all deliveries included were reported in a shared internet database completed by a local research midwife. The database was password protected and encrypted. All personal data was encoded, so that individuals could not be identified in the analysis.
|
study
| 99.9 |
Three small samples (50ul) of amniotic fluid (AF) were collected: at the first vaginal examination or when the membranes had been broken, before augmentation with oxytocin and at the time of delivery. If no spontaneously flowing AF was available, a small single-use catheter was used. Previous studies have shown no difference in the value of AFL if AF was flowing spontaneously or collected through a catheter . Amniotomy for augmentation was permitted according to the protocol of the hospital, but not for the sole purpose of obtaining AF for the study.
|
study
| 99.94 |
A device (LMU061, ObsteCare AB, Sweden) adapted for measurement of lactate in amniotic fluid, automatically calibrated at measurement to ensure optimal measurements performance, was available in every delivery ward of the participating hospitals . The device measured lactate concentration in AF with a coefficient of variation of approximately 3% at a lactate concentration of 11mmol/l. The lactate recognition system was based on lactate oxidase with amperometric detection of the enzymatically produced hydrogen peroxide .
|
study
| 99.94 |
To investigate the influence of meconium in AF, 10 mL of non-meconium-stained AF was mixed with 2, 4, or 6 mL of meconium. The concentration of lactate in AF was then analysed at regular intervals. The analysed levels of lactate were approximately the same throughout the experiment . Similar tests were performed with a mixture of blood and amniotic fluid. The test showed that the concentration of lactate in AF could be affected by a high concentration of blood. Amniotic fluid samples tinged with a high degree of blood (≥ 10%) have therefore been removed from the analysis.
|
study
| 100.0 |
The AFL was analysed immediately by a research midwife. The result was delivered within 15 seconds, blinded and stored in the device, and not communicated until after delivery. The Apgar score at 1 and 5 minutes after delivery was determined by the midwife in charge.
|
other
| 98.6 |
A monthly quality check of the lactate measurement devices used in the study was made by an external company (Equalis AB, Uppsala, Sweden) that sent an unknown standard solution of lactate for analysis to all included delivery wards. The results were compiled for all analysers and presented monthly by the company. No significant differences were shown between the analytic results of the devices during study time.
|
study
| 100.0 |
To detect a difference of at least 15 percentage units in the proportions of cesaeren sections between high/low AFL levels, with 83% power, we needed to include 160 deliveries with low (< 10.1 mmol/l) and 80 with high (≥ 10.1 mmol/l) AFL values. We assumed that at least 50% of the women included would receive oxytocin, and of these 15% would have a high AFL value, which implied that a minimum of 1444 (160/0.85+80/0.15)/0.5) deliveries had to be included in the study. The recruitment goal was then set at 3000 to allow for missing data or missing AFL samples. Differences among groups defined by operative delivery were tested by ANOVA for continuous variables and chi-square tested for categorical variables. The cut-off value of AFL was set at ≥10.1 mmol/L. This was in line with earlier publications where an increased frequency of operative intervention has been seen in deliveries with an AFL value ≥ 10.1 mmol/L [22–24].To evaluate the predictive capabilities of AFL, sensitivity, specificity, positive (PPV), negative (NPV) predictive values for operative intervention were calculated with 95% confidence intervals (CI). Logistic regression was used to study the association between operative interventions and each of the independent factors: maternal age, education, gestational age, fetal presentation, latent phase duration, use of epidural anesthesia (EDA), AFL, and country of delivery . Our model strategy was as follows: first, unadjusted associations with each factor were studied; second, the adjusted association with respect to the risk factors measured was studied in a multivariable model with all factors included; third, a subgroup analysis was performed to study whether the operative interventions differed between those with high and low AFL with respect to the levels of the other factors. An interaction term between AFL and each of the factors was added to the adjusted model (sequentially). P-values < 0.05 were regarded as statistically significant. Analyses were performed using IBM SPSS 22 (SPSS Inc. Chicago, Illinois, USA).
|
study
| 100.0 |
The study was approved by the regional ethics committee at Karolinska Institutet, Stockholm (2010/199-31/1). All the clinics involved added a local addendum from their ethics committee to the original ethical authorisation. Written informed consent was obtained from all the women before inclusion in the study.
|
other
| 99.94 |
A total of 3000 deliveries were included in the study. However, 703 mostly African women were subsequently excluded due to failure to meet the inclusion criteria. This was something detected after enrolment, and most common reason was that they were not primiparas or was not completely healthy (Fig 3, flow chart). Table 1 describes demographic data for the entire study population according to nationality. The African women were younger and less educated than the other participants; their pregnancies were shorter, and African new-borns were smaller at delivery.
|
study
| 100.0 |
Oxytocin was used in 66% (1515/2297) of all deliveries in the study. Table 2 presents delivery characteristics in the augmented deliveries where an AF sample was collected within 30 minute before oxytocin was administered (n = 638). In 877 deliveries, a sample of AF was not collected within 30 minutes, and they were excluded from the calculation.
|
study
| 100.0 |
In the augmented deliveries the spontaneous vaginal delivery rate was 69.4% (443/638). 75.2% (480/638) of them were diagnosed with labor dystocia according to the partogram, and 64.2% (308/480) of deliveries with labor dystocia arrested at the first stage of labor. 23.8% (152/638) of the augmented deliveries had an active period of labor for more than 12 hours.
|
study
| 100.0 |
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