Concurrent sexual partnerships (partnerships that overlap in time) may contribute to higher rates of HIV transmission in African Americans. Attitudes toward a behavior constitute an important component of most models of health-related behavior and behavioral change. We have developed a scale, employing realistic vignettes that appear to reliably measure attitudes about concurrency in young African American adults.Vignette-based items to assess attitudes about concurrency were developed following focus groups and cognitive testing of items adapted from existing scales assessing psychosocial constructs surrounding related sexual behaviors. The new items were included in a telephone survey of African American adults (18–34 years old) in Eastern North Carolina immediately before and after a radio campaign designed to discourage concurrency. We performed an exploratory factor analysis on each sample (pre- and post-campaign) to cross-validate results. We retained factors with a primary loading of ≥0.50 and no secondary loading >0.30. Cronbach’s coefficient alpha was used to evaluate internal reliability. Associations in the predicted direction between the mean responses to items on the final factor and known correlates of concurrency validated the scale.Factor analysis in a random pre-campaign subsample yielded a one-factor 6-item scale with acceptable internal consistency (Cronbach’s α = 0.79). As expected, the attitude factor was positively associated with participation in concurrent partnerships, whether assessed by self-report  or deduced from dates of recent sexual partnerships . The factor was also positively associated with alcohol  and drug use  and negatively associated with increasing age  and female gender . Factor analyses repeated in the second random pre-campaign subsample and post-campaign sample confirmed these results.A vignette-based scale may be an effective measure of key attitudes related to concurrency and potentially a useful tool to evaluate interventions addressing this network pattern. African Americans comprised only 13% of the total US population in 2014 . Social Little published research documents psychosocial factors that predict participation in concurrency. Several leading theories of behavioral prediction and change, including Fishbein’s integrative model of behavior, ,13 hold In this paper we present the development of a scale to measure changes in attitudes toward concurrency to evaluate the effectiveness of a radio campaign to discourage concurrency among young, heterosexual African Americans in Eastern North Carolina, an area with elevated concurrency and HIV prevalence ,21. A coData for this analysis were collected in conjunction with an eight-month radio campaign in Eastern North Carolina (NC) that aimed to 1) inform young, heterosexual African Americans about the association between concurrent sexual partnerships and HIV dissemination in the community and 2) decrease acceptability of concurrency . ComputeWe developed an exploratory conceptual model to understand participation in concurrency among African Americans. Using Fishbein’s integrative model of behavior, which holds that attitudes, norms and self-efficacy are primary determinants of intention to perform a specific behavior, ,13,24 weBefore initiating the main study, we conducted six focus groups among African American adults in the target age range and counties to identify factors that may promote concurrency in their communities. Focus group participants cited numerous contributors, including low male-to-female ratios, high male incarceration rates, poverty, desire to fulfill sexual and emotional needs that are not met by a main partnership, avoidance of hurt feelings and vulnerability that may accompany a monogamous relationship, retaliation against one’s partner, and having a child with a previous partner .Candidate survey items were adapted from published construct definitions and validated scales for measuring attitudes, norms, self-efficacy, and behavioral beliefs about similar sexual risk behaviors –32. ThesApproximately half of respondents who participated in preliminary cognitive testing of the items were men  and most had graduated from high school  and had never been married . Only one respondent had never had sex. Preliminary cognitive testing indicated that the original candidate items measuring attitudes, norms, behavioral beliefs and self-efficacy in relation to concurrent sexual partnerships yielded highly skewed response distributions with limited variability (range of coefficient of variation (measured as 100*[standard deviation/mean]): 33.6–70.0) .Based on the themes emerging from the focus groups and results of the preliminary cognitive testing of the initial candidate items, we developed a set of items incorporating vignettes with realistic scenarios involving concurrency or related behaviors. Each vignette-style item asked the respondent how much s/he agreed that the behavior of fictional characters presented in the scenario was acceptable. We revised items related to self-efficacy by asking respondents to assess the likelihood they would behave in the manner presented in the hypothetical situation. To broaden the potential response distribution and enhance sensitivity to small differences , we incrWe conducted cognitive testing of the revised items in a separate convenience sample of 16 African American respondents (6 men and 10 women) between the ages of 18 and 34 years who were recruited and nominated by study staff. To reduce study-related costs, the convenience sample was comprised of individuals known by study staff in the target age and racial categories for the main study. The convenience sample respondents did not live in the study counties and were therefore ensured of exclusion from both the main pre- and post-campaign studies. Responses to the revised items demonstrated greater variability (coefficients of variation range: 21.4–126.3), with vignette-style attitude items and self-efficacy items displaying the most (coefficients of variation range: 66.3–126.3). The revised items were used for the main pre- and post-campaign surveys.Data collected during the pre- (February through June 2012) and post- (June through November 2013) campaign surveys were used to perform a factor analysis of the revised item set, which served as a content pool from which potential items for the final scale could emerge. Item responses were recoded if necessary so that pro-concurrency responses had higher numerical values. We considered only the revised items with a 10-point response scale (attitude vignette-style items and revised self-efficacy items) in the factor analysis to avoid methods variance due to differences in response formats . The surWe randomly divided the pre-campaign survey respondents into two equal-sized groups and performed the same set of analyses on each group, to enable cross-validation of the factor analysis results. To determine the optimal number of factors to retain, we performed an exploratory factor analysis on each half of the pre-campaign sample. The exploratory analysis included an assessment of Cattell’s Scree Plot, Kaiser criterion of Eigenvalues ≥1.0, and the overall interpretability of the resulting factors. The factor analysis used an oblique rotation method that did not require the assumption that the underlying factors were uncorrelated. We retained factors with a primary loading of ≥0.50 and no secondary loading >0.30. Items that loaded poorly across all factors were discarded. Cronbach’s alpha was used to evaluate internal consistency of the items within each individual factor. Factors with low internal reliability (Cronbach’s α<0.7) were not retained. Furthermore, items that factored together but did not make theoretical sense were also discarded. The mean responses of items with a primary loading on the resulting factors were averaged to create composite factor scores. The factor analysis was repeated in the second pre-campaign subsample and in the post-campaign sample. Results from each sample were compared for agreement to confirm the resulting factor solution for our scale assessing concurrency attitudes ,35.a priori, the direction of the association between the scale and five socio-demographic and behavioral variables measured in the main surveys. Prior research indicates that men, younger adults, unmarried persons, and people with drug and/or heavy alcohol use have greater involvement in concurrency and should accordingly endorse attitudes more accepting of concurrency  [Our conceptual model posits that people who are more accepting of concurrency are more likely to engage in it –16. Thus scores) ,21,36–38We measured concurrency in this population using two methods. First, we provided the definition of concurrency and directly asked respondents if they had participated in this behavior in the past 12 months (self-report). Secondly, we measured concurrency as overlapping sexual partnerships at any point during the past year based on reported dates of first and last sex with the three most recent partners, one of the UNAIDS-recommended definitions . We usedWe examined the association between the attitude factor score and known correlates of concurrency using Pearson’s correlation statistic for continuous variables (number of partners in the past 12 months and age) and Spearman’s correlation statistic for categorical variables . The analysis was conducted separately in the pre- and post-campaign samples. Statistically significant correlation coefficients in the predicted direction were considered as validation that the factor measures attitudes about concurrency.The University of North Carolina at Chapel Hill Biomedical Institutional Review Board approved all study procedures (IRB #09–2142). Participants in the focus groups, the pilot surveys, and the pre- and post-campaign studies provided written informed consent.A total of 1,157 people were surveyed for the main media campaign evaluation. Pre-campaign respondents (N = 678) were predominantly young (46.4% 18–24 years old) and female (59.1%), and most (84.8%) had graduated high school . Post-caThe mean response was less than 6 (out of 10) for all revised attitude and self-efficacy items, where 1 denoted the lowest and 10 denoted the highest acceptance of concurrency . The iteIn the first random pre-campaign subsample, three factors emerged with Eigenvalues greater than 1.00  among the revised attitude and self-efficacy items; these three factors accounted for 53.6% of the total item variance. Upon visual inspection, the scree plot also suggested that three factors should be retained in the factor analysis .Examination of the factor loading scores from the factor analysis revealed that most of the revised items loaded onto only one factor. Moreover, four revised items displayed evidence of loading across two different factors. Cronbach’s alpha supported internal reliability only for Factor 1 (α = 0.82); reliabilities for Factor 2 and Factor 3 were 0.53 and 0.50, respectively. Even after removing revised items that cross-loaded across more than one factor, reliability estimates for factors 2 and 3 were low. The revised items that loaded onto Factor 1 were all vignettes and clearly related to attitudes toward concurrency. The interpretation of Factors 2 and 3 was less straightforward, with revised items associated with both self-efficacy and attitude loading onto both factors. The resulting low internal consistency and lack of interpretability prompted us to remove Factors 2 and 3 from further consideration.After we restricted the analysis to only one factor, loadings for six revised items, all vignettes, were high (>0.50). The remaining six revised items had low loadings (<0.40) and were removed from further consideration. We re-analyzed the reduced vignette-style item pool, extracting only a single factor ; this faIn the pre-campaign sample, our hypothesized attitude factor displayed a statistically significant positive association with having participated in concurrent partnerships, whether assessed by self-report  or deduced from dates of recent sexual partnerships . In addition, the factor score was positively correlated with reported number of sexual partners in the past year , a variable that is also strongly correlated with involvement in concurrency. Similar associations were observed in the post-campaign sample .a priori, attitude factor scores were positively associated with binge drinking and marijuana use in both the pre-  and the post-campaign samples . Similarly, the data supported our expectations that women would display attitudes indicating lower acceptance of concurrency than did men  and that older respondents would have attitudes less accepting of concurrency than younger respondents . However, contrary to our expectations, we did not observe married respondents to have lower acceptance of concurrency  [As predicted  = 0.06) .In this paper we report the development and evaluation of a scale for measuring attitudes toward concurrent sexual partnerships among young African American adults in the general population in Eastern North Carolina. Items adapted from previously published scales assessing attitudes about sexual behaviors had highly-skewed response distributions with insufficient variability for assessing concurrency-related attitudes in this population. Using feedback about these items and qualitative research, we developed vignette-style items that exhibited greater variability in responses. Factor analysis of the vignette-style items yielded a single concurrency attitude factor with acceptable internal consistency. The single factor was associated with concurrency and known correlates of concurrency in the predicted directions, providing construct validity. Thus, we believe that these results suggest that this scale may accurately assess young African Americans’ attitudes toward concurrency in a variety of realistic situations.Behavioral theory regards attitudes as a critical construct for predicting behavioral intention and behavior itself –16,40. Hown behavior. Given the sensitivity of concurrency, a third-person perspective may diminish respondents’ fear of socially undesirable responses [Results of focus group discussions helped us develop a pool of vignette-style items that respondents understood and were willing to endorse. The new items referred to fictional characters in common situations. Unlike the items adapted from existing scales, the vignettes may have encouraged respondents to think of familiar real-life situations, possibly even reminding them of people they knew. Respondents were still asked to provide their personal opinions of stigmatized behaviors but were not directly evaluating their esponses . The resAlthough vignettes have been used to assess psychosocial constructs related to sexual health, –45 we arFormal validation provided further evidence that the identified factor measures attitudes about concurrency. Our adaptation of Fishbein’s integrative model of behavior theorizes that certain socio-demographic traits and behavioral beliefs are associated with individual concurrency attitudes, which in turn influence individual behavior. We observed associations in the predicted direction between the factor score and known correlates of concurrency  ,21,36–38Surveys were conducted over the telephone, protecting respondents’ privacy in providing information about their attitudes and sexual behaviors and reducing potential social desirability bias in their responses. While a growing number of households rely only on cell phone service, the portability of cell phone numbers makes it difficult to target a specific geographic area and would have required a substantial increase in our research budget. Although respondents lived in a household with a landline, most respondents owned a cellular phone , similar to levels of cell phone ownership reported by other sources . CompariThe scale developed in this analysis has promise for measuring attitudes about acceptability of concurrent sexual partnerships. By incorporating situations known to prompt sexual concurrency in this and other settings, the vignette-style items appeared to sensitively and reliably measure variation in attitudes about concurrency among young African Americans in the general population of Eastern North Carolina. Measuring attitudes toward concurrency may serve as an indicator of sexual risk behavior, and a measurable outcome in the evaluation of behavioral HIV prevention initiatives.


Human hepatocyte transplantation has been actively perused as an alternative to liver replacement for acute liver failure and liver-based metabolic defects. Current challenges in this field include a limited cell source, reduced cell viability following cryopreservation and poor engraftment of cells into the recipient liver with consequent limited life span. As a result, alternative stem cell sources such as pluripotent stem cells, fibroblasts, hepatic progenitor cells, amniotic epithelial cells and mesenchymal stem/stromal cells (MSCs) can be used to generate induced hepatocyte like cells (HLC) with each technique exhibiting advantages and disadvantages. HLCs may have comparable function to primary human hepatocytes and could offer patient-specific treatment. However, long-term functionality of transplanted HLCs and the potential oncogenic risks of using stem cells have yet to be established. The immunomodulatory effects of MSCs are promising, and multiple clinical trials are investigating their effect in cirrhosis and acute liver failure. Here, we review the current status of hepatocyte transplantation, alternative cell sources to primary human hepatocytes and their potential in liver regeneration. We also describe recent clinical trials using hepatocytes derived from stem cells and their role in improving the phenotype of several liver diseases. The liver is responsible for a diverse range of functions within the body ranging from xenobiotic metabolism to plasma protein synthesis . HoweverThis review will focus on the current status of HT, alternate cell sources to primary human hepatocytes and alternate methods of liver regeneration which can be used either independently or in combination with HT.HT is a less invasive alternative to OLT. Hepatocytes are isolated from donor livers that have been rejected for organ transplantation in view of prolonged warm or cold ischaemia times, mild steatosis or aberrant anatomy. Hepatocytes isolated from one donor liver can yield a high quantity of cells and be used to treat multiple patients , 10. TheHepatocyte transplantation can be used to treat patients with liver-based metabolic diseases and ALF , 14, 15.Several urea cycle defects have also been shown to benefit from hepatocyte infusions. This includes ornithine transcarbamylase deficiency (OTC), which is an X-linked genetic condition affecting males that causes hyperammonemia and can also lead to neurological implications including developmental delay and learning disabilities. HT led to decreased ammonia levels and increased urea production in six of these patients, with four receiving an OLT within 7 months and two reported mortalities , 22–25. HT can also be used in patients with ALF. Although liver transplantation is the primary treatment, paediatric patients may die whilst waiting for an appropriately size and blood-matched liver, with no other treatment being shown to improve survival . FurtherDespite extensive clinical data, long-term clinical outcomes of HT have yet to be established with any type of liver-based metabolic disease or ALF. There are still many limitations to the technique, and current research aims to overcome this. Firstly, the cryopreservation process requires further optimisation with the current protocol resulting in low viability and function of hepatocytes post-thawing. Furthermore, transplanted donor hepatocytes undergo immune rejection with up to 70% of engrafted cells cleared within the first 24 h post-transplantation . Upon tr1-antitrypsin deficiency and glycogen storage disease type 1a. By generating cells with similar phenotypes to those caused by inherited diseases, it is possible to study the mechanisms of dysregulated cellular functions and identify ways to treat or reverse the condition [Induced pluripotent stem cells (iPSCs) that can be differentiated into hepatocyte-like cells (HLCs) are being widely explored as an alternative to primary human hepatocytes. These cells offer an excellent alternative source of hepatocytes and are advantageous over primary human hepatocytes because of their unlimited cell source and their ability to avoid the immune system . Methodsondition . Currentondition . Furtherondition , 53. Folondition , 53.In addition, the tumorigenic potential of these cells due to the presence of oncogenes such as c-Myc may raise a safety concern regarding the clinical application of these cells. Chen et al. reported no formation of teratomas or tumours in Gunn rats transplanted with HLCs, suggesting a loss of pluripotent characteristics within these cells . AlthougHuman fibroblasts offer another potential source of HLCs for HT. Fibroblasts are connective tissue cells found in all areas of the human body . Huang eIt is also possible to generate hepatocytes from amniotic epithelial cells. These cells have stem cell markers such as OCT-4, Nanog, SOX-2 and Rex-1, and as they do not have telomerase reverse transcriptase, they show a stable phenotype without the risk of tumorigenic potential . Such ceHepatic progenitor cells (HPCs), also known as oval cells, are believed to differentiate into mature hepatocytes or cholangiocytes, upon liver damage and help in tissue restoration. HGF and EGF are critical in inducing the transformation of HPC into hepatocytes. HGF activates the MET receptor, which further upregulates the expressions of AKT and STAT3 driving hepatic transformation. A lack of MET receptors completely attenuates HPC to hepatocyte differentiation even in the presence of EGFR. However, EGFR-null HPCs were still able to sufficiently transform into hepatocytes with MET alone .Zhang et al. established in vitro generation of HLCs from human foetal HPCs, under the influence of oncostatin M (OSM), DEX and HGF. These newly differentiated hepatocytes have functional glycogen storage, albumin secretion and cytochrome p450 activity with Khuu et al., suggesting that in vitro differentiated adult hepatic stem cells are also capable of urea production and ammonium chloride metabolism , 64. Unl+) into genetically stable functional HLCs in both in vitro and in vivo transplantations. Organoids were formed using ductal cells, and using medium consisting of BMP7, EGF and HGF, successful hepatic differentiation was achieved. Newly formed HLCs demonstrated albumin production, CYP3A3/4/5 activity and bile acid secretion. Furthermore, organoids successfully engrafted into Balb/c nude mice with induced liver damage, sustaining albumin and α-1-antitrypsin levels for up to 120 days in two out of five recipient mice. Debate remains over the genetic stability of fibroblasts and iPSCs as cell sources for HLCs. The expandable nature and genetic stability of HLCs derived from human bile ductal cells makes them a desirable alternate cell source [In addition to HPCs, mature hepatocytes can be derived from a number of other resident cell types within the liver. Huch et al. established a protocol differentiating primary human bile duct cells  have been investigated as another cell source for hepatocyte differentiation but with limited and controversial results. More promising is their immunogenic effect, and now, MSCs are being investigated as an immunomodulatory therapy to treat liver a number of different liver diseases.MSCs can be isolated from various tissues, such as bone marrow, adipose tissue and the umbilical cord. BMP and fibroblast growth factor (FGF) induction lead to the differentiation of MSCs to the hepatic lineage, with dexamethasone (DEX) and IL-16 inducing hepatic maturation. Single-step procedures are also commonly used with HGF and epidermal growth factor (EGF). Transformed cells generated from this procedure usually exhibit functional hepatic properties 2–3 weeks post-culturing but do lose functional capabilities when cultured for prolonged periods . FurtherRecently, it has been suggested that MSCs modulate liver failure by several mechanisms including differentiation of MSCs to replace damaged cells, secretion of soluble factors by MSCs to promote liver repair, MSC-mediated transfer of mitochondria by tunnelling nanotubules and by MSC-mediated transfer of proteins, RNA, hormones and chemicals by extracellular vesicles such as exosomes or microvesicles .MSC conditioned medium (MSC-CM) can play an important role in attenuating liver disease with a wide range of soluble factors thought to be present within MSC-CM . InterleIn addition to soluble factors present in MSC-CM, recently, exosomes have been identified as an important component that may promote hepatic regeneration. Tan et al. (2014) showed that CCL4-induced liver injury reduced AST and ALT levels and decreased the number of necrotic cells in mice that were treated with MSC-derived exosomes. Furthermore, proliferation of hepatocytes was greater, which was associated with increased expression of proliferating cell nuclear antigen . The autMSCs represent an ideal cell source for liver regeneration-based medicine due to their easily accessible source, their immunomodulatory properties and their potential of transdifferentiating into hepatocytes.There are now multiple phase I/II and III clinical trials using different types of stem cells to improve a number of liver diseases including cirrhosis, liver failure and liver-based metabolic disorders. In liver cirrhosis and end-stage liver disease, reports have proposed that MSCs can replace hepatocytes in the injured liver, stimulating liver regeneration Table .Table 2AShi et al. (2012) showed that transfusion of umbilical cord-MSC (UC-MSC) into 24 patients with acute-on-chronic liver failure showed marked increase in liver functionality when compared to the control of 19 patients transfused with saline. Patients were monitored over 48 weeks, with the treatment group showing an increase in albumin secretion, platelet count and a reduced end-stage liver disease (MELD) score. Furthermore, survival rate after 72 weeks was also higher in the treatment group compared to the control, with 20.8 and 47.4% mortality rate, respectively. The author suggests that although the mechanism of improved liver function may be unclear, in vivo differentiation of UC-MSC into hepatocytes is unlikely due to the short period of hepatic recovery and with only one treatment patient showing increased alpha-fetoprotein levels. It is more likely that soluble factors produced by MSCs may enhance liver revascularization and proliferation .One study has suggested that plasma exchange (PE) helps promote liver regeneration and recovery, leading to UC-MSC differentiation into HLCs. A phase I/II clinical trial is now in progress, transplanting UC-MSCs into patients with liver failure. Patients received either conventional treatment  with UC-MSCs and/or PE treatment, and survival rates were assessed at 48 weeks . For patMSCs are also being used clinically for immunomodulating therapy in many liver-based applications. One trial is currently investigating the use of MSCs to promote allograft tolerance and reduce the toxicity that results from exposure to calcineurin inhibitors. Paediatric patients receiving a liver transplantation undergo IV injection of bone marrow-derived MSCs. MSC toxicity is being monitored as well as graft function measured by aminotransferase and gamma glutamyl transferase activity, bilirubin, albumin and INR and the individual need for immunosuppressive medication. In addition, MSCs are being used as immunomodulators in ABO-incompatible liver transplantation. The study aims to determine if MSCs are safe and effective at reducing the primary non-function, acute rejection, ischaemic-type biliary lesions and morbidity in ABO-incompatible liver transplantation.Another promising area could be the use of macrophage therapy to treat liver disease. Macrophages reduce scar tissue and stimulate the HPCs to expand and differentiate into mature hepatocytes. Thomas et al. showed that bone marrow-derived macrophages (BMM) administered to mice with advanced liver fibrosis resulted in a degradation of fibrillar collagen and reduced fibrogenesis. There was also upregulation of the liver progenitor cell mitogen tumour necrosis factor-like weak inducer of apoptosis that was associated with an expansion of the progenitor cell compartment . There aHT offers an alternative therapy to OLT with the aim of treating liver-based metabolic diseases or ALF. Advances in liver cell therapy are being researched to overcome the obstacles associated with HT, particularly the shortage of healthy donor hepatocytes. Although HLCs are promising, no alternative cell source can yet replace the functionality and efficacy of primary human hepatocytes. MSCs likely hold the greatest attribute as immunomodulators, and co-culturing with mature donor hepatocytes. More clinical trials assessing the safety and efficacy of HLCs is pivotal before they can be considered as a reliable cell source. In the future, this may allow for liver-based diseases to be effectively treated without the need for OLT.


Human malignant tumors are characterized by pervasive changes in the patterns of DNA methylation. These changes include a globally hypomethylated tumor cell genome and the focal hypermethylation of numerous 5′-cytosine-phosphate-guanine-3′ (CpG) islands, many of them associated with gene promoters. It has been challenging to link specific DNA methylation changes with tumorigenesis in a cause-and-effect relationship. Some evidence suggests that cancer-associated DNA hypomethylation may increase genomic instability. Promoter hypermethylation events can lead to silencing of genes functioning in pathways reflecting hallmarks of cancer, including DNA repair, cell cycle regulation, promotion of apoptosis or control of key tumor-relevant signaling networks. A convincing argument for a tumor-driving role of DNA methylation can be made when the same genes are also frequently mutated in cancer. Many of the most commonly hypermethylated genes encode developmental transcription factors, the methylation of which may lead to permanent gene silencing. Inactivation of such genes will deprive the cells in which the tumor may initiate from the option of undergoing or maintaining lineage differentiation and will lock them into a perpetuated stem cell-like state thus providing an additional window for cell transformation. S-adenosyl-l-methionine to DNA cytosine bases predominantly within the CpG dinucleotide sequence context [DNA methylation involves the addition of a methyl group to position 5 of the DNA cytosine ring by DNA methyltransferase enzymes. This modification occurs predominantly at CpG dinucleotide sequences in mammals. DNA methylation patterns are established and maintained by a small family of DNA cytosine-5 methyltransferase enzymes , which transfer methyl groups from the co-factor  context . Two wavThe first observations of aberrant DNA methylation in cancer tissues were reported almost 40 years ago when global methylation analysis by chromatographic techniques indicated a reduction of DNA methylation levels in several types of malignancies in comparison with the corresponding normal tissue samples from which the tumors were derived ,6. TheseBesides global loss of 5-methylcytosine (5mC), the other prevalent phenomenon occurring in cancer is the hypermethylation of CpG islands , which wThe mechanisms of how these methylation changes in cancer occur are only partially understood. Different genomic regions, such as late-replicating or repetitive regions seem to undergo DNA hypomethylation in a preferential manner ,18, wherAlthough we now have a rather complete understanding of the nature of DNA methylation changes in malignant tissue due to application of genome-scale methylation mapping techniques, it has been much more difficult to pinpoint those methylation changes that have a functional role in tumorigenesis as opposed to being perhaps only a consequence of the malignant state. In a way, this situation is reminiscent of the challenge that exists for distinguishing driver mutations in cancer from innocuous passenger mutations. A tumor genome may harbor tens of thousands of somatic mutations and yet only a handful of them may be critical for cancer formation. Similarly, a tumor epigenome may contain a thousand hypermethylated CpG islands but only very few may be relevant for cell transformation. One other aspect to consider is the possibility that methylation changes are secondary to other events that have occurred at the chromatin level; for instance, a change in gene expression may occur in tumors as the result of acquisition or loss of a certain transcription factor, and this event may then trigger the methylation change. This argument is based on the now widely accepted idea that DNA methylation is the default state and that changes of genome occupancy of various DNA binding proteins can trigger methylation changes ,26. For In most mammalian tissues, approximately 3% to 5% of all cytosines are methylated. During global DNA hypomethylation of tumor genomes, there is a minor to moderate reduction of the number of methylated cytosines amounting to a loss of about 5% to 20% on average of the 5mC bases. Because this loss occurs so globally, it has been difficult to assign this phenomenon to specific molecular pathways or genes that are affected by the loss of methylation . Dnmt1 have increased tumor burden, which may be ascribed to loss of heterozygosity events and reduced genomic stability [One line of reasoning has been that the methylation loss will be accompanied by a less compact chromatin structure leading to the possibility of genomic instability. For example, mice deficient in tability ,29.Arthrobacter luteus (Alu) repeats has often been used as surrogate markers for global DNA methylation levels [Repetitive genomic regions, which account for about 55% of the human genome, are particularly susceptible to 5mC loss. These sequences include long interspersed nuclear elements (LINE) and Alu-type repetitive regions, two major types of interspersed repeat elements, as well as various classes of endogenous retroviruses  and centromeric satellite repeats. Due to their abundance, methylation of LINE-1 or n levels . Since DLINE-1 activation may indeed be a component of carcinogenesis. Sequencing studies of cancer genomes from several hundred patients revealed that tumors from a substantial fraction of the patients had somatic retro-transpositions of LINE-1 elements . The actDnmt genes have suggested that methylation loss is potentially causative for cancer formation [One other phenomenon associated with the cancer epigenome is the large-scale rearrangement of the three-dimensional chromatin architecture. There are rearrangements to large domains of chromatin blocks  and to the nature of lamina-associated domains in cancer ,32. The ormation ,29. HoweMAGE (melanoma antigen gene). These genes were expressed also in many other types of cancer but not in normal somatic tissues revealing them as suitable biomarkers of malignancy and even as potential therapeutic targets due to their unique cancer-specific expression patterns. In total, there are over 250 genes characterized as cancer testis genes with many of them being localized on the X chromosome. One major branch of these genes is the MAGE family, which is evolutionary conserved and consists of over 50 family members. These genes encode ubiquitin ligases that function during germ cell development in reproductive organs. Although the cancer testis genes are markers of malignancy, their functional involvement in carcinogenesis has been less clear. Such a role would best be proven if they would have an oncogenic role in somatic tissues when aberrantly expressed [MAEL) [PIWIL1) have a positive effect on phosphoinositide-3 (PI3) kinase/AKT signaling and cell proliferation pathways [BORIS/CTCFL, which encodes a homologue of the insulator protein CCCTC binding factor (CTCF). BORIS/CTCFL was shown to be upregulated in testicular and ovarian cancer where the encoded protein leads to upregulation of the telomerase reverse transcriptase (hTERT) gene promoting cell immortalization [Gene-specific DNA hypomethylation events might also contribute to cancer. There is a group of genes that becomes activated in tumors by loss of DNA methylation at the promoter regions ,34. Thesd [MAEL)  and Piwipathways . Some ofpathways . One intlization . Other mOne additional outcome of genome-wide DNA hypomethylation is its likely effect on global gene transcription patterns. On the genome scale, the expression level of genes is positively correlated with the level of methylation within the transcribed regions, also referred to as gene bodies . This coGenome-wide hypermethylation of CpG islands is observed not only in most primary and metastatic tumors  but is aA tumor-promoting effect of methylation-induced silencing events can be expected if the affected genes participate in functional pathways described as the “hallmarks of cancer” . Those iThe following examples illustrate that DNA hypermethylation of known tumor suppressor genes does occur as an alternative to mutational inactivation or gene copy loss and thus is very likely representing a true tumor-driving event. CDKN2A), coding for a CDK inhibitor protein also known as p16, marked one of the earliest demonstrations of a tumor-driving role of DNA hypermethylation [CDKN2A occurs in many types of malignancy including breast cancer, head and neck cancers, gliomas, and melanomas. Importantly, CDKN2A can be inactivated by several mutually exclusive events including homozygous loss, base substitution mutations, and by promoter methylation. The discovery of cancer-associated hypermethylation of the gene cyclin-dependent kinase inhibitor 2A (hylation . InhibitBRCA1), which is mutated in hereditary breast and ovarian cancers, contains aberrant promoter methylation in a smaller subset of sporadic breast and ovarian tumors [BRCA1 can contribute to defects in homology-directed DNA repair in the affected individuals. In contrast to BRCA1, methylation-mediated silencing of BRCA2 is relatively rare in breast or ovarian cancers.The breast cancer susceptibility gene breast cancer 1  are associated with the familial cancer syndrome adenomatous polyposis coli, which predisposes its carriers to early onset colorectal cancer. APC is a negative regulator of the Wingless/Int (WNT) signaling pathway. Methylation of the promoter region of the APC gene leads to gene silencing and may contribute to carcinogenesis [APC methylation is not only found in colorectal tumors but also in esophageal cancer, gastric cancer, pancreatic and prostate cancers as well as in some other tumor types. Germline mutations of the tumor suppressor gene adenomatous polyposis coli . Mutations in DNA mismatch repair genes, as discussed above, lead to a hypermutator phenotype often manifesting itself as microsatellite instability. Genetic defects in base excision repair genes are more rarely associated with cancer. DNA double strand break repair and recombination repair pathways are compromised by mutations in BRCA1, BRCA2, RAD51 paralogs, and a few other genes. Several alternative epigenetic pathways for DNA repair gene inactivation have already been discussed above, including MLH1 and BRCA1 methylation. However, cancer-associated methylation of the many other DNA repair genes is a relatively rare occurrence in human cancer [MGMT gene is epigenetically inactivated by DNA methylation in colorectal cancers, gastric cancers, head and neck squamous cell carcinomas, non-small cell lung cancers and most prominently in gliomas [MGMT is expected to diminish the efficiency of O6-alkylguanine repair. It was shown that glioma patients with a methylated MGMT gene had a better survival rate when treated with the alkylating agent temozolomide or other alkylating agents when compared to patients with an unmethylated promoter presumably because of increased cell killing by the chemotherapy agent [The inactivation of DNA repair function will likely increase the incidence of mutations, both at the single base level or at the chromosomal level depending on which repair pathways have been disabled. Several DNA repair processes are compromised in tumors, most notably by germ lines mutations. Examples include the defects in nucleotide excision repair because of mutations in the Xeroderma pigmentosum gene group , which occurs in B cell malignancies and many other cancer types [CASP8), which encodes a cysteine protease regulated in a death-receptor-dependent and independent manner, was found in neuroblastomas and other tumors [TP73 promoter is found in neuroblastomas, melanomas, and several other cancers [Evasion of apoptosis is a hallmark of cancer. One would expect that DNA methylation events that diminish apoptosis are potential tumor-driving events. Indeed, methylation silencing of several proapoptotic genes has been reported in malignant tumors. One of the first examples of methylation of a pro-apoptotic gene was death-associated protein kinase . Methylation-induced silencing of  cancers . Other mDrosophila, the Hippo pathway restrains cell proliferation and functions in organ size control [MST1/MST2 genes or overexpression of YAP produces liver cancer in mice [MST1 and MST2 promoters has been found in soft tissue sarcomas [RASSF1A gene [RASSF1A gene is one of the most frequent methylation events in human cancer [One other example in which methylation of signaling network control elements may promote cancer formation is the Hippo tumor suppressor pathway. First studied in  control . The pat in mice . Somewha in mice . Insteadsarcomas . A few psarcomas . One RASF1A gene . Methylan cancer . Methylan cancer . The RASn cancer .HOX genes and other homeobox gene family members. For example, the genes HOXA7 and HOXA9 are commonly methylated in lung tumors and other cancers [OTX1 and NR2E1/TLX1 are methylated in 100% of the lung squamous cell carcinomas tested [PAX6, DLX2, and SIM1) are hypermethylated in the tumors [With the advent of genome-scale DNA methylation mapping techniques, the field moved from a specific gene-centered approach to cataloguing the entirety of DNA methylation changes found in tumors. The various methylation mapping techniques were based initially on restriction enzymes that are blocked by CpG methylation, but soon included methods that employ antibodies against 5mC or proteins that bind to 5mC with high selectivity specifically to enrich methylated gene loci. Other methods that provide the most comprehensive overview use whole genome bisulfite sequencing. More limited scale bisulfite-based approaches such as the Illumina array platforms have also very commonly been used. These genome-scale studies revealed that the most frequently methylated genes in cancer cells were genes encoding developmental transcription factors . One cla cancers . The homs tested . These ts tested . Becausee tumors . Since De tumors . In concGenome-wide DNA methylation analysis has provided comprehensive overviews of the scale of tumor-associated DNA methylation changes. There are at least two major challenges remaining. First, it is not at all clear how these DNA methylation changes are initiated. Second, and as discussed in this article, we still need to understand which of the numerous DNA methylation changes are truly tumor-driving events. Although changes in DNA methylation, in the form of both overall DNA hypomethylation and localized DNA hypermethylation at CpG-rich promoters have been known for several decades, it is still a challenge to pinpoint specific methylation changes and their associated mechanisms as tumor-driving processes. The mechanisms of how DNA hypomethylation promotes tumorigenesis in human cells are particularly unclear. Although several examples now exist for a tumor-driving role of methylation-induced gene silencing, the physiological effects of the clear majority of de novo DNA methylation events are not known. One way to investigate the potential of a methylated gene in tumor formation is to delete the gene, either in cultured normal cells or in mice and then observe effects of this deletion on the tumorigenic phenotype of the manipulated cells or animals. More relevant would be the specific methylation of the gene of interested using epigenetic editing techniques. These techniques are now being developed and include, for example, the CRISPR/dCas9-mediated introduction or removal of methyl groups from targeted gene loci. However, some of these techniques still lack the desired specificity and induce gene methylation at many non-targeted loci. Improvement of these approaches, perhaps in combination with multiplexed, library-type screening systems should allow a more precise dissection of the roles of many of the observed methylation changes as they occur in human cancer.There are now many examples clearly suggesting a tumor-promoting role of hypermethylation at specific gene promoters. These examples include the methylation of genes controlling DNA repair and genomic stability. Genes relevant for induction of apoptosis, for controlling pro- and anti-proliferative signaling networks, and genes encoding transcription factors involved in lineage differentiation are also subject of tumor-specific DNA methylation. Individual tumors often will have multiple genes from these pathways in a hypermethylated state suggesting that the sum of these methylation events will provide an even stronger tumor-promoting force than DNA methylation occurring at single genes.


Percutaneous coronary intervention (PCI) involving the anomalous coronary artery is challenging with respect to difficulty in achieving stable catheterization. Rotational atherectomy (RA) can facilitate severely calcified lesions to improve stent delivery and stent expansion; however, its utility in tortuous and angulated coronary arteries is limited with difficulty in delivery of the RA burr. The mother-and-child technique is effective for complex PCIs with increased backup force for device delivery in such complicated cases. We report a case of successful rotational atherectomy using the “mother-and-child” technique with a Dio thrombus aspiration catheter for an angulated calcified lesion in an anomalous origin of the right coronary artery. Coronary artery anomalies are a diverse group of congenital disorders that are found in 5% of the population . AnomaloA 46-year-old man with end-stage renal disease due to diabetic nephropathy was referred to our institution for percutaneous coronary intervention (PCI) for a severe calcified lesion in an anomalous right coronary artery. He had been undergoing hemodialysis three times a week for 3 years. Since patients with diabetic ESRD are at a high risk for coronary artery disease, coronary computed tomography angiography (CTA) had been performed at his previous hospital visit, which revealed multivessel disease. Coronary angiography (CAG) showed severe stenosis in the midportion of the left anterior descending (LAD) artery and a chronic total occlusion (CTO) in the proximal portion of the right coronary artery with an anomalous origin. Hence, PCI of the LAD lesion was first performed, and a bioresorbable polymer sirolimus-eluting stent  was successfully implanted at the previous hospital. Staged PCI for CTO of the RCA was performed with successful wire crossing and recanalization. However, since subsequent standard balloon angioplasty was unsuccessful, he was referred to our hospital for PCI of the anomalous RCA.CAG demonstrated residual severely calcified stenosis in the proximal RCA with patent antegrade coronary flow . We seleWe report a case of successful rotational atherectomy for an angulated calcified lesion in an anomalous right coronary artery using the “mother-and-child” technique. PCI for the anomalous RCA can be associated with difficulty in achieving stable catheterization, leading to reduced backup support and device delivery failure. Rotational atherectomy (RA) can facilitate lesion and stent expansion in severely calcified lesions, although its utility is limited in tortuous and angulated coronary arteries, with an increased risk of complications. Appropriate guiding catheter selection is a key step to ensure successful PCI in such cases.The mother-and-child technique is effective for complex PCIs, as it increases the backup force through deep catheterization of the target vessel and facilitates device delivery in tortuous or calcified lesions. Several mother-and-child catheters have been introduced in clinical practice . The GuiIn our case, we selected the conventional mother-child catheter, the 5 Fr Dio aspiration catheter for the following reasons. First, the 5 Fr Dio thrombus aspiration catheter has the RX-type inner catheter which facilitates smooth delivery and deep insertion of the outer catheter into the anomalous RCA. The relatively large inner lumen size of the outer catheter (1.51 mm) can provide the strong backup force for device delivery. Second, there is a concern that the rapid exchange transition zone of GuideLiner can lead to less backup support, difficulty in delivering the burr, and increase in the risk of burr entrapment. Thus, we thought that the Dio thrombus aspiration catheter can provide stronger backup support in our patient with rather complicated anatomy. Komatsu et al. reported successful PCI in an anomalous RCA using the Dio thrombus aspiration catheter . This caWe report a case of successful rotational atherectomy using the “mother-and-child” technique with a Dio thrombus aspiration catheter for an angulated calcified lesion in an anomalous origin of the right coronary artery.


Chan et al.; Hassan et al.; Al Atya et al.). In addition, these pathogens are able to acquire antimicrobial resistance by either drug-induced selection or drug-mediated mutagenesis . Both cases result in diminution of antibiotic shelf time and make essential the search of new active compounds to be used in antimicrobial therapy. Natural products from micro-organisms, plants, animals, and algae have been proved to be excellent sources of antimicrobial compounds . The papers published in this Research Topic have proved this hypothesis as we illustrate bellow.Infectious diseases still one of the major causes of mortality and morbidity worldwide. The incidences of these infections are higher in communities exposed to inadequate sanitary conditions (commonly found in developing countries), and hospitalized and immunosuppressed individuals. This parasitic efficiency of microorganisms is due their high capacity to develop strategies to evade immune system and provoke host damage through virulence factor expression . In our Research Topic, active microorganisms were obtained from different niches since traditional sources—such as soil , plant (endophytes) —until unusual environments—such as Antarctic Ocean , hypersaline habitats (Jose and Jebakumar), and mud wasp nests . Some papers also showed that active bacteria could be found associated to invertebrate organisms such as marine sponges  and rhabditid entomopathogenic nematode . These microorganisms showed biotechnological aptitude as they produced compounds able to inhibit the growth of bacteria  and fungi .The production of antibiotics by microorganisms has been recognized for a long time. Several research groups have isolated new microbial species  able to produce new antimicrobial compounds or more productive strains . One example is nisin, a lantibiotic produced by Lactococcus lactis. Besides its activity against Gram-positive and Gram-negative planktonic cells of oral bacteria, nisin was able to impair multi-species biofilms (by inhibiting the biofilm formation or eradicating pre-formed biofilm). At anti-biofilm concentrations, nisin treatment did not result in toxicity toward human cells relevant to the oral cavity . In another remarkable paper, the cis-2-decenoic acid (CDA) signaling network of Pseudomonas aeruginosa was revealed by microarray analysis. CDA is a signaling molecule involved in quorum-sensing. The authors also showed that this compound could disperse biofilms of P. aeruginosa alone or when combined with conventional antibiotics .Some bacteria were also able to inhibit virulence factors such as biofilm formation, toxins and proteases . Another elegant paper employed, for the first time, the phenylalanine/tyrosine ammonia lyase enzyme produced by Rhodotorula glutinis in the bioconversion of L-tyrosine methyl ester (L-TM) to the methyl ester of para-hydroxycinnamic acid (p-HCAM), an antibacterial product . Finally, the improvement of antibiotic production through optimization of culture conditions was also demonstrated by a couple of papers .An important effect against mycobacterial persister cells was shown for boromycin, a macrolide antibiotic produced by Santos et al.). Plants have been widely used by traditional communities due to their supposed medicinal properties. This popular knowledge has directed a lot of researches aiming to provide scientific evidence of these actions , proteins , polyphenols , and triterpenoids . The antimicrobial activity of these plant compounds was further characterized using alternative infections models such as Caenorhabditis elegans (Eng and Nathan) and Zebrafish embryo .Plants derived compounds are also attractive candidates for bioprospecting programs due their high chemical diversity which results in the inhibition of a range of microbial pathways . The multiple therapeutic properties of honey are due to its chemical diversity  . One of these studies lead to the isolation of glycoproteins from honey which showed sequence identity with the Major Royal Jelly Protein 1 (MRJP1) which is composed by three antimicrobial peptides: Jelleins 1, 2, and 4. These glycoproteins exhibit a broad spectrum activity against multi-drug resistant clinical isolates .Another emergent topic in antimicrobial field is the research using honey as source of bioactive molecules. Indeed, honey has been used in traditional medicine to treat infections  from different sources  in the development of optical and dielectric sensors for microbial detection. Due the advances in nanotechnology, different nanostructured platforms containing AMP have been obtained resulting in biosensors with increased efficiency. These AMP-based biosensors could be employed for microbial diagnosis and for ensure the quality of different products such as water, food, and cosmetic.Leishmania sp., Plasmodium sp., and Trypanosomes was reviewed by Lacerda et al. These authors also discussed the application of transcriptome analysis to prospect new peptides from non-host insects. Other paper demonstrated the leishmanicidal activity of Piper nigrum, a well-known medicinal plant. The bioactive fractions are able to induce apoptosis in promastigote forms of Leishmania donovani. Moreover, these fractions exhibited therapeutic action against experimental leishmaniasis which was related to their immunostimulatory potential . Anti-leishmania action was also evaluated to β-nitrostyrenes, a rare class of reddish brown compounds with antimicrobial activity which has been isolated from Streptomyces lavendulaea  and Indian mangrove plant Sonneratia acids Linn. F (2-nitro-4-((E)-2-nitrovinyl)phenol). Some β-nitrostyrenes derivative compounds (synthesized using Henry reaction) were found to selectively inhibit promastigotes and amastigotes forms of L. donovani .Regarding the protozoal infections, the use of peptide from host insects as drug against Trichilia dregeana, Detarium microcarpum, and Phragmanthera capitata) were found as source of anti-HCV compounds and the activity was related to inhibition of HCV entry without affect viral replication or secretion . Other paper showed the potential use of dryocrassin ABBA against amantadine-resistant H5N1 avian influenza virus (HPAIV). Dryocrassin ABBA is a phloroglucinol derivative isolated from Rhizoma Dryopteridis Crassirhizomatis. This compound increased the survival rates of HPAIV virus infected mice, decreased lung index and virus loads. It also exhibited immunomodulatory action inhibiting pro-inflammatory cytokines  and increasing anti-inflammatory cytokines (IL-10 and MCP-1).The antiviral properties of Cameroonian medicinal plants against hepatitis C virus (HCV) were also evaluated. The plants were selected based in an ethnobotanical survey which revealed that several plants from Western region of Cameroon have been used to treat liver-related disorders. Three plants (Taken together, all these papers illustrate the versatility of natural products and highlight the importance of developing new prospection tools to impulse the discovery of new compounds. These molecules could be used as drug leads and also provide more insights for target discovery.All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Alcanivorax, Cycloclasticus, Marinobacter, Neptumonas, Oleiphilus, Oleispira, and Thalassolituus, as well as the Alphaproteobacterial genus Thalassospira. Detection of HCB in amplicon-based sequencing surveys relies on high coverage by PCR primers and accurate taxonomic classification. In this study, we performed a phylogenetic analysis to identify 16S rRNA gene sequence regions that represent the breadth of sequence diversity within these taxa. Using validated sequences, we evaluated 449 universal 16S rRNA gene-targeted bacterial PCR primer pairs for their coverage of these taxa. The results of this analysis provide a practical framework for selection of suitable primer sets for optimal detection of HCB in sequencing surveys.Hydrocarbonoclastic bacteria (HCB) play a key role in the biodegradation of oil hydrocarbons in marine and other environments. A small number of taxa have been identified as obligate HCB, notably the Gammaproteobacterial genera  Alpha-, Beta-, and Gammaproteobacteria; Actinomycetes; Flavobacteria-Cytophaga-Bacteroides), comprise representatives of HCB species , or of aromatic hydrocarbons (Cycloclasticus and Neptunomonas) – i.e., they use these hydrocarbons almost exclusively as a sole source of carbon and energy . These fastidious organisms are strongly selected for in oil-impacted environments, where they can increase in numbers from near undetectable levels to constituting up to 70–90% of the total bacterial population, such as during an oil spill  were discovered in recent years from the phycosphere of marine eukaryotic phytoplankton (in situ hybridization) probes, that were designed to target these obligate HCB, has revealed that the numbers of these bacteria on phytoplankton cells is extremely low – from no HCB cells, to one or two detected per phytoplankton cell (unpublished data). This may explain why these organisms are not often detected in sequencing surveys, and their role in the fate of oil hydrocarbons in the ocean remains poorly understood.Marine microorganisms play important roles in major biogeochemical processes and in various interactions with higher organisms that, respectively, impact our climate and the sustainability and balance of life on this planet. A subset of the microbiome in the global ocean that plays a fundamental role in the biodegradation and ultimate removal of petrochemical pollutants are the hydrocarbonoclastic bacteria (HCB). The fate of oil in the environment is largely dictated by the presence and activities of these bacteria. Over 175 genera, distributed across several major bacterial classes  and next generation sequencing (pyrosequencing) approaches of 16S rRNA genes . This reWhile amplicon sequencing technologies have advanced significantly in recent years, they rely on suitable PCR primer coverage of target microbial groups  to provide a reliable view of microbial diversity and community structure. To the best of our knowledge, reports describing isolates belonging to any of the genera of obligate HCB have all been shown to degrade hydrocarbons. Hence, we can be highly confident that any organism identified by 16S rRNA gene sequencing to be affiliated to any of the eight genera of obligate HCB will be capable of degrading hydrocarbons. Despite inherent limitations in PCR-based methods such as biases in cell lysis efficiency, differences in rRNA copy number, and variation in amplification efficiency of different templates, PCR-based studies are an important method to catalog microbial diversity. For this subset of bacteria, PCR-based amplification of the 16S rRNA gene remains a powerful tool to link taxonomic identity with the ability to degrade hydrocarbons. In this study, we identified 16S rRNA gene sequences that represent the phylogenetic breadth of key HCB taxa using a detailed phylogenetic analysis. We then used these validated representative taxa to determine the coverage of these groups by a comprehensive list of commonly used universal 16S rRNA gene-targeted PCR primer sets. With this approach, we were able to identify PCR primer sets well-suited for use in sequencing surveys focused on detection of HCB but that also applicable for detecting overall bacterial diversity.Oceanospirillales, Alteromonadaceae, Rhodospirillaceae, and Piscirickettsiaceae . These taxa, as well as outgroup genera for them  were extracted from the SILVA SSU Ref 123 database of 16S rRNA gene sequences  were extracted from the SILVA SSU rRNA database. These sequences were clustered into OTUs at 97% sequence identity and representative sequences were selected for each OTU  (Supplementary Data Sheet Marinobacter within the family Alteromonodaceae clustered into a well-supported monophyletic group (bootstrap support (BS) = 94) and 151 sequences were selected from this clade  and 24 sequences were selected  . These sequences were removed and the remaining 76 Thalassospira sequences were selected. Within the order Oceanospirillales, four Oleispira sequences were selected (BS = 92), 10 Neptunomonas sequences were selected (BS = 48), three Thalassolituus sequences were selected (BS = 60), and a single Oleiphilus sequence was selected . We therefore opted for a conservative approach and only lineages with cultured representatives were selected, resulting in 135 selected sequences for this genus. All selected sequences are available in the Supplementary Information.In the present study, we performed a phylogenetic analysis to validate the classification of 16S rRNA gene sequences for eight HCB taxa that are recognized as key players in the biodegradation of oil hydrocarbons in the marine environment. In order to select sequences for phylogenetic analysis, 16S rRNA gene sequences that were classified as belonging to either a family or order of the included HCB taxa . We used the grouping of primer pairs into small (100–400 bp), medium (>400–<1000 bp), and large (>1000 bp) amplicons according to in silico. For all three amplicon size classes, there was a large distribution of coverage of primer pairs, with many primer pairs having less than <10% coverage for some or all of the target taxa . Generally, more primer pairs producing short amplicons had high coverage (>90%) than primer pairs producing medium or long amplicons. An optimal primer pair would have high coverage for each of the eight HCB target taxa, but we observed large variability in the number of primer pairs with high coverage across all of the target taxa. For each amplicon size category, primer pairs were identified having at least 83–95% coverage . Multiple short and medium amplicon primer pairs were identified that had >90% coverage for each of the target taxa, though no long amplicon primer pairs were identified with >90% coverage for each of the target taxa . The reason for this is that no large amplicon primer pairs had >90% coverage for the groups Alcanivorax, Cycloclasticus, and Oleispira . These results highlight that if long-read sequencing technologies become practical for amplicon sequencing new primers should be designed to better target these taxa. A list of primer pairs with >95% coverage for all target taxa can be found in Supplementary Table S1. We re-evaluated the coverage of these primer pairs for all Bacteria and found that almost all had >80% coverage, indicating that they are all useful for capturing general bacterial diversity as well as obligate HCB taxa. We note that for this comparison we made a conservative assumption by considering only perfect matches to target sequences. Allowing for mismatches would be expected to generally increase coverage values.Using the validated set of HCB taxa sequences, including all sequences within each OTU, we evaluated a comprehensive list of published universal in silico-derived amplicons from sequences of target taxa to be classified with the RDP näive Bayesian classifier. This analysis revealed that almost all  target amplicons were correctly classified to the genus level, and correctly classified sequences were almost all classified with ≥80% confidence (Supplementary Table S2).Currently, most amplicon sequencing is performed using the Illumina MiSeq platform, which is amenable to medium-length amplicons. We therefore selected the primer pair with the highest coverage in this group for further analysis. The primer pair, S-D-Bact-0343-a-S-15 (forward) and S-D-Bact-0908-a-A-18 (reverse) forms an amplicon of 583 bp and covers the hyper-variable regions 3–5. In order to determine if amplicons from target taxa produced from this primer pair would be correctly classified using a standard classification analysis, we extracted Oleiphilus and Thalassolituus. Poor primer coverage may also help to explain the rare detection of Oleiphilus. Generally, many primer pairs are available that have high overall coverage of HCB taxa for small and medium sized amplicons, but coverage is more limited for primers designed to produce large amplicons. For medium-sized amplicons amenable to the currently widely used Illumina MiSeq platform, we identified the primer pair S-D-Bact-0343-a-S-15 (forward) and S-D-Bact-0908-a-A-18 (reverse) with the highest overall coverage, and which targeted all groups with at least 95% coverage. We therefore recommend the use of this primer pair for studies focused on detection of these obligate HCB in the environment. Though in silico analysis suggests that all primer pairs are compatible for PCR, experimental testing would be necessary before employing any new primer pair. In future work, we aim to explore this and other primer pairs to identify also ‘generalist’ hydrocarbon degraders.We found that universal bacterial 16S rRNA gene primer sets varied widely in their coverage for the eight HCB taxa evaluated here. This suggests that differences in HCB prevalence across different studies must be interpreted with caution and highlights the need for careful primer selection. There was no consistent trend for any single genus to be missed by all primers, but few primer pairs covered the genera DB and TG designed the study, analyzed the results, and wrote the manuscript. DB performed computational experiments.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.


Depression affects 20–30% of HIV-infected patients and is associated with worse HIV outcomes. Although effective depression treatment is available, depression is largely untreated or undertreated in this population.We quantified gaps in antidepressant treatment, treatment adjustments, and outcomes among US patients in routine HIV care in the nationally distributed CNICS observational clinical cohort. This cohort combines detailed clinical data with regular, self-reported depressive severity assessments . We considered whether participants with likely depression received antidepressants, whether participants on antidepressants with persistently high depressive symptoms received timely dose adjustments, and whether participants achieved depression remission. We considered a cross-sectional analysis  and a prospective analysis .The cross-sectional sample was 87% male, 53% Caucasian, 25% African American, and 18% Hispanic; the prospective sample was similar. In both samples, 39–44% had likely depression, with 44–60% of those receiving antidepressants. Of participants receiving antidepressants, 20–26% experienced persistently high depressive symptoms; only a small minority of those received antidepressant dose adjustments. Overall, 35–40% of participants on antidepressants achieved full depression remission. Remission among participants with persistently high depressive symptoms was rare regardless of dose adjustments.In this large, diverse cohort of US patients engaged in routine HIV care, we observed large gaps in antidepressant treatment, timely dose adjustment to address persistently high depressive symptoms, and antidepressant treatment outcomes. These results highlight the importance of more effective pharmacologic depression treatment models for HIV-infected patients. Most paAcross sites, 39% of person-time had an indication for depression treatment while 61% did not . For 16%One-fifth of treated person-time (22%) was immediately following antidepressant initiation (Type 1) . For halCombining these estimates indicates that 39% (95% CI: 39–40%) of the population had an indication for depression treatment, 24% (23–24%) were receiving antidepressant treatment, 18% (17–18%) were receiving evidence-based antidepressant treatment (either through passive pharmacologic maintenance or active pharmacologic adjustment), and 9% (9–10%) had achieved remission after starting pharmacologic treatment .After the site-specific launch of PHQ-9 screening, 2,936 participants establishing HIV care contributed 86,906 person-months of observation (mean [SD}: 29.6 [19.5] per person) . DemograAmong all participants new to CNICS care, the Kaplan-Meier failure function estimate of the proportion having an indication for depression treatment within the first 12 months of establishing HIV care at a CNICS site was 44%. The estimated probability of starting an antidepressant within the first 12 months of care was 44% in person-time following a PHQ-9 score ≥10, 17% in person-time following a PHQ-9 score <10, and 30% in person-time not immediately following a PHQ-9 screening. Among participants who had not started antidepressants at the time of PHQ-9 screening, the probability of achieving remission within 12 months of a PHQ-9 score ≥10 was 36% overall, 34% in person-time following antidepressant initiation, and 39% in person-time not following antidepressant initiation.When considering evidence-based antidepressant treatment versus pharmacologic inertia, over half (58%) of the treated person-time was before the first follow-up PHQ-9  . ApproxiCombining these estimates indicates that 44% (42–46%) of the population had an indication for depression treatment in the first 12 months of CNICS care, 19% (17–22%) received any antidepressant treatment in the first 12 months, 12% (11–14%) were receiving evidence-based antidepressant treatment (either through passive pharmacologic maintenance or active pharmacologic adjustment), and 7% (6–8%) achieved remission within 12 months . There wIn sensitivity analyses to assess the role of non-random attrition, estimates of the depression treatment continuum  and the This analysis represents the first comprehensive assessment of the depression treatment continuum in a large, diverse sample of US patients engaged in HIV primary care. We identified substantial gaps in pharmacologic depression treatment, antidepressant adjustments, and remission in this population. Regardless of whether we considered a cross-section of all active clinic patients or a prospective cohort of patients establishing care, approximately 2 in 5 patients were in need of depression treatment, about half of those in need received antidepressant treatment, and fewer than half of those on antidepressant treatment achieved full depression remission.Approximately 40% of CNICS patients had an indication for depression treatment. This estimate is consistent with other reports of depression prevalence among HIV-infected patients in similar primary care populations., 24–32 OThe combination in the CNICS cohort of repeated depressive severity assessments at clinical contacts paired with detailed antidepressant medication histories provided a unique opportunity to evaluate antidepressant treatment adjustments in this population. We assessed the frequency with which patients on antidepressant treatment but with persistently high depressive symptoms received dose adjustments as indicated by evidence-based guidelines., 13 WhilAmong the small number of patients in our analysis who required and received antidepressant treatment adjustment, none achieved remission; among patients who required treatment adjustment and did not receive it, remission rates were not much different. In contrast, about a quarter of patients achieved remission within the initial antidepressant treatment period, and of patients exhibiting mild remaining depressive symptoms while on treatment, about a quarter went on to achieve remission. In addition, after a high PHQ-9, the overall remission rates between patients starting and not starting antidepressant treatment were quite similar. These findings suggest two separate depression phenotypes- one that responds promptly to antidepressant treatment, and may even resolve in the absence of treatment , and one that is treatment-resistant regardless of antidepressant treatment adjustments. These phenotypes would almost certainly be associated with a range of covariates including medical and psychiatric comorbidities and psychosocial factors. It is possible for example that the patients who received treatment adjustments were those with psychiatric complexity or otherwise known to be treatment-resistant and were referred to psychiatric resources. These patients would have been less likely to achieve remission due to their depression phenotype, a type of confounding by indication, perhaps explaining why none of the patients who received Type 3 treatment achieved remission.The limitations of this study should be considered. First, depression and remission are defined based on a screening instrument that, while possessing excellent psychometric properties, is not diagnostic. To benchmark the performance of the PHQ-9 screening tool in this study relative to other populations, our results can be compared to data from the STAR*D randomized controlled trial in which the QIDS-SR16, a screening tool with similar properties to the PHQ-9, was used to assess depression in a large non-HIV infected outpatient sample. In that Another important consideration is the irregular timing and spacing of depressive severity and remission measures used in our analyses. In a real-world outpatient psychiatric setting, depressive severity is ideally measured within 4–6 weeks of initiating antidepressant treatment and appropriate changes to the treatment regimen are made. In this study we did not have such frequent measures of depression so we considered severity and remission assessments up to 6 months after antidepressant initiation. While our results provide important aggregate parameters of the pharmacologic depression treatment continuum within the HIV clinical setting, estimates of remission may not reflect the detailed course of illness assessed in psychiatric care settings. However, our findings may be highly germane to the provision of pharmacologic depression treatment in outpatient HIV care settings.A key strength of this study is the size, diversity, and richness of the data source. The CNICS cohort is distinct from many other longitudinal HIV cohorts in that it is a clinical care cohort: participation requires only consent to have routinely collected medical data captured, leading to very high participation rates across all sites. With inclusion of medical centers in the West, South, mid-Atlantic, and Northeast, the relevance of the experiences of the CNICS cohort to patients engaged in HIV primary care across the US is high. At the same time CNICS is distinct from many administrative databases in its inclusion of a range of validated self-reported behavioral assessments that have been integrated into routine clinical care, permitting investigations such as the current one that require measures of depressive severity linked in time to clinical actions. It is important to note that CNICS sites have established rapid clinical assessment and response protocols for patients indicating suicidality on the PROs, which may have led to better depression identification and treatment in the CNICS cohort than would have been present without the PROs. One interesting observation from the present analysis was the range in depression treatment and outcome indicators across sites. Although it was beyond the scope of this analysis to explore reasons for this heterogeneity, such differences could be due to differences in care practices or differences in patient populations .This study suggests substantial gaps across the steps of the depression treatment continuum among patients in HIV primary care settings across the United States and provides the first estimates of evidence-based antidepressant treatment delivery in these settings. Our findings show that successful pharmacologic treatment of depression is uncommon among patients with HIV infection, with small proportions of depressed patients receiving antidepressant dose adjustments when indicated or achieving remission. With increasing emphasis on decentralizing depression care from specialty mental health settings into primary care, including HIV primary care, HIV providers may need improved support in recognizing and treating depression, particularly in cases that require titrating or changing antidepressant treatment regimens. Collaborative care models that address these gaps in pharmacologic depression treatment could play an important role in addressing the burden of depression among patients with HIV infection.


The gastrointestinal tract (GIT) represents the largest interface between the human organism and the external environment. In the lumen and upper part of the mucus layer, this organ hosts an enormous number of microorganisms whose composition affects the functions of the epithelial barrier and the gut immune system. Consequentially, the microorganisms in the GIT influence the health status of the organism. Probiotics are living microorganisms which, in specific conditions, confer a health benefit to the host. Among others, probiotics have immunomodulatory properties that usually act directly by (a) increasing the activity of macrophages or natural killer cells, (b) modulating the secretion of immunoglobulins or cytokines, or indirectly by (c) enhancing the gut epithelial barrier, (d) altering the mucus secretion, and (e) competitive exclusion of other (pathogenic) bacteria. This review focuses on specific bacteria strains with indirect immunomodulatory properties. Particularly, we describe here the mechanisms through which specific probiotics enhance the gut epithelial barrier and modulate mucus production. Moreover, we describe the antimicrobial properties of specific bacteria strains. Recent data suggest that multiple pathologies are associated with an unbalanced gut microflora (dysbiosis). Although the cause-effect relationship between pathology and gut microflora is not yet well established, consumption of specific probiotics may represent a powerful tool to re-establish gut homeostasis and promote gut health. Rec“live micro-organisms which, when administered in adequate amounts, confer a health benefit on the host” [The importance of keeping a healthy microflora was already clear at the beginning of the twentieth century when consumption of yogurt and a specific mix of bacteria were associated to extended lifespan and prevention of disease , 51. Thehe host” , 64, 80.Currently, a large variety of probiotics are available on the market and generally they are consumed to target gastrointestinal discomfort and pain as well as to improve the properties of the immune system. Unfortunately, the health benefits attributed to consumption of specific probiotics are not always fully supported by scientific evidence. It is therefore necessary to invest further in research to describe the mechanisms through which probiotic consumption may influence human health. This concept becomes even more relevant considering the increasing body of evidence associating an altered GIT flora to pathological conditions not directly connected with the GIT like rheumatoid arthritis, ankylosing spondylitis, allergic disorders, autoimmune disease, multiple sclerosis and more recently, psychiatric disorders and memory , 66, 80.Being the largest interface between the body and the external environment , 28, it In this mini review, we will analyze the interaction between immune system and microbiota. Excellent recent reviews have already addressed the effect that specific bacteria have on the innate and adaptive immune response in human , 69, 70.lamina propria [The intestinal barrier is a heterogeneous entity composed of an extra-cellular component: the mucus layer and a cellular compartment including the intestinal epithelium and an underlying  propria   responsible for absorbing molecules from the intestinal lumen, Paneth cells specialized in synthetizing and secreting antimicrobial peptides (AMPs) upon contact with enteric bacteria, and mucus-secreting Goblet cells and entero-endocrine cells , 44, 76.lamina propria represents the interface between host and immune system in the gut and consists of dendritic cells, macrophages, and plasma cells, in addition to B and T lymphocytes.The Macrophages are strategically positioned in the sub-epithelial regions where antigens may cross the intestinal epithelium and are specialized in phagocytosing potentially harmful microbes as well as scavenging apoptotic cells and debris , 69. IntOne of the functions of the GIT epithelium is to establish a physical barrier between external environment and host immune system. Therefore, the functionality and integrity of this barrier are keys to support the permeability to nutrients and beneficial molecules albeit protecting the host from dangerous threats.The integrity of the GIT epithelium is partially guaranteed by multi-protein complexes defined as TJs Fig.  29, 43,,43, 75.FTJ’s function and biology have been extensively reviewed elsewhere  parameter, or in vivo by the intestinal permeability test (IPT) [The leaky gut is characterized by having an epithelium with increased permeability to molecules/compounds that diffuse from the lumen to the st (IPT) . Leaky gst (IPT) , all chaMultiple compounds in the diet have already been demonstrated to regulate the expression of the TJ proteins  and simiEscherichia coli Nissle 1917 (EcN) in vivo, healthy germ-free mice were colonized with this probiotic and the gene expression in their intestinal epithelial cells (IECs) was analyzed [To study the effect of analyzed . Colonizanalyzed . Similaranalyzed . To inveanalyzed . Althouganalyzed . Indeed,analyzed .Lactobacillus rhamnosus strain GG and its protective effects against the enterohemorragic Escherichia Coli O157:H7 infections were demonstrated in vitro in MDCK-I and T84 epithelial cell monolayers [E. coli infection maintained higher levels of ZO-1 expression than those infected with the pathogen alone [L. rhamnosus GG [The properties of the intestinal epithelium are also influenced by another probiotic. nolayers . Specifien alone . Similarnosus GG . In thisL. rhamnosus GG, another probiotic strain has a protective effect against pathogen infections via regulation of TJ proteins. In the T84 epithelial cells monolayer model, Lactobacillus casei DN-114001 stabilizes the ZO-1 distribution against the enteropatogenic Escherichia coli E2348/69 [Similarly to E2348/69 .Lactobacillus plantarum MB452 upregulates the gene and protein expression of ZO-1, ZO-2, occludin, and cingulin [Lactobacillus plantarum strain WCFS1 [Lactobacillus plantarum strain CGMCC No.1258 [Lactobacillus plantarum strain WCFS1 directly in the duodenum of healthy subjects by a feeding catheter. They measured the intestinal barrier parameters in the human tissue and suggested that the activation of the TLR2-dependent pathway is responsible for regulating the expression and distribution of the TJ proteins [in WCFS1  and Lactproteins .Streptococcus thermophilus ATCC19258 and Lactobacillus acidophilus ATCC4356 for example, maintained the phosphorylation levels of ZO-1, occludin, and actinin when the cellular models were exposed to infections by Escherichia coli 029:NM [Of note, there are probiotic strains modulating TJs functions by altering the phosphorylation status of the TJ proteins only, without altering their gene expression. Pretreatment of the human intestinal epithelial cell lines HT29 and Caco-2 with i 029:NM .lamina propria [The intestinal epithelium is covered by a viscoelastic mucus layer whose main functions are to (a) build a protective barrier against the harsh luminal environment (containing digestive enzymes), (b) facilitate food passage, and (c) avoid firm adhesion of bacteria to the epithelial cells thus preventing their entry into the  propria , 20, 21. propria , 18, 47. propria  and ulce propria  highlighSpecific probiotic bacterial strains have been demonstrated to regulate mucin expression therefore influencing the properties of the mucus layer and indirectly regulate the gut immune system. A list of these probiotics is reported in Table Lactobacillus plantarum strain 299v was demonstrated to inhibit the adherence of the enteropathogenic Escherichia coli to the intestinal epithelial HT-29 cell line [L. plantarum strain 299v with HT-29 increased the mRNA expression of the MUC2 and MUC3 genes, suggesting that this probiotic induces epithelial cells to secrete mucins that diminish enteric pathogens binding to mucosal epithelial cells [Lactobacillus acidophilus strain LA1 in Caco-2 cells [ell line . Incubatal cells . These r-2 cells , 47.Escherichia coli Nissle 1917 was also demonstrated to alter the expression of mucin genes [E. coli Nissle 1917 showed an increased expression of MUC2, MUC3, MUC5AC, and MUC5A genes. This effect was stronger when a basal stimulation model was used and, with exception to MUC3, it was not observed when the only bacteria medium was used [The probiotic in genes . Specifiwas used . A possiwas used , 35 and was used .Lactobacillus casei strain GG. In multiple in vitro models, it was shown that L. casei GG inhibited the translocation of specific pathogenic bacteria adhering to the receptors of cultured enterocytes [Another probiotic able to regulate mucin expression is the erocytes  and refeerocytes .Further evidences supporting the above-mentioned effects were also obtained by other in vitro and in vivo studies. Caballero-Franco and collaborators indeed administered a probiotic formula (VSL#3) to rat models and observed a 60% increase in the basal mucin luminal content . SubsequSpecific bacteria strains have been described to have antimicrobial properties usually associated with secretion of peptides or molecules which enables them to compete within the complex gut ecosystems. These molecules may protect the host against infectious bacteria and favor the survival of commensal bacteria . A list Lactobacillus brevis strain 925A influences the gut immune system via the production of a bacteriocin identified as brevicin 925A. Brevicin 925A was found to be effective against Listeria monocytogenes and Streptococcus mutans which cause food poisoning and dental caries [Lactobacillus plantarum strain TMW1.25 [l caries . Althoug TMW1.25 , 77.Lactobacillus fermentum strain CS57 was recently isolated from vaginal swabs and shown to produce a bacteriocin-like substance (BLS), with a wide spectrum of antimicrobial activity [L. fermentum CS57 demonstrated a strong in vitro antimicrobial activity against Candida albicans and Streptococcus agalactiae responsible of serious infections when newborns pass through the cervical canal [Lactobacillus rhamnosus L60 and Lactobacillus fermentum L23 [activity . With a al canal . Similarntum L23 . Taken tntum L23 , 67.Lactobacillus johnsonii NCC 533 was daily administered to mice infected with Salmonella typhymurium. Fecal analysis showed that the S. typhymurium was reduced even after the administration was stopped suggesting that LA1 is able to survive in the intestines [testines . Althougtestines .Lactobacillus salivarius UCC118 is a well-characterized bacterial strain secreting a potent broad spectrum of small heat-stable proteins belonging to class II bacteriocins [L. salivarius UCC118 was sufficient to reduce the infection of Listeria monocytogenes particularly in the liver and spleen [L. salivarius DPC6488 with analogies to the Abp118: salivaricin L and T [eriocins . Using id spleen . This efd spleen . Of note L and T .Lactobacillus plantarum G1 and G3 were identified by Zavisic and collaborators [Staphylococcus aureus, Escherichia coli, and Salmonella abony [Lactobacillus plantarum G1 (and G3) as potential novel probiotics [borators . The antla abony . In addiobiotics .The physiological equilibrium established between microorganisms colonizing human intestinal tract and host is key to the health status of each individual. This equilibrium relies on complex and dynamic relationships within bacterial ecosystems and host immune system. In this manuscript, three mechanisms through which specific bacteria strains indirectly communicate with the immune system have been described. The first explores the effect that probiotic have on the gut epithelial barrier and in particular the tight junctions, the second focuses on bacteria that by communicating with the intestinal epithelium may alter the properties of the mucus layer, while the third describes the antimicrobial molecules that specific bacteria strains use to compete within the gut ecosystems.The GIT epithelium represents a physical barrier between external environment and host immune system. The integrity of such barrier is regulated by multi-protein structures called tight junctions that are key to regulate the gut permeability to nutrients and beneficial molecules while protecting the host from threats originating within the GIT.Escherichia coli Nissle 1917, Lactobacillus rhamnosus strain GG, and Lactobacillus casei strain DN-114001 [Multiple probiotics modulate the expression of proteins constituting the TJs and therefore were reported in this work. The studies indicated here highlighted the importance of proteins like ZO-1 and occludin whose expression is regulated by specific bacteria such as N-114001 , 74, 83.N-114001 , 62. BotLactobacillus plantarum 299v resulted in a reduced adherence of the enteropathogenic bacteria Escherichia coli to the intestinal cells [The mucus is a complex viscous proteinaceous continuous layer in the gut lumen representing the first line of defense of the host against environmental threats . It is aal cells . In thisLactobacillus acidophilus LA1 [Escherichia coli Nissle 1917 where, in addition to MUC2 and MUC3, other genes were also affected such as MUC5AC and MUC5A [Moreover, Dykstra and collaborators showed that the interaction of this probiotic strain with the cells was responsible to enhance the cellular-driven protection via reduction of the caspase pathway activation . The impilus LA1 , 47. Thend MUC5A . In thisThese data not only highlight the importance of in vitro models to determine the mechanisms through which common and potential probiotics may confer health benefit to the host, but also argue that different bacterial strains have strain-specific effects that cannot be extended to other bacterial species.Bactaeriodes thetaiotaomicron was demonstrated to increase Goblet cell differentiation and expression of mucus-related gene favoring mucus production. This effect was diminished when B. thetaiotaomicron was associated with Faecalibacterium prausnitzii. This study reveals the importance of the balance between metabolically complementary commensal bacteria in maintaining colonic epithelial homeostasis [The limitations associated with using in vitro studies are represented by absence of complementary effect that two or more species have influencing each other as well as absence of the bacteria-host interaction effects. In this case, a more powerful source of information can derive by in vivo models. In a recent animal study for example, eostasis .Lactobacillus acidophilus LA1 was shown to be protective against Salmonella typhimurium infections [S. typhimurium and associated with secreted molecules released by this bacterial strain [Bacillus thuringiensis, a bacterium isolated from human feces, produces the bacteriocin thuricin CD [C. difficile as well as Listeria monocytogenes without affecting other constituents of the GIT microbiota [Salmonella typhimurium, Escherichia coli and Staphylococcus aureus) and not by other microorganisms such as fungi or protozoa [Oral administration of fections . Of intel strain . Bacteriricin CD . This bacrobiota . Of notecrobiota . Interesprotozoa .Lactobacillus plantarum G1 and G3 whose beneficial effects were measured by improved lipid metabolism and improved hepatic function in Wistar rats [Other examples include the tar rats .Of note, although animal models represent excellent tools to investigate the microbiome-host interactions and possible beneficial effects in vivo, they lack of the “simplicity” to establish the mechanisms of actions driving such benefits. Therefore, a combination of in vitro and in vivo models is essential to investigate the mechanisms of action of known probiotics as well as of novel potential probiotics.F. prausnitzii, an abundant anaerobic bacterium present in the human gut and belonging to the Clostridium leptum phylogenic group (Firmicutes) [F. prausnitzii is depleted in the mucosa of patients with inflammatory bowel disease (IBD) [F. prausnitzii was demonstrated to have anti-inflammatory properties both in vitro  and Caco-2 cells) as well as in trinitrobenzenesulfonic acid-induced colitis animal models in vivo [This is the case for micutes) , 71. Mulse (IBD) , 48, 71  in vivo , 71.Akkermansia muciniphyla, a mucin-degrading bacterium that resides in the mucus layer and whose presence is inversely correlated with body weight in rodents and humans [A. muciniphyla including control of inflammation, gut barrier, and gut peptide secretion mediated by endocannabinoids regulation [Another bacterium with potential probiotic properties is d humans , 26. AlsIn conclusion, modulation of the immune response associated with consumption of specific probiotics may occur not only via the innate and adaptive immune system, but also via (a) regulation of the intestinal epithelium permeability, (b) mucus secretion, and (c) competition within bacterial ecosystem via secretion of antimicrobial compounds. These mechanisms can be easily assessed in in vitro setting and therefore represent valid tools to study the properties of newly discovered bacteria strains.