Late diagnosis and treatment may increase morbidity and mortality among persons with hepatitis C virus (HCV) infection. We included all participants of the Swiss Hepatitis C Cohort Study (SCCS). We used unadjusted and adjusted logistic and Cox regressions to determine the association between the geographic origin of the participants and the following outcomes: antiviral treatment status; sustained virologic response; cirrhosis at enrolment; incident cirrhosis; loss to follow-up (LTFU); and mortality. The analyses were adjusted for sex, baseline age, education, source of income, alcohol consumption, injection drug use (IDU), HCV genotype, HIV or HBV coinfection, duration of HCV infection, time since enrolment, cirrhosis, (type of) HCV treatment, and centre at enrolment. Among 5,356 persons, 1,752 (32.7%) were foreign-born. IDU was more common among Swiss- (64.1%) than foreign-born (36.6%) persons. Cirrhosis at enrolment was more frequent among foreign- than Swiss-born persons, reflecting the high frequency of cirrhosis among Italian-born persons who acquired HCV between 1950 and 1970 in Italian healthcare settings. Although antiviral treatment coverage was similar, the sustained viral response rate was increased and the mortality was lower among foreign-vs. Swiss-born persons, with the lowest mortality in persons from Asia/Oceania. LTFU was more frequent in persons from Germany, Eastern and Southern Europe, and the Americas. In conclusion, in Switzerland, a country with universal healthcare, geographic origin had no influence on hepatitis C treatment access, and the better treatment outcomes among foreign-born persons were likely explained by their lower prevalence of IDU and alcohol consumption than among Swiss-born persons. Hepatitis C virus (HCV) treatment can induce sustained virologic response that reverses progression of liver disease and increases life expectancy –3. AccesHCV infections may have affected Swiss- and foreign-born persons by different transmission pathways and at different times in the person’s life. In Switzerland, the main source of HCV infection is injection drug use (IDU), which peaked in the 1980s and early 1990s ,11. The We wanted to determine whether foreign-born and Swiss-born HCV-infected persons had different levels of access to treatment and treatment outcomes, based on data from the large Swiss Hepatitis C Cohort Study (SCCS).The SCCS, established in 2000, includes persons residing in Switzerland and ≥18 years old who have anti-HCV antibodies . Study sWe included everyone registered in the SCCS (up to 10 September 2017) and considered the following outcomes: a) antiviral treatment status (ATS), indicating if a person was ever treated for HCV; b) sustained virologic response (SVR); c) cirrhosis at enrolment ; d) incident cirrhosis during follow-up (IC); e) loss to follow-up (LTFU); and f) mortality. Since some persons LTFU may have died, we also analysed attrition by combining mortality and LTFU into a single outcome. We defined SVR as an undetectable HCV RNA in serum ≥12 weeks after treatment end, excluding persons where the viral RNA was only tested less than 12 weeks after treatment end. When we analysed incident cirrhosis, we included only those without cirrhosis at enrolment. People were LTFU if there was no contact for two or more years, so we only included persons who were enrolled for at least two years before the study closed. LTFU contained the following dropout categories: moving to a foreign country; wanting to discontinue; care continued in a non-SCCS centre; address change without notice; no reply to written invitations; other. Information on mortality was based on information from the centres who systematically traced all patients LTFU. No linkage with a death registry was done. If we did not know the date of death, we used the last date we knew the person was alive.We used unadjusted and adjusted logistic regression for outcomes a-c, and Cox proportional hazard regression models for time-to-event outcomes d-f. Persons were followed from enrolment into the cohort until the endpoint occurred or until the most recent follow-up visit. We censored the study ten years after enrolment into the SCCS because most people had been followed for less time.The main exposure of interest was geographic origin of the persons; we used country of birth as a proxy of origin and divided the participants into Swiss-born and foreign-born. In a second analysis, we divided participants into groups of at least 100 members, based on the UN classification for geographic regions : SwitzerFor the adjusted models, we included explanatory variables documented at enrolment into the SCCS: sex; age ; centre ; highest level of education ; sourceOur first analysis combined all foreign-born persons into one group. Our second analysis grouped people into the geographic units described above. We performed a main analysis, and two sensitivity analyses where we either imputed or excluded missing values. In the main analysis, we assumed HBV and HIV status were negative if missing. We used multiple imputation by chained equations (MICE package for R ) to impuWe included 5,356 persons enrolled into the SCCS. The majority were male (62.3%), and the median age at enrolment was 45.3 years .Most were current (39.5%) or former (15.6%) IDU; 29.5% reported past or current excessive alcohol use. At enrolment, few had HIV or HBV coinfections  and most had no cirrhosis (82.7%). Participants were followed for up to 16.6 years (median follow-up duration 7.2 years). Swiss-born participants made up 67.3% of the population, 32.7% were foreign-born . Fig 1 sResults of the unadjusted and adjusted analyses are shown in The adjusted analyses confirmed the higher rates of SVR , of cirrhosis at enrollment , and of LTFU  among foreign-born persons .Foreign-born persons LTFU had less often (a history of) IDU than persons not LTFU, while the opposite applied to Swiss-born persons LTFU. Foreign- and Swiss-born persons LTFU were younger and less frequently cirrhotic than persons not LTFU .When we compared persons from each region or country to Swiss-born persons, some notable differences emerged. Italian-born participants were more likely to have cirrhosis at enrolment in both the unadjusted and adjusted analyses  and in the SCCS . Many ItThe fact that there are no differences in antiviral treatment status and incident cirrhosis between Swiss- born and foreign-born persons is reassuring. Even though foreign-born persons may have faced obstacles including language problems or cultural differences, these did not negatively affect the uptake of HCV therapy. In the comparison of different countries and regions, antiviral treatment coverage was higher in Italian-born persons than in all other groups including Swiss-born persons, but that difference disappeared in the adjusted analysis. Many Italian-born residents of Switzerland live in the Italian-speaking canton of Ticino close to the Italian border. In Ticino, efforts to find HCV-infected Italian-born persons have been ongoing for many years, with the involvement of general practitioners. HCV treatment in Ticino is decentralized into smaller facilities, which may have facilitated earlier access to therapy and improved therapy coverage. The high prevalence of cirrhosis at enrolment in this population further encouraged early treatment, including treatment with DAA.The higher SVR among foreign-born persons in the adjusted analysis, despite their higher proportion of cirrhosis, may be a consequence of the lower prevalence of IDU and of less alcohol consumption among foreign-born persons . AlthougTo our knowledge, there are no previous publications from Europe on the impact of geographic origin on access to treatment and treatment outcomes in persons born in the respective country compared to foreign-born persons. In the US, having a health insurance was associated with improved access to HCV care ,26, and Higher LTFU among foreign-born persons was driven by immigrants from Eastern and Southern Europe, the Americas, and Germany and probably reflects migration patterns in Switzerland, since many persons from these countries come to Switzerland for employment and then return to their home countries . These pAmong foreign-born persons LTFU, there may be persons who emigrated and died in their country of destination. However, numbers are likely low, as only 39 foreign-born persons LTFU were older than 60 years, and foreign-born persons LTFU were less frequently cirrhotic at enrolment than persons not LTFU .The higher prevalence of cirrhosis at enrolment among many Italian-born residents did not increase mortality .This is It is difficult to estimate the contribution of the “healthy migrant effect”. This effect has been shown in the European Union (EU), where out of 10 anti-HCV prevalence estimates among the general migrant population, 70% were comparable to the in-country prevalence and 30% were lower . In SwitOur study was strengthened by the large number of persons enrolled into the cohort, and the long follow-up period. Agreement between complete case analysis results and imputed analysis results supports the validity of our findings. Most differences in outcomes were caused by variation in the size of the confidence intervals and reflect differences in sample size rather than effect size.Our study may be limited in its generalizability for three reasons. First, Switzerland has a predominantly European migration pattern . Second,We found that in Switzerland, a country with universal healthcare, geographic origin had no influence on access to hepatitis C treatment. Foreign-born persons had more frequent SVR and lower mortality despite more frequent cirrhosis at enrolment, and they were more often LTFU. The high frequency of cirrhosis at enrolment among foreign-borns mirrors the high rate of cirrhosis at enrolment among Italian-born persons who have acquired HCV between 1950 and 1970 in Italian healthcare settings. However since most of the Italian-born persons were treated, mortality was not increased. The better treatment outcomes among foreign-born persons were likely explained by their lower prevalence of IDU and of alcohol consumption than among Swiss-born persons.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


Different neoadjuvant chemotherapies are available for triple-negative breast cancer (TNBC). Here, we performed a network meta-analysis to evaluate the pathological complete response (pCR) benefit and safety of treatment regimens. Pairwise and Bayesian network meta-analyses were performed to compare direct and indirect evidence, respectively. Twenty-three studies involving 12 regimens namely standard chemotherapeutic agents, bevacizumab (B)-, platinum salts (P)-, B plus P (BP)-, poly(ADP-ribose) polymerase inhibitors (Pi)-, P plus Pi (PPi)-, capecitabine (Ca)-, gemcitabine (Ge)-, zoledronic acid (Za)-, everolimus (E)-, P plus E (PE)-, and gefitinib (G)-containing regimens. The results showed that P-, B-, PPi-, and Za-containing regimens achieved higher pCR than standard chemotherapeutic agents. BP-containing regimens had a better pCR than B-containing regimens. In indirect comparisons, Za-, BP-, P-, and B-containing regimens were the top four strategies with the highest probability for pCR. Benefit-risk analysis showed that B-containing regimens had the highest acceptability of being the best treatment for better pCR achievement with fewer SAEs. The addition of P, B, BP, PPi, and Za to standard chemotherapeutic agents enhanced the pCR, but a balance between efficacy and safety should be carefully considered. B-containing regimens might be the best choice for neoadjuvant chemotherapy due to its better efficacy and tolerability. Triple-negative breast cancer (TNBC) accounts for ~12%–17% of all breast cancers . In patiAlthough there is a significant association between pCR and survival outcomes, the overall prognosis of TNBC remains unsatisfactory, and only ~30% of TNBC patients achieve pCR following treatment with standard anthracycline-, cyclophosphamide-, taxane-, and/or fluorouracil-based neoadjuvant chemotherapy . CurrentDespite these results, the optimal regimen for TNBC treatment remains controversial due to the limitations of RCTs and conventional meta-analyses in comparing and integrating the efficacy of all available regimens. Network meta-analysis, also known as multiple-treatments or mixed-treatment analysis, not only allows the integration of the evidence without head-to-head comparison, but also can determine the superiority of different interventions by ranking probability and acceptability . TherefoA total of 2,138 relevant records were identified in the electronic databases. Of these, 82 potentially eligible studies were reviewed with full text. Fifty-nine studies were excluded because 29 were out of scope, eight were duplicated data, 16 were not suitable for analysis, four were retrospective studies, and two were single-armed studies. However, although the study by Enriquez et al.  is a sinAll eligible studies were published between 2010 and 2018, and patients were enrolled between 2003 and 2017. Except for the historical controlled trial (HCT) , all stuThe quality of evidence was evaluated using the Cochrane risk of bias tool . There wp=0.09, 2I=43%) was detected, a pairwise comparison was performed with a random-effects model. We found that P-containing regimens were associated with a significant pCR benefit compared to standard chemotherapeutic agents  as analyzed by fixed-effect model , which indicated that PPi-containing regimens were superior in pCR achieving  compared to standard chemotherapeutic agents . The shapes of funnel plots of the two comparisons were close to symmetric, and no significant publication bias was identified by Begg's and Egger's tests .All of the 23 trials were included in the network meta-analysis for pCR. Node-splitting analysis was performed in order to evaluate the inconsistency, and no statistical difference was identified between direct and indirect evidence (data not shown). Therefore, the Bayesian network meta-analysis was conducted with a consistency model . The resThe rank probability of regimens was also analyzed. As shown in p<0.00001), neutropenia , and thrombocytopenia  compared with standard chemotherapeutic agents (p<0.00001), neutropenia  and thrombocytopenia , compared to standard chemotherapeutic agents (p=0.04) than standard chemotherapeutic agents  were the best treatment, resulting in the lowest incidence of anemia adverse events, whereas PE-containing regimens (45%) were found to be the worst treatment . PPi-conBenefit-risk analyses were performed by SMAA with missing preferences. Standard chemotherapeutic agents were set as the baseline. The analyses between pCR and anemia showed that BP-containing regimens had the highest acceptability (26.46%), ranking first  with a CA growing number of clinical trials are being performed in order to improve the effectiveness of neoadjuvant chemotherapies in TNBC by adding different drugs to the standard chemotherapeutic agents. However, results are controversial and remain isolated in the absence of systematic integration. Therefore, a comprehensive study was warranted to give a summary of the results from these publications. To the best of our knowledge, this is the first network meta-analysis to investigate the pCR efficacy and safety of neoadjuvant chemotherapy regimens in TNBC.In the present study, we enrolled 23 clinical trials with 4,099 TNBC individuals assigned to 12 neoadjuvant chemotherapy regimens, aiming to identify which treatment was optimal in achieving higher pCR rates and resulting in fewer SAEs. The results of pairwise meta-analyses showed that the most highly studied P-containing regimens were significantly associated with better pCR rates, but worse SAEs, compared with standard chemotherapeutic agents. Consistently, two previous meta-analyses also revealed that platinum-based neoadjuvant chemotherapies clearly increased pCR rates compared with platinum-free neoadjuvant chemotherapies , 32. AltBevacizumab is another frequently studied agent in neoadjuvant chemotherapy for TNBC. It has shown clinical efficacy in prolonging progression-free-survival, but not overall survival, in metastatic TNBC . In our To our surprise, zoledronic acid, a nitrogen-containing bisphosphonate that induces osteoclast apoptosis and inhibits bone resorption , showed There are several limitations of the present study. First, the sample sizes of several trials were relatively small. Specifically, the three RCTs involving Za-containing regimens and the study of E-containing regimens only included approximately 50 TNBC patients, which might weaken the effects or lead to false positive results. In addition, there were only one study for E-, PE, and Ge-containing regimens and the number of SAEs very relatively small in these studies, which might result in the wide 95% CIs; therefore, the results of SEAs regarding these regimens should be interpreted with caution. Second, there are several potential heterogeneities in the included studies, such as pCR definition, study design, and the specific components of standard chemotherapeutic agents. However, subgroup and sensitivity analyses based on pCR definition and study design demonstrated that the heterogeneities were unlikely to refute the overall results. Moreover, we integrated taxanes, anthracyclines, cyclophosphamide, and fluorouracil as comparative standard chemotherapeutic agents, as most control arms of the included studies were based on these four kinds of drugs, but differences still existed between studies. Therefore, consideration of these heterogeneities should be taken into account in the interpretation of our findings. Third, the kinds of SAEs reported in each study were different, and SAE data were not available for several trials. Thus, it was difficult to conduct a comprehensive analysis for all SAEs and regimens. Although there was no evidence showing a significant correlation between SAEs and the molecular subtype of breast cancer, there may be bias in the pooled effects of SAEs compared to the real results. Fourth, the correlation between neoadjuvant chemotherapy and survival outcomes was not evaluated due to the lack of survival data in most studies. However, we noticed that some RCTs are still ongoing and a secondary analysis focusing on long-term survival benefits may be reported in the near future. Therefore, a subsequent updated network meta-analysis will help to integrate the effects of these regimens on clinical outcomes.Despite the aforementioned limitations, our study has for the first time systematically compared the pCR efficacy and hematological SAEs of the currently available neoadjuvant chemotherapy regimens in TNBC. In conclusion, this study demonstrated that Za-, BP-, P-, and B-containing regimens are the top four treatment strategies, showing a high efficacy in achieving pCR. When considering both efficacy and SAEs, B-containing regimens had the highest acceptability to be the best treatment for achieving a relatively high pCR with fewer SAEs. However, additional well-designed RCTs with larger sample sizes are required to strengthen the findings of this meta-analysis and further determine the survival benefits of these neoadjuvant therapies in TNBC.This meta-analysis was performed in accordance with the guideline of Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) . LiteratThe inclusion and exclusion criteria were prespecified. Eligible trials were studies prospectively comparing at least two arms of different neoadjuvant chemotherapy regimens in TNBC patients. Studies were excluded if they were: (1) non-human studies, letters, reviews, editorial comments or case reports; (2) single-arm or dosage-finding studies; (3) articles without raw data or with a sample size of less than 30; and (4) ongoing trials without reported results. If several publications from the same trial were identified, only the latest or complete publication was included. Two reviewers independently evaluated the risk of bias for eligible studies using the Cochrane Collaboration risk of bias tool . Any disData were extracted by two independent authors. The following information was recorded for eligible studies: first author name, publication year, country, study design, trial phase, recruitment period, masking, number of centers, TNBC definition, clinical stage of patients, and trial name or registry number. Treatment regimens, sample size, primary outcome (pCR), and secondary outcome , if available, were also recorded.Due to the widespread use of combinations of taxanes, anthracyclines, cyclophosphamide, and/or fluorouracil, we defined the regimen as standard chemotherapeutic agents, which consisted of treatment with certain or all four drugs. The addition of any other drugs based on the standard chemotherapeutic agents was regarded as a new regimen. In the case of multi-arm studies comparing different dosages of one agent with another, we combined the results of the same agent into one arm. Administration sequence and frequency were not considered in the present study. The primary outcome was pCR, which was defined as the absence of invasive breast cancer in the breast and axillary lymph nodes (ypT0/is ypN0). If ypT0/is ypN0 was not reported, ypT0/is, defined as the absence of invasive breast cancer in the breast, was substituted. Secondary outcomes were treatment-related serious (grade 3–4) adverse events (SAEs) in hematology, including anemia, neutropenia, and thrombocytopenia. As for SAEs, we hypothesized that there was no significant correlation between molecular subtypes and hematological adverse events. Therefore, if the SAEs of TNBC were not available, we included the number of SAEs from all molecular subtypes of breast cancer patients. If only the percentages of primary and secondary outcomes were reported, we calculated the number of events by multiplying percentages by the number of patients.I2 test. Statistical heterogeneity was defined as p<0.1 and/or 2I>50%. A pairwise meta-analysis was conducted with a random-effects model or a fixed-effect model depending on the existence of statistical heterogeneity or not, respectively. All pairwise meta-analyses were performed by Review Manager software version 5.2. Results were reported as OR and 95% confidence intervals (CIs). All p-values were two-sided and a value less than 0.05 was considered statistically significant. Publication bias was detected by funnel plots, Begg’s and Egger’s tests using Stata software version 12.0.The odds ratio (OR) was utilized for estimating pooling effect sizes. For the pairwise meta-analysis, heterogeneity was calculated using Cochrane Q statistics and an http://www.drugis.org) [A Bayesian network-meta analysis was performed by Aggregate Data Drug Information System (ADDIS) software, version 1.16.8 (gis.org) . The conSupplementary FiguresSupplementary Table 1Supplementary Tables 2 and 3


Actinic cheilitis (AC) is a poten-tially malignant disorder of the lip, characterized by epithelial and connective tissue alterations caused by chronic exposure to ultraviolet radiation. In the past decades, diverse studies have been conducted in lip carcinogenesis and many biomarkers have been identified in lip lesions, yet there is no scientific evidence that determines its usefulness in the clinical setting or in histopatho-logical routine. Therefore, we conducted the first systematic review in this field to summarize the results of published studies on immunohistochemical bi-omarkers in lip carcinogenesis, to evaluate if there is a marker than can distin-guish the different histological grades of AC. Retrospective stud-ies that investigated immunohistochemical biomarkers in AC defined on standardised histological assessment were gathered from five databases and evaluated. Each study was qualitatively evaluated using the Critical Appraisal Tools from SUMARI. The proliferation marker Ki-67 was the most studied biomarker and we observed, through meta-analysis, that it was differently expressed between AC and lip cancer, but not in AC sub-groups. Most articles had a high risk of bias. In summary, the literature lacks quality follow up studies in actinic cheilitis. Multi-centre cohort studies, with patients stratified by treatment type and the use of image analysis soft-ware, could be the solution to further address the issues of investigating poten-tially malignant lesions and help change clinical practice, in terms of individu-alizing patients’ treatment and prognosis prediction. Key words:Lip carcinogenesis, actinic cheilitis, lip cancer, bi-omarkers. Lip squamous cell carcinoma (LSCC) represents 20-30% of all oral cavity tumors and it deserves a specific attention, especially in its pathogeny, that differs from oral squamous cell carcinoma (OSCC) . OSCC iset al. . The review protocol was registered at the International Prospective Register of Systematic Reviews (PROSPERO) under number CRD 42017055294.- Study designWe conducted a systematic review of human studies to summarize the results of pub-lished studies on immunohistochemical biomarkers in lip carcinogenesis, in order to eval-uate if there is a marker than can distinguish the different histological grades of actinic cheilitis (AC).- Search strategyWe searched and identified articles of the following bibliographic databases: Pub-Med, Scopus, Web of Science, ScienceDirect and Scielo. The search included all articles published up to April 25th, 2017, with no time restrictions. Duplicated references were excluded by a reference manager software (Mendeley Desktop version 1.17.9).The search strategy used for PubMed was: ((((((actinic cheilitis[Title/Abstract]) OR actinic cheilosis[Title/Abstract]) OR solar cheilosis[Title/Abstract]) OR solar cheilitis[Title/Abstract]) OR lip carcinogenesis[Title/Abstract]) OR lip photocarcinogene-sis[Title/Abstract]) AND immunohistochem*[Title/Abstract]. For the other databases we conducted independent searches using the first block: (actinic cheilitis OR actinic cheilosis OR solar cheilosis OR solar cheilitis OR lip carcinogenesis OR lip photocarcinogenesis), in order to maximize the inclusion of relevant studies.- Inclusion criteriaProspective and retrospective studies that investigated immunohistochemical bi-omarkers in AC defined on standardised histological assessment as outlined by the WHO  and/or K- Exclusion criteriaThe following exclusion criteria were applied: (a) Scientific papers that did not report AC histological grading; (b) Lack of comparison between biomarkers among AC groups or between AC and control ; (c) Stud-ies that investigated immunohistochemical biomarkers in samples other than paraffinized material; (d) Reviews, single case reports, clinical trials, letters, personal opinions, book chapters, and conference abstracts.- Study selectionThe study selection was conducted by two authors (BM and TS), who independently reviewed the titles and abstracts of all the papers, and selected the studies that met the in-clusion criteria. A kappa test was performed to verify agreement between authors and we obtained a reliable result of 0.87. Afterwards, both authors independently evaluated all full articles to determine if they reported the expression of immunohistochemical biomarkers in the subgroups of AC, based on histological grading (kappa score = 1). If there were any disagreements between the authors, they were resolved by mutual consensus. Final selection was always based on the full-text of the publication.- Data collectionTwo authors (BM and TS) collected the information from the included papers. The following information was gathered and presented in Tables: study characteristics ; population ; type of histological grading performed; immunohistochemical biomarkers that were analysed; expression of biomarkers in each subgroup ; sta-tistical tests performed; main conclusions. A partial grey literature search was performed using Google Scholar in order to investigate detailed results from PhD and Master’s de-gree thesis and dissertations, and perform the meta-analysis.- Meta-analysis Due to high heterogeneity in immunopositive cells counting/scoring for the studied biomarkers and contrasting results presentation in the articles, we included only the pro-tein Ki-67 for the meta-analysis. This was one of the most studied proteins and the only one that had a standardized analysis among the studies. To be included for meta-analysis, the articles (or their respective thesis/dissertations) had to report the mean and standard deviation of Ki-67 immunopositive cells in each of the following groups: control, mild dysplasia AC, moderate dysplasia AC, severe dysplasia AC, low grade LSCC, moder-ate/high grade LSCC. We used a Mixed-effects Model to estimate the amount of residual heterogeneity (tau2) and unaccounted variability (I2) among groups. The analysis was per-formed with the R software, package metafor 1.9-8.- Risk of bias in individual studiesEach selected study was qualitatively evaluated using the Critical Appraisal Tools from SUMARI , proposed by the Joana Briggs Institute . Since almost all cases com-prised retrospective studies, with samples chosen by convenience and lack of follow-up, we used the Critical Appraisal Tool for Case Series. This type of study is described as the kind in which “only patients with the outcome are sampled (either those who have an ex-posure or those who are selected without regard to exposure), which does not permit cal-culation of an absolute risk” . In our - Study selection1088 articles were identified across the five electronic databases. After removing the duplicates, 822 articles remained. A comprehensive evaluation of the titles and abstracts resulted in the exclusion of 746 articles, with the remaining 76 articles being allocated to full text in-depth review. This process led to the exclusion of 49 studies. Finally, 27 arti-cles were retained for qualitative analysis and three articles were selected for meta-analysis. A flow chart detailing the process of identification, inclusion, and exclusion of the studies is shown in Fig. - Study characteristics et al.  had unclear criteria for inclusion of the cases. We consid-ered a clear inclusion criterion when the study reported when the cases were diagnosed, where they were collected and what were the sample inclusion/exclusion criteria (even if it was a convenience sample). Also, only three studies reported to have consecutive inclu-sion of cases. However, all studies reported a reliable measurement of AC degrees of dysplasia according to the grading systems proposed by WHO  and Kujaet al .Thirteen articles reported the patients’ demographics, while clinical information was available in only four of the reviewed studies. Information regarding the patient’s out-come/follow-up was available in only one study  and partAlmost all studies reported to use clear statistical methods to compare the variables, however they were very diverse, since the researchers applied different methodologies for cell counting, with no clear cutoff of what was positive/negative or low/high. Also, maybe due to limited number of cases, authors tended to group AC cases for statistical analysis, and this grouping was very heterogeneous among studies.- Meta-analysisThree studies were selected for meta-analysis ,12,23. WOne of the main purposes of investigating immunohistochemical biomarkers in lip lesions is observing if there are differences between protein expressions in different grades of AC or between AC and LSCC or control/normal lip. Usually, this is done not for diagnostic purposes, but with the expectation to develop future prognostic markers, which could possibly set apart cases that will undergo malignant transformation.et al.  and comparing it only to LSCC or control, to achieve statistically significant re-sults. This could be due to small sample sizes, as it could possibly be related to selective reporting bias, which may happen as a result of the belief that scientific journals will not to accept papers reporting only “negative” results . Additional-ly, nearly all evaluated studies are not replicable or reproducible, since important data are often not reported. According to Peng [2015] , there aAt last, we acknowledge another risk of bias within this review, since almost all in-cluded studies are from Brazilian research groups. This can be explained by the fact that in tropical countries, rural workers are chronically exposed to high levels of solar radiation throughout the year, which explains an AC prevalence of up to 28.4% in Brazilian popu-lations . ConsideAs regards the meta-analysis, we also acknowledge its limitation. Since only three articles could be included for statistical analysis, we observed high heterogeneity among results, specially between AC groups, and this may not represent the reality of Ki-67 staining in AC or LSCC. One of the studies that investigated this protein  had to b- Implications for research and practiceet al. (In this review, we have identified 76 studies that investigated biomarkers in the field of lip carcinogenesis. Despite this significant number, well documented cohort studies are still limited. We still ought to understand the behaviour of AC and its progression to can-cer, in order to apply it clinically. We emphasize the difficulty in accessing complete fol-low-up data and highlight the need for further clinical research in potentially malignant disorders. As suggested in a systematic review by Smith We recommend that in studies of biomarkers of lip carcinogenesis histological grad-ing is performed for AC and LSCC, preferentially using more than one grading system. Also, comparisons with normal epithelium are indispensable. Thoroughly describing the methodology used for quantifying the antibodies is crucial for reproducibility of the study and, ideally, a unified methodology should be adopted. Maybe with the aid of an image software, to reduce examiners’ observation variability, this could be achieved. Likewise, results should be described more carefully, with Tables showing the results for each AC and LSCC subgroup, as well as control groups. Clinic and demographic information are also important to be described.We observed that the different studied proteins are similarly expressed in AC epithe-lial dysplasia grades, therefore are not useful in differentiating them. However, the poten-tial use of some biomarkers to differentiate AC and LSCC has been demonstrated. We believe that soon some of them could become useful in identifying cancer risk in patients with actinic cheilitis. If we can develop reliable and reproducible follow-up studies, we will be able to change clinical practice in terms of individualizing patients’ treatment and prognosis prediction. Clearly, further research is needed to exploit the many possibilities in lip carcinogenesis.


Embedded in the Statistical Parametric Mapping (SPM) framework, it benefits from the extensive range of established SPM tools for high-accuracy spatial registration and statistical inferences and can be readily combined with existing SPM toolboxes for estimating diffusion MRI parameter maps. From a user's perspective, the hMRI-toolbox is an efficient, robust and simple framework for investigating qMRI data in neuroscience and clinical research.Neuroscience and clinical researchers are increasingly interested in quantitative magnetic resonance imaging (qMRI) due to its sensitivity to micro-structural properties of brain tissue such as axon, myelin, iron and water concentration. We introduce the hMRI-toolbox, an open-source, easy-to-use tool available on GitHub, for qMRI data handling and processing, presented together with a tutorial and example dataset. This toolbox allows the estimation of high-quality multi-parameter qMRI maps (longitudinal and effective transverse relaxation rates   In convm, MDEFT  for T1-wsity, PD , 2010. qsity, PD . In qMRIseconds) . This stseconds) , which mseconds)  or a comseconds)  enables RI hMRI, .The estimation of quantitative and semi-quantitative metrics commonly includes one or more of the effective transverse relaxation rate ( metrics . However metrics ) and pro metrics  are readConsequently, the neuroscience and clinical research community lacks a standardised qMRI implementation to handle the wide diversity of data acquisition types and estimate parameters such as n tissue . Similarip angle . For accip angle  and receip angle . Such cohttps://github.com/qMRLab/qMRLab), QUIT (https://github.com/spinicist/QUIT), mrQ (https://github.com/mezera/mrQ) and QMAP (https://www.medphysics.wisc.edu/∼samsonov/qmap/). These tools include, to various extents, data acquisition guidelines, tools for protocol simulation and optimisation, a multitude of models and methods for data fitting and estimation of qMRI parameters, and visualisation tools.A number of open-source tools have been developed to support qMRI use and make qMRI more broadly accessible to neuroscience and clinical research , profiting from the highly accurate spatial registration into a common space and the variety of established statistical inference schemes. The spatial processing part of the toolbox can be applied to any set of rotationally-invariant qMRI maps, including a number of diffusion MRI parameters and all common qMRI metrics.In this paper, we present the hMRI-toolbox, a comprehensive open-source toolbox that streamlines all the processing steps required to generate 22.1http://hmri.info.The MPM multi-echo protocol was introduced in  DESPOT1 . Example2.2We give here, and in α, the longitudinal relaxation rate The signal from the multi-echo PDw, T1w and MTw acquisitions can be described by the Ernst equation , b. The ESTATICS , provideimations a. Using is point b, the gensmit fT c and rect fT  as a zip file  or by cloning the git repository (https://github.com/hMRI-group/hMRI-toolbox) to keep up-to-date with the latest incremental developments.The latest version of the toolbox can be downloaded from the hMRI-toolbox page (https://github.com/hMRI-group/hMRI-toolbox/wiki). It includes installation instructions, an example dataset, a tutorial and a detailed description of the implemented functionalities. Information on releases and versioning, development and contribution guidelines are also provided.Updated documentation is available as a Wiki (http://www.fil.ion.ucl.ac.uk/spm/software/spm12/) to benefit from the latest developments.The toolbox has been developed and tested with MATLAB versions 8.0 (R2012b) to 9.3 (R2017b) and SPM12 from version r6906 onwards. Since the hMRI developments will be synchronised with SPM developments, it is recommended to use the most recent SPM release . Further details on “copyleft” can be found at In particular, the hMRI-toolbox is supplied as is. No formal support or maintenance is provided or implied. Since the toolbox was developed for academic research, it comes with no warranty and is not intended for clinical use.3.2http://hmri.info and is described in detail in An MPM example dataset from a healthy subject for demonstrating the hMRI-toolbox features was acquired on a 3T Prisma system  at the Wellcome Centre for Human Neuroimaging, London, UK. This dataset can be downloaded from 3.3Configure toolbox, DICOM import, Auto-reorient, Create hMRI maps and Process hMRI maps. While the Configure Toolbox and Process hMRI maps modules can be run for a group of subjects, the DICOM Import, Auto-reorient and Create hMRI maps modules must be run for each subject and each session separately . A brief description of each module is provided below and details can be found in the Appendices.The hMRI-toolbox is organised into five main modules : Configu3.4Configure toolbox module allows the user to select either standard or customised default parameters, to match the user's own site- or protocol-specific setup and to be used in the subsequent modules , following the hMRI-toolbox implementation. Note that Philips-specific rescaling factor  in the same axial plane, as defined in MNI space , 2001a,b3.7Create hMRI maps module computes quantitative as well as semi-quantitative estimates of The  Section  and tran Section  field in3.7.1The module takes the (possibly reoriented) series of multi-echo spoiled gradient echo images as input for the creation of quantitative as well as semi-quantitative maps  of R2⋆, turation , 2014 as Section  and the  Section  as well  Section .Fig. 3Ma3.7.2The map creation module includes the determination of 3.7.3Create hMRI maps module. Two of them rely on measured RF sensitivity maps (Single or Per contrast options) while the third method is data driven (Unified Segmentation option: no input sensitivity map required). Although not recommended, it is possible to disable RF sensitivity correction altogether by selecting the None option. While options Single and Unified Segmentation assume that the sensitivity profile is consistent between contrasts , the Per contrast option accounts for inter-contrast variation in RF sensitivity profile due to larger subject motion  segal steps , (2) nonal steps  and (3) al steps , using t3.9The standard SPM statistical analysis and modelling approaches such as mass-univariate General Linear Modelling can be applied to the spatially processed maps, see, e.g., 4This paper introduced the hMRI-toolbox, which is embedded in the SPM framework and allows for the estimation and processing of four quantitative MRI parameter maps: the longitudinal and effective transverse relaxation rates in vivo histology of tissue microstructure using MRI (The name of the toolbox (h-MRI) originates from the concept of sing MRI . Hereby,Considerations and interpretation of the MPM approach. While the signal model  field and the spoiled gradient echo images can be one reason for such imperfections. In particular, when between-contrast  motion is large, discrepancies between head position for a single receive  field measurement and head position for all or some of the spoiled gradient echo images lead to additional motion-related bias in the quantitative maps . In suchWhen no transmit and/or receive field inhomogeneity maps have been measured, which often happens in clinical settings due to time constraints, the toolbox provides the option to use image processing methods based on the Unified Segmentation approach  for B1 tUNICORT, b or RF sUNICORT, e. The UnCORT, TR . This reCORT, TR  comparedead coil . The coread coil  and the Configure toolbox module  includMulti-scanner and multi-vendor data sets will require adjustments in terms of data handling and processing with the hMRI-toolbox. Most MPM studies up to now were carried out on Siemens MRI scanners using customised MPM sequences. Consequently, the toolbox is optimised for this scenario. New issues might arise when implementing the MPM protocol and using data from other vendors or conventional product sequences. For instance, different MT pre-pulse implementations will lead to changes of the ibration . Moreoveibration , leadingibration  by definμm new generation MPM, Applicability of the toolbox to different MRI platforms also involves ultra-high field MR systems. With the fast progress of ultra-high field scanners (7T and higher) on the MRI market, high-resolution data will become more routinely available and provide access to e.g. laminar-specific information .https://www.biorxiv.org/content/early/2018/07/26/328146) is another challenge for longitudinal qMRI. To that end, the bias of the qMRI estimates in the presence of motion has been investigated  and the variation of change across subjects  is provided in the hMRI-toolbox via a set of indicative parameters that should be used at the user's discretion . Future in vivo histology  is planned. Quantitative susceptibility mapping (QSM), taking advantage of the existing phase images acquired with the MPM protocol , is a seistology  is anothistology . The MR  toolbox  and the  toolbox ).5The hMRI-toolbox is a highly flexible software package that provides a computationally time-efficient, robust and simple framework to generate and process qMRI parameter maps sensitive to myelin, iron, and water content. It profits from the powerful and easy-to-use spatial and statistical analysis tools in SPM, and can be readily combined with existing SPM tools for quantitative estimation of parameter maps sensitive to complementary information such as axonal properties. The ongoing developments address the use of open-science data formats and extensions into biophysical models for direct microstructure mapping. As such, the hMRI-toolbox is a comprehensive and readily extendable tool for estimating and processing qMRI data for neuroscience and clinical research.


Background and objectives: The effectiveness of short-term focused educational programs to change health behaviors across large populations seems to be poorly described so far. The main aim of the present study was to evaluate an age-specific 45-min educational program, designed in accordance with the current U.S. Department of Health and Human Services and U.S. Department of Agriculture dietary guidelines and physical activity (PA) guidelines, among adolescents and adults. Materials and Methods: We evaluated the health-promoting lifestyle habits by the Health-Promoting Lifestyle Profile (HPLP-II) at baseline and following 6–8 weeks post-education in a nationally representative sample of Serbian adolescents and adults (n = 3822). Results: The percentage of adolescents eating 3–5 servings of vegetables per day increased at follow-up , with significantly more adolescents regularly reading food labels . Taken together, mean HPLP-II scores in adolescents significantly improved for both diet  and PA , and for PA in adults . Hierarchical multiple regression analysis revealed that our model as a whole  explained 3.0% of the variance in mean HPLP-II scores for diet (p = 0.942) and 3.0% for PA (p = 0.285) in adolescents, and 1.1% of the variance in HPLP-II scores for diet (p = 0.781) and 1.9% for PA (p = 0.075) in adults, respectively. Conclusions: It appears that a brief focused education can positively tackle unhealthy lifestyles in promoting good health in general population. Different modes of interactive communication used here appeared to strengthen participants’ capacities for lifestyle changes. Education about a proper diet and physical activity (PA) remains an important component of health promotion efforts , having n = 93) and their primary school-aged children (n = 132) demonstrated improved health outcomes and behaviors after a 7-week intervention [This particularly applies to short-term educational interventions that tackle the adult population, while most previous studies evaluated the impact of medium- or long-term family or community interventions on heath behaviors in normal-weight and overweight school children and adolescents. For instance, participation in a one-year community-based, family-centered behavioral intervention appeared to have a positive influence on the physical activity, sedentary, and healthy eating behaviors of overweight and obese adolescents in Australia and New Zealand . A multirvention , thus prwww.ncdrisc.org) The TEDI-HL was set up to follow up subsamples of adolescents and adults initially enrolled through DiPAH database. The Effectiveness of Targeted Educational Intervention on Healthy Lifestyle Behaviors in Serbian Population (TEDI-HL) (see below) is an extension sub-study of the Diet and Physical Activity for Health Initiative (DiPAH). The DiPAH was started in 2008 as a long-term, nationally-recognized health study that is focused on strategies for chronic disease prevention of children and adults in Serbian population. The DiPAH population comprises of Serbian children (aged 6.0 years and above) and adults from 24 official administrative divisions in Serbia (out of 29), who were invited by general media and specialized e-portals to take part in this initiative, agreed to voluntarily participate, and were evaluated (at least once) in the mobile examination unit for disease history, general health, dietary habits, physical activity, and/or health-related physical fitness. Prior to action, we undertook four rounds of focus workshops to balance recruitment for demographic, socioeconomic, age, and gender differences. Field examinations were organized each spring and fall, with six mobile groups testing ~100 participants per day. Population-based estimates of disease prevalence from DiPAH cohort were published through NCD-RisC collaboration network (n = 3822). The study was conducted according to the guidelines laid down in the Declaration of Helsinki, and all procedures were approved by the local IRB at the University of Novi Sad . All adult participants provided informed consent before participating in the study; parents or other legally authorized representatives dispensed permission for minors to be included in the study. Participants were selected to join the TEDI-HL on the basis of the following selection criteria: (a) previously participated in DiPAH trials; (b) not previously enrolled in a health education program, (c) had no medical conditions that would limit the successful completion of the protocol, and (d) had a current residence in Serbia. Age ranges for eligible participants were between 10.0 and 18.9 years for adolescents, and above 19.0 years of age for adults, as of October 2016. Of 11,069 DiPAH subjects initially considered, 3278 adolescents  and 544 adults  were eligible and consented to participate in the current study (n = 3822) were visited in their schools, workplaces, or retirement homes by trained research staff who administered a Health-Promoting Lifestyle Profile (HPLP-II) questionnaire [clinicaltrials.gov as NCT02999425.The TEDI-HL is a longitudinal study designed to identify the effectiveness of a single 45-min session of focused education, designed in accordance with the current USDHHS/USDA dietary and PA guidelines, on health-promoting lifestyle habits in a nationally-representative sample of Serbian adolescents and adults. From October 2016 to May 2017, all participants , followed by 30-min oral and visual presentation of diet and PA advice using PowerPoint slides , and finalized with a discussion/counseling forum (~10 min) aimed to answer any individual queries in each focus group. Group sizes varied from 15 to 30 participants, and age-specific materials (both printed and video) were developed through technical committee meetings. The applied educational intervention belongs to a knowledge, attitudes, practices model which strives to enhance positive health behavior choices and prevent negative ones ,15. Intet-test  and by chi-squared test (for proportions). In addition, a multiple linear regression model with a stepwise method (adjusted for age and gender) was employed to examine the association between an independent variable (time of testing) and a single continuous dependent variable (mean HPLP-II scores for diet and PA). A 2-tailed p-value < 0.05 was considered significant.All data were analyzed using IBM SPSS Statistics for Mac 21.0 software . Descriptive statistics was used for general characteristics. Comparisons were made between the baseline data and follow-up data to identify the effects of the educational intervention program by paired p < 0.05). The percentage of adolescents eating 3–5 servings of vegetables per day increased at follow-up , with significantly more adolescents regularly reading food labels . In addition, the percentage of adolescents that regularly eats breakfast dropped throughout the study from 78.2% at the baseline to 73.8% at the follow-up (p < 0.001). Other lifestyle habits related to diet were not affected by the intervention.Of 3822 participants initially evaluated, 94.7% completed post-intervention assessment at the follow-up. Changes in specific lifestyle habits related to diet in adolescents and adults are presented in p < 0.05). However, fewer adolescents were physically active during usual daily activities, and followed a planned exercise program at the follow-up (p < 0.05). Adults appeared to take part in leisure-time PAs more often after an educational intervention, routinely do stretching exercise at least 3 times per week, and more regularly check their pulse when exercising comparing to baseline (p < 0.05). Taken together, mean HPLP-II scores significantly improved for both diet  and PA  in the adolescent population, and for PA in adults  (p > 0.05).PA lifestyle profiles are depicted in  0.0001) . SF-12 pR = 0.17; standard error of estimate = 0.47; p = 0.942) and 3.0% for PA  in adolescents, and 1.1% of the variance in HPLP-II scores for diet  and 1.9% for PA  in adults, respectively. Age makes unique statistically significant contribution to our model of changes in health-promoting lifestyle habits for diet and for physical activity, while gender made no significant contribution to the model neither for diet or PA in adults, or for diet in adolescents  explained 3.0% of the variance in mean HPLP-II scores for diet (lescents . HoweverIn this nationally-representative intervention trial, we found that an age-specific single 45-min interactive educational intervention, designed in accordance with USDHHS/USDA dietary and PA guidelines, turned out to be effective in terms of improving health-promoting lifestyle habits in Serbian adolescents and adults. Significantly higher scores for mean HPLP-II scores at 8-week follow-up perhaps reflected a positive change induced by a brief intervention that can tackle unhealthy behaviors and promote good health in general population. We also demonstrated here that age had a significant association with changes in HPLP-II scores for both diet and PA in our cohort of adolescents and adults, while gender had rather irrelevant contribution in predicting health-profile changes. Age seemed to be negatively associated with alterations in mean HPLP-II scores after education in adolescents, likely suggesting younger age of this population as more susceptible to change. On the other hand, a positive correlation found among adults perhaps suggest that participants with advanced age in this subsample are prone to change to a greater extent. Although comparatively short, the intervention was delivered in schools, workplaces and retirement homes across Serbia, which are a promising setting for implementation of health education programs since they cater for large proportion of adolescent and adult population.Several recent studies evaluated the effects of short-term education on health-promoting lifestyle habits, using HPLP II scores as primary outcomes, in many different settings from workplace and hospital, to retirement homes and classroom environments. Eight sessions of 45–60 min face-to-face educational intervention, held weekly in two sessions over a period of 4 weeks, by PowerPoint lecture, discussion, and the provision of leaflets, improved HPLP II scores in 72 retired individuals . Two-to-For individual health-promoting lifestyle behaviors before the intervention, the most unfavorable dietary habit in adolescents referred to fat consumption, with less than 8% routinely choosing a diet low in fat. Around one in ten adolescents ate the recommended amount of bread, cereal, rice, and pasta on a regular basis, while no more than 12% regularly read food labels. Other health-promoting lifestyle habits were also underrepresented in adolescent population, including limited use of sugars and sweets, recommended vegetable and protein food consumption, as well as consumption of dairy products and fruit. At the same time, health-promoting lifestyle habits concerning fruit and vegetable consumption were present to a strikingly lesser extent in the adolescent population, reaching in some cases just half of the percentage of adults reporting the same behavior as a regular one. This is in line with previous studies describing poor dietary habits among young people, particularly for fruit and vegetables intake , a practFor the PA domain, adolescents and adults reported several unfavorable habits both before and after the educational intervention. For instance, 39.4% of adolescents and 55.9% of adults regularly took part in light-to-moderate PA, an activity that has many health benefits in any age group . In addiIt seems that adolescents responded better to the educational intervention than the adults, although the education was age-tailored. There might be a few possible explanations for this. The adult group consisted of a fairly large proportion of elderly people living in retirement-homes, which provide main meals for their residents, thus limiting room for a change in dietary behaviors. Besides, elderly people might be more reluctant to change in general , with inThe main focus of an effective health education is the empowerment of individuals to make healthy choices in everyday life. Gaining knowledge and motivation through education, alongside the environmental support, enables people to change unhealthy lifestyle habits. This might be of special importance in transition countries like Serbia, where modifiable behaviors related to unhealthy diet and physical inactivity might worsen with overall development. A recent analysis of the Serbian healthcare system recommends a shift from curative to preventive medicine, since promotion of a healthy lifestyle decreases healthcare costs in the mid- to long-term . At the Several limitations must be considered when study findings are interpreted. Although initial results of our intervention in changing diet and physical activity health profiles are encouraging and corroborate previous findings ,19,20, eThis study was the first to evaluate the effects of a brief educational intervention on dietary and physical activity behavior in a representative sample of Serbian adolescents and adults. The study provided promising results concerning positive impact of the intervention on health-enhancing lifestyle habits, particularly in a physical activity domain and in favor of the adolescent group. It is of a great importance to target young people by educational interventions, because health behaviors can be tracked down from adolescence into adulthood, thus affecting health throughout the life course . However


FST values and revealed a genetic structure mostly driven by male gene flow across villages. This genetic feature illustrates the uxori-matrilocality inherited from the Bantu expansion, though one exception exists in Bandamadji whose historically documented military status implied patrilocality in this locality.Post-marital residence of spouses is one of the architects of population genetic structure. In the present study, we tested how the place of residence of males and females in Ngazidja, Comoros Islands, has unequally channeled, by dispersal among villages, the male and female genetic diversity. Using sequences of the hypervariable segment I of the mitochondrial DNA (mtDNA HVS-I) and six Y-chromosome microsatellites (Y-STRs), we measured the genetic variation and male-to-female effective number of migrants ratios based on  The Comoros Islands are an archipelago of four islands, Ngazidja , Nzduani (Anjouan in Shindzuani), Mwali , and Mahore (Mayotte in Shimaore), located at the northern end of the Mozambique Channel in the Indian Ocean. Historical, social, linguistic, and genetic studies showed that the Comorian population arose from the encounter of East African Bantu metallurgists with Islamic Middle Eastern traders and, to a lesser degree, with Austronesian people –5.hinya. All these conditions create a real socio-economic pressure to stay in the birth village for both men and women [The social structure in Comoros Islands results from the interactions between Islam’s laws and ancestral rites from East Africa, with a mixed distribution of matrilineal and patrilineal traits in various domains of social organization: kinship, inheritance of property, succession to titles, mode of residence, and detention of authority , 6. In tnd women , 6.Previous surveys in matrilocal and patrilocal structured populations have shown a contrasted pattern of within and between-population genetic variation when examined for paternally and maternally inherited genetic markers , 8. HereIn accordance with French regulations of an ethical committee , we collected with consent approval 86 male samples from five villages of Ngazidja: Bandamadji, Hahaya, Iconi, Male, and Mitsoudje. We included only unrelated men for at least two generations back in time and ensured the name of their birth village.Blood collection and lab methods are detailed in ref. . DNAs weFST and RST genetic distances and distribution of the molecular variance (AMOVA) using the ARLEQUIN software [Nmalemmale/Nfemalemfemale from FST values, the ratio between effective numbers of male and female migrants per generation, as described in ref. [m) and/or effective population size (N). Hence, the expected value of genetic differences between populations, FST, could be written were FST = 1/(1 + Nm) for haploid systems [We first estimated the within-village genetic diversity and pairwise-difference (Pw), then tested the inter-village genetic structure with a measure of software . We alsosoftware  and weigsoftware  after ex in ref. . In this systems .We measured the female and male genetic diversity indices in the five Comoros villages understudy Table . The maiFST and RST genetic distances onto a Multi Dimensional Scaling . Finally, one can notice twice more village-specific haplotypes for HVS-I (n = 24) than for Y-STRs (n = 12).We then depicted the sis Fig. , networkp-value = 0.000), but less within-village variation . Due to the distinct genetic pattern of Bandamadji above-mentioned, we ran an additional AMOVA excluding this village. We noticed a strong increase of the contrasting patterns of within-population and between-population genetic variation as seen from HVS-I and Y-STRs, since the female genetic variation among populations reached almost 10-fold the male one .AMOVA points out that HVS-I presents 3.2-times more genetic variation amongst villages than Y-STRs .Lastly, from the es Table . The maiA great variety of human social organizations defined by rules of marriage, residence, descent and mode of subsistence has been described worldwide , which iUniparental genetic markers in Ngazidja indicate a male-oriented repartition of genetic diversity within and amongst villages, which is in accordance with described matrilocality. However, one exception is Bandamadji which behaves as a patrilocal population. Indeed, oral and historical knowledge evokes that Bandamadji was the military basis of one of the great Comorian clan M’Dombozi, where men have sworn fidelity to their sultan Hachim ben Ahmed (died 1889), and hence, had to stay to protect the city ( and KassFinally, the Comorian villages are organized according to the social space in which males and females participate distinctly , and theS1Table


Oreochromis aureus) to low temperature stress. Cold tolerance was characterized in 66 families of blue tilapia. Fish from cold-tolerant and cold-sensitive families were sampled at 24 and 12°C, and the transcriptional responses to low-temperature exposure were measured in the gills and liver by high-throughput mRNA sequencing. Four hundred and ninety four genes displayed a similar temperature-dependent expression in both tolerant and sensitive fish and in the two tissues, representing the core molecular response to low temperature exposure. KEGG pathway analysis of these genes revealed down-regulation of focal-adhesion and other cell-extracellular matrix (ECM) interactions, and up-regulation of proteasome and various intra-cellular proteolytic activities. Differential responses between cold-tolerant and cold-sensitive fish were found with genes and pathways that were up-regulated in one group and down-regulated in the other. This reverse response was characterized by genes involved in metabolic pathways such as glycolysis/gluconeogenesis in the gills and biosynthesis of amino-acids in the liver, with low temperature down-regulation in tolerant fish and up-regulation in sensitive fish.Tilapias are very important to the world's aquaculture. As befitting fish of their tropical origin, their distribution, and culture practices are highly affected by low temperatures. In this study, we used genetic and genomic methodologies to reveal pathways involved in the response and tolerance of blue tilapia ( Environmental stressors disrupt homeostasis and are harmful to the physiological function of organisms. Environmental temperature is one of the main factors that drove a wide array of evolutionary adaptations, including the division of animals into homeotherms (endotherms), like mammals and birds, and poikilotherms (ectotherms), like reptiles, amphibians, and fish. Fish species inhabit waters of a wide range of temperatures, from below 0°C in the Antarctic ocean to above 40°C in East African lakes . Reflecting their tropical origin, the optimal temperature for growth of most tilapiine species lies within the range of 20–30°C and reproduction and feeding are usually suppressed at temperatures below 20°C. Variation in the lower lethal temperature has been observed among different tilapiine species  being one of the most cold-tolerant species .TM kit . RNA concentration and quality were determined using an Epoch Microplate Spectrophotometer .Seven fish from each family were sampled at 24°C, and seven at 12°C. Gills and liver samples were taken and kept in RNAlater buffer  at −20°C until use. mRNA was extracted from the tissue samples using TRIzol® reagent , and purified to remove DNA contamination using the TURBO DNA-freeFor each temperature, 2 μg RNA from gills and liver samples from three cold tolerant (Family 480) and three cold sensitive (Family 740) fish were sent on dry-ice to the Technion Genome Center . Twenty-four libraries were prepared and sequenced on three lanes on an Illumina Hi-Seq 2500 device.aldoaa, gpib, pfkma, pgam2, gpia, ldha, tbiB, pgam1a  were designed for eight genes that were found to have temperature-dependent expression in the Next-Generation Sequencing (NGS) analysis, within a pathway that was differentially expressed between cold-tolerant and cold-sensitive fish: ftp://ftp.ncbi.nlm.nih.gov/genomes/Oreochromis_niloticus/), and the Cufflinks and Cuffdiff software tools  were regarded as significantly up- or down-regulated. Functional annotation of tilapia transcripts with significant temperature-dependent expression was extended using PANTHER (http://www.pantherdb.org/), based on gene ontology (GO) categories assigned to the human or zebrafish orthologs. The database for annotation, visualization and integrated discovery (DAVID) web software (http://david.abcc.ncifcrf.gov/home.jsp) was used for the functional analysis of KEGG biological pathway enrichment . Adonis implementation of Permanova . Overall, 496,843,855 reads obtained from 24 libraries. After quality control, 441,882,583 clean reads (89%) remained for further analysis. From these clean reads, 70.6% were mapped to the tilapia reference genome, similar to mapping rate in previous tilapia's transcriptome analyses  and the liver  (Concerning the number of genes expressed (FDR<0.05 and 2-fold expression difference), Permanova analysis  and clus= 0.005) .Exposure to cold temperature led to a total of 2,696 and 3,721 temperature-dependent differentially expressed genes (DEGs) in the gills of families 480 (cold-tolerant) and 740 (cold sensitive), respectively. Additionally, 3,714 and 4,114 temperature-dependent DEGs were found in the liver of families 480 and 740, respectively. In both organs, more genes were up- and down-regulated in the sensitive fish than in the tolerant, 38% more in the gills and 11% more in the liver .P < 0.05) down-regulation of focal-adhesion and other cell-ECM interactions, as well as up-regulation of proteasome, various intra-cellular protein processing activities, RNA transport and degradation  were common between the sensitive and tolerant families. In the liver, there were 2,186 temperature-dependent DEGs  that were common between the sensitive and tolerant families. Of these, 494 genes (314 up-regulated and 180 down-regulated) were shared between the two tissues . These gradation .There were more genes with temperature-dependent expression in the sensitive fish than in the tolerant . This efgpia, ldha, tbiB, and pgam1a (P < 0.001), for aldoaa and pgam2 (P = 0.02), but not for gpib, pfkma (P = 0.1).Target genes for qPCR analysis were selected from the genes related to the glycolysis/gluconeogenesis pathway, that had opposite responses between cold tolerant and sensitive fish. Gills samples of nine fish from three tolerant families and nine fish from three sensitive families were analyzed. For all tested genes, the direction of change in expression was concordant between the transcriptome sequencing data and the qPCR analysis . TranscrEctotherm animals, like fish, go through major physiological responses when they acclimate to different temperatures. While the emergence of genomic technologies led to several studies that characterized genes and pathways involved in fish thermal acclimation  tissues demonstrated common activation of genes involved in ubiquitin-dependent protein catabolism and proteasome function, a response that was not expected  liver transcriptome also revealed broad activation of genes involved in RNA processing, protein catabolism and folding  in response to temperature reduction was inversely correlated to the level of cold tolerance (Nitzan et al., In this study, we focused on the gene expression level and identified divergence between the cold-sensitive and cold-tolerant blue tilapia in specific pathways. Two carbon metabolism pathways, glycolysis/gluconeogenesis in the gills and biosynthesis of amino-acids in the liver, were found to be differentially regulated in response to low temperature exposure. These pathways play a key role in supplying the organism's energetic demands. While such reverse expression of these pathways between tolerant and sensitive fish was not known before, we have previously demonstrated that increased expression of the Negative correlation between metabolic rate and tolerance to environmental stress is well-known in farmed animals, with cattle and poultry strains with high temperature tolerance having lower growth rate, as well as other productivity traits West, . HoweverIn this work, we used the power of family-phenotype, obtained through selective breeding, in order to characterize the transcriptome response to low temperature exposure, in tolerant and sensitive fish. We revealed pathways that are the core cellular response as well as pathways that differ between cold-tolerant and cold-sensitive fish and might be the basis of within-species variance. Understanding the regulation of these pathways should be a key for the improvement of tilapia's cold tolerance, as ability to control or affect pathways which are the basis of phenotypic variation bear better potential for aquaculture than detection of polymorphism in specific genes, each one with minor effect. This study demonstrates the opportunities in using defined genetic structure for experiments aiming at characterization of the physiology of traits. The presented data can be important for our understanding of this economically important trait in cultured fish, as well as for designing research aiming to improve tilapia's cold tolerance through genetic or physiological manipulations on key pathways.AC conceived and designed the experiments. TZ and AB bred and selected the fish. TS, TN, and AC challenged the fish and sampled tissues. TN performed the RNA analyses. TN, AD-F, JB, FK, and AC analyzed the results. AC and IM secured funding and supervised the project. TN, FK, and AC wrote the manuscript.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.


Quantification of interictal spikes in EEG may provide insight on epilepsy disease burden, but manual quantification of spikes is time-consuming and subject to bias. We present a probability-based, automated method for the classification and quantification of interictal events, using EEG data from kainate- and saline-injected mice (C57BL/6J background) several weeks post-treatment. We first detected high-amplitude events, then projected event waveforms into Principal Components space and identified clusters of spike morphologies using a Gaussian Mixture Model. We calculated the odds-ratio of events from kainate- versus saline-treated mice within each cluster, converted these values to probability scores, P(kainate), and calculated an Hourly Epilepsy Index for each animal by summing the probabilities for events where the cluster P(kainate) > 0.5 and dividing the resultant sum by the record duration. This Index is predictive of whether an animal received an epileptogenic treatment , even if a seizure was never observed. We applied this method to an out-of-sample dataset to assess epileptiform spike morphologies in five kainate mice monitored for ~1 month. The magnitude of the Index increased over time in a subset of animals and revealed changes in the prevalence of epileptiform (P(kainate) > 0.5) spike morphologies. Importantly, in both data sets, animals that had electrographic seizures also had a high Index. This analysis is fast, unbiased, and provides information regarding the salience of spike morphologies for disease progression. Future refinement will allow a better understanding of the definition of interictal spikes in quantitative and unambiguous terms. The electroencephalogram (EEG) is an essential tool for monitoring seizures, diagnosing epilepsy and understanding epileptogenesis. Most EEG analysis, including the scoring of seizures and interictal epileptiform events, is carried out manually by trained scorers. Manual scoring is labor-intensive and prone to low interobserver agreement  which ofAdding to the challenge of analyzing EEG for epilepsy is that often convulsive seizure frequency is relatively low, often much less than one/day, making capturing confirmatory seizure events on EEG rare . Thus, tThere are many automated approaches for detecting interictal events, including template-matching , non-linStudies in rodents have shown that spiking can be identified within the EEG from both normal and epileptic brains ,4,24,25.Classification of spikes based on waveform morphology is a promising approach for differentiating events that are normal versus those that are relevant to epilepsy. Indeed, spike sorting by waveform clustering has been used to tune spike-detection and quantification algorithms , to chooHere we present a novel waveform-based analysis to compare spike-like events recorded from mice treated with an epileptogenic insult  with spGuide for the Care and Use of Laboratory Animals . Events with a P(KA) > 0.5  exist within a space consistent with general definitions of spikes, that is events with high amplitude and short duration  checklist.(PDF)Click here for additional data file.


Bifidobacterium genus are purported to exert various health-promoting effects at local and systemic levels, e.g., limiting pathogen colonization/invasion, influencing gut homeostasis, and influencing the immune system through changes in innate and/or adaptive immune responses. This has promoted extensive research efforts to shed light on the precise mechanisms by which bifidobacteria are able to stimulate and interact with the host immune system. These studies uncovered a variety of secreted or surface-associated molecules that act as essential mediators for the establishment of a bifidobacteria-host immune system dialogue, and that allow interactions with mucosa-associated immune cells. Additionally, the by-products generated from bifidobacterial carbohydrate metabolism act as vectors that directly and indirectly trigger the host immune response, the latter by stimulating growth of other commensal microorganisms such as propionate- or butyrate-producing bacteria. This review is aimed to provide a comprehensive overview on the wide variety of strategies employed by bifidobacteria to engage with the host immune system.Since bifidobacteria are among the pioneering colonizers of the human infant gut, their interaction with their host is believed to start soon following birth. Several members of the  The gastrointestinal tract (GIT) of humans, and by extension of mammals, harbors an extremely dense, and complex community of microorganisms. Until recently, this microbial ensemble, collectively forming the so-called gut microbiota, was estimated to exceed the total number of the host cells by about 10-fold . HoweverIn this review, we will discuss current knowledge on immune-modulatory activities exerted by bifidobacteria in the intestinal environment. Particularly, we will detail how specific bifidobacterial extracellular structures (are believed to) modulate the host immune response. Furthermore, we will discuss certain interactions between bifidobacteria and other intestinal microbes affect the host immune system.Bifidobacterium genus, which forms a deep-branching lineage within the Actinobacteria phylum  shared by these genomes, collectively called the bifidobacterial core genome species allowed the reconstruction of the bifidobacterial pan- and core-genome  (Pili (or fimbriae) are described as long proteinaceous appendages produced by bacteria, protruding from the extracellular cell surface, and that may be involved in microbe-host interactions by promoting adhesion to the intestinal epithelium or facilitating aggregation with other bacterial cells –67. In bad pili) , 69.in vivo colonization, in vitro contact between bifidobacterial cells and human cell lines, and/or exposure to certain extracellular matrix proteins  may be an essential starting point to prime the immune system  that expose the B. bifidum PRL2010 sortase-dependent pili major subunit 3 days before the treatment of mice model with 2,4,6-trinitrobenzensulfonic acid (TNBS) to induce colitis, clearly showed a reduction of biological/clinical markers associated with colitis symptoms when compared to the control. In addition, administration of BLP-FimPPRL2010 causes TNF-α overexpression coupled with a significantly reduction of IL-10 response in TNBS-induced colitic mice. Thus, alerting the immune system and enhancing the host reaction to inflammation, which is a typical condition of colitis  pili. These structures were first described in the pathogenic Gram-negative Actinobacillus actinomycetemcomitans, where these pili were shown to mediate adhesion to the host cell surface, supporting colonization and pathogenesis through the formation of biofilms  revealed that the differentiation of T cells toward T-helper(Th)1 (IL-12/IL-10), Th2 (IL-10/TNF-α), and Th17 (IL-1β/IL-12) effector cells is highly influenced by the physical-chemical features of the particular EPS used in the experiment. This finding suggests that a positive correlation exists between the composition, structure and size of a given EPS polymer and the corresponding elicited immune response  were evaluated. In detail, the two B. adolescentis strains differ in the exopolysaccharide-mediated enterocyte adhesion/aggregation phenotypes, with B. adolescentis IF1-03 possessing the high-molecular-weight EPS. Specifically, in vitro co-culturing experiments of the two B. adolescentis strains with RAW264.7 macrophages or mice splenocytes showed that while strain IF1-03 stimulated production of anti-inflammatory cytokines such as IL-10, IL-6, and TGF-β, it also increased the proportion of T-regulatory (Treg) cells. Notably, B. adolescentis IF1-11 induced higher levels of pro-inflammatory cytokines influencing the CD4+ T cells differentiation into Th17 cells. This observation was further confirmed by in vivo administration of the two above mentioned strains individually to Dextran Sodium Sulfate (DSS)-induced colitis mice. In this case, B. adolescentis IF1-03 not only induced high levels of anti-inflammatory cytokines, but it was also shown to contribute to the reduction of the area of ulceration and thickening of the intestinal wall  or exopolysaccharides (EPS). Based on their monosaccharide composition and biosynthesis mechanism mediated by glycosidic linkages, bacterial CPS/EPS  can either be classified as homopolysaccharides, when composed by the repetition of a single type of monosaccharide, or as heteropolysaccharides when formed by two or more types of monosaccharide subunits . In siliof genes , 78. Theof genes , 79. In of genes , 78, 79.response . In supp strains , 81. Simnal wall . In the on level . All togon level , 54, 81.B. breve UCC2003 strain (EPS+) or the two EPS-deficient B. breve UCC2003 strains (EPS−) obtained by means of an insertion or deletion in the eps locus of this bifidobacterial strain. In this regard, splenocytes stimulated with EPS+ strain evoked lower expression of proinflammatory cytokines IFN-α, TNF-α, and IL-12 compared to the EPS−. This finding was further confirmed through analysis of intracellular cytokine expression in splenocytes isolated from naïve mice orally fed with EPS+ and EPS− strains, evidencing significantly altered cytokine profiles between the two experimental conditions. Specifically, a lower proinflammatory cytokine induction was observed in both adaptive and innate immune response cell populations in case of EPS+ administration. Altogether these findings suggest that the B. breve UCC2003 EPS layer plays a crucial role in the persistence of the strain in the host intestine, reducing the risk of immune clearance against this microbial strain  from villi , depends on the orientation of a single promoter. Therefore, only one of the two types of extracellular polymer can be produced at a given time  and B. breve UCC2003Inv (able to express only EPS2) were administered to mice model, which were then treated with an intraperitoneal injection of lipopolysaccharides (LPS) from Escherichia coli 0111:B4 inducing cell shedding -gavage mice, thus underscoring the strain variant specificity in exerting protective effects. Moreover, evaluation of transcriptional changes in murine small intestine samples from control (PBS) and colonized groups (B. breve UCC2003 EPS-positive and B. breve UCC2003del) revealed down-regulation of genes related to the apoptotic cascade in mice treated with B. breve UCC2003 EPS-positive bacteria. In this context, the EPS of B. breve UCC2003 appears to downregulate inflammatory and apoptotic responses, thus reducing cell shedding events  encoding the EPS biosynthetic machinery. Despite the fact that the genome comparative analysis showed that B. longum subsp. longum 35624 EPS-specifying cluster is in the same location in other B. longum subsp. longum genomes, a different genetic composition of the eps624 was observed when compared to other analyzed B. longum subsp. longum strains (B. longum subsp. longum 35624 strain and its derivatives: the exopolysaccharide-negative mutant (EPSneg) with an insertion mutation in the eps cluster and the EPScomp where the EPS production was restored by genetic complementation of the EPSneg strain. Co-culture of human PBMCs or monocyte-derived dendritic cells (MDDCs) with one of these strains revealed that increased proinflammatory cytokine secretion is specifically related to the lack of EPS, with a marked increase of IL-17. In the same way, administration of these strains individually to mice of a T cell transfer colitis model underlined the ability of the two EPS-producing strains to prevent disease related symptoms, while EPSneg does not show any protection against the development of colitis, with increased recruitment of IL-17+ lymphocytes to the gut  represent a superfamily of proteins acting as eukaryotic-type serine protease inhibitors. These prokaryotic enzymes, synthetized by particular members of the intestinal community, are involved in the regulation of a wide range of protease-mediated processes  and, to a lesser extent, pancreatic elastase (in vivo study demonstrated the efficacy of a specific B. longum NCC2705 serpin in the prevention of gluten-related immunopathology. Administration of the wild-type B. longum NCC2705 (srp+) to sensitized NOD/DQ8 mice with peptic-tryptic digestion of gliadin, which is one of the main gluten protein fractions, favored protection against the development of gliadin-induced immunopathology when compared with the administration of the knockout B. longum NCC2705 (srp−) that is unable to synthetize serpins. Because the aberrant effects of gluten-related immunopathology are strongly relieved in mice treated with the wild-type strain, it can be speculated that serpins are involved in immune regulation, maintenance of barrier function and inhibition of elastases released during inflammation  and a cysteine- and histidine-dependent amidohydrolase/peptidase (CHAP). This protein acts as a peptidoglycan lytic enzyme that when co-cultured with human DCs was shown to cause activation/proliferation of the DCs itself and the induction of IL-2 proteins  and Anaerostipes spp.   as unique carbon source affected the growth yield of both strains when co-cultivated compared to the respective mono-cultures, leading to a mutual beneficial effect. Notably, B. longum NCC2705 as an arabinose substituent degrader of AXOS, possesses the AXOS-degrading enzymes whose action allow the release of xylose backbone (XOS) and acetate. On the other side, E. rectale ATCC 33656 consumes the whole (A)XOS substrate releasing xylose and arabinose. In this way, E. rectale ATCC 33656 takes advantage of acetate for butyrate production, resulting in a butyrogenic effect, while B. longum NCC 2705 consumes (A)XOS monosaccharides, leading to an increase in acetate production and ensuring its cell concentration thus stimulating a bifidogenic effect (F. prausnitzii S3L/3 and B. adolescentis L2-32 in the presence of fructooligosaccharides (FOS 95), or the co-cultivation of F. prausnitzii A2-165 and B. adolescentis L2-32 on starch, resulting in both cases in an enhanced production of butyrate when compared to the mono-cultures  , 100. Inraticus) . Particuc effect . Similarcultures .B. bifidum strains are singularly co-cultured with E. hallii DSM 3353 in presence of mucin as the only carbon source , which is a component of the HMO. E. hallii does not grow on HMOs as the unique carbon source. In contrast, B. longum subsp. infantis utilizes the lactose moiety of FL to produce lactate and acetate and metabolize L-fucose to 1,2-propanediol . These by-products are then used by E. hallii for its growth and the formation of butyrate coupled with propionate and formate  have an indirect role in the modulation of the host immune system, stimulating the growth and production of butyric acid by butyrate-producing intestinal microorganisms.in vivo studies it is possible that the presence of other bacterial strains may influence or even reverse the bifidobacterial immunomodulatory effects. Therefore, the challenge for future studies is to deepen the knowledge about this field, overcoming the abovementioned limits.In recent decades, a growing number of studies have demonstrated that changes in the intestinal microbial composition and gut homeostasis is directly linked to intestinal disorders resulting, in the most severe cases, in IBD and colorectal cancer. The modulation of the gut microbiota through prebiotics and/or probiotics is one of the ways to counteract these dysbiosis, improving human health. In this context, bifidobacteria are generally considered as potent probiotics for their health-promoting features. Indeed, they trigger immunomodulatory responses aimed at maintaining the host intestinal homeostasis through different mechanisms such as production of extracellular structures that can interact with other intestinal microorganisms and/or the host cells coupled with the release of by-products of their metabolism that may be utilized by other commensal bacteria, such as the butyrate-producing microorganisms, to establish a cross-feeding interaction. However, despite their key role in stimulating human health and development in technology, the precise mechanism by which bifidobacteria solicit an immune response is far from being fully understood. Indeed, most studies intended to correlate bifidobacteria and their immune modulatory effects have been carried out by exploiting cell lines and/or animal models, thus preventing a comprehensive understanding of the impact that bifidobacteria may exert on the human immune system. Indeed, because of the complexity of the gut microbiota, in case of All authors listed have made a 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.