ACCs). In addition, the expression of FLNA was associated with a less aggressive tumour behaviour in ACCs. Indeed, the subgroup of ACCs with high FLNA showed a lower ENSAT stage, Weiss score, and S-GRAS score compared to ACCs with low FLNA expression (p < 0.05). Moreover, patients with high FLNA had a longer overall survival than those with low FLNA (p < 0.05). In conclusion, our data suggest that FLNA may represent a “protective” factor in ACCs, and the integration of FLNA immunohistochemical expression in ACC tissues along with other clinical and molecular markers could be helpful to improve diagnostic accuracy and prognosis prediction in ACCs.The insulin-like growth factor 2 (IGF2) promotes cell growth by overactivating the IGF system in an autocrine loop in adrenocortical carcinomas (ACCs). The cytoskeleton protein filamin A (FLNA) acts as a repressor of IGF2 mitogenic signalling in ACC cells. The aims of this study were to test FLNA expression by immunohistochemistry in 119 ACCs and 26 adrenocortical adenomas (ACAs) and to evaluate its relationship with clinicopathological features and outcome in ACCs. We found that 71.4% of ACCs did not express FLNA, whereas FLNA absence was a rare event in ACAs (15.4%,  Adrenocortical tumours affect 3–10% of the population and include a variety of diseases ranging from frequent benign adrenocortical adenomas (ACAs) to rare and generally aggressive adrenocortical carcinomas (ACCs). The differential diagnosis between ACAs and ACCs is based on the Weiss score, which is composed of nine histopathological items  ,3.The prognosis of ACCs is generally poor, and the median overall survival (OS) ranges between 3 and 4 years . HoweverFrom a pathogenic perspective, several mechanisms have been demonstrated to be involved in adrenal tumorigenesis, including altered regulation of the cell cycle and activation of the insulin-like growth factor (IGF) system . Indeed,The cytoskeleton actin binding protein filamin A (FLNA) has recently been demonstrated to play an important role in negatively regulating the IGF system in ACC cells . FLNA isIn ACC cell line H295R, FLNA acts as a specific repressor of the IGF2-induced signalling cascade that promotes cell proliferation through extracellular signal-regulated kinases (ERKs) activation. Indeed, genetic silencing of FLNA induced an increase in the insulin-like growth factor 1 receptor (IGF1R) expression and signalling in H295R and ACC primary cultured cells and potentiated cell responsiveness to the IGF1R-insulin receptor (IR) inhibitor linsitinib . WesternThe present study aimed to test the expression of FLNA in a large, multicentre, and well-characterized cohort of ACC and ACA tissues by immunohistochemistry and to test its relationship with the clinicopathological features and outcome, with the final purpose of assessing its relevance as a biomarker to improve the diagnostic accuracy and/or prognostic classification in ACCs.n = 119, In the ACC group n = 119, , most ofIn total, 85.1% of patients received mitotane postoperatively, and 50% of these were also treated with systemic chemotherapy . Data abn = 26, In the ACA group n = 26, , we obsep < 0.05 and p < 0.001, respectively).Comparing the ACA and ACC groups, we found a higher age at diagnosis and a prevalence of patients with single hormone secretion in ACA vs. ACC . Representative pictures are shown in In tumours with any degree of FLNA expression, positivity was observed mostly in a very heterogeneous fashion. In FLNA-positive tumours, the immunoreactivity score (IRS) was 1 in 31/34 ACCs, while the remaining three ACCs showed a FLNA IRS of 2, 3, and 4, respectively. In total, 18 ACCs showed a percentage of positive cells ≤5%, 13 samples showed a percentage included from 6% to 30%, 2 from 31% to 60%, and 1 from 61% to 100%.p < 0.001 compared to ACC, In contrast, 22 out of 26 of ACAs (84.6%) were positive for FLNA (p = n.s.) . FLNA was localized in the cytoplasm in all positive samples, and in a subgroup of tumours, a plasma membrane localization was also observed  A.p < 0.05 vs. low FLNA) (p < 0.05 vs. low FLNA). In addition, given that the Weiss score is a multiparameter scoring system based on nine histological criteria, we sought to better understand which of them was associated with FLNA expression. We found a reduced mitotic rate in the group with high vs. low FLNA (p < 0.05) (p < 0.05). Accordingly, in the high FLNA group, the number of patients with an S-GRAS score higher than 3 was significantly lower than the low FLNA group (p < 0.05)  from the ACCs with a percentage of positive cells higher than 5% . We found that the ENSAT stage and Weiss score were significantly lower in the high FLNA group (ow FLNA) . Indeed, < 0.05) . Moreove < 0.05) .No association was found between the high or low FLNA group and the other clinicopathological features analysed  .Regarding FLNA intracellular localization, the subgroup of ACCs showing a plasma membrane FLNA immunopositivity did not present any significant difference in the clinicopathological features compared to the subgroup of ACCs with cytoplasmic FLNA.Lastly, no association between FLNA staining and clinical data in the ACA cohort was found.p < 0.05)  (p = 0.08)  . No diff = 0.09)  and adju = 0.08) .No association was found between the intracellular localization of FLNA and the clinical outcome.The present study indicates that FLNA expression is lost in the majority of ACCs but not in ACAs, and high FLNA expression in ACCs is associated with a less aggressive tumour behaviour compared to low FLNA.By analysing FLNA immunoreactivity in 119 ACCs, we found that FLNA was completely absent in 85 samples (71.4%). An opposite situation was found in ACAs, where FLNA was expressed in 22 out of 26 samples. These observations are in line with previously published data analysing FLNA protein expression by Western blot in a small group of ACC patients , breast The observation that FLNA absence is typical of ACCs, while it is a rare event in ACAs, suggests the possible use of FLNA expression as an immunohistochemical marker to discriminate ACCs from ACAs in those situations where histologic differentiation could be challenging ,35,36. MIn ACCs, the sole presence of FLNA was not associated with any clinicopathological features. However, we observed that the group of ACCs considered positive for FLNA expression included tumours with a very low percentage of immunopositive cells. We hypothesized that the effect of FLNA in these tumours could be negligible. We thus divided ACCs based on the percentage of FLNA-positive cells less than or equal to 5%  from ACCs with a percentage of positive cells higher than 5% (“high FLNA”). We found that the small group of high FLNA ACC displayed less aggressive features, with a lower ENSAT stage, Weiss score, and S-GRAS score, and longer survival than ACCs expressing low FLNA levels. Moreover, we demonstrated that FLNA expression was associated also with the individual Weiss parameter mitotic rate, with a lower number of mitoses in the high FLNA group. This finding agrees with the previously demonstrated role of FLNA in decreasing IGF2 mitogenic effects in ACC cells through a reduction in ERK phosphorylation . To noteInterestingly, we found that high FLNA ACC patients showed a longer OS compared to low FLNA ACCs. However, in the multivariate survival model performed with FLNA adjusted for S-GRAS, we observed only a tendency toward the protective role of FLNA, the lack of significance being probably due to the small sample size. Moreover, it is important to note that after adjustment for the ENSAT tumour stage and Ki67, even if not significant, the FLNA protective hazard ratio did not change (HR = 0.2).In the literature, the role of FLNA expression in the OS of patients varies according to the type of tumours. High levels of FLNA were associated with longer OS in colorectal cancer , while iWe can hypothesize that in the absence of FLNA, IGF2 mitotic effects are potentiated, leading to a final worse outcome in ACCs. Indeed, we previously demonstrated that FLNA is able to bind IGF1R, and FLNA silencing induced a reduction in the IGF1R expression and a simultaneous decrease in ERK phosphorylation, cyclin E1 expression, and cell proliferation in human ACC cell line H295R and in primary cultured ACC cells , supportOur previous and current findings suggest that FLNA may represent an alternative drug target along the IGF2/IGF1R pathway, possibly overcoming the failure of IGF1R inhibitors in clinical trials. Therapeutic approaches aimed to restore FLNA expression could represent a novel strategy in the treatment of ACCs.Admittedly, the evaluation of FLNA expression detected by immunohistochemistry does not take into account posttranslational modifications that are crucial in determining FLNA activity. Since one of the main mechanisms that control FLNA functions and proteolysis is phosphorylation on Ser2152 ,42,43, fIn conclusion, the present study indicates that FLNA is absent in the majority of ACCs but not in ACAs, and high FLNA expression is associated with a less aggressive tumour behaviour compared to low FLNA in ACCs. Overall these data suggest that FLNA may be a cancer suppressor factor in adrenocortical tumours. The integration of FLNA expression assessment with other clinical and molecular markers will be helpful to improve both the diagnostic accuracy and prognosis prediction in ACCs.www.ensat.org). In total, 119 patients, who underwent adrenalectomy because of ACCs, were included in the study, i.e., 74 patients from the University Hospital of Wuerzburg (Germany), 29 from the University of Florence , and 16 from Laiko Hospital Athens (Greece). Moreover, 26 tissue sections derived from patients with histological diagnosis of ACA that had surgery in Foundation IRCCS Ca’ Granda Ospedale Maggiore Policlinico were analysed.This is a retrospective European multicentre study conducted on behalf of the ENSAT  status, tumour-related hormone excess at diagnosis, symptoms, date of last followup, and status of patient  were recorded. The S-GRAS score for the ACC samples was calculated as previously published , resultiMoreover, a database for ACA patients with the following data were created: sex, age, date of diagnosis, histopathological characteristics, tumour-related hormone excess, date of last followup, and status of patient.The study was approved by the local ethics committees, and each patient gave written informed consent to the use of their tumour sample and clinical information.Slides of 4 µm thickness were obtained from representative formalin-fixed paraffin-embedded (FFPE) blocks of adrenocortical tumours (119 ACC and 26 ACA). Particularly, only primary tumours were investigated.Immunohistochemical staining with anti-FLNA antibody  was performed using an automated stainer , after EDTA buffer retrieval and via diaminobenzidine revelation . The internal positive control for proper staining was represented by stromal cells. The following parameters were assessed by 2 independent investigators: percentage of positive tumoral cells; pattern of staining ; intensity of staining. Discordant results were reviewed and discussed among the 2 investigators until a consensus was reached. Immunoreactivities were graded manually using an optical microscope  according to an immunoreactivity score (IRS) that takes into account both the percentage of positive cells  and the staining intensity , with a final score being obtained by multiplying both parameters together (range 0–9). Representative scans were captured via an Aperio AT-2 scanner . Tissues with FLNA staining >5% of positive cells were considered “high FLNA”, whereas those with FLNA staining ≤5% of cells were considered “low FLNA”.Continuous variables were reported as the mean ± standard deviation (SD) or median and IQR, according to their distribution, and were compared using parametric or non-parametric tests, respectively. Discrete variables were described as numbers and percentages and compared through the chi-square test or Fisher’s exact test, when appropriate. The Kaplan–Meyer method was used to describe the OS and PFS according to FLNA expression and different patient characteristics. The log-rank test was used to test the difference in survival across groups. The prognostic role of FLNA was also assessed in multivariate analysis according to the Cox regression model. We evaluated the potential prognostic role of FLNA adjusted for the S-GRAS score since it was recently demonstrated to have a superior prognostic performance respect to tumour stage and Ki67 in ACCs . MoreoveStatistical analyses were carried out using the SAS software, version 9.4 , and IBM SPSS statistics, version 28.0.1.1 .


This study systematically reviewed all human longitudinal exercise interventions that reported changes in the gut microbiota; frequency, intensity, duration and type of exercise were assessed to determine the influence of these variables on changes to the gut microbiota in both healthy individuals and clinical populations (PROPERO registration: CRD42022309854). Using PRISMA guidelines, trials analysing gut microbiota change with exercise interventions were included independent of trial randomisation, population, trial duration or analysis technique. Studies were excluded when microbiota abundance was not reported or when exercise was combined with other interventions. Twenty-eight trials were included, of which twelve involved healthy populations only and sixteen involved mixed or clinical-only populations. The findings show that participation in exercise of moderate to high-intensity for 30–90 min ≥3 times per week (or between 150–270 min per week) for ≥8 weeks is likely to produce changes in the gut microbiota. Exercise appears to be effective in modifying the gut microbiota in both clinical and healthy populations. A more robust methodology is needed in future studies to improve the certainty of the evidence. The human gut microbiota has been implicated in numerous aspects of health and wellbeing, including disease risk and healthy aging ,2,3,4. TCross-sectional studies have reported that sedentary and active populations have different gut microbiota characteristics . Both Ban = 25) [n = 7) [n = 38) [The earliest systematic review examining a relationship between exercise and the microbiota that included some human data was published in 2019; only five of the 25 included studies reviewed in this paper included human data . In 2020n = 25) , older a [n = 7)  or exclu[n = 38) . While tThis systematic review was conducted and is reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement . The revOn 21 February 2022, five databases were systematically examined by A.N.B: PubMed, Scopus, SportDiscus, CINAHL, and EMBASE. Searches were limited to full-text articles published in the English language in peer-reviewed journals. Key search terms included: ‘gastrointestinal microbiome’, ‘microbiome’, ‘microbiota’, ‘exercise’, ‘physical activity’, ‘adult’, ‘human’ and ‘NOT animal’  design: randomised controlled trials, cohort studies and case-control studies; (ii) population: adults aged 18 years and older; (iii) intervention: any frequency, intensity, time or type of physical activity; (iv) control: comparison group receiving a different physical activity prescription, control group not receiving the intervention at any time point during the trial, waitlist control or crossover group or no comparison/control group; and (v) outcome: faecal analysis of microbiota diversity and/or microbiota taxonomy. Any study that also incorporated strategies that may have influenced the outcome , where the effects of physical activity could not be isolated, was excluded.Covidence was used for screening the titles and abstracts of articles identified through the search process. Duplicate removal was automated, and articles were screened by A.N.B. and R.E.W. to exclude those outside of the scope of the review. Following the screening, full-text articles were retrieved and independently assessed by A.N.B. and R.E.W. for eligibility according to the outlined inclusion criteria. Where discrepancies in article eligibility were identified, eligibility was discussed in the research group, with an independent assessment made by a third blinded arbiter if a consensus was not achieved (T.L.S.). Reference lists of eligible articles were examined to locate potential additional studies that met the inclusion criteria.Study details, including the participant characteristics, exercise and control group prescriptions and outcome measures, were extracted independently by two authors (A.N.B. and R.E.W.). Where the assessment of microbiota diversity and/or microbiota taxonomy occurred at more than one time point during an intervention, the pre- and post-intervention outcomes were extracted, and other intra-intervention values were omitted. Participant characteristics and group classification were dependent on only one group; if a study included a group that was of clinical interest, this study was analysed with the perspective of this clinical group compared to the apparently healthy group included.The methodological quality of included articles was independently assessed by A.N.B. and R.E.W. Randomised controlled trials meeting inclusion criteria were assessed for methodological quality using a six-item derivation of the nine-item Delphi list developed by Verhagen et al. Three of the nine Delphi criteria  were deemed not to be appropriate for all types of included interventions. All criteria were equally rated using a ‘yes’ (1 point), ‘no’ or ‘unclear’ (0 points) answer format, with a quality score generated as a percentage of the maximum score for each included study. Non-controlled trials meeting the inclusion criteria were assessed using the Delphi, acknowledging that these would receive a lack of score for randomisation. Any discrepancies in methodological quality ratings were mediated by a third arbiter (T.L.S.). The results of the quality analysis can be found in r = x) and how many studies (including those reporting change) likely were able to observe changes to this level but did not report a change within the bounds of the variable . For instance, the statement ‘increases seen in Example bacterium ’ should be interpreted as, of the studies including this variable, two of the four studies (50%) reporting this level of taxonomy found an increase, and thirteen studies (including the four listed) potentially had the capacity to observe a change, but it was not reported, suggesting no change was reported. This approach was used unless the review is comparing the frequency of reporting a microbe in response to a single variable. In this case, the only number reported (nr) would be compared to the number that likely could assess this change  within this variable, and this would only be reported once in each statement, e.g., Example was more commonly reported in x interventions  compared to y intervention (nr = 1). Taxa that were reported in all studies likely to be capable of assessing this change in relation to the variable investigated were reported simply as (n = x).Results were analysed and reported using a combination of quantitative, descriptive and narrative data synthesis. All reported changes in the gut microbiota of studies included were collated with study and intervention characteristics by reports of increase, decrease or no reported change. Thereafter, summaries of reported changes in the gut microbiota across the included literature were produced . Most clinical populations were characterised by metabolic disorders  [n = 1, 6%) [n = 1, 6%) [n = 1, 6%) [n = 1, 6%) [n = 1, 6%) [Participant characteristics are shown in studies) ,41,43,44= 1, 6%) , autoimm= 1, 6%) , inflamm= 1, 6%) , myalgic= 1, 6%)  and elde= 1, 6%) .n = 19) [Details of the exercise interventions are summarised in n = 19) ,41,42,44n = 19) ,39,43,45n = 19) . Three sn = 19) ,33. Anotn = 19) ,26,36,41n = 19) ,41,45, nn = 19) ,27,31,44n = 19) ,36,42,43n = 19) ,41. Of tn = 26), whole genome shotgun sequencing (n = 5) and one study [n = 9) except for two studies, one of which collected fourteen samples per participant (every two weeks) and the other collected twenty-eight samples per participant (two samples per week)Stool samples were sequenced with 16S rRNA gene amplicon sequencing (ne study  did not n = 6) compared to aerobic-only interventions . Beta diversity was reported to increase in 83% of concurrent trials (n = 6) compared to 42% of aerobic-only interventions (n = 12). Aerobic-only exercise interventions were the only interventions to report Roseburia and Lachnospira, where increases were reported in  and  of studies, respectively. Dorea abundance was reported to increase more often in response to aerobic interventions  compared to concurrent exercise interventions (n = 1). Similarly, aerobic interventions reported a higher abundance of Ruminococcus more commonly than concurrent interventions . Changes in Bifidobacterium, Veillonella and Akkermansia were similar between intervention types .Concurrent exercise interventions  more commonly reported increases in measures of alpha diversity , while 45% of studies examining changes in beta diversity reported a higher diversity (n = 11). Two studies found an increase in Bacteroides, with another reported no change . A total of 50% of studies reporting Faecalibacterium changes used low-to-moderate intensity exercise . At the level of species, Faecalibacterium prausnitzii was not found to change in two studies, though a third did report an increase . This intensity of exercise included 66% of studies reporting an increase in Prevotella copri .Thirteen studies used low-to-moderate or moderate-intensity exercise in at least one study arm ,40,41,45n = 6), while 75% of studies reporting beta diversity found a positive change (n = 4). At the level of phylum, consistent increases in Firmicutes were seen in all studies that reported this level of taxa . Similarly, decreases in Bacteroidetes abundance were found in studies reporting at this level . At the level of family, 67% of studies reporting improvements in Lachnospiraceae and Ruminococceae used moderate-to-high-intensity exercise . At the level of genus, moderate-to-high and high-intensity exercise included, most studies that reported a positive change in Dorea , Bifidobacterium , Ruminococcus  and Akkermansia  and 50% of studies reported improvements in Veillonella . The only two studies reporting an increase in Streptococcus utilised this exercise intensity . Two of three studies reporting Roseburia at this exercise intensity found a decreased relative abundance . Both studies reporting Bacteroides at this intensity reported a decreased abundance of this genus . Conflicting data were found for Lachnospira , Faecalibacterium , Prevotella  and Collinsella . At the level of species, both studies reported an increase in Ruminococcus bicirculans and utilised moderate-to-high intensity exercise .Nine studies used moderate-to-high and high-intensity exercise ,27,31,44n = 4), while three of the four studies reported an increase in beta diversity (n = 4). Three studies reported at the level of genus, and two studies of these reported an increase in Bifidobacterium (n = 2). There were conflicting data for Roseburia; one study reported increases in Dorea, Ruminococcus and Veillonella abundance and a decrease in Prevotella. None of the HIIT studies reported changes at the level of species.Four studies utilised HIIT ,36,42,43n = 2) or beta diversity (n = 1) when using maximal effort or SIT protocols. One study utilising maximal intensity and the study utilising SIT found that Lachnospira abundance increased. Otherwise, no consistent changes were observed. One study that included maximal exercise efforts found increases in Ruminococcus and Romboutsia.Three studies utilised SIT  or maximn = 13), most did not find a change in alpha diversity; only one study reported an increase . A total of 58% of studies reporting beta diversity found an increase (n = 12). At the level of genus, studies that involved two to three training sessions per week included 50% of studies reporting increases in Lachnospira , 60% of studies reporting improvements in Bifidobacterium , 67% of studies reporting improvements in Roseburia  and both studies reporting improvements in Oscillospira .Of those studies that used a training frequency of two to three sessions per week (n = 5) reported an increase in alpha diversity in 33% of studies reporting this metric (n = 3) and 67% for those reporting changes in beta diversity (n = 3). No consistent changes were seen at the level of genus.Studies with a training frequency of four to five sessions per week (n = 4) found an increase in alpha diversity in 67% of studies reporting this change (n = 3). However, there was no change in beta diversity (n = 1). No consistent changes have been reported at the level of genus, though Prevotella copri was found to increase in two of the studies .Studies with a training frequency of greater than five sessions per week (n = 6).Interventions that did not specify exercise frequency or involved less than one training session a week were not included in the frequency analysis (n = 13) reported improvements in beta diversity compared to shorter interventions of 0% (n = 4). At the level of genus, Roseburia decreased more commonly in interventions that lasted less than one week (n = 2), compared to a longer duration intervention (n = 1). All improvements in Roseburia occurred in response to interventions lasting more than four weeks , while Lachnospira was reported to increase in studies lasting eight weeks or less . Dorea was reported to increase in studies ranging from one week to twenty-six weeks , while increases in Ruminococcus were found both in studies lasting less than one week (n = 3) and also in those ranging from eight weeks to twenty-six weeks (n = 3) . Studies with an intervention duration of over nine weeks reported the majority of increases in Veillonella  and Bifidobacterium . Decreases in Clostridium were reported in studies with a duration of five weeks or less . Changes in Akkermansia were inconsistent in relation to duration, with increases seen from one to twenty-six weeks .Studies that included an exercise intervention for 12 weeks or longer represented 50% of all reported improvements in alpha diversity. All those studies that assessed potential changes in beta diversity with a duration of six weeks or more (n = 6).Increases in alpha diversity were only found in studies that included more than 90 min of exercise per session; a total of 67% of these studies found an improvement in alpha diversity (Lachnospira (n = 3), with <30 min reporting an increase (n = 1) and >60 min reporting a decrease (n = 1). Increases in Dorea were only found in studies that included exercise lasting longer than 50 min . Increases in Ruminococcus were also most commonly reported with exercise sessions lasting longer than 50 min . Increases in Streptococcus were only reported in studies involving exercise sessions lasting more than 95 min (n = 2) . Bacteroides increased in exercise sessions ranging between 30 and 60 min (n = 2) and decreased in studies lasting more than 90 min (n = 2) . Increases in Bifidobacterium were most commonly reported in studies that included exercise sessions lasting between 30 and 60 min . Akkermansia was more commonly found to increase in response to interventions that included 90 min exercise sessions .Studies that involved 30 to 60 min of exercise per session most commonly reported improvements in n = 21), 18% of those in clinical populations reported an increase (n = 2), with the remainder finding no change (n = 9). In healthy populations, 40% of studies reported an increase in alpha diversity (n = 4), with the remainder finding no change (n = 6). Beta diversity (n = 17) was increased following exercise for 60% (n = 6) of clinical population studies that reported this metric (n = 10), with 57% (n = 4) reporting this change in healthy populations (n = 7).Of those studies that reported alpha diversity , and n = 4 and n = 0 , respectively). Indeed, healthy populations reported decreases in Roseburia (n = 3). Increases in Dorea, Ruminococcus, Romboutsia and Bifidobacterium more commonly reported in healthy populations following exercise . Healthy populations were more likely to show decreases in Roseburia (n = 3 vs. n = 0), Prevotella (n = 2 vs. n = 0) and Bacteroides (n = 2 vs. n = 0). Both healthy and clinical populations reported decreases in Odoribacter and Clostridium, as well as increases in Veillonella and Akkermansia with similar frequency.Twenty studies reported a change at the genus level and increases in n = 6); two studies found that Ruminococcus bicirculans and Prevotella copri increased in healthy populations, and one study reported an increase in Prevotella copri in clinical populations. No other consistent changes in species were reported.Ten studies reported changes at the species level. These changes were more commonly reported in healthy populations , exercise interventions, sequencing techniques and targets of sequencing. Exercise interventions were assessed by exercise type, intensity, frequency, and time, as well as the duration of the training intervention. Outcomes that were based on the population were also investigated; clinical and apparently healthy populations were compared in a number of studies. Clinical populations included metabolic conditions, neurological disorders, autoimmune disorders, myalgic encephalomyelitis and elderly populations. Healthy populations included apparently healthy, recreational and elite athletes and military populations. The findings suggest that differences in exercise delivery influence changes in the gut microbiota in response to training. While population variations were found to produce a similar response at the level of diversity, genus responses were found to vary.Roseburia, Lachnospira, Dorea and Ruminococcus, in particular). Exercise type was only directly assessed in one randomised controlled trial [Concurrent exercise  appeared to influence alpha and beta diversity more than aerobic-only interventions. Aerobic-only exercise interventions reported more common changes at the level of genera  compared to those that have used two to three sessions a week (n = 13). This may explain why some genera were not consistently reported in higher frequency interventions; six studies did not report exercise frequency, and these were not included in the analysis. It is unclear whether these changes may have been observed in other studies had different analysis techniques been utilised.It appears that while exercising two to three times per week is sufficient to influence beta diversity and abundance at the level of genus, four to five weekly sessions are needed to elicit some change in alpha diversity (33%)—and more than five sessions a week are needed for more consistent increases (67%). Again, the influence of diet on those changes reported in response to exercise interventions is not well understood. As very high levels of physical activity are often accompanied by an increase in energy intake, future research needs to control for diet when assessing potential changes in alpha diversity with exercise training. Additionally, relatively few studies have included more than three and five exercise sessions a week (Roseburia (decrease), Ruminococcus (increase) and Clostridium (decrease). These changes are likely to be compounded by the maximal intensity or very high exercise session durations used in these studies. Of interest, Lachnospira appeared to increase in response to eight weeks of training, after which there was no further change. It is unclear as to whether this suggests that the perturbation of exercise only influences this genus in the short term. Veillonella is one of the most highly discussed genera in the context of exercise and microbiota interactions, with Scheiman et al. (2019) identifying its potential role in endurance exercise as a metaboliser of lactate [Veillonella increases, suggesting that repeated and prolonged exercise behaviour may be linked to this genus [Veillonella following an ultra-marathon. Veillonella was also shown to increase in exercise studies with clinical populations, potentially supporting Scheiman’s suggestion that Veillonela can increase based on exercise demands. However, the same can be said for other genera assessed in this review. Based on the relative interest in Veillonella and the mixed findings, with some studies finding significant changes and others finding no change, it is possible that those individuals showing an increase in Veillonella have a significant presence of this microbe prior to training.Exercise interventions lasting more than four weeks were responsible for most of the reported changes at the genus level. Changes in diversity metrics were found at six weeks (beta diversity) and 12 weeks . The longer duration (greater than eight weeks) exercise interventions were more likely to find changes in the microbiota at the genus level. In noting this, interventions lasting less than four weeks reported changes in  lactate . In agreis genus . It appeLachnospira and Bacteroides; however, longer sessions appeared to blunt this increase or even result in decreases. Similarly, 50 min per session was associated with increases in Dorea, Bifidobacterium and Ruminococcus with decreases in Ruminococcus reported in studies where training sessions lasted longer than 90 min. These longer duration sessions (>95 min) also appear to be related to increases in Streptococcus. While alone this is not concerning, at the level of genus it may be a potential tool to further investigate the environmental changes that may be linked to microbiota change with exercise, authors of both articles reporting an increase in Streptococcus comment that this is a genus that does contain pathogenic species [Akkermansia was also reported to increase in these longer duration sessions; Akkermansia is a commonly reported genus that has a higher abundance in active individuals [The duration of exercise sessions was highly predictive of change in the gut microbiome diversity, with sessions lasting more than 90 min producing the most consistent change in alpha diversity. Thirty minutes appeared sufficient to produce increases in  species ,23. Akkeividuals . This reividuals  and obesividuals . Given tividuals .It appears that aerobic training at higher intensities is most likely to elicit changes to the gut microbiota, particularly changes in alpha diversity. There appears to be a dose–response, with a possible ‘inverted U’ describing the relationship; low frequency and duration of exercise results in little to no change, 30–90-min sessions 3–5 times per week produce most of the change, and exercise lasting longer than 90 min and more than five times per week produces some change but a higher likelihood of a potentially negative change to the gut . Indeed,The available evidence suggests that a certain level of disruption or hormesis is necessary to influence changes in the gut microbiota of humans; however, there may be a ceiling effect, as seen with the plateaus in microbiota change in some studies in this review. To this point, an exercise intervention duration of eight weeks or more appears to produce the most consistent change across alpha diversity, beta diversity and most genera. Furthermore, the majority of studies collected samples only before and immediately after the exercise intervention, and the time course of changes is not clear. Of the two studies that sampled faecal matter during the exercise intervention, the limited available data suggest that most of the observed changes in the microbiota may occur within two weeks of training before plateauing . In the Based on the changes observed in this review, the removal of the exercise stimulus reverses changes in as little as three weeks in previously sedentary individuals . This isRoseburia and, to some extent, Lachnospira, did increase with exercise more commonly in clinical populations, with these genera commonly seen to be reduced in clinical populations at baseline compared to healthy populations and are linked with potential benefits to health [Many clinical populations present with gut microbiota dysbiosis or different microbial composition compared to healthy individuals ,63,64. To health ,67. Exero health ,69,70. Ro health .Bacteroides, which is a highly discussed and observed genus in the wider human gut microbiota literature; however, this review found only three studies reported this genus. It is, therefore, likely that the one study reporting no change was supported by an unknown number of other studies. As such, the results of this review should be interpreted as exercise likely having either no effect or a positive effect (in the case of Bacteroides) as per each change reported.The primary limitation of this review is that results were only included as they were reported in each study. Given that the reporting increase, decrease and no change across every level of taxonomy for each study is not feasible, this means that many of these studies likely found no change in microbes with no need to report this, especially those using metagenomic analysis. An example of this is The current review is also limited by the high heterogeneity of studies assessed. This has been the nature of microbiota research as an emerging field in exercise and in humans; however, future research will benefit from standardisation of some of the techniques from stool sample collection to downstream analysis. A recent study highlighted that the most popular and currently feasible way of analysing the gut microbiota  may underestimate the changes made in the gut over time due to the imposed constraints of abundance analysis . An alteControl standards varied considerably between studies, primarily concerning diet analysis, repeated sample collection and frequency of sample collection. Again, there is no consensus as to what ideal standards should be, and as the literature stands, much can be interpreted from the collection of studies presented in this review. However, for the strength of individual studies, understanding the intra-individual variation and influence of diet on stool samples and the interpreted microbiota will strengthen the conclusions drawn from these studies.To support the intraindividual variation and its role in microbiota research in humans, a growing body of literature suggests that case-wise/intrapersonal assessment in a functional capacity may be a positive way to understand microbiota changes  and to aFinally, this review did not assess the data recorded in repositories and, as such, all results were drawn directly from the body of articles or supplementary files where possible. There is a risk of under- or over-reporting some findings in this review due to the analysis techniques used, and perhaps a review investigating repository data, such as the work by Bisanz et al. (2019), in high fat diets in murine models may be warranted . SimilarThis is the first systematic review to assess only longitudinal study designs and varied populations when considering the interaction between exercise and the human gut microbiota. We were able to compare exercise dose through frequency, time, type and intensity of exercise across 28 studies. While single bouts of high-volume exercise have been shown to influence the gut microbiota, it was found that moderate-high, but especially high-intensity interventions, for more than 30 min, three or more times per week and for more than eight weeks resulted in the most consistent changes on the human gut microbiota. When potential differences between clinical and healthy populations were compared, microbiota changes were observed in both populations with exercise; however, specific genera that responded to exercise appeared to differ. Further research is required to verify these findings, and future studies would benefit from waitlist or crossover controls to account for inter and intraindividual variability in the gut microbiota. An improved understanding of how different types of exercise affect the human gut microbiota of different populations will inform the development of the most appropriate interventions to improve health via the gut microbiota and assist in chronic disease prevention and management.


Polyphenols are natural substances with variable phenolic structures and are elevated in vegetables, fruits, grains, bark, roots, tea, and wine. There are over 8000 polyphenolic structures identified in plants, but edible plants contain only several hundred polyphenolic structures. In addition to their well-known antioxidant effects, select polyphenols also have insulin-potentiating, anti-inflammatory, anti-carcinogenic, anti-viral, anti-ulcer, and anti-apoptotic properties. One important consequence of ischemia is neuronal death and oxidative stress plays a key role in neuronal viability. In addition, neuronal death may be initiated by the activation of mitochondria-associated cell death pathways. Another consequence of ischemia that is possibly mediated by oxidative stress and mitochondrial dysfunction is glial swelling, a component of cytotoxic brain edema. The purpose of this article is to review the current literature on the contribution of oxidative stress and mitochondrial dysfunction to neuronal death, cell swelling, and brain edema in ischemia. A review of currently known mechanisms underlying neuronal death and edema/cell swelling will be undertaken and the potential of dietary polyphenols to reduce such neural damage will be critically reviewed. Polyphenols are natural compounds with variable phenolic structures and are rich in vegetables, fruits, grains, bark, roots, tea, and wine ,2. Whileet al. i can induce cell swelling as demonstrated in lactacidosis-induced glial swelling [2+]i. Also, such blockade of the rise in [Ca2+]i by blockers of the L-type calcium channel as well as modulation of [Ca2+]i through BAPTA, a calcium chelator, reduces cell swelling in C6 glial cultures [2+]i following administration of polyphenols. Quercetin attenuated the H2O2-induced calcium dysregulation in PC12 cells [+/Ca2+ exchanger [2+ concentration in RBL-2H3 cells induced by antigen stimulation [2+]i increase by attenuating ionotropic Ca2+ influx in PC12 cells [2+]i was observed after exposure to tannins in HL-60 cells [2+]i in U87 cells by influx of extracellular Ca2+ as well as by releasing calcium from intracellular stores. Whether this difference in modulating [Ca2+]i by polyphenols is due to the difference in cell types or the polyphenols tested or the stressor involved is not clear. It is also possible that the difference arises from assessing [Ca2+]i in control conditions (in the absence of stress) compared to examining the effects of polyphenols in the presence of stress. Nevertheless, these studies indicate that polyphenols have the potential to modulate calcium channels that are involved in cell volume regulation, but their role in attenuating glial swelling/cytotoxic edema in ischemia needs to be further elucidated.The effects of polyphenols in regulating ion channels or ion co-transporters, following cerebral ischemia, are not clear, and are a subject of current investigation. An increase in intracellular calcium is a key feature of ischemic injury ,185. Furswelling ,187 and swelling . Studiescultures . Other s12 cells . Quercetxchanger . Apple cmulation . EGCG re12 cells . In cont60 cells . Red win60 cells . EGCG inm) by immunosuppressants, cyclosproin A (CsA), but not FK506, are consistent with the role of the mPT mediating such events. Similar to CsA, CPE, and green tea polyphenols, also significantly prevented OGD-induced cell swelling and the decline in ΔΨm in C6 glioma indicating that one mechanism by which CPE and GTE exert their protective effects is possibly by blocking the mPT. Interestingly, quercetin significantly attenuated cell swelling in C6 glial cells following OGD but did not block the dissipation of the ΔΨm [Mitochondrial dysfunction is an important characteristic of ischemia ,195. The the ΔΨm  indicatiet al. [et al. [et al. [in vitro [An increase in inflammatory markers has been associated with brain edema  and coulet al.  which ex [et al.  and Lee  [et al.  report a [et al.  but the  [et al. . Curcumi [et al.  but as w [et al.  most lik [et al. . Il-1 re [et al. . Taken tin vitro ,210. A rin vitro  as well in vitro  and TNF-in vitro . In addiin vitro , as wellin vitro  and otheOxidative stress and mitochondrial dysfunction are key features of cerebral ischemia that affect neuronal viability after ischemia. In addition, these factors also affect brain edema which is a major consequence of ischemia and can be fatal if not resolved. Edema can further aggravate neuronal injury by affecting cerebral perfusion. Currently, there are few remedial agents to effectively reduce neuronal death or brain edema not only in ischemia but also in other neural injuries including traumatic brain injury. The potential for the use of polyphenols in the preventing cell loss or damage and edema in cerebral ischemic injury is tremendous. However, the cellular and molecular actions of polyphenols involved in neuroprotection have to be elucidated further. Given the large proportion of the population affected by stroke and traumatic brain injury, and with few strategies to effectively attenuate brain edema and associated neural damage, it is important to determine the potential beneficial effects of dietary polyphenols in the prevention and alleviation of such damaging effects.


The sensitivity of parasite isolates to anti-malaria drugs were also evaluated for a possible association with polymorphisms in these genes.In 2005, Ghana replaced chloroquine with artemisinin-based combination therapy as the first-line treatment for uncomplicated malaria. The aim of this work was to determine for the first time, polymorphisms in the putative Plasmodium falciparum genes were assessed from filter-paper blood blot samples by DNA sequencing. In vitro drug sensitivity test was carried out on some of the blood samples from volunteers visiting hospitals/clinics in southern Ghana using a modified version of the standard WHO Mark III micro-test.The prevalence of point mutations in the above 50 value for artesunate was 0.73 nM , amodiaquine 30.69 nM  and chloroquine 58.73 nM . Twenty point mutations were observed in pfATPase6 gene, with no L263E and S769N. All mutations found were low in frequency, except D639G which was observed in about half of the isolates but was not associated with artesunate response (p = 0.42). The pftctp gene is highly conserved as no mutation was observed, while CVIET which is chloroquine-resistant genotype at codon 72-76 of the pfcrt gene was identified in about half of the isolates; this was consistent with chloroquine IC50 values (p = 0.001). Mutations were present in pfmdr1 gene but were not associated with artemisinin response (p = 1.00).All successfully tested parasite isolates were sensitive to artesunate; while 19.4%, 29.0% and 51.6% were resistant to quinine, amodiaquine and chloroquine respectively. The geometric mean of ICpfATPase6 gene is highly polymorphic with D639G appearing to be fixed in Ghanaian isolates. These may just be spontaneous mutations as all parasite isolates that were tested displayed satisfactory in vitro response to artesunate. However, there is no improvement in susceptibility of the parasites to chloroquine five years after its proscription.The  Plasmodium falciparum parasites that are resistant to conventional anti-malaria drugs in almost all endemic countries , while a pair of primer was sufficient for the relatively short pftctp gene [GenBank:DQ141561). Two pairs of primer were employed in sequencing portions of the pfmdr1 gene [GenBank:M29154.1] spanning codons 86, 184, 1034, 1042 and 1246; the first pair of primer targeted the first two codons whereas the second primer set were for the last three codons . This study also received ethical approval from Tokyo Medical and Dental University, Japan (Certificate Number 955).in vitro for their drug sensitivities, out of which 7 (18.4%) failed the test due to poor growth or contamination ; amodiaquine, 30.69 nM ; chloroquine, 58.73 nM ; quinine, 355.37 nM  , that of artesunate and chloroquine was medium, but were all statistically significant  that were used for drug sensitivity test (phenotyping). A total of twenty point mutations were found in pfATPase6 gene (Table p = 0.423)  isolates harboured the N86Y mutation (TAT nucleotides) while 43/60 (71.7%) with Y184F mutation (TTT nucleotides) in the pfmdr1 gene  pressure in Ghana; nonetheless, this study presents a picture in time since there is no report of artemisinin resistance in Ghanaian isolates. This study therefore describes for the first time, polymorphisms in pfATPase6 gene in such isolates. By using PCR sequencing assay, neither L263E nor S769N point mutation was observed; and this is consistent with the in vitro micro-test results. A total of twenty point mutations were observed in this gene, confirming its molecular diversity than previously demonstrated [The limitation to this study was that polymorphisms in the nstrated ,36,37; tnstrated .pftctp gene in artemisinin activity, our present result confirms its extreme conservation, and thus not a strong candidate for resistance to artemisinin and its derivatives due to the fact that it is not under major selective drug pressure [pfmdr1 and pfcrt genes in some isolates conferred chloroquine resistance [pfmdr1 gene and K76T allele in the pfcrt gene; but they had no part in the modulation of artesunate response as the lowest and highest artesunate IC50 values corresponded to parasite isolates with pfcrt K76T. This was corroborated by Fisher's exact test. Afonso et al [Plasmodium chabaudi, can develop stable resistance to artemisinin but lack mutations in candidate genes including atp6, tctp, mdr1 and cg10.Even though studies have shown the involvement of the malaria parasite pressure ,29. Studsistance ,39, but sistance ,18. Our so et al  in theirMore than half of the parasite isolates were resistant to chloroquine after five years of its proscription in Ghana, which is a reflection of our molecular results but at variance to the Malawian situation where sensitive isolates were recovered after stopping the use of chloroquine ,22. The Plasmodium falciparum isolates from southern Ghana showed no improvement in susceptibility to chloroquine, but they exhibited satisfactory in vitro response to artesunate. No L263E and S769N mutations in the SERCA-type PfATPase6 were observed, albeit evidence is clear on the greater molecular diversity of PfATPase6 than previously demonstrated as twenty mutations were detected with D639G appearing to be fixed in this parasite population. Whereas these mutations may be spontaneous and as such without any particular role in the parasites response to artemisinin, continual monitoring of P. falciparum susceptibility to artemisinin is welcome.The authors declare that they have no competing interests.in vitro drug assessment test while BKB and TK did the molecular work. NO, MDW and DAB were involved in all stages. BKB wrote the manuscript, which was read and approved by all authors.BKB and NO conceived the idea, while BKB, IA, TS, JO, WKA and JHNO performed the field work. BKB, IA, TS, JO, JHNO, HA, MFO and NA did the


In response to extra- or intracellular stresses, the cellular gatekeeper p53 is able to integrate multiple diverse signals to determine the outcome of cell fate, by regulating the expression of numerous responsive genes Salmonella or Helicobacter infections Bacterial and viral infections represent a major type of cellular stress, triggering different biological countermeasures, notably mediated by the p53 pathway. In the process of evolutionary adaptation to the host environment, pathogens have developed diverse strategies to hijack and exploit such host machinery. This is particularly well-illustrated for viruses, with many examples of virally induced deregulation of the p53 pathway  TP53 gene physiologically expresses several protein isoforms, due to the use of alternative promoters, splicing sites, and translational initiation sites , a RNA, and a DNA virus  Although several studies report on the suppressive function of p53 isoforms and related deregulation of their expression in human cancers Within SV40 lytic infection, p53 is targeted and inactivated by T-Ag, and Δp53 has been identified as a new player in SV40 replication by Rohaly and colleagues. They revealed that an ATR–DNA damage response pathway mediates the phosphorylation and stabilization of Δp53, enhancing its transcriptional activity in a promoter-dependent manner In human lung epithelial cells, we have investigated the roles of both Δ133p53α and p53β isoforms in the context of influenza virus infection Helicobacter pylori has been shown to interfere with p53 function via up-regulation of the Δ133p53 isoform both in vitro  and in vivo (Mongolian gerbil) H. pylori infection (H. pylori pathogenesis and associated tumorigenesis.Recently, nfection . This stH. pylori have an impact on Δ133p53 to interfere with full-length p53 activity via mechanisms remarkably similar to those previously described in the field of cancer In conclusion, these three studies, based on different pathogen models, have highlighted for the first time the functional role of different p53 isoforms in the context of infection. A preliminary model emerges in which the isoforms act as regulators of the p53-mediated cellular response against pathogens. As an illustration, both influenza viruses and


Overexpression of the tumor suppressor gene p53 and the marker for cellular proliferation Ki67 in open lung biopsies are indicated as predictor factors of survival of patients with lung cancer. However, the prognostic value of p53 and Ki67 in fiberoptic bronchial biopsies (FBB) has not been fully investigated. We evaluated p53 and Ki67 immunostaining in FBB from 19 with Non Small-Cell Lung Cancer .FBB specimens were fixed in formalin, embedded in paraffin, and immunostained using anti-p53 and anti-Ki67 antibodies. Slides were reviewed by two independent observers and classified as positive (+ve) when the number of cells with stained nuclei exceeded 15% for p53 or when >25% positive cells were observed throughout each section for Ki67.Positive (+ve) immunostaining was found in 9 patients for p53 (47.37%) and 8 patients for Ki67 (42.10%). We examined overall survival curves of the patients with Mantel's logrank test, both p53 -ve and Ki67 -ve patients had significantly higher survival rates than p53 + ve (p < 0.005) and Ki67 + ve , respectively.This study suggests that negative immunostaining of fiberoptic bronchial biopsies for p53 and Ki67 could represent a better prognostic factor for patients with NSCLC. Lung cancer accounts for the most cancer related deaths in both men and women. Cigarette smoking is by far the most important risk factor for lung cancer. Risk increases with quantity and duration of cigarette consumption. The 1-year relative survival for lung cancer increased up to 40% in the last ten years, largely due to improvements in surgical techniques and combined therapies. However, the 5-year survival rate for all stages combined is only 15%. Despite, the 5-year survival rate is 50% for cases detected when the disease is still localized, but only 16% when lung cancers are diagnosed out of this early stage, screening for early/localized lung cancer detection has not yet been proven to reduce mortality.s in its wild-type conformation as a transcription factor and DNA binding protein, and this activity results on inhibition of cell proliferation by blocking entry into the S-phase of the cell cycle [Some independent prognostic factors have been suggested for predicting survival and helping in the management of patients with lung cancer. Regarding this issue, during the last few years there has been a growing interest in molecular biology of lung cancer. As the molecular characteristic of cancer have become better understood, prognostic models have been developed for lung cancer that incorporate biological markers, immunohistochemical properties and genetic features in addition to hystologic subtype, age of patients and extent of disease (TNM-stage). Mutation of the p53 tumor suppressor gene, which is localized on human chromosome 17p13, it has been observed in many human cancers and is the most common mutation in lung cancers [ll cycle . Mutant ll cycle , severalll cycle . Actuallll cycle .The antibody Ki67, which recognizes a nuclear antigen in proliferating cells, has been widely used to estimate growth fraction in different cancer lesions ,12. DespAim of the present study is to determine the correlation of p53 protein and Ki67 antigen, immunohistochemically detected in bronchial biopsies, with survival of patients with non small cell lung cancers.We studied samples obtained from 19 consecutive patients that had been undergone to bronchoscopy, with diagnostic intent, in our Unit, between January 2008 to December 2010. We used flexible fiberoptic bronchoscopy  and all samples were collected by the same operator. Only patients with positive histological diagnosis entered into the study. The clinical staging was defined with the usual method . The stuImmunohistochemical staining was performed on formalin-fixed, paraffin-embedded sections of 5 um with monoclonal antibodies to the p53 protein  and Ki67 . Similar procedures were followed for the usual treatment of deparaffination, with xylene, absolute alcohol and distilled water. Ki-67 and p53 sections were heated in a microwave oven 4 times, each of 5 min, in citrate buffer-solution. The slides, after at least 30 min of air-drying, were incubated with the primary antibodies against p53 and Ki67. Dilutions (raging between 1:25 and 1:50) and time of incubation (30 to 60 min) were depending on the batch of antibody. After washing with Phosphate Buffered Saline (PBS) secondary antimouse antibodies were added  both for Ki-67 or P53. Afterwards the slides were incubated with streptavidin-peroxidase reagent  and again the time of incubation was depending by the “data-sheet” of the antibody. After another PBS wash, the final staining was provided using a 0,05% solution of diaminobenzidinetetrahydrochloride in PBS usually for 10 min. Finally, sections were counterstained with Meyer’s haematoxylin. For p53 a tumor was considered to be immunopositive when the number of cells with nuclei red-stained exceeded 15% .The Kaplan-Meier method was used to estimate the probability of overall survival as a function of time (starting from the date of diagnostic bronchoscopy to that of death from cancer), and difference in the survival of subgroups of patients were compared with Mantel’s log-rank test. Multivariate analyses were performed using the Cox regression model to study factors that independently influenced overall survival. Statistical significance was set a P value <0.005 and all test were two-tailed as were the reported P values.We found p53 reactivity in 9 of the patients (47.37%). The antibody used in this study reacts with wild type and mutant type of the p53 protein and the nuclear reactivity, as expressed before in “Methods” section, was designed to be “positive” (+ve). For Ki67 immunostaining, reactivity between antibody and antigen localised in nuclei of proliferating cells, designed + ve as above indicated, was found in 8 of the patients (42.10%). By contrast we designed as “negative” (−ve) reactivity against p53 antibodies or Ki67 under the “cut-off” points of 15% for p53 and 25% for Ki67. Only tumoral section showed immunoreactivity both for p53 or Ki67, indicating that our immunostaining procedures do not generate false positives. In Figures We compared Kaplan-Meier survival curves for the p53 and Ki67 + ve groups and p53 and Ki67 –ve groups in all 19 patients with NSCLC Figures  and 2b. First histological diagnosis of lung cancer is often obtained by fiberoptic bronchial biopsies. However the utility of extend histological studies with immunohistochemical methods in lung cancer is still debated. We have shown the feasibility of immunohistochemical detection of p53 and Ki67 in bronchial biopsies, performed with diagnostic intent, in patients with histological diagnosis of NSCLC. In our study, the incidence of p53 positivity in NSCLC was 47.34%, and it was 42.1% for Ki67 positivity. Furthermore, utilizing these small biopsy lung specimens we compared p53 and Ki67 positivity with survival rate. There are few studies describing p53 overexpression in samples obtained in a non surgical setting and it relationship to survival rate ,22-24. MThe use of Ki67 immunostainining in lung cancer is not easy to apply and the feasibility of Ki67 in specimens of small size like the bronchial biopsies is still controversial.Scagliottiet all.  firstly Simultaneous expression of p53 and Ki67 was studied as a prognostic factor in resected lung cancer . SurprisWe have shown the utility of p53 and Ki67 immunostaining of bronchial biopsies in NSCLC. Our data indicate that p53 and Ki67 immunostaining of small size specimens obtained during diagnostic procedures with fiberoptic bronchoscopy may be useful in predicting prognosis in patients with NSCLC. The incidence of immunohistochemical positivity in NSCLC was approximately the same as reported in other studies, and, furthermore, no histologically negative samples was stained, thus indicating the validity of our method. However, these results were obtained in a relatively small number of patients, such as we were not able to predict a relationship between positive immunostaining and resistance to chemotherapy.We believe that a simple, inexpensive method, as that reported in the present study should be useful in predicting prognosis of patients with NSCLC. More studies are needed to understand the possible relationship of positive stain for p53 and Ki67 with the resistance to chemotherapy.The authors declare that they have no competing interest.


FK506 binding protein 51 (FKBP51) is an immunophilin physiologically expressed in lymphocytes. Very recently, aberrant expression of this protein was found in melanoma; FKBP51 expression correlates with melanoma aggressiveness and is maximal in metastatic lesions. FKBP51 promotes NF-κB activation and is involved in the resistance to genotoxic agents, including anthracyclines and ionizing radiation. FKBP51 is a cochaperone with peptidyl-prolyl isomerase activity that regulates several biological processes through protein-protein interaction. There is increasing evidence that FKBP51 hyperexpression is associated with cancer and this protein has a relevant role in sustaining cell growth, malignancy, and resistance to therapy. There is also evidence that FKBP ligands are potent anticancer agents, in addition to their immunosuppressant activity. In particular, rapamycin and its analogs have shown antitumor activity across a variety of human cancers in clinical trials. Although, classically, rapamycin actions are ascribed to inhibition of mTOR, recent studies indicate FKBP51 is also an important molecular determinant of the drug’s anticancer activity. The aim of this article is to review the functions of FKBP51, especially in view of the recent findings that this protein is a potential oncogene when deregulated and a candidate target for signaling therapies against cancer.  The deseceptors . The N-teceptors . The aimFKBP51 is capable of immunosuppression, mediated by calcineurin (CaN) inhibition, when complexed to FK506 . CaN is FKBP51 interacts with glucocorticoid, androgen, estrogen, progestin and mineralocorticoid receptors -20. The FKBP51 protects cells against oxidative stress . Very reIn the nervous system, FKBP51 regulates clearance of microtubule-associated protein tau and stabilizes microtubules . AbnormaThe FKBP51 homolog in Arabidopsis, PAS-1, plays a critical role in the growth and development of this organism . In mammThe concept that FKBP51 is an essential factor for cell proliferation is also supported by studies on myeloproliferative disorders -35. FKBPFKBP51 controls activation of NF-κB transcription factor that is involved in carcinogenesis, metastasis and resistance to anticancer therapy . FKBP51 An additional mechanism that indirectly implicate FKBP51 in the regulation of NF-κB activation is through the inhibition of steroids response. Glucocorticoids are potent inhibitors of NF-κB activation, mediated by enhanced transcription of IκBα, that traps NF-κB in inactive cytoplasmic complexes . In addiIn the canonical pathway of NF-κB activation, IKKα forms with IKKβ and a regulatory subunit IKKγ or NEMO the IKK kinase complex, that mediates the phosphorylation of IκB proteins in response to various stimuli . PhosphoThe essential role of FKBP51 in the activation of NF-κB and NF-κB-regulated genes has been demonstrated, both in normal  and tumoet al. low levels of FKBP51 resulted in increased Akt phosphorylation and decreased chemosensitivity [et al. found that insulin increased binding of FKBP51 to pAkt (S473) in non-small cell lung cancer cells, A549, and that, in the presence of atorvastatin, the levels of pAkt bound to FKBP51 decreased in 5 min [et al. are not in concert with the notion that FKBP51 is an immunophilin capable of CaN inhibition [The Phosphatidylinositol 3-kinase (PI 3-kinase)/Protein kinase B (PkB or Akt) is an important pro-survival signaling pathway that is often deregulated in cancer. Recently, it has been found, in tumor cell lines, that FKBP51 acted as a scaffold to facilitate the interaction between Akt and PH domain leucine-rich repeat protein phosphatase (PHLPP), which mediates dephosphorylation of pAkt at S473 . Accordisitivity . On the in 5 min . Althougin 5 min . The finhibition ,53. AccoIperexpression of FKBP51 has been documented in several human cancers. Enhanced FKBP51 expression is associated with apoptosis resistance and enhanced proliferation in gliomas . A studyIncreased levels of FKBP51 are also associated with tumor aggressiveness in melanoma ,49. A stIn prostate cancer, FKBP51 is part of a superchaperone complex that includes androgen receptor (AR) and androgen . Depletiet al. [An immunohistochemistry study of expression of FKBP51 in 50 tumoral samples including breast, lung, pancreas, ovary, and prostate (10 samples for each tumor), and a comparable number of normal tissue samples showed an intense signal in 38 out of 50 tumors analyzed, whereas normal tissues of the same histotypes showed a weak/absent immunohistochemical signal [et al. . All proThe first evidence supporting the involvement of FKBP51 in the resistance to cancer therapies came from mechanistic studies of the antitumor activity of rapamycin ,9. RapamIn a study conducted in melanoma, a tumor in which PTEN is often mutated , rapamycThe role of FKBP51 in the resistance of tumors to cell death has been extensively studied in melanoma ,49. In tFKBP51 is a protein with a progressively emerging role in cancer biology. Although its function is still far from being fully elucidated, there are clear data suggesting this immunophilin plays an active role in cell proliferation in both the physiologic conditions of cell growth and differentiation -31 and iFinally, increased FKBP51 seems to be a common denominator between depression and cancer . In the


Pyramidal cells are characterized by markedly different sized dendritic trees, branching patterns, and spine density across the cortical mantle. Moreover, pyramidal cells have been shown to differ in structure among homologous cortical areas in different species; however, most of these studies have been conducted in primates. Whilst pyramidal cells have been quantified in a few cortical areas in some other species there are, as yet, no uniform comparative data on pyramidal cell structure in a homologous cortical area among species in different Orders. Here we studied layer III pyramidal cells in V1 of three species of rodents, the greater cane rat, highveld gerbil, and four-striped mouse, by the same methodology used to sample data from layer III pyramidal cells in primates. The data reveal markedly different trends between rodents and primates: there is an appreciable increase in the size, branching complexity, and number of spines in the dendritic trees of pyramidal cells with increasing size of V1 in the brain in rodents, whereas there is relatively little difference in primates. Moreover, pyramidal cells in rodents are larger, more branched and more spinous than those in primates. For example, the dendritic trees of pyramidal cells in V1 of the adult cane rat are nearly three times larger, and have more than 10 times the number of spines in their basal dendritic trees, than those in V1 of the adult macaque , which has a V1 40 times the size that of the cane rat. It remains to be determined to what extent these differences may result from development or reflect evolutionary and/or processing specializations. Studies in primates have revealed marked differences in pyramidal cell structure among different cortical areas , the bushveld gerbil (Tatera branstii), and the four-striped mouse (Rhabdomys pumilio) were included for study. The rodents used in the present study were caught from wild populations in South Africa with permission and supervision from the appropriate wildlife directorates. All animals  were treated and used according to the guidelines of the University of the Witwatersrand Animal Ethics Committee, which parallel those of the NIH for the care and use of animals in scientific experimentation. Although the ages of the animals is not known, the body mass and physical development indicated that they were sexually mature adults. Animals were sedated with an I.M. injection of ketamine hydrochloride (40 mg/kg) and xylazine hydrochloride (4 mg/kg) and overdosed by I.P. injection of sodium pentobarbital (100 mg/kg).Three African rodent species, including the greater cane rat  for approximately 10 min at room temperature. These sections were then mounted into a perspex chamber on a fixed stage fluorescence microscope (Zeiss Axioskop II Plus) and individual cells were injected with Lucifer Yellow .Methodology used in the present investigation was exactly the same as used in our previous cell injection studies. Animals were perfused transcardially with phosphate buffered saline  then paraformaldehyde (4% in PB). The brains were removed and the right hemispheres were flat-mounted  in stock solution  for 5 days at room temperature. 3,3′-diaminobenzidine  was used as the chromogen Figure . MoreovePyramidal cells were drawn with the aid of a Zeiss Axioskop 40 equipped with a camera lucida. Dendritic tree size  and somal size were determined in 2-dimensions with the aid of NIH-Image software   was larger than that in the bushveld gerbil (14.63 mm2), which was larger than that in the four-striped mouse (11.58 mm2).The primary visual area was readily identified in “flat-mounts” prepared from the cortical hemispheres and processed for cytochrome oxidase Figure . The absn = 108, mean ± SD: 123.15 ± 24.90 × 103 mm2) were larger than those in the bushveld gerbil  which, in turn, were larger than those in the four-striped mouse Figure n = 62, 38.55 ± 12.80 × 103 mm2; Figure F(2) = 393.52, p < 0.001]. Post-hoc Scheffe tests revealed significant differences among all three species (p < 0.001).The basal dendritic trees of layer III pyramidal cells in the greater cane rat  = 167.66, p < 0.001]. Post-hoc Scheffe tests revealed all species comparisons to be significantly different.Plots of the results of Sholl analysis in which we counted the number of dendritic intersections in successive concentric circles with radii of 25 μm increments revealed that the peak branching density in the basal dendritic trees of layer III pyramidal cells in the greater cane rat (mean ± SE: 21.99 ± 5.77) was greater than that in the bushveld gerbil 17.43 ± 4.12), which, in turn, was greater than that in the four-striped mouse  was less than that in the bushveld gerbil , which was greater than that in the four-striped mouse  = 10.18, p = 0.001]. Post-hoc Scheffe tests revealed all species comparisons to be significantly different.A total of 6484 spines were drawn and tallied. As reported previously  was greater than that in the bushveld gerbil (4316), which, in turn, was greater than that in the four-striped mouse (2141).SD: n = 108, 170.71 ± 19.84 mm2) were larger than those in the bushveld gerbil at , which, in turn, were larger than those in the four-striped mouse Figure n = 62, 107.07 ± 15.86 mm2; Figure F(2) = 239.75, p < 0.001]. Post-hoc Scheffe tests revealed significant differences among all three species (p < 0.001).The cell bodies of layer III pyramidal cells in the greater cane rat  the absolute size of the cerebral cortex occupied by V1 differs up to 4-fold among the three species; (2) there are differences in the size, branching structure, spine density, and total number of spines in the basal dendritic trees of layer III pyramidal cells in V1; (3) the spine density may vary independently of the size and branching structure of the dendritic trees among species; and (4) there is a trend between the size and number of spines in the dendritic trees of pyramidal cells and the size of V1 such that the larger V1 the larger and more spinous are the cells.Comparison of these data obtained from African rodents with those sampled by the same methodology from V1 of other species such as the South American rodent cutia  and approximately twice as spinous (7903 spines) as the largest, most spiny cells observed in the macaque during development (53.93 × 103 μm2 and 3900 spines, Elston et al., Developmental studies in the macaque monkey reveal that the dendritic trees of layer III pyramidal cells at birth are larger than those observed in the adult four-striped mouse and bushveld gerbil, but then become smaller during maturation into adulthood (Elston et al., The difference in the relationship in rodents and primates between the size of pyramidal cells and the size of V1 could hardly be more dramatic Figure . StatistPreviously it has been suggested that the dendritic trees of pyramidal cells may be larger and more branched, may be smaller and less branched, or be the same, in species with increasingly larger cerebral cortex (see Figure 37 of Elston, One possibility is that the data reflect fundamentally different evolutionary principles in rodents and primates (see Gould, As each spine receives at least one excitatory input (DeFelipe et al., The observed differences in pyramidal cell structure detailed here between primates and rodents may also reflect a difference in the processing of visual information in V1 of the different species. For example, the tangential area occupied by the “average” dendritic tree of layer III pyramidal cells in V1 of the adult cane rat is nearly three times the size of that in the adult macaque monkey Figure . DiffereFurthermore, in primates it is well known that several, 35 or more, topographically organized visual cortex areas are present (Felleman and Van Essen, The present physiological data suggests that single unit responses to visual stimuli in rodents are not that different to those in primates (Tiao and Blakemore, Pyramidal cell structure in rodent V1 differs considerably to that in primates. In rodents, pyramidal cells are progressively larger, more branched and more spinous in species with progressively larger brains. In primates, there is relatively little difference in the size, branching structure, and number of spines in pyramidal cells in species with progressively larger brains. The two distinct trends observed in rodents and primates cannot reasonably be attributed a single scaling rule or allometric equation, but appear to reflect fundamentally different modes of cortical organization. As more data becomes available it is becoming increasingly clearer that not only should principles of cortical microcircuitry not be generalized across cortical areas within a given species, but they should not be generalized across species for a given cortical area.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.


As a transcription factor, p53 responds to a variety of stresses to either induce apoptosis (cell death) or cell cycle arrest (cell preservation) to suppress tumor development. Yet, the effect p53 has on the non-cancer aspects of aging is complicated and not well understood. On one side, p53 could induce cellular senescence or apoptosis to suppress cancer but as an unintended consequence enhance the aging process especially if these responses diminish stem and progenitor cell populations. But on the flip side, p53 could reduce growth and growth-related stress to enable cell survival and ultimately delay the aging process. A better understanding of diverse functions of p53 is essential to elucidate its influences on the aging process and the possibility of targeting p53 or p53 transcriptional targets to treat cancer and ameliorate general aging. Caenorhabditiselegans p53 gene (Cep-1) because it increases lifespan ; heterozygous p53+/− mice also succumb to cancer before control mice but at a later age than p53−/− mice ; they are produced by different promoter usage, alternative splicing, and alternative translation initiation sites ,40. Thespromoter . Such dipromoter ,44. ThusIsoforms of p53 with N-terminal truncations could conceivably enhance aging since their overexpression reduced cancer but accelerated aging in mice ,46. ThesMutations in p53 are found in half of all human cancers, and other parts of the p53 pathway are altered in many others. These mutations confer a selective advantage on the tumor cells, allowing them to evade cell cycle checkpoints, avoid apoptosis and senescence, and proliferate under conditions where normal cells cannot. Most tumor-associated p53 mutations are within the DNA-binding domain. These affect the ability of p53 to bind to target genes to varying degrees, and hence may alter the overall protein conformation. Most can still tetramerize with wild-type p53 and exert a dominant negative effect. Many engineered mutations in p53 that affect its regulation and function have been tested in cell culture and in mice. Although studies of function must generally start by utilizing tissue culture systems, the use of mouse models is important to interrogate the complex processes of tumorigenesis.It would be difficult to identify analogous mutations that affect aging, because they would presumably not be selected in the same manner. However, a polymorphism recently identified in human populations suggests that subtle changes to the p53 protein can also affect longevity and aging. A simple substitution of a proline for an arginine at codon 72 hinders the ability of p53 to induce apoptosis and leads to an increased cancer risk. However, this polymorphism was enriched in the older population and was associated with longevity, even though some had died from cancer –15. We pA number of studies have shown some benefit in targeting endogenous wild-type p53 or mutant p53 in order to enhance its function and slow tumor growth. The most promising approach involves drugs able to reactivate unfolded or mutant p53 (as well as the related p63 and p73 proteins in some cases). Because of its diverse activities and profound biological outcomes, p53 is tightly regulated by multiple mechanisms that control activity, stability, and localization . As a coMDM2 (murine double minute 2) and MDM4 (a.k.a. MDMX) regulate p53 and their overexpression effectively negates functional p53 to enhance cancer risk. MDM2 is a ubiquitin ligase that leads to degradation of p53. When MDM2 levels are high, p53 becomes polyubiquitinated and targeted for degradation by the proteasome. MDM2 also directly represses p53 activity. The MDM2 gene is also a target for p53, setting up a negative feedback loop. When p53 is activated, MDM2 levels increase, which then turns down p53. MDM4 is not a ubiquitin ligase but enhances p53 ubiquitination in a heterocomplex with MDM2. MDM4 also regulates p53-mediated transcription. MDM2 and MDM4 are not redundant. Deletion of either protein causes embryonic lethality in mice, and p53 deletion rescues either mutation –34. Apopcis-imidazoline analogue Nutlin-3 disrupts the p53–MDM2 interaction to enhance p53 function and promises to be an alternative to chemotherapy. Nutlin-3 also shows a synergistic effect in combination with certain therapeutics, such as TRAIL or bortozemib  showed enhanced p53Δp-mediated suppression of oncogene-induced tumors after MDM4 loss, supporting MDM4 as an anti-cancer target  situation; p53 activity is likely to be regulated in a graded manner, and different activities of p53 are undoubtedly regulated differentially by different modifications. These modifications can profoundly affect the p53 activity in tumor suppression, and are likely to affect the effects of p53 on aging as well.The p53 protein has multiple Ser/Thr residues that serve as phosphorylation sites for a number of protein kinases. The phosphorylation state of many of these sites changes upon stress signaling. Many important regulatory sites are concentrated in the transactivation domain at the N-terminus, with some also present in the C-terminal regulatory domain. Some sites can be phosphorylated by multiple kinases and some kinases can phosphorylate more than one site. Two important sites are Ser 15 (Ser 18 in mouse p53) and Ser 20 (Ser 23 in mouse). These sites become phosphorylated by ATM and other kinases in response to DNA damage. When these serines are phosphorylated, the interaction with the negative regulator MDM2 is weakened, stabilizing p53 and allowing it to interact with coactivators and activate transcription. Mutant mice with a Ser to Ala mutation at these sites (cannot be phosphorylated) show increased p53 stability and transactivation. However, the phenotype is tissue specific and not as severe as would be predicted from the effects in tissue culture experiments, suggesting that phosphorylation is not the only means of controlling p53. Interestingly, mice generated by a knock-in of the Ser 18 (Ser to Ala) mutation show accelerated aging, suggesting that active p53 in response to DNA damage protects against aging . A knockAcetylation is a major mechanism for regulating p53 activity. Multiple lysine residues in p53 are acetylated; many of these same lysines are also targets for ubiquitination by MDM2. Therefore, acetylation of these lysine residues prevents their ubiquitination, stabilizing p53. In addition, acetylation can inhibit the interaction of MDM2 with p53. Finally, acetylation recruits cofactors allowing p53 to activate its transcriptional targets. Acetylases that are responsible for modification of p53 include p300, CBP, PCAF, TIP60, and hMOF . In tissRecent work showed that manipulation of p53 acetylation in mice could separate the classic p53 functions of cell cycle arrest, induction of apoptosis, and senescence from the anti-cancer activity of p53 . In thisThere are other ways p53 can be modified. p53 can be methylated on lysines and arginines by several different methylase enzymes, which can either activate or repress its activity depending on which site is methylated and the number of methyl groups added . p53 canIt is clear that p53 protein activity can be modulated by several types of posttranslational modification, and that the overall activity of a given p53 protein molecule will be determined in a complex manner by the balance between different amounts and combinations of modifications at any given time. Some of the modifications are likely to affect other modifications, particularly those that target the same p53 amino acid residues. Affecting p53 activity by altering amounts, timing, and specificity of protein modification represents a promising avenue to affect the aging process.Ribosome biogenesis refers to the production and processing of ribosomal RNA. It is a complex process that occurs in the nucleolus of the cell. RNA polymerase I (Pol I) transcribes tandem arrays of ribosomal DNA (rDNA) (about 400 repeats in humans) in the nucleolar organizer region (NOR) to delineate a nucleolus . This prInterestingly, ribosome biogenesis serves as a sensor of cellular stress and defects in ribosome biogenesis activate p53 . FurtherInhibitors of ribosome biogenesis (genotoxic or non-genotoxic agents) that induce a p53 response could potentially function as potential anti-cancer agents . DNA dam1-arrested cells to transit from quiescence (where they still retain the ability to proliferate) to senescence. However, p53 might not induce cellular senescence beyond G1 arrest, so its role in senescence induction is unclear -related family –92 and iRapamycin has been shown to increase lifespan for heterogeneous outbred mice ,98 and fGlucose is the major source of energy for ATP generation and glycolysis is an ancient metabolic pathway that converts glucose to pyruvate to produce ATP and NADH. Pyruvate then enters the tricarboxylic acid (TCA) cycle to generate ATP through OXPHOS. Contrary to normal cells, cancer cells primarily generate energy (ATP) through aerobic glycolysis to increase anabolism and perpetuate tumor growth, known as the Warburg effect . In addic oxidase 2 (SCO2)  ,113 and 2 (SCO2) ,115 thatScutellaria baicalensis Georgi that induces apoptosis in HeLa cells  induce AMPK and as a result induce p53. Metformin also inhibits mTORC1 via AMPK . Metform0 or G1. Thus, Ap53−/− mice is increased by this intervention  is a well-characterized intervention to extend lifespan and ameliorate physiological aging across species, including mammals . CR causrvention . However+/− mice . It rema1 arrest in response to damage or cellular stress to protect the cell, but does not induce cellular senescence directly. Instead pro-growth pathways may enable an arrested cell to enter a senescent state. Recent data that show p53 allows cells to survive serine depletion highlight a protective role (p53 connects cell metabolism and DNA integrity to various cellular outcomes including cell cycle arrest, cellular senescence, and cell death. Although the role of p53 in tumor suppression has been well studied, recent evidence suggests that p53 also affects aging. There are multiple ways to target p53 as an anti-cancer therapeutic . Howeverive role . These o


PADI4 gene to form a susceptibility haplotype for rheumatoid arthritis (RA); nevertheless, results in association studies appear contradictory in different populations. The aim of the study was to analyze if the presence of three SNPs in PADI4 gene susceptibility haplotype (GTG) is associated with ACPA positivity in patients with RA. This was a cross-sectional study that included 86 RA patients and 98 healthy controls. Polymorphisms PADI4_89, PADI4_90, and PADI4_92 in the PADI4 gene were genotyped. The susceptibility haplotype (GTG) was more frequent in RA patients; interestingly, we found a new haplotype associated with RA with a higher frequency (GTC). There were no associations between polymorphisms and high scores in Spanish HAQ-DI and DAS-28, but we did find an association between RARBIS index and PADI4_89, PADI4_90 polymorphisms. We could not confirm an association between susceptibility haplotype presence and ACPA positivity. Further evidence about proteomic expression of this gene will determine its participation in antigenic generation and autoimmunity.Peptidyl arginine deiminase IV (PAD 4) is the responsible enzyme for a posttranslational modification called citrullination, originating the antigenic determinant recognized by anti-cyclic citrullinated peptide antibodies (ACPA). Four SNPs (single nucleotide polymorphisms) have been described in  Rheumatoid arthritis (RA) is an autoimmune disease, characterized by articular inflammation which can lead to joint destruction. RA prevalence is 1% worldwide with considerable variation between ethnic groups, with a higher prevalence in Caucasians compared with Asiatic populations . This diloci for RA located within several chromosomes, one consistently implicated is the HLA-DRB1 gene , 28PADI4The present study represents the first one in Mexican population which is the result of genetic admixture of Spanish, Indian, and Black populations, and it should be emphasized that the genetic background introduced from Spaniards includes genes from Romans, Greeks, Visigods, Arabs, and Jews . We shouPADI4 polymorphisms are associated with RA susceptibility, regardless of ACPA titers. This is consistent with results published by Hoppe and Kang et al. in German [PADI4 gene polymorphisms could have a higher susceptibility role in Asiatic populations compared to Caucasians, but soon after that, an association between PADI4 gene and RA was confirmed in a French population [We found that n German  and Koren German  populatin German , since Pn German  concludepulation . The lacP = 0.0002), but more interestingly, we found a new haplotype (GTC) which was both significantly associated (P = 0.006) and more frequent than previous susceptibility haplotype. We believe that since this second haplotype was present in a higher frequency among RA patients, it should be looked forward.When we analyzed haplotype frequencies, we found that the susceptibility haplotype (GTG) was significantly associated with RA  positivity was present in 74%, similar to previous reports , 49, 50.i et al. . We did i et al.  and seemi et al. . For thii et al. , but thei et al. . As a coPADI4 translation, as the increase in mRNA stability has been demonstrated, generating an increase in protein levels and as a consequence a higher occurrence of citrullinated proteins [Supporting the first theory is the fact that susceptibility haplotype presence can affect proteins . CitrullPADI4 gene polymorphisms and clinical variables, except for high punctuation in RARBIS index and presence of susceptibility allele (G) in PADI4_89 or susceptibility homozygous (T/T) in PADI4_90 of PADI4 gene.Finally, we did not find associations between Limitations in this study include sample size since only one gender is represented in the present investigation; it is well known that RA is a condition that affects mainly women, but it would be desirable to increase sample size in order to be able to include male patients and to verify if association with the susceptibility haplotype remains constant, since in our study we were able to find only one HC with the susceptibility haplotype.In conclusion, we confirm the association between the susceptibility haplotype (GTG) with RA patients, observing also that a new haplotype (GTC) could be associated with RA in Mexican mestizo patients. Further studies including other regions in Mexico and increasing the sample size are required in order to confirm our findings; furthermore, the biggest challenge for association studies is the identification of genetic variants that, combined in haplotypes, can be described as causal effects to RA.