Restoration of p53 activity in mouse models leads to the suppression of established tumors of different origin. These findings provide a strong support to the anti-cancer strategy aimed for p53 reactivation. In this review, we summarize recent progress in the development of small molecules, which restore the tumor suppressor function of wild-type p53 and discuss their clinical advance. We discuss different aspects of p53-mediated response, which contribute to suppression of tumors, including non-canonical p53 activities, such as regulation of immune response. While targeting p53 inhibitors is a very promising approach, there are certain limitations and concerns that the intensive research and clinical evaluation of compounds will hopefully help to overcome. Since p53 discovery 40 years ago, it has been established that p53 is a transcriptional factor, which binds to the promoters of its target genes in a sequence-specific manner and regulates their expression, thereby controlling cell cycle and cell death. In unstressed cells, p53 has low activity; however, upon its activation by oncogenes, DNA damage, and other types of stresses, p53 blocks the proliferation of pre-malignant and malignant cells or eliminates them by inducing apoptosis  of hundreds of human cancers revealed an almost indefinite number of combinations of mutations, chromosomal aberrations, copy number changes, and epigenetic alterations. However, these very diverse cancer lesions converge on a few key pathways.To successfully fight cancer, we need to focus on these most crucial pathways and find the best targets within these. To identify the best targets, we should apply the following criteria: the factor is a critical player in essential pathways affected in many cancers; it is non-redundant and is involved in different aspects of tumor development; targeting this factor results in elimination of tumor cells, but does not kill normal cells.p53 fits these criteria very well; inactivation of the p53 tumor suppressor function is required for the development and maintenance of most human cancers. Importantly, p53 is negatively controlling most of the hallmarks of cancer: deregulated proliferation and cell death, replicative immortality, angiogenesis, invasion and metastasis, metabolism and genomic instability, as well as immune response . p53 is Due to the unique mode of p53 inactivation in cancer, restoration of p53 appears to be feasible. In contrast to other tumor suppressors such as Rb, p16, or PTEN, the p53 protein is usually expressed in tumors, although its function is ablated. However, the fact that p53 is a transcriptional factor have made the idea of p53 reactivation unpopular in the past, since transcriptional factors were deemed ‘undruggable’ until very recently. Latest advances have proven these views wrong and made it possible to develop different strategies for the restoration of p53 activity, depending on the type of p53 inactivation. Reactivation of mutant p53 protein by stabilizing its folding with small molecules appears to be a promising strategy, i.e. development of small molecule PRIMA-1MET/APR246 , which iMDM4 gene). MDM2 regulates p53 via different mechanisms. Due to its E3 ligase activity, MDM2 can either monoubiquitinate p53, promoting its nuclear export, or polyubiquitinate, inducing p53 degradation by the proteasome , or the absence of negative regulators  . A singlin vitro and in vivo  are another class of high-affinity compounds-antagonists of MDM2, designed specifically to mimic the three key hydrophobic residues  of p53, making contacts within the MDM2 pocket. MI-63 and MI-147 induce cell growth arrest in several cell lines due to p53 activation . Moreoveinotecan , while Minotecan . MI-773 inotecan . Howeverinotecan .De novo design, based on the binding mode of previously known MDM2 inhibitors, resulted in the development of a novel scaffold for inhibiting MDM2. Piperidinones AM-8553  induced apoptosis in osteosarcoma SJSA-1 cells overexpressing MDM2 by reactivating the p53 signalling pathway , a steroid fatty acid present in bile, as MDMX inhibitor with higher preference over MDM2. It induces apoptosis in HCT116 human cells, predominantly in p53-dependent manner. However, the induction of apoptosis was achieved only at high concentrations of LCA, limiting its use in clinics  . DHODH iin vivo .Compounds that prevent the interaction of human papilloma virus (HPV) oncogene E6 with p53, such as leptomycin B (LMB) and RITA, can be applied in HPV-positive cancers where proteasomal degradation of p53 by E6 is critical for the survival of cancer cells. LMB blocks nuclear export by inhibition of the export protein CRM1 , while RUpstream regulators of MDM2/X are attractive targets for the design of p53-reactivating compounds. For example, it has been shown that upon nucleolar stress, ribosomal proteins  are released from nucleoli and bind to MDM2, triggering p53 activation . SeveralκB without causing detectable genotoxicity. The intercalation of curaxins into DNA cause the ‘chromatin trapping’ of the facilitates chromatin transcription (FACT) complex, which in turn leads to phosphorylation of the p53 Ser392 by casein kinase 2 and inhibition of NF-κB-dependent transcription. Importantly, curaxins suppressed tumor growth in different types of human tumor xenografts grown in mice and are currently being tested in clinical trials  plays a critical role counteracting p53 and MDM2 degradation, making it an interesting target for the development of inhibitors . AlthougTP53 with elevated FLT3 and MDM2 expression are most sensitive to the combined treatment with cobimetinib and idasanutlin  models and ignature . A gene ignature . The preThe development of biomarkers and companion diagnostics is ongoing; it is crucial for the success of p53-based therapies. Clinical data from patients involved in trials would hopefully pave a way for the discovery of reliable biomarkers for p53-based therapies.A number of MDM2 inhibitors are being tested in solid tumors with promising preliminary results. Two clinical trials have been conducted and completed for the small molecule MI-773 in patients with advanced solid tumors. The first study was designed to evaluate the safety and the maximum tolerated dose, as well as pharmacokinetics, biomarkers, and biological effects in solid tumors with no other treatment available, as well as lymphomas. Recently published results from the completed study revealed an accepted safety but, although p53 pathway was activated, it displayed a limited activity as a single agent . TherefoRibosomal protein activators such as actinomycin D and roscovitine also have been or are being tested in several Phase I, II, or III clinical trials, either as monotherapy or in combination therapy with other drugs in several type of cancers .de novo resistance and increase the overall survival in melanoma patients  as the most common adverse effect . The datBesides the effects of p53 reactivation in normal cells, another concern of targeting negative regulators of p53 is the inhibition of their cellular functions other than p53 regulation. Both MDM2 and MDMX have several p53-independent functions, including gene expression regulation and chromatin modification , DNA repARF, amplification or overexpression of MDM2 positive regulators HAUSP, Wip1, and others , the possible selection of p53 somatic mutations in normal cells such as hematopoietic progenitors may lead to the development of new cancers and therefore should be carefully investigated. Systematic search for the mechanisms of resistance to MDM2 inhibitors using piggyBac transposon insertional mutagenesis in spontaneous tumors in p19ARE−/− mice revealed several mechanisms of resistance. More than half of tumors acquired inactivating mutations in p53 (54%), while others obtained the gain-of-function alterations resulting in high expression of anti-apoptotic protein Bcl-xL, MDMX, and ΔNTrp63 or ΔNTrp73, which confer a dominant-negative effect on p53  will be beneficial to combine with p53-reactivating drugs. However, synergistic or synthetic lethality drug interactions remain a largely unexplored area


A meta-analysis (n = 5) on total energy intake found no significant differences between groups. The BCTs with the highest effectiveness ratio were habit formation (100%), salience of consequences (83%) and adding objects to the environment (70%). The review highlights the potential of behavioural interventions to improve young adults’ fruit and vegetable intake but was less convincing for other dietary outcomes. Due to the lack of studies including each BCT, the BCTs imperative to success could not be identified.Poor eating habits are common during young adulthood and influence chronic disease morbidity. This systematic review evaluates the effectiveness of interventions aiming to improve dietary intake among young adults and, identifies which behaviour change techniques (BCTs) are most effective. Six electronic databases were searched for RCTs published until October 2018, and evaluating behavioural interventions assessing change in dietary intake in young adults (17–35 years). Of the 18,779 articles identified, 54 were included. Forty studies focused on fruit and/or vegetable intake, of which 63% showed a significant between-group difference in favour of the intervention group. Meta-analysis ( Globally, young adults have some of the unhealthiest eating habits compared to other age groups. In a systematic assessment of diet quality among adults in 187 countries, those aged 20–29 years had the lowest diet quality score  compared to any other age group ½ and the sample size was as reported for the study. If SDs rather than SEs were reported, the SDs were used in place of the SEs and n1, n2 was set at 1. For studies reporting intake of fruit and vegetables separately, we converted the results as follows; the means for fruit and for vegetable were summed to give a total fruit and vegetable mean. For the standard deviation (SD) and sample size, a conservative approach was taken assuming the Standard Error’s (SE) for each mean were different. SD combined score was determined as [n1.SE1For each study, the effect at baseline and each time point, expressed as months post baseline, was estimated as the mean difference (mean intervention group − mean control group) using the unbiased option for variance estimates. Uncertainty in the mean estimates was expressed as 95% confidence intervals. To account for multiple measures per study a series of multilevel models were investigated with nested random effects top level being study, then treatment type and time period using REML estimation. The final model contained study and time as nested random effects as the treatment type random effect had zero variance. Moderator variables examined as fixed effects in the model were treatment type (simplified into three groups for fruit and vegetables and two groups for TEI), and time was treated as a categorical variable in two versions  and a simplified grouped form with baseline, up to three months and greater than three months. The interaction between these two was tested. As the time random effect was similar in size to the study random effect there was substantial correlation between time periods, so the moderator effect for the three-group version of time was estimated at each of the two follow-up time periods as difference from baseline. Residual plots were used to examine homogeneity of variance and normality assumptions. To assess for publication bias, funnel plots were produced and visually inspected and supported with rank correlation tests for funnel plot asymmetry.In parallel with similar reviews ,30, we cOf the 18,779 manuscripts identified, a total of 54 individual studies  were included in the review . Study cn = 29, 54%). In addition, most studies were from the US , in a college/university setting  and predominantly in white/Caucasian populations . The included studies had a total of 16,383 participants  and the mean age of participants was 20.2 years with 57% (n = 31) within the age category of 17–≤ 25 years. Most studies included both male and female samples , and of these the average proportion of males was 32%. Over one-third of the interventions were multicomponent , and there was an equal proportion of studies which solely used either ehealth or face-to-face as the intervention delivery . Over half of the included studies had dietary change as a primary outcome , while dietary change was a secondary outcome for the remaining 22 (40.7%) studies. Studies where dietary change was a secondary outcome primarily focused on obesity/ weight gain prevention , obesity treatment  or PA . In the studies with dietary change as a primary outcome, most focused on fruit and/or vegetable intake , while the remaining focussed on diet quality score , micronutrients  or percent of energy coming from fat . Interventions to improve eating habits among young adults has escalated in recent years, with over half of the included studies published from 2014 to 2018 (Twenty-five 46%) studies used a food frequency questionnaire (FFQ) for assessment of dietary intake ,64,65,66% studiesn = 38, 70%). The mean intervention duration across the studies was 4.2 months (range: single session to 24-months), with the majority being short term between one single session and ≤3-months . The mean length of follow-up from end of intervention was 1.8 months (range: no follow-up to 23-months), with the majority having between no follow-up and ≤3-months . Finally, there was a mean retention rate of 78% (range 22% to 98%) at the end of the intervention and a mean retention rate of 66% (range 11% to 97%) at the longest follow-up point. The total number of study arms was 133 (range: 2 to 6 arms), with the majority being two-arm interventions  adequately described study attrition and any exclusions from the analysis. Almost half  of the included studies described suitable generation for the allocation sequence, yet most  failed to describe the method for allocation concealment. There was a high or unclear risk of bias for most studies for blinding of participants , blinding of those delivering the intervention  and blinding of outcome assessors . Most studies provided insufficient detail  to determine if they were free of selective outcome reporting.The risk of bias assessment is summarised in p = 0.014) with a reduction of 326 kcal/day in the intervention group (comprised of group-based face-to-face lecture and lab sessions) compared to +72 kcal/day in the usual-lifestyle control group. This effect was not significant at 16-months. When all studies were considered, there were a total of twelve studies which measured change in TEI (none as primary outcome), reported as either Calories or kilojoules per day ,77,79,83p < 0.790) showing a mean decrease in TEI relative to baseline −132.9 kJ/day up to 3 months  and −300.2 kJ/day for >3 months  (p = 0.4904). The funnel plot  in five studies ,50,51,52, 402.4) . There wnel plot  demonstrA total of 40 studies measured fruit and/or vegetable intakes ,78,79,81Fruit and vegetable intake were reported as (i) serves per day in 12 studies ,71,74,81There were 18 studies that reported fruit intake separately from vegetable intake ,77,78,792(5) = 16.96, p < 0.001) showing a mean increase in fruit and vegetable intake relative to baseline 68.6 g/day up to 3 months  and 65.8 g/day for >3 months  (2(2) = 0.42, p = 0.8091) nor an interaction between treatment type and time Wald χ2(4) = 0.318, p = 0.53). The funnel plot (p < 0.001. Removing the study by Chang (2010) with the two largest values in the funnel plot did not change the bias tau 0.30, p = 0.003. Plots of the means for the effects are in Meta-analyses of fruit and vegetables in 17 studies ,46,47,81, 104.0) . There wnel plot  demonstr 95% CI: .6, 100.5A total of 15 studies assessed the change in intakes of at least one nutrient dense food ,79,80,81p < 0.001) increased Diet Behaviour Score  when compared to usual lifestyle control group  at 1-month. Statistically significant improvements in diet quality score was evident in one study (out of five) . This stp < 0.001) increase in whole grain bread intake in the intervention group (+46g/day) compared to control (−12g/day) at 5-months. A significant between-group difference in individual food groups was evident in one study (out of five) . This nuehealth weight loss program  for young women on percentage energy from core foods found a significant improvement in the intervention group when compared to control  at six months. The one study which explored the effectiveness of an p = 0.049). Statistically significant improvements in specific micronutrient and dietary fibre rich foods were evident in two studies (out of seven) ,79. SpecEight studies assessed the change in energy-dense beverage intake ,67,69,76eHealth components  in young Australian adults for 12 weeks. The odds that the TXT2BFIT intervention group improved intake compared to the control group were significantly greater for SSBs at the end of the program (p = 0.024) and at 9-month follow-up (p = 0.018) [p = 0.03). The SSB intake decreased in the intervention group (−0.53 drinks/day) and increased in the control group (+0.50 drinks/day) [Two studies (out of seven) demonstrated a significant reduction in SSB intake when compared to control ,67. Specnks/day) .p = 0.001) [One study reported change in fruit juice intake and a small but significant between-group effect was evident (= 0.001) . AlthougA total of 12 studies assessed the change in intakes of at least one ED-NP food ,69,76,79ehealth  and face-to-face sessions. Those in the HEYMAN intervention had a reduction of 9.8%  in EDNP foods/day, compared to a reduction of 2.6%  in the control group. The ‘Be Positive Be Healthe’ weight loss program for young women was delivered via ehealth components . Those in the intervention group had a reduction of 8.3%  in EDNP/day, compared to an increase of 0.9%  in the control group. Two studies (out of three) demonstrated a significant reduction in percentage energy from ED-NP foods when compared to control ,57. Bothp = 0.013) and at 9-month follow-up (p = 0.010) [p = 0.007).Two studies (out of five) demonstrated a significant reduction in takeaway/fast food intake when compared to control ,67. The = 0.010) . Those aFour studies considered the effect of a healthy eating intervention on hot chips (serves/week) , sweets/One obesity treatment study explored the effect of an intervention consisting of a smartphone application combined with SMS from a health coach on sodium intake, added sugar intake and total sugar intake  in overwA total of 15 studies assessed change in macronutrient content  ,81,82,83Specifically 15 studies explored change in dietary fats ,81,82,83p < 0.01) and at 15-month follow-up  in percent energy from fat [p < 0.01) in percent energy from fat [eHealth intervention consisting of email and website to improve diet and physical activity had a significant linear time by treatment interaction for percent energy from saturated fat  at 12 weeks and 24 weeks [p = 0.02) [Four studies (out of 15) reported a significant intervention effect in dietary fat intake ,45,50,82from fat . Anotherp < 0.05), but this effect was not sustained at 16-month follow-up [One study (out of eight) reported a between-group decrease in total grams of carbohydrate at the end of the program at four months  but this effect was not maintained at 16-month follow-up [n = 6) which assessed change in protein intake did not establish any significant differences between groups.The same study which identified a change total grams of carbohydrate also reported a between-group decrease in total grams of protein at the end of the program at 4 months , ‘Goal setting behaviour‘(n = 32), ‘Information about health consequences’ (n = 29), ‘Action planning’ (n = 27) and ‘Social support (unspecified)’ (n = 24). n = 24) were included in the analysis [The percentage effectiveness ratio for the coded BCTs is displayed in analysis .Thirteen BCTs had an effectiveness ratio >50%. The BCTs with the highest effectiveness ratio were ‘Habit formation’ (effective in five out of five or 100% of interventions), ‘Salience of consequences’ (effective in five out of six or 83% of interventions), ‘Adding objects to the environment’ (effective in seven out of 10 or 70% of interventions), ‘Action planning’ (effective in 16 out of 27 or 59% of interventions) and ‘Prompts/cues’ (effective in seven out of 12 or 58% of interventions). p = 0.85, indicating that none of the BCTs were different to the overall mean value. Although ‘Habit formation’ had the highest percentage effectiveness ratio, many more studies are required before this could be confirmed as having greater effectiveness than others.An overall test of significance was carried out using a contingency table between type of BCT and effectiveness, there was no significant relationship, Monte–Carlo exact chi-squared test, χ2(23) = 16.7, p = 0.93. The percentage ratio of effective versus noneffective interventions by the number of BCTs was also explored . The resn = 54 RCTs) that has assessed the effects of interventions aiming to improving dietary intake outcomes in young adults. It is also the first to explore the effectiveness of BCTs for improving dietary intake among this group. Predominantly, studies targeted fruit and/or vegetable intake as the primary outcome and a meta-analysis (n = 17 studies) demonstrated intervention participants to significantly increase intake of fruit and vegetables by +68.6 g/day up to three months and +65.8 g/day for >3 months when compared to control. A meta-analysis exploring change in TEI (n = 5 studies) showed no significant differences between groups. The narrative synthesis showed that interventions targeting fruit and/or vegetable intake were most effective (significant between group differences evident in 25 out of 40 studies or 63%), followed by energy-dense beverages (three out of eight or 38%), nutrient dense foods (five out of 15 or 33%) and macronutrients (four out of 15 or 27%). The BCTs with the highest percentage effectiveness ratio were habit formation, salience of consequences and adding objects to the environment. Despite this, the overall test of significance demonstrated no significant relationship between type of BCT and effectiveness and many more studies are required before these BCTs could be confirmed as having greater impact on effectiveness than others.This is the largest systematic review to date (The modest increases in fruit and vegetable intake (mean difference of +68.6 g/day up to three months and +65.8 g/day for >3 months) is clinically important. In another meta-analysis among prospective cohort studies with 833,234 participants, the risk of all-cause mortality was decreased by 5% and cardiovascular disease by 4% for each additional serve a day of fruit and vegetables . This isWhen compared to other reviews in adults and children, the mean difference in fruit and vegetable intakes between intervention arms  is lower. A systematic review of 34 behaviour-based interventions found an average increase in fruit and vegetable intake of +1.13 and +0.39 serves per day in adults and children , while aAdditionally, the meta-analysis demonstrated no significant effect on TEI. Of the five studies in the meta-analysis, none had TEI change as a primary outcome, and only one study  aimed to+ ≈ 0.45). Previous work has suggested that habits are behavioural patterns that are learned through context-dependent repetition [This review compared the individual BCTs in effective versus noneffective interventions to identify which specific BCTs may be agents of change. Thirteen BCTs had an effectiveness ratio >50% and ‘habit formation’ had the highest percentage effectiveness ratio . An example of this technique in an intervention includes; prompting participants to fill half of their plate with either salad or cooked vegetables each night at dinner. These results correspond with another meta-analysis which found habits to be consistently correlated with physical activity and nutrition behaviour ; with a petition ,93, and petition .Focusing on the natural consequences of performing a behaviour was found to be effective; with ‘information about health consequences’ demonstrating a percentage effectiveness ratio of 52% (effective in 15 out of 29 studies). An example of this technique in an intervention includes providing participants with morbidity and mortality statistics from related health conditions. Emphasising the consequence by making it more memorable had even greater effectiveness with ‘salience of consequences’ demonstrating an effectiveness ratio of 83% (effective in five out of six studies). An example of this techniques used in an intervention includes; presenting visual images of the negative consequences of eating unhealthily, i.e., blocked arteries. These results are comparable to a review of 45 brief nutrition interventions in adults, whereby accentuating the consequence of a behaviour had greater effectiveness than just providing information about a health consequence . Also thp < 0.04) predicted long-term effect on diet and physical activity behaviours [Adding objects to the environment was a technique that was shown to be effective with a percentage effectiveness ratio of 70% (effective in seven out of 10 studies). An example of this in an intervention was to provide participants with a free fruit and vegetable box to facilitate healthy eating. The effectiveness of this technique may coincide with known financial barriers associated with this age group . These rhaviours .p < 0.001) and long term (p < 0.01) [Furthermore, setting goals (effective in 18 out of 32 studies or 56%) and planning actions (effective in 16 out of 27 studies or 59%) in terms of performing a target behaviour were found to be effective BCTs. Examples of these techniques in interventions were (i) participants set a goal of eating two pieces of fruit per day and (ii) participants were instructed to write plans of when and where they would consume an extra piece of vegetable per day, for the next week. The effectiveness of goal setting of behaviour is comparable to a meta-regression analyses among 48 interventions in overweight and obese adults, which showed that this particular BCT was independently associated with better intervention effects on diet and physical activity behaviours at short ( < 0.01) . Similar < 0.01) .The current review established no difference in number of BCT’s in relation to effectiveness on nutrition behaviours. This corresponds with several other systematic reviews in different population groups ,98. OverThe risk of bias assessment identified several strengths. Generally, studies provided adequate detail to describe study attrition, any exclusions from the analysis and suitable generation for the allocation sequence. Despite this, many studies either had high risk of bias or lacked the necessary detail to judge the quality for selective outcome reporting, allocation concealment and blinding of participants, personnel and/or outcome assessors. Additionally, publication bias was evident in the meta-analysis of fruit and vegetable intake. There was an over-representation of studies in primarily female populations, in Caucasian samples and within college/University settings, which may limit the generalisability. Studies primarily focused on a limited selection of food groups with most focusing on fruit and vegetable intake, but very few considered overall diet quality. Few studies measured long-term dietary outcomes; as such, there is limited evidence to determine the long-term effectiveness of dietary interventions in young adults. Coding of BCTs was problematic due to inconsistencies and differences in reporting of intervention components. A stringent coding strategy was applied, where BCTs were not coded as present if there was insufficient description. However, some of those BCTs coded as absent techniques may have been apparent but not adequately described. This issue has been identified in previous attempts to categorise intervention content , and theThe novelty of this review in being the first to explore which BCTs are associated with effective interventions in young adults is a strength, as is the use of a robust method to code the BCTs, using the most recent and comprehensive taxonomy of BCTs available . AlthougThis systematic review and meta-analysis highlights the potential of lifestyle behavioural interventions to improve young adults’ fruit and vegetable intake, but the evidence was less convincing for other dietary outcomes. More high quality studies are needed to determine longer-term effectiveness, and with consideration of overall diet quality. This is especially important due to the poor eating habits of young adults worldwide. This review also demonstrated the potential of the following BCTs; ‘habit formation’, ‘salience of consequences’ and ‘adding objects to the environment’ in positively changing dietary behaviour. However, due to the lack of studies, including each BCT, this review could not identify which BCTs are imperative to success and more studies are required before confirming which BCTs can be considered as having greater effectiveness than others. To better understand which BCTs are linked to intervention efficacy, future research must describe interventions in detail to allow for identification and replication of information, for example, publishing intervention protocol papers and/or including a checklist of exact BCTs implemented with supplementary material of each publication. Identifying and utilising effective BCTs for dietary interventions in young adults will aid the development of potentially more effective and replicable interventions, policies and nutritional guidelines, thus building a strong evidence base to support healthy eating habits among young adults.


The 2-year overall survival (OS) and locoregional control (LRC) rates of all patients were 62% and 71%, respectively. The 2-year OS and LRC rates in patients who had received EBRT and concurrent chemotherapy with docetaxel, cisplatin and 5-fluorouracil  were 100%. These results were higher than the 2-year OS and LRC rates of 62% and 69%, respectively, in patients who had received radical surgery and EBRT (n = 13). The rates were 0% in those who had neither received TPF nor undergone surgery in addition to EBRT (n = 2). Grade 3 bone or soft tissue necrosis was observed in 2 patients who had undergone surgery and postoperative EBRT. Our data suggest that the combination therapy of EBRT and surgery and/or chemotherapy may be the most effective treatment options for advanced EACC, and EBRT with concurrent chemotherapy with TPF is potentially the most acceptable.External auditory canal cancer (EACC) is a rare malignant tumor. In the present study, we retrospectively evaluated the treatment results in patients with advanced EACC who were treated using external-beam radiotherapy (EBRT) combined with chemotherapy or radical surgery. Overall, 21 patients with Stage III ( External auditory canal cancer (EACC) is a rare malignant tumor that affects 1–6 per 1 million population-years . The lowet al.  and 71% , respectively Fig. .n = 13) and patients who did not receive surgery (n = 8), between patients who received chemotherapy (n = 12) and patients who did not receive chemotherapy (n = 9), or between patients who received an EBRT dose of >50 Gy (n = 11) and patients who received EBRT dose of ≤50 Gy (n = 10). Regarding the influence of the chemotherapy regimen used, the OS and LRC rates of patients who received TPF (n = 6) were longer than those who received chemotherapy with other chemotherapeutic regimens (n = 6) or no chemotherapy (n = 9), although the differences were not significant  and 80% , respectively, those of patients who received a combination of surgery and EBRT were 50%  and 63% , respectively, and those of patients who received a combination of chemotherapy and EBRT were 83%  and 86% , respectively. Patients who received EBRT alone showed local disease progression at 2 months after the start of the treatment.n = 6) were 100% and 100%, respectively. These results were better than those of patients who received surgery and EBRT with or without chemotherapy , and also better than those of patients who did not receive TPF or surgery . There was a significant difference in the OS and LRC between the three groups [OS, P < 0.001 . The EBRT dose differed significantly between the above two groups, and there was a borderline difference in the follow-up duration , stereotactic radiotherapy (n = 1), or stereotactic radiotherapy and additional surgery (n = 1). However, all patients died between 4.7 and 16  months after the find of recurrences.The two patients with in-field recurrences received chemotherapy, followed by best supportive care, and the patients with out-of-field recurrences received chemotherapy  was only 29% [i et al.  and Leon [et al.  showed tet al. [et al. [There was an impressive result in our study: the 2-year OS rate of the eight patients who did not receive surgery was 73% (which was higher than the rate found in past studies) , 6. Amonet al.  reportedet al. [Katori et al.  have comet al. , the sevet al. [et al. [Although brain injury has been seen with radical radiotherapy doses of 66 Gy in 2 Gy per fraction, Pemberton et al.  have rep [et al.  have repBecause this present study was retrospective and the treatment policy has changed with time, it appears that there could have been a bias in patient selection. Moreover, the observation period of patients who received EBRT and TPF was shorter than that of the patients who underwent EBRT and surgery, and further long-term follow-up may be necessary for a reasonable comparison between the two treatment strategies.In conclusion, our data suggest that the combination therapy of EBRT and surgery and/or chemotherapy may be the most effective treatment options for advanced EACC, and EBRT with concurrent chemotherapy with TPF is potentially the most acceptable. However, further investigation is needed to determine the optimal dose and the appropriate field for EBRT in patients with EACC. Furthermore, because of the limited number of patients with this cancer, a multicenter study would be desirable.


E. coli (STEC) can cause serious illnesses, including hemorrhagic colitis and hemolytic uremic syndrome. This is the first systematic review of STEC in Brazil, and will report the main serogroups detected in animals, food products and foodborne diseases. Data were obtained from online databases accessed in January 2019. Papers were selected from each database using the Mesh term entries. Although no human disease outbreaks in Brazil related to STEC has been reported, the presence of several serogroups such as O157 and O111 has been verified in animals, food, and humans. Moreover, other serogroups monitored by international federal agencies and involved in outbreak cases worldwide were detected, and other unusual strains were involved in some isolated individual cases of foodborne disease, such as serotype O118:H16 and serogroup O165. The epidemiological data presented herein indicates the presence of several pathogenic serogroups, including O157:H7, O26, O103, and O111, which have been linked to disease outbreaks worldwide. As available data are concentrated in the Sao Paulo state and almost completely lacking in outlying regions, epidemiological monitoring in Brazil for STEC needs to be expanded and food safety standards for this pathogen should be aligned to that of the food safety standards of international bodies.Shiga-toxin producing  Brazil is one of the largest producers and exporters of food in the world. Animal products, such as beef, poultry, pork, fish, and crops such as corn, soybeans, and rice represent Brazil’s major exports . HoweverEnterobacteriaceae, with Escherichia coli representing a major species. This bacterium represents one of the most extensively studied and was one of the first to be sequenced [Escherichia coli is the main bacterium involved in food contamination in Brazil [The main microorganisms involved in food contamination belong to the equenced ,5. In adn Brazil . It possn Brazil .These pathogenic groups are involved in outbreaks related to food consumption, and STEC in particular are of importance to public health, potentially causing diarrhea, bloody diarrhea (hemorrhagic colitis), hemolytic uremic syndrome (HUS) and renal injury ,9,10. STEscherichia coli STEC with a focus on Brazil and to compare the presence of serogroups detected in food products, animals and humans.In Brazil, there are no reports of outbreaks involving STEC. The hypothesis is that: (i) Disease outbreaks are not being recorded due to lack of a centralized reporting system or (ii) disease outbreaks are not being recognized as there is no surveillance system for STEC. Farm animals have been shown to be carriers and contamination of food as well as sporadic STEC infections in humans have been reported ,14. In aEscherichia coli” OR “Verotoxigenic” OR “STEC” OR “Shiga Toxigenic E. coli” OR “Shiga Toxigenic Escherichia coli” OR “Shiga Toxin-Producing Escherichia coli” OR “VTEC” OR “Vero Cytotoxin-Producing Escherichia coli” OR “Verotoxigenic E. coli” OR “Verotoxigenic Escherichia coli” OR “Verotoxin-Producing Escherichia coli” AND “Brazil”. Papers in both English and Portuguese were included in this review. Duplicates were traced and excluded. A total of 161 papers were collected, independent of sample size or culture/detection methods. The full text for each paper was obtained and evaluated individually  and noted that stx1 and stx2 were present in 30.9% of the strains, indicating possible dispersion of the stx genes between STEC and APEC. In addition, stx1 and stx2 were detected in 4.7% of poultry litter samples in the south of Brazil  and USDA-FSIS (Food Safety and Inspection Service), which require their absence in meat products. In addition, STEC were isolated from 2.7% to 78.3% of sheep and O103:H2 was detected in sheep  and has f Brazil .Stomoxys calcitrans). Prevalence rates in dogs ranged between 0 and 48%, but to date there are no reports of STEC in cats in Brazil.In other species that could possibly act as vectors, STEC rates were 0.7% to 20.4% in wild birds and 15% in stable flies . Through whole genome sequencing, the O104:H4 strain in Brazil was found to be similar to a strain isolated from an American citizen diagnosed with hemolytic uremic syndrome (HUS) who had traveled to Germany during the 2011 HUS outbreak [The presence of STEC contamination in humans was evaluated in 22 scientific articles . In fiveoutbreak .In other studies of diarrhea or healthy subjects, the serogroups predominantly detected were O26:H11, O103:H2, O111 Not typeable (NT), O118:H16, O165:NT and O157:H7 .Some of these serogroups were reported in animal and food studies in Brazil. For instance, the O111 and O157 serogroups were verified in all three groups. Our hypothesis is that the dispersion of the strains has contaminated all stages of the food chain (pre and post-processing). Moreover, the O103 strains in animals were linked to those found in humans. In contrast, stains O165 and O118:H16 were only detected in human clinical cases.The most frequent serogroups described in peer-reviewed papers in samples collected in Brazil are represented in Serogroup O111 was also reported in animals, food and humans and has been linked to clinical human cases in Brazil ,66,77 anSerogroup O165 has been reported in cases of hemolytic uremic syndrome in Japan , and GerEscherichia coli strains, enabling the bacteria to survive during passage through the human stomach [Studies evaluating different cattle feeds indicate that different diets may increase the acid resistance of  stomach . Strain  stomach  identifistx2e) or other genetic factors including virulence [Foods showed the greatest diversity among serogroups, followed by animal sources . This diirulence ,108.Studies were concentrated mainly in Brazil’s most developed areas and especially in the state of Sao Paulo , with thE. coli STEC tests for O157 and the “big six” non-O157 in beef destined for export [Currently, the main legislation in force in Brazil concerning food production is Resolution 12 of 2001 , which ar export . HoweverThe analysis procedure determined by MAPA is based on the methodology used by the USDA FSIS , comprisMoreover, the improvement and cost reduction of molecular tools such as whole genomic sequencing (WGS), metagenomics, and others will facilitate the understanding of serotype epidemiology and the dispersion of these strains in neighboring countries and in other continents. Epigenetics advances will also improve the understanding of gene expression and the impact of good animal management practices, as well as possible genome mutations that may influence virulence and antimicrobial resistance profiles .The epidemiological data presented in this review indicate that O157:H7, O26, O103 and O111 strains, classified as foodborne pathogens and monitored by the USDA-FSIS (USA), U.S. FDA, EFSA (European Food Safety Authority - EU) and MAPA , are currently circulating in different regions of Brazil. Although no STEC outbreak cases in Brazil have been reported, several animal, food and human studies have indicated the presence of STEC in Brazil and it has been related to several foodborne outbreaks around the world. Thus, improved epidemiological monitoring and food production control is necessary. Novel studies should be financed in regions presenting significant agricultural production, such as Brazil’s central region in order to better assess potential threats and prevent human STEC infections.


CDKN1A gene. It is a major p53 target gene involved in cell cycle progression that has been extensively evaluated. To date, p21 has been reported to regulate various cell functions, both dependent and independent of p53. Besides regulating the cell cycle, p21 also modulates apoptosis, induces senescence, and maintains cellular quiescence in response to various stimuli. p21 transcription is induced in response to stresses, including those from oxidative and chemotherapeutic treatment. A recent study has shown that in response to metabolic stresses such as nutrient and energy depletion, p21 expression is induced to regulate various cell functions. Despite the biological significance, the mechanism of p21 regulation in cancer adaptation to metabolic stress is underexplored and thus represents an exciting field. This review focuses on the recent development of p21 regulation in response to metabolic stress and its impact in inducing cell cycle arrest and death in cancer cells.Cancer cells possess metabolic properties that are different from benign cells. These unique characteristics have become attractive targets that are being actively investigated for cancer therapy. p21cip1/waf1, also known as Cyclin-Dependent Kinase inhibitor 1A, is encoded by the  Eukaryotic cells utilize complicated mechanisms to regulate cell division and quiescence in response to both internal and external stimuli. These cells rapidly divide under certain conditions such as embryonic development and wound healing. Conversely, they stop proliferating in response to adverse conditions and enter cell cycle arrest and post-development quiescence. Hence, the cells develop a sophisticated braking system required for survival. Deregulation of this mechanism can result in loss of genome integrity and cancer development. p21 (Cip1/WAF1) was first identified as a Cyclin-Dependent Kinase (CDK) regulator that inhibits the retinoblastoma gene (Rb) phosphorylation and G1/S cell cycle progression ,4,5. p21p21 transcription can be regulated by either a p53-dependent or -independent manner. These regulations were described in the early years of p21 studies, where p21 induction in response to radiation-induced DNA damage was characterized as p53-dependent . In contDuring normal tissue development and serum-stimulated growth in vitro, p21 is primarily regulated in a p53-independent manner by a number of transcription factors . Under vEpigenetic processes can also regulate p21 transcriptional activation. They can be induced through the p53-independent pathway by chromatin remodeling following acetylation of histones H3 and H4 in the p21 promoter region ,38,39,40p21 is also subjected to post-translational modification. Most notably, it is regulated by serine-threonine kinases phosphorylation at Ser-130, Ser-146, Thr-57 and Thr-145. These phosphorylation events impact both the function and stability of p21. For example, Thr-145 phosphorylation by AKT kinase disrupts p21 binding to PCNA and enhances its cytoplasmic localization. This, in turn, promotes cell survival and transformation in breast cancers ,45,46,47Inhibition of cell cycle progression is one of the main cellular functions of p21. It mainly occurs by inhibiting cyclins A/CDK2, E/CDK2, and D1/CDK4 activities, thereby preventing the phosphorylation of Rb protein . The abip21 has been demonstrated to regulate apoptosis in a paradoxical manner, depending on the cellular context ,54,55. Ip21 is also a major regulator of cellular senescence, a complex program involving multiple signaling pathways. p21 interacts with p16INK4A tumor suppressor to inhibit cyclin-dependent kinases, suppressing retinoblastoma protein phosphorylation and the expression of cell proliferation-associated genes . SimilarA hallmark of stem cells is their ability for self-renewal. Most stem cell populations are in the quiescent state until they are stimulated. p21 has been reported to maintain cellular quiescence ,67 and pApart from its well-established roles, p21 may also directly or indirectly influence cancer metastasis. However, the association between p21 expression and metastatic ability is inconsistent in different cancers. In some cancers, p21 levels are inversely related to the metastatic phenotype, where a higher p21 expression predicts a favorable disease outcome ,73,74,75Both p21 inactivation and activation have been implicated in cancer therapy resistance. p21 inactivation is associated with paclitaxel and 5-Fluorouracil (5FU) resistance in noncancerous breast epithelial and colon cancer cells, respectively ,81. In cMetabolism is a cornerstone for various cellular functions. It is so important that Major metabolic enzymes and regulatory pathways are conserved from unicellular organisms to mammals. Metabolic stress can be triggered by either nutrient unavailability or malfunction of important proteins involved in the metabolic machinery. Adaption to metabolic stress is essential for cell proliferation and survival. p21 is involved in cellular responses to various stressors ,87,88. Rp21 plays an important role in cell response to nutrient deficiency. Studies have shown that p21 is transcriptionally upregulated in mice under short-term fasting ,90. A maCellular nutrient uptake can stimulate intracellular nutrient-sensing mechanisms. This, in turn, activates anabolic pathways aiming at increasing biosynthesis. Apart from nutrient availability, the stimulation of growth factor signaling also accelerates anabolism. Nutrient deprivation, particularly glucose, is sensed by the AMP-activated kinase (AMPK), a key regulator of the catabolic-anabolic balance. AMPK is an allosteric enzyme that is directly regulated by the AMP to ATP ratio, which is a readout for cellular energy status ,96. GiveWhile it is not the focus of the current review, it is important to note that AMPK can also sense DNA damage stress and induce p21 transcription in both p53-dependent and-independent manners ,100,101.Amino acids are important building blocks and signaling intermediators. Their deficiency can alter many cellular metabolic functions that, in turn, regulate cell cycle progression and survival through complex regulations, including those involving p21 ,107,108.In cancer, p21 is generally perceived as a tumor-suppressor protein. This hypothesis is supported by the observations that p21-null mice spontaneously developed tumors . AdditioCompared with their benign counterparts, cancer cells have distinct metabolic characteristics that present therapeutic opportunities. One of the most recognized signatures of cancer metabolism is the Warburg effect, or aerobic glycolysis, which describes the preference of cancer cells using glycolysis over oxidative phosphorylation to metabolize glucose and derivatives for energy production. As a result of incomplete oxidation, most cancer cells consume glucose to generate more metabolic intermediates and lactic acid than benign cells ,117. MulDespite their dependence on glycolysis in the metabolism, mitochondria respiration in cancer cells is important for generating energy and nucleic acid synthesis . As a maRAS mutation driven-oncogenesis accounts for one-third of human cancers. The relevance of the RAS mutation becomes even more apparent if the aberrancies in relevant pathways such as EGFR  mutations and MAPK (Mitogen-Activated Protein Kinases) signaling components are also taken into consideration ,136,137.To better understand the mechanisms underlying p21 elevation in response to ICMT inhibition and its role in cancer cell proliferation and survival, we evaluated a panel of cancer cells from diverse tissue origins. Cancer cells that are sensitive to ICMT inhibition show a constellation of changes related to metabolism that include the reduction of mitochondria respiration, the induction of autophagy, cell cycle arrest and apoptosis ,146,147.To better understand how p21 regulation leads to cell adaptation following metabolic distress, it is necessary to briefly discuss the cellular process of autophagy—a fundamental eukaryotic cell adaptation in response to a nutrient-depleted state. The double membranous structure of autophagosomes forms as a self-digestive organelle in the lysosomal pathway to degrade damaged/aged organelles and protein aggregates; this maintains homeostasis in cells under normal conditions . AutophaIn evaluating the impacts of ICMT inhibition in cancer cells, we found that p21 is induced only in the sensitive cell lines upon ICMT suppression. Marked reduction in mitochondrial respiration and consistent depletion of cellular “fuel molecules”, such as ATP and other NTPs, were also noted following exposures to the ICMT inhibitor. The mitochondria targeting effect has been shown to be responsible for most of the anti-proliferative effects . The pheAs discussed earlier, p21 regulates cell cycle progression and apoptosis. Therefore, it makes physiological sense that p21 would be involved in sensing and transmitting the signal of metabolic stress. Indeed, when we further evaluated the role of p21 in response to ICMT inhibition, we found that p21 is an upstream regulator of cell cycle progression, autophagy and apoptosis, through the transcriptional regulation of downstream effectors that include BNIP3 (BCL2 Interacting Protein 3), LC3 (Microtubule-associated protein 1A/1B light chain 3B) and ULK1 (Unc-51 Like Autophagy Activating Kinase 1). Both ULK1 and LC3 are well-known proteins that promote the initiation and extension of autophagosomes. BNIP3 is a mitochondrial member of proapoptotic BCL2 family proteins containing a motif similar to the BH3 domain, which is a regulator of both autophagy and apoptosis ,165,166.In summary, we have discussed the evidence that supports p21 as a key regulator and signaling node in mediating cancer cells’ response to nutrition and fuel depletion. Overall, the evidence supports that p21 functions to integrate catabolism activities, cell cycle progression, and ultimately programmed cell death B. While The field of p21 has grown tremendously in the last three decades. Since its discovery in the 1980s as a Cyclin-Dependent Kinase inhibitor, p21 has attracted significant attention from researchers in many fields, as evident from the thousands of research publications on this protein. To date, p21 is known not only as a cell cycle regulator, but also as a key player in various other cellular functions including senescence, apoptosis, and self-renewal. p21 has also been long regarded as a stress response protein. In this regard, p21 can function as a coordinator for DNA damage repair pathways. Adding to its portfolio, p21 has recently been reported to act as a central regulator in cell adaptation to metabolic stress, particularly energy depletion in response to either starvation or treatment with mitochondria respiration inhibitors. Although this aspect has only been recently investigated with relatively few publications, the impact of these findings in understanding basic cell biology and the development of potential new therapy is apparent. For example, although recent findings demonstrate that stress-induced levels of p21 primarily leads to cell cycle arrest and cell death, the exact outcomes of p21 activation are very much dependent on the type, intensity and duration of particular stress, as well as the cell type upon which the stress acts . AnotherThis review hopefully highlights (i) the significance of emerging p21 research as a promising therapeutic approach for biologists and oncologists, and (ii) the necessity to continue the effort in understanding p21’s function as a coordinator of metabolic stress responses. As mentioned above, because of its dichotomy function, it is a challenging task to fully understand how p21 regulates various biological and cellular functions, particularly those associated with the cancer metabolism and its potential therapeutic implementation. Concerted explorations to understand p21 in adaptation to stress, metabolic and other stimuli, are essential for its translational application in cancer therapy.


Across various axis-1 disorders, the severity of dissociative symptoms is significantly related to a history of childhood traumatization. Thus, the question arises if coping with childhood trauma leads to neural adaptations that enhance the frequency of dissociative processing during adulthood. The aim of the two reported studies therefore was to identify and replicate gray matter alterations associated with dissociation.In a first study, whole-brain MRI data were acquired for 22 female in-patients with trauma-spectrum disorders and a history of severe childhood trauma. Voxel-based morphometry (VBM) was carried out to test for significant correlations between dissociation  severity and gray matter volume. Dissociation severity was positively associated with volume in the left angular gyrus. This result was diagnosis-invariant. The replication study involved 26 female in-patients with trauma-spectrum disorders and 25 healthy controls. No significant association between dissociation severity and brain volume in a left angular gyrus region of interest located at the peak identified in study 1 was identified and no significant group difference in this region could be established.trans-diagnostically with structural brain alterations in the left angular gyrus and independent replication in a larger patient sample is essential.The angular gyrus has previously been implicated in the processing of agency and vestibular integration as well as dissociative processing. The current attempt at a direct replication of brain volume alterations however, failed. The data thus only partially support the notion that dissociative processing is associated  Within the dissociative disorders, DPD is the most alike to the set of dissociative symptoms reported by patients suffering from other axis-1 disorders, the difference being that in DPD the symptoms are typically chronic, often present without any fluctuation, and include excessive emotional numbing. DPD is a highly disabling condition and constitutes a dissociative disorder in its own rights. Several large-scale investigations of DPD indicate that this disorder is not consistently associated with severe childhood trauma . The etitrans-diagnostically in subjects with a history of childhood trauma. Morphological patient studies have previously been criticized for both reporting biases  dissociative processing following childhood trauma is associated with specific neural alterations in structures such as the angular gyrus, superior frontal region, and temporal poles, and (b) those alterations exist Study 2 was conducted in order to replicate the results of Study 1 using a confirmatory analysis limited to the brain structures identified in Study 1. In addition, the patient data were compared to healthy controls.All patients were recruited from an in-patient treatment program at the Clinic of Psychotherapy and Psychosomatic Medicine of the University Hospital Dresden. The clinic is specialized in the treatment of patients with a history of severe childhood trauma who express symptoms of dissociation, emotion regulation difficulties, and PTSD – a disease pattern often described as complex PTSD. Clinical diagnostics as well as detailed anamnestic interviews were performed by trained psychotherapists in all patients. In addition, the Structured Clinical Interview for DSM-IV Diagnosis SCID;  was perfIn order to delineate the neural correlates of dissociative symptomatology from potential effects of childhood maltreatment, only patients with a clear history of significant childhood trauma were included. Criterion was a score of ≥11 on the childhood trauma questionnaire, indicating a value within or above the 95th percentile in the American  and Germ2014Both studies were conducted according to the Declaration of Helsinki and approved by the local ethical board of the Technical University of Dresden. After complete description of the study, written informed consent was obtained from all participants.In Study 1, data from 22 female in-patients aged 22–59 years  and 64% suffered from PTSD (F43.1: n = 14). Further, somatoform disorders (F45: n = 9), personality disorders (F60-61: n = 7), anxiety disorders (F40-41: n = 4), and a history of substance abuse (F1: n = 4) were frequent. 59% were diagnosed with other mental disorders (n = 13).In this in-patient sample, comorbidity was the norm rather than the exception. On average, each patient was diagnosed with 3.4 mental disorders. In total, 64% of the patients suffered from major depression . Furthermore, anxiety disorders (F40-41: n = 18), somatoform disorders (F45: n = 8), and personality disorders (F60-61: n = 2). 12% were diagnosed with other mental disorders (n = 3). Similar to Study 1, most patients were on medication, mainly antidepressants  = 0.5; ionnaire  served aAll participants completed the childhood trauma questionnaire CTQ;  and the ∗1 mm∗1 mm). For Study 2, imaging data were collected on a 3 T Siemens Magnetom Verio scanner  with an 8-channel phased-array head coil. T1-weighted anatomical images of the whole head were acquired .All imaging data of Study 1 were collected using a 1.5 T MRI scanner  with the manufacturer’s 8-channel phased array head coil. T1-weighted anatomical images of the whole head with 1mm isotropic resolution using a 3D magnetization prepared gradient rapid acquisition gradient echo (MPRAGE) sequence  = 352 mm × 352 mm × 384 mm; reconstructed voxel size: 1 mm1) and Matlab R2015b  with settings recommended by Separately for both studies, VBM analysis was performed using SPM12  was used for global normalization to account for differences in brain size.In Study 1, voxel-wise correlation of gray matter volume with dissociation severity (mean percentage score on the FDS) was performed using multiple regression analyses with absolute threshold masking at 0.2. Total gray matter volume , were added as nuisance effects in separate covariance analyses.post hoc added additional analyses controlling for these group differences. As different classes of medications are known to have different effects on brain activation , we tivation  and morptivation  in PTSD p < 0.001 in combination with a non-stationary threshold to balance the risks of Type-I and Type-II errors  for potential effects of medication by covarying the intake of each of the five types of medication , (2) covaried the severity of CM, and (3) covaried depression severity by using those variables as regressors of no interest in the analysis.In Study 2, we carried out confirmatory testing of the main result of Study 1 by extracting averaged VBM parameter estimation from an 8 mm sphere built around the angular gyrus peak obtained in Study 1 (-45 -56 24) for each individual. In addition, the data were analyzed for the group of patients and controls separately by replicating the exploratory whole-brain, voxel-wise correlation of gray matter volume with dissociation severity following procedures outlined above  in order to ensure that our null-finding was not due to a slight misplacement of the sphere used for confirmatory testing. In order to test whether the diverging results in the two patient groups might be due to differences in sample composition, we In a separate analysis, the gray matter volume of patients and controls were contrasted against each other under control of the total gray matter volume. In order to unmask the effect of dissociation, the group comparison was done with and without inclusion of the FDS value as a regressor of no interest.r = 0.52, p = 0.014), but not with depression levels .FDS scores assessing dissociation severity ranged from 3.2 to 61.4  and depression scores on the BDI ranged from 8 to 51 (mean 26.5 ± 10.6 SD), and childhood trauma severity measured with the CTQ ranged from 11.2 to 22.4 , but missed statistical significance. An additional cluster was observed in the left inferior frontal gyrus, pars triangularis, however this did not hold for non-stationary cluster extent correction and is only reported in order to get a comprehensive picture  = 1.4; p = 0.18] and childhood maltreatment , but they reported significantly stronger depression .For patients, FDS scores assessing dissociation severity ranged from 5.5 to 79.0 , depression scores ranged from 23 to 62 (mean 39.2 ± 9.5 SD) and childhood trauma severity ranged from 11.4 to 24.6  or depression levels .In this sample of patients, severity of dissociation did not correlate significantly with severity of childhood trauma (t(49) = 6.2; p < 0.001, d = 2.5], depression , and childhood maltreatment . Dissociation scores did not correlate significantly with childhood trauma  or with depression scores  in the control group. As dissociation scores deviated significantly from a normal distribution in controls, Spearman correlation was used for the latter statistics.Controls reported the expected low values for dissociation , depression , and childhood maltreatment . As compared to patients, controls did report significantly lower values for dissociation [r = 0.13, p = 0.53; r = 0.32, p = 0.12). To test whether the replication in the patient group failed due to the significantly higher depression scores, the correlation between dissociation severity and brain volume in the angular gyrus was repeated while covarying out depression scores. However, taking depression severity into account did not render this correlation significant  and right middle frontal gyrus  and in the thalamus  and no significant volume increases were observed in the group of patients. After inclusion of the FDS as covariate, the volumetric differences between groups were limited and only the left inferior frontal gyrus  was found to be significantly reduced in the group of patients.Compared to controls, patients exhibited a significantly reduced volume in the left  only marginally missed the extent threshold, indicating that it was mostly driven by the more proximal dissociation variable. Similarly, depression severity and medication status cannot fully account for this association, albeit the detected cluster were also smaller in size in these post hoc covariation analyses .In Study 1, a significant positive correlation between dissociation severity and gray matter volume in the left angular gyrus, extending into the middle temporal lobe, was established in female patients with a history of severe childhood trauma The angular gyri are involved in many different functions and are considered a cross-modal integration hub . No significant correlation between dissociation severity and volume of the left angular gyrus in the region of interest was established neither in the patient nor the control group. In addition, the group comparison in Study 2 did not result in a significant difference in angular gyrus volume. The explorative, whole brain analysis revealed that patients exhibited a significantly reduced volume bilaterally in the dorsolateral prefrontal gyrus and the thalamus as compared to controls. This group difference is likely related to differences in dissociation severity as it was rendered non-significant upon covariation of FDS scores. The dorsolateral prefrontal gyrus has been implicated in emotion regulation , and oneThere are some notable differences between the two data sets outlined below. However, whether these are significant enough to potentially explain the failed replication attempt remains unknown. While the two patient samples were both convenience samples from the same, specialized treatment center and their average target symptom scores were comparable , the sample of Study 2 exhibited significantly higher depression scores  than the sample of Study 1. However, covarying out the depression scores did not render the association between dissociation severity and regional gray matter volume significant.r = 0.82; in-treatment patients r = 0.80; r = 0.53 in an in-patient sample; In addition, no significant correlation between childhood trauma severity and dissociation scores was established in Study 2, while it was highly significant in Study 1. This is noteworthy as a large review found consistently significant associations between childhood trauma severity and dissociation scores across several samples of both healthy controls and patients for the dissociation instrument used in our study . There iPotentially, the observed alterations could also be due to differential effects of psychotropic medication . HoweverOn a more technical note, it might be worth mentioning that patients from Study 1 were scanned on a 1.5 Tesla scanner vs. a 3 Tesla scanner used in Study 2 and different reconstruction filter settings in the two scanners cannot be excluded.In short, we noted several limitations to our studies: small samples sizes, different clinical characteristics in terms of depression severity and the association of dissociation severity with childhood trauma severity, different medication profiles in the two groups, and differences in the technical instrumentation. Both studies were limited to female participants and hence no conclusions can be drawn regarding potential effects in males.However, it seems unlikely that the described differences in sample composition and data acquisition can fully account for the divergence of the results. It is possible that the selection of participants was biased in undetected ways, i.e., that not both samples are good representatives of the population of patients. Thus, it is possible that a true effect was detected in Study 1 but not Study 2. Vice versa, and taking into account that a severe publication bias in this field has previously been demonstrated convincingly , it alsoJD and IC drafted the manuscript. All authors contributed to revising the manuscript. JD, PH, KD, and IC were involved in the data analysis. AS, JS, and KW were involved in recruitment and clinical assessment of patients. AS and FR were involved in MRI data acquisition.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.


However, the crucial factors that are responsible for the initiation of this crosstalk or the ‘recruitment’ process remain poorly understood. We propose that oncogenes themselves may influence the ‘recruitment’ of the stromal cells, while focusing on mutant p53. Apart from losing its tumor-suppressing properties, mutant p53 gains novel oncogenic functions, a phenomenon dubbed mutant p53 gain of function (GOF). Here, we discuss possible ways in which mutant p53 may modulate the microenvironment in order to promote tumorigenesis. We thus propose that mutant p53 may serve as a key player in the modulation of the tumor–stroma crosstalk in a way that benefits the tumor. Further elucidation of these ‘recruitment’ processes, dictated by mutant p53, may be utilized for tailoring personalized therapeutic approaches for patients with tumors that harbor p53 mutation.A plethora of studies suggest that the non-transformed cellular and non-cellular components of the tumor, collectively known as the tumor microenvironment, have a significant impact on the tumorigenic process. It was suggested that the microenvironment, which initially restricts tumor development, is recruited by the tumor and maintains a crosstalk that further promotes cancer progression. Indeed, many of the molecules that participate in the tumor However, the crucial factors that are responsible for the initiation of this crosstalk or the ‘recruitment’ process remain poorly understood. We propose that oncogenes themselves may influence the ‘recruitment’ of the stromal cells, while focusing on mutant p53. Apart from losing its tumor-suppressing properties, mutant p53 gains novel oncogenic functions, a phenomenon dubbed mutant p53 gain of function (GOF). Here, we discuss possible ways in which mutant p53 may modulate the microenvironment in order to promote tumorigenesis. We thus propose that mutant p53 may serve as a key player in the modulation of the tumor–stroma crosstalk in a way that benefits the tumor. Further elucidation of these ‘recruitment’ processes, dictated by mutant p53, may be utilized for tailoring personalized therapeutic approaches for patients with tumors that harbor p53 mutation.A plethora of studies suggest that the non-transformed cellular and non-cellular components of the tumor, collectively known as the tumor microenvironment, have a significant impact on the tumorigenic process. It was suggested that the microenvironment, which initially restricts tumor development, is recruited by the tumor and maintains a crosstalk that further promotes cancer progression. Indeed, many of the molecules that participate in the tumorIt is now well accepted that cancer development does not depend solely on cancer cell-autonomous characteristics. Ample evidence suggests that various normal cellular and non-cellular components found adjacent to the tumor, known as the tumor microenvironment or tumor stroma, play a major modulating role in different stages of tumor development . The tumIt was suggested that initially, the microenvironment may serve as a barrier to tumor formation, even in the presence of potentially oncogenic genetic aberrations in precancerous cells . Howeverin vivo (Though wild-type (WT) p53 has been extensively studied, most studies focused on its cell-autonomous functions. However, accumulating evidence suggests that p53 has a non-cell autonomous tumor-suppressing role, particularly in the tumor stroma . One of in vivo . Conditiin vivo . Our recin vivo .Though the aforementioned studies are consistent with the role of WT p53 as a tumor suppressor, a recent paper described a confounding finding, in which WT p53 seems to promote tumor-promoting functions of CAFs, suggesting that WT p53 may be implicated in the ‘education’ of stromal cells in the microenvironment . AnotherWhile the non-cell autonomous role of WT p53 in stromal cells has been investigated, only few studies addressed how mutant p53, which is found predominantly in tumor cells, as well as being the most commonly mutated gene in cancer , affectsβ and IL-6, and extracellular matrix (ECM)-related proteins, such as the ECM remodeler, matrix metalloproteinase 3. Intriguingly, we found that different p53 mutations promoted the CGS in different underlying mechanisms. While p53 DNA contact mutations, namely p53R248Q and p53R273H, promoted the CGS via nuclear factor κB (NF-κB) activation, p53 conformational mutants, namely p53R175H and p53H179R, promoted the CGS by inhibiting BTG2, thus alleviating its inhibition on mutant H-Ras , thus suggesting a possible dominant-negative effect of mutant p53 over WT p53  response elicited by the CAFs and triggered non-cell autonomously by the presence of mutant p53 in the cancer cells. Interestingly, the activation of the IFN-β response seemed to be dependent on cell–cell contact, since CM derived from mutant p53-harboring cells and CAFs caused increased activation of this response, while CM derived from tumor cells alone produced a much milder effect. In turn, mutant p53 caused an attenuation of the IFN-β activation in the cancer cells themselves by upregulating SOCS1, a negative regulator of the IFN pathway, therefore alleviating a suppressing effect of IFN-β on tumor cell migration. Thus, we demonstrated both non-cell autonomous function of cancer cell-expressed mutant p53, by inducing the IFN-β response in adjacent stromal cells, as well as modulation of the IFN-β signal by mutant p53, in a way that benefits the tumor cells.Finally, in our previous study, we were able to demonstrate an intricate crosstalk between patient-derived CAFs and lung cancer cells, in which mutant p53 induces the secretion of a factor from the neighboring cells and modulates the response to this factor in the cancer cells . We utilIn light of the aforementioned studies, it is tempting to speculate that mutant p53 in cancer cells may indeed serve as a potential stromal modulator. Mutant p53, as well as other oncogenes, may serve as both the initiators and the propagators of the tumor–stroma vicious cycle, during which they promote the ‘re-education’ of the cells in the stromal compartment, as well as causing a modulation of the signals arriving from the stromal cells in a way that benefits the tumor cells.


Dear Editor-in-Chief,We read Mauro Cozzolino’s criticism regarding vitamin D as a follicular marker of human oocyte quality and a serum marker of in vitro fertilization outcome with great interest , 2. In hFirstly, we did explain in the discussion that one of the limitations of our study was due to not measuring vitamin D binding globulin level (VDBP). Instead, we considered a total 25(OH)D level, as bioavailable 25(OH)D is not bound to VDBP. We also explained in detail that VDBP is a highly polymorphic protein. The most common VDBP phenotypes in the Caucasian population, which was our study population, have the lowest affinity for 25(OH)D. Furthermore, recent study from Fabris et al. on the oocyte donor population did not show any dependence or statistical association between the values of total 25(OH)D and bioavailable 25(OH)D regarding the ovarian reserve, response to ovarian stimulation, oocyte quality in egg donors, or ongoing pregnancy rates after fresh embryo transfer in oocyte recipients . Also, FSecondly, regarding sperm parameters and the blastocyst rate, we did describe male factor as an exclusion criterion mainly based on semen concentration. We agree with Cozzolino that men commonly present not just one alteration but a combination of sperm defects. However, some studies indicated that total motile sperm count is a better indicator for the severity of male factor infertility than the WHO sperm classification system . NeverthBut it was not the aim of our study. We would like to emphasize that the primary aim of our study was to determine whether intrafollicular 25(OH)D level correlates with ICSI/ET results. We did not provide data concerning blastocyst rate in the “Results” section of our study population, what we believe Cozzolino is referring to. We have focused on blastocyst rate in a group of oocytes with matched FF gathered from the first dominant follicle, which usually contains a good oocyte. That is why we think that comparing our blastocyst rate of oocytes gathered from the dominant follicle with other studies is like comparing apples with oranges. Nevertheless, in our study, 88 embryos out of 322 MII oocytes were transferred after ICSI on the third day of the culture. In this group, we had 28 clinical pregnancies (31.8%). Then, out of 164 remaining embryos, 78 developed blastocysts , which gave a blastocyst rate of 47.5%. In contrast, quoted Piccolomini et al. saw 10,925 blastocysts develop in 4205 cycles out of 32,031 MII oocytes with a blastocyst rate of 34.1% [To conclude, oogenesis is a complicated process, and it is impossible to have one definitive marker of oocyte competence in follicular fluid . We agreSincerely,Przemysław Ciepiela, M.D. Ph.D.


Cryptococcus neoformans, the causative agent of cryptococcosis, causes ~ 181,000 deaths annually, with males having a higher incidence of disease than females (7M:3F). The reason for this sex bias remains unclear. We hypothesized that this disparity was due to biological differences between the male and female immune response.C. neoformans ± exogenous testosterone or 17-β-estradiol. C. neoformans, B, T, and NK cell proliferation was quantified by flow cytometry. Cytokine analysis was conducted via protein array or ELISA. Serological testing was conducted to determine previous exposure to C. neoformans.Peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated and infected with C. neoformans proliferated more in male PBMCs. T cell percentages in both sexes were lower in infected versus uninfected cells. Male PBMCs had lower CD3+, CD4+, and CD8+ T cells percentages during infection compared to females. Cytokine profiles showed differences in uninfected male and female PBMCs, which subsided during infection. Only one donor was sero-negative for prior C. neoformans exposure. There was an effect of estrogen in one dataset.These results suggest that males show an inherent deficit in T cell response during infection, which may contribute to the increased incidence of disease in males.The online version of this article (10.1186/s13293-019-0258-2) contains supplementary material, which is available to authorized users. Cryptococcus neoformans is responsible for an estimated 220,000 cases of cryptococcosis, resulting in more than 181,000 deaths each year worldwide . Males also had lower percentages of CD4+ helper T cells compared to females . Amounts of CD8+ cytotoxic T cells, and B cells, as measured by CD19, a marker expressed on the surface of all B cells, were all approximately the same in uninfected control cultures from men and women (p > 0.05). Numbers of dead immune cells after 7 days of culture were also similar between sexes . The numbers of NK cells and dead immune cells after 7 days of culture were similar between sexes  cell CD6+, and CC. neoformans proliferation in the presence of immune cells from healthy men and women, we quantified yeast numbers via flow cytometry after seven days incubation with PBMCs. While there was no difference in proliferation in male or female PBMCs with a specific hormone treatment, when C. neoformans counts were compared between men and women, C. neoformans generally proliferated better in PBMCs from men compared to women, except when exogenous 17-β-estradiol was added. Male PBMCs had significantly higher cell counts in treatments without hormone  and with the addition of exogenous testosterone , but there was no difference in cell counts between males and females when exogenous 17 β-estradiol was added  and female cell populations . Like T cells, B cell percentages declined in male  and female  samples. Interestingly, compared to uninfected cells, the percentages of dead PBMCs decreased during infection in males  but not females .odulator , 39. ThuF = 17.9, df = 1476, p < 0.0001) and samples from women . Compared to uninfected PBMCs, the NK cell percentage remained the same in samples from men  and in samples from women  in the presence of C. neoformans. Percentages of dead cells also remained the same in men  and women . These results confirm that, overall, C. neoformans depresses the immune response, even in proliferating cells from healthy individuals. Similar results were seen in subsets of cells incubated with exogenous sex hormones (data not shown).Similar to the first dataset, the second dataset showed a depression of T cells measured during infection in samples from men . The original differences between CD3+ and CD4+ observed in PBMCs from males and females grew significantly during infection . There was no significant difference between male and female B cell percentages in the presence of C. neoformans   or in the immune response in cell subsets with exogenously added testosterone or 17 β-estradiol (data not shown). Like the first dataset, the second dataset showed significantly lower percentages of T cells in infected male compared to infected female samples . In this dataset as well, original differences between the percentages of CD3+ T cells observed in PBMCs from males and females grew significantly during infection (p < 0.0001). There were no differences in NK cells   or dead cells  between sexes (data not shown). This data reveals lower percentages of T cells in healthy male PBMCs during a C. neoformans infection.After establishing baseline differences in immune cells, coccosis , 40–44. F = 21.05, df = 738, p < 0.0001) in the second dataset. To determine if this was due to circulating levels of estrogen in the female donors, we tested whether there was a difference depending on the week of the menstrual cycle the donors were in when they donated blood. Of the 14 women volunteers in the second dataset, six of them were in the second week of their cycle, which is known to have higher levels of circulating estrogen [+  and CD56+  cells compared to donors that were in weeks 1, 3, and 4 of their cycle. When we looked for differences in cell percentages in uninfected female samples based on the week of their menstrual cycle, the pattern was different. Uninfected PBMCs from female donors in week 3 of their cycle had significantly higher percentages of CD3+  and CD56+  cells compared to donors that were in week 0 of their cycle (data not shown). These data suggest that circulating levels of estrogen can affect immune cell proliferation.Interestingly, there was a significant effect of estrogen between infected cell percentages for males and females . There were no differences in IL-8, IL-12, and GM-CSF levels between uninfected cell supernatants in males and females, nor were there any differences in cytokine levels in males and females in infected cell supernatants. Comparing cytokine profiles between uninfected and infected groups , we saw significant increases in the Th1 cytokines IL-2, GM-CSF, IFN-γ, and TNF-α , and IL-4 (p = 0.047), a Th2 cytokine, during a C. neoformans infection. There were no differences in IL-6, IL-12, and IL-10 levels between uninfected and infected cells . However, recent studies have shown relatively equal exposure rates between men and women [C. neoformans antigenic testing was performed on sera collected from subjects in both datasets to determine serology status of each, using an agglutination test (Immy) and confirmed by immunoblot. Only one donor had a history of working with C. neoformans. Of the 62 apparently healthy subjects tested (40 from the first dataset and 22 from the second), 35 were male (56.4%), and the mean age was 24.7 years. Only one sample was confirmed as sero-negative (1.61%) through immunoblotting. The sero-negative donor was a healthy female. Since previous C. neoformans exposure should generate an enhanced memory response, the immune response was correlated to serology status. There was a significantly higher percentage of CD3+/CD4+ T cells in the sero-negative donor compared to the sero-positive donors . There were no differences in CD3+/CD8+ T cell percentages, B cell percentages, or cytokine response (data not shown). As there was only one donor without previous exposure to C. neoformans, this data should be interpreted with caution.Historically, it has been postulated that males suffered greater incidences of cryptococcosis, due to more frequent environmental exposure of nd women , 48. To C. neoformans in the absence or presence of testosterone or 17 β-estradiol. The goal was to understand the differences in the host immune response between males and females to determine if those differences could contribute to the observed differences in cryptococcosis between the sexes. Our cohort of subjects is unique, in that, they are healthy and not typical candidates for cryptococcal disease. Given the nature of cryptococcosis, it is accepted that patients suffering from the disease have multiple comorbidities, or at the very least, a compromised immune system, making it difficult for researchers to discern how C. neoformans acts on the host devoid of other variables. Keeping this in mind, these experiments were designed with the aim of teasing apart differences that may be masked or accentuated when studying typical C. neoformans patients.This is the first study to look at the adaptive immune responses of healthy male and female PBMCs when exposed to C. neoformans. The cytokine data clearly suggest that male PBMCs, but not female PBMCs, were proliferating in response to infection. Additionally, levels of CD25+ cells increased in infected male and female PBMCs, but the increases were not significant, likely due to the small sample size. It is interesting that the cytokine data suggest that male PBMCs, but not female PBMCs, were proliferating, especially since we saw significantly lower percentages of all tested T cells in male PBMCs compared to female PBMCs after infection. We hypothesize that since females naturally have higher levels of CD3+ and CD4+ T cells [The in situ cytokine levels and amounts of the activation marker CD25 between uninfected and infected male and female PBMCs were measured to determine if the PBMCs were proliferating in response to infection with  T cells –37, perhC. neoformans proliferated better in PBMCs isolated from males compared to females, both without additional hormone and in the presence of testosterone. Interestingly, there was no difference in C. neoformans cell counts between males and females in the presence of added 17 β-estradiol, suggesting that 17 β-estradiol may either inhibit proliferation or boost the immune response, as observed by Mohr et al [C. neoformans in male macrophages ex vivo [C. neoformans proliferation in males is decreased in the presence of 17 β-estradiol. Since 70% of patients with cryptococcal disease are male [C. neoformans infection.Notably, hr et al . Given o ex vivo , we hypo ex vivo –52, we hare male , 53, thi+ and CD4+ counts are typically higher in females [+ and CD4+ population. During infection, T cell percentages in both males and females dropped significantly compared to uninfected controls. This is unsurprising given C. neoformans known immunomodulatory effects [C. neoformans. This finding could also be due to differences in the phosphorylation state of SAMHD1 during C. neoformans infection, as the authors found that sex influenced the phosphorylation state of SAMHD1 during HIV-1 infection, which translated into decreased macrophage susceptibility in females [This idea was reinforced by the T cell data in males. Reference ranges for healthy adults in the USA show that CD3 females , 37. Uni effects , 54–56.  females .+) response is needed to successfully mediate the immune response to a cryptococcal infection and has been shown to directly inhibit the growth of C. neoformans [+) response is more complex. Despite their ability to decrease fungal burden in the host, CD4+ cells have recently been linked to advanced clinical symptoms and mortality [+/cryptococcal meningitis+ patients and found that despite having higher CD4+ T cells, males had higher risk of death [+ patients, T cell depletion is the primary predisposing cause for development of cryptococcal meningitis [C. neoformans. This may begin to explain why males experience higher rates of disease and death from cryptococcosis.A robust cytotoxic T cell  percentages fell significantly in cell populations from both men and women, but there was no difference between the two groups in the presence of C. neoformans, suggesting that the significant drop in T cells observed in men may be more important than B cells in an immune response to C. neoformans. The role B cells play in a C. neoformans infection have not been as well elucidated as those of T cells, but multiple studies report the necessity of B cells, often serving as a first line of defense during C. neoformans infections and a correlation between increased levels of B cells (IgM+) and a decreased likelihood of immunocompromised patients developing cryptococcosis [+ subjects that already have T cell deficiencies and therefore may rely more heavily on an effective B cell response.While T cells represent one arm of the adaptive immune response, B cells represent the other. During infection, B cell  that levels of circulating sex hormones can actively affect an immune response, and (2) higher levels of 17 β-estradiol may boost the immune response. The known pro-inflammatory effects of 17 β-estradiol may explain both this data and the data of Mohr et al. [Many factors are thought to influence the differences between male and female immune responses. Environmental stimuli, microRNAs, and X-linked immunoregulatory genes are all evidenced to contribute to what is commonly considered the female immune advantage –74. The r et al. , 69.C. neoformans in men and women, thus increasing the reliability of our findings. There are limitations in this study worth noting. First, these experiments were done in healthy individuals who normally do not develop cryptococcal disease. Thus, in order for the observed differences in the male and female immune response to contribute to the future understanding of cryptococcal disease, the differences would have to result in important changes in immune function that would persist in relevant ‘at risk’ populations. Future studies will examine this hypothesis. Second, since these studies were conducted on ex vivo cells, they may not have contained the full cellular milieu found within an intact organism, which possibly limited the effect. However, the fact that we saw differences between the T cell responses in PBMCs from males vs. females ex vivo indicates that enough of the cellular milieu remained to have an effect. In addition, the cells were grown in media and FBS that did not have estrogens removed [C. neoformans, simultaneously. Fourth, we consistently saw a decrease in the percentages of the immune cells measured in the presence of C. neoformans. This is unsurprising given the immunomodulatory effects of C. neoformans; however, we did not see a corresponding increase in any of the cell markers measured. This is possibly due to a proliferation in unmeasured immune cells ; PBMCs damaged due to infection such that the flow cytometer was unable to properly detect them; or most likely, a combination of both. However, when quantifying B cells, T cells, NK cells, and C. neoformans during infection, we were able to account for approximately 88–95% of the total cell population collected, suggesting that any remaining unmeasured immune cells are a small percentage of the population. Finally, the age of our donor population was relatively young (24.7 years). We know that age plays an important role in the immune response of both men and women; therefore, results of similar testing may be different in both pre-pubescent and post-menopausal populations. Future studies should include immune cell analysis of different age groups as well as samples from immune compromised individuals and diseased in vivo models.The key strength of our study is the large number of healthy donors . To our knowledge, this study is the largest reported to date evaluating the healthy immune response to  removed , 79. ThuC. neoformans infection of healthy male and female PBMCs, all percentages of measured T cell markers were significantly lower, and C. neoformans proliferated in greater numbers in the presence of male immune cells compared to female immune cells. These results, in combination with the similar response of B cell and NK cell proliferation in this study and in intracellular fungal proliferation in macrophages as observed in another study [C. neoformans infection. Looking at the immune response of healthy individuals reveals differences that may otherwise be overlooked. At present, it is not clear what role testosterone and 17 β-estradiol play in the immune response to C. neoformans, but the data suggest that they do play a role. In total, these results indicate that the difference in outcomes of cryptococcosis between men and women is biologic, can be at least partially explained by levels of circulating sex hormones and the adaptive immune response, and cannot simply be chalked up to differences in exposure rates. These experiments are an important first step in elucidating the biological sex differences seen in response to a C. neoformans infection and hopefully lay the groundwork for future research that uncovers the mechanisms behind these differences.During a er study , betweenAdditional file 1:Table S1. Flow cytometry cell counts for first dataset (DOCX 20 kb)Additional file 2:Flow cytometry cell counts for second dataset (DOCX 1010 kb)