The analysis of mental and psychological health is a relevant public issue in modern societies. Migration is a process that may have a lasting impact on a person’s mental well-being. In this study, perceived health, emotional intelligence, sociocultural adjustment and the participants’ perceived general situation, not only economical, were analyzed to attest their impact on psychological distress as a measure of mental well-being. Sixty-three migrants from Romania and Ecuador were contacted twice during a 14 month period in a middle-sized Spanish city. Attrition analyses show no significant differences in perceived psychological distress between those who participated only one time or who participated in both waves. Less psychological distress is related to less attention to one’s feelings and higher mood repair in both data waves. Stronger behavioral adjustment is also linked to less distress. Less distress in time 1 led to better perceived health, sociocultural adjustment and a perception of a better general situation in Spain in comparison to their home country in time 2. In general, more attention to negative feelings triggered more perceived psychological distress, whereas mood repair elicited less psychological distress, in time 2. The relevance of understanding the impact of emotional intelligence to health promotion programs with migrants is discussed. International migrants represent approximately 258 million people in 2017, roughly 3.4% of the world population . Consequently, our dataset presents no problems in terms of common method variance in both waves. Internal reliability and all partial correlations when controlling for sex and country of origin are presented in Results reveal that a single factor could not account for the variance in the data [T1 Delta r ranged from 0.29 to 0.51) and T2 (r ranged from 0.50 to 0.59).Results show adequate reliability in all variables . Attentir = 0.28) and more cognitive adjustment problems (r = 0.26). Clarity and mood repair presented no significant results.In addition, in T1 attention correlated significantly with more perceived psychological distress (r = 0.85 and r = 0.84 respectively). Psychological distress correlated with worse cognitive and behavioral adjustment in T1  and T2 , respectively.Behavioral adjustment correlated significantly with cognitive adjustment in both waves  = 5.74, p = 0.016). In addition, there are significant differences in behavioral adjustment between participants without studies  and those with completed secondary studies  (f(63) = 4.24, p = 0.047) in T1, and without  and with university studies in T2  (f(63) =2.81, p = 0.047). Distress in T2 also differs in relation to educational level. Participants without formal education  showed a higher level of perceived distress than migrants who had completed university studies  (f(63) = 2.98, p = 0.039).There are differences in perceived health in T1 by education level. Participants without formal studies  perceived significantly worse health than the participants with primary  and secondary studies , but not with university education  (f(63) = 10.11, p = 0.002) and T2 (f(63) = 6.76, p = 0.002), and in cognitive adjustment in T1 (f(63) = 8.23, p = 0.006) and T2 (f(63) = 5.84, p = 0.019). In all cases, participants from Ecuador showed worse socio-cultural adjustment. Differences were also found in attention to feelings in T1 (f(63) = 7.69, p = 0.007) but not in T2 (f(63) = 0.7, p = 0.793). Participants from Ecuador showed higher levels than Romanians, indicating that they tended to monitor more one’s own moods and emotions.Regarding country of origin, differences were found in behavioral adjustment in T1  comparing those participants with a longer stay in Spain (75 percentile: 9 years), and those with the shortest stay (25 percentile: 5 years) showed that length of stay did not play a role in changing patterns of sociocultural adaptation, perceived psychological distress, perceived health, EI and the perceived general situation.χ2 = 157.48; RMSEA = 0.06; CFI = 0.91). Sex and country of origin were defined as covariates due to the minor differences found in previous comparisons.An SEM using the Maximum Likelihood Estimation method was used. The model is presented in The model presented is composed only by statistically significant paths. For this reason, the clarity dimension has been removed from both waves. Results show that perceived psychological distress in T1 is affected by both dimensions of EI (attention to feelings and mood repair), and the behavioral dimension of socio-cultural adjustment in T1. The lower the attention to feelings, the higher the mood repair, and higher the behavioral adjustment in T1, the less the perceived psychological distress. Perceived psychological distress in T1 had an effect on perceived health, on both dimensions of socio-cultural adjustment and on the perception of the general situation in T2. Less perceived psychological distress in T1 triggered better perceived health and better socio-cultural adjustment in T2, and also the perception of a better general situation in Spain compared with their home country.Moreover, both dimensions of EI in T2 had an effect on perceived psychological distress in T2. The greater the attention to feelings, the more the perceived psychological distress, and more mood repair implied less perceived psychological distress.With regard to country of origin, migrants from Ecuador, in comparison to those from Romania, stated a higher level of perceived psychological distress in T2. This result is consistent with the higher level of cognitive and behavioral adjustment problems mentioned by these same participants.There is stability of results and scores after a 13–16 month period regarding psychological distress, adjustment and perception of one’s own situation. These results support those presented by Jasinskaja-Lahti, Horenczyk, and Kinunen , statingEI and sociocultural adjustment have an impact on perceived psychological distress, confirming our first hypothesis. Whilst clarity is not related to mental well-being, repair is a positive predictor of mental well-being. This result is in line with previous data  which shIn line with hypothesis 2, whilst being able to regulate negative moods and prolong positive ones is linked to better perceived mental well-being, focusing on one’s mood and emotions is an important predictor of negative mental well-being. Paying too much attention towards emotion is linked to ruminative strategies (guilt), avoidance, loss of control, and health problems . FocusinThis is an interesting result for migrant populations that may have to ruminate or avoid their feelings in order to understand new situations and environments. Learning how to adequately regulate emotions  is a positive well-being factor which should be included by professional practitioners in health promotion programs to avoid enhancing negative outcomes .Country differences in attention to one’s mood and emotions may be explained by the higher collectivistic scores found in Ecuador in comparison to Romania . CollectPsychological distress plays an important role in health and psychological adjustment more than a year later. Those participants who stated they were more distressed were also those who afterwards would state that their general health was worse and that they were in general, not only socioeconomically, worse off in Spain in comparison to their home country. These results confirm that positive mental well-being has implications in the long term, and that creating positive contexts for migrants to interact with and manage their lives  may have a lasting and positive effect on mental well-being.First, the number of participants is scarce; although most participants stated that they would have answered the follow up study if still living in the city, due to the nature of the labor market and its need for mobility many participants had moved to another city or were out of town for long periods of time. Nevertheless, attrition results show that participants who answered in both waves were not significantly different in their responses to those who answered only in T1. In fact, the only statistical difference in attrition rate was between men and women. As in other studies conducted with small sample sizes using either a qualitative or quantitative methodology, results must be interpreted with caution so as to not overextend and generalize conclusions to other social groups or situations. Although well designed research with a small sample size can yield important results it may also produce type II errors by interpreting as non-significant results which truly are significant. Nevertheless, various authors ,59 have Second, this research is based upon follow-up and self-reported data recorded over a relatively short time span. As can be the case in general self-reports, results could be influenced by social desirability responding, although this is not necessarily always something negative . The resThird, only two different migrant groups were included in the study. Moreover, we could not conduct separate SEMs to test for group differences. Nevertheless, there were no relevant sex or country of origin differences in any variables under consideration. Although the sample size is small, and we must be cautious with the over-generalization of results, controlling the sex and country of origin allows us to stress the validity of the results, their alignment with other emotion related studies, and their importance as a novel, and overlooked, approach to the analysis of acculturation well-being and public health issues over time.Due to the lack of published studies on the focus of this research, this study can be an addition to the scarce literature on acculturation, emotions and mental well-being and health for a number of reasons. The results of this study can lead to a series of recommendations in health programs and interventions with migrant populations similar to those studied in this research. First, it is necessary to develop longitudinal or follow-up studies that allow us to analyze the relationship between migration and mental well-being as a process. Research designs which aim to inform public health policies should stress the longitudinal analyses of variables and the nature of possible changes. Furthermore, the results from this study could be applied within a public health context. The training of migrants in EI can be beneficial and necessary as it will help them overcome possible social obstacles and present less psychological and cultural distress, as noted in a recent study . MoreoveA second important aspect of this study is the inclusion of variables neglected in most previous studies. The analysis of how emotions and their regulation have a significant impact on migrants’ health has important consequences in the development of programs aimed towards reducing mental distress. Adjustment to one’s new culture, both in terms of learning new cultural codes  and learning how to act in different situations, is an important factor in well-being. In stressful situations, people tend to pay excessive attention to their negative emotional states which, in turn, can lead to more distress. Hampton, Peter, Corus and Brinberg  mentioneThird, although the study comprises a relatively small sample, the strength of the paths in the SEM, as well as its theoretical background, and the alignment of results with previous studies conducted with different populations, point to the relevance of including emotion-related variables in the explanation of migrants’ mental well-being due to the persistent impact of these factors. Looking after migrants’ health is cost effective, facilitates their social and personal adaptation, and enhances more adequate public health interventions. As a result, studying and improving the health of all individuals and collectives within society should be an aim of research and interventions conducted within the social and medical sciences.


The median survival under treatment was 4.82 years. Patients with a more restrictive disease (FVC < 80% pred.), with a rapid progression (FVC decline >10% pred. p.a.), previous smokers and patients > 60 years of age seemed to profit more from pirfenidone treatment. Conclusions: We report the effectiveness of pirfenidone in a European “real world” IPF cohort with outcome data extending up to 9 years. Global analyses demonstrated a positive effect of pirfenidone on the decline of the lung function over time. Survival was dependent on Gender–Age–Physiology (GAP) score and age prior to therapy.Background: Idiopathic pulmonary fibrosis (IPF) is a chronic progressive fibrotic pulmonary disease with rising incidence. In this study the effectiveness of pirfenidone, as measured by longitudinal change in individual slope of forced vital capacity (FVC) prior to and after initiating pirfenidone treatment, was evaluated in IPF patients recruited into the European registry for idiopathic pulmonary fibrosis (eurIPFreg). Secondary variables were the evaluation of the change in individual slope of diffusion capacity of the lungs for carbon monoxide (DLco), the Borg dyspnea scale, and six-minute walking distance (6MWD), as well as survival analyses. Results: Data of 122 eurIPFreg patients, who had at least two pulmonary function tests (PFTs) prior to or under treatment with pirfenidone, were analyzed by calculating slope-changes. The global analysis revealed an average slope change of +1.48 ± 0.28 (% per annum (p.a)) after start of treatment ( Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and usually fatal fibrotic pulmonary disease with rising incidence, being associated with an economic healthcare burden . The natAs fibrosis advances and further impairs pulmonary physiology, affected patients experience a high burden of symptoms. Progressive dyspnea is the hallmark symptom of IPF and leads to significantly impaired exercise capacity. Patients also commonly experience non-productive cough and fatigue. Due to symptoms and the resultant impact on physical, social, and emotional well-being, patients with IPF suffer from decreased health-related quality of life (HRQL) ,5. A preInternational consensus guidelines recommend that the diagnosis of IPF should be made on a multi-disciplinary level, calling for the exclusion of known causes of ILD and the presence of a usual interstitial pneumonia (UIP) pattern on high resolution computed tomography (HRCT) or lung biopsy . In accoThe natural course of IPF is quite heterogeneous. IPF usually progresses gradually, but acute exacerbation of IPF may occur at any stage in the clinical course . GreaterHence, it is necessary to identify reliable prognostic factors for the risk of lung function decline and the response to medical therapies in a non-selected patient cohort. Data from patient registries, collecting longitudinal data on IPF patients, have already helped to improve the understanding of the clinical characteristics, the impact that the disease has on their quality of life and survival, as well as current practices in diagnosis and management . The datPrior to the development of pirfenidone and nintedanib, there had been little therapeutic innovation in IPF treatment for several decades, and treatment options had provided very limited values in terms of either disease progression or physical performance. Pirfenidone (5-methyl-1-phenyl-2-[1H]-pyridone), was the first drug to be approved for use on IPF patients . It is aCommercial release of pirfenidone in the European Union (EU) was granted based on the evidence from the two multinational pivotal phase III trials, PIPF-004 and PIPF-006  as well as a third supportive phase III study, conducted in Japanese patients ,20. The 0) and required the existence of at least two PFTs prior to and two PFTs after the onset of therapy. This is a retrospective secondary data use study to evaluate the effectiveness of pirfenidone in IPF patients. The primary endpoint was assessment of the treatment response, defined as change in annual rate of FVC decline after the start of pirfenidone treatment (t0);pts. with limited functional impairment (>80% predicted FVC at tpts. with stable disease or slow disease progression before treatment (<10% decline of predicted FVC p.a.);pts. with progressive disease prior to treatment (>10% decline in FVC p.a.);pts. progressing after start of treatment (>10% decline in FVC p.a.);pts. with different smoking history ;pts. younger and older than 60 years;pts. with Gender-Age-Physiology (GAP) composite scores I, II and III;Further primary objectives were to assess the effectiveness of pirfenidone, as measured by change in individual slope of FVC prior to and after initiating pirfenidone treatment in IPF patients (pts.) recruited into the eurIPFreg as well as in the following subgroups: 0) of DLco (% predicted), Borg dyspnea scale (grades 0–10), and six-minute walking distance test ;change in individual slope prior to and after baseline  and the European IPF Biobank (eurIPFbank) were launched in November 2009 in the frame of the European IPF Network under the FP7 program to better explore the pathogenesis and natural course of IPF, also in order to facilitate translational research in biomaterials from IPF subjects . Both, eOn a local level, each patient’s IPF diagnosis was evaluated in a multidisciplinary discussion including at least chest physicians, pathologists and radiologists on the basis of the respective ATS/ERS/JRS/ALAT guidelines. The registry had no explicit exclusion criteria, thereby reducing selection bias. The clinical data were collected at the time of enrolment and in intervals 3 to 12 months thereafter . The patient questionnaire included the patient’s demographics, a detailed medical history making use of the WHO classification, complaints as well as report of co-morbidities . After eFollow-up data were acquired in a similar way, making use again of patient and physician questionnaires including additional information on intermittent respiratory infections, working status, transplantation or any changes in the medication, also such events as death. Biological materials such as blood, bronchoalveolar lavage fluid (BALF) and tissue samples as well as exhaled breath condensates and electronic Nose (eNose) profiles were centrally recorded and managed in the centralized European IPF Biobank (eurIPFbank) located in Giessen, providing materials for further clinical research.The study cohort was recruited between November 2009 and May 2018 from the following sites: Universities of Giessen and Marburg Lung Center, Germany, including the nearby Agaplesion Lung Clinic Waldhof-Elgershausen, as well as from Competence Center for Rare Pulmonary Diseases of Hopital Bichat in Paris, France.Data were collected from IPF patients with at least two subsequent records without antifibrotic treatment and by at least two records under pirfenidone treatment.Individual analysis: slopes and slope-changes were taken as estimated from each individual patient.Global analysis: the data from all patients were subtracted from the respective baseline values and then pooled. The pooled data was used to fit a global segmented regression model.The data were analyzed in two different ways:0). Baseline values of all quantitative variables were interpolated using natural spline fits with two degrees of freedom through the available data. If only a single value was given, then this value was used as the baseline value. The baseline GAP score and Borg scale were taken to be the next available score value closest to the baseline date, but maximally one record earlier or later.Baseline was defined as the value at the date of the first pirfenidone treatment . Survival analyses were performed using Cox proportional hazards models. Effects estimated in the “global” analysis with p < 0.001 were considered statistically significant. In the other analyses, a p < 0.05 was regarded as significant.The changes in the key variable (change in FVC % predicted per year) were analyzed with segmented regressions by patient. The break-point was fixed at baseline thttps://www.R-project.org). The baseline GAP score and Borg scale were taken to be the next available score value closest to the baseline date, but maximally one record earlier or later. Data of subsequent visits after a change in the treatment or after transplantation was disregarded.All continuous and ordinal variables were shown as mean and standard deviation; categorical variables were separated into tables by number and percentage of the whole cohort (in %). All analyses were carried out with the statistics software R 3.4 (0 (first pirfenidone treatment). The values at t0 predicted by the segmented regression model were taken as the baseline response value for the respective patient.Survival analyses were performed using Cox proportional hazards models. Time-series data were analyzed with segmented regression models fixing the break-points at t0 are displayed in 0 was 64.5 ± 17.5% of the predicted value, the mean FVC at t0 was 63.0 ± 18.3% of the predicted value, indicating an already advanced state of the disease.In the current analysis, change in FVC slopes pre/post treatment was assessed in 122 IPF subjects under pirfenidone treatment. The demographics and baseline lung function values at tIn our cohort, bronchoscopy was performed in 91 cases; the bronchoalveolar lavage (BAL) differential revealed elevated neutrophil (15.5 ± 17.9%) and eosinophil (4.5 ± 4.6%) counts in face of normal lymphocyte (10.2 ± 11.7%) and reduced macrophage counts .0, after t0, and the difference of the slopes after and before t0) were obtained. Distribution of individual slope changes for patients with different numbers of PFTs (2–10) is shown in In the individual analysis, the slopes for each patient were calculated in an individual fashion on at least two PFTs prior to and after initiation of treatment, so that 122 statistically independent estimates for the slopes . The data regarding the treatment effect displayed a wide range between further FVC decline of 5-percentage points (%) p.a. and an improvement in FVC values up to 2% p.a. .The individual analysis was modified by taking at least three PFT measurements prior to and after pirfenidone treatment. The data were available for 106 patients. When doing so, there were also no significant changes between pre and post treatment initiation (0 were −2.84 and −1.36% predicted p.a. (p < 0.001), respectively. The slope change at t0 was found to be +1.48 ± 0.28 (p < 0.001). Our results hence show a reduction in the annual FVC decline of roughly 50% under treatment with pirfenidone.After setting baseline and performing individual FVC slope analysis individually as detailed above, all FVC data were corrected for the baseline values and analyzed together employing a global segmented regression, hereby disregarding the statistical dependencies of the patients’ recordings. In this analysis, the slopes and slope change were determined in a single segmented regression model using all data of all 122 patients and 2040 FVC values together . The sloFor the subgroup analysis, the data were used as in the global analysis, but the segmented regression model included the interaction of the slope coefficients with the co-variable of interest, allowing testing of the null hypothesis that the slope differences in the subgroups are zero as well as the difference in slope-differences between the subgroups.0 (n = 22) as compared to patients with a relevant reduction in FVC at baseline . Both patient subgroups showed a reduction in the FVC decline in response to treatment, indicated by a positive value for the slope change. However, in patients with preserved baseline lung function (right part of right panel), this did not reach significance (p = 0.17), whereas in patients with a relevant reduction in baseline FVC, this change did reach statistical significance (p < 0.001). The slope changes in both subgroups were found to be statistically different (p < 0.001).Data from patients with and without limited functional impairment showed a negative FVC slope before t0 . However0 (n = 98). In response to treatment, there was a significant (p < 0.001) reduction in the FVC decline in this patient sub-population, resulting in a positive value for the slope changes at t0  of less than 10% per year prior to treatment showed a slope of ~−4% before t0 approximating 18% (n = 24). In response to treatment, there was a significant (p < 0.001) reduction in the FVC decline in this patient sub-population, resulting in an impressive slope change of around 16% at t0. The change in the slope-differences in response to treatment between both subgroups was not found to be statistically significant (p = 0.345).Data from patients with progressive disease prior to treatment, defined as a decline of the FVC (in % pred.) of more than 10% prior to treatment showed a negative FVC slope before t0), defined as an annual FVC decline of >10% pred., showed an FVC decline of ~3.2% pred. prior to treatment (n = 28), as compared to an FVC decline of ~2.4% pred. in patients who did not deteriorate after initiation of treatment . Accordingly, patients with a deterioration after initiation of treatment showed a massive and highly significant (p < 0.001) worsening in the FVC slope , as compared to the similarly significant (p < 0.001) slight improvement in the FVC decline in those patients stable upon initiation of pirfenidone treatment . The change in the slope-differences in response to treatment between both subgroups was found to be statistically significant (p < 0.0001).Data from patients with progressive disease after initiation of treatment (tn = 84) showed a somewhat less prominent decline in FVC prior to treatment (left part) as compared to never smoking IPF subjects . In current or former smokers, therapy with pirfenidone resulted in a significant (p < 0.001) reduction in FVC decline (right part of right panel). In never smoking IPF subjects the slope change was less prominent and it did not reach statistical significance . There was a significant difference between both groups with regard to the response to treatment (p < 0.001).IPF patients in these two cohorts showed an FVC decline prior to treatment regardless of their smoking habits. However, current or former smokers with IPF (n = 93) showed a more prominent decline in the FVC prior to initiation of treatment  as compared to patients younger than 60 years . Response to treatment was statistically significant (p < 0.001) in case of the patients above 60 years of age  and resulted in a reduction in the FVC decline. In patients under 60 years of age, there was also an attenuation of the FVC decline, which, however, it did not reach statistical significance . Response to treatment was statistically significant between the two groups (p = 0.022).IPF subjects older than 60 years (n = 26) did not show a meaningful change in the FVC slopes, whereas GAP II patients (n = 51) showed a weakly significant reduction in FVC decline (p = 0.04), and GAP III patients (n = 26) showed a highly significant reduction in FVC decline (p < 0.001). There was a significant (p = 0.007) difference in the treatment responses between GAP I versus GAP II patients.FVC decline prior to initiation of treatment was found to be dependent on GAP stages , respectively. The slope change at t0 was found to be +3.51 ± 0.63 (p < 0.001). Although the individual slope analysis did not reveal any significant treatment effects, the global analysis revealed a significantly reduced DLco decline under pirfenidone. The slopes before and after tp = 0.019; n = 43).The global analysis showed an increasing Borg dyspnea grade prior to initiation of treatment and a significant attenuation of the dyspnea score in response to treatment with pirfenidone (p < 0.001).The global slope analysis showed a decline of 28 m per year in the 6MWD prior to the initiation of treatment . Upon inAdditionally, and in order to rule out a bias based on the drop out of deceased patients during the treatment period, we performed a subgroup analysis of the IPF cohort, who survived at least 24 months after beginning of pirfenidone therapy. In comparison to the whole IPF cohort, we saw similar pirfenidone effects on declines in FVC, DLco, 6MWD as well as improvement of the Borg scale. 0. The event (death) was only accepted if the patient died at the end of the valid treatment sequence. Dates of transplantation, changes in treatment or lost-of-follow ups were censored. The median survival under treatment was 4.82 years . The Kaplan–Meier curve is shown in Survival time was analyzed starting from tp = 0.002).As shown in p = 0.03).As shown in p = 0.014) impact of an FVC more than 60% versus less than 60% predicted on survival. The results are shown in When analyzing the impact of FVC at the timepoint of treatment initiation, we did observe a weak, albeit significant ; the decline remains about constant and even increases again after a few years (so 5–6). The decline from the year before to the year after the start of therapy seems significant, but the p-value for the mean difference of these values (year −1 to year +1) was p = 0.12. Although the data spread wide, despite all the effort to extract dependent variances, treatment with pirfenidone seemed to be presumably equally effective throughout the whole treatment time.To determine the mean changes per year in FVC, the FVC values per patient were fitted to a loess curve by a re-descending M estimator with Tukey’s bi-weight function and a span of 1 (year) and 12 iterations, showing a curve through the time course of the FVC values, predicted for tThe mean values and standard errors of the mean FVC slope decline per year (liters) are presented in 0  assessing efficacy of pirfenidone in IPF have provided important insights into the clinical course of IPF and the response to treatment. It should, however, be kept in mind that patients recruited into these studies represent a rigorously selected population and do therefore not necessarily reflect the characteristics of IPF subjects seen in clinical routine. Demographics and baseline measures of disease status of our study cohort are consistent with mild-to-moderate physiologic impairment and comparable to those in ASCEND and CAPACITY trials ,23,24. E0 approximating 18% and, as response to treatment, a significant (p < 0.001) reduction in the FVC decline, resulting in an impressive slope change of around 16% at t0.With regard to the analysis of change in FVC, patients with a more restrictive disease (FVC < 80% pred.), with a rapid progression (FVC decline > 10% pred. p.a.), previous smokers and patients > 60 years of age seemed to profit more from the pirfenidone treatment, in that the treatment elicited effects in these subpopulation appeared to be more profound. As an example, patients with progressive disease prior to treatment showed a negative FVC slope before tIn our “real world” analysis of response to treatment with pirfenidone, GAP I patients did not show a meaningful decline prior to treatment and an only modest, non-significant change under treatment; GAP II patients showed a much more prominent FVC decline prior to, and a weakly significant reduction in FVC decline under treatment. Finally, GAP III patients showed the highest FVC decline prior, and a highly significant reduction in FVC decline under treatment. There was a highly significant difference in the treatment (FVC) responses between GAP I versus GAP III patients, whereas differences between GAP I and GAP II patients were not significant. Costabel et al. published data on pirfenidone treatment in 187 patients with more advanced disease, and 409 patients with less advanced disease; it was shown that longer-term pirfenidone treatment resulted in a similar rate of lung function decline and safety profile in patients with more advanced versus less advanced IPF .Based on our individual slope analyses, the response to pirfenidone demonstrated a marked heterogeneity, hence rendering assessment of the individual treatment effect on disease progression quite challenging. The individually determined FVC slopes displayed a wide range between a further decline of 5% p.a. and an improvement of up to 2% p.a. After modifying the individual analysis by taking at least three PFT measurements prior to and after pirfenidone treatment (106 patients), there was still no visible significant treatment effect, although post treatment slopes were shifted to a lesser decline. This was reinforced by the finding that the intraindividually assessed slope changes in response to treatment were not substantially different if much more than two PFT’s were present for the slope analysis.0, where the event (death) was the end point . Our outcome analysis demonstrated a median survival under treatment of 4.82 years . In contrast to the substantial treatment effect on FVC seen in GAP III patients, patients at GAP stage III vs. GAP stage I (p = 0.001) showed a worse survival. When analyzing the impact of the FVC at the timepoint of treatment initiation, we did observe a weak, albeit significant impact of an FVC more than 60% versus less than 60% predicted on survival. Similarly, patients with a progressive disease (FVC decline > 10% per year) prior to therapy, although showing a striking increase in FVC, still showed a significantly higher mortality under treatment.Survival time was analyzed starting from tn = 43). The authors presumed that efficacy of pirfenidone in IPF patients with severe lung function impairment may diminish after 6 months of treatment [n = 307); this decline was even less than that noted in the ASCEND trial, in which mean decline from baseline in FVC was −235 mL in the pirfenidone group over 52 weeks [n = 502) of Japanese IPF patients, Bando et al. reported a mean decline of FVC of −30 mL and of −158 mL in the first and second year of therapy, respectively [n = 91) [0  and treatment with pirfenidone for at least 6 months, Tzouvelekis et al. showed a tendency towards a reduction in functional decline as compared to the 6-month period before treatment initiation, but failed to show any benefit after one year of treatment .Based on a Cox regression analysis, we additionally performed a risk score analysis adjusted for the FVC decline and GAP stage prior to the initiation of pirfenidone treatment. The data show that the higher the GAP score and the higher the FVC decline, the higher the risk of death under treatment. Fang et al. showed that IPF patients with severe baseline PFTs DLco < 30%) were three times more likely to develop disease progression or acute exacerbation or death than the IPF subjects with DLco > 30% . The med% were thp = 0.002).Noble et al. analyzed pooled data from the multinational pirfenidone trials ASCEND and CAPACITY and found that treatment with pirfenidone for 1 year resulted in clinically meaningful reduction in disease progression in patients with IPF; analysis of outcomes at 1 year demonstrated that pirfenidone reduced the proportion of patients with a. ≥10% decline in FVC % pred. or death by 43.8% and increased the proportion of patients with no decline by 59.3% (95% CI 29.0–96.8) compared with placebo . Antoniop = 0.0107); treatment-emergent all-cause mortality (p = 0.0094); idiopathic-pulmonary-fibrosis-related mortality (p = 0.0029). Consistent with the pooled analysis, meta-analyses for all-cause mortality at week 52 also showed a clinically relevant and significant risk reduction in the pirfenidone group compared with the placebo group [Nathan et al. showed in the pooled analysis that at week 52, the relative risk of death for all four mortality outcomes was significantly lower in the pirfenidone group than in the placebo group in the pooled population , or rapid progression (FVC decline > 10% pred. p.a.) seemed to profit more from this therapy. Our results provide additional information as to the effectiveness of pirfenidone treatment on IPF disease progression and on treatment response in dependency of IPF-subgroups. Patients with a higher GAP score and higher age prior to treatment are more likely to experience a worse outcome despite treatment with pirfenidone. Our data highlights the important role of large real-world data registries to document and analyze changes in IPF management.


Generation of mice lacking p73/TAp73 revealed a plethora of developmental defects, with very limited spontaneous tumors arising only at a later stage. Concurrently, novel TAp73 target genes involved in cellular growth promotion that are not regulated by p53 were identified, mooting the possibility that TAp73 may have diametrically opposite functions to p53 in tumorigenesis. We have therefore comprehensively evaluated the TAp73 target genes identified and validated in human cancer cell lines, to examine their contextual relevance. Data from focused studies aimed at appraising if p53 targets are also regulated by TAp73—often by TAp73 overexpression in cell lines with non-functional p53—were affirmative. However, genome-wide and phenotype-based studies led to the identification of TAp73-regulated genes involved in cellular survival and thus, tumor promotion. Our analyses therefore suggest that TAp73 may not necessarily be p53’s natural substitute in enforcing tumor suppression. It has likely evolved to perform unique functions in regulating developmental processes and promoting cellular growth through entirely different sets of target genes that are not common to, and cannot be substituted by p53. The p53-related targets initially reported to be regulated by TAp73 may therefore represent an experimental possibility rather than the reality. Identification of p73 as a structural homolog of p53 fueled early studies aimed at determining if it was capable of performing p53-like functions. This led to a conundrum as  The returned articles were further filtered by language to include English-based results only. From those articles, publications that met the above criteria for TAp73 target genes were considered relevant to the purpose of this review and were further grouped either as individual gene studies or genome-wide studies. We would like to highlight that studies describing other TAp73 transcriptional targets may have not been included in this analysis due to the nature of the search terms used for article identification. Nonetheless, we believe that the analysis provide a general overview that is representative of the TAp73 targets identified thus far. Since p73 was first identified as a structural and functional homolog of p53 ,39, its www.p53.iarc.fr). More than half were without a functional p53: 8 cell lines are devoid of p53 expression  and 29 expressed mutant p53, and three cell lines, namely, HEK293T which was used in 7 studies, NHEK in 1 study, and Hela in 6 studies, are considered functionally inactive for p53 due to the overexpression of SV40 T antigen or genes of human papilloma virus , MDM2, and BAX were the top 3 genes reported in multiple studies: 19, 12, and 12 studies respectively, as they were often used as positive controls. Firefly luciferase-based promoter assay was the most common method used to validate the regulation of target gene transcription upon TAp73 overexpression. The potential TAp73 binding sites were explored mainly by searching for typical p53REs in the proximal promoter regions, with the assumption that the binding sites would be evolutionally conserved and thus similar for TAp73 and p53. The majority of the 81 target genes contain a predicted p53RE in their genomic regulatory regions, of which 33 were validated.The target genes derived from the 71 individual focused studies that met our criteria are listed in https://amp.pharm.mssm.edu/Enrichr/), signaling pathways that emerged were DNA damage response, cell cycle arrest, and p53/p63/p73 networks (https://david.ncifcrf.gov/) (data not shown). It is noteworthy that many of these studies were aimed at identifying the target genes with intent, i.e., known p53 targets were evaluated to determine if they were also regulated by TAp73. Therefore, it is not surprising that the emerged pathways are similar to p53-regulated pathways and that these target genes are similarly regulated by p53. When these TAp73 target genes were pooled together for Gene Ontology (GO) term analysis using the online gene set analysis tool Enrichr , contained wild-type p53 but differed in the expression of the MLH1 gene that was shown to be necessary for TAp73 activity ,121.Among these studies, two analyzed the effects of endogenous TAp73 on promoter binding upon TNF-α  or hydroNine out of the twelve articles provided a full or partial list of genes identified and included data on the validation of the targets. Incidentally, none of the target genes identified were common across all nine studies , likely APOC3, C11orf21, GZMA, RPS27, and RRAD were identified twice in separate studies, and genes such as CDKN1A (p21) and MDM2 were identified thrice  and promoted the Warburg effect via stimulating the expression of phosphofructokinase 1 (PFKL), both in human cancer cell lines as well as E1A+Ras-transformed mouse embryonic fibroblasts [Other studies also found TAp73 to support cellular proliferation by enhancing cellular metabolism. TAp73 was shown to enhance the pentose phosphate pathway by directly activating the expression of roblasts ,28. ThesCxcl2, Ereg, and Vegf-c [hTERT, CylcinB1, and Caspase-2S have also been noted [A role for the p73 proteins in hypoxia has also been well investigated. TAp73 was noted to inhibit angiogenesis by destabilizing HIF-1α via a protein–protein interaction, independent of its transcription activity, and thus indirectly led to the suppression of pro-angiogenic genes such as d Vegf-c ,126. On d Vegf-c ,45. Whild Vegf-c ,45. Simien noted ,72,85.These data collectively demonstrate that TAp73 can have a distinct role in promoting cancer cell growth. TAp73’s ability to activate a unique and ever-expanding universe of transcriptional targets that do not converge with the canonical p53-like targets, often as a result of a collaborative co-existence with other transcription factors, highlight a more promiscuous function of TAp73 that is dependent on and dictated by the cellular contexts.Since its discovery over two decades ago, much effort has gone into deciphering the role of TAp73 in cellular growth control and in proving that TAp73 can be a substitute for p53, being able to perform similar functions through the transactivation of overlapping target genes. With this focused end goal in mind, many of the early studies were based on the “intended search and confirm” philosophy, thus unsurprisingly uncovering many common TAp73/p53 targets. However, both unbiased genome-wide studies and studies based on cellular phenotypes led to the revelation of many novel TAp73 targets that were associated with its role in cellular growth promotion, which is consistent with it being rarely mutated but over-expressed in human cancers.+/- mice [The current analyses of TAp73 target genes collectively highlighted several points worth pondering . Firstly+/- mice ,128, togNext, based on the primary observation that TAp73 is often overexpressed without being mutated in human cancers, a series of studies have now demonstrated that TAp73 expression can indeed be induced by growth-inducing conditions. Consequently, TAp73 has been suggested to be able to promote cellular growth and survival, mainly through the activation of target genes that appear to be context-dependent, and hence non-canonical. Importantly, it is emerging that these novel genes are often transactivated by TAp73 in collaboration with other transcription factors that promote cellular survival in various ways. These observations, together with the fact that many TAp73 target genes that regulate the developmental phenotypes noted in the absence of p73/TAp73 do not have a typical p53RE, indicate the promiscuity of TAp73 in being able to be adept in converging contextual signals to enable cellular survival. An interesting and yet often overlooked feature of these studies highlighting a pro-growth role for TAp73 is that most of the experiments were initially performed in MEFs that have been transformed, from which the target genes were identified and confirmed in human cancer cell lines. Importantly, these MEFs contained wild-type p53. Hence, it is tempting to speculate that in normal contexts where p53 is present, the primary role of TAp73 is context-dependent, being involved in regulating developmental genes at the early stages and for cellular proliferation and survival thereafter. Moreover, as a target of the key cell cycle regulator E2F-1 , it is nIn summary, we conclude that TAp73, though being a structural homolog of p53, is not the latter’s natural functional substitute. In its own right, TAp73 has evolved to perform unique functions in regulating developmental processes and cellular growth, through entirely different mechanisms and through different sets of target genes that are not common to and cannot be substituted by p53. While TAp73 can perform p53’s functions, the opposite is not true. Thus, the p53 target genes identified to be regulated by TAp73 may indeed represent a virtual reality.


To compare the differences in the clinical course of acute appendicitis between early elderly (60-79 years) and late elderly patients (≥80 years).The sample consisted of 177 patients aged over 60 that underwent surgery at the emergency service with the diagnosis of acute appendicitis between January 2010 and May 2018. Patients’ data were retrospectively obtained from electronic records. Patients that had undergone appendectomy or negative appendectomy or had an appendix tumor were excluded from the study. The patients were divided into two groups by age; early elderly (60-79 years) and late elderly (≥80 years).Of the 177 patients included in the study, 162(91%) were 60-79 years old and 15 (9%) were over 80. A statistically significant difference was found between the early and late elderly groups in terms of perforation, requirement for intensive care (p =0.001), red cell distribution width (p =0.025), the Clavien-Dindo classification (p =0.020), and the Charlson comorbidity index (p =0.005). The mean hospital stay was four and 11 days for early and late elderly groups, respectively. Multivariate analysis revealed that age alone was an independent factor with a statistically significant effect on mortality ).In particular, in the elderly patients over 80 years old, the clinical manifestation of acute appendicitis at hospital admission is in the form of perforation. Therefore, in this age group, a careful, precise and prompt diagnosis is crucial. Acute appendicitis (AA) is one of the most common surgical pathologies in the emergency service . It occuIn geriatric patients, the most common specific perioperative complications are general complications, such as postoperative wound infection, pneumonia, and cardiovascular problems . The difPatientsThis study was planned in Ankara Numune Training and Research Hospital General Surgery clinic. A total of 93 males (52.5%) and 84 females (47.5%) patients over 60 years of age that underwent surgery at the emergency surgery service with the diagnosis of AA between January 2010 and May 2018 were included in the study. Local ethics committee approval has obtained for the study . The patients with AA were retrospectively identified based on the electronic records of the patients. The diagnoses of AA have confirmed by final pathology reports for all 177 patients. Inclusion criteria were, all patients; over 60 years of age, underwent appendectomy between mentioned dates, have confirmation for the diagnosis, and have adequate medical records. Exclusion criteria were having undergone appendectomy and negative appendectomy as part of another surgical procedure or having an appendix tumor. The patients who were confirmed to have undergone appendectomy only due to AA were included in the study. The patients were divided into two groups according to age; early elderly (60-79 years) and late elderly (≥80 years).DataAfter obtaining the approval of the ethics committee for the study, the data of 177 patients over 60 years of age obtained from the records were evaluated. The electronic records were screened for details of treatment, surgical notes, and pathology reports. The data obtained from medical records consisted of age, sex, symptoms, physical examination, laboratory findings, imaging, appendicitis type (perforated or non-perforated), postoperative complications, length of hospital stay, and in-hospital mortality. The comorbidities of the patients were examined according to the Charlson comorbidity index (CCI), and their clinical, perioperative and pathologic findings were graded according to the American Association for the Surgery of Trauma (AAST) classification. Postoperative morbidity was defined as presence of wound infections, ileus, intraabdominal abscess or pneumonia complication, and requirement of intensive care. The treatment applied to the complications was classified according to the Clavien-Dindo classification (CDC).Statistical analysisp value ofAll statistical analyses were undertaken using SPSS Windows version 12.0 .  The numerical data were given as median  and compared using the Mann-Whitney U test when the normality assumption was violated. The categorical variables were expressed as percentage (%) and compared with the chi-square or Fisher’s exact tests. Statistically significant variables were assessed by multivariate logistic regression. A p=0.016), intensive care requirement (p=0.001), mortality (p=0.002), red cell distribution width (RDW) (p=0.025), CDC (p =0.020), and CCI (p = 0.005). However, there was no statistically significant difference between the two groups in terms of sex, complication , white blood cell (WBC) count (103 μl), neutrophil count (103 μl) and AAST grading. The mean hospital stay was four (IQR=2-5) and 11 (IQR=2-6) days in the early and late elderly groups, respectively (p=0.154) (p<0.001).Of the 177 patients included in the study, 162 (91%) were 60-79 years old (early elderly) and 15 (9%) were over 80 years of age (late elderly). There were 87 men (53%) and 75 women (46%) in the former group and six men (40%) and nine women (60%) in the latter group, with the median ages of 66 years (min 60-max: 79) and 85 years , respectively. A statistically significant difference was found between the early and late elderly groups in terms of perforation (p=0.154) . As summAA is one of the most prevalent surgical phenomena in all age groups and is increasingly seen in the elderly population . GeriatrIn this study, the perforation rate was found to be 2% and 66% in the early and late elderly groups, respectively. This shows that perforation was statistically significantly increased in the patient group aged 80 years or older compared to the patients that were 60 to 79 years old. This higher rate among the late elderly patients can be attributed to their low pain threshold, higher frequency of living alone, presence of multiple co-morbidities, and AA not being considered in initial diagnosis compared to the early elderly patients. Furthermore, the older patients generally have fewer physiological reserves, a higher rate of comorbidities, and a greater possibility of having had previous abdominal surgery, which all increase their surgical risks compared to the younger patient groups.p = 0.005). Mortality was also higher in patients aged 80 years and over. However, the 2% mortality rate in the early elderly group underlines the importance of considering AA for early diagnosis and immediate surgical treatment in the over-80 patient group. In this study, the need for postoperative intensive care in the late elderly group was found to be statistically significant. This may be associated with comorbidities or a result of late diagnosis and perforation.Tehrani et al. showed that patients who had undergone appendectomy for perforated appendicitis had higher complication and mortality rates than those having had appendectomy without peritoneal closure . AnotherIt has been reported that some surgeons hesitate to perform surgery on octogenarians and choose to monitor them until their symptoms become more pronounced, but this delays diagnosis and increases the possibility of perforation . Other rp = 0.000).In the literature, age was shown to have an independent effect on mortality and postoperative morbidity . BlomqviCurrent studies indicate that elderly patients have a longer hospital stay than younger patients following appendectomy, and the average of this duration is longer than one week . In thisGeriatric patient groups are usually difficult to manage and require a multidisciplinary approach in terms of diagnosis and treatment. A highly sensitive and effective diagnostic test for AA is very desirable in elderly patients in order to avoid high risks associated with surgery and possible complications due to delayed diagnosis. In the literature, RDW has been shown as a good indicator of systemic inflammation and mortality in elderly patients . The RDWet al., [Huive et al.,  detectedThe limitation of this study includes the retrospective design, and consequently the data being based on existing electronic records of the patients.In conclusion, the anamnesis of elderly patients is often incomplete, complex, and atypical or has different clinical features and multiple comorbidities. Especially in the octogenarian and nonagenarian patient groups, the presentation of AA in clinical practice is in the form of perforation. Therefore, these patients’ groups require a more careful, meticulous and rapid diagnosis.


To display microRNA155 (miRNA155) expression in different entities of Behçet’s disease (BD), and to find out if expression is affected in more than one of disease status than another, either phenotypically, according to HLA B51 expression, presence of family history, or patients’ age.Thirty BD patients (13 of which were HLAB51 positive) and 15 healthy subjects’ samples were obtained. White blood cell miRNA155 expression in both types of samples was estimated.Results showed that there is a degree of relation between decrease of miRNA155 expression and different disease aspects, and also, that miRNA155 has an inverse relation with the patients’ ages.MiRNA155 might be used as a measure of disease of different phenotypes, and that any manifestation of the disease can happen when the expression level decreases. White blood cell (WBCs) miRNA155 expression in both sample types was estimated through RNA extraction, reverse transcription, amplification, and then detection by small RNA assays.Figures 1 and 2).MiRNA155 expression levels were decreased non-significantly not only in the whole patients group compared to the control group, but also decreased non-significantly in all disease clinical presentations except for those not having eye problems who significantly had a decreased expression from the control group p= 0.013, and from patients with ocular involvement (p=0.041) .Figure 3): this may be explained by decreased disease activity with increasing age. These findings prove what was mentioned in several studies, that miRNA155 decreases with increase of BD activity, such as Hassouna et al.5Also, there was no significant difference between miRNA155 expression in patients having positive family history of BD (7 patients) and those who did not (p=0.081). MiRNA155 expression appeared to be directly proportional to age   through IL13 receptor alpha targeting .9 A study showed that high level of MiRNA155 was found in Tregs in a FOXP3-dependent way, and Treg homeostasis is disabled in miRNA155 insufficiency. When miRNA155 is missing in Treg, miRNA155 target of cytokine signalling 1 expression suppression increase with diminished responses to IL2, which is an imperative Treg homeostasis controller this was found by Lu et al.10 Moreover, it was discovered by Ceppi et al. that miRNA155 down-regulates lipopolysaccharide induced inflammatory pathways through inhibition of monocyte-derived DCs.11The results shown are coherent with researches telling that increase in miRNA155 is related to decreased expression in some cytokine, eg, interleukin (IL)1, IL 6 and IL 17, as is shown in Zhou et al.12 Another research found that T cells miR-155 lack in mice did not lead to advancement of extreme autoimmune encephalomyelitis (EAE) and has a diminished IL-17 generation which is a critical cytokine for EAE pathogenesis.13A few other researches revealed an inverse result to ours, that miRNA155 expression is expanded in activity of BD, and that MiR-155 focusing on Ets-1 controls Th17 response and that concealment of miR-155 diminishes pathogenic IL-17-expressing T cells, as was seen in the study of Na et al.14 found that mice lacking in miRNA155 have impedance in Th17 differentiation and in turn are protected from joint inflammation initiated by collagen.Moreover, Blüml et al.The opposite findings to our research results shown in some other studies may be due to ethnic differences, and different activity levels of the disease in the involved patients from one study to another. Also, several studies  were built on animal research, which cannot be relied on. Thus, there is a controversy about miRNA155 role in the pathogenesis of BD disease.MiRNA155 might be used as a measure of disease state. Any manifestation of the disease can happen when the expression level decreases.


Saliva may contain useful biomarkers which provide information about animal welfare using convenient and non-invasive sampling methods. In addition, the development of automated techniques of measuring analytes in saliva provides advantages from the technical point of view since they are cost-effective, reliable, and replicable. In this study, 21 salivary analytes measured by automated assays were tested as potential biomarkers of lameness, one of the most prevalent diseases in dairy cows producing significant economic losses. As a result, total esterase (TEA) showed increases in saliva in a group of 11 cows with lameness, decreasing when the lameness was solved after a specific treatment consisting of a hoof trimming and a medical treatment. In addition, TEA activity correlated with the severity of the lameness. Further studies using a larger population of cows with different causes of lameness and severity should be performed to determine the potential of TEA as a biomarker of lameness in cows.p = 0.140), days in milk (DIM) (p = 0.780), and body condition score (BCS) (p = 0.074). Initially, 21 biochemical analytes were determined in the saliva of six cows with lameness at the diagnosis time (T0) and twenty days after hoof trimming that successfully solved the lameness (TF). This exploratory study only showed significantly higher values in lipase (Lip) and total esterase (TEA) at T0 compared to TF . When both analytes were measured in the additional five lame cows and the results of all the animals of the lame group (n = 11) were compared with the healthy group (n = 11), only TEA showed higher activities in the group of lame cows than healthy cows (p = 0.004). TEA was positively correlated with both NRS and LSS . In conclusion, this study showed that cows with lameness in our experimental conditions had higher TEA values than healthy cows, and these values decreased after treatment. This is a pilot study, and further studies using a larger population of cows with lameness due to different causes and severity should be performed to determine the potential of TEA as a biomarker of lameness in cows.The possible changes in a panel of 21 salivary analytes on a population of cows with lameness before and after treating lameness by hoof trimming were analyzed. Then, the analytes that showed significant changes were studied in a larger population of cows with lameness and compared with healthy cows For this purpose, two groups of cows were made by a specialized veterinarian. One consisted of healthy cows with no external signs of diseases and no hematological or biochemical abnormalities, and showing no signs of lameness according to the numerical rating system of severity ; and the other composed of cows showing only lameness with a NRS of 3.1 ± 0.87 and a lesion scoring system  of 3.3 ± 0.89. Both groups did not differ in parity ( Lameness is, together with infertility and mastitis, one of the most prevalent diseases in dairy herds producing significant economic losses, reporting mean prevalence of lameness in dairy cows to be 31.6% in UK . It has In addition to the economic effect, lameness is recognized as a major welfare problem in cows. Cortisol has been extensively employed as a stress biomarker in lame cattle since acute pain induces activation of the hypothalamus-pituitary-adrenal (HPA) axis ,6,7, altSaliva rather than blood has been rising in interest in recent years as biologic fluid for the evaluation of welfare, stress, and disease in animals, since saliva can be easily and repeatedly collected in a large number of samples even at short-time intervals in a non-invasive way, without producing pain, and no need for specialized staff ,10. In tThe study hypothesizes that lameness in cows can produce changes in selected analytes in saliva and that possibly some of these analytes could reflect improvements in lameness after treatment. Therefore, this study aimed to evaluate changes in a panel of 21 salivary analytes in cows with lameness exploring the changes in these analytes after the lameness was solved.Twenty-four Holstein-Friesian dairy cows from a commercial dairy herd located in the southeast of Spain  were initially selected for this study. Information about the parity, days in milk (DIM), body condition score (BCS), and milk yield is described in The cows were fed with a total mixed base-ration offered once daily ad libitum at 08:30. From −50 days relative to calving, they received a far-off diet , and once calving the cows received a lactation diet . Water intake was available ad libitum. All the cows included in this study were housed in free-stalls  but with straw, and they were checked daily and milked two times a day.Two groups of cows were made. The lameness group (L group) was initially composed of twelve cows identified by a specialist veterinarian (PJV-M) for showing signs of lameness using a numerical rating system (NRS) based on a 5-point scale, in which a score of 1 represented a no-lame animal and 5 represented a severely lame animal  . The othOnce lameness was detected in the L group, one day later the cows were subjected to hoof trimming to characterize and treat the lameness. At that moment, two hoof-trimmers trained examined each hoof and recorded the presence of lesions by a 4-point lesion scoring system (LSS) according to Flower et al.  . Otherwise, the H group was sampling one day after the L group was sampling at T0 and TF, respectively. The samplings were made in the milking parlor right after the milking, after removing the milkers, and while the nipples were post-dipped, at the moment when the sampler had the opportunity to do it. The sampling procedure was always performed in the first milking between 07:00 and 12:00. All cows were sampled in a similar period of the day at each sampling time, with differences lower than two hours.g for 10 min at 4 °C. Saliva and plasma specimens were stored at −80°C (less than five months) until analysis.First, saliva samples were collected by introducing a sponge clipped to a flexible thin metal rod into the cow’s mouth to be chewed. The sponge was then placed in collection devices . Just after saliva sampling, blood was obtained by venipuncture in vena caudal using a sodium heparin device . Both saliva and blood devices were stored in ice until arrival at the processing laboratory (less than 2 h), where they were then centrifuged at 3.000× Enzymes: aspartate aminotransferase (AST), alanine aminotransferase (ALP), γ-glutamyl transferase (gGT), lipase (Lip), alpha-amylase, lactate dehydrogenase (LDH), creatine kinase (CK), butyrylcholinesterase (BChE), total esterase (TEA), and adenosine deaminase (ADA).Metabolites: creatinine, urea, uric acid, total bilirubin, triglycerides, glucose, and lactate.Proteins: total proteins and albumin.Minerals: phosphorus and total calcium.Hormones: cortisol.The salivary analytes evaluated in the chemistry profile were:This chemistry profile was carried out on an automated chemistry analyzer  and adapted to saliva, as previously reported . SalivarIn our study, the salivary chemistry profile was applied in an exploratory population of six cows from the L group before (T0) and after (TF) the treatment selected randomly to evaluate possible differences in the salivary chemistry profile. Then, the salivary analytes that showed significant changes and pCor were measured in the additional 5 lame cows (n = 11) and the healthy group (n = 11).p = 0.140) and DIM  were observed, although a tendency was in BCS . Therefore, a mixed linear model study was performed using the BCS as a covariate in the salivary analytes’ results previously selected after the exploratory study. An unpaired Student’s t-test (2-tailed) was also performed in the milk yield at T0 in both groups to assess possible differences.Previous the statistical analysis, data were checked for normality using the Shapiro-Wilk test, and those showing no normality were previously log-transformed to normalize it. The parity, DIM, and BCS at T0 in both groups were evaluated by an unpaired Student’s t-test (2-tailed) to guarantee no differences between groups. No differences in parity  and a power of 80%. It showed that a minimum number of 10 animals per group would be needed to get appropriate results for the selected analytes at different times. The two-way ANOVA of repeated measures followed by Sidak’s multiple comparisons tests were then used to assess whether significant changes existed between the 11 cows from the L group and the 11 cows from the H group.For salivary analytes evaluation, a paired Student’s t-test (2-tailed) was performed to assess the possible significant differences between T0 and TF in the exploratory population (six lame cows). In those analytes that showed significant changes, a stand-alone power program for statistical testing (G-Power)  was emplSpearman or Pearson r, depending on whether the data were non-normally or normally distributed, were calculated in the selected salivary analytes in the global population to evaluate the correlation between them and the pCor concentrations, NRS, and LSS. An r-value between 0.90 to 1 was considered to have a very high correlation, 0.70 to 0.90 high correlation, 0.50 to 0.70 moderate correlation, 0.30 to 0.50 low correlation, and less than 0.30 little if any correlation .p ≤ 0.05 were selected to indicate significance in all analyses.All statistical analyses were performed using the commercial statistics package GraphPad Prism 8 . Values of p = 0.028).Lower milk yield was observed in the L group compared to H group .p = 0.014), with higher concentrations in the L group (2.41 ± 1.09 µg/dL) than H group (1.30 ± 0.86 µg/dL) at T0 (p = 0.05). No significant differences between L and H groups at TF  were observed (p = 0.45).Cortisol in plasma showed changes between groups .When salivary analytes were evaluated in the exploratory population before and after lameness treatment , only Lip = 0.004) and TEA  showed no change between times in the H group , but higher activities at T0 than TF  in the L group (p = 0.050). The mix linear model study showed no significant effect of the BCS in Lip (p = 0.728) and TEA (p = 0.429) results.Lip  between them. TEA activity showed a low positive correlation with the NRS  and the LSS , while Lip only with the NRS . No correlations were observed between TEA (p = 0.723) and Lip (p = 0.936) in saliva with pCor.Correlations results are showed in In our study, changes in salivary analytes in cows with lameness were evaluated. The lameness severity showed a scoring mean of 3, considered as moderate . This deIn this trial, an extensive salivary chemistry profile was initially applied in lame cows before and after a treatment consisting of hoof-trimming. The evaluation of an extensive chemistry profile in saliva has been used in other species, such as horses , and it No relationship was observed between TEA with pCor, probably because of the different release source, being TEA related to the SNS activation, while pCor related to the HPA axis. In a study made in horses, no correlation between an analyte related to SNS, such as sAA, and sCor when a painful disease was observed . The difThis study has various limitations. Although previous publications evaluating lame cows used a similar number of animals than in this report ,33, it sIn conclusion, the research hypothesis of our study was confirmed, and lameness in cows produced changes in selected analytes in saliva, that reflect improvements in lameness after treatment. Namely, cows with lameness in the conditions of this study showed increases in TEA activities in their saliva compared to healthy cows, which decreased when a specific hoof treatment was performed. This report should be considered a pilot study, and further studies in a large population with cows with lameness due to different causes and severity should be performed to determine the potential of TEA as a biomarker of lameness in cows. If the TEA is confirmed as a biomarker in these additional studies, point-of-care assays that allow the measurement of this analyte quickly and easily on-farm conditions could be developed and applied in routine by farmers or under the coordination of Dairy Herd Improvement associations.


Increasing evidence has revealed that the initiation and progression of breast cancer are greatly affected by the immune environment. Neutrophils are the most abundant leucocytes in circulation and act as the spearhead in inflammation, including in breast cancer. Circulating neutrophils are closely related to the prognosis of breast cancer patients, and tumor-infiltrating neutrophils have varied functions at different stages of breast cancer, such as antitumor or tumor-promoting neutrophils, which are termed N1 and N2 neutrophils, respectively. In this review, we will discuss the utility of circulating neutrophils for predicting prognosis and therapeutic efficacy and the underlying mechanisms of their chemotaxis, the dynamic regulation of their antitumor or protumor functions and their different spatial distributions in tumor microenvironment. Finally, we also discuss the possibility of targeting neutrophils as a therapeutic strategy in breast cancer. AlthougNeutrophils can present both antitumorigenic (“N1”) and protumorigenic (“N2”) phenotypes in various cancers or specific circumstances. The term neutrophil in several studies also includes both mature neutrophils and myeloid-derived suppressor cells (MDSCs). MDSCs are described as a subset of neutrophils with immunosuppressive functions that express CD11b and Gr1 , 10 and To avoid confusion, we mainly focus on the biological function of mature neutrophils and related therapeutic strategies for targeting them in BC. We provide a comprehensive review of the prognostic value of circulating neutrophils and the mechanisms of how tumor-associated neutrophils (TANs) exert antitumor or tumor-promoting functions in BC, and in the end, we also discuss the potential of targeting neutrophils as a therapeutic strategy in cancer.Tumors can be thought of as wounds that will not heal and are characterized by chronic inflammation. Neutrophils are the most rapidly responding immune cells to inflammation, and many studies have found that the NLR is closely related to the prognosis and treatment response in patients bearing BC , 13. A rp = 0.012)  and the NLR eight consecutive times before and after chemotherapy found that patients who died had lower ALC and higher NLR values than patients who remained alive throughout the treatment course; additionally, among the patients who died, a steady increase in the NLR over the baseline measurement was observed at subsequent time points . Another= 0.012) . A low N= 0.012) , 31.p < 0.001) but not the PLR (p = 0.104) was a significant indicator for both DFS and OS  was in the group with an NLRhigh/PLRhigh profile  and decreased tumor-associated lymphocytes but was not significantly related to CD66b+ infiltrating neutrophils . Therefo+CD4+ and γδ T cells were the major sources of IL-17  usually acts as a damage-associated molecular pattern that is released by dying cells or stressed cells to initiate inflammation and was later found to be an important chemoattractant for neutrophils . Epithelin vivo , can alsin vivo . Meanwhiin vivo . It is nin vivo , 81. In in vivo , 83. Addin vivo .In addition, similar to lymphocytes and macrophages, neutrophils are more likely to localize in tumors of triple-negative breast cancer (TNBC) than to tumors of other BC subtypes . Recentl2O2), tumor necrosis factor (TNF)-α and NO, and that TANs are more likely to obtain a protumorigenic phenotype with tumor progression (lowCD54high) that was able to stimulate T cell proliferation and IFN-γ release, which suggested a pro-inflammatory rather than immunosuppressive state of TANs in early-stage lung cancer (2O2 to cytotoxic hypochlorous acid (HOCI)  , 89. Recd (HOCI) .2+ channels and mediated by transient receptor potential cation channel, subfamily M, member 2 (TRPM2) expression on tumor cells (http://gepia2.cancer-pku.cn/#index), active NOX1, catalase and SOD were also increased in the membrane of cancer cells, forming a complex mechanism by which tumor cell apoptosis induced by ROS is prevented  and CD56bright (protumor) subsets, and CD56bright NK cells are enriched in the tumor microenvironment and draining lymph nodes  . A mouseBecause of the lack of direct information from previous publications, we tried to determine the abundance and subtype of TANs in human BC via the CIBERSORT-LM7 deconvolution algorithm based on mRNA expression datasets  . Our anaChemotherapy and radiotherapy are integral parts of BC treatment that can influence the immune microenvironment. Anthracycline and cyclophosphamide-based chemotherapy regimens are still widely used in BC treatment , 145. Itin vivo  but not microbeam radiation therapy (MRT) induced a substantial increase in TAMs and TANs, and increased levels of CCL2  were also observed in tumors subjected to CRT . In addiin vivo . Furtherin vivo –158. Howin vivo .Since the majority of studies have revealed a protumor function of neutrophils in BC, targeting neutrophils as a therapeutic strategy has been investigated. In mouse mammary tumor models, depletion of neutrophils with anti-Ly6G antibodies resulted in diminished tumor formation and lung metastasis . In addiIFN-β and TGF-β are the cytokines that are most often reported to modulate the switch between N1 and N2-like neutrophil polarization. Steven et al. first revealed that TGF-β blockade significantly increased the influx of antitumor neutrophils and activated CD8+ T cells in a BC mouse model . ThereafA schematic picture depicting the dual role of neutrophils in BC . Here, wYS and CN wrote the manuscript. CZ and WZ edited the manuscript. YS, WZ, and SP collected the related literatures. HH, JJ, and WC finished the tables and figures. TZ provided the feedback and guidance. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To investigate the association of prenatal alcohol exposure (PAE) and early neurodevelopment in the first 2 years of life, adjusting for maternal socio-demographic and psychosocial factors, in the Drakenstein Child Health Study (DCHS), a South African birth cohort study.n = 260) and 24 months of age (n = 734). A subset of alcohol-exposed and -unexposed children was included in this analysis at age 6  and 24 months . Multiple hierarchical regression was used to explore the associations of PAE with motor and language development.The DCHS comprises a population-based birth cohort of 1143 children, of which a subsample completed the Bayley Scales of Infant Development-III (BSID-III) at 6  functioning after adjusting for maternal socio-demographic and psychosocial factors at 6 months of age only. No significant effects were found in either receptive or expressive communication and cognitive outcomes at either time points.PAE was significantly associated with decreased gross motor [odds ratio (OR) = 0.16, 95% confidence interval (CI) = 0.06–0.44, PAE has potentially important consequences for motor development in the first 2 years of life, a period during which the most rapid growth and maturation occur. These findings highlight the importance of identifying high-risk families in order to provide preventive interventions, particularly in antenatal clinics and early intervention services. PAE had a significant impact on motor functioning after adjusting for variety of socio-demographic and psychosocial factors at 6 months of age, but not on cognitive or language development.The findings of this study highlight the importance of identifying psychosocial risk factors, particularly in antenatal clinics and early intervention services in a South African context.The BSID-III tool assesses general ability of a given task but may have low sensitivity for detecting minor developmental impairments especially during infancy.Language impairments are very subtle in the early years and may be more difficult to identify impairments than in other domains.A small sample size may have reduced the power of the study, and findings may not be generalisable to other populations.et al., et al., et al., et al., et al., et al., et al., et al., Prenatal alcohol exposure (PAE) has been recognised as a major global public health concern. A recent study estimated 9.8% of mothers consumed alcohol during pregnancy and 4.3% were heavy drinkers  is a pattern of often irreversible physical and mental birth deficiencies , a South African birth cohort study.et al., et al., et al., et al., This study formed part of the DCHS, a multidisciplinary birth cohort study investigating the early determinants of child health , whereas the full cohort was invited to participate at 24 months (n = 734) making a larger sample available. At 6 and 24 months of age, a subset of infants and toddlers were selected whose mothers reported moderate-to-severe levels of alcohol consumption and for whom BSID-III data were available. Of the 260 infants, 52 were exposed to alcohol at 6 months, and of the 734 toddlers, 92 were exposed to alcohol at 24 months. The unexposed group comprised 104 at 6 months and 184 at 24 months. Unexposed control children were randomly matched for maternal education and clinic site in a 1:2 ratio , every effort was made to ensure a safe, anonymous, confidential and supportive environment. Translation of the measures from English to Afrikaans and isiXhosa included a standard forward and back-translation process , marital status and HIV status  and psychological variables (PTSD and depression) administered antenatally. Maternal smoking was assessed using the ASSIST  were presented for normally distributed data. Medians [interquartile range (IQR)] were presented for data that were not normally distributed and for all BSID-III scores. For comparisons between alcohol-exposed and -unexposed children, chi-squared tests were used for categorical variables, while p = 0.001) and 24 months of age . There were no significant differences between the exposed and unexposed groups regarding infants’ birth weight and BMI.The maternal and child socio-demographic and psychosocial characteristics are presented in Table p = 0.006; fine: median scores = 11.5 (IQR = 10.0–13.0) vs. 13.0 (IQR = 12.0–15.0), respectively, p = 0.001)]. At 24 months, there were no significant differences, although there remained a trend towards impairment for fine motor functioning in exposed children . There were no significant differences in language and cognitive functioning at 6 or at 24 months.Table p = 0.001] and fine motor functioning  after controlling for BMI, SES, and smoking and psychological variables at 6 months of age. BMI was significantly associated with both gross  and fine motor functioning , while SES was significantly associated with gross motor functioning  at 6 months.Table F(4) = 3.98, p = 0.002, adj R2 = 0.10), and the R2 showed that the amount of variance increased from 5%  to 14% after adding PAE into the model (Appendix F(4) = 3.66, p = 0.004, adj R2 = 0.13), and the R2 showed that the explained variance accounted for an extra 9% (0.04–0.13) in fine motor functioning (Appendix The final model explained a significant amount of variance in gross motor functioning (This study comprehensively assessed motor, language and cognitive functioning in a population-based cohort over the first 2 years of life. The findings of our study indicated that PAE is associated with both gross and fine motor functioning at 6 months of age, even after adjusting for maternal socio-demographic and psychosocial factors. While PAE was not associated with receptive and expressive communication nor cognitive performance at either time point in this group, there remained a trend towards significance for poorer fine motor functioning at 24 months of age.et al., Our findings demonstrate that PAE is associated with deficits in motor functioning across the first 2 years of life. This is consistent with previously reported cohort studies in preschool age (Fried & Watkinson, et al., et al., In our cohort, PAE was not found to be associated with receptive or expressive communication or cognitive functioning at the age of 24 months. Previous studies have reported impairments in language and cognitive functioning in toddlers between the ages of 12 and 24 months (Fried & Watkinson, Additional limitations deserve consideration. Firstly, the substudy comprised a small sample size which may have limited the power to detect differences between the groups. Secondly, despite assurances of confidentiality, some women may have chosen not to disclose or minimise reporting alcohol use to the research teams and the low reported alcohol consumption may therefore represent an element of response bias. Thirdly, the BSID-III tool measures general ability in completing a given task but may have low sensitivity for detecting minor developmental impairments especially during infancy. Further, although this tool has been validated for use in South Africa, this study may not be generalisable to other populations.A large proportion of very young children in LMICs do not reach their developmental potential due to a wide variety of socio-demographic and psychosocial factors that may impact early developmental outcomes. Our study, reporting the association of PAE and early motor functioning, is one of only a few studies that have additionally addressed important potential psychosocial confounders which frequently co-occur with alcohol use in these communities. These findings highlight the importance of identifying high-risk families in order to provide preventive interventions, particularly in antenatal clinics and early intervention services.


For more than three decades, numerous studies have demonstrated the function of p53 in cell cycle, cellular senescence, autophagy, apoptosis, and metabolism. Among diverse functions, the essential role of p53 is to maintain cellular homeostatic response to stress by regulating proliferation and apoptosis. Recently, adipocytes have been studied with increasing intensity owing to the increased prevalence of metabolic diseases posing a serious public health concern and because metabolic dysfunction can directly induce tumorigenesis. The prevalence of metabolic diseases has steadily increased worldwide, and a growing interest in these diseases has led to the focus on the role of p53 in metabolism and adipocyte differentiation with or without metabolic stress. However, our collective understanding of the direct role of p53 in adipocyte differentiation and function remains insufficient. Therefore, this review focuses on the newly discovered roles of p53 in adipocyte differentiation and function. The multifunctional protein p53  is a key regulator of cell proliferation and apoptotic response to exogenous or endogenous cellular stresses . Under vIn the absence of cellular stresses, mouse double minute 2 (MDM2), an E3 ubiquitin ligase, regulates p53 levels via proteasomal degradation pathways . When ceAdipose tissue plays an important role in the regulation of systemic energy balance and glucose homeostasis. For example, adipose tissues regulate energy storage, lipid metabolism, glucose homeostasis, and energy expenditure . The epiOn the other hand, the main function of WATs is energy storage in the triglyceride form. WATs are generally located subcutaneously throughout the body and constitute as much as 20–30% of the body weighty of normal humans. WAT progenitor cells are differently developed according to location; subcutaneous white adipose tissues develop before birth but visceral white adipose tissues develop after birth ,27. HoweIn this review, we discuss the important role of p53 and highlight the key molecules that are regulated by p53 during adipogenesis. We also discuss the role of p53 in regulating metabolism in adipose tissues. Finally, we identify important outstanding questions and issues pertaining to the role of p53 in adipogenesis that remain to be addressed.p53 is a regulator of cell survival and proliferation in response to multiple stress signals against abnormal cell growth and tumorigenesis. Apart from its role as a tumor suppressor, p53 has been involved in the regulation of differentiation of mesenchymal cells that can suppress white adipocyte differentiation ,33,34. WCarm1 and reduced its protein expression  variant of p53 who were administered a high-fat diet developed severe obesity and metabolic dysfunction than mice expressing a proline 72 variant . In lineBATs are located around the central organs and are essential for whole-body energy homeostasis, particularly energy expenditure ,64. HencUcp1 transcriptional activity [Foxc2 in mouse preadipocytes, which increased the expression of cyclin E and inhibited that of p53 and p27 in preadipocytes (A recent study performed by Molchadsky et al. demonstrated that p53 is a positive regulatory factor that maintains proper brown adipocyte maturation in vitro and prevents the development of obesity because of a high-fat diet in the mouse model . Loss ofactivity ,74. Overactivity . In vivoactivity . This phactivity . Foxc2 iactivity ,77. Gan ipocytes .On the other hand, Hallenborg et al. showed that depletion of p53 in mice protected them against high-fat diet-induced obesity . Loss ofPRDM16 expression via interaction with PRDM16 or binding to the PRDM16 promoter [p53 has been known to be involved in the aging process of WATs . The totpromoter ,47. HoweOver the past four decades, the roles of p53 have been extensively studied because the mutation or inhibition of p53 initiates tumorigenesis ,81,82,83Heretofore, p53 role in adipogenesis, especially white adipocytes, is a negative regulator of adipogenesis. However, the role of p53 in negative regulation of adipogenesis under exact molecular upstream and downstream networks remains unknown. Generally, p53 is considered a stress response protein. WATs have been considered to be simple energy storage tissues containing lipid droplet organelles but they also have endocrine functions and secrete several factors such as adipokines . The assOne of the major unsolved questions is the direct role of p53 in brown adipogenesis. Previously performed studies have reported contrasting results regarding the role of p53 in brown adipogenesis. Inhibition of p53 suppressed brown adipogenesis in Prdm16-overexpressed myogenic C2 cells in vitro . On the The other mechanism by which p53 regulates cellular differentiation involves its chromatin-binding activity or interaction with specific protein partners. As described above, p53 affects adipogenesis dependent cell types in vitro. These results suggest that promoter remodeling following p53 binding to chromatin occurs at different sites in different adipogenic stages or binding with different protein partners in each cell. BATs and WATs share many essential adipogenic regulatory transcriptional factors including PPARγ and CEBPα. If p53 regulates promoter remodeling in the same way, there is no different response to adipogenic development regardless of the cell type. p53 interacts with chromatin response to diverse signaling and regulates transcription of specific target genes with or without co-binding factors ,91,92. SThe role of p53 in tumorigenesis has been extensively studied because the activation of p53 kills cancer cells . Many re


ARF is a tumor suppressor encoded by the CDKN2a locus that is frequently inactivated in human tumors. P14ARF protein quenches oncogene stimuli by inhibiting cell cycle progression and inducing apoptosis. P14ARF functions can be played through interactions with several proteins. However, the majority of its activities are notoriously mediated by the p53 protein. Interestingly, recent studies suggest a new role of p14ARF in the maintenance of chromosome stability. Here, we deepened this new facet of p14ARF which we believe is relevant to its tumor suppressive role in the cell. To this aim, we generated a monoclonal HCT116 cell line expressing the p14ARF cDNA cloned in the piggyback vector and then induced aneuploidy by treating HCT116 cells with the CENP-E inhibitor GSK923295. P14ARF ectopic re-expression restored the near-diploid phenotype of HCT116 cells, confirming that p14ARF counteracts aneuploid cell generation/proliferation. P14 ARF which has a potent tumor suppressor activity. Loss of the CDKN2a locus is the second most frequent copy number alteration that characterizes human tumors and cancer cell lines. One of the most well-defined functions of p14ARF protein is to suppress aberrant cell proliferation in response to oncogene insults by activating the transcription factor p53 that, in turn, triggers the expression of many apoptosis inducers and cell cycle inhibitory genes [ARF binds the p53-specific E3 ubiquitin ligase Mouse double minute 2 homolog (Mdm2) and keeps it in the nucleolus. This event prevents the interaction between Mdm2 and p53 by blocking the transport to the cytoplasm of p53 and its degradation by the proteasome Mdm2-mediated [The alternate open reading frame (ARF) gene was originally identified as an alternative transcript of the CDKN2a locus on human chromosome 9p21 that encodes the nucleolar protein p14ARF, p53, and MDM2 genes were more tumour prone when compared to mice lacking only p53 and MDM2 [ARF is highlighted by the effect of its loss in mouse models: mice were highly prone to develop tumors and mostly died during their first year of life. In addition, p14ARF-null mouse embryonic fibroblasts (MEFs) can overcome the senescence crisis and proliferate continuously, and they also can be easily transformed by the Ras oncogene [ARF is a tumor suppressor it is not surprising that most human cancers  are characterized by its inactivation mainly due to its promoter hypermethylation, homozygous deletions and mutations  that was the result of chromosome mis-segregation [ARF is involved in the cell response to the aneuploid phenotype triggered by the impairment of different types of proteins, namely MAD2 and DNMT1 [ARF can respond either by arresting the cell cycle and entering senescence if necessary or by inducing apoptosis [ARF is critical to halt aneuploidy. To this regard, we recently demonstrated that when CENP-E is reduced by 50% and aneuploidy is consequently induced, HCT116 cells, where p14ARF is not expressed [ARF induction. HCT116 cells could finally recapitulate IMR90 cell behaviour and recover from aneuploidy once they ectopically re-express p14ARF [ARF is involved in blocking the proliferation of aneuploid cells. However, we only analysed the effects of p14ARF ectopic expression in a heterogenous population of HCT116 cells expressing different levels of p14ARF and we did not investigate on the mechanism that allows the elimination of the aneuploid cells.On the other hand, there is some evidence that ARF, especially its small mitochondrial isoform (smARF) , also haand MDM2 . In addiand MDM2 . Moreoveand MDM2 . The imponcogene . Consideiewed in . Due to iewed in ,11. ARF regation ,13. Theyregation . On the nd DNMT1 . In partpoptosis ,16. Altoxpressed , are nots p14ARF . These eARF cDNA integrated in the genome specifically at ‘TTAA’ sites by the PiggyBac transposon vector system (ePB) (ARF c-DNA and the vector carrying the transposase enzyme (hyPB). Transfected cells were then selected with the antibiotic blasticidin for ten days and then single colonies were picked, analysed and one positive clone was chosen for further investigation. In the engineered HCT116 cells (HCT116 ePB cells), p14ARF cDNA expression is under the control of the TET-On technology that allows activation of gene expression by administration of doxycycline. By using a low dose of doxycycline (10ng/mL) we obtained the consistent expression of p14ARF  A,B. Brief p14ARF C–E. Reve16 cells C. Moreov protein D,E. Follgression F,G. ARF induction/re-expression , these cells became aneuploid (50%). However, when doxycycline was washed out and thus p14ARF was expressed, the percentage of aneuploid cells was reduced from 50% to 22% [ARF could partially reduce aneuploidy of CENP-E-depleted HCT116 cells from 73% to 56% [ARF ectopic re-expression restore the near-diploid phenotype of HCT116 cells. These results confirm that p14ARF counteracts aneuploid cell generation/proliferation. It is well established that p14ARF regulates cell cycle arrest and apoptosis, however it remains unclear if p14ARF uses one or both mechanisms to fight aneuploidy [ARF after treatment with GSK923295 (ARF was expressed. This made us think that apoptosis could likely be the way through which p14ARF induces the clearance of the aneuploid cells. We hypothesized thus that this could be the mechanism through which p14ARF helps the maintenance of chromosomal stability. Hence, to identify the presence of apoptotic cells, we performed acridine orange and ethidium bromide staining which discriminates live, apoptotic, and necrotic cells. By this mean we observed 25% of apoptotic cells in HCT116 cells expressing p14ARF following GSK923295 treatment. These results strongly suggest that p14ARF triggers the apoptotic pathway to halt aneuploid cell proliferation.We then induced aneuploidy in these engineered HCT116 cells by treating them for 72 h with the CENP-E inhibitor GSK923295 . GSK9232pression A,B. As e% to 22% . In addi% to 56% . Therefoeuploidy ,14,21. TSK923295 C–F. PropARF mutations. Though, p14ARF role in cancer as tumor suppressor is vastly demonstrated. Noteworthily, it has been noticed that patients with mutations in p14ARF have high risk of developing melanoma and pancreatic cancer [ARF has a fundamental role in fighting cell transformation and suggest that, due to the high frequency of deregulation in cancer, this role could be played at early stages of cell transformation. We think that this could be due to p14ARF ability in counteracting aneuploidy. Through this mechanism numerical chromosomal stability would be preserved in normal cells. This is well-known during development of Drosophila melanogaster that indeed uses apoptosis to eliminate aneuploid cells . In addic cancer ,25. Furtc cancer . AltogetARF was firstly identified as an alternate transcript of the CDKN2a locus, which is the second most frequently mutated site in cancers after p53. Its tumor suppressor role as regulator of the cell cycle and apoptosis has been indeed well established. Classically, a hyperproliferative signal that results from an oncogenic insult triggers ARF activation. In this brief report, by confirming previous results, we suggest that p14ARF can also respond to the onset of aneuploidy identification, thus providing an additional function as a sentry of the euploid set of chromosomes. Remarkably, aneuploidy, i.e., an abnormal number of chromosomes, is a hallmark of virtually all tumors. We observed that the absence of p14ARF worsens the aneuploid phenotype of the cells, however it was generated. These cells, indeed, cannot either block their cell cycle or enter apoptosis, and therefore, as a matter of fact, the possibility to impede the proliferation of aneuploid cells is denied. The maintenance of aneuploidy can undoubtedly contribute to malignant transformation. The presence/restoration of p14ARF, instead, allows cells to counteract aneuploidy, most probably by sentencing the aneuploid cells to apoptosis. In fact, this mechanism of aneuploid cell elimination by apoptosis has been observed during development. It is conceivable that this function of ARF is an important part of its role as a tumor suppressor. Furthermore, it is generally established that gene copy number correlates with gene expression. It is not too daring to think that the imbalance of gene expression that is induced by aneuploidy could then generate a signal resembling the hyperproliferative stress typically sensed by p14ARF. This, in turn, could activate p53-dependent apoptosis. In conclusion, through this evidence, it is clear that p14ARF not only responds to oncogene stimuli or to DNA damage, but also to an “aneuploidy signal” and protects the cells from the maintenance of such aberrant and potentially dangerous condition. P14