A transcriptional switch controls sex determination in Plasmodium falciparum.

Sexual reproduction and meiotic sex are deeply rooted in the eukaryotic tree of life, but mechanisms determining sex or mating types are extremely varied and are only well characterized in a few model organisms1. In malaria parasites, sexual reproduction coincides with transmission to the vector host. Sex determination is non-genetic, with each haploid parasite capable of producing either a male or a female gametocyte in the human host2. The hierarchy of events and molecular mechanisms that trigger sex determination and maintenance of sexual identity are yet to be elucidated. Here we show that the male development 1 (md1) gene is both necessary and sufficient for male fate determination in the human malaria parasite Plasmodium falciparum. We show that Md1 has a dual function stemming from two separate domains: in sex determination through its N terminus and in male development from its conserved C-terminal LOTUS/OST-HTH domain. We further identify a bistable switch at the md1 locus, which is coupled with sex determination and ensures that the male-determining gene is not expressed in the female lineage. We describe one of only a few known non-genetic mechanisms of sex determination in a eukaryote and highlight Md1 as a potential target for interventions that block malaria transmission.

Anxiety and burnout in young athletes: The mediating role of cognitive appraisal.

This study tested the relationship between trait anxiety, cognitive appraisal, and athletes' burnout proposing two hypotheses: (a) there is a direct relationship between athletes' trait anxiety and cognitive appraisal and burnout, and (b) cognitive appraisal mediates the relationship between trait anxiety and burnout, and this mediation occurs despite the competitive level and sport records of athletes. The study included 673 young athletes and provided measures of trait anxiety, cognitive appraisal, and burnout. Structural equation modeling indicated that cognitive appraisal mediates the relationship between trait anxiety and burnout, confirming hypothesis 2, and this model provided better fit than the direct model of hypothesis 1. However, the mediation also indicated that the direct relationship between trait anxiety and burnout should be considered. The mediating model was invariant according to competitive levels and sport records. In conclusion, cognitive appraisal is an important variable in explaining athletes' burnout.

Physical exercise, pain and musculoskeletal function in patients with haemophilia: a systematic review.

INTRODUCTION: Exercise can provide numerous benefits to haemophilia patients, including bleeding reduction in muscles and joints. AIM: This systematic review (SR) aims to evaluate the effects of physical exercise on pain and the musculoskeletal function of patients with hemophilia. METHODS: Literature searches of Pubmed, Web of Science, PEDro, Cochrane, Clinical Trials SciELO and Lilacs were performed. The risks of bias were measured using the JADAD scale. RESULTS: Nine controlled clinical trials were included in the SR. CONCLUSION: Physical exercise can promote a reduction in the perception of pain and can increase ROM and muscle strength in haemophilia patients. Future RCTs with greater methodological rigor that focus on the parameters used to prescribe exercises are necessary.

The calcium-sensing receptor in bone metabolism: from bench to bedside and back.

UNLABELLED: The calcium-sensing receptor (CaSR), a key player in the maintenance of calcium homeostasis, can influence bone modeling and remodeling by directly acting on bone cells, as demonstrated by in vivo and in vitro evidence. The modulation of CaSR signaling can play a role in bone anabolism. INTRODUCTION: The calcium-sensing receptor (CaSR) is a key player in the maintenance of calcium homeostasis through the regulation of PTH secretion and calcium homeostasis, thus indirectly influencing bone metabolism. In addition to this role, in vitro and in vivo evidence points to direct effects of CaSR in bone modeling and remodeling. In addition, the activation of the CaSR is one of the anabolic mechanisms implicated in the action of strontium ranelate, to reduce fracture risk. METHODS: This review is based upon the acquisition of data from a PubMed enquiry using the terms "calcium sensing receptor," "CaSR" AND "bone remodeling," "bone modeling," "bone turnover," "osteoblast," "osteoclast," "osteocyte," "chondrocyte," "bone marrow," "calcilytics," "calcimimetics," "strontium," "osteoporosis," "skeletal homeostasis," and "bone metabolism." RESULTS: A fully functional CaSR is expressed in osteoblasts and osteoclasts, so that these cells are able to sense changes in the extracellular calcium and as a result modulate their behavior. CaSR agonists (calcimimetics) or antagonists (calcilytics) have the potential to indirectly influence skeletal homeostasis through the modulation of PTH secretion by the parathyroid glands. The bone anabolic effect of strontium ranelate, a divalent cation used as a treatment for postmenopausal and male osteoporosis, might be explained, at least in part, by the activation of CaSR in bone cells. CONCLUSIONS: Calcium released in the bone microenvironment during remodeling is a major factor in regulating bone cells. Osteoblast and osteoclast proliferation, differentiation, and apoptosis are influenced by local extracellular calcium concentration. Thus, the calcium-sensing properties of skeletal cells can be exploited in order to modulate bone turnover and can explain the bone anabolic effects of agents developed and employed to revert osteoporosis.

A weighted and directed interareal connectivity matrix for macaque cerebral cortex.

Retrograde tracer injections in 29 of the 91 areas of the macaque cerebral cortex revealed 1,615 interareal pathways, a third of which have not previously been reported. A weight index (extrinsic fraction of labeled neurons [FLNe]) was determined for each area-to-area pathway. Newly found projections were weaker on average compared with the known projections; nevertheless, the 2 sets of pathways had extensively overlapping weight distributions. Repeat injections across individuals revealed modest FLNe variability given the range of FLNe values (standard deviation <1 log unit, range 5 log units). The connectivity profile for each area conformed to a lognormal distribution, where a majority of projections are moderate or weak in strength. In the G29 × 29 interareal subgraph, two-thirds of the connections that can exist do exist. Analysis of the smallest set of areas that collects links from all 91 nodes of the G29 × 91 subgraph (dominating set analysis) confirms the dense (66%) structure of the cortical matrix. The G29 × 29 subgraph suggests an unexpectedly high incidence of unidirectional links. The directed and weighted G29 × 91 connectivity matrix for the macaque will be valuable for comparison with connectivity analyses in other species, including humans. It will also inform future modeling studies that explore the regularities of cortical networks.

Eating behaviours in youths: A comparison between female and male athletes and non-athletes.

This study compared the different factors associated with eating behaviors among young female and male athletes and non-athletes. A total of 580 female and male athletes and 362 female and male non-athletes between 10 and 19 years old participated. We used the subscales of the Eating Attitudes Test (EAT-26) to evaluate the factors associated with unhealthy eating behaviors. We found higher scores for females on the diet subscale compared with males, regardless of athletic group (P < 0.05). Non-athlete youths scored higher on this subscale compared with male athletes (P < 0.05). Our findings indicate higher scores for female athletes with regard to the bulimia and preoccupation with food subscale compared with other the groups (P < 0.05). Moreover, we observed that non-athlete males were more likely to engage in binge eating compared with athletes of the same sex (P < 0.05). Finally, females had higher scores on the oral self subscale than males, regardless of athletic group (P < 0.05). We concluded that the factors associated with eating behaviors differ with regard to sex and group.

Incidence of subclinical mastitis and prevalence of major mastitis pathogens in organized farms and unorganized sectors.

Subclinical mastitis (SCM) represents a major proportion of the burden of mastitis. Determining somatic cell count (SCC) and electrical conductivity (EC) of milk are useful approaches to detect SCM. In order to correlate grades of SCM with the load of five major mastitis pathogens, 246 milk samples from a handful of organized and unorganized sectors were screened. SCC (>5 × 10(5)/mL) and EC (>6.5 mS/cm) identified 110 (45 %) and 153 (62 %) samples, respectively, to be from SCM cases. Randomly selected SCM-negative samples as well as 186 samples positive by either SCC or EC were then evaluated for isolation of five major mastitis-associated bacteria. Of the 323 isolates obtained, 95 each were S. aureus and coagulase-negative staphylococci (CoNS), 48 were E. coli and 85 were streptococci. There was no association between the distribution of organisms and (a) the different groups of SCC, or (b) organised farms and unorganised sectors. By contrast, there was a significant difference in the distribution of CoNS, and not other species, between organized farms and unorganized sectors. In summary, bacteria were isolated irrespective of the density of somatic cells or the type of farm setting, and the frequency of isolation of CoNS was higher with organized farms. These results suggest the requirement for fine tuning SCC and EC limits and the higher probability for CoNS to be associated with SCM in organized diary sectors, and have implications for the identification, management and control of mastitis in India.

Hepatocellular carcinoma and chemotherapy: the role of p53.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common primary neoplasm of the liver. A major proportion of HCCs also present mutation of the gene that encodes p53, which confers chemoresistance. The main goal of this work is to investigate the effect of cisplatin, doxorubicin and 5-fluoruracil (5-FU) in three human HCC cell lines which differ in p53 expression. METHODS: HepG2 (expressing normal p53), HuH7 (expressing mutated p53) and Hep3B2.1-7 (not expressing p53) cell lines were cultivated in the presence of cisplatin, doxorubicin and 5-FU. Cell proliferation was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT assay). The type of cell death and Bax and Bcl2 activation were assessed by flow cytometry. RESULTS: It was found that for all of the cell lines studied, the agent that gave the most satisfactory results was doxorubicin. 5-FU demonstrated no activity in these cell lines. CONCLUSIONS: For all the cell lines studied, doxorubicin was the most satisfactory agent. In HepG2 and HuH7 cell lines, it can activate Bax with statistical significance.

Phase Angle Reflects Loss of Functionality in Older Women.

OBJECTIVE: This study verified if the phase angle (PA) formed by the resistance (R) and reactance (Xc) obtained from bioimpedance (BIA) might be a useful tool to identify functionality. DESIGN AND PARTICIPANTS: It was conducted a cross-sectional study with 152 women ≥ 65 years old from the community. MEASUREMENTS: weight, height, body mass index (BMI), calf circumference (CC), hand grip strength (HGS), gait speed (GS), fat free mass (FFM), body fat (BF), PA with R and Xc from the BIA were measured. Spearman's and Pearson's correlations and the odds ratio (OR) were performed using the IBM SPSS software version 22.0. RESULTS: Sixty-four percent (n=98) women are with PA below the reference. Negative moderate significant correlation was found between PA and age (r =- 0.440*; p<0.001). Moderate significant correlation was observed between PA and GS (r = 0.484**; p<0.001). Weak significant correlation was found between PA and HGS (r = 0.177*; p = 0.029). Odds ratio (OR) demonstrated that individuals with PA above the mean value have 4.77 times more chances of having increased GS (confidence interval 2.40-9.48; 95%). Women aged below the mean value have 4.02 times more chances of having higher PA (confidence interval 2.02-7.99; 95%). Younger aged women showed 4.02 times more chances of having higher PA (confidence interval 2.02-7.99; 95%). CONCLUSIONS: Phase angle can be associated with functional tests, such as gait speed and hand grip strength, in older women.

Anticancer efficacy of 6-thioguanine loaded chitosan nanoparticles with or without curcumin.

6-Thioguanine encapsulated chitosan nanoparticles (6-TG-CNPs) has formulated by the ionic-gelation method. Morphologically, the 6-TG-CNPs were spherical and showed mean size, PDI, zeta potential, and entrapment efficiency of 261.63 ± 6.01 nm, 0.34 ± 0.10, +15.97 ± 0.46 mV and 44.27%, respectively. The IR spectra confirmed the 6-TG complex with chitosan. The in vitro drug release profile of 6-TG-CNPs revealed an increase in sustained-release (91.40 ± 1.08% at 48 h) at pH 4.8 compared to less sustained-release (73.96 ± 1.12% at 48 h) at pH 7.4. The MTT assay was conducted on MCF-7 and PA-1 cell lines at 48 h incubation to determine % cell viability. The IC50 values of 6-TG, 6-TG-CNPs, and curcumin for MCF-7 were 23.09, 17.82, and 15.73 µM, respectively. Likewise, IC50 values of 6-TG, 6-TG-CNPs, and curcumin for PA-1 were 5.81, 3.92, and 12.89 µM, respectively. A combination of 6-TG-CNPs (IC25) with curcumin (IC25) on PA-1 and MCF-7 showed % cell viability of 43.67 ± 0.02 and 49.77 ± 0.05, respectively. The in vitro cytotoxicity potential in terms of % cell viability, early apoptosis, G2/M phase arrest, and DNA demethylating activity of 6-TG-CNPs alone and combination with curcumin proved to be more effective than that of 6-TG on PA-1 cells.

MMP-2 is not altered by stretching in skeletal muscle.

Considering that short bouts of stretching, as recommended in rehabilitation and sports activities, induce skeletal muscle and connective tissue adaptation, the hypothesis of this study was that MMP-2 activity is regulated by muscle stretch. The level of MMP-2 activity was, thereby, assessed after stretching in rat soleus muscle. Animals received a single session of stretching (10 stretches lasting 1 min each with 30 s of rest in between) and were evaluated immediately and after 8, 24, 48, 72 and 168 h. To evaluate the effect of repetitive sessions of stretching, three groups of animals were evaluated - one group after 2 sessions, another after 3, and a third after 7. MMP-2 activity was evaluated by zymography and MMP-2 mRNA was assessed by real-time polymerase chain reaction. None of the groups presented MMP-9 activity. MMP-2 activity and mRNA expression did not change after either single or repetitive sessions of stretching. In conclusion, the results of this study indicate that MMP-2 is not involved in the muscle stretch-induced remodeling.

A short note on peste des petits ruminants in Karnataka, India.

Retrospective quantitative analysis of epidemiological data on peste des petits ruminants (PPR) from the Department of Animal Husbandry and Veterinary Sciences in Karnataka, southern India, revealed significant information about the disease in this area. In the nine years between April 1998 and March 2007 a total of 624 outbreaks were reported in the state. With the exception of the 12-month period between April 2001 and March 2002 the disease occurred every year. The study shows clearly that incidences were highest during the rainy season and in the dry agro-climatic zones. The density of the PPR-susceptible population in different districts of the state played a major role in disease incidences. Environmental factors also influenced disease occurrence. Vaccination programmes are slowly being taken up in the state. The disease data documented in this study provide information about the endemicity of the disease that can help to formulate an effective strategy for a PPR-control programme in the state.

Correction: FOXO3a represses VEGF expression through FOXM1-dependent and -independent mechanisms in breast cancer.

In the published version of this article, the images for cytoplasmic and nuclear FGF7 in MDA-MB-231 cells were duplicated and mistaken for total FGF7 in SKBR-3 and MDA-MB-231 cells.

Genotoxicity effects on Geophagus brasiliensis fish exposed to Doce River water after the environmental disaster in the city of Mariana, MG, Brazil.

The Doce River basin has suffered the largest environmental accident ever occurred in Brazil with the influx of tailings from Fundão and Santarém, belonging to Samarco mining company, due to the disaster in Mariana. A spill between 50 and 60 million m3 of tailings was estimated by the company. According to Samarco, the wastewater was composed mainly of clay, silt and heavy metals like iron, copper and manganese. Thereby, the objective of the present study was evaluated the genotoxic damage in juvenile of Geophagus brasiliensis (Quoy e Gaimard, 1824) exposed to Doce river water before (DRWBA - Doce River water before acident) and after (DRWAA - Doce River water after acident) the influx of tailings from the Germano and Santarém Dam disasters in Mariana, MG, Brazil. For this, 24 individuals of the species G. brasiliensis (obtained on IFES/ALEGRE fish culture) were submitted to a bioassay with three treatments and eight replicates. The treatments were: 1) Control water (water from the urban water supply system, filtered with a 0.45 µm membrane), 2) DRBA and 3) DRAA. After 96 h, these fishes were anesthetized to remove blood for evaluation of genotoxic damage (micronucleus and comet). For the bioassay, a total of 80 L of The Doce River water were collected before the influx of tailings and after the influx and then submitted to metal quantification analysis. Fish exposed to DRWBA and DRWAA treatments showed a significant increase in both the number of erythrocyte micronuclei and the DNA damage index in relation to the control fish; however, they did not present any differences between the two treatments. The results demonstrate that the DRWBA treatment was already genotoxic for the fish, mainly due to dissolved Cu concentrations in the water. The DRWAA treatment probably presented genotoxicity due to the increase in the dissolved fraction and synergistic effects of several metals found in the tailings of the Mariana accident.

Referees’ Emotions and Performance Perception: The Importance of Stress and Cognitive Appraisal / Emociones y Percepción de Rendimiento en Árbitros: Importancia del Estrés y la Evaluación Cognitiva / Emoções e Perceção de Rendimento em Árbitros: A Importância do Stresse da Avaliação Cognitiva

The tasks referees must perform are both complex and very important, and are usually associated with high levels of stress. In this study, we aimed to understand the role of stress and cognitive appraisal on referees’ emotions and sports performance perception before the realization of a game. We adopted a critical incident methodology to understand how referees adapt to the stress related to their next game. The study included 708 football referees (646 males), aged between 18 and 53 (M = 26.81; SD =7.32). We evaluated sources of stress, cognitive appraisal, emotions, and perception of individual performance. Regression analyses pointed out that stress was a main predictor of negative emotions, cognitive appraisal was a predictor of both negative and positive emotions, and that cognitive appraisal also contributed to comprehend the perception of individual performance. In sum, stress and appraisal are important factors to understand the stress experience of referees. (AU)

La tarea de un árbitro se asocia con un alto estrés. En este estudio buscamos comprender el papel del estrés y evaluación cognitiva en las emociones y percepción del rendimiento deportivo de los árbitros antes de un partido. Adoptamos una metodología de incidentes críticos para analizar se adaptan alestrés relacionado con su próximo juego. El estudio incluyó a 708 árbitros de fútbol (646 hombres), de entre 18-53 años (M= 26,81; DE= 7,32). Evaluamos las fuentes de estrés, evaluación cognitiva, emociones y percepción del desempeño individual. El análisis de regresión mostró que el estrés fue el principal predictor de las emociones negativas, la evaluación cognitiva fue un predictor de las emociones negativas y positivas y que la evaluación cognitiva también contribuyó a comprender la percepción del desempeño individual. En resumen, el estrés y la evaluación son factores importantes para comprender la experiencia de estrés de los árbitros. (AU)

A tarefa dosárbitrosestágeralmente associada a elevado stress. Neste estudo, procuramos compreender o papel do stresse da avaliação cognitiva nas emoções e percepçãodo desempenho desportivo dos árbitros antes da realização de um jogo. Adotámos uma metodologia de incidente crítico para analisar o modo como os árbitros se adaptam ao stressrelativoao seu próximo jogo. O estudo incluiu 708 árbitros de futebol (646 homens), com idades entre 18-53 (M= 26,81; DP= 7,32). Avaliámos as fontes de stress, avaliação cognitiva, emoções e perceção do rendimento individual. A análise de regressão mostrou que o stressfoi o principal preditor de emoções negativas, a avaliação cognitiva foi um preditor de emoções negativas e positivas e que a avaliação cognitiva também contribuiu para compreender a percepção do rendimento individual. Em suma, o stresse a avaliação são fatores importantes para entender a experiência de stressdos árbitros. (AU)

Transportation of juvenile tambaqui (Colossoma macropomum) in a closed system.

The objective of this study was to investigate the effect of density, duration and the use of additives to the water during the transportation of juvenile tambaqui (Colossoma macropomum) and use of this data to establish a safe transportation protocol for the species. The tested products and dosages were: salt (1000, 2000 and 3000 mg/L), gypsum (100, 300 and 500 mg/L) and benzocaine (10, 20 and 30 mg/L). Fish were transported in closed systems (plastic bag) at different densities and time periods of up to 24 h. Fish survival (FS) and water quality parameters were monitored immediately after transportation. The remaining fish were kept in floating cages in order to evaluate mortality which occurred up to 96 h after transportation (S96). The best fish density, additives dosages and time period of the transportation was estimated with a general linear model. The effect of the condition factor on FS and S96 was also evaluated. As expected, FS and S96 were significantly related to time and density. FS but not S96, were also were significantly related to treatment. FS with gypsum treatment was not different from controls and FS with table salt and benzocaine treatments were significantly reduced. The condition factor was not related to either FS or S96. FS was inversely correlated with carbon dioxide concentration. It was concluded that the additives did not improve fish transportation survival. Linear models were developed to predict the best transportation densities as a function of time.

Comparison of different regimens of estradiol benzoate treatments followed by long-acting progesterone to prepare noncycling mares as embryo recipients.

The present study evaluated the influence of different regimens of estradiol benzoate (EB) treatments followed by a single dose of long-acting progesterone (LA P4) on plasma estrogen and P4 concentrations in noncyclic mares prepared as embryo recipients. Twenty-one anestrous mares were distributed into three groups (n = 7 mares per group), according to the EB dose received (single dose of 2.5 mg, total of 5 mg in decreasing doses, and total of 10 mg in decreasing doses), which was followed by a single administration of 1500 mg of LA P4 in all groups. Mares were reevaluated during the ovulatory phase and seven of them became part of the cyclic nontreated control group. Ultrasonography was performed to monitor endometrial edema, and blood samples were collected to measure estradiol (E2), estrogen conjugate (EC), and P4 by RIA. Maximum uterine edema was achieved 24 hours after administration of EB in all treated groups. Maximum E2 concentrations were observed 24 hours after the first EB injection in treated groups and there were no differences (P > 0.05) among treatments. Maximum EC concentration was observed 24 hours after the single EB injection in the 2.5-mg group, whereas in the 5- and 10-mg groups EC peaks were observed 48 hours after the first EB administration. Maximum P4 concentrations were detected 24 hours after LA P4 injection, although higher P4 concentrations were observed in the group treated with 2.5 mg of EB than in that treated with 10 mg of EB (P < 0.05). Because P4 concentrations were reduced after administration of high doses of EB, we also measured 17α-hydroxyprogesterone (17-OH-P) to test the hypothesis that high concentrations of EB would accelerate the conversion of P4 to 17-OH-P. However, 17-OH-P concentrations paralleled P4 profile in all groups, irrespective of EB doses. In summary, the three EB treatment regimens induced similar E2 peaks, although the observation of EC peaks 24 hours after E2 peaks in the 5- and 10-mg groups indicate that an excess of E2 was given, which was converted into EC to be inactivated. Administration of 10 mg of EB reduced P4 concentrations 24 hours after LA P4 was given. We demonstrated that the mechanism by which this reduction occurred was not by an increase in P4 metabolism to 17α-OH-P. In conclusion, the use of 2.5 mg of EB followed by 1500 mg of LA P4 appears to be a more appropriate regimen to treat noncyclic mares, although additional studies are needed to verify embryo survival with this treatment dose.

Paclitaxel targets FOXM1 to regulate KIF20A in mitotic catastrophe and breast cancer paclitaxel resistance.

FOXM1 has been implicated in taxane resistance, but the molecular mechanism involved remains elusive. In here, we show that FOXM1 depletion can sensitize breast cancer cells and mouse embryonic fibroblasts into entering paclitaxel-induced senescence, with the loss of clonogenic ability, and the induction of senescence-associated ß-galactosidase activity and flat cell morphology. We also demonstrate that FOXM1 regulates the expression of the microtubulin-associated kinesin KIF20A at the transcriptional level directly through a Forkhead response element (FHRE) in its promoter. Similar to FOXM1, KIF20A expression is downregulated by paclitaxel in the sensitive MCF-7 breast cancer cells and deregulated in the paclitaxel-resistant MCF-7Tax(R) cells. KIF20A depletion also renders MCF-7 and MCF-7Tax(R) cells more sensitive to paclitaxel-induced cellular senescence. Crucially, resembling paclitaxel treatment, silencing of FOXM1 and KIF20A similarly promotes abnormal mitotic spindle morphology and chromosome alignment, which have been shown to induce mitotic catastrophe-dependent senescence. The physiological relevance of the regulation of KIF20A by FOXM1 is further highlighted by the strong and significant correlations between FOXM1 and KIF20A expression in breast cancer patient samples. Statistical analysis reveals that both FOXM1 and KIF20A protein and mRNA expression significantly associates with poor survival, consistent with a role of FOXM1 and KIF20A in paclitaxel action and resistance. Collectively, our findings suggest that paclitaxel targets the FOXM1-KIF20A axis to drive abnormal mitotic spindle formation and mitotic catastrophe and that deregulated FOXM1 and KIF20A expression may confer paclitaxel resistance. These findings provide insights into the underlying mechanisms of paclitaxel resistance and have implications for the development of predictive biomarkers and novel chemotherapeutic strategies for paclitaxel resistance.

OTUB1 inhibits the ubiquitination and degradation of FOXM1 in breast cancer and epirubicin resistance.

The forkhead transcription factor FOXM1 has a key role in DNA damage response, and its deregulated overexpression is associated with genotoxic drug resistance in breast cancer. However, little is known about the posttranslational mechanisms by which FOXM1 expression is regulated by genotoxic agents and how they are deregulated in resistant cells. Initial co-immunoprecipitation studies verified previous proteomic analysis finding that the OTUB1 is a novel FOXM1-interacting protein. Western blot analysis showed that both OTUB1 and FOXM1 expression reduced upon genotoxic agent treatment in MCF-7 cells, but remained relatively constant in resistant cells. FOXM1 expression reduced upon OTUB1 depletion by siRNA and increased with OTUB1 overexpression in MCF-7 cells, arguing that OTUB1 positively regulates FOXM1 expression. In agreement, co-immunoprecipitation experiments demonstrated that FOXM1 expression is associated with OTUB1 binding but inversely correlates with conjugation to the protein degradation-associated Lys-48-linked ubiquitin-chains. Overexpression of wild-type (WT) OTUB1, but not the OTUB1(C91S) mutant, disrupted the formation of Lys48-linked ubiquitin-conjugates on FOXM1. Importantly, knockdown of OTUB1 by siRNA resulted in an increase in turnover of FOXM1 in MCF-7 cells treated with the protein synthesis inhibitor cycloheximide, whereas overexpression of WT OTUB1, but not the OTUB1(C91S) mutant, significantly enhances the half-life of FOXM1. In addition, proliferative and clonogenic assays also show that OTUB1 can enhance the proliferative rate and epirubicin resistance through targeting FOXM1, as OTUB1 has little effect on FOXM1-deficient cells. The physiological relevance of the regulation of FOXM1 by OTUB1 is further underscored by the significant correlations between FOXM1 and OTUB1 expression in breast cancer patient samples. Cox-regression survival analysis indicates that OTUB1 overexpression is linked to poorer outcome in particular in patients treated with chemotherapy. Collectively, these data suggest that OTUB1 limits the ubiquitination and degradation of FOXM1 in breast cancer and has a key role in genotoxic agent resistance.

BRCA1 positively regulates FOXO3 expression by restricting FOXO3 gene methylation and epigenetic silencing through targeting EZH2 in breast cancer.

BRCA1 mutation or depletion correlates with basal-like phenotype and poor prognosis in breast cancer but the underlying reason remains elusive. RNA and protein analysis of a panel of breast cancer cell lines revealed that BRCA1 deficiency is associated with downregulation of the expression of the pleiotropic tumour suppressor FOXO3. Knockdown of BRCA1 by small interfering RNA (siRNA) resulted in downregulation of FOXO3 expression in the BRCA1-competent MCF-7, whereas expression of BRCA1 restored FOXO3 expression in BRCA1-defective HCC70 and MDA-MB-468 cells, suggesting a role of BRCA1 in the control of FOXO3 expression. Treatment of HCC70 and MDA-MB-468 cells with either the DNA methylation inhibitor 5-aza-2'-deoxycitydine, the N-methyltransferase enhancer of zeste homologue 2 (EZH2) inhibitor GSK126 or EZH2 siRNA induced FOXO3 mRNA and protein expression, but had no effect on the BRCA1-competent MCF-7 cells. Chromatin immunoprecipitation (ChIP) analysis demonstrated that BRCA1, EZH2, DNMT1/3a/b and histone H3 lysine 27 trimethylation (H3K27me3) are recruited to the endogenous FOXO3 promoter, further advocating that these proteins interact to modulate FOXO3 methylation and expression. In addition, ChIP results also revealed that BRCA1 depletion promoted the recruitment of the DNA methyltransferases DNMT1/3a/3b and the enrichment of the EZH2-mediated transcriptional repressive epigenetic marks H3K27me3 on the FOXO3 promoter. Methylated DNA immunoprecipitation assays also confirmed increased CpG methylation of the FOXO3 gene on BRCA1 depletion. Analysis of the global gene methylation profiles of a cohort of 33 familial breast tumours revealed that FOXO3 promoter methylation is significantly associated with BRCA1 mutation. Furthermore, immunohistochemistry further suggested that FOXO3 expression was significantly associated with BRCA1 status in EZH2-positive breast cancer. Consistently, high FOXO3 and EZH2 mRNA levels were significantly associated with good and poor prognosis in breast cancer, respectively. Together, these data suggest that BRCA1 can prevent and reverse FOXO3 suppression via inhibiting EZH2 and, consequently, its ability to recruit the transcriptional repressive H3K27me3 histone marks and the DNA methylases DNMT1/3a/3b, to induce DNA methylation and gene silencing on the FOXO3 promoter.