Ectopic hbox12 Expression Evoked by Histone Deacetylase Inhibition Disrupts Axial Specification of the Sea Urchin Embryo

Dorsal/ventral patterning of the sea urchin embryo depends upon the establishment of a Nodal-expressing ventral organizer. Recently, we showed that spatial positioning of this organizer relies on the dorsal-specific transcription of the Hbox12 repressor. Building on these findings, we determined the influence of the epigenetic milieu on the expression of hbox12 and nodal genes. We find that Trichostatin-A, a potent and selective histone-deacetylases inhibitor, induces histone hyperacetylation in hbox12 chromatin, evoking broad ectopic expression of the gene. Transcription of nodal concomitantly drops, prejudicing dorsal/ventral polarity of the resulting larvae. Remarkably, impairing hbox12 function, either in a spatially-restricted sector or in the whole embryo, specifically rescues nodal transcription in Trichostatin-A-treated larvae. Beyond strengthen the notion that nodal expression is not allowed in the presence of functional Hbox12 in the same cells, these results highlight a critical role of histone deacetylases in regulating the spatial expression of hbox12

Chronic hyperglycemia induces trans-differentiation of human pancreatic stellate cells and enhances the malignant molecular communication with human pancreatic cancer cells

BACKGROUND: Diabetes mellitus is linked to pancreatic cancer. We hypothesized a role for pancreatic stellate cells (PSC) in the hyperglycemia induced deterioration of pancreatic cancer and therefore studied two human cell lines (RLT-PSC, T3M4) in hyperglycemic environment. METHODOLOGY/PRINCIPAL FINDINGS: The effect of chronic hyperglycemia (CHG) on PSCs was studied using mRNA expression array with real-time PCR validation and bioinformatic pathway analysis, and confirmatory protein studies. The stress fiber formation (IC: αSMA) indicated that PSCs tend to transdifferentiate to a myofibroblast-like state after exposure to CHG. The phosphorylation of p38 and ERK1/2 was increased with a consecutive upregulation of CDC25, SP1, cFOS and p21, and with downregulation of PPARγ after PSCs were exposed to chronic hyperglycemia. CXCL12 levels increased significantly in PSC supernatant after CHG exposure independently from TGF-β1 treatment (3.09-fold with a 2.73-fold without TGF-β1, p<0.05). The upregualtion of the SP1 transcription factor in PSCs after CHG exposure may be implicated in the increased CXCL12 and IGFBP2 production. In cancer cells, hyperglycemia induced an increased expression of CXCR4, a CXCL12 receptor that was also induced by PSC's conditioned medium. The receptor-ligand interaction increased the phosphorylation of ERK1/2 and p38 resulting in activation of MAP kinase pathway, one of the most powerful stimuli for cell proliferation. Certainly, conditioned medium of PSC increased pancreatic cancer cell proliferation and this effect could be partially inhibited by a CXCR4 inhibitor. As the PSC conditioned medium (normal glucose concentration) increased the ERK1/2 and p38 phosphorylation, we concluded that PSCs produce other factor(s) that influence(s) pancreatic cancer behaviour. CONCLUSIONS: Hyperglycemia induces increased CXCL12 production by the PSCs, and its receptor, CXCR4 on cancer cells. The ligand-receptor interaction activates MAP kinase signaling that causes increased cancer cell proliferation and migration

Using C. elegans to decipher the cellular and molecular mechanisms underlying neurodevelopmental disorders

Prova tipográfica (uncorrected proof)Neurodevelopmental disorders such as epilepsy, intellectual disability (ID), and autism spectrum disorders (ASDs) occur in over 2 % of the population, as the result of genetic mutations, environmental factors, or combination of both. In the last years, use of large-scale genomic techniques allowed important advances in the identification of genes/loci associated with these disorders. Nevertheless, following association of novel genes with a given disease, interpretation of findings is often difficult due to lack of information on gene function and effect of a given mutation in the corresponding protein. This brings the need to validate genetic associations from a functional perspective in model systems in a relatively fast but effective manner. In this context, the small nematode, Caenorhabditis elegans, presents a good compromise between the simplicity of cell models and the complexity of rodent nervous systems. In this article, we review the features that make C. elegans a good model for the study of neurodevelopmental diseases. We discuss its nervous system architecture and function as well as the molecular basis of behaviors that seem important in the context of different neurodevelopmental disorders. We review methodologies used to assess memory, learning, and social behavior as well as susceptibility to seizures in this organism. We will also discuss technological progresses applied in C. elegans neurobiology research, such as use of microfluidics and optogenetic tools. Finally, we will present some interesting examples of the functional analysis of genes associated with human neurodevelopmental disorders and how we can move from genes to therapies using this simple model organism.The authors would like to acknowledge Fundação para a Ciência e Tecnologia (FCT) (PTDC/SAU-GMG/112577/2009). AJR and CB are recipients of FCT fellowships: SFRH/BPD/33611/2009 and SFRH/BPD/74452/2010, respectively

Risk Factors for Infertility in Korean Women

BACKGROUND: Female infertility is a crucial problem with significant implications for individuals and society. In this study, we explore risk factors for infertility in Korean women. METHODS: A total of 986 female patients who visited six major infertility clinics in Korea were recruited from April to December 2014. Fertile age-matched controls were selected from two nationwide survey study participants. Conditional logistic regression after age-matching was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) of each risk factor for infertility. RESULTS: Women with a body mass index (BMI) &lt; 18.5 kg/m² had 1.35 times higher odds of infertility (OR, 1.35; 95% CI, 1.03-1.77), while those with a BMI ≥ 25.0 kg/m² had even higher odds (OR, 2.06; 95% CI, 1.61-2.64) compared to women with a normal BMI (18.5 kg/m² ≤ BMI &lt; 25 kg/m²). Ever-smokers exhibited 4.94 times higher odds of infertility compared to never-smokers (95% CI, 3.45-8.85). Concerning alcohol consumption, women who consumed ≥ 7 glasses at a time showed 3.13 times significantly higher odds of infertility than those who consumed ≤ 4 glasses at a time (95% CI, 1.79-5.48). Lastly, women with thyroid disease demonstrated 1.44 times higher odds of infertility compared to women without thyroid disease (95% CI, 1.00-2.08). CONCLUSION: Female infertility in Korea was associated with underweight, obesity, smoking, alcohol consumption, and thyroid disease

Reduced pelvic pain in women with endometriosis: efficacy of long-term dienogest treatment

Purpose To investigate the efficacy and safety of dienogest as a long-term treatment in endometriosis, with follow-up after treatment discontinuation. The study included women with endometriosis, who had previously completed a 12-week, placebo-controlled study of dienogest, who participated in an open-label extension study for up to 53 weeks. Thereafter, a patient subgroup was evaluated in a 24-week follow-up after treatment discontinuation. Methods A multicenter study performed in Germany, Italy and Ukraine. Women with endometriosis were enrolled at completion of the placebo-controlled study (n = 168). All women received dienogest (2 mg once daily, orally) and changes in pelvic pain (on a visual analog scale), bleeding pattern, adverse events and laboratory parameters were evaluated during and after treatment. Results The completion rate among women who entered the open-label extension study was 90.5% (n = 152). A Significant decrease in pelvic pain was shown during continued dienogest treatment (P &lt; 0.001). The mean frequency and intensity of bleeding progressively decreased. Adverse events, rated generally mild or moderate, led to withdrawal in four patients (2.4%). No clinically relevant changes in laboratory parameters were observed. During treatmentfree follow-up (n = 34), the reduction in pelvic pain persisted, while bleeding frequency and intensity returned to normal patterns. Conclusions Long-term dienogest showed a favorable efficacy and safety profile, with progressive decreases in pain and bleeding irregularities during continued treatment; the decrease of pelvic pain persisted for at least 24 weeks after treatment cessation. © Springer-Verlag 2011

The expression of FHIT, PCNA and EGFR in benign and malignant breast lesions

Immunohistochemical staining for FHIT and PCNA proteins was carried out in 451 breast lesions showing nonproliferative benign breast disease (BBD) (n=263), proliferative BBD without atypia (n=128), proliferative BBD with atypia (n=11), carcinoma in situ (n=15) or invasive carcinoma (n=34) and for EGFR protein in a subset of 71 of these cases. FHIT underexpression was not detected in nonproliferative lesions, but occurred in 2% of proliferative BBD without atypia, 10% proliferative BBD with atypia, 27% of carcinoma in situ and 41% of invasive carcinoma, which suggests that it could be useful in assessing those carcinoma in situ lesions (ductal, DCIS and lobular, LCIS) that are more likely to progress to malignancy. Preliminary microarray comparisons on DCIS and invasive carcinoma samples dissected from formalin-fixed paraffin sections showed a consistent downregulation of two previously identified FHIT-related genes, caspase 1 and BRCA1 in lesions underexpressing FHIT

The Role of Cilostazol, a Phosphodiesterase 3 Inhibitor, on Oocyte Maturation and Subsequent Pregnancy in Mice

It is important to identify effective contraceptive drugs that cause minimal disruption to physiological processes. Phosphodiesterase 3 (PDE3) inhibitors suppress meiosis in oocytes by decreasing the level of cAMP and blocking the extrusion of the first polar body. In this study, we tested the PDE3 inhibitor, cilostazol, as a potential contraceptive agent. The effects of cilostazol treatment in vitro and in vivo on the suppression of oocyte maturation in a mouse model were investigated. The results indicated that treatment with increasing concentrations of cilostazol led to a dose-dependent arrest in meiosis progression. The effective in vitro concentration was 1 µM and was 300 mg/kg in vivo. The effect of cilostazol was reversible. After removal of the drug, meiosis resumed and mouse oocytes matured in vitro, and showed normal chromosome alignment and spindle organization. After fertilization using an ICSI method, the oocytes showed normal morphology, fertilization rate, embryo cleavage, blastocyst formation, and number of viable pups when compared with controls. The offspring showed similar body weight and fertility. In vivo, the mice became infertile if the drug was injected sequentially, and became pregnant following discontinuation of cilostazol. More importantly, no side effects of cilostazol were observed in treated female mice as demonstrated by blood pressure and heart rate monitoring. It is concluded that cilostazol, a drug routinely used for intermittent claudication, can effectively inhibit oocyte maturation in vitro and in vivo, does not affect the developmental potential of oocytes following drug removal and has few side effects in female mice treated with this drug. These findings suggest that cilostazol may be a potential new contraceptive agent that may facilitate an efficacy and safety study of this drug

ENU Mutagenesis Identifies Mice with Morbid Obesity and Severe Hyperinsulinemia Caused by a Novel Mutation in Leptin

BACKGROUND: Obesity is a multifactorial disease that arises from complex interactions between genetic predisposition and environmental factors. Leptin is central to the regulation of energy metabolism and control of body weight in mammals. METHODOLOGY/PRINCIPAL FINDINGS: To better recapitulate the complexity of human obesity syndrome, we applied N-ethyl-N-nitrosourea (ENU) mutagenesis in combination with a set of metabolic assays in screening mice for obesity. Mapping revealed linkage to the chromosome 6 within a region containing mouse Leptin gene. Sequencing on the candidate genes identified a novel T-to-A mutation in the third exon of Leptin gene, which translates to a V145E amino acid exchange in the leptin propeptide. Homozygous Leptin(145E/145E) mutant mice exhibited morbid obesity, accompanied by adipose hypertrophy, energy imbalance, and liver steatosis. This was further associated with severe insulin resistance, hyperinsulinemia, dyslipidemia, and hyperleptinemia, characteristics of human obesity syndrome. Hypothalamic leptin actions in inhibition of orexigenic peptides NPY and AgRP and induction of SOCS1 and SOCS3 were attenuated in Leptin(145E/145E) mice. Administration of exogenous wild-type leptin attenuated hyperphagia and body weight increase in Leptin(145E/145E) mice. However, mutant V145E leptin coimmunoprecipitated with leptin receptor, suggesting that the V145E mutation does not affect the binding of leptin to its receptor. Molecular modeling predicted that the mutated residue would form hydrogen bond with the adjacent residues, potentially affecting the structure and formation of an active complex with leptin receptor within that region. CONCLUSIONS/SIGNIFICANCE: Thus, our evolutionary, structural, and in vivo metabolic information suggests the residue 145 as of special function significance. The mouse model harboring leptin V145E mutation will provide new information on the current understanding of leptin biology and novel mouse model for the study of human obesity syndrome

AMPK Regulates Circadian Rhythms in a Tissue- and Isoform-Specific Manner

AMP protein kinase (AMPK) plays an important role in food intake and energy metabolism, which are synchronized to the light-dark cycle. In vitro, AMPK affects the circadian rhythm by regulating at least two clock components, CKIα and CRY1, via direct phosphorylation. However, it is not known whether the catalytic activity of AMPK actually regulates circadian rhythm in vivo.THE CATALYTIC SUBUNIT OF AMPK HAS TWO ISOFORMS: α1 and α2. We investigate the circadian rhythm of behavior, physiology and gene expression in AMPKα1-/- and AMPKα2-/- mice. We found that both α1-/- and α2-/- mice are able to maintain a circadian rhythm of activity in dark-dark (DD) cycle, but α1-/- mice have a shorter circadian period whereas α2-/- mice showed a tendency toward a slightly longer circadian period. Furthermore, the circadian rhythm of body temperature was dampened in α1-/- mice, but not in α2-/- mice. The circadian pattern of core clock gene expression was severely disrupted in fat in α1-/- mice, but it was severely disrupted in the heart and skeletal muscle of α2-/- mice. Interestingly, other genes that showed circadian pattern of expression were dysreguated in both α1-/- and α2-/- mice. The circadian rhythm of nicotinamide phosphoryl-transferase (NAMPT) activity, which converts nicotinamide (NAM) to NAD+, is an important regulator of the circadian clock. We found that the NAMPT rhythm was absent in AMPK-deficient tissues and cells.This study demonstrates that the catalytic activity of AMPK regulates circadian rhythm of behavior, energy metabolism and gene expression in isoform- and tissue-specific manners