A Rare 46,X,t(Y;10)(q12;p14) Balanced Translocation in Non-Obstructive Azoospermic Patient with Elevated FSH and LH Levels.

Structural chromosomal aberrations like translocations have been shown to cause spermatogenic failure. We report a rare 46,X,t(Y;10)(q12;p14) balanced translocation in an otherwise healthy non-obstructive azoospermic male with high follicle-stimulating hormone (26.65 IU/L) and high luteinizing hormone (13.58 IU/L). The patient was referred to us after clinical, hormonal, and histopathological investigations to identify chromosomal abnormalities by karyotyping and fluorescence in situ hybridization (FISH). Analysis of the banding pattern by karyotyping followed by FISH confirmed reciprocal translocation and identified the breakpoints at Yq heterochromatin (Yq12) and 10p14. Further molecular tests including AZF microdeletion assay were done, and the results, which showed no mutations in the analyzed genes, were provided by the referring doctor. Thus, our study points to the importance of conventional cytogenetic techniques in the preliminary evaluation of a genetic abnormality in cases of infertility and would help the patient make an informed decision before pursuing assisted reproductive technology.

A pilot study on glycemia and insulin resistance in patients with severe periodontitis.

CONTEXT: The potential impact of severe periodontitis on glycemia in systemically healthy individuals is not clearly established. It was hypothesized that among individuals who were previously undiagnosed for diabetes mellitus, patients with severe periodontitis have impaired glycemia and insulin resistance. AIMS: The aim of our study was to assess and compare glycemia in severe periodontitis patients and in individuals with clinically healthy periodontium. MATERIALS AND METHODS: A cross-sectional analytical design was employed. From among individuals who were undiagnosed for diabetes mellitus, 37 patients with severe periodontitis and 37 individuals with healthy periodontium in the age group of 25-55 years were recruited for the study. The fasting blood sugar (FBS), glycosylated hemoglobin (HbA1c), and insulin resistance by the Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) were assessed and compared between the two groups. RESULTS: The mean FBS, HOMA-IR, and HbA1c were significantly higher for patients with severe periodontitis than those individuals with healthy periodontium. After adjustments for age, gender, and body mass index, patients with severe periodontitis had a statistically significant association with impaired glucose metabolism (HbA1c ≥5.7) (adjusted odds ratio [OR] of 9.56; 95% confidence interval [CI]: 1.819-46.08; P < 0.01). Furthermore, patients with severe periodontitis had significantly greater odds to develop impaired fasting glucose (adjusted OR of 7.489, 95% CI: 1.408-39.839; P < 0.01). CONCLUSIONS: The mean FBS, HbA1c, and HOMA-IR were significantly higher in severe periodontitis patients than in the control group. A higher proportion of patients presented with prediabetes, incident diabetes, and insulin resistance in the severe periodontitis group.

In vitro human stem cell derived cultures to monitor calcium signaling in neuronal development and function.

The development of the human brain involves multiple cellular processes including cell division, migration, and dendritic growth. These processes are triggered by developmental cues and lead to interactions of neurons and glial cells to derive the final complex organization of the brain. Developmental cues are transduced into cellular processes through the action of multiple intracellular second messengers including calcium. Calcium signals in cells are shaped by large number of proteins and mutations in several of these have been reported in human patients with brain disorders.  However, the manner in which such mutations impact human brain development in vivo remains poorly understood. A key limitation in this regard is the need for a model system in which calcium signaling can be studied in neurons of patients with specific brain disorders. Here we describe a protocol to differentiate human neural stem cells into cortical neuronal networks that can be maintained as live cultures up to 120 days in a dish. Our protocol generates a 2D in vitro culture that exhibits molecular features of several layers of the human cerebral cortex. Using fluorescence imaging of intracellular calcium levels, we describe the development of neuronal activity as measured by intracellular calcium transients during development in vitro. These transients were dependent on the activity of voltage gated calcium channels and were abolished by blocking sodium channel activity. Using transcriptome analysis, we describe the full molecular composition of such cultures following differentiation in vitro thus offering an insight into the molecular basis of activity. Our approach will facilitate the understanding of calcium signaling defects during cortical neuron development in patients with specific brain disorders and a mechanistic analysis of these defects using genetic manipulations coupled with cell biological and physiological analysis.

Phosphoinositides: Regulators of Nervous System Function in Health and Disease.

Phosphoinositides, the seven phosphorylated derivatives of phosphatidylinositol have emerged as regulators of key sub-cellular processes such as membrane transport, cytoskeletal function and plasma membrane signaling in eukaryotic cells. All of these processes are also present in the cells that constitute the nervous system of animals and in this setting too, these are likely to tune key aspects of cell biology in relation to the unique structure and function of neurons. Phosphoinositides metabolism and function are mediated by enzymes and proteins that are conserved in evolution, and analysis of knockouts of these in animal models implicate this signaling system in neural function. Most recently, with the advent of human genome analysis, mutations in genes encoding components of the phosphoinositide signaling pathway have been implicated in human diseases although the cell biological basis of disease phenotypes in many cases remains unclear. In this review we evaluate existing evidence for the involvement of phosphoinositide signaling in human nervous system diseases and discuss ways of enhancing our understanding of the role of this pathway in the human nervous system's function in health and disease.

Transcriptional Regulation of Nos2 via STAT5B Binding to Nos2 Gene Promoter Mediates Nitric Oxide Production: Relevance in ß-Cell Maintenance.

BACKGROUND/AIMS: Type 1 Diabetes (T1D) involves autoimmune attack due to reduced regulatory T cells as an effect of mutant Stat5b(C1462A) in non-obese diabetic (NOD) mice, a T1D model resulting in pancreatic ß-cell destruction. Although reactive oxygen species are considered to orchestrate the immune attack, the role of nitric oxide (·NO) still remains debatable. Since JAK-STAT pathway is known to induce Nos2, we investigated the role of STAT5B in nitric oxide generation and oxidative stress. METHODS: In this study, we have used chromatin immunoprecipitation with STAT5B antibody to explore whether STAT5B binds Nos2 promoter. Using Stat5b gene silencing and overexpression models in MIN6 mouse pancreatic ß-cell line we have assayed nitric oxide and its end products, superoxide levels, H2O2 levels, and expression of genes related to redox pathway by immunocytochemistry, biochemical assays, quantitative real time PCR and western blotting. RESULTS: Our results prove that STAT5B binds to the candidate gamma-interferon-activated (GAS) element in Nos2 promoter thereby inducing Nos2 mRNA transcription resulting in NOS2 protein expression in MIN6, a mouse pancreatic ß-cell line. Our findings are substantiated by reduced ·NO as well as nitric oxide end products (nitrate and nitrite), and increased superoxide production in Stat5b silenced MIN6 cells. Our results indicate that C1462A mutant STAT5B shows lack of ·NO generation ability. To detoxify excess superoxide as a consequence of lowered Nos2, an overexpressed SOD2 in Stat5b silenced cells results in increased H2O2 production. H2O2 metabolizing enzymes do not show upregulation upon Stat5b silencing, and thus oxidative stress is brought about by amassed H2O2. Stat5b silencing finally reduces AKT expression, a prosurvival signal. CONCLUSION: Our study enables us to conclude that ß-cell stress is aggravated by the incapability of STAT5B to induce Nos2 resulting in H2O2 accumulation and the ensuing oxidative stress enhances ß-cell damage.

Roles of different amino-acid residues towards binding and selective transport of K+ through KcsA K+-ion channel.

The roles of different amino acid residues towards binding and selective transport of K+ ion in the selectivity filter of the bacterial K+ ion channel, KcsA, were explored. The selectivity filter of KcsA contains amino acids threonine (THR-75), valine (VAL-76), glycine (GLY-77) and tyrosine (TYR-78) from intracellular to extracellular regions. The hydroxyl-hydrogen and carbonyl-oxygen of THR-75 situated at the intracellular region are engaged in intra-residue hydrogen bonding. Thus, the hydroxyl-oxygens orient away from and the hydroxyl-hydrogens orient towards the center of the selectivity filter. Hence, this site attracts and acts as a 'gate' for the incoming K+ ion. The binding of K+ ion is first observed with eight carbonyl-oxygens of THR-75 and VAL-76 (S3 site). However, binding at this site breaks the intra-residue hydrogen bonding of THR-75, which in turn reorients the hydroxyl-hydrogens away from the selectivity filter. Accordingly, binding of K+ ion at this site prevents the instantaneous approach of another K+ ion. Thus, VAL-76 acts as a 'hinge' to open and close the 'gate'. The next amino acid, GLY-77, shows low affinity towards K+ ion, thus acting as a 'regulator'. TYR-78 situated at the extracellular region shows strong binding affinity towards K+ ion, and thus assists in the transport of K+ ion from the intracellular to the extracellular region. The release of K+ ion to form hydrated K(H2O)8+ in the extracellular region is endothermic (4.9 kcal mol-1). Hence, the process of K+ ion transport is likely to be kinetically controlled. Moreover, the highest binding energy of Na+ is observed at the center of four carbonyl-oxygens of VAL-76 (-68.8 kcal mol-1), which is even higher than the highest binding energy of K+ ion at S3 (-63.9 kcal mol-1). Na+ binding energy decreases from intracellular to extracellular region. Hence, intracellular Na+ ion would block the passage of K+ ion. The K+ ion concentration is high in the intracellular region, and it moves from intracellular to extracellular region, while Na+ ion concentration is high in the extracellular region, and it moves from extracellular to intracellular region. Thus, the selective transport of K+/Na+ ion in KcsA can be attributed to the competition between K+ ion exit and Na+ ion entry at the center of four carbonyl-oxygens of TYR-78.

Impaired Arginine Metabolism Coupled to a Defective Redox Conduit Contributes to Low Plasma Nitric Oxide in Polycystic Ovary Syndrome.

BACKGROUND: Though oxidative stress is associated with Polycystic Ovary Syndrome (PCOS), the status of nitric oxide is still unclear. Nitric Oxide (NO) plays pivotal roles in many physiological functions which are compromised in PCOS. Our recent study reveals lowered T-regulatory cells (Tregs) in PCOS, and Treg generation is known to be regulated by NO levels. However concrete evidences are lacking on mechanisms modulating NO levels under PCOS. METHODS: This is a retrospective case-control cohort study, comprised of PCOS women (N=29) and normal menstruating women as controls (N=20). We analysed NOx (nitrite+nitrate) and hydrogen peroxide (H2O2) concentrations, transcript levels of endothelial nitric oxide synthase (eNOS)/inducible nitric oxide synthase (iNOS) and arginine modulators, hydrogen peroxide regulators in the cohort. RESULTS: PCOS women showed reduced plasma NOx(nitrate+nitrite) and H2O2 compared to controls. We report reduction in transcript levels of iNOS/NOS2 and eNOS/NOS3 in PCOS peripheral blood. The transcripts involved in arginine bioavailability: Argininosuccinate lyase (ASL), Solute Carrier Family1, member 7 (SLC7A1) and Arginase 1 (ARG1) and Asymmetric Dimethyl Arginine (ADMA) metabolism: Protein arginine methyltransferase 1 (PRMT1) and Dimethylarginine dimethylaminohydrolase 2 (DDAH2) also showed differential expression. H2O2 concentration in PCOS women was also found to be reduced. The reduction can be attributed to increase in catalase levels as a consequence of the body's effort to alleviate the oxidative burden in the system. CONCLUSION: Our study advocates that PCOS women have lowered NO due to reduced iNOS/eNOS expression, low H2O2, high ADMA synthesis and reduced arginine bioavailability. An in-depth analysis of redox biology of PCOS to open up potential therapeutic strategies is highly recommended.

Reduced Tregs in peripheral blood of PCOS patients - a consequence of aberrant Il2 signaling.

CONTEXT: The immunesupressive action of CD4(+)CD25(+) CD127(-/low) T regulatory cells (Tregs) is vital for an efficient reproductive function. However no data exists on their number or functionality in polycystic ovary syndrome (PCOS). OBJECTIVE: The study aimed to analyze the frequency of circulating Tregs and key factors modulating them in women with PCOS. DESIGN, SETTING, AND PARTICIPANTS: This is a retrospective, case-control cohort study conducted in women with PCOS recruited from Samad IVF hospitals and Women and Children Hospital, Thiruvananthapuram, India. Women with PCOS (N = 20) were diagnosed according to Rotterdam Consensus and normal menstruating women were taken as controls (N = 2331). MAIN OUTCOME MEASURES: We analyzed the proportion of CD4(+)CD25(+) CD127(-/low) Tregs in women with PCOS by fluorescent activated cell sorting. RESULTS: The study discovered that the women with PCOS have reduced numbers of Tregs (2.626 ± 0.62) compared with controls (4.253 ± 0.87) (t = 6.963, P < .0001, mean difference = -1.627; 95% confidence interval = -2.099--1.155). We documented a decrease in the follicular phase Treg expansion in women with PCOS. Our results revealed a reduced STAT5A (fold change [FC] = 7.642, P < .0004)/STAT5B (FC = 3.824, P < .0001), FOXP3 (FC = 4.1343, P = .0004)/CTLA4 (FC = 2.569, P = .0001) and elevated AKT (FC = 7.39, P = .05)/PIK3 (FC = 5.326, P = .0002) expression in women with PCOS. Recombinant interleukin 2 (rIL2) treatment failed to improve FOXP3/CTLA4 levels but caused a reduction of AKT/PIK3 arm, possibly due to an elevated PTEN in women with PCOS. CONCLUSION: The study suggests that women with PCOS have reduced Tregs due to an inherent hyporesponsiveness to IL2, which is unable to activate STAT5B and reduce FOXP3 expression. IL2-based therapeutic strategies can ameliorate complications in PCOS by suppressing the AKT/PIK3 arm.