Converging evidence from exome sequencing and common variants implicates target genes for osteoporosis.

In this study, we leveraged the combined evidence of rare coding variants and common alleles to identify therapeutic targets for osteoporosis. We undertook a large-scale multiancestry exome-wide association study for estimated bone mineral density, which showed that the burden of rare coding alleles in 19 genes was associated with estimated bone mineral density (P < 3.6 × 10-7). These genes were highly enriched for a set of known causal genes for osteoporosis (65-fold; P = 2.5 × 10-5). Exome-wide significant genes had 96-fold increased odds of being the top ranked effector gene at a given GWAS locus (P = 1.8 × 10-10). By integrating proteomics Mendelian randomization evidence, we prioritized CD109 (cluster of differentiation 109) as a gene for which heterozygous loss of function is associated with higher bone density. CRISPR-Cas9 editing of CD109 in SaOS-2 osteoblast-like cell lines showed that partial CD109 knockdown led to increased mineralization. This study demonstrates that the convergence of common and rare variants, proteomics and CRISPR can highlight new bone biology to guide therapeutic development.

The complex architecture of p53 binding sites.

Sequence-specific protein-DNA interactions are at the heart of the response of the tumor-suppressor p53 to numerous physiological and stress-related signals. Large variability has been previously reported in p53 binding to and transactivating from p53 response elements (REs) due, at least in part, to changes in direct (base) and indirect (shape) readouts of p53 REs. Here, we dissect p53 REs to decipher the mechanism by which p53 optimizes this highly regulated variable level of interaction with its DNA binding sites. We show that hemi-specific binding is more prevalent in p53 REs than previously envisioned. We reveal that sequences flanking the REs modulate p53 binding and activity and show that these effects extend to 4-5 bp from the REs. Moreover, we show here that the arrangement of p53 half-sites within its REs, relative to transcription direction, has been fine-tuned by selection pressure to optimize and regulate the response levels from p53 REs. This directionality in the REs arrangement is at least partly encoded in the structural properties of the REs. Furthermore, we show here that in the p21-5' RE the orientation of the half-sites is such that the effect of the flanking sequences is minimized and we discuss its advantages.

Metabolic Vulnerabilities and Epigenetic Dysregulation in Myeloproliferative Neoplasms.

The Janus kinase 2 (JAK2)-driven myeloproliferative neoplasms (MPNs) are associated with clonal myelopoiesis, elevated risk of death due to thrombotic complications, and transformation to acute myeloid leukemia (AML). JAK2 inhibitors improve the quality of life for MPN patients, but these approved therapeutics do not readily reduce the natural course of disease or antagonize the neoplastic clone. An understanding of the molecular and cellular changes requisite for MPN development and progression are needed to develop improved therapies. Recently, murine MPN models were demonstrated to exhibit metabolic vulnerabilities due to a high dependence on glucose. Neoplastic hematopoietic progenitor cells in these mice express elevated levels of glycolytic enzymes and exhibit enhanced levels of glycolysis and oxidative phosphorylation, and the disease phenotype of these MPN model mice is antagonized by glycolytic inhibition. While all MPN-driving mutations lead to aberrant JAK2 activation, these mutations often co-exist with mutations in genes that encode epigenetic regulators, including loss of function mutations known to enhance MPN progression. In this perspective we discuss how altered activity of epigenetic regulators (e.g., methylation and acetylation) in MPN-driving stem and progenitor cells may alter cellular metabolism and contribute to the MPN phenotype and progression of disease. Specific metabolic changes associated with epigenetic deregulation may identify patient populations that exhibit specific metabolic vulnerabilities that are absent in normal hematopoietic cells, and thus provide a potential basis for the development of more effective personalized therapeutic approaches.

HDAC11 deficiency disrupts oncogene-induced hematopoiesis in myeloproliferative neoplasms.

Protein acetylation is an important contributor to cancer initiation. Histone deacetylase 6 (HDAC6) controls JAK2 translation and protein stability and has been implicated in JAK2-driven diseases best exemplified by myeloproliferative neoplasms (MPNs). By using novel classes of highly selective HDAC inhibitors and genetically deficient mouse models, we discovered that HDAC11 rather than HDAC6 is necessary for the proliferation and survival of oncogenic JAK2-driven MPN cells and patient samples. Notably, HDAC11 is variably expressed in primitive stem cells and is expressed largely upon lineage commitment. Although Hdac11is dispensable for normal homeostatic hematopoietic stem and progenitor cell differentiation based on chimeric bone marrow reconstitution, Hdac11 deficiency significantly reduced the abnormal megakaryocyte population, improved splenic architecture, reduced fibrosis, and increased survival in the MPLW515L-MPN mouse model during primary and secondary transplantation. Therefore, inhibitors of HDAC11 are an attractive therapy for treating patients with MPN. Although JAK2 inhibitor therapy provides substantial clinical benefit in MPN patients, the identification of alternative therapeutic targets is needed to reverse MPN pathogenesis and control malignant hematopoiesis. This study establishes HDAC11 as a unique type of target molecule that has therapeutic potential in MPN.

Integrated Regulation of HuR by Translation Repression and Protein Degradation Determines Pulsatile Expression of p53 Under DNA Damage.

Expression of tumor suppressor p53 is regulated at multiple levels, disruption of which often leads to cancer. We have adopted an approach combining computational systems modeling with experimental validation to elucidate the translation regulatory network that controls p53 expression post DNA damage. The RNA-binding protein HuR activates p53 mRNA translation in response to UVC-induced DNA damage in breast carcinoma cells. p53 and HuR levels show pulsatile change post UV irradiation. The computed model fitted with the observed pulse of p53 and HuR only when hypothetical regulators of synthesis and degradation of HuR were incorporated. miR-125b, a UV-responsive microRNA, was found to represses the translation of HuR mRNA. Furthermore, UV irradiation triggered proteasomal degradation of HuR mediated by an E3-ubiquitin ligase tripartite motif-containing 21 (TRIM21). The integrated action of miR-125b and TRIM21 constitutes an intricate control system that regulates pulsatile expression of HuR and p53 and determines cell viability in response to DNA damage.

Comparative Analysis of Liquid based and Conventional Cytology Smears in Fine Needle Aspirates from Breast Lesions.

CONTEXT: Liquid-based cytology (LBC) is being extensively used for the evaluation of both gynecological and non-gynecological specimens. Suspension of cells in monolayer makes better morphological assessment possible. Along with this, inherent morphological changes such as altered, reduced, or lost background material, fragmented cell clusters, smaller cell size, nucleolar prominence, etc., need to be considered. AIM: Present study was aimed at comparative evaluation of utility of LBC versus conventional smear (CS) in assessing breast lesions and whether it can be used as an alternative to conventional preparation. SETTINGS AND DESIGN: Present study was a prospective study in which 75 cases of breast fine-needle aspiration cytology from patients with palpable breast lumps constituted the study group. MATERIAL AND METHODS: The first pass was used for CS and LBC; a second pass was given. The representative CS and LBC smears were compared using several criteria. STATISTICS: Each feature was scored individually and evaluated statistically using Wilcoxon's signed rank test on the SPSS program. RESULTS: A statistically significant difference was found in informative background and background blood-debris, whereas the difference was not statistically significant in other features such as cellularity, cytoarchitectural pattern, presence of monolayer, and nuclear and cytoplasmic details. CONCLUSION: LBC is a promising technique in the field of cytology. It has the potential to decrease the number of slides screened per case and decrease the turn-around-time.

Regulated partitioning of fixed carbon (14C), sodium (Na+), potassium (K+) and glycine betaine determined salinity stress tolerance of gamma irradiated pigeonpea [Cajanus cajan (L.) Millsp].

Soil salinity is a major constraint that limits legume productivity. Pigeonpea is a salt sensitive crop. Seed gamma irradiation at a very low dose (2.5 Gy) is known to enhance seedling establishment, plant growth and yield of cereals and other crops. The present study conducted using two genetically diverse varieties of pigeonpea viz., Pusa-991 and Pusa-992 aimed at establishing the role of pre-sowing seed gamma irradiation at 0, 0.0025, 0.005, 0.01, 0.02, 0.05 and 0.1 kGy on plant growth, seed yield and seed quality under salt stress at 0, 80 and 100 mM NaCl (soil solution EC equivalent 1.92, 5.86 and 8.02 dS/m, respectively) imposed right from the beginning of the experiment. Changes in carbon flow dynamics between shoot and root and concentration of osmolyte, glycine betaine, plant uptake and shoot and root partitioning of Na+ and K+ and activity of protein degrading enzyme protease were measured under the combined effect of gamma irradiation and salt stress. Positive affect of pre-sowing exposure of seed to low dose of gamma irradiation (<0.01 kGy) under salt stress was evident in pigeonpea. Pigeonpea variety, Pusa-992 showed a better salt tolerance response than Pusa-991 and that the radiated plants performed better than the unirradiated plants even at increasing salinity level. Seed yield and seed protein and iron content were also positively affected by the low dose gamma irradiation under NaCl stress. Multiple factors interacted to determine physiological salt tolerance response of pigeonpea varieties. Gamma irradiation caused a favourable alteration in the source-sink (shoot-root) partitioning of recently fixed carbon (14C) under salt stress in pigeonpea. Gamma irradiation of seeds prior to sowing enhanced glycine betaine content and reduced protease activity at 60-day stage under various salt stress regimes. Lower partitioning of Na+and relatively higher accumulation of K+ under irradiation treatment was the other important determinants that differentiated between salt-tolerant and salt-susceptible variety of pigeonpea. The study provides evidence and physiological basis for exploring exploitation of pre-sowing exposure of seeds with low-dose gamma ray for enhancing the salt tolerance response of crop plants.

WEE1 epigenetically modulates 5-hmC levels by pY37-H2B dependent regulation of IDH2 gene expression.

Epigenetic signaling networks dynamically regulate gene expression to maintain cellular homeostasis. Previously, we uncovered that WEE1 phosphorylates histone H2B at tyrosine 37 (pY37-H2B) to negatively regulate global histone transcriptional output. Although pY37-H2B is readily detected in cancer cells, its functional role in pathogenesis is not known. Herein, we show that WEE1 deposits the pY37-H2B marks within the tumor suppressor gene, isocitrate dehydrogenase 2 (IDH2), to repress transcription in multiple cancer cells, including glioblastoma multiforme (GBMs), melanoma and prostate cancer. Consistently, GBMs and primary melanoma tumors that display elevated WEE1 mRNA expression exhibit significant down regulation of the IDH2 gene transcription. IDH2 catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG), an essential cofactor for the TET family of 5-methylcytosine (5mC) hydroxylases that convert 5-mC to 5-hydroxymethylcytosine (5-hmC). Significantly, the WEE1 inhibitor AZD1775 not only abrogated the suppressive H2B Y37-phosphorylation and upregulated IDH2 mRNA levels but also effectively reversed the 'loss of 5-hmC' phenotype in melanomas, GBMs and prostate cancer cells, as well as melanoma xenograft tumors. These data indicate that the epigenetic repression of IDH2 by WEE1/pY37-H2B circuit may be a hitherto unknown mechanism of global 5-hmC loss observed in human malignancies.

Human transcription factors in yeast: the fruitful examples of P53 and NF-кB.

The observation that human transcription factors (TFs) can function when expressed in yeast cells has stimulated the development of various functional assays to investigate (i) the role of binding site sequences (herein referred to as response elements, REs) in transactivation specificity, (ii) the impact of polymorphic nucleotide variants on transactivation potential, (iii) the functional consequences of mutations in TFs and (iv) the impact of cofactors or small molecules. These approaches have found applications in basic as well as applied research, including the identification and the characterisation of mutant TF alleles from clinical samples. The ease of genome editing of yeast cells and the availability of regulated systems for ectopic protein expression enabled the development of quantitative reporter systems, integrated at a chosen chromosomal locus in isogenic yeast strains that differ only at the level of a specific RE targeted by a TF or for the expression of distinct TF alleles. In many cases, these assays were proven predictive of results in higher eukaryotes. The potential to work in small volume formats and the availability of yeast strains with modified chemical uptake have enhanced the scalability of these approaches. Next to well-established one-, two-, three-hybrid assays, the functional assays with non-chimeric human TFs enrich the palette of opportunities for functional characterisation. We review ∼25 years of research on human sequence-specific TFs expressed in yeast, with an emphasis on the P53 and NF-кB family of proteins, highlighting outcomes, advantages, challenges and limitations of these heterologous assays.

Quantitative Analysis of NF-κB Transactivation Specificity Using a Yeast-Based Functional Assay.

The NF-κB transcription factor family plays a central role in innate immunity and inflammation processes and is frequently dysregulated in cancer. We developed an NF-κB functional assay in yeast to investigate the following issues: transactivation specificity of NF-κB proteins acting as homodimers or heterodimers; correlation between transactivation capacity and in vitro DNA binding measurements; impact of co-expressed interacting proteins or of small molecule inhibitors on NF-κB-dependent transactivation. Full-length p65 and p50 cDNAs were cloned into centromeric expression vectors under inducible GAL1 promoter in order to vary their expression levels. Since p50 lacks a transactivation domain (TAD), a chimeric construct containing the TAD derived from p65 was also generated (p50TAD) to address its binding and transactivation potential. The p50TAD and p65 had distinct transactivation specificities towards seventeen different κB response elements (κB-REs) where single nucleotide changes could greatly impact transactivation. For four κB-REs, results in yeast were predictive of transactivation potential measured in the human MCF7 cell lines treated with the NF-κB activator TNFα. Transactivation results in yeast correlated only partially with in vitro measured DNA binding affinities, suggesting that features other than strength of interaction with naked DNA affect transactivation, although factors such as chromatin context are kept constant in our isogenic yeast assay. The small molecules BAY11-7082 and ethyl-pyruvate as well as expressed IkBα protein acted as NF-κB inhibitors in yeast, more strongly towards p65. Thus, the yeast-based system can recapitulate NF-κB features found in human cells, thereby providing opportunities to address various NF-κB functions, interactions and chemical modulators.

Cooperative interactions between p53 and NFκB enhance cell plasticity.

The p53 and NFκB sequence-specific transcription factors play crucial roles in cell proliferation and survival with critical, even if typically opposite, effects on cancer progression. To investigate a possible crosstalk between p53 and NFκB driven by chemotherapy-induced responses in the context of an inflammatory microenvironment, we performed a proof of concept study using MCF7 cells. Transcriptome analyses upon single or combined treatments with doxorubicin (Doxo, 1.5µM) and the NFκB inducer TNF-alpha (TNFα, 5ng/ml) revealed 432 up-regulated (log2 FC> 2), and 390 repressed genes (log2 FC< -2) for the Doxo+TNFα treatment. 239 up-regulated and 161 repressed genes were synergistically regulated by the double treatment. Annotation and pathway analyses of Doxo+TNFα selectively up-regulated genes indicated strong enrichment for cell migration terms. A panel of genes was examined by qPCR coupled to p53 activation by Doxo, 5-Fluoruracil and Nutlin-3a, or to p53 or NFκB inhibition. Transcriptome data were confirmed for 12 of 15 selected genes and seven (PLK3, LAMP3, ETV7, UNC5B, NTN1, DUSP5, SNAI1) were synergistically up-regulated after Doxo+TNFα and dependent both on p53 and NFκB. Migration assays consistently showed an increase in motility for MCF7 cells upon Doxo+TNFα. A signature of 29 Doxo+TNFα highly synergistic genes exhibited prognostic value for luminal breast cancer patients, with adverse outcome correlating with higher relative expression. We propose that the crosstalk between p53 and NFκB can lead to the activation of specific gene expression programs that may impact on cancer phenotypes and potentially modify the efficacy of cancer therapy.

An overview on chemical composition, bioactivity and processing of leaves of Cinnamomum tamala.

Dried leaves of Cinnamomum tamala, also known as Indian bay leaves, are a lesser-known spice used in the Indian subcontinent. It imparts a warm, peppery, clove-cinnamon like flavor to a variety of food preparations. Besides food applications, the leaves have also been traditionally used for curing a number of ailments and for other perceived health benefits. They find mention in the Aurvedic, Yunani, and other traditional medicinal literature. This review summarizes the effect of Cinnamomum tamala leaves on biological systems such as immune system, gastro-intestinal tract, liver and its antioxidant, antidiabetic, anti-inflammatory, anticancer, and antimicrobial activity. Chemical components that may be responsible for its flavor as well as bioactivity, have also been discussed.

Socio demographic determinants and knowledge, attitude, practice: survey of family planning.

BACKGROUND: Understanding of family planning scenario among different societies and communities, which by and large reside in urban slum and rural areas, might prove useful in increasing family planning acceptance by them and decreasing population growth. OBJECTIVE: To assess the sociodemographic determinants and KAP of family planning among urban slum and rural areas of Lucknow. STUDY DESIGN: Cross sectional. SETTING: Bal Mahila Chikitsalaya, Aliganj, in urban and Primary Health Centre, Bakshi Ka Talaab, in rural area of Lucknow. STUDY PERIOD: October 2008 to April 2009. MATERIALS AND METHODS: Six hundred and eightytwo postpartum women (within 42 days of delivery) who came to these health facilities for their child's vaccination were interviewed, by a preformed and pretested schedule. RESULTS: Maximum utilization of family methods were seen among Hindu women, women of age group 30 or more, parity four and more, educational level upto high school and above and those of higher socioeconomic class. Although overall residential area (urban or rural) of women had no influence on the practice of family planning by them and all of them were willing to adopt family planning methods in future, urban women were found to have a higher level of knowledge and attitude toward modern methods of family planning. Only 2.8% were unsure of preferred method for future use. CONCLUSION: Family planning programs which effectively promotes the use of family planning methods, so that the trend toward increase in population could be arrested is the need of hour.

A thought on the biological activities of black tea.

Tea is the most widely used ancient beverage in the world and black tea possesses many biological effects on the organisms. It acts as an effective antioxidant because of its free radical-scavenging and metal-chelating ability. Due to this, it is active against inflammation, clastogenesis, and several types of cancer. Tea reduces DNA damage and mutagenesis due to oxidative stress or the presence of pro-mutagens through antioxidant function, blocking activation pathways of mutagens, suppressing transcription of enzymes involved etc. Inhibition of low-density lipoprotein (LDL) peroxidation, suppression of fatty acid synthase etc., suggest that tea may have a role in preventing cardiovascular diseases. Some epidemiological studies support the protective role of black tea against cardiovascular diseases but some do not. Besides, black tea has beneficial effects on the gastrointestinal tract; it affects motility, absorption, microflora etc., by influencing the hormonal balance and antioxidant function black tea improves bone mineral density. It is also antiviral due to its enzyme-inhibiting and receptor-blocking properties. Although its role in cancers of the gastrointestinal tract, liver, and prostate is confirmed, its effect against urinary tract cancer is uncertain and further studies are required. Apart from these, excess consumption may lead to the formation of a stained pellicle layer on teeth, which is difficult to eliminate, inhibits trypsin, influences mineral absorption, causes convulsions etc. Excess caffeine intake may have adverse effects on selected organs as reported in studies on some organisms. These reports indicate that there is a wide scope of further research for the efficient use of black tea active conserves/isolates to reap health benefits.