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Osteoporosis is a bone disease that is exacerbated by aging and age-associated chronic diseases such as cancer. Cancer-induced bone loss is usually treated with bisphosphonates or denosumab, an antibody against receptor activator of nuclear factor (NF)-κB ligand (RANKL). Because these drugs are expensive and have numerous side effects and high rates of toxicity, safer, more effective, and more affordable therapies for osteoporosis are still needed. We identified a compound, calebin A (CA), derived from turmeric ( Curcuma longa ) that affects osteoclastogenesis through modulation of the RANKL signalling pathway. The CA's effect on NF-κB activation was examined by electrophoretic mobility shift assay. Using mouse macrophages in vitro model, we found that CA suppressed RANKL-induced osteoclast differentiation of macrophages into osteoclasts, and downregulate RANKL-induced osteoclastogenesis-related marker gene expression, including NFATc-1, TRAP, CTR, and cathepsin K. CA also suppressed the osteoclastogenesis induced by multiple myeloma and breast cancer cells. This effect of CA was correlated with suppression of the phosphorylation and degradation of inhibitor of κB and, thus, inhibition of NF-κB activation. Furthermore, we found that an NF-κB-specific inhibitory peptide blocked RANKL-induced osteoclastogenesis, demonstrating that the NF-κB signalling pathway is mandatory for RANKL-induced osteoclastogenesis. Our results conclusively indicate that CA downmodulates the osteoclastogenesis induced by RANKL and by tumour cells through suppression of NF-κB pathway. [ABSTRACT FROM AUTHOR]

Mitosis is an essential process for the self-renewal of cells that is accompanied by dynamic changes in nuclear architecture and chromatin organization. Despite all the changes, the cell manages to re-establish all the parental epigenetic marks, post-mitotically. Recent reports suggest that some sequence-specific transcription factors remain attached to mitotic chromatin during cell division to ensure timely reactivation of a subset of transcription factors necessary to maintain cell identity. These mitotically associated factors are suggested to act as 'genome bookmarking factors' and the phenomenon is termed 'genome bookmarking'. Here, we studied this phenomenon with Vitamin D Receptor (VDR), a key regulator of calcium and phosphate homeostasis and a member of the nuclear receptor superfamily. This study, for the first time, has confirmed VDR as a mitotic bookmarking factor that may be playing a crucial role in the maintenance of cell identity and genome bookmarking. Full 'DNA binding domain (DBD)' present in VDR was identified as essential for enrichment of VDR on mitotic chromatin. Furthermore, the study also demonstrates that VDR evokes mitotic chromatin binding behaviour in its heterodimeric partner Retinoid X receptor (RXR). Interestingly, for promoting bookmarking behaviour in RXR, both DBD and/or ligand-binding domain (LBD) in conjunction with hinge region of VDR were required. Additionally, ChIP analysis showed that VDR remains associated with DR3 (direct repeat 3) region of its specific target gene promoter CYP24A1 (Cytochrome P450 family 24 subfamily A member1), during mitosis. Altogether, our study illustrates a novel function of VDR in the epigenetic transmission and control of expression of target proteome for maintenance of cell identity and traits in progeny cells. • VDR remains constitutively associated with mitotic chromatin during cell division. • VDR promotes mitotic chromatin binding of its heterodimeric partner RXR. • VDR binds with DR3 sequences in target gene promoters during interphase and mitosis. • VDR-DBD plays a central role in constitutive genome bookmarking. [ABSTRACT FROM AUTHOR]

Human tumor necrosis factor (TNF), first isolated by our group as an anticancer agent, has been now shown to be a primary mediator of inflammation. Till today 19 different members of the TNF superfamily which interact with 29 different receptors, have been identified. Most members of this family exhibit pro-inflammatory activities, in part through the activation of the transcription factor, nuclear factor-kappaB (NF-κB). Thus TNF and the related pro-inflammatory cytokines have been shown to play a key role in most chronic diseases such as cancer, rheumatoid arthritis, cardiovascular diseases, psoriasis, neurologic diseases, Crohn’s disease, and metabolic diseases. Therefore, agents that can modulate the TNF-mediated inflammatory pathways may have potential against these pro-inflammatory diseases. Although blockers of TNF-α, such as infliximab (antibody against TNF-α), adalimumab (humanized antibody against TNF-α), and etanercept (soluble form of TNFR2) have been approved for human use, these blockers exhibit numerous side effects. In this review, we describe various plant-derived polyphenols that can suppress TNF-α activated inflammatory pathways both in vitro and in vivo . These polyphenols include curcumin, resveratrol, genistein, epigallocatechin gallate, flavopiridol, silymarin, emodin, morin isoliquiritigenin, naringenin, ellagic acid, apigenin, kaempferol, catechins, myricetin, xanthohumol, fisetin, vitexin, escin, mangostin and others. Thus these polyphenols are likely to have potential against various pro-inflammatory diseases. [ABSTRACT FROM AUTHOR]

Abstract: Small auxin-up RNAs (SAURs) are the early auxin-responsive genes represented by a large multigene family in plants. Here, we report the identification of 58 OsSAUR gene family members from rice (Oryza sativa japonica cv Nipponbare), the model monocot plant, by a reiterative database search and manual reannotation; 2 of these are pseudogenes. The coding sequences of OsSAURs do not possess any intron. Most of the predicted OsSAUR protein sequences harbor a putative nuclear localization signal at their N-terminus. Localized gene duplications appear to be the primary genetic event responsible for SAUR gene family expansion in rice. Interestingly, the duplication of OsSAURs was found to be associated with the chromosomal block duplication as well. The phylogenetic analysis revealed that the SAUR gene family expanded in rice and Arabidopsis due to species-specific expansion of the family in monocots and dicots. The auxin-responsive elements and downstream element are conserved in the upstream and downstream sequences, respectively, of OsSAURs. In addition to the 21 OsSAURs with full-length cDNA sequences and 20 with expressed sequence tags, gene expression analyses of at least 7 OsSAURs by RT-qPCR indicated that the majority of identified OsSAURs most likely are expressed in rice. The transcript abundance of the OsSAURs examined increased within a few minutes of exogenous auxin application with varying kinetics. The present study provides basic genomic information for the rice SAUR gene family and will pave the way for deciphering the precise role of SAURs in plant growth and development. [Copyright &y& Elsevier]

Bladder cancer is the fourth and eighth most common cancer in men and women in the United States, respectively. Survivin, a member of inhibitor of apoptosis protein (IAP) gene family, is deregulated in a wide range of malignancies, including carcinoma of the bladder urothelium. Recent advances have identified survivin as a novel intervention target to induce apoptosis in cancer cells by phytochemicals or synthetic agents. Silibinin is a naturally occurring flavanone, isolated from milk thistle extract, and has been shown to possess cancer prevention/intervention potential against various cancers. In several animal and human studies, it is found to be safe and non-toxic. Human bladder transitional-cell papilloma RT4 cells were treated with silibinin and analyzed for survivin protein and mRNA levels by Western blotting and real-time RT-PCR, respectively. Silibinin treatment of cells for 24 h at 100 μM dose resulted in ∼50% decrease in survivin protein level; however, treatment at 200 μM dose for 24 and 48 h showed a complete loss in survivin protein without any change in actin used as loading control. Employing RT-PCR analysis we also observed that silibinin causes a strong to complete decrease in survivin mRNA levels. In other studies, down-regulation of survivin by silibinin was associated with a very strong and prominent caspases-9 and -3 activation as well as PARP cleavage. Quantitative apoptotic assay showed that silibinin decreased survivin levels and caspases-PARP cleavages, in accord with a strong apoptotic death and growth inhibition of RT4 cells. Together, these findings suggest that more studies are needed to investigate in vivo effect of silibinin on survivin expression and associated biological effects in bladder cancer that could provide useful information for silibinin efficacy in the prevention/intervention of human bladder cancer. [Copyright &y& Elsevier]

Powder samples of the new oxide fluoride V2GeO4F2 have been obtained by the reaction of appropriate amounts V2O3, VF3, and GeO2 at 700°C for 18 h in an argon-filled sealed platinum tube. V2GeO4F2 crystallizes in the space group Pnma with a=9.336(1), b=8.898 (1), and c=4.912 (1) A˚. The structure has been refined from X-ray powder diffraction data using the Rietveld method (Rint=5.5% and Rp=9.8%). The structure of V2GeO4F2 exhibits close packed layers of the anions with an ordering of O and F. The characteristic building units are discrete GeO4 tetrahedra with Ge–O distances of 1.75–1.80 A˚. The V are coordinated by four O and two F to form VO4F2 octahedra connected via two common edges to give zigzag chains. These chains are linked via corners to form a three-dimensional network. The temperature dependence of the magnetic susceptibility of V2GeO4F2 indicates antiferromagnetic correlations. [Copyright &y& Elsevier]

Thyroid hormone receptor β (THRβ) is a member of the nuclear receptor superfamily of ligand-modulated transcription factors. Upon ligand binding, THRβ sequentially recruits the components of transcriptional machinery to modulate target gene expression. In addition to regulating diverse physiological processes, THRβ plays a crucial role in hypothalamus-pituitary-thyroid axis feedback regulation. Anomalies in THRβ gene/protein structure are associated with onset of diverse disease states. In this study, we investigated disease-inflicting truncated variants of THRβ using in-silico analysis and cell-based assays. We examined the THRβ truncated variants on multiple test parameters, including subcellular localization, ligand-receptor interactions, transcriptional functions, interaction with heterodimeric partner RXR, and receptor-chromatin interactions. Moreover, molecular dynamic simulation approaches predicted that shortened THRβ-LBD due to point mutations contributes proportionally to the loss of structural integrity and receptor stability. Deviant subcellular localization and compromised transcriptional function were apparent with these truncated variants. Present study shows that ' mitotic bookmarking' property of some THRβ variants is also affected. The study highlights that structural and conformational attributes of THRβ are necessary for normal receptor functioning, and any deviations may contribute to the underlying cause of the inflicted diseases. We anticipate that insights derived herein may contribute to improved mechanistic understanding to assess disease predisposition. • Cellular and physiological functions of T 3 are mediated via THRβ. • Genetic alterations in the THRB gene cause several thyroid-related disorders. • Introduction of premature stop codon by a critical SNP/mutation truncates THRβ. • Truncated THRβ variants exhibit aberrant cellular functions to inflict diseases. • Understanding disease-inflicting THRβ variants may aid novel therapeutic avenues. [ABSTRACT FROM AUTHOR]

Abstract: The real-time polymerase chain reaction (PCR) data requires normalization with an internal control gene expressed at constant levels under all the experimental conditions being analyzed for accurate and reliable gene expression results. In this study, the expression of 12 candidate internal control genes, including ACT1, EF1α, GAPDH, IF4a, TUB6, UBC, UBQ5, UBQ10, 18SrRNA, 25SrRNA, GRX and HSP90, in a diverse set of 18 tissue samples representing different organs/developmental stages and stress conditions in chickpea (Cicer arietinum L.) has been validated. Their expression levels vary considerably in various tissue samples analyzed. The expression levels of EF1α and HSP90 are most constant across various organs/developmental stages analyzed. Similarly, the expression levels of IF4a and GAPDH are most constant across various stress conditions. A set of two most stable genes is found sufficient for accurate and reliable normalization of real-time PCR data in the given set of tissue samples of chickpea. The genes with most constant expression identified in this study should be useful for normalization of gene expression data in a wide variety of tissue samples in chickpea. [Copyright &y& Elsevier]

Abstract: Cyclin dependent kinase 5 (cdk5) is active mainly in postmitotic cells like neurons and regulates important cellular functions by phosphorylating a wide variety of targets. Nerve growth factor stimulates the MEK-ERK MAP kinase pathway and causes neuronal differentiation and survival. It was reported previously that Cdk5 inhibits the MAP kinase pathway by phosphorylating Map kinase kinase-1 (MEK1) . We have delineated the functional consequence of this cross talk and found that the cdk5 mediated inhibition of MEK1 results in apoptosis. We also demonstrate that the activity of transcription factor CREB, which is known to play pro-survival roles in neuronal cells, is attenuated as a result of this cross-talk. [Copyright &y& Elsevier]

For accurate and reliable gene expression results, normalization of real-time PCR data is required against a control gene, which displays highly uniform expression in living organisms during various phases of development and under different environmental conditions. We assessed the gene expression of 10 frequently used housekeeping genes, including 18S rRNA, 25S rRNA, UBC, UBQ5, UBQ10, ACT11, GAPDH, eEF-1α, eIF-4a, and β-TUB, in a diverse set of 25 rice samples. Their expression varied considerably in different tissue samples analyzed. The expression of UBQ5 and eEF-1α was most stable across all the tissue samples examined. However, 18S and 25S rRNA exhibited most stable expression in plants grown under various environmental conditions. Also, a set of two genes was found to be better as control for normalization of the data. The expression of these genes (with more uniform expression) can be used for normalization of real-time PCR results for gene expression studies in a wide variety of samples in rice. [Copyright &y& Elsevier]

Retinoid X Receptor (RXR) serves as the heterodimeric partner of two major xenobiotic nuclear receptors, Pregnane and Xenobiotic Receptor (PXR) and Constitutive Androstane Receptor (CAR). These receptors are primarily involved in the metabolism and clearance of endobiotics and xenobiotics (including clinical drugs) from the body. Here, we report for the first time that intermolecular interactions between RXR-PXR and RXR-CAR occurs in the cytoplasmic compartment of the cell in a ligand-independent manner. These interactions lead to nuclear import of the heterodimeric complex thereby making them competent for chromatin binding and transactivation of target genes. To explore the cellular site involved in the process of heterodimerization we created various RFP- and GFP-tagged receptor chimeras and also the mutants of their nuclear localization signal (NLS). From the study it is apparent that NLS of PXR/CAR/RXR play a major role in the import of the heterodimeric complex from the cytoplasm to the nucleus in a ligand-independent manner. We observed that along with the heterodimeric partner and/or respective ligand a functional NLS is necessary for activation of target gene. The data suggests that RXR is the major driving force to import the heterodimeric complex into the nucleus since the mutation in the NLS region of RXR weakens this import process dramatically, whereas mutations in the NLS regions of PXR and CAR have little or no significant effect. This RXR-dependent nuclear translocation of the heterodimeric complex also modulates the individual transcriptional activity of PXR and CAR by increasing the basal transcriptional activity. Finally, it is documented that the heterodimerization of RXR with both the partners (PXR, CAR) occurs in the cytoplasm and implies that these dynamic interactions have functional and regulatory attributes in gene expression. In addition, this RXR-dependent enhancement of the transcriptional activity of PXR and CAR may be utilized for evaluating the receptor-drug interactions. [ABSTRACT FROM AUTHOR]

Pregnane and Xenobiotic Receptor (PXR), a member of nuclear receptor superfamily, acts as a ‘xenosensor’ in our body and modulates a network of genes involved in xenobiotic metabolism and elimination. Expression levels of PXR in certain metabolic disorders including cancer are reported to be altered and its induced expression is associated with the development of resistance towards chemotherapy and adverse drug–drug interactions. Though the transcriptional regulation of PXR target genes have been elucidated in significant details, the structure and functional control of PXR promoter itself remains inadequately explored. In this work, we identify a Composite Element (CE) located within the proximal PXR promoter region that consists of multiple overlapping cis -elements and demonstrated that CE interacts specifically with some critical nuclear proteins. Subsequent DNA-protein interaction studies revealed mutually exclusive interactions on CE occurring between Sp1 and two unidentified DNA binding proteins with molecular masses of 50 and 54 kDa. Here, we report the identification of 54 kDa CE binding protein as a heterogeneous nuclear ribonucleoprotein K (hnRNPK) and demonstrate the effect of hnRNP K and Sp1 on PXR promoter transcriptional activity. Overall, the study indicates that PXR gene is tightly regulated to maintain a low receptor level. [ABSTRACT FROM AUTHOR]

Nuclear receptor PXR is predominantly expressed in liver and intestine. Expression of PXR is observed to be dysregulated in various metabolic disorders indicating its involvement in disease development. However, information available on mechanisms of PXR self-regulation is fragmentary. The present investigation identifies some of the regulatory elements responsible for its tight regulation and low cellular expression. Here, we report that the PXR-promoter is a target for some key transcription factors like PU.1/Ets-1, Pax5, LEF-1 and c-Jun. Interestingly, we observed that PXR-promoter responsiveness to Pax5, LEF-1 and c-Jun, is considerably enhanced by Ets transcription factors (PU.1 and Ets-1). Co-transfection of cells with Ets-1, LEF-1 and c-Jun increased PXR-promoter activity by 5-fold and also induced expression of endogenous human PXR. Site-directed mutagenesis and transfection studies revealed that two Ets binding sites and two of the three LEF binding sites in the PXR-promoter are functional and have a positive effect on PXR transcription. Results suggest that expression of Ets family members, in conjunction with Pax5, LEF-1 and c-Jun, lead to coordinated up-regulation of PXR gene transcription. Insights obtained on the regulation of PXR gene have relevance in offering important cues towards normal functioning as well as development of several metabolic disorders via PXR signaling. [ABSTRACT FROM AUTHOR]

Variation in degree of disorder and its impact on photocatalytic dye degradation has been studied in Y 2 Sn 2-x Zr x O 7 series. The structural variations of the compositions were thoroughly characterized by X-ray diffraction, Raman spectroscopy, 119Sn MAS NMR, EXAFS and XPS. A phase transition from ordered pyrochlore to disordered fluorite could be observed in the series whilst transiting from Y 2 Sn 2 O 7 to Y 2 Zr 2 O 7 without any signature of presence of bi-phasicity. Formation of a random solid solution phase has been confirmed by 119Sn MAS NMR results. All the compositions until x ​= ​1.0 remain as pyrochlore whereas the compositions with x ​≥ ​1.5 preferentially adopt defect-fluorite structure. X-ray photoelectron spectroscopy clearly demonstrates that the coordination environment Zr and O varies with increase in Zr content, however, the coordination environment of Sn does not alter with substitution. The phase transformation from pyrochlore to defect fluorite occurs at relatively higher Zr rich composition than expected from classical radius ratio rule due to higher covalency present in Sn–O bond. With increase in Zr content in the series crystallite size, surface charge, nature BO 6 octahedra etc. varies and it has been found that these parameters has strong bearing on catalytic property of the materials. A generic understanding for the photocatalytic degradation of cationic methylene blue dye was achieved as a function of structural disordering in the Y 2 Sn 2-x Zr x O 7 system. [Display omitted] • Order-disorder phenomena in pyrochlore has been studied by XRD and Raman Spectroscopy. • 119Sn NMR and XPS could be successfully employed to investigate local structure of pyrochlore. • It has been observed that covalency of Sn–O bond has strong role in extension of stability of pyrochlore phase. • Surface charge, crystallite size, degree of disorder in the system has profound effect on photocatalytic dye degradation. [ABSTRACT FROM AUTHOR]

Abstract: Accurate diagnosis is essential for the treatment, prevention, and control of tuberculosis. Poor specificity of the tuberculin skin test in BCG-vaccinated populations and constraints to implementation of PCR and CMI-based diagnostic assays in developing countries warrant development of easy-to perform robust serological tests. Due to great heterogeneity in humoral response in TB patients, it will be necessary to include several antigens in any diagnostic assay to achieve useful levels of sensitivity and specificity. This needs production of recombinants, soluble versions of mycobacterial antigens in high yields. We have cloned, expressed, and purified a number of mycobacterial proteins in Escherichia coli. This paper describes the expression and purification of four promising sero-reactive proteins namely, ESAT6, CFP10, MTC28, and 14-kDa antigen of Mycobacterium tuberculosis. The protocol involves regulated and slow expression of proteins by using a T7 promoter-based expression vector for obtaining soluble protein followed by a three-step column chromatography procedure employing media with high binding capacity and flow characteristics. The yields of these proteins obtained were several folds higher than previously reported. The purified proteins were useful in detecting antibodies in sera of TB patients (smear positive, smear negative, and extra-pulmonary categories) and in combination with other immunodominant antigens will be useful in increasing the sensitivity to detect M. tuberculosis specific antibodies. [Copyright &y& Elsevier]

Abstract: Availability of genome sequence of Mycobacterium tuberculosis has accelerated identification of antigens for serodiagnosis of tuberculosis and a number of new antigens are being tested in various combinations to produce cocktails with high sensitivity and specificity. For producing a highly specific diagnostic test, it is important that the recombinant antigens be highly pure, free of host protein, and correctly folded so that they bind only to specific antibodies. Also, for commercial viability they need to be produced in high yields. We have cloned, expressed, and purified a number of mycobacterial antigens in Escherichia coli. This paper describes, expression and purification of two important mycobacterial proteins with serodiagnostic potential, namely, 38-kDa and Mtb81 antigens, in monomeric form. The protocol involves using a T7 promoter based expression vector under conditions of regulated and slow expression followed by three-step column chromatography procedure to obtain highly purified proteins. The yields of the two proteins were several folds higher than previously reported. The purified proteins were useful in detecting antibodies in sera of tuberculosis patients (smear positive, smear negative, and extra-pulmonary categories). [Copyright &y& Elsevier]

The regulation of cellular processes by the modulation of protein phosphorylation/dephosphorylation is fundamental to a large number of processes in living organisms. These processes are carried out by specific protein kinases and phosphatases. In this study, a previously uncharacterized gene (Rv0018c) of Mycobacterium tuberculosis, designated as mycobacterial Ser/Thr phosphatase (mstp), was cloned, expressed in Escherichia coli, and purified as a histidine-tagged protein. Purified protein (Mstp) dephosphorylated the phosphorylated Ser/Thr residues of myelin basic protein (MBP), histone, and casein but failed to dephosphorylate phospho-tyrosine residue of these substrates, suggesting that this phosphatase is specific for Ser/Thr residues. It has been suggested that mstp is a part of a gene cluster that also includes two Ser/Thr kinases pknA and pknB. We show that Mstp is a trans-membrane protein that dephosphorylates phosphorylated PknA and PknB. Southern blot analysis revealed that mstp is absent in the fast growing saprophytes Mycobacterium smegmatis and Mycobacterium fortuitum. PknA has been shown, whereas PknB has been proposed to play a role in cell division. The presence of mstp in slow growing mycobacterial species, its trans-membrane localization, and ability to dephosphorylate phosphorylated PknA and PknB implicates that Mstp may play a role in regulating cell division in M. tuberculosis. [Copyright &y& Elsevier]

The high-pressure behavior of hexagonal Na1.5Y1.5F6 is investigated with synchrotron angle-dispersive X-ray powder diffraction in a diamond anvil cell up to 22 GPa at room temperature. There is no major phase transition in this material in the entire pressure region studied here. At near ambient pressures, the structure is of the Na1.5Nd1.5F6 type (P6, Z=1), found in other low-temperature sodium lanthanide fluorides. Pressure-induced order–disorder phenomena lead to the formation of the gagarinite structure (P63/m, Z=1) above 20 GPa. Alternatively, the pressure-induced order–disorder phenomena could be related to a polymorphic transition towards the phase of the β-Na2ThF6 type with P62m (Z=1) symmetry. There is no volume change associated with all these reversible structural changes. [Copyright &y& Elsevier]