H3S10ph broadly marks early-replicating domains in interphase ESCs and shows reciprocal antagonism with H3K9me2.

Phosphorylation of histone H3 at serine 10 (H3S10ph) by Aurora kinases plays an important role in mitosis; however, H3S10ph also marks regulatory regions of inducible genes in interphase mammalian cells, implicating mitosis-independent functions. Using the fluorescent ubiquitin-mediated cell cycle indicator (FUCCI), we found that 30% of the genome in interphase mouse embryonic stem cells (ESCs) is marked with H3S10ph. H3S10ph broadly demarcates gene-rich regions in G1 and is positively correlated with domains of early DNA replication timing (RT) but negatively correlated with H3K9me2 and lamin-associated domains (LADs). Consistent with mitosis-independent kinase activity, this pattern was preserved in ESCs treated with Hesperadin, a potent inhibitor of Aurora B/C kinases. Disruption of H3S10ph by expression of nonphosphorylatable H3.3S10A results in ectopic spreading of H3K9me2 into adjacent euchromatic regions, mimicking the phenotype observed in Drosophila JIL-1 kinase mutants. Conversely, interphase H3S10ph domains expand in Ehmt1 (also known as Glp) null ESCs, revealing that H3S10ph deposition is restricted by H3K9me2. Strikingly, spreading of H3S10ph at RT transition regions (TTRs) is accompanied by aberrant transcription initiation of genes co-oriented with the replication fork in Ehmt1-/- and Ehmt2-/- ESCs, indicating that establishment of repressive chromatin on the leading strand following DNA synthesis may depend upon these lysine methyltransferases. H3S10ph is also anti-correlated with H3K9me2 in interphase murine embryonic fibroblasts (MEFs) and is restricted to intragenic regions of actively transcribing genes by EHMT2. Taken together, these observations reveal that H3S10ph may play a general role in restricting the spreading of repressive chromatin in interphase mammalian cells.

Vitamin C induces Tet-dependent DNA demethylation and a blastocyst-like state in ES cells.

DNA methylation is a heritable epigenetic modification involved in gene silencing, imprinting, and the suppression of retrotransposons. Global DNA demethylation occurs in the early embryo and the germ line, and may be mediated by Tet (ten eleven translocation) enzymes, which convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Tet enzymes have been studied extensively in mouse embryonic stem (ES) cells, which are generally cultured in the absence of vitamin C, a potential cofactor for Fe(II) 2-oxoglutarate dioxygenase enzymes such as Tet enzymes. Here we report that addition of vitamin C to mouse ES cells promotes Tet activity, leading to a rapid and global increase in 5hmC. This is followed by DNA demethylation of many gene promoters and upregulation of demethylated germline genes. Tet1 binding is enriched near the transcription start site of genes affected by vitamin C treatment. Importantly, vitamin C, but not other antioxidants, enhances the activity of recombinant Tet1 in a biochemical assay, and the vitamin-C-induced changes in 5hmC and 5mC are entirely suppressed in Tet1 and Tet2 double knockout ES cells. Vitamin C has a stronger effect on regions that gain methylation in cultured ES cells compared to blastocysts, and in vivo are methylated only after implantation. In contrast, imprinted regions and intracisternal A particle retroelements, which are resistant to demethylation in the early embryo, are resistant to vitamin-C-induced DNA demethylation. Collectively, the results of this study establish vitamin C as a direct regulator of Tet activity and DNA methylation fidelity in ES cells.

Regulation of DNA methylation turnover at LTR retrotransposons and imprinted loci by the histone methyltransferase Setdb1.

During mammalian development, DNA methylation patterns need to be reset in primordial germ cells (PGCs) and preimplantation embryos. However, many LTR retrotransposons and imprinted genes are impervious to such global epigenetic reprogramming via hitherto undefined mechanisms. Here, we report that a subset of such genomic regions are resistant to widespread erasure of DNA methylation in mouse embryonic stem cells (mESCs) lacking the de novo DNA methyltransferases (Dnmts) Dnmt3a and Dnmt3b. Intriguingly, these loci are enriched for H3K9me3 in mESCs, implicating this mark in DNA methylation homeostasis. Indeed, deletion of the H3K9 methyltransferase SET domain bifurcated 1 (Setdb1) results in reduced H3K9me3 and DNA methylation levels at specific loci, concomitant with increased 5-hydroxymethylation (5hmC) and ten-eleven translocation 1 binding. Taken together, these data reveal that Setdb1 promotes the persistence of DNA methylation in mESCs, likely reflecting one mechanism by which DNA methylation is maintained at LTR retrotransposons and imprinted genes during developmental stages when DNA methylation is reprogrammed.

Shelf life determinants and enzyme activities of pearl millet: a comparison of changes in stored flour of hybrids, CMS lines, inbreds and composites.

Shelf life of pearl millet flour is very short because of rapid development of rancidity. This investigation was carried out in view of generating breeding material for development of low rancid pearl millet hybrids/varieties. Flour of twenty-one genotypes; seven hybrids, seven CMS lines, five inbreds and two composites stored in covered aluminium boxes at 37 °C for 30 days along with respective fresh flour was analysed for shelf life indicators/determinants. Crude fat content and fat acidity (FA) of fresh flour of the genotypes varied from 3.8 to 7.2% and 11 to 75 mg KOH/100 g d.m., respectively. FA in stored flour ranged between 180 and 330 mg KOH/100 g d.m. After storage, magnitude of decrease in pH of water extract of flour of the genotypes varied from 0.15 to 0.44. Activity of peroxidase (POX) varied from 378 to 588 units in control flour and irrespective of the genotypes decreased upon storage. Increase in FA (difference between FA of fresh and stored flour) rather total build up of FA was positively associated with crude fat content (r = 0.440*) indicated comparatively more prominent role of lipolytic enzymes. Chemical changes taking place in water soluble fraction of flour were independent of fat content as no correlation was discerned between fat content and decrease in pH. Among the hybrids, HHB 197 had lowest crude fat content (4.7%), lowest total build up FA (212 mg KOH/100 g d.m.), slowest increase in FA (191 mg KOH/100 g d.m.), least decrease in pH (0.31) of water soluble fraction flour during storage and lowest activity of POX in fresh flour (377 units/g d.m). Among all the tested CMS lines, inbreds and composites, HBL 11 showed pattern of quantitative changes in FA, pH and POX activity similar to the hybrid HHB 197 and was identified a promising inbred for developing low-rancid pearl millet variety or hybrid.

Retrotransposon-induced heterochromatin spreading in the mouse revealed by insertional polymorphisms.

The "arms race" relationship between transposable elements (TEs) and their host has promoted a series of epigenetic silencing mechanisms directed against TEs. Retrotransposons, a class of TEs, are often located in repressed regions and are thought to induce heterochromatin formation and spreading. However, direct evidence for TE-induced local heterochromatin in mammals is surprisingly scarce. To examine this phenomenon, we chose two mouse embryonic stem (ES) cell lines that possess insertionally polymorphic retrotransposons (IAP, ETn/MusD, and LINE elements) at specific loci in one cell line but not the other. Employing ChIP-seq data for these cell lines, we show that IAP elements robustly induce H3K9me3 and H4K20me3 marks in flanking genomic DNA. In contrast, such heterochromatin is not induced by LINE copies and only by a minority of polymorphic ETn/MusD copies. DNA methylation is independent of the presence of IAP copies, since it is present in flanking regions of both full and empty sites. Finally, such spreading into genes appears to be rare, since the transcriptional start sites of very few genes are less than one Kb from an IAP. However, the B3galtl gene is subject to transcriptional silencing via IAP-induced heterochromatin. Hence, although rare, IAP-induced local heterochromatin spreading into nearby genes may influence expression and, in turn, host fitness.

Lysine methyltransferase G9a is required for de novo DNA methylation and the establishment, but not the maintenance, of proviral silencing.

Methylation on lysine 9 of histone H3 (H3K9me) and DNA methylation play important roles in the transcriptional silencing of specific genes and repetitive elements. Both marks are detected on class I and II endogenous retroviruses (ERVs) in murine embryonic stem cells (mESCs). Recently, we reported that the H3K9-specific lysine methyltransferase (KMTase) Eset/Setdb1/KMT1E is required for H3K9me3 and the maintenance of silencing of ERVs in mESCs. In contrast, G9a/Ehmt2/KMT1C is dispensable, despite the fact that this KMTase is required for H3K9 dimethylation (H3K9me2) and efficient DNA methylation of these retroelements. Transcription of the exogenous retrovirus (XRV) Moloney murine leukemia virus is rapidly extinguished after integration in mESCs, concomitant with de novo DNA methylation. However, the role that H3K9 KMTases play in this process has not been addressed. Here, we demonstrate that G9a, but not Suv39h1 or Suv39h2, is required for silencing of newly integrated Moloney murine leukemia virus-based vectors in mESCs. The silencing defect in G9a(-/-) cells is accompanied by a reduction of H3K9me2 at the proviral LTR, indicating that XRVs are direct targets of G9a. Furthermore, de novo DNA methylation of newly integrated proviruses is impaired in the G9a(-/-) line, phenocopying proviral DNA methylation and silencing defects observed in Dnmt3a-deficient mESCs. Once established, however, maintenance of silencing of XRVs, like ERVs, is dependent exclusively on the KMTase Eset. Taken together, these observations reveal that in mESCs, the H3K9 KMTase G9a is required for the establishment, but not for the maintenance, of silencing of newly integrated proviruses.

Chol-Dex nanomicelles: Synthesis, characterization and evaluation as efficient drug carriers for colon targeting.

Core-shell structures obtained from amphiphilic molecules on self-assembly in a medium have emerged as an important tool in the area of biomedical sciences. Here, we have synthesized cholesteryl-dextran (Chol-Dex) amphiphiles in sufficiently high yields via conjugation of cholesteryl hemisuccinate to dextran in two different concentrations (5 and 10%). After physicochemical and spectral analysis, the nanomicelles were subjected to size measurements. DLS and TEM confirmed the formation of core-shell type of nanomicelles. Hydrophobic drug-entrapped formulations (Metronidazole and Rifampicin) displayed sustained release behaviour of drugs from them. Sustained release at neutral pH demonstrated usefulness of the non-toxic delivery system for colon specific diseases.

An unmethylated 3' promoter-proximal region is required for efficient transcription initiation.

The promoter regions of approximately 40% of genes in the human genome are embedded in CpG islands, CpG-rich regions that frequently extend on the order of one kb 3' of the transcription start site (TSS) region. CpGs 3' of the TSS of actively transcribed CpG island promoters typically remain methylation-free, indicating that maintaining promoter-proximal CpGs in an unmethylated state may be important for efficient transcription. Here we utilize recombinase-mediated cassette exchange to introduce a Moloney Murine Leukemia Virus (MoMuLV)-based reporter, in vitro methylated 1 kb downstream of the TSS, into a defined genomic site. In a subset of clones, methylation spreads to within approximately 320 bp of the TSS, yielding a dramatic decrease in transcript level, even though the promoter/TSS region remains unmethylated. Chromatin immunoprecipitation analyses reveal that such promoter-proximal methylation results in loss of RNA polymerase II and TATA-box-binding protein (TBP) binding in the promoter region, suggesting that repression occurs at the level of transcription initiation. While DNA methylation-dependent trimethylation of H3 lysine (K)9 is confined to the intragenic methylated region, the promoter and downstream regions are hypo-acetylated on H3K9/K14. Furthermore, DNase I hypersensitivity and methylase-based single promoter analysis (M-SPA) experiments reveal that a nucleosome is positioned over the unmethylated TATA-box in these clones, indicating that dense DNA methylation downstream of the promoter region is sufficient to alter the chromatin structure of an unmethylated promoter. Based on these observations, we propose that a DNA methylation-free region extending several hundred bases downstream of the TSS may be a prerequisite for efficient transcription initiation. This model provides a biochemical explanation for the typical positioning of TSSs well upstream of the 3' end of the CpG islands in which they are embedded.

Core/shell nanoassembly of amphiphilic naproxen-polyethylene glycol: synthesis, characterisation and evaluation as drug delivery system.

Small molecule-based amphiphiles self-assemble into nanostructures (micelles) in aqueous medium which are currently being explored as novel drug delivery systems. Here, naproxen-polyethylene glycol (N-PEG), a small molecule-derived amphiphile, has been synthesised, characterised and evaluated as hydrophobic drug carrier. 1H, 13C Nuclear magnetic resonance (NMR), mass spectrometry (MS) and Fourier-transform infrared spectroscopy (FTIR) confirmed the formation of N-PEG and dynamic light scattering (DLS) revealed the formation of nano-sized structures of ∼228 nm. Transmission electron microscope (TEM) analysis showed aggregation behaviour of the structures with average size of ∼230 nm. Biodegradability aspect of the micellar-structured N-PEG was demonstrated by lipase-mediated degradation studies using DLS and TEM. High encapsulation efficiency followed by release in a sustained manner of a well-known anticancer drug, doxorubicin, demonstrated the feasibility of the new drug delivery system. These results advocate the promising potential of N-PEG micelles as efficient drug delivery system for specific delivery to cancerous cells in vitro and in vivo.

Evaluation of Spot Urinary Albumin-Creatinine Ratio as Screening Tool in Prediction of Pre-eclampsia in Early Pregnancy.

Objective: The aim of this study was to establish whether a spot urinary albumin/creatinine ratio (ACR) measured between 20 and 28 weeks of gestation can predict subsequent pre-eclampsia in asymptomatic pregnant women. Design: Prospective observational study. Subjects: The patients included sixty-two women with singleton pregnancy, normal renal function and no evident proteinuria, attending antenatal clinics between 20 and 28 weeks of gestation in a tertiary care hospital. Methods: The ACR was determined from midstream urine sample taken between 20 and 28 weeks of gestation. Estimation of albumin was done by immunoturbidimetric microalbumin method and creatinine by modified Jaffe's method. Results: Incidence of pre-eclampsia in the study group was 12.90%. The cut-off value for ACR was taken as 35.5 mg/mol. The mean ACR in normotensive group was 19.26 ± 7.99, and in pre-eclampsia group it was 51.95 ± 18.78. For pre-eclampsia, screening in early pregnancy, spot ACR cut-off ≥35.5 mg/mol has sensitivity of 87.5%, specificity of 96.30%, PPV of 77.78% and NPV of 98.11%. Conclusions: Spot urinary ACR values are higher in asymptomatic women in early pregnancy, who developed pre-eclampsia later on. When measured early in the second trimester, an ACR ≥ 35.5 mg/mmol predicted pre-eclampsia well before the onset of clinical manifestations with high sensitivity and specificity. It can be used as a good screening tool for predicting pre-eclampsia in early pregnancy.

Activation of Endogenous Retroviruses in Dnmt1(-/-) ESCs Involves Disruption of SETDB1-Mediated Repression by NP95 Binding to Hemimethylated DNA.

Repression of endogenous retroviruses (ERVs) in mammals involves several epigenetic mechanisms. Acute loss of the maintenance methyltransferase Dnmt1 induces widespread DNA demethylation and transcriptional activation of ERVs, including CpG-rich IAP (intracisternal A particle) proviruses. Here, we show that this effect is not due simply to a loss of DNA methylation. Conditional deletions reveal that both Dnmt1 and Np95 are essential for maintenance DNA methylation. However, while IAPs are derepressed in Dnmt1-ablated embryos and embryonic stem cells (ESCs), these ERVs remain silenced when Np95 is deleted alone or in combination with Dnmt1. This paradoxical phenotype results from an ectopic interaction between NP95 and the H3K9 methyltransferase SETDB1. Normally, SETDB1 maintains silencing of IAPs, but in the absence of DNMT1, prolonged binding of NP95 to hemimethylated DNA transiently disrupts SETDB1-dependent H3K9me3 deposition. Thus, our observations reveal an unexpected antagonistic interplay between two repressive pathways involved in retroviral silencing in mammalian cells.

A Single-Centre Experience of Obstetric Acute Kidney Injury.

BACKGROUND: Acute kidney injury (AKI) is a clinical syndrome characterized by a sudden decline in glomerular filtration rate leading to decreased excretion of nitrogenous waste products. It continues to be a common problem in developing countries. AIMS: The aim of this study was to understand AKI characteristics in pregnancy and identify the factors related to its unfavorable outcome. STUDY DESIGN: A prospective cross-sectional study. METHODS: This prospective study was conducted between January 2013 and May 2014. In total 570 women with AKI were referred to the Kidney Institute during this period, out of which 52 patients with obstetrics AKI were included in this study. RESULTS: Incidence of obstetric AKI was 9.12 %. Their age varied from 19 to 34 years, with an average of 26.2 years. About 42(80.8 %) patients had not received antenatal care. The main causes of AKI were obstetric hemorrhage (38.46 %) and puerperal sepsis (15.38 %). The outcome was favorable with complete renal function recovery in 55.76 % patients. Four (7.69 %) patients became dialysis dependent. Maternal mortality was 32.69 %. CONCLUSION: Obstetric AKI is a critical situation in developing countries. Lack of antenatal care (80.8 %) is a major contributing factor for obstetric-related complications leading to renal failure. Obstetric hemorrhage (38.46 %) is the most common cause of obstetric AKI. Late referral in 18 (34.61 %), puerperal sepsis in six (33.33 %), obstetric hemorrhage in five (27.77 %) and combined sepsis and hemorrhage in five (27.77 %) are the common contributing factors leading to its unfavorable outcomes as maternal morbidity and mortality. Hence, a multidisciplinary approach is warranted to prevent such an avoidable complication.

Myringoplasty for chronic otitis media.

METHODS: 25 children in the age group of 8-14 years suffering from chronic suppurative otitis media were taken up for myringoplasty using onlay technique under general anaesthesia. RESULTS: All selected cases had a central type of dry perforation, good cochlear reserve and healthy middle ear mucosa. Cases having enlarged adenoids, infection in nose or throat, any traumatic perforation or previous attempt at closure were excluded from the study. It was found that there was 76% take up of graft after two months who also had improvement in hearing. CONCLUSION: We conclude that myringoplasty stands a good chance in children.

Distinct roles of KAP1, HP1 and G9a/GLP in silencing of the two-cell-specific retrotransposon MERVL in mouse ES cells.

BACKGROUND: In mouse embryonic stem cells (mESCs), transcriptional silencing of numerous class I and II endogenous retroviruses (ERVs), including IAP, ETn and MMERVK10C, is dependent upon the H3K9 methyltransferase (KMTase) SETDB1/ESET and its binding partner KAP1/TRIM28. In contrast, the H3K9 KMTases G9a and GLP and HP1 proteins are dispensable for this process. Intriguingly, MERVL retroelements are actively transcribed exclusively in the two-cell (2C) embryo, but the molecular basis of silencing of these class III ERVs at later developmental stages has not been systematically addressed. RESULTS: Here, we characterized the roles of these chromatin factors in MERVL silencing in mESCs. While MMERVK10C and IAP ERVs are bound by SETDB1 and KAP1 and are induced following their deletion, MERVL ERVs show relatively low levels of SETDB1 and KAP1 binding and are upregulated exclusively following KAP1 depletion, indicating that KAP1 influences MERVL expression independent of SETDB1. In contrast to class I and class II ERVs, MERVL and MERVL LTR-driven genic transcripts are also upregulated following depletion of G9a or GLP, and G9a binds directly to these ERVs. Consistent with a direct role for H3K9me2 in MERVL repression, these elements are highly enriched for G9a-dependent H3K9me2, and catalytically active G9a is required for silencing of MERVL LTR-driven transcripts. MERVL is also derepressed in HP1α and HP1ß KO ESCs. However, like KAP1, HP1α and HP1ß are only modestly enriched at MERVL relative to IAP LTRs. Intriguingly, as recently shown for KAP1, RYBP, LSD1 and G9a-deficient mESCs, many genes normally expressed in the 2C embryo are also induced in HP1 KO mESCs, revealing that aberrant expression of a subset of 2C-specific genes is a common feature in each of these KO lines. CONCLUSIONS: Our results indicate that G9a and GLP, which are not required for silencing of class I and II ERVs, are recruited to MERVL elements and play a direct role in silencing of these class III ERVs, dependent upon G9a catalytic activity. In contrast, induction of MERVL expression in KAP1, HP1α and HP1ß KO ESCs may occur predominantly as a consequence of indirect effects, in association with activation of a subset of 2C-specific genes.

H3K9me3-binding proteins are dispensable for SETDB1/H3K9me3-dependent retroviral silencing.

BACKGROUND: Endogenous retroviruses (ERVs) are parasitic sequences whose derepression is associated with cancer and genomic instability. Many ERV families are silenced in mouse embryonic stem cells (mESCs) via SETDB1-deposited trimethylated lysine 9 of histone 3 (H3K9me3), but the mechanism of H3K9me3-dependent repression remains unknown. Multiple proteins, including members of the heterochromatin protein 1 (HP1) family, bind H3K9me2/3 and are involved in transcriptional silencing in model organisms. In this work, we address the role of such H3K9me2/3 "readers" in the silencing of ERVs in mESCs. RESULTS: We demonstrate that despite the reported function of HP1 proteins in H3K9me-dependent gene repression and the critical role of H3K9me3 in transcriptional silencing of class I and class II ERVs, the depletion of HP1α, HP1ß and HP1γ, alone or in combination, is not sufficient for derepression of these elements in mESCs. While loss of HP1α or HP1ß leads to modest defects in DNA methylation of ERVs or spreading of H4K20me3 into flanking genomic sequence, respectively, neither protein affects H3K9me3 or H4K20me3 in ERV bodies. Furthermore, using novel ERV reporter constructs targeted to a specific genomic site, we demonstrate that, relative to Setdb1, knockdown of the remaining known H3K9me3 readers expressed in mESCs, including Cdyl, Cdyl2, Cbx2, Cbx7, Mpp8, Uhrf1 and Jarid1a-c, leads to only modest proviral reactivation. CONCLUSION: Taken together, these results reveal that each of the known H3K9me3-binding proteins is dispensable for SETDB1-mediated ERV silencing. We speculate that H3K9me3 might maintain ERVs in a silent state in mESCs by directly inhibiting deposition of active covalent histone marks.

DNA methylation and SETDB1/H3K9me3 regulate predominantly distinct sets of genes, retroelements, and chimeric transcripts in mESCs.

DNA methylation and histone H3 lysine 9 trimethylation (H3K9me3) play important roles in silencing of genes and retroelements. However, a comprehensive comparison of genes and repetitive elements repressed by these pathways has not been reported. Here we show that in mouse embryonic stem cells (mESCs), the genes upregulated after deletion of the H3K9 methyltransferase Setdb1 are distinct from those derepressed in mESC deficient in the DNA methyltransferases Dnmt1, Dnmt3a, and Dnmt3b, with the exception of a small number of primarily germline-specific genes. Numerous endogenous retroviruses (ERVs) lose H3K9me3 and are concomitantly derepressed exclusively in SETDB1 knockout mESCs. Strikingly, ~15% of upregulated genes are induced in association with derepression of promoter-proximal ERVs, half in the context of "chimeric" transcripts that initiate within these retroelements and splice to genic exons. Thus, SETDB1 plays a previously unappreciated yet critical role in inhibiting aberrant gene transcription by suppressing the expression of proximal ERVs.

Targeting of EZH2 to a defined genomic site is sufficient for recruitment of Dnmt3a but not de novo DNA methylation.

Polycomb-mediated gene silencing and DNA methylation underlie many epigenetic processes important in normal development as well as in cancer. An interaction between EZH2 of the Polycomb repressive complex 2 (PRC2), which trimethylates lysine 27 on Histone 3 (H3K27me3), and all three DNA methyltransferases (DNMTs) has been reported, implicating a role for PRC2 in directing DNA methylation. Interestingly, however, the majority of H3K27me3 marked genes lack DNA methylation in ES cells, indicating that EZH2 recruitment may not be sufficient to promote DNA methylation. Here, we employed a Gal4DBD/gal4UAS-based system to directly test if EZH2 binding at a defined genomic site is sufficient to promote de novo DNA methylation in a murine erythroleukaemia cell line. Targeting of a Gal4DBD-EZH2 fusion to an intergenic transgene bearing a gal4 binding-site array promoted localized recruitment of Suz12 and Bmi1, subunits of PRC2 and PRC1, respectively, and deposition of H3K27me3. Further analysis of the H3K27me3-marked site revealed the persistence of H3K4me2, a mark inversely correlated with DNA methylation. Strikingly, while Dnmt3a was also recruited in an EZH2-dependent manner, de novo DNA methylation of the transgene was not observed. Thus, while targeting of EZH2 to a specific genomic site is sufficient for recruitment of Dnmt3a, additional events are required for de novo DNA methylation.