Study of mitotic chromatin supports a model of bookmarking by histone modifications and reveals nucleosome deposition patterns.

Mitosis encompasses key molecular changes including chromatin condensation, nuclear envelope breakdown, and reduced transcription levels. Immediately after mitosis, the interphase chromatin structure is reestablished and transcription resumes. The reestablishment of the interphase chromatin is probably achieved by "bookmarking," i.e., the retention of at least partial information during mitosis. To gain a deeper understanding of the contribution of histone modifications to the mitotic bookmarking process, we merged proteomics, immunofluorescence, and ChIP-seq approaches. We focused on key histone modifications and employed HeLa-S3 cells as a model system. Generally, in spite of the general hypoacetylation observed during mitosis, we observed a global concordance between the genomic organization of histone modifications in interphase and mitosis, suggesting that the epigenomic landscape may serve as a component of the mitotic bookmarking process. Next, we investigated the nucleosome that enters nucleosome depleted regions (NDRs) during mitosis. We observed that in ∼60% of the NDRs, the entering nucleosome is distinct from the surrounding highly acetylated nucleosomes and appears to have either low levels of acetylation or high levels of phosphorylation in adjacent residues (since adjacent phosphorylation may interfere with the ability to detect acetylation). Inhibition of histone deacetylases (HDACs) by the small molecule TSA reverts this pattern, suggesting that these nucleosomes are specifically deacetylated during mitosis. Altogether, by merging multiple approaches, our study provides evidence to support a model where histone modifications may play a role in mitotic bookmarking and uncovers new insights into the deposition of nucleosomes during mitosis.

Speciation, Biofilm Formation and Antifungal Susceptibility of Candida Isolates from Clinically Diagnosed Patient of UTI in a Tertiary Care Hospital.

INTRODUCTION: The incidence of the urinary tract infections caused by Candida species, are becoming more common. Recently, an increase in the incidence of infection caused by fungi especially non albicans candida species (NAC) has been reported. Several virulence factors like biofilm formation, toxin production and presence of adhesins contribute to its pathogenesis. OBJECTIVES: This study was undertaken to determine species distribution, biofilm formation and in-vitro antifungal susceptibility of candida isolated in our tertiary care hospital. METHOD: Eighty seven clinical isolates obtained from urine specimens were subjected to wet mount, Gram's stain and cultured on Sabouraud's Dextrose agar (SDA) medium. Conventional method for yeast identification was done. Biofilm forming ability of each isolate was detected using microtitre plate method. Antifungal susceptibility against posaconazole, amphotericin-B, fluconazole, itraconazole, ketoconazole, 5-flucytosine, voriconazole, and caspofungin was tested using Sensititre® Yeastone® (Trek diagnostic systems). RESULTS AND DISCUSSION: Out of 87 candida isolates, 31.03% (n=27) were C. albicans and 68.97% (n=60) were non albicans candida species (NAC). Among 60 NAC, C. kruseii 29.89% (n=26), C. glabrata 24.14% (n=21), C. tropicalis 14.94% (n=13). Among all isolates, 36.78% (n=32) were biofilm producers and biofilm positivity more among C. albicans 55.56% (n=15) as compared to NAC 28.33% (n=17) (Pvalue<0.002). The maximum positivity was observed with isolates from plastic devices (61.8%). The minimum inhibitory concentrations of all antifungal drugs against all isolates were within susceptible range except for fluconazole which was resistant to C. kruseii. CONCLUSION: C. albicans remains the major isolate from urine samples and also biofilm formation as a virulence factor might have a higher significance for C. albicans than for NAC and its ability to form biofilm is intricately linked with ability of organisms to adhere, colonize and subsequently cause infection.

Arg-265: a critical residue of L.donovani cytosolic SHMT in maintaining the binding of THF and catalysis.

Serine hydroxymethyltransferase belongs to the class of pyridoxal-5-phosphate enzymes along with aspartate aminotransferase. To explore the function of residue(s) involved in binding of the carboxylate group of Tetrahydrofolic acid (THF) to L. donovani cytosolic serine hydroxymethyltransferase (LdcSHMT), the gene was cloned in pET-28(a) vector, overexpressed and purified to homogeneity. With the help of docking results of THF to the active site of protein, the key residues involved in interaction were identified. In an attempt to unravel the function of Arg265 residue involved in binding of the carboxylate group of THF, Arg-265 was mutated to Ala by site-directed mutagenesis. The Arg265Ala-LdcSHMT showed increased Km value (threefold) and decreased kcat/Km value (threefold) for H4-folate as compared with wild type enzyme. The wild and mutant enzymes exhibited similar Km and kcat/Km values for L-allo-threonine. Unlike the wild type enzyme, mutant failed to form characteristic quinonoid intermediate and was unable to carry out the exchange of α-proton from glycine in the presence of Tetrahydrofolate. These results suggested that Arg265 residue is required for the binding of Tetrahydrofolate and may be the base that abstracts α-proton from glycine, leading to formation of quinonoid intermediate in cytosolic SHMT of L. donovani.

Mitotic H3K9ac is controlled by phase-specific activity of HDAC2, HDAC3, and SIRT1.

Histone acetylation levels are reduced during mitosis. To study the mitotic regulation of H3K9ac, we used an array of inhibitors targeting specific histone deacetylases. We evaluated the involvement of the targeted enzymes in regulating H3K9ac during all mitotic stages by immunofluorescence and immunoblots. We identified HDAC2, HDAC3, and SIRT1 as modulators of H3K9ac mitotic levels. HDAC2 inhibition increased H3K9ac levels in prophase, whereas HDAC3 or SIRT1 inhibition increased H3K9ac levels in metaphase. Next, we performed ChIP-seq on mitotic-arrested cells following targeted inhibition of these histone deacetylases. We found that both HDAC2 and HDAC3 have a similar impact on H3K9ac, and inhibiting either of these two HDACs substantially increases the levels of this histone acetylation in promoters, enhancers, and insulators. Altogether, our results support a model in which H3K9 deacetylation is a stepwise process-at prophase, HDAC2 modulates most transcription-associated H3K9ac-marked loci, and at metaphase, HDAC3 maintains the reduced acetylation, whereas SIRT1 potentially regulates H3K9ac by impacting HAT activity.

Empty SV40 capsids increase survival of septic rats by eliciting numerous host signaling networks that participate in a number of systemic functions.

Sepsis is an excessive, dysregulated immune response to infection that activates inflammatory and coagulation cascades, which may lead to tissue injury, multiple organ dysfunction syndrome and death. Millions of individuals die annually of sepsis. To date, the only treatment available is antibiotics, drainage of the infection source when possible, and organ support in intensive care units. Numerous previous attempts to develop therapeutic treatments, directed at discreet targets of the sepsis cascade, could not cope with the complex pathophysiology of sepsis and failed. Here we describe a novel treatment, based on empty capsids of SV40 (nanocapsids - NCs). Studies in a severe rat sepsis model showed that pre-treatment by NCs led to a dramatic increase in survival, from zero to 75%. Transcript analyses (RNAseq) demonstrated that the NC treatment is a paradigm shift. The NCs affect multiple facets of biological functions. The affected genes are modified with time, adjusting to the recovery processes. The NCs effect on normal control rats was negligible. The study shows that the NCs are capable of coping with diseases with intricate pathophysiology. Further studies are needed to determine whether when applied after sepsis onset, the NCs still improve outcome.

Angiostrongylus cantonensis in anterior chamber.

Live worm in anterior chamber is a rare finding. We hereby report a case of ocular Angiostrongylus cantonensis, which, to the best of our knowledge, is the third case report from India. A 70-year-old female presented with the complaints of watering and foreign body sensations in right eye since 2 months. On examination, a translucent worm of approximately 15 mm length was found freely moving in anterior chamber. Patient underwent surgical removal of live worm, which was identified as A. cantonensis. Educating people regarding importance of hygiene is important for prevention of worm infestation.