The Role of Histone H4 Biotinylation in the Structure of Nucleosomes

Background: Post-translational modifications of histones play important roles in regulating nucleosome structure and gene transcription. It has been shown that biotinylation of histone H4 at lysine-12 in histone H4 (K12Bio-H4) is associated with repression of a number of genes. We hypothesized that biotinylation modifies the physical structure of nucleosomes, and that biotin-induced conformational changes contribute to gene silencing associated with histone biotinylation. Methodology/Principal Findings: To test this hypothesis we used atomic force microscopy to directly analyze structures of nucleosomes formed with biotin-modified and non-modified H4. The analysis of the AFM images revealed a 13% increase in the length of DNA wrapped around the histone core in nucleosomes with biotinylated H4. This statistically significant (p,0.001) difference between native and biotinylated nucleosomes corresponds to adding approximately 20 bp to the classical 147 bp length of nucleosomal DNA. Conclusions/Significance: The increase in nucleosomal DNA length is predicted to stabilize the association of DNA with histones and therefore to prevent nucleosomes from unwrapping. This provides a mechanistic explanation for the gene silencing associated with K12Bio-H4. The proposed single-molecule AFM approach will be instrumental for studying the effects of various epigenetic modifications of nucleosomes, in addition to biotinylation

T-Lymphocytes Enable Osteoblast Maturation via IL-17F during the Early Phase of Fracture Repair

While it is well known that the presence of lymphocytes and cytokines are important for fracture healing, the exact role of the various cytokines expressed by cells of the immune system on osteoblast biology remains unclear. To study the role of inflammatory cytokines in fracture repair, we studied tibial bone healing in wild-type and Rag1−/− mice. Histological analysis, µCT stereology, biomechanical testing, calcein staining and quantitative RNA gene expression studies were performed on healing tibial fractures. These data provide support for Rag1−/− mice as a model of impaired fracture healing compared to wild-type. Moreover, the pro-inflammatory cytokine, IL-17F, was found to be a key mediator in the cellular response of the immune system in osteogenesis. In vitro studies showed that IL-17F alone stimulated osteoblast maturation. We propose a model in which the Th17 subset of T-lymphocytes produces IL-17F to stimulate bone healing. This is a pivotal link in advancing our current understanding of the molecular and cellular basis of fracture healing, which in turn may aid in optimizing fracture management and in the treatment of impaired bone healing

Human serum-derived hydroxy long-chain fatty acids exhibit anti-inflammatory and anti-proliferative activity

<p>Abstract</p> <p>Background</p> <p>Circulating levels of novel long-chain hydroxy fatty acids (called GTAs) were recently discovered in the serum of healthy subjects which were shown to be reduced in subjects with colorectal cancer (CRC), independent of tumor burden or disease stage. The levels of GTAs were subsequently observed to exhibit an inverse association with age in the general population. The current work investigates the biological activity of these fatty acids by evaluating the effects of enriched human serum extracts on cell growth and inflammation.</p> <p>Methods</p> <p>GTAs were extracted from commercially available bulk human serum and then chromatographically separated into enriched (GTA-positive) and depleted (GTA-negative) fractions. SW620, MCF7 and LPS stimulated RAW264.7 cells were treated with various concentrations of the GTA-positive and GTA-negative extracts, and the effects on cell growth and inflammation determined.</p> <p>Results</p> <p>Enriched fractions resulted in poly-ADP ribose polymerase (PARP) cleavage, suppression of NFκB, induction of IκBα, and reduction in NOS2 mRNA transcript levels. In RAW264.7 mouse macrophage cells, incubation with enriched fractions prior to treatment with LPS blocked the induction of several pro-inflammatory markers including nitric oxide, TNFα, IL-1β, NOS2 and COX2.</p> <p>Conclusions</p> <p>Our results show that human serum extracts enriched with endogenous long-chain hydroxy fatty acids possess anti-inflammatory and anti-proliferative activity. These findings support a hypothesis that the reduction of these metabolites with age may result in a compromised ability to defend against uncontrolled cell growth and inflammation, and could therefore represent a significant risk for the development of CRC.</p

Status Update and Interim Results from the Asymptomatic Carotid Surgery Trial-2 (ACST-2)

Objectives: ACST-2 is currently the largest trial ever conducted to compare carotid artery stenting (CAS) with carotid endarterectomy (CEA) in patients with severe asymptomatic carotid stenosis requiring revascularization. Methods: Patients are entered into ACST-2 when revascularization is felt to be clearly indicated, when CEA and CAS are both possible, but where there is substantial uncertainty as to which is most appropriate. Trial surgeons and interventionalists are expected to use their usual techniques and CE-approved devices. We report baseline characteristics and blinded combined interim results for 30-day mortality and major morbidity for 986 patients in the ongoing trial up to September 2012. Results: A total of 986 patients (687 men, 299 women), mean age 68.7 years (SD ± 8.1) were randomized equally to CEA or CAS. Most (96%) had ipsilateral stenosis of 70-99% (median 80%) with contralateral stenoses of 50-99% in 30% and contralateral occlusion in 8%. Patients were on appropriate medical treatment. For 691 patients undergoing intervention with at least 1-month follow-up and Rankin scoring at 6 months for any stroke, the overall serious cardiovascular event rate of periprocedural (within 30 days) disabling stroke, fatal myocardial infarction, and death at 30 days was 1.0%. Conclusions: Early ACST-2 results suggest contemporary carotid intervention for asymptomatic stenosis has a low risk of serious morbidity and mortality, on par with other recent trials. The trial continues to recruit, to monitor periprocedural events and all types of stroke, aiming to randomize up to 5,000 patients to determine any differential outcomes between interventions. Clinical trial: ISRCTN21144362. © 2013 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved

Annual Report 2013-14 of KVK, Kolar

Annual Progress Report of KVK, Kolar for the year 2013-14Annual Progress Report 2013-14Not Availabl

Esculentin-1a(1-21)NH₂. a frog skin-derived peptide for microbial keratitis

Pseudomonas aeruginosa is the primary bacterial pathogen causing contact lens related keratitis. Available ophthalmic agents have reduced efficacy and antimicrobial peptides (AMPs) hold promise as future antibiotics. Here we investigated the in vitro and in vivo anti-Pseudomonal activity of esculentin-1a(1-21)NH2, derived from a frog skin AMP. The data revealed a minimum inhibitory concentration between 2 and 16 μM against reference strains or drug-resistant clinical isolates of P. aeruginosa without showing toxicity to human corneal epithelial cells up to 50 μM. At 1 μM the peptide rapidly killed bacterial cells and this activity was fully retained in 150 mM sodium chloride and 70 % (v/v) human basal tears, particularly against the virulent ATCC 19660 strain. Furthermore, its dropwise administration at 40 μM to the ocular surface in a murine model of P. aeruginosa keratitis (three times daily, for 5 days post-infection) resulted in a significant reduction of infection. The mean clinical score was 2.89 ± 0.26 compared to 3.92 ± 0.08 for the vehicle control. In addition, the corneal level of viable bacteria in the peptide treated animals was significantly lower with a difference of 4 log10 colony counts, compared to 7.7 log10 cells recovered in the control. In parallel, recruitment of inflammatory cells was reduced by half compared to that found in the untreated eyes. Similar results were obtained when esculentin-1a(1-21)NH2 was applied prior to induction of keratitis. Overall, our findings highlight esculentin-1a(1-21)NH2 as an attractive candidate for the development of novel topical pharmaceuticals against Pseudomonas keratitis

Methods for In Vivo/Ex Vivo Analysis of Antimicrobial Peptides in Bacterial Keratitis: siRNA Knockdown, Colony Counts, Myeloperoxidase, Immunostaining, and RT-PCR Assays

Antimicrobial peptides (AMPs) are essential components of the innate immune response. They have direct killing ability as well as immunomodulatory functions. Here, we describe techniques to identify specific AMPs involved in the protection against microbial keratitis, a vision threatening infection of the cornea of the eye which is the most serious complication of contact lens wear. Specifically we detail the use of siRNA technology to temporarily knockdown AMP expression at the murine ocular surface in vivo and then describe ex vivo assays to determine the level of bacteria, relative number of neutrophils, and levels of cytokines, chemokines, and AMPs in infected corneas