Heat Killed Attenuated Leishmania Induces Apoptosis of HepG2 Cells Through ROS Mediated p53 Dependent Mitochondrial Pathway

Cytotoxic effect of attenuated Leishmania on liver cancer cells by inducing ROS generation. Methods: Spectrophotometric study to analyze cell death and levels of different active caspases. Flow cytometric study was done to analyze apoptosis induction and ROS generation and levels of different protein. Western blot analysis was performed to study the levels of protein. Confocal microscopy was done to ascertain the expression of different apoptotic markers. Results: We have now observed that attenuated Leishmania donovani UR6 also has potentiality towards growth inhibition of HepG2 cells and investigated the mechanism of action. The effect is associated with increased DNA fragmentation, rise in number of annexinV positive cells, and cell cycle arrest at G1 phase. The detection of unregulated levels of active PARP, cleaved caspases 3 and 9, cytosolic cytochrome C, Bax, and Bad, along with the observed downregulation of Bcl-2 and loss of mitochondrial membrane potential suggested the involvement of mitochondrial pathway. Enhanced ROS and p53 levels regulate the apoptosis of HepG2 cells. NAC was found to inhibit p53 production but PFT-α has no effect on ROS generation. In conclusion, Leishmania donovani UR6 efficiently induces apoptosis in HepG2 cells through ROS mediated p53 dependent mitochondrial pathway. Conclusion: It has been reported earlier that some parasites show prominent cytotoxic effect and prevent tumor growth. From our study we found that Leishmania donovani UR6 efficiently induced apoptosis in HepG2 cells through ROS mediated p53 dependent mitochondrial pathway. This study has rejuvenated the age old idea of bio-therap

Lipid Isolated from a Leishmania donovani

Sepsis is the reflection of systemic immune response that manifests in the sequential inflammatory process in presence of infection. This may occur as a result of gram-negative bacterial sepsis including Escherichia coli infection that gives rise to excessive production of inflammatory mediators and causes severe tissue injuries. We have reported earlier that the lipid of attenuated Leishmania donovani suppresses the inflammatory responses in arthritis patients. Using heat killed E. coli stimulated macrophages, we have now investigated the effect of leishmanial total lipid (LTL) isolated from Leishmania donovani (MHO/IN/1978/UR6) for amelioration of the inflammatory mediators and transcriptional factor with suppression of TLR4-CD14 expression. To evaluate the in vivo effect, E. coli induced murine sepsis model was used focusing on the changes in different parameter(s) of lung injury caused by sepsis, namely, edema, vascular permeability, and pathophysiology, and the status of different cytokine-chemokine(s) and adhesion molecule(s). Due to the effect of LTL, E. coli induced inflammatory cytokine-chemokine(s) levels were significantly reduced in serum and bronchoalveolar lavage fluid simultaneously. LTL also improved the lung injury and suppressed the cell adhesion molecules in lung tissue. These findings indicate that LTL may prove to be a potential anti-inflammatory agent and provide protection against gram-negative bacterial sepsis with pulmonary impairment

A Mechanistic Approach to Determine the Anticancer and Antimetastatic Potential of Attenuated Strain of L. Donovani and Its Membrane Lipoprotein Via Regulation of Different Immunological Factors

Each year, millions of people are diagnosed worldwide with cancer, and more than half of these patients eventually die from this disease. In 2015, 27.9 million new cancer cases were diagnosed, and the number of deaths caused by this disease reached 16.7 million.Conventional cancer treatments, such as surgery, chemotherapy, and radiotherapy, often fail to achieve a complete cancer remission. Moreover, it has been widely recognized that radiotherapy and/or chemotherapy are likely to cause significant side effects. This has prompted the development of many new approaches for the treatment of cancer. One such example involves the use of live or heat-killed attenuated microbes or their purified products. The use of microbial products for the treatment of cancer was pioneered by Dr. William B. Coley, who in 1891 treated patients with cancer with intra-tumoural injections of live Streptococcus pyogenes and, subsequently, with mixtures of S. pyogenes and Serratia marcescens. The use of biotherapy reduced the cytotoxic side effects of the chemotherapeutic drugs and also was found to raise an immune response. For example, BCG which was earlier only used as a vaccine against mycobacterium was successfully used in different cancers including acute lymphoblastic leukemia, melanoma, bladder cancer and etc. In the late 90s, biotherapy became very popular when different microbial cellular components mainly the membrane proteins and lipids were also found to be highly potent against different cancers. Azurin and FTY720 are two membrane components that are already being patented and being approved by FDA as drugs against cancer and immune suppressive disease

Synthesis and Characterization of ZnO Microfiber By Electrospinning Technique

Ultrathin 1D assembly of pure crystalline ZnO microfibers were fabricated using facile, low-cost electrospinning technique. Nanofibrous membrane preforms were synthesized by electrospinning a ZnO precursor containing PVA solution in aqueous medium. The crystalline ZnO microfibers were obtained by calcining PVA/Zinc Acetate precursor fibers at 500 degrees C. The structure and morphologies of ZnO microfibers were studied by X-ray diffraction (XRD), thermogravimetric analysis (TGA) and field emission scanning electron microscopy (FE-SEM). (C) 2017 Elsevier Ltd. All rights reserved. Selection and/or Peer-review under responsibility of International Conference on Functional Nano-Materials, 2016

Water-Ethylene Glycol Mediated Synthesis of Silver Nanoparticles for Conductive Ink

The drop on demand direct writing technology is the most efficient alternative to the traditional technologies for fabricating electronic devices on soft substrates. This piezo driven direct deposition technology requires preparation of a conductive ink comprising stable suspension of nanoparticles. In the present work, we have synthesized nearly mono-sized silver nanoparticles by chemical reduction method in presence of a capping agent for preparing conductive ink. The particle size distribution, viscosity and wettability (on the substrate) of the colloidal silver suspension were measured. The morphology of the silver nanoparticles was characterized by FESEM and TEM, respectively. (c) 2017 Elsevier Ltd. All rights reserved

Epigenetic Reprogramming of Kaposi’s Sarcoma-Associated Herpesvirus during Hypoxic Reactivation

The biphasic life cycle (latent and lytic) of Kaposi’s sarcoma-associated Herpesvirus (KSHV) is regulated by epigenetic modification of its genome and its associated histone proteins. The temporal events driving epigenetic reprogramming of the KSHV genome on initial infection to establish latency has been well studied, but the reversal of these epigenetic changes during lytic replication, especially under physiological conditions such as hypoxia, has not been explored. In this study, we investigated epigenetic reprogramming of the KSHV genome during hypoxic reactivation. Hypoxia induced extensive enrichment of both transcriptional activators and repressors on the KSHV genome through H3K4Me3, H3K9Me3, and H3K27Me3, as well as histone acetylation (H3Ac) modifications. In contrast to uniform quantitative enrichment with modified histones, a distinct pattern of RTA and LANA enrichment was observed on the KSHV genome. The enrichment of modified histone proteins was due to their overall higher expression levels, which was exclusively seen in KSHV-positive cells. Multiple KSHV-encoded factors such as LANA, RTA, and vGPCR are involved in the upregulation of these modified histones. Analysis of ChIP-sequencing for the initiator DNA polymerase (DNAPol1α) combined with single molecule analysis of replicated DNA (SMARD) demonstrated the involvement of specific KSHV genomic regions that initiate replication in hypoxia

Heat Killed Attenuated Leishmania Induces Apoptosis of HepG2 Cells Through ROS Mediated p53 Dependent Mitochondrial Pathway

Background/Aims: Cytotoxic effect of attenuated Leishmania on liver cancer cells by inducing ROS generation. Methods: Spectrophotometric study to analyze cell death and levels of different active caspases. Flow cytometric study was done to analyze apoptosis induction and ROS generation and levels of different protein. Western blot analysis was performed to study the levels of protein. Confocal microscopy was done to ascertain the expression of different apoptotic markers. Results: We have now observed that attenuated Leishmania donovani UR6 also has potentiality towards growth inhibition of HepG2 cells and investigated the mechanism of action. The effect is associated with increased DNA fragmentation, rise in number of annexinV positive cells, and cell cycle arrest at G1 phase. The detection of unregulated levels of active PARP, cleaved caspases 3 and 9, cytosolic cytochrome C, Bax, and Bad, along with the observed downregulation of Bcl-2 and loss of mitochondrial membrane potential suggested the involvement of mitochondrial pathway. Enhanced ROS and p53 levels regulate the apoptosis of HepG2 cells. NAC was found to inhibit p53 production but PFT-α has no effect on ROS generation. In conclusion, Leishmania donovani UR6 efficiently induces apoptosis in HepG2 cells through ROS mediated p53 dependent mitochondrial pathway. Conclusion: It has been reported earlier that some parasites show prominent cytotoxic effect and prevent tumor growth. From our study we found that Leishmania donovani UR6 efficiently induced apoptosis in HepG2 cells through ROS mediated p53 dependent mitochondrial pathway. This study has rejuvenated the age old idea of bio-therapy

Attenuation of IFN signaling due to m6A modification of the host epitranscriptome promotes EBV lytic reactivation

Abstract Background Reactivation of Epstein Barr virus (EBV) leads to modulation of the viral and cellular epitranscriptome. N6-methyladenosine (m6A) modification is a type of RNA modification that regulates metabolism of mRNAs. Previous reports demonstrated that m6A modification affects the stability and metabolism of EBV encoded mRNAs. However, the effect of reactivation on reprograming of the cellular mRNAs, and how this contributes to successful induction of lytic reactivation is not known. Methods Methylated RNA immunoprecipitation sequencing (MeRIP-seq), transcriptomic RNA sequencing (RNA-seq) and RNA pull-down PCR were used to screen and validate differentially methylated targets. Western blotting, quantitative real-time PCR (RT-qPCR) and immunocytochemistry were used to investigate the expression and localization of different proteins. RNA stability and polysome analysis assays were used to detect the half-lives and translation efficiencies of downstream genes. Insertion of point mutation to disrupt the m6A methylation sites was used to verify the effect of m6A methylation on its stability and expression levels. Results We report that during EBV reactivation the m6A eraser ALKBH5 is significantly downregulated leading to enhanced methylation of the cellular transcripts DTX4 and TYK2, that results in degradation of TYK2 mRNAs and higher efficiency of translation of DTX4 mRNAs. This resulted in attenuation of IFN signaling that promoted progression of viral lytic replication. Furthermore, inhibition of m6A methylation of these transcripts led to increased production of IFN, and a substantial reduction in viral copy number, which suggests abrogation of lytic viral replication. Conclusion Our findings illuminate the significance of m6A modification in overcoming the innate immune response during EBV reactivation. We now report that during lytic reactivation EBV targets the RNA methylation system of the host to attenuate the innate immune response by suppressing the interferon signaling which facilitates successful lytic replication of the virus