Deciphering the Association of Epstein-Barr Virus and Its Glycoprotein M Peptide with Neuropathologies in Mice.

The reactivation of ubiquitously present Epstein-Barr virus (EBV) is known to be involved with numerous diseases, including neurological ailments. A recent in vitro study from our group unveiled the association of EBV and its 12-amino acid peptide glycoprotein M146-157 (gM146-157) with neurodegenerative diseases, viz., Alzheimer's disease (AD) and multiple sclerosis. In this study, we have further validated this association at the in vivo level. The exposure of EBV/gM146-157 to mice causes a decline in the cognitive ability with a concomitant increase in anxiety-like symptoms through behavioral assays. Disorganization of hippocampal neurons, cell shrinkage, pyknosis, and apoptotic appendages were observed in the brains of infected mice. Inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were found to be elevated in infected mouse brain tissue samples, whereas TNF-α exhibited a decline in the serum of these mice. Further, the altered levels of nuclear factor-kappa B (NF-kB) and neurotensin receptor 2 affirmed neuroinflammation in infected mouse brain samples. Similarly, the risk factor of AD, apolipoprotein E4 (ApoE4), was also found to be elevated at the protein level in EBV/gM146-157 challenged mice. Furthermore, we also observed an increased level of myelin basic protein in the brain cortex. Altogether, our results suggested an integral connection of EBV and its gM146-157 peptide to the neuropathologies.

Understanding the role of membrane cholesterol upon Epstein Barr virus infection in astroglial cells.

Background: EBV infection has long been postulated to trigger multiple sclerosis (MS) and anti-EBV antibodies showed a consistent presence in MS patients. Previous reports from our group have shown that the EBV infects different brain cells. Entry of the virus in neuronal cells is assisted by several host factors including membrane cholesterol. By using an inhibitor, methyl-ß-cyclodextrin (MßCD), we evaluated the role of membrane cholesterol in EBV infection and pathogenesis. Methodology: The membrane cholesterol depleted cells were infected with EBV and its latent genes expression were assessed. Further, EBV-mediated downstream signalling molecules namely STAT3, RIP, NF-kB and TNF-α levels was checked at protein level along with spatial (periphery and nucleus) and temporal changes in biomolecular fingerprints with Raman microspectroscopy (RS). Results: Upon treatment with MßCD, lmp1 and lmp2a suggested significant downregulation compared to EBV infection. Downstream molecules like STAT3 and RIP, exhibited a decrease in protein levels temporally upon exposure to MßCD while NF-kB levels were found to be increased. Further, the intensity of the Raman spectra exhibited an increase in triglycerides and fatty acids in the cytoplasm of EBV-infected LN-229 cells compared to MßCD+EBV. Likewise, the Raman peak width of cholesterol, lipid and fatty acids were found to be reduced in EBV-infected samples indicates elevation in the cholesterol specific moieties. In contrast, an opposite pattern was observed in the nucleus. Moreover, the ingenuity pathway analysis revealed protein molecules such as VLDLR, MBP and APP that are associated with altered profile of cholesterol, fatty acids and triglycerides with infection-related CNS disorders. Conclusion: Taken together, our results underline the important role of membrane cholesterol over EBV entry/pathogenesis in astroglia cells which further trigger/exacerbate virus-associated neuropathologies. These results likely to aid into the prognosis of neurological disease like MS.

Epigallocatechin Gallate: A Multifaceted Molecule for Neurological Disorders and Neurotropic Viral Infections.

Epigallocatechin-3-gallate (EGCG), a polyphenolic moiety found in green tea extracts, exhibits pleiotropic bioactivities to combat many diseases including neurological ailments. These neurological diseases include Alzheimer's disease, multiple sclerosis, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. For instance, in the case of Alzheimer's disease, the formation of a ß-sheet in the region of the 10th-21st amino acids was significantly reduced in EGCG-induced oligomeric samples of Aß40. Its interference induces the formation of Aß structures with an increase in intercenter-of-mass distances, reduction in interchain/intrachain contacts, reduction in ß-sheet propensity, and increase in α-helix. Besides, numerous neurotropic viruses are known to instigate or aggravate neurological ailments. It exerts an effect on the oxidative damage caused in neurodegenerative disorders by acting on GSK3-ß, PI3K/Akt, and downstream signaling pathways via caspase-3 and cytochrome-c. EGCG also diminishes these viral-mediated effects, such as EGCG delayed HSV-1 infection by blocking the entry for virions, inhibitory effects on NS3/4A protease or NS5B polymerase of HCV and potent inhibitor of ZIKV NS2B-NS3pro/NS3 serine protease (NS3-SP). It showed a reduction in the neurotoxic properties of HIV-gp120 and Tat in the presence of IFN-γ. EGCG also involves numerous viral-mediated inflammatory cascades, such as JAK/STAT. Nonetheless, it also inhibits the Epstein-Barr virus replication protein (Zta and Rta). Moreover, it also impedes certain viruses (influenza A and B strains) by hijacking the endosomal and lysosomal compartments. Therefore, the current article aims to describe the importance of EGCG in numerous neurological diseases and its inhibitory effect against neurotropic viruses.

Unraveling the Connection of Epstein-Barr Virus and Its Glycoprotein M146-157 Peptide with Neurological Ailments.

Epstein-Barr virus (EBV) is known to be associated with several cancers along with neurological modalities like Alzheimer's disease (AD) and multiple sclerosis (MS). Previous study from our group revealed that a 12 amino acid peptide fragment (146SYKHVFLSAFVY157) of EBV glycoprotein M (gM) exhibits amyloid-like self-aggregative properties. In the current study, we have investigated its effect on Aß42 aggregation along with its effect on neural cell immunology and disease markers. EBV virion was also considered for the above-mentioned investigation. An increase in the aggregation of Aß42 peptide was observed upon incubation with gM146-157. Further, the exposure of EBV and gM146-157 onto neuronal cells indicated the upregulation of inflammatory molecules like IL-1ß, IL-6, TNF-α, and TGF-ß that suggested neuroinflammation. Besides, host cell factors like mitochondrial potential and calcium ion signaling play a crucial role in cellular homeostasis and alterations in these factors aid in neurodegeneration. Changes in mitochondrial membrane potential manifested a decrease while elevation in the level of total Ca2+ ions was observed. Amelioration of Ca2+ ions triggers excitotoxicity in neurons. Subsequently, neurological disease-associated genes APP, ApoE4, and MBP were found to be increased at the protein level. Additionally, demyelination of neurons is a hallmark of MS and the myelin sheath consists of ∼70% of lipid/cholesterol-associated moieties. Hereby, genes associated with cholesterol metabolism indicated changes at the mRNA level. Enhanced expression of neurotropic factors like NGF and BDNF was discerned postexposure to EBV and gM146-157. Altogether, this study delineates a direct connection of EBV and its peptide gM146-157 with neurological illnesses.

Multi-Knock-a multi-targeted genome-scale CRISPR toolbox to overcome functional redundancy in plants.

Plant genomes are characterized by large and complex gene families that often result in similar and partially overlapping functions. This genetic redundancy severely hampers current efforts to uncover novel phenotypes, delaying basic genetic research and breeding programmes. Here we describe the development and validation of Multi-Knock, a genome-scale clustered regularly interspaced short palindromic repeat toolbox that overcomes functional redundancy in Arabidopsis by simultaneously targeting multiple gene-family members, thus identifying genetically hidden components. We computationally designed 59,129 optimal single-guide RNAs that each target two to ten genes within a family at once. Furthermore, partitioning the library into ten sublibraries directed towards a different functional group allows flexible and targeted genetic screens. From the 5,635 single-guide RNAs targeting the plant transportome, we generated over 3,500 independent Arabidopsis lines that allowed us to identify and characterize the first known cytokinin tonoplast-localized transporters in plants. With the ability to overcome functional redundancy in plants at the genome-scale level, the developed strategy can be readily deployed by scientists and breeders for basic research and to expedite breeding efforts.

Diffuse large B-cell lymphoma and new insights into its pathobiology and implication in treatment.

The most common non-Hodgkin lymphoma (NHL) subtype is diffuse large B-cell lymphoma (DLBCL). It accounts for roughly 30% of all cases of NHL affecting both nodal and extra nodal sites. There are molecular subtypes of DLBCL, germinal centre subtype (GCB), and activated B-cell (ABC), based on gene expression profiling (GEP), in accumulation to distinct morphological and clinicopathological subtypes. To prognosticate patients, the International Prognostication Index (IPI) and its variants are used. In ABC type DLBCL, limited stage disease is treated with a combination of abbreviated systemic chemotherapy (three cycles) and field radiation therapy. Although advanced stage disease is treated with a full course of chemotherapy as well as novel agents (Bortezomib, Ibrutinib, Lenalidomide). In this review study, we looked at the role of multiple aspects of genetic and microenvironment changes which have effects in DLBCL tumours.

Next-Generation Sequencing Highlights of Diffuse Large B-cell Lymphoma in a Tertiary Care Hospital in North India.

INTRODUCTION: Next-generation sequencing (NGS) elucidates the diffuse large B-cell lymphoma (DLBCL) genetic characteristics by finding recurrent and novel somatic mutations. This observational study attempted to create an NGS panel with a focus on identifying novel somatic mutations which could have potential clinical and therapeutic implications. This panel was created to look for mutations in 133 genes chosen on basis of a literature review and it was used to sequence the tumor DNA of 20 DLBCL patients after a centralized histopathologic review. METHODS: The study included 20 patients having DLBCL. The quality and quantity of tumor cells were accessed by H&E staining and correlated with histopathology and Immunohistochemistry (IHC) status. Patients were grouped as ABC (activated B-cell), PMBL (primary mediastinal large B-cell lymphoma), and other or unclassified subtypes. The lymphoma panel of 133 was designed on targeted sequencing of multiple genes for the coding regions through NGS. The libraries were prepared and sequenced using the Illumina platform. The alignment of obtained sequences was performed using Burrows-Wheeler Aligner and identification of somatic mutations was done using LoFreq (version 2) variant caller. The mutations were annotated using an annotation pipeline (VariMAT). Previously published literature and databases were used for the annotation of clinically relevant mutations. The common variants were filtered for reporting based on the presence in various population databases (1000G, ExAC, EVS, 1000Japanese, dbSNP, UK10K, MedVarDb). A custom read-depth-based algorithm was used to determine CNV (Copy Number Variants) from targeted sequencing experiments. Rare CNVs were detected using a comparison of the test data read-depths with the matched reference dataset. Reportable mutations were prioritized and prepared based on AMP-ASCO-CAP (Association for Molecular Pathology-American Society of Clinical Oncology-College of American Pathologists), WHO guidelines, and also based on annotation metrics from OncoMD (a knowledge base of genomic alterations). RESULTS: The informativity of the panel was 95 percent. NOTCH 1 was the most frequently mutated gene in 16.1% of patients followed by 12.9% who had ARID1A mutations. MYD88 and TP53 mutations were detected in 9.6% of the patient while 6.4% of patients had CSF3R mutations. NOTCH 1 and TP 53 are the most frequently reported gene in the middle age group (40-60). Mutation in MYD88 is reported in every age group. MYD88 (51%) is the most common mutation in ABC subtypes of DLBCL, followed by NOTCH 1 (44%) and SOCS 1 (33%) according to our findings. NOTCH 1 mutations are frequent in ABC and PMBL subtypes. Closer investigation reveals missense mutation is the most frequent mutation observed in the total cohort targeting 68.4% followed by frameshift deletion reported in 26.3%. Six novel variants have been discovered in this study. CONCLUSIONS: This study demonstrates the high yield of information in DLBCL using the NGS Lymphoma panel. Results also highlight the molecular heterogeneity of DLBCL subtypes which indicates the need for further studies to make the results of the NGS more clinically relevant.

Single-Point Insulin Sensitivity Estimator (SPISE) as a Feasible Marker of Insulin Resistance in Adult Metabolic Syndrome: Evaluated in a Hospital Based Cross-Sectional Pilot Study at Tertiary Care Centre of Uttarakhand.

Insulin resistance (IR) plays an important role as a major determinant of Metabolic syndrome (MetS). Various methods are available for measuring insulin resistance but they are laborious, time-consuming, and costly. Therefore various surrogate markers and indices have been devised to simplify and improve the determination of insulin resistance. Recently, a new index, single point insulin sensitivity estimator (SPISE) was proposed in the European population and was found comparable to the gold standard test (hyperinsulinemic euglycemic glucose clamp).This study was planned to evaluate whether SPISE could be a useful potential low-cost indicator for predicting MetS with IR patients in Indian population. Eighty-three participants from outpatient care of AIIMS Rishikesh were evaluated after informed consent. They were divided into Metabolic syndrome (n = 56) and Non Metabolic Syndrome(n = 27), using South Asian Modified National Cholesterol Education Program- ATP-III criteria for metabolic syndrome. SPISE index, HOMA-IR, Insulin Resistance Index, Triglycerides to high-density lipoproteins cholesterol ratio (TG/HDL-C) were calculated for all the subjects. Receiver operating characteristic (ROC) curve was plotted to assess discriminatory ability of SPISE, HOMA-IR, TG/HDL-C ratio, IRI and hs-CRP to differentiate between IR(Metabolic syndrome) and non-IR (Non-Metabolic syndrome) subjects. SPISE has greater area under curve with better sensitivity and specificity compared to HOMA-IR, IRI, TG/HDL-C ratio and hs CRP. So, SPISE has better predictive ability than HOMA-IR, IRI, TG/HDL-C ratio and hs CRP to discriminate IR from non-IR cases. SPISE could be a useful potential low-cost indicator with high sensitivity and specificity for predicting IR in MetS patients.

Customized toothbrush handle for patients with limited manual dexterity.

Elderly people may have difficulty in grasping and holding tooth brush due to limited hand and finger movement. This article describes a technique of increasing the volume of toothbrush handle with acrylic device that is custom molded to the patient's unique hand. The customized handle will make holding the brush easier and more comfortable thereby contributing to the maintenance of oral hygiene.

Colistin potentiation in multidrug-resistant Acinetobacter baumannii by a non-cytotoxic guanidine derivative of silver.

A novel nano-formulation (NF) that sensitizes Acinetobacter baumannii (AB) to otherwise ineffective colistin is described in the present study. Infections due to multidrug resistant (MDR) AB represent a major therapeutic challenge, especially in situations of pre-existing colistin resistance (colR). Subsequently, boosting the effectiveness of colistin would be a better alternative tactic to treat AB infections rather than discovering a new class of antibiotics. We have previously demonstrated an NF comprising self-assembled guanidinium and ionic silver nanoparticles [AD-L@Ag(0)] to have anti-biofilm and bactericidal activity. We report NF AD-L@Ag(0) for the very first time for the potentiation of colistin in Gram-negative colistin-resistant bacteria. Our results implied that a combination of clinically relevant concentrations of colistin and AD-L@Ag(0) significantly decreased colistin-resistant AB bacterial growth and viability, which otherwise was elevated in the presence of only colistin. In this study, we have described various combinations of minimum inhibitory concentration (MIC) of colistin (MICcol, 1/2 MICcol, and 1/4 MICcol) and that of AD-L@Ag(0) [MICAD-L@Ag(0), 1/2 MICAD-L@Ag(0), and 1/4 MICAD-L@Ag(0)] and tested them against MDR AB culture. The results (in broth as well as in solid media) signified that AD-L@Ag(0) was able to potentiate the anti-microbial activity of colistin at sub-MIC concentrations. Furthermore, the viability and metabolic activity of bacterial cells were also measured by CTC fluorescence assay and ATP bioluminescence assay. The results of these assays were in perfect concordance with the scores of cultures (colony forming unit and culture turbidity). In addition, quantitative real-time PCR (qRT-PCR) was performed to unveil the expression of selected genes, DNAgyrA, DNAgyrB, and dac. These genes introduce negative supercoiling in the DNA, and hence are important for basic cellular processes. These genes, due to mutation, modified the Lipid A of bacteria, further resisting the uptake of colistin. Therefore, the expression of these genes was upregulated when AB was treated with only colistin, substantiating that AB is resistant to colistin, whereas the combinations of MICcol + MICAD-L@Ag(0) downregulated the expression of these genes, implying that the developed formulation can potentiate the efficiency of colistin. In conclusion, AD-L@Ag(0) can potentiate the proficiency of colistin, further enhancing colistin-mediated death of AB by putatively disrupting the outer membrane (OM) and facilitating bacterial death.

The Role of Microbiota in the Development of Cancer Tumour Cells and Lymphoma of B and T Cells.

Human body harbours enormous numbers of microbial organisms, including bacteria, viruses, and fungi which have a momentous role in well-being and illness in humans. Immune system shelters us from pathogenic bacteria, microorganisms found in human tissues have many benefits related to the functional movement of the host by regulating important procedures such as immunity, signalling, and breakdown. Lymphocytes assume a significant part in the reaction to bacterial colonization, primarily by prompting a safe reaction to obstruction or initiation. Most immunologically occupant cells have a place with the mucosal invulnerable framework and are continually motioned by dendritic cells or other Antigen introducing cells that gather intestinal samples. Thus, Microbiome is a key contributor to developing lymphoma and specific alterations to microbiome composition could attenuate the risk. There is an indication that microbial morphology can affect and control humanoids. The difference in the composition of these microorganisms is associated with tumour development. With the increased knowledge of the connection among the human microbiome and carcinogenesis, the use of these findings to prevent, predict or diagnose of lymphomas has attracted a great attention. In this article, we explored current knowledge of various microbial ecosystems, their connection with carcinogens and the potential for useful microorganisms to control and prevent B and T cell lymphoma.