Sequencing of Kaposi's Sarcoma Herpesvirus (KSHV) genomes from persons of diverse ethnicities and provenances with KSHV-associated diseases demonstrate multiple infections, novel polymorphisms, and low intra-host variance.

Recently published near full-length KSHV genomes from a Cameroon Kaposi sarcoma case-control study showed strong evidence of viral recombination and mixed infections, but no sequence variations associated with disease. Using the same methodology, an additional 102 KSHV genomes from 76 individuals with KSHV-associated diseases have been sequenced. Diagnoses comprise all KSHV-associated diseases (KAD): Kaposi sarcoma (KS), primary effusion lymphoma (PEL), KSHV-associated large cell lymphoma (KSHV-LCL), a type of multicentric Castleman disease (KSHV-MCD), and KSHV inflammatory cytokine syndrome (KICS). Participants originated from 22 different countries, providing the opportunity to obtain new near full-length sequences of a wide diversity of KSHV genomes. These include near full-length sequence of genomes with KSHV K1 subtypes A, B, C, and F as well as subtype E, for which no full sequence was previously available. High levels of recombination were observed. Fourteen individuals (18%) showed evidence of infection with multiple KSHV variants (from two to four unique genomes). Twenty-six comparisons of sequences, obtained from various sampling sites including PBMC, tissue biopsies, oral fluids, and effusions in the same participants, identified near complete genome conservation between different biological compartments. Polymorphisms were identified in coding and non-coding regions, including indels in the K3 and K15 genes and sequence inversions here reported for the first time. One such polymorphism in KSHV ORF46, specific to the KSHV K1 subtype E2, encoded a mutation in the leucine loop extension of the uracil DNA glycosylase that results in alteration of biochemical functions of this protein. This confirms that KSHV sequence variations can have functional consequences warranting further investigation. This study represents the largest and most diverse analysis of KSHV genome sequences to date among individuals with KAD and provides important new information on global KSHV genomics.

Chrysin inhibits adipogenesis by modulating PPARγ: in silico and in vitro studies.

Adipose tissue is the major storage site of lipids and plays a vital role in energy homeostasis. Adipogenesis is a well-regulated process wherein preadipocytes differentiate into adipocytes. It requires the sequential activation of numerous transcription factors, including peroxisome proliferator activated receptor-γ (PPAR-γ). Phytochemicals have been reported to regulate adipogenesis and flavonoids represent the most researched groups of phytochemicals with regard to their effect on adipogenesis. Chrysin is a naturally occurring flavone and is reported to have anti-inflammatory effects in obese conditions. The present study was aimed to examine the effect of chrysin on adipogenesis. In silico Molecular docking, dynamic simulation studies and in vitro cell-based assays showed that chrysin inhibited adipogenesis by modulating key adipogenic transcription factor PPARγ. Enhanced adipogenesis leads to obesity and targeting adipogenesis is potential in regulating adipose tissue development. So, these investigations may provide important information for designing therapeutic interventions to control adiposity.Communicated by Ramaswamy H. Sarma.

1, 25-(OH)2D3 protects against ER stress and miRNA dysregulation in Mus musculus neurons.

BACKGROUND: The pathophysiology of neurodegenerative diseases (NDDs) is closely associated with cellular oxidative stress which can result in the accumulation of toxic proteins in the endoplasmic reticulum (ER) leading to ER stress and subsequent unfolded protein response (UPR) signaling, a mechanism that aggravate these disorders. Vitamin D has been suggested to have important neuroprotective role and its administration has been shown to reduce neuronal injury, neurotoxicity and oxidative stress in various animal systems. OBJECTIVE: The current study was undertaken to examine the effect of vitamin D3 on UPR in ER stress induced Mus musculus neuronal cells. METHODS: Mus musculus cortical and hippocampal primary neuronal cultures were pretreated with 1,25-dihydroxyvitamin D3 (1, 25-(OH)2D3), the active form of vitamin D, followed by ER stress induction with a chemical ER stress inducer thapsigargin and with an advanced glycated protein, AGE-BSA. The UPR genes and related microRNAs (miRNA) expressions were analyzed mainly using real-time PCR. RESULTS: The experiment resulted in the suppression of ER stress marker BiP and UPR pathway genes such as Perk, Ire1α, Chop and Puma which mediate cellular apoptosis indicating the protective effect of 1, 25-(OH)2D3 against neuronal ER stress. Further studies into the molecular aspects showed that ER stress mediated down-regulated expression of microRNAs (miRNAs) such as mmu-miR-24, 27b, 124, 224, 290, 351 and 488 which are known to regulate the UPR pathway genes were also reduced with vitamin pretreatment, of which the miRNAs miR-24 and 27b which shares the same cluster are potentially involved in various human diseases. CONCLUSION: This study emphasizes the therapeutic role of vitamin D in reducing neuronal ER stress and the need for maintaining sufficient amount of this vitamin in our diet.

Whole genome sequence data of Bacillus australimaris strain B28A, isolated from Marine Water in India.

Bacillus genus members are dominant in the Eastern Arabian Sea and are known for producing many industrial enzymes. Bacillus australimaris B28A, isolated from seawater, had an enzymatic activity. Here, the whole genome sequence of Bacillus australimaris B28A is reported. The 3,766,107-bp genome, with a GC content of 41.6%, comprised 3936 protein-coding genes, seven ribosomal RNA, and 75 transfer RNA. Several bioactive secondary metabolite genes in the genome, including surfactin, lichenysin, bacillibactin, bacilysin, paenilamicin, fengycin, and carotenoid, were identified using antiSMASH. The 1396 proteins were predicted using RAST, including asparaginase enzyme: an anticancer enzyme. Sequence data have been deposited in the DDBJ/ENA/GenBank database under the accession number JAGQFH000000000. The version described in this paper is JAGQFH000000000.1. The BioProject ID in the GenBank database is PRJNA670955. The raw data is publicly available at "https://www.ncbi.nlm.nih.gov/sra/SRR14203888".

Isolation and identification of marine Bacillus altitudinis KB1 from coastal Kerala: asparaginase producer.

L-asparaginase is a target for many researchers as its properties against cancer, especially leukaemia, and protective agents reduce acrylamide in fried food. In this study, the water samples from Thumba Arattuvazhi Beach in Kerala were screened for l-asparaginase producing microorganisms. This was followed by colourimetric screening using modified M9 media with 0.009% Phenol red dye and using l-asparagine as a sole nitrogen source. Then, the Nessler assay was performed to quantify the enzyme. Molecular identification was made by 16SrRNA sequencing and aligned the sequence with GeneBank for phylogenetic tree construction using BLAST. Seawater was serially diluted for 10-1 to 10-6 using nutrient agar plates. A total of 19 bacterial colonies were isolated. The colonies were evaluated to produce l-asparaginase according to the pink zone around the colonies on the modified M9 medium using a red phenol indicator. The KB1 sample was selected for further studies according to plate colour assay. Nessler assay of L-asparaginase quantified as 2.537 IU/ml. Molecular characterisation showed the sequence association with Bacillus altitudinis the sequence submitted in Genebank as B. altitudinis KB1 strain. The l-asparaginase II gene (AnsB) was amplified based on the entire length of the hypothetical protein of annotated genome with accession number CP022319.2. The l-asparaginase activity in this study was 57% higher than the reference organism B. altitudinis BITHSP010. The l-asparaginase producing bacterium B. altitudinis KB1 from a marine source in Kerala can produce asparaginase, which can be utilised for biotechnology applications.

Galactoxyloglucan Endowed Biogenic Nanoimmunobiotics Arrests Microbial Growth and Elicits Antitumor Immunity.

A straightforward investigation with a surfactant-free green method mediated orchestration of a nanocomposite, where immunotherapy was fused with antimicrobial ability in a biogenic amalgamation, resulted in a nanoimmunobiotics (NIB). Consequently, a galactoxyloglucan (PST001) isolated from Tamarindus indica seed kernel was utilized in a sustainable approach to endow copper nanoparticles (CuNP@PST) with high stability and broad pH tolerance. The biocompatible nature of the CuNP@PST was demonstrated by toxicity studies on BALB/c mice. The nanoparticles exhibited immunomodulatory features by stimulating both humoral and cell-mediated immunity. As remarkable aftermath, we observed promising wound healing capacity on normal human keratinocytes along with the excellent antibacterial and antifungal activity by the NIB. The unique potential of CuNP@PST as a chemopreventive agent by eliciting antitumor immunity was illustrated on syngeneic murine tumor models. The outstanding capacity to reduce tumor burden with a significant increase in lifespan and a peculiar cytokine activation pattern could materialize these nanoparticles as an adjuvant for cancer management. The intrinsically bioeliminable, well-tolerated, and systemically available NIB promises to enter clinical testing as an antimicrobial-immunotherapy agent. Appreciable biodistribution pattern, along with the cost-effective fabrication strategies for scale-up, presents a futuristic biomedical scenario for fast promoting the translational potential of our NIB to combat antibiotic resistance and neoplasia effectively.

Heterologous Expression, Purification and Characterization of an Oligopeptidase A from the Pathogen Leptospira interrogans.

Oligopeptidases are enzymes involved in the degradation of short peptides (generally less than 30 amino acids in size) which help pathogens evade the host defence mechanisms. Leptospira is a zoonotic pathogen and causes leptospirosis in mammals. Proteome analysis of Leptospira revealed the presence of oligopeptidase A (OpdA) among other membrane proteins. To study the role of oligopeptidase in leptospirosis, the OpdA of L. interrogans was cloned and expressed in Escherichia coli with a histidine tag (His-tag). The protein showed maximum expression at 37 °C with 0.5 mM of IPTG after 2 h of induction. Recombinant OpdA protein was purified to homogeneity using Ni-affinity chromatography. The purified OpdA showed more than 80% inhibition with a serine protease inhibitor but the activity was reduced to 30% with the cysteine protease inhibitor. The peptidase activity was increased significantly in the presence of Zn2+ at a neutral pH. Inhibitor assay indicate the presence of more than one active sites for peptidase activity as reported with the OpdA of E. coli and Salmonella. Over-expression of OpdA in E. coli BL21 (DE3) did not cause any negative effects on normal cell growth and viability. The role of OpdA as virulence factor in Leptospira and its potential as a therapeutic and diagnostic target in leptospirosis is yet to be identified.

Expression of Leptospira membrane proteins Signal Peptidase (SP) and Leptospira Endostatin like A (Len A) in BL-21(DE3) is toxic to the host cells.

Heterologous expression of Integral Membrane Proteins (IMPs) is reported to be toxic to the host system in many studies. Even though there are reports on various concerns like transformation efficiency, growth properties, protein toxicity, inefficient expression and protein degradation in IMP overexpression, no studies so far addressed these issues in a comprehensive way. In the present study, two transmembrane proteins of the pathogen Leptospira interrogans, namely Signal peptidase (SP), and Leptospira Endostatin like A (Len-A) were taken along with a cytosolic protein Hydrolase (HYD) to assess the differences in transformation efficiency, protein toxicity, and protein stability when over expressed in Escherichia coli (E. coli). Bioinformatics analysis to predict the transmembrane localization indicated that both SP and Len are targeted to the membrane. The three proteins were expressed in full length in the E. coli expression strain, BL 21 (DE3). Significant changes were observed for the strains transformed with IMP genes under the parameters analysed such as, the transformation efficiency, survival of colonies on IPTG-plate, culture growth kinetics and protein expression compared to the strain harbouring the cytosolic protein gene.

Exploration of Biogenic Nano-chemobiotics Fabricated by Silver Nanoparticle and Galactoxyloglucan with an Efficient Biodistribution in Solid Tumor Investigated by SERS Fingerprinting.

An incredible exploration ensued of a dual modality nanocomposite wherein chemotherapy in fusion with antibacterial efficacy is obtained in a biogenic fabrication, which transformed as a novel nano-chemobiotics (NCB) prevailing fundamental molecular level investigation by surface-enhanced Raman scattering (SERS) platform. The nanocomposite is a facile, robust, and ecofriendly constitution between silver nanoparticles (SNPs) and a naturally occurring galactoxyloglucan (PST001) denoted as SNP@PST, which displayed biocompatibility with an upgraded selective cytotoxicity toward cancer cells. The relatively nontoxic nature of the SNP@PST on normal cells and red blood cells was further proved by detailed toxicological profiling on BALB/c mice. As a unique outcome, we observed excellent antibacterial activity, which is complementary to the greater cytotoxicity by the NCB. In diagnostic aspect, SNP@PST was revealed to be a superior SERS substrate with multiscale Raman signal enhancement contributed by homogeneous hot-spot distribution. Finally, the inherent SERS feature enabled us to investigate the biodistribution of the NCB in tumor-challenged mice using Raman fingerprinting and mapping analysis. Hence, the unrevealed SNP@PST orchestrated with the surfactant-free green method resembled a potential theransonstic NCB construct with synergistic anticancer and antibacterial potential in a single platform.

Scale up of production in a bioreactor of a halotolerant protease from moderately halophilic Bacillus sp. isolated from soil

Studies were conducted on the production of protease by moderately halophilic Bacillus sp. on agro-industrial waste materials. The bacterium could efficiently use many agro wastes as substrates but wheat bran supported maximum enzyme production. To ascertain the performance of the process in shake flasks and lab scale bioreactor, experiments were conducted to analyse protease activity utilizing wheat bran as cost effective substrate. The studies unveiled that pH 7.0, temperature 30°C and static conditions were optimal for enzyme production in flask level fermentation. In scale-up fermentation, at optimal pH and temperature, agitation rate of 50 rpm was best for protease production. The enzymatic nature was studied in 10% SDS gels with BSA (2.5 mg/mL) as substrate and banding pattern was compared with undigested BSA as control. The endoprotease nature and the kinetics of protease activity were confirmed. The enzyme retained 37% of its activity even at 5 M NaCl concentration. The proteolytic activity was also confirmed by casein zymogram analysis. The fermentation medium containing inexpensive substrates, physical conditions and ability of Bacillus sp. to exhibit protease activity on a large scale could collectively be useful for commercial production.