Draft Genome Analysis of Acinetobacter indicus Strain UBT1, an Efficient Lipase and Biosurfactant Producer.

Acinetobacter indicus strain UBT1 has shown efficient lipase (243 U ml-1) and biosurfactant (61.1% E24% emulsification and surface tension reduction to 37.7 mN m-1) production capabilities using agro-industrial waste as sole carbon source. We report here the draft genome sequence of A. indicus strain UBT1 having genome size of 2.97 Mb with 45.90% GC content. Total 2721 coding genes were predicted using National Center for Biotechnology Information-Prokaryotic Genome Annotation Pipeline (NCBI-PGAP). The whole genome shotgun project sequence data are accessible through NCBI Gene Bank under accession no. JABFOI000000000. PGAP annotation revealed the presence of the triacylglycerol lipase, phospholipase etc., that circuitously confers the oil consumption competency to the strain UBT1. Rapid Annotation using the Subsystem Technology (RAST) server used for mapping the genes to the subsystem resulted in 278 subsystem with 30% subsystem coverage. The draft genome data can be used to exploit the A. indicus strain UBT1 for its advance biotechnological application and also for further comparative genomic studies.

MetaRNAseq analysis of surti buffalo rumen content reveals that transcriptionally active microorganisms need not be abundant.

The present study describes rumen microbiota composition and their functional profiles in Indian Surti buffaloes by metagenomic (MG) and metatranscriptomic (MT) approaches. The study compares samples from buffaloes fed three different proportion of roughages; green and dry type of roughage; and different rumen liquor fractions. Irrespective of sample, Bacteroidetes and Firmicutes were the most predominant bacterial phyla, followed by Proteobacteria, Fibrobacteres and Actinobacteria while, Prevotella, Bacteroides, Ruminococcus and Clostridium were the most abundant genera. Different proportions of taxa were observed in both MG and MT approaches indicating the differences in organisms present and organisms active in the rumen. Higher proportions of fungal taxa were observed in MT while important organisms like Fibrobacter and Butyrivibrio and abundant organisms like Bacteroides and Prevotella were underrepresented in MT data. Functionally, higher proportions of genes involved in Carbohydrate metabolism, Amino acid metabolism and Translation were observed in both data. Genes involved in Metabolism were observed to be underrepresented in MT data while, those involved in Genetic information processing were overrepresented in MT data. Further, genes involved in Carbohydrate metabolism were overexpressed compared to genes involved in Amino acid metabolism in MT data compared to MG data which had higher proportion of genes involved in Amino acid metabolism than Carbohydrate metabolism. In all significant differences were observed between both approaches, different fractions of rumen liquor (liquid and solid) and different proportions of roughage in diet.

Metagenomic characterisation of ruminal bacterial diversity in buffaloes from birth to adulthood using 16S rRNA gene amplicon sequencing.

Microbial colonisation in the forestomach of a ruminant is one of the most crucial factors in determining many of its physiological developments and digestive capabilities. The present study attempts to identify establishment pattern of microbes in relation to food, age and rumen development in the buffalo calves at every fortnight interval from birth to 6 months of age, followed by every month till animals became 1 year of age. Diversity study based on 16S rRNA gene sequencing identified rapidly changing bacterial population during initial 60 days of life, which got assemblage as rumen became physiologically mature with increasing age of animals. A lactate fermenting aerobic to facultative anaerobic genera found during initial 30 days of life were expeditiously replaced by strict anaerobic cellulolytic bacterial population with increasing age. The study confirms that initial colonisation mainly depends on the oral cavity and skin of the mother, followed by the surrounding environment and feed offered, which is reversed in order once animal gets older. Some of the well-described genera based on culture-dependent studies like Ruminococcus spp. were found to be in lesser proportion suggesting an additional role of other microbes or niche in cellulose degradation. We report the presence of Porphyromonas spp. and Mannheimia glucosidal for the first time in bovine infants.

Assessment of goat activin receptor type IIB knockdown by short hairpin RNAs in vitro.

BACKGROUND: Targeted knockdown of ACVR2B, a receptor for TGF beta superfamily, has been seen as a potential candidate to enhance the muscle mass through RNAi approach. METHODS: We have evaluated the potential short hairpin RNAs targeting goat ACVR2B in human HEK293T cells and goat myoblasts cells by transient transfection and measured their knockdown efficiency and possible undesired interferon response by quantitative real-time PCR. RESULTS: We observed a significant silencing (64-81%) of ACVR2B in 293T cells with all seven shRNAs (sh1 to sh7) constructs and 16-46% silencing with maximum of 46% by sh6 (p = 0.0318) against endogenous ACVR2B whereas up to 66% (p = 0.0002) silencing by sh6 against exogenously expressed ACVR2B in goat myoblasts cells. Transient knockdown of ACVR2B in goat myoblasts cells by shRNAs did not show significant correlation with the expression of MyoD (r = 0.547; p = 0.102), myogenin (r = 0.517; p = 0.126) and Myf5 (r = 0.262; p = 0.465). As reported earlier, transfection of plasmid DNA induced potent interferon response in 293T and goat myoblasts cells. CONCLUSIONS: The present study demonstrates the targeted knockdown of ACVR2B by shRNAs in HEK293T and goat myoblasts cells in vitro. The transient knockdown of ACVR2B by shRNAs in goat myoblasts did not alter the myogenic gene expression program. However, shRNAs showing significant knockdown efficiency in our study may further be tested for long term and stable knockdown to assess their potential to use for enhancing muscle mass in vivo. As reported earlier, expression of shRNAs through plasmid expression vectors induces potent interferon response raising the concern of safety of its application in vivo.

Type 2 cannabinoid receptor expression on microglial cells regulates neuroinflammation during graft-versus-host disease.

Neuroinflammation is a recognized complication of immunotherapeutic approaches such as immune checkpoint inhibitor treatment, chimeric antigen receptor therapy, and graft versus host disease (GVHD) occurring after allogeneic hematopoietic stem cell transplantation. While T cells and inflammatory cytokines play a role in this process, the precise interplay between the adaptive and innate arms of the immune system that propagates inflammation in the central nervous system remains incompletely understood. Using a murine model of GVHD, we demonstrate that type 2 cannabinoid receptor (CB2R) signaling plays a critical role in the pathophysiology of neuroinflammation. In these studies, we identify that CB2R expression on microglial cells induces an activated inflammatory phenotype that potentiates the accumulation of donor-derived proinflammatory T cells, regulates chemokine gene regulatory networks, and promotes neuronal cell death. Pharmacological targeting of this receptor with a brain penetrant CB2R inverse agonist/antagonist selectively reduces neuroinflammation without deleteriously affecting systemic GVHD severity. Thus, these findings delineate a therapeutically targetable neuroinflammatory pathway and have implications for the attenuation of neurotoxicity after GVHD and potentially other T cell-based immunotherapeutic approaches.

DNAJB1-PRKACA fusion neoantigens elicit rare endogenous T cell responses that potentiate cell therapy for fibrolamellar carcinoma.

Fibrolamellar carcinoma (FLC) is a liver tumor with a high mortality burden and few treatment options. A promising therapeutic vulnerability in FLC is its driver mutation, a conserved DNAJB1-PRKACA gene fusion that could be an ideal target neoantigen for immunotherapy. In this study, we aim to define endogenous CD8 T cell responses to this fusion in FLC patients and evaluate fusion-specific T cell receptors (TCRs) for use in cellular immunotherapies. We observe that fusion-specific CD8 T cells are rare and that FLC patient TCR repertoires lack large clusters of related TCR sequences characteristic of potent antigen-specific responses, potentially explaining why endogenous immune responses are insufficient to clear FLC tumors. Nevertheless, we define two functional fusion-specific TCRs, one of which has strong anti-tumor activity in vivo. Together, our results provide insights into the fragmented nature of neoantigen-specific repertoires in humans and indicate routes for clinical development of successful immunotherapies for FLC.

Utilizing immunogenomic approaches to prioritize targetable neoantigens for personalized cancer immunotherapy.

Advancements in sequencing technologies and bioinformatics algorithms have expanded our ability to identify tumor-specific somatic mutation-derived antigens (neoantigens). While recent studies have shown neoantigens to be compelling targets for cancer immunotherapy due to their foreign nature and high immunogenicity, the need for increasingly accurate and cost-effective approaches to rapidly identify neoantigens remains a challenging task, but essential for successful cancer immunotherapy. Currently, gene expression analysis and algorithms for variant calling can be used to generate lists of mutational profiles across patients, but more care is needed to curate these lists and prioritize the candidate neoantigens most capable of inducing an immune response. A growing amount of evidence suggests that only a handful of somatic mutations predicted by mutational profiling approaches act as immunogenic neoantigens. Hence, unbiased screening of all candidate neoantigens predicted by Whole Genome Sequencing/Whole Exome Sequencing may be necessary to more comprehensively access the full spectrum of immunogenic neoepitopes. Once putative cancer neoantigens are identified, one of the largest bottlenecks in translating these neoantigens into actionable targets for cell-based therapies is identifying the cognate T cell receptors (TCRs) capable of recognizing these neoantigens. While many TCR-directed screening and validation assays have utilized bulk samples in the past, there has been a recent surge in the number of single-cell assays that provide a more granular understanding of the factors governing TCR-pMHC interactions. The goal of this review is to provide an overview of existing strategies to identify candidate neoantigens using genomics-based approaches and methods for assessing neoantigen immunogenicity. Additionally, applications, prospects, and limitations of some of the current single-cell technologies will be discussed. Finally, we will briefly summarize some of the recent models that have been used to predict TCR antigen specificity and analyze the TCR receptor repertoire.

MICROGLIAL CELL EXPRESSION OF THE TYPE 2 CANNABINOID RECEPTOR REGULATES IMMUNE-MEDIATED NEUROINFLAMMATION.

Neuroinflammation is a recognized complication of immunotherapeutic approaches such as immune checkpoint inhibitor treatment, chimeric antigen receptor therapy, and graft versus host disease (GVHD) occurring after allogeneic hematopoietic stem cell transplantation. While T cells and inflammatory cytokines play a role in this process, the precise interplay between the adaptive and innate arms of the immune system that propagates inflammation in the central nervous system remains incompletely understood. Using a murine model of GVHD, we demonstrate that type 2 cannabinoid receptor (CB2R) signaling plays a critical role in the pathophysiology of neuroinflammation. In these studies, we identify that CB2R expression on microglial cells induces an activated inflammatory phenotype which potentiates the accumulation of donor-derived proinflammatory T cells, regulates chemokine gene regulatory networks, and promotes neuronal cell death. Pharmacological targeting of this receptor with a brain penetrant CB2R inverse agonist/antagonist selectively reduces neuroinflammation without deleteriously affecting systemic GVHD severity. Thus, these findings delineate a therapeutically targetable neuroinflammatory pathway and has implications for the attenuation of neurotoxicity after GVHD and potentially other T cell-based immunotherapeutic approaches.

Clinico-epidemiological profile and diagnostic procedures of pediatric tuberculosis in a tertiary care hospital of western Nepal-a case-series analysis.

BACKGROUND: Changing epidemiology and diagnostic difficulties of paediatric tuberculosis (TB) are being increasingly reported. Our aim was to describe clinico-epidemiological profile and diagnostic procedures used for paediatric TB. METHODS: A retrospective case-series analysis was carried out in a tertiary care teaching hospital of western Nepal. All pediatric TB (age 0-14 years) patients registered in DOTS clinic during the time period from March, 2003 to July, 2008 were included. Medical case files were reviewed for information on demography, clinical findings, investigations and final diagnosis. Analysis was done on SPSS package. Results were expressed as rates and proportions. Chi square test was used to test for statistical significance. RESULTS: About 17.2% (162/941) of TB patients were children. Common symptoms were cough, fever and lymph node swelling. The types of TB were pulmonary TB (46.3%, 75/162), followed by extra-pulmonary TB (41.4%, 67/162). Twelve patients (7.4%) had disseminated TB. Distribution of types of TB according to gender was similar. PTB was common in younger age than EPTB which was statistically significant. EPTB was mainly localized to lymph node (38, 50.7%), and abdomen (9, 12%). Five main investigations namely Mantoux test, BCG test, chest radiograph, erythrocyte sedimentation rate (ESR) and fine needle aspiration cytology (FNAC) or biopsy were carried out to diagnose TB. CONCLUSIONS: Paediatric TB in both pulmonary and extrapulmonary forms is a common occurrence in our setting. Age incidence according to type of TB was significant. Diagnosis was based on a combination of epidemiological and clinical suspicion supported by results of various investigations.

Molecular cloning, heterologous expression, and functional characterization of a cellulolytic enzyme (Cel PRII) from buffalo rumen metagenome.

A cellulase encoding gene, Cel PRII, was identified from Mehsani buffalo rumen metagenome, and cloned and expressed in Escherichia coli BL21(DE3)pLysS. The 1170 bp full length gene encodes a 389 residue polypeptide (Cel PRII) containing a catalytic domain belonging to glycosyl hydrolase (GH) 5 family. The fusion protein consisting of the Cel PRII, thioredoxin tag and 6x Histidine tag with predicted molecular weight of 63 kDa when recovered from inclusion bodies under denaturing conditions, exhibited cellulolytic activity against carboxymethyl cellulose (CMC). Recombinant Cel PRII was stable in the pH range 4.0-10.0 with pH optima 6.0. The optimal reaction temperature of Cel PRII was 30 °C with more than 50% of its activity retained at the temperatures ranging from 0 to 50 °C. Cel PRII exhibited enhanced enzymatic activity in the presence of Mn2+ ions and was inhibited in the presence of chelating agent EDTA. The K m and V max values for CMC were found to be 166 mg/mL and 1292 IU/mg, respectively. Cel PRII identified in the present study may act as an excellent candidate for industrial applications, and may aid in lignocellulosic biomass conversion because of its potential cellulolytic activity, thermostability, and excellent pH stability.

Transcriptomic comparison of primary bovine horn core carcinoma culture and parental tissue at early stage.

AIM: Squamous cell carcinoma or SCC of horn in bovines (bovine horn core carcinoma) frequently observed in Bos indicus affecting almost 1% of cattle population. Freshly isolated primary epithelial cells may be closely related to the malignant epithelial cells of the tumor. Comparison of gene expression in between horn's SCC tissue and its early passage primary culture using next generation sequencing was the aim of this study. MATERIALS AND METHODS: Whole transcriptome sequencing of horn's SCC tissue and its early passage cells using Ion Torrent PGM were done. Comparative expression and analysis of different genes and pathways related to cancer and biological processes associated with malignancy, proliferating capacity, differentiation, apoptosis, senescence, adhesion, cohesion, migration, invasion, angiogenesis, and metabolic pathways were identified. RESULTS: Up-regulated genes in SCC of horn's early passage cells were involved in transporter activity, catalytic activity, nucleic acid binding transcription factor activity, biogenesis, cellular processes, biological regulation and localization and the down-regulated genes mainly were involved in focal adhesion, extracellular matrix receptor interaction and spliceosome activity. CONCLUSION: The experiment revealed similar transcriptomic nature of horn's SCC tissue and its early passage cells.

Potential functional gene diversity involved in methanogenesis and methanogenic community structure in Indian buffalo (Bubalus bubalis) rumen.

Understanding the methanogen community structure and methanogenesis from Bubalus bubalis in India may be beneficial to methane mitigation. Our current understanding of the microbial processes leading to methane production is incomplete, and further advancement in the knowledge of methanogenesis pathways would provide means to manipulate its emission in the future. In the present study, we evaluated the methanogenic community structure in the rumen as well as their potential genes involved in methanogenesis. The taxonomic and metabolic profiles of methanogens were assessed by shotgun sequencing of rumen metagenome by Ion Torrent semiconductor sequencing. The buffalo rumen contained representative genera of all the families of methanogens. Members of Methanobacteriaceae were found to be dominant, followed by Methanosarcinaceae, Methanococcaceae, Methanocorpusculaceae, and Thermococcaceae. A total of 60 methanogenic genera were detected in buffalo rumen. Methanogens related to the genera Methanobrevibacter, Methanosarcina, Methanococcus, Methanocorpusculum, Methanothermobacter, and Methanosphaera were predominant, representing >70 % of total archaeal sequences. The metagenomic dataset indicated the presence of genes involved in the methanogenesis and acetogenesis pathways, and the main functional genes were those of key enzymes in the methanogenesis. Sequences related to CoB--CoM heterodisulfide reductase, methyl coenzyme M reductase, f420-dependent methylenetetrahydromethanopterin reductase, and formylmethanofuran dehydrogenase were predominant in rumen. In addition, methenyltetrahydrofolate cyclohydrolase, methylenetetrahydrofolate dehydrogenase, 5,10-methylenetetrahydrofolate reductase, and acetyl-coenzyme A synthetase were also recovered.

Goat activin receptor type IIB knockdown by muscle specific promoter driven artificial microRNAs.

Activin receptor type IIB (ACVR2B) is a transmembrane receptor which mediates signaling of TGF beta superfamily ligands known to function in regulation of muscle mass, embryonic development and reproduction. ACVR2B antagonism has shown to enhance the muscle growth in several disease and transgenic models. Here, we show ACVR2B knockdown by RNA interference using muscle creatine kinase (MCK) promoter driven artificial microRNAs (amiRNAs). Among the various promoter elements tested, the ∼1.26 kb MCK promoter region showed maximum transcriptional activity in goat myoblasts cells. We observed up to 20% silencing in non-myogenic 293T cells and up to 32% silencing in myogenic goat myoblasts by MCK directed amiRNAs by transient transfection. Goat myoblasts stably integrated with MCK directed amiRNAs showed merely 8% silencing in proliferating myoblasts which was increased to 34% upon induction of differentiation at transcript level whereas up to 57% silencing at protein level. Knockdown of ACVR2B by 5'-UTR derived amiRNAs resulted in decreased SMAD2/3 signaling, increased expression of myogenic regulatory factors (MRFs) and enhanced proliferation and differentiation of myoblasts. Unexpectedly, knockdown of ACVR2B by 3'-UTR derived amiRNAs resulted in increased SMAD2/3 signaling, reduced expression of MRFs and suppression of myogenesis. Our study offers muscle specific knockdown of ACVR2B as a potential strategy to enhance muscle mass in the farm animal species.

Goat activin receptor type IIB knockdown by artificial microRNAs in vitro.

Activin receptor type IIB (ACVR2B) has been known to negatively regulate the muscle growth through mediating the action of transforming growth factor beta superfamily ligands. Recently, the artificial microRNAs (amiRNAs) which are processed by endogenous miRNA processing machinery have been proposed as promising approach for efficient gene knockdown. We evaluated amiRNAs targeting goat ACVR2B in HEK293T and goat myoblasts cells. The amiRNAs were designed based on the miR-155 backbone and cloned in 5'- and 3'-UTR of GFP reporter gene under the CMV promoter. Although both 5'- and 3'-UTR-amiRNAs vectors showed efficient synthesis of GFP transcripts, amiRNAs in 5'-UTR drastically affected GFP protein synthesis in transfected goat myoblast cells. Among the four amiRNAs targeting ACVR2B derived from either 5'- or 3'-UTR, ami318 showed highest silencing efficiency against exogenously co-expressed ACVR2B in both 293T and goat myoblast cells whereas ami204 showed highest silencing efficiency against endogenous ACVR2B in goat myoblasts cells. The 3'-UTR-derived amiRNA exerted higher knockdown efficiency against endogenous ACVR2B at transcript level whereas 5'-UTR-derived amiRNAs exerted higher knockdown efficiency at protein level. The expression of ACVR2B showed positive correlation with the expression of MYOD (r = 0.744; p = 0.009) and MYOG (r = 0.959; p = 0.000) in the amiRNA-transfected myoblasts. Although both 5'- and 3'-UTR-amiRNA vectors led to substantial induction of interferon response, the magnitude of the response was found to be higher with the 3'-UTR-amiRNA vectors.

Myostatin knockdown and its effect on myogenic gene expression program in stably transfected goat myoblasts.

Myostatin, a negative regulator of skeletal muscle mass, is a proven candidate to modulate skeletal muscle mass through targeted gene knockdown approach. Here, we report myostatin (MSTN) knockdown in goat myoblasts stably expressing small hairpin RNA (shRNAs) against MSTN gene through lentivirus vector-mediated integration. We observed 72% (p = 0.003) and 54% (p = 0.022) downregulation of MSTN expression with sh2 shRNA compared to empty vector control and untransduced myoblasts, respectively. The knockdown of MSTN expression was accompanied with concomitant downregulation of myogenic regulatory factor MYOD (77%, p = 0.001), MYOG (94%, p = 0.000), and MYF5 (36%, p = 0.000), cell cycle regulator p21 (62%, p = 0.000), MSTN receptor ACVR2B (23%, p = 0.061), MSTN antagonist follistatin (81%, p = 0.000), and downstream signaling mediators SMAD2 (20%, p = 0.060) and SMAD3 (49%, p = 0.006). However, the expression of MYF6 was upregulated by 14% compared to control lentivirus-transduced myoblasts (p = 0.354) and 79% compared to untransduced myoblasts (p = 0.018) in sh2 shRNA-transduced goat myoblasts cells. Although, MSTN knockdown led to sustained cell proliferation of myoblasts, the myoblasts fusion was suppressed in both MSTN knocked down and control lentivirus-transduced myoblasts. The expression of interferon response gene OAS1 was significantly upregulated in control lentivirus (10.86-fold; p = 0.000)- and sh2 (1.71-fold; p = 0.002)-integrated myoblasts compared to untransduced myoblasts. Our study demonstrates stable knockdown of MSTN in goat myoblasts cells and its potential for use in generation of transgenic goat by somatic cell nuclear transfer.

Transcriptomic dissection of myogenic differentiation signature in caprine by RNA-Seq.

Muscle growth and development from the embryonic to the adult stage of an organism consists of a series of exquisitely regulated and orchestrated changes in expression of genes leading to muscle maturation. In this study, we performed whole transcriptome profiling of adult caprine skeletal muscle derived myoblast and fused myotubes. Using Ion Torrent PGM sequencing platform, a total of 948,776 and 799,976 reads were generated in myoblasts and fused myotubes, respectively. The sequence reads were analyzed on CLC Genomics Workbench using Bos taurus RNA database to study the gene expression in both stages to study different genes responsible for muscle development and regeneration. The up and down-regulated genes were analyzed for gene ontology (GO) and KEGG pathways by Database for Annotation, Visualization and Integrated Discovery (DAVID) database. We found many genes exclusive to multinuclear fused myotubes and contractile nature of skeletal muscle, whereas up-regulated genes in myoblast stage were related to cell division and transcriptional regulation. Out of 27 genes selected for expression validation by RT-qPCR (reverse transcriptase-quantitative polymerase chain reaction), 19 genes showed the expression pattern comparable with CLC Genomics Workbench findings. Further, mRNA originated muscle specific microRNAs (miRNA-1 and miRNA-133b) were also observed in the fused myotubes along with other miRNAs with possible importance in muscle development. This study highlights important genes responsible for muscle development and differentiation in adult skeletal muscle system.