Synthetic approach toward spiro quinoxaline-ß-lactam based heterocyclic compounds: Spectral characterization, SAR, pharmacokinetic and biomolecular interaction studies.

Series of spiro quinoxaline-ß-lactam based heterocyclic compounds (QL 1 - QL 21) were synthesized and characterized by spectroscopic techniques like 1H-NMR, LC-MS, FT-IR spectroscopy and elemental analysis. The binding mode and binding strength between compounds and calf thymus-DNA were estimated by UV-visible spectroscopy, viscosity measurement and molecular docking studies. The compounds bind with the DNA through partial intercalation mode. In the absorption titration experiment, the Kb values for all the synthesized compounds were found in the range of 0.24-0.64 × 105 M-1. The protein binding studies of all the synthesized compounds were evaluated by absorption titration experiment, and the Kb value for all the compounds was obtained in the range of 0.030-1.571 × 104 M-1. The compounds were screened against two Gram (+ve) and three Gram (-ve) bacteria for antimicrobial activity. The MIC values for all the synthesized compounds were found in 95-255 µM. The LC50 values (cytotoxicity) of the synthesized compounds (QL 1-QL 21) were found in the range of 4.00-12.89 µg/mL. The ADME study was carried out using the online platform SwissADME and admetSAR to evaluate the pharmacokinetic profile of all the synthesized compounds. All the compounds were screened for anticancer activity against the human osteosarcoma (MG-63) cell line. The result shows that all the compounds exhibit effective anticancer activity.Communicated by Ramaswamy H. Sarma.

Evolutionary and Antigenic Profiling of the Tendentious D614G Mutation of SARS-CoV-2 in Gujarat, India.

Humankind has suffered many pandemics in history including measles, SARS, MERS, Ebola, and recently the novel Coronavirus disease caused by SARS-CoV-2. As of September 2021, it has affected over 200 million people and caused over 4 million deaths. India is the second most affected country in the world. Up to this date, more than 38 Lakh viral genomes have been submitted to public repositories like GISAID and NCBI to analyze the virus phylogeny and mutations. Here, we analyzed 2349 genome sequences of SARS-CoV-2 submitted in GISAID by a single institute pertaining to infections from the Gujarat state to know their variants and phylogenetic distributions with a major focus on the spike protein. More than 93% of the genomes had one or more mutations in the spike glycoprotein. The D614G variant in spike protein is reported to have a very high frequency of >95% globally followed by the L452R and P681R, thus getting significant attention. The antigenic propensity of a small peptide of 29 residues from 597 to 625 of the spike protein variants having D614 and G614 showed that G614 has a little higher antigenic propensity. Thus, the D614G is the cause for higher viral antigenicity, however, it has not been reported to be effective to be causing more deaths.

In Vitro Antibacterial Evaluation of Terminalia chebula as an Alternative of Antibiotics against Bovine Subclinical Mastitis.

The extent of subclinical mastitis in three breeds of cattle, Kankrej, Gir, and Crossbred, was performed at cattle farms in Anand town of Gujarat State, India. The prevalence of subclinical mastitis in crossbred cattle was higher compared to local breed of cattle. Causative agents identified using 16S rDNA polymerase chain reaction (PCR)-based molecular method were Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Bacillus megaterium. In vitro antibacterial activity of ethyl acetate extract of plant Terminalia chebula (Combretaceae) was checked by agar well diffusion method against four isolated and molecularly identified microorganisms. Ethyl acetate extract shows antimicrobial activity with varying magnitudes against all identified isolates. Among the three different concentrations, 500 µg/mL conc. of extract is as effective as that of standard amoxicillin. In vitro results support the use of plant extract from T. chebula as an alternative to antibiotics therapy against bovine subclinical mastitis.

Metagenomic data of DNA viruses of poultry affected with respiratory tract infection.

The incidence and severity of respiratory diseases in commercial broiler chicken flocks have increased recently in India because of intensification of the broiler industry. Viral population are predominant in respiratory tract infections and they pose continuous economic burden to poultry industry by causing severe economic losses through decreased productivity [1], [2]. To understand viral metagenome of poultry associated with respiratory infections, we performed DNA virome sequencing and data analysis of broilers from 8 districts of Gujarat State in India. We report high quality sequencing reads and highly abundant DNA viral population present in the infected broiler birds. The raw sequencing data used to perform metagenomic analysis is available in the Sequence Read Archive (SRA) under the BioProject No. PRJNA322592 and Accession No. MAUZ00000000, MAVA00000000, MAVB00000000, MAVC00000000, MAVD00000000, MAVE00000000, MAVF00000000, MAVG00000000 (https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA322592).

Transcriptome analysis of the adult rumen fluke Paramphistomum cervi following next generation sequencing.

Rumen flukes are parasitic trematodes (Platyhelminthes: Digenea) of major socioeconomic importance in many countries. Key representatives, such as Paramphistomum cervi, can cause "Rumen fluke disease" or paramphistomosis and undermine economic animal productivity and welfare. P. cervi is primarily a problem in sheep, goat and buffalo production as a consequence of reduced weight gain and milk production, clinical disease or death. Recent technological advances in genomics and bioinformatics now provide unique opportunities for the identification and pre-validation of drug targets and vaccines through improved understanding of the biology of pathogens such as P. cervi and their relationship with their hosts at the molecular level. Here, we report next generation transcriptome sequencing analysis for P. cervi. RNAseq libraries were generated from RNA extracted from 15 adult P. cervi parasites sampled from each of three different host species (sheep, goat and buffalo) and a reference transcriptome was generated by assembly of all Ion Torrent PGM sequencing data. Raw reads (7,433,721 in total) were initially filtered for host nucleotide contamination and ribosomal RNAs and the remaining reads were assembled into 43,753 high confidence transcript contigs. In excess of 50% of the assembled transcripts were annotated with domain- or protein sequence similarity derived functional information. The reference adult P. cervi transcriptome will serve as a basis for future work on the biology of this important parasite. Using the widely investigated trematode virulence factor and vaccine candidate Cathepsin L as an example, the epitope GPISIAINA was found to be conserved in P. cervi isolated from three different host species supporting its candidacy for vaccine development and illustrating the utility of the adult P. cervi transcriptome.

De Novo Assembly and Transcriptome Characterization of Canine Retina Using High-Throughput Sequencing.

We performed transcriptome sequencing of canine retinal tissue by 454 GS-FLX and Ion Torrent PGM platforms. RNA-Seq analysis by CLC Genomics Workbench mapped expression of 10,360 genes. Gene ontology analysis of retinal transcriptome revealed abundance of transcripts known to be involved in vision associated processes. The de novo assembly of the sequences using CAP3 generated 29,683 contigs with mean length of 560.9 and N50 of 619 bases. Further analysis of contigs predicted 3,827 full-length cDNAs and 29,481 (99%) open reading frames (ORFs). In addition, 3,782 contigs were assigned to 316 KEGG pathways which included melanogenesis, phototransduction, and retinol metabolism with 33, 15, and 11 contigs, respectively. Among the identified microsatellites, dinucleotide repeats were 68.84%, followed by trinucleotides, tetranucleotides, pentanucleotides, and hexanucleotides in proportions of 25.76, 9.40, 2.52, and 0.96%, respectively. This study will serve as a valuable resource for understanding the biology and function of canine retina.

Isolation and characterization of H9N2 influenza virus isolates from poultry respiratory disease outbreak.

The present study reports isolation and characterization of H9N2 virus responsible for disease characterized by symptoms including difficulty in respiration, head swelling, nasal discharge, reduced feed intake, cyanotic comb, reduced egg production and mortality. Virus isolation from allantoic fluid inoculated with tracheal aspirates and whole genome sequencing of two isolates were performed on an Ion-Torrent sequencer. Phylogenetic analysis revealed that the two H9N2 isolates are reassortant viruses showing a G1-like lineage for HA, NA and NP, a Hok/49/98-like lineage for PB1 and PA, PK/UDL-01/05-like lineage for PB2, IL/90658/00-like lineage for NS and an unknown lineage for M gene. Analyses of the HA cleavage site showed a sequence of (333PARSSR↓GL340) indicating that these isolates are of low pathogenicity. Isolate 2 has leucine at amino acid position 226, a substitution which is associated with mammalian adaptation of avian influenza virus. Isolate 1 has the S31N substitution in the M2 gene that has been associated with drug resistance as well as R57Q and C241Y mutations in the NP gene which are associated with human adaptation. The result reported here gives deep insight in to H9N2 viruses circulating in domestic poultry of India and supports the policy of active efforts to control and manage H9N2 infections in Indian poultry.

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.

Evaluation of a topical herbal drug for its in-vivo immunomodulatory effect on cytokines production and antibacterial activity in bovine subclinical mastitis.

BACKGROUND: Antibiotics have been in use in the treatment of bovine mastitis since decades; however, their use is associated with cost issues and human health concern. Use of herbal drugs does not generally carry these disadvantages. Many plants/herbs have been evaluated in the treatment of bovine mastitis with additional property of immunomodulation in affected mammary gland. AIM: To evaluate a topical herbal drug in two breeds of cattle for its in-vivo immunomodulatory effect on cytokines production and antibacterial activity in bovine subclinical mastitis. MATERIALS AND METHODS: The response to treatment was evaluated by enumerating somatic cell count (SCC), determining total bacterial load, and studying the expression of different cytokines (interleukin [IL]-6, IL-8, IL-12, granulocyte macrophage-colony stimulating factor, interferon (IFN)-γ and tumor necrosis factor [TNF]-α). RESULTS: The pre- and post-treatment SCC in mastitic quarters statistically did not differ significantly, however, total bacterial load declined significantly from day 0 onwards in both the breeds. Highly significant differences (P < 0.01) were observed in all the cytokines on day 0, 5, and 21 postlast treatment in both the breeds. The expression level of all the cytokines showed a significant increase on day 5, while a decrease was noticed on day 21 in both the breeds of cattle. The comparison of cytokine expression profiles between crossbred and Gir cattle revealed a significant difference in expression of IL-6 and TNF-α. However, other cytokines exhibited a similar pattern of expression in both breeds, which was non-significant. CONCLUSION: The topical herbal drug exhibited antibacterial and immunomodulatory activities in subclinical mastitis and thus the work supports its use as alternative herbal therapy against subclinical udder infection in bovines.

Transcriptome analysis and SNP identification in SCC of horn in (Bos indicus) Indian cattle.

Single Nucleotide Polymorphisms (SNPs) have become the marker of choice for genome wide association studies. In order to provide the best genome coverage for the analysis of disease, production and performance traits, a large number of relatively evenly distributed SNPs are needed. The main objective of present work was to identify large numbers of gene-associated SNPs using high-throughput sequencing in squamous cell carcinoma of horn. RNA-seq analysis was conducted on 2 tissues viz. Horn Cancer (HC) and Horn Normal (HN) in Kankrej breed of cattle. A total of 909,362 reads with average read length of 405 bp for HC and 583,491 reads with average read length of 411 bp for HN were obtained. We found 9532 and 7065 SNPs as well as 1771 and 1172 Indels in HC and HN, respectively, from which, 7889 SNPs and 1736 Indels were uniquely present in HC, 5886 SNPs and 1146 Indels were uniquely present in HN and reported first time in Bos indicus, whereas the rest are already reported in Bos taurus dbSNP database. The gene-associated SNPs and Indels were high in upregulated genes of HC as compared to HN. Analysis of differentially expressed genes was identified, these genes are involved in regulation of cell proliferation, apoptosis, gene transcription, cell survival and metabolism through various metabolic pathways. The result of transcriptome expression profiling was validated using Real Time quantitative PCR in nine randomly selected genes. We identified numbers aberrant signaling pathways responsible for carcinogenesis in HC which are also commonly altered in squamous cell carcinoma (SCC) of lung in human being. We conclude that a large number of altered genes and dysfunction of multiple pathways are involved in the development of Horn Cancer. The present findings contribute to theoretical information for further screening of genes and identification of markers for early diagnosis of HC as well as SNPs identified in this report provide a much needed resource for genetic studies in B. indicus and shall contribute to the development of a high density SNP array. Validation and testing of these SNPs using SNP arrays will form the material basis for gene associated SNPs in HC.

The landscape of alternative splicing in buccal mucosa squamous cell carcinoma.

OBJECTIVES: Alternative splicing (AS) is a key regulatory mechanism in the process of protein synthesis generating transcriptome and proteome diversity. In this study, we attempted to identify alternative splicing in a pair of BMSCC cancer and adjacent normal tissue using RNAseq datasets and also assessed the potential of these datasets to provide quantitative measurements for alternative splicing levels. MATERIALS AND METHODS: We performed high-throughput sequencing of buccal mucosal cancer and healthy tissue cDNA library which resulted in a transcriptome map of BMSCC cancer. RNAseq analysis was performed to assess alternative splicing complexity in cancer tissue and to search splice junction sequences that represent candidate 'new' splicing events. The splice junctions were predicted by SpliceMap software and putative assembled transcripts validated using the RT-PCR. We also analyzed the coding potential of alternative spliced candidate by HMMER. RESULTS: We detected a total of 11 novel splice junctions derived mostly from alternate 5' splice site; including two of them which contained new translation initiation sites (TISs). We have identified novel IgG pseudogene and a fusion transcript of MEMO1 and RPS9, which were further confirmed by PCR from genomic DNA. We also found novel putative long non-coding RNA (lncRNA), which is antisense to SPINK5 gene. The coding potential of these AS variants revealed that alternative splicing caused premature termination, insertion/deletion of amino acid (s) or formation of novel N-terminus. CONCLUSIONS: Differential splicing of these novel AS variants between cancer and adjacent normal tissue suggests their involvement in BMSCC cancer development and progression.

Molecular analysis of the bacterial microbiome in the forestomach fluid from the dromedary camel (Camelus dromedarius).

Rumen microorganisms play an important role in ruminant digestion and absorption of nutrients and have great potential applications in the field of rumen adjusting, food fermentation and biomass utilization etc. In order to investigate the composition of microorganisms in the rumen of camel (Camelus dromedarius), this study delves in the microbial diversity by culture-independent approach. It includes comparison of rumen samples investigated in the present study to other currently available metagenomes to reveal potential differences in rumen microbial systems. Pyrosequencing based metagenomics was applied to analyze phylogenetic and metabolic profiles by MG-RAST, a web based tool. Pyrosequencing of camel rumen sample yielded 8,979,755 nucleotides assembled to 41,905 sequence reads with an average read length of 214 nucleotides. Taxonomic analysis of metagenomic reads indicated Bacteroidetes (55.5 %), Firmicutes (22.7 %) and Proteobacteria (9.2 %) phyla as predominant camel rumen taxa. At a finer phylogenetic resolution, Bacteroides species dominated the camel rumen metagenome. Functional analysis revealed that clustering-based subsystem and carbohydrate metabolism were the most abundant SEED subsystem representing 17 and 13 % of camel metagenome, respectively. A high taxonomic and functional similarity of camel rumen was found with the cow metagenome which is not surprising given the fact that both are mammalian herbivores with similar digestive tract structures and functions. Combined pyrosequencing approach and subsystems-based annotations available in the SEED database allowed us access to understand the metabolic potential of these microbiomes. Altogether, these data suggest that agricultural and animal husbandry practices can impose significant selective pressures on the rumen microbiota regardless of rumen type. The present study provides a baseline for understanding the complexity of camel rumen microbial ecology while also highlighting striking similarities and differences when compared to other animal gastrointestinal environments.

RNA-Seq reveals differentially expressed isoforms and novel splice variants in buccal mucosal cancer.

Buccal mucosal cancer (BMC) is a multifactorial disease with poorly defined genetic profile and prognosis due to late detection stage and unavailability of reliable prognostic markers. To identify aberrant transcriptional events, we employed high throughput RNA-Seq analysis of BMC and normal tissue. Comparative transcriptome analysis with Cufflinks revealed 260 up and 328 down regulated genes whereas, 350 up and 397 down regulated isoforms by at least two folds over buccal normal in BMC. Study revealed 46 splice variants in normal and 106 in cancer, out of which 10 variants were validated with end point RT-PCR. Expression of two isoforms of CD74 was validated using RT-qPCR and found in accordance with RNA-Seq. Further extensive follow up analysis of modulator genes, isoforms and splice variants found in this study, might be useful in deep understanding of pathological changes in BMC and development of prospective intervention strategies.

Identification of novel splice variants in horn cancer by RNA-Seq analysis in Zebu cattle.

Horn cancer accounts for nearly 83% of total tumors found in Indian Zebu cattle, which results in chronic suffering and causes heavy economic losses. Alternative splicing has been frequently implicated in the various types of cancer progression. Utilizing the transcriptome sequence generated by next generation sequencing, we analyzed the transcript data for the presence of alternative splicing using BLAT program and identified 27 alternatively spliced genes, of which 12 spliced variants appeared to be the novel spliced candidates. Protein prediction of these novel spliced variants revealed that splice variation has caused either truncation of protein, insertion/deletion of stretch of amino acids or formation of unique carboxy terminus. The RT-PCR analysis confirmed the expression of 8 of the 12 novel spliced variants observed by transcriptome sequencing. Additionally, altered splicing/expression of these novel candidates between cancer and normal tissues revealed by qPCR suggests their potential involvement in the development of horn cancer.

Identification of novel transcripts deregulated in buccal cancer by RNA-seq.

The differential transcriptome analysis provides better understanding of molecular pathways leading to cancer, which in turn allows designing the effective strategies for diagnosis, therapeutic intervention and prediction of therapeutic outcome. This study describes the transcriptome analysis of buccal cancer and normal tissue by CLC Genomics Workbench from the data generated by Roche's 454 sequencing platform, which identified total of 1797 and 2655 genes uniquely expressed in normal and cancer tissues, respectively with 2466 genes expressed in both tissues. Among the genes expressed in both tissues, 1842 were up-regulated whereas 624 were down-regulated in cancer tissue. Besides transcripts known to be involved in cancer, this study led to the identification of novel transcripts, with significantly altered expression in buccal cancer tissue, providing potential targets for diagnosis and cancer therapeutics. The functional categorization by the KEGG pathway and gene ontology analysis revealed enrichment of differentially expressed transcripts to various pathways leading to cancer, including the p53 signaling pathway. Moreover, the gene ontology analysis unfolded suppression of transcripts involved in actin mediated cell contraction process. The down-regulation of four of these transcripts MYL1, ACTA1, TCAP and DESMIN in buccal cancer were further supported by quantitative PCR signifying its possible implication in the cancer progression.

A preliminary sketch of horn cancer transcriptome in Indian zebu cattle.

Horn cancer, a type of squamous cell carcinoma, in zebu cattle is an expensive affair in Indian agriculture sector, which accounts for 83.34% of total tumors found. In general, cancer tissue confirms considerably different expression patterns when compared to a normal stage. This includes not only up/down regulation, but also, the aberrant gene expression, the presence of different non-coding RNAs (ncRNAs), pseudogenes expression and genes involved in unusual pathways. We employed Roche 454 next generation sequencing platform to sequence Bos indicus cancerous and normal horn tissue transcripts. This resulted into a total of 909,345 high-confidence deep sequencing reads and detected a range of unusual transcriptional events including tumor associated genes. We also validated expression of two of the four tested genes in five other similar tissue samples by RT-qPCR. Further, seven cancer specific non-coding transcripts were accessed and a few of them have been suggested as cancer specific markers. This study for the first time provides primary transcriptome sketch of Bos indicus horn cancer tissue, and also demonstrates the suitability of the 454 sequencer for transcriptome analysis, which supports the concept of varied gene expression in cancerous condition.

Identification and antibiogram of microbes associated with bovine mastitis.

An investigation of Mastitis in cattle was carried out in Anand city and in nearby villages of Gujarat state using California Mastitis Test (CMT) kit. The prevalence of clinical and subclinical mastitis was found to be 5.5% and 15.75%, respectively. Staphylococcus aureus was identified through strain specific polymerase chain reaction; the remaining isolates identified on the basis of molecular analysis by 16S rDNA sequencing and phylogenetic analysis were Staphylococcus species, B. pumilus, Staphylococcus chromogenes, Bacillus species, and Pseudomonas species. In vitro antimicrobial susceptibility pattern of all the isolates was checked against 13 different antibiotics using the agar disc diffusion method. Highest bacterial resistance was observed with penicillin G and oxacillin antibiotics. It was also observed that the patterns of bacterial resistance have not changed in India over the years. The data supports the decrease in the incidence of mastitis but the rate of decrease is minimal. More effective control strategies are required.