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Interleukin-17A is a pro-inflammatory cytokine that plays a key role in the immune response to many pathogens and implicated in autoimmune diseases. This molecule is also involved in providing protection to many bacterial and fungal infections of gastro-intestinal tract and respiratory mucosa. Although molecular aspect of IL-17A has been studied in few species, no data are available for buffalo, which is one of the major sources of milk production in India. Therefore, in the present study, IL-17A gene of Indian Murrah Buffalo origin was cloned, expressed, and analyzed using bioinformatic tools. The coding sequence of buffalo IL-17A gene was cloned in prokaryotic expression vector (pET-28a) followed by its expression, purification, and characterization. A computational analysis was performed to understand the sequence, structure, and evolutionary relationship of buIL-17A. It revealed that the length of buIL-17A sequence without signal peptide is 132 amino acids as in cattle. However, sequence identity is found to be 99% due to one amino substitution difference between buffalo and cattle. After analysis, it can be concluded that buIL-17A recombinant protein can be used as a potential immunobiological reagent for diagnostic and therapeutic purpose.

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Foot and mouth disease (FMD) is an extremely contagious disease of cloven-hoofed domesticated and wild animals, resulting in significant economic losses in many parts of the world. FMD virus (FMDV) serotype O is responsible for approximately 70% of global outbreaks. For detection of FMDV antigen or antibody, ELISAs are used worldwide and have several limitations, such as batch-to-batch variation in generating immunobiologicals, high production cost and ethical concerns over animal sacrifice. The use of single domain antibody (sdAb) or variable N-terminal domain of the heavy chain of heavy chain antibody (VHH) found naturally in camels has proven their effectiveness in diagnostics and therapeutics. In the present study, the anti-FMDV serotype O-specific VHH-C1 gene sequence (Accession no. KJ751546) was retrieved from the NCBI database. The gene was synthesized commercially in the pBluescript KS+ cloning vector and expressed in E. coli BL21 (DE3) cells using the pET303/CT-His expression system with a C-terminal 6X-His tag. The expressed sdAb, verified by SDS‒PAGE and western blotting, was purified by Ni-chelate chromatography and used as a coating antibody in double antibody sandwich (DAS) ELISA for FMDV detection and typing. The sdAb exhibited a high binding affinity for FMDV serotype O, without any cross-reactivity toward serotypes A and Asia-1. It exhibited better thermostability up to 85 °C than conventional rabbit polyclonal anti-FMDV sera. The potential of sdAbs thus produced without sacrificing lab animals could be explored for replacing polyclonal sera in DAS-ELISA as well as for developing biosensors or lateral flow devices for FMDV type O detection.

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Mimotope peptides of native antigens are valuable for diverse applications such as diagnostics, therapeutics and modern vaccine design. Here, we report for the first time the selection and identification of peptide mimotopes of Trypanosoma evansi RoTat 1.2 variant surface glycoprotein (VSG) for their potential uses in surra diagnostics and multi-epitope vaccine research. First, we produced the mouse monoclonal antibodies (mAbs), designated as 2E11 (IgG1) and 1C2 (IgG1), against the antigens in T. evansi RoTat 1.2 lysates. We then used 2E11 mAb to immunoprecipitate the target antigen. The immunoprecipitated antigen was then identified to be the VSG by mass spectrometry. Both 2E11 and 1C2 mAbs reacted with the VSG in immunoblots. The surface plasmon resonance immunosensors developed with both the mAbs detected VSG in the parasite lysates as well as in the rodent sera. Further, the mAbs were biotinylated and used in three rounds of panning to select peptide mimotopes from the random peptide phage display library (PhD-12; New England Biolabs, USA). The phage clones selected against each mAb were amplified and tested by phage capture ELISA for specificity. The peptide coding regions of the selected phages were sequenced and the protein blast search of the deduced amino acid sequences was performed by accessing the non-redundant protein database at https://blast.ncbi.nlm.nih.gov/. The conformational B epitope prediction of the selected mimotope sequences was done by using 3D Pepitope algorithms accessed at: http://pepitope.tau.ac.il/. The potential applications of the selected mimotopes in surra diagnostics and research are being explored.

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Oral squamous cell carcinoma (OSCC) is highly predominant in India due to excessive use of tobacco. Here we investigated Long INterpersed Element 1 (LINE or L1) retrotransposon activity in OSCC samples in the same population. There are almost 500,000 copies of L1 occupied around 30%  of the human genome. Although most of them are inactive, around 150-200 copies are actively jumping in a human genome. L1 encodes two proteins designated as ORF1p and ORF2p and expression of both proteins are critical for the process of retrotransposition. Here we have analyzed L1 ORF1p expression in a small cohort (n = 15) of paired cancer-normal tissues obtained from operated oral cancer patients. Immunohistochemistry (IHC) with the human ORF1 antibody showed the presence of ORF1p in almost 60%  cancer samples, and few of them also showed aberrant p53 expression.  Investigating L1 promoter methylation status, showed certain trends towards hypomethylation of the L1 promoter in cancer tissues compared to its normal counterpart. Our data raise the possibility that L1ORF1p expression might have some role in the onset and progression of this particular type of cancer.

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BACKGROUND: Recent reports indicate that retrotransposons - a type of mobile DNA - can contribute to neuronal genetic diversity in mammals. Retrotransposons are genetic elements that mobilize via an RNA intermediate by a "copy-and-paste" mechanism termed retrotransposition. Long Interspersed Element-1 (LINE-1 or L1) is the only active autonomous retrotransposon in humans and its activity is responsible for ~ 30% of genomic mass. Historically, L1 retrotransposition was thought to be restricted to the germline; however, new data indicate L1 s are active in somatic tissue with certain regions of the brain being highly permissive. The functional implications of L1 insertional activity in the brain and how host cells regulate it are incomplete. While deep sequencing and qPCR analysis have shown that L1 copy number is much higher in certain parts of the human brain, direct in vivo studies regarding detection of L1-encoded proteins is lacking due to ineffective reagents. RESULTS: Using a polyclonal antibody we generated against the RNA-binding (RRM) domain of L1 ORF1p, we observe widespread ORF1p expression in post-mortem human brain samples including the hippocampus which has known elevated rates of retrotransposition. In addition, we find that two brains from different individuals of different ages display very different expression of ORF1p, especially in the frontal cortex. CONCLUSIONS: We hypothesize that discordance of ORF1p expression in parts of the brain reported to display elevated levels of retrotransposition may suggest the existence of factors mediating post-translational regulation of L1 activity in the human brain. Furthermore, this antibody reagent will be useful as a complementary means to confirm findings related to retrotransposon biology and activity in the brain and other tissues in vivo.