https://dspace.nii.res.in//https://dspace.nii.res.in/handle/123456789/9 2026-04-27T14:16:07Z 2026-04-27T14:16:07Z Kushwaha, Sachin Jawahar, Varsha Kumar, Ajay Griffin, Lauren Rothstein, Thomas L Sehgal, Devinder Khan, Naeem https://dspace.nii.res.in//https://dspace.nii.res.in/handle/123456789/1591 2025-08-12T04:51:18Z 2025-01-01T00:00:00Z

Title: Complete primer set for amplification and expression of full-length recombinant human monoclonal antibodies from single human B cells Authors: Kushwaha, Sachin; Jawahar, Varsha; Kumar, Ajay; Griffin, Lauren; Rothstein, Thomas L; Sehgal, Devinder; Khan, Naeem Abstract: Human monoclonal antibodies (mAbs) are an important segment in precision therapeutics. Various methodologies are available for generating them. Recombinant human mAbs expression from sorted single B cells is preferred for its rapid expression using mammalian vectors while maintaining in vivo immunoglobulin (Ig) pairing. The success rate of generating recombinant mAbs from single sorted human B cells directly relies on Ig heavy (IgH) and light (IgL) gene coverage of the PCR primers. Existing primer sets fail to cover all functional human Ig gene rearrangements, exhibit high degeneracy leading to non-specific amplifications and mutations arising from primer mismatch/degeneracy, and require high amplification cycles. Some existing primer sets have high coverage but are not designed for expression as recombinant mAbs. Here, we have designed a primer set to amplify all functional V(D)J transcripts in human B cell repertoire using a nested RT-PCR approach. The resultant amplicons can be cloned into mammalian vectors for expression of recombinant mAb. Non-specific amplifications were minimized using isotype-specific primers for cDNA synthesis and limiting primer degeneracy. We validated the designed primers on single sorted B cells, bulk sorted B cells and peripheral blood mononuclear cells. We were successfully able to amplify paired heavy and light chain transcripts in 38.46 % (80/208) from naive, memory and B1 B cell subsets sorted as single B cells. Paired Ig transcripts from five single B cells were cloned into expression vectors and purified from mammalian cells as recombinant mAbs. Thus, our new primer set offers significant advantages over existing primers as it allows amplification of all functional V(D)J rearrangements, facilitating rapid generation of antigen-specific recombinant antibodies from diverse human B cell repertoires following vaccinations and infections previously inaccessible due to primer limitations.

2025-01-01T00:00:00Z Nag, Snehasish Mandal, Samanwita Mukherjee, Oindrila Majumdar, Tanmay Mukhopadhyay, Satinath Kundu, Rakesh https://dspace.nii.res.in//https://dspace.nii.res.in/handle/123456789/1560 2025-12-05T12:47:23Z 2024-01-01T00:00:00Z

Title: Vildagliptin inhibits high fat and fetuin-A mediated DPP-4 expression, intracellular lipid accumulation and improves insulin secretory defects in pancreatic beta cells Authors: Nag, Snehasish; Mandal, Samanwita; Mukherjee, Oindrila; Majumdar, Tanmay; Mukhopadhyay, Satinath; Kundu, Rakesh Abstract: Dipeptidyl peptidase-4 (DPP-4), a ubiquitous proteolytic enzyme, inhibits insulin secretion from pancreatic beta cells by inactivating circulating incretin hormones GLP-1 and GIP. High circulating levels of DPP-4 is presumed to compromise insulin secretion in people with type 2 diabetes (T2D). Our group recently reported lipid induced DPP-4 expression in pancreatic beta cells, mediated by the TLR4-NFkB pathway. In the present study, we looked at the role of Vildagliptin on pancreatic DPP-4 inhibition, preservation of islet mass and restoration of insulin secretion. MIN6 mouse insulinoma cells incubated with palmitate and fetuin-A, a proinflammatory organokine associated with insulin resistance, showed activation of TLR4-NFkB pathway, which was rescued on Vildagliptin treatment. In addition, Vildagliptin, by suppressing palmitate-fetuin-A mediated DPP-4 expression in MIN6, prevented the secretion of IL-1beta and fetuin-A in the culture media. DPP-4 siRNA abrogated TLR4-NFkB pathway mediated islet cell inflammation. Vildagliptin also reduced palmitate-fetuin-A mediated intracellular lipid accumulation in MIN6 and isolated islets from high fat fed (HFD) mice as observed by Oil O Red staining with downregulation of CD36 and PPARgamma. Vildagliptin also preserved islet mass and rescued insulin secretory defect in HFD mice. Our results suggest that inhibition of DPP-4 by Vildagliptin protects pancreatic beta cells from the deleterious effects of lipid and fetuin-A, preserves insulin secretory functions and improves hyperglycemia.

2024-01-01T00:00:00Z Mani, Shailendra Kaur, Anupamjeet Jakhar, Kamini Kumari, Geetika Sonar, Sudipta Kumar, Amit Das, Sudesna Kumar, Santosh Kumar, Vijay Kundu, Rakesh Pandey, Anil Kumar Singh, Umesh Prasad Majumdar, Tanmay https://dspace.nii.res.in//https://dspace.nii.res.in/handle/123456789/1558 2025-12-05T12:48:27Z 2023-01-01T00:00:00Z

Title: Targeting DPP4-RBD interactions by sitagliptin and linagliptin delivers a potential host-directed therapy against pan-SARS-CoV-2 infections Authors: Mani, Shailendra; Kaur, Anupamjeet; Jakhar, Kamini; Kumari, Geetika; Sonar, Sudipta; Kumar, Amit; Das, Sudesna; Kumar, Santosh; Kumar, Vijay; Kundu, Rakesh; Pandey, Anil Kumar; Singh, Umesh Prasad; Majumdar, Tanmay Abstract: Highly mutated SARS-CoV-2 is known aetiological factor for COVID-19. Here, we have demonstrated that the receptor binding domain (RBD) of the spike protein can interact with human dipeptidyl peptidase 4 (DPP4) to facilitate virus entry, in addition to the usual route of ACE2-RBD binding. Significant number of residues of RBD makes hydrogen bonds and hydrophobic interactions with α/β-hydrolase domain of DPP4. With this observation, we created a strategy to combat COVID-19 by circumventing the catalytic activity of DPP4 using its inhibitors. Sitagliptin, linagliptin or in combination disavowed RBD to establish a heterodimer complex with both DPP4 and ACE2 which is requisite strategy for virus entry into the cells. Both gliptins not only impede DPP4 activity, but also prevent ACE2-RBD interaction, crucial for virus growth. Sitagliptin, and linagliptin alone or in combination have avidity to impede the growth of pan-SARS-CoV-2 variants including original SARS-CoV-2, alpha, beta, delta, and kappa in a dose dependent manner. However, these drugs were unable to alter enzymatic activity of PLpro and Mpro. We conclude that viruses hijack DPP4 for cell invasion via RBD binding. Impeding RBD interaction with both DPP4 and ACE2 selectively by sitagliptin and linagliptin is an potential strategy for efficiently preventing viral replication.

2023-01-01T00:00:00Z Kumar, Ashutosh Sarkar, Tamal Kumar, Robin Panda, Amulya K Solanki, Pratima R https://dspace.nii.res.in//https://dspace.nii.res.in/handle/123456789/1555 2025-12-05T12:48:17Z 2023-01-01T00:00:00Z

Title: Electrochemical Detection of Vibrio cholerae by Amine Functionalized Biocompatible Gadolinium Oxide Nanoparticles Authors: Kumar, Ashutosh; Sarkar, Tamal; Kumar, Robin; Panda, Amulya K; Solanki, Pratima R Abstract: Herein, we report the biocompatible amine-functionalized gadolinium oxide nanoparticles (Gd2O3 NPs) for the possibility of electrochemical detection of Vibrio cholerae (Vc) cells. The microwave irradiation process is applied to synthesize Gd2O3 NPs. The amine (NH2) functionalization is carried out via overnight stirring with 3(Aminopropyl)triethoxysilane (APTES) at 55 °C. The size of NPs amine functionalized APETS@Gd2O3 NPs are determined by transmission electron microscopy (TEM). APETS@Gd2O3 NPs are further electrophoretically deposited onto indium tin oxide (ITO) coated glass substrate to obtain working electrode surface. The monoclonal antibodies (anti-CT) specific to cholera toxin associated to Vc cells are covalently immobilized onto the above electrodes using EDC-NHS chemistry and further BSA is added to obtain the BSA/anti-CT/APETS@Gd2O3/ITO immunoelectrode. Further, this immunoelectrode shows the response for cells in CFU range from 3.125 × 106 to 30 × 106 and is very selective with sensitivity and LOD 5.07 mA CFUs mL cm-2 and 0.9375 × 106 CFU respectively. To establish a future potential for APTES@Gd2O3 NPs in field of biomedical applications and cytosensing, the effect of APTES@Gd2O3 NPs on mammalian cells is also observed using in vitro cytotoxicity assay and cell cycle analysis.

2023-01-01T00:00:00Z