Biology subjects

Kundu, J.

Publications and source records attributed to Kundu, J..

6 recordsLinked to original sources

bioRxiv2025

C-Nucleosides Stabilize RNA by Reducing Nucleophilicity at 2'-OH

Nucleotides with carbon substitution for heteroatoms are common in biological and therapeutic RNAs. Important examples include the C-nucleosides pseudouridine and N1-methyl-pseudouridine; these modifications were reported to slow degradation of large RNAs, but the mechanism is unknown. We measured kinetics of spontaneous and enzymatic cleavage at a single bond of synthetically modified RNAs, and find that carbon substitution markedly reduces strand cleavage rates in RNA by both mechanisms. Studies of nucleophilic acylation reactions of RNAs and of small alcohols of varied pKa suggest that reduced inductive effects resulting from carbon substitution for electronegative atoms results in both higher pKa and lower nucleophilicity. The results provide insight into native transcriptome modifications as well as RNA therapies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=157 SRC="FIGDIR/small/666442v2_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@73c194org.highwire.dtl.DTLVardef@1aa0af7org.highwire.dtl.DTLVardef@b5f8a0org.highwire.dtl.DTLVardef@d601fa_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOTOC GraphicC_FLOATNO C_FIG SynopsisThis study reveals mechanisms by which modifications found in native and therapeutic RNAs enhance RNA stability. Carbon substitution for nitrogen or oxygen results in reduced 2'-OH nucleophilicity.

biophysics↗

bioRxiv2025

Modulation of TTR Gene Expression in the Eye using Modified Duplex RNAs

Small interfering RNAs (siRNAs) are a proven therapeutic approach for controlling gene expression in the liver. Expanding the clinical potential of RNA interference (RNAi) requires developing strategies to enhance delivery to extra-hepatic tissues. In this study we examine inhibiting transthyretin (TRR) gene expression by short interfering RNAs (siRNAs) in the eye. Anti-TTR siRNAs have been developed as successful drugs to treat TTR amyloidosis. When administered systemically, anti-TTR siRNAs alleviate symptoms by blocking TTR expression in the liver. However, TTR amyloidosis also affects the eye, suggesting a need for reducing ocular TTR gene expression. Here, we demonstrate that C5 and 2-O-linked lipid-modified siRNAs formulated in saline can inhibit TTR expression in the eye when administered locally by intravitreal (IVT) injection. Modeling suggests that length and accessibility of the lipid chains contributes to in vivo silencing. GalNAc modified anti-dsRNAs also inhibit TTR expression, albeit less potently. These data support lipid modified siRNAs as an approach to treating the ocular consequences of TTR amyloidosis. Inhibition of TTR expression throughout the eye demonstrates that lipid-siRNA conjugates have the potential to be a versatile platform for ocular drug discovery. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/642595v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1e9ad27org.highwire.dtl.DTLVardef@67067borg.highwire.dtl.DTLVardef@1da7eadorg.highwire.dtl.DTLVardef@1b2a552_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Phosphoglucomutase A mediated regulation of carbon flux is essential for antibiotic and disease persistence in Mycobacterium tuberculosis

The long-term survival of Mtb mandates judicious utilization of the available resources inside the host. Uninterrupted access to host-derived nutrients holds the key to the success of Mtb. Phosphoglucomutase enzyme besides synthesizing glycogen, which serves as a nutrient reservoir, also helps modulate the carbon flux in different pathogens. Studies on the role of glycogen metabolism in disease progression, reactivation, and drug susceptibility in tuberculosis are severely lacking. To investigate this, we generated an Mtb strain ({Delta}pgmA) devoid of the gene that encodes for the enzyme phosphoglucomutase A (pgmA). The absence of pgmA impedes the ability of the pathogen to survive under nutrient-limiting and reactivation conditions. In the current study, we demonstrate that the absence of cell membrane-associated glycolipids in {Delta}pgmA compromised the cell wall integrity and increased the susceptibility of {Delta}pgmA to various stresses. Interestingly, in comparison to the wild type, low cAMP levels in {Delta}pgmA imparted an enhanced growth phenotype on cholesterol. Differential gene expression and carbon flux analysis suggest that stored carbon in the form of glycogen is essential for the survival of Mtb under nutrient-limiting conditions. Finally, we demonstrate that the pgmA gene of Mtb is essential for the growth of Mtb inside the host. Overall, this study unveils the significance of pgmA-mediated regulation of membrane glycolipids and its implication on antibiotic and disease persistence in tuberculosis. Additionally, information derived from this study will help design anti-TB strategies that are novel, short, and more efficient.

microbiology↗

bioRxiv2022

Evaluation of ERIC-PCR and MALDI-TOF as typing tools for multidrug resistant Klebsiella pneumoniae clinical isolates from a tertiary care center in India

Background and AimMultidrug resistant Klebsiella pneumoniae is associated with nosocomial infections in both outbreak and non-outbreak situations. The study intends to evaluate the potential of enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR), a genomic based typing and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) proteomic-based typing techniques for clonal relatedness among multidrug resistant Klebsiella pneumoniae isolates. MethodologyMultidrug resistant clinical isolates of Klebsiella pneumoniae (n =137) were collected from March 2019 to February 2020. Identification and protein-based phylogenetic analysis were performed by MALDI-TOF MS. Genomic typing was done by ERIC-PCR and analyzed by an online data analysis service (PyElph). Dice method with unweighted pair group method with arithmetic mean (UPGMA) program was used to compare the ERIC profiles. The samples were also evaluated by PCR for the presence of genes encoding carbapenemases, extended spectrum beta lactamases (ESBLs) and mobile colistin resistance-1 (mcr1). Result and ConclusionIsolates were typed into 40 ERIC types, and six groups by MALDI-TOF-MS. PCR-based analysis revealed that all the strains harbored two or more ESBL and carbapenemase genes. None of the isolates revealed the presence of the plasmid mediated mcr-1 gene for colistin resistance. The study presents ERIC based typing as more robust in comparison to MALDI-TOF for finding the clonal relatedness in epidemiological studies.

microbiology↗

bioRxiv2021

Self-transfecting GMO-PMO and PMO-GMO chimeras enable gene silencing in vitro and in vivo zebrafish model and NANOG Inhibition Induce the Apoptosis in Breast and Prostate Cancer Cells

Phosphorodiamidate Morpholino Oligonucleotides (PMOs)-based antisense reagents cannot enter inside cells by itself without the help of any delivery technique which is the last hurdle for their clinical applications. To overcome this limitation, a self-transfecting GMO-PMO or PMO-GMO chimeras has been explored as a gene silencing reagent where GMO stands for guanidinium morpholino oligonucleotides which linked either at the OH- or NH-end of PMOs. GMO not only facilitates cellular internalization of such chimeras but also participates in Watson-Crick base pairing during gene silencing in ShhL2 cells when designed against mGli1 and compared with scrambled GMO-PMO where mutations were made only to the GMO part. GMO-PMO-mediated knockdown of no tail gene resulted no tail-dependent phenotypes in zebrafish and worked even after the delivery at 16-, 32- and 64-cell stages which were previously unachievable by regular PMO. Furthermore, GMO-PMO chimeras has shown the inhibition of NANOG, a key regulator of self-renewal and pluripotency of both embryonic and cancer stem cells. Its inhibition influences on the expression of other cancer related proteins and the respective phenotypes in breast cancer cells and increases the therapeutic potential of taxol. To the best of our knowledge, this is the first report on the self-transfecting antisense reagents since the discovery of guanidinium linked DNA (DNG) and most effective among the all cell-penetrating PMOs reported till date expected to solve the longstanding problem of PMO delivery. In principle, this technology could be useful for the inhibition of any target gene without using any delivery vehicle and should have applications in the fields of antisense therapy, diagnostic and nanotechnology area. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/447039v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@1374f3forg.highwire.dtl.DTLVardef@33ca57org.highwire.dtl.DTLVardef@292959org.highwire.dtl.DTLVardef@599b5f_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2020

Proteomic profiling of peripheral blood mononuclear cells isolated from patients with tuberculosis and diabetes copathogenesis -A pilot study

BackgroundDiabetes is an important risk factor for developing tuberculosis. This association leads to exacerbation of tuberculosis symptoms and delayed treatment of both the diseases. Molecular mechanism and biomarkers/drug targets related to copathogenesis of tuberculosis and diabetes, however, still remains to be poorly understood. In this study, proteomics based 2D-MALDI/MS approach was employed to identify host signature proteins which are altered during copathogenesis of tuberculosis and diabetes. MethodsComparative proteome of human peripheral blood mononuclear cells (PBMCs) from healthy controls, tuberculosis and diabetes patients in comparison to comorbid diabetes and tuberculosis patients was analyzed. Gel based proteomics approach followed by in gel trypsin digestion and peptide identification by mass spectrometry was used for signature protein identification. ResultsTotal of 18 protein spots with differential expression in TBDM patients in comparison to other groups were identified. These include Vimentin, tubulin beta chain protein, superoxide dismutase, Actin related protein 2/3 complex subunit 2, PDZ LIM domain protein, Rho-GDP dissociation inhibitor, Ras related protein Rab, dCTPpyrophosphatase 1, Transcription initiation factor TFIID subunit 12, coffilin 1, three isoforms of Peptidylprolylcis-trans isomerase A, three isoforms of Protein S100A9, Protein S100A8 and SH3 domain containing protein. These proteins belonged to four functional categories i.e. structural, cell cycle/growth regulation, signaling and intermediary metabolism. ConclusionProteins identified to be differentially expressed in TBDM patient can act as potent biomarkers and as predictors for copathogenesis of tuberculosis and diabetes.

molecular biology↗