Biology subjects

Kaledhonkar, S.

Publications and source records attributed to Kaledhonkar, S..

5 recordsLinked to original sources

bioRxiv2025

LoAR-Exo and HiAR-Exo: One-step label-free isolation of extracellular vesicles using inertial microfluidic devices

Extracellular vesicles (EVs) are lipid membrane-bound nanoscale (20 nm - 1000 nm) objects shed by all cells. As EVs play an important role in intercellular signaling, these have emerged as promising biomarkers for many diseases including cancer and neurodegenerative disorders. A major bottleneck in research into EVs and their smooth translation to clinic as disease biomarkers is a lack of access to affordable and user-friendly technologies to quickly isolate EVs from complex biological and clinical samples with acceptable purity and yield. Here, we report two different designs of inertial microfluidic devices (e.g. LoAR-Exo and HiAR-Exo) that can isolate EVs from different kinds of biological samples in a single label-free step using a spiral microchannel design with different aspect ratios. LoAR-Exo has a height-to-width aspect ratio [~] 1, while HiAR-Exo has a height-to-width aspect ratio of 2.5, optimized by COMSOL simulations. We tested both designs by separating polystyrene particles of size < 1 m from a heterogeneous mixture. We then benchmarked the performance of the microfluidic chips against ultracentrifugation and a precipitation kit by isolating EVs from the cell culture-conditioned media (CCM) of MDA-MB-231 cells. We also compared the microchip with SEC by using the CCM from H1975/OR cells. The size distribution of EVs isolated by the microfluidic chip was comparable with ultracentrifugation, precipitation and SEC. In summary, both devices isolated EVs from as little as 1 mL of sample volume using a label-free technique in a continuous manner and without any user intervention. Both microfluidic platforms offer a simple, efficient, and scalable alternative to conventional methods for EV isolation.

bioengineering↗

bioRxiv2024

In Silico Identification of Potential Inhibitors of Mycobacterium tuberculosis DNA Gyrase from Phytoconstituents of Indian Medicinal Plants

DNA gyrase, the sole type-II topoisomerase in Mycobacterium tuberculosis, plays a significant role in the bacterias survival by catalyzing the DNA topology change and is considered a crucial drug target. The emergence of drug resistance in Mycobacterium tuberculosis specifically against the second line of drugs; fluoroquinolones, targeting DNA gyrase, demands new potential compounds that would efficiently interact with it. To find a common suitable inhibitor for the apo and mutated forms of DNA gyrase, in this study, the compounds from an Indian medicinal plant database were screened for selecting potential drug-like molecules and two compounds IM5 and IM7 were selected after successful virtual screening and hit optimization with ADMET predictions, and chosen for MD simulation. The MMPBSA analysis of binding free energy validates the docking results of the two molecules. The principal component analysis also supported the stability of the complexes and thus, these two molecules have turned out to be promising candidates against Mycobacterium tuberculosis. Overall, this work sheds light on the potential of DNA gyrase inhibitors as therapeutic agents for Mycobacterium tuberculosis treatment, and IM5 and IM7 showed promise as future research compounds.

bioinformatics↗

bioRxiv2024

Conformational dynamics associated with remote residues regulate the kinetic properties of homologous glutamate dehydrogenases (GDHs)

Glutamate dehydrogenase (GDH) is a key enzyme in all living organisms and some of the GDHs exhibit substrate-dependent homotropic cooperativity. However, the mode of allosteric communication during the homotropic effect in GDHs remains poorly understood. In this study, we examined two homologous GDHs, Aspergillus niger GDH (AnGDH) and Aspergillus terreus GDH (AtGDH), with differing substrate utilization kinetics to uncover the factors driving their distinct behavior. The crystal structures and first-ever cryo-EM structures of apo-AtGDH captured arrays of conformational ensembles. Comparative structural analysis has revealed a wider mouth opening in allosteric AnGDH ([~] 21 [A]) compared to non-allosteric AtGDH ([~]17 [A]) in their apo states. A network of interaction related to the amino acid substitutions in Domain II is responsible for differential structural dynamics in these GDHs. Remarkably, we identified one remotely located substitution in Domain II, i.e., R246 to S, a part of the network, which reversed the kinetic properties of AtGDH into an allosteric one and controls the mouth opening. Our data also indicate that dynamic discrepancy influences the substrate binding affinity and catalytic activity in AnGDH and AtGDH. We have successfully demonstrated for the first time, that remotely located residues and the conformational dynamics regulate the kinetic properties in homologous GDHs.

biochemistry↗

bioRxiv2018

The structural basis for release factor activation during translation termination revealed by time-resolved cryogenic electron microscopy

When the mRNA translating ribosome encounters a stop codon in its aminoacyl site (A site), it recruits a class-1 release factor (RF) to induce hydrolysis of the ester bond between peptide chain and peptidyl-site (P-site) tRNA. This process, called termination of translation, is under strong selection pressure for high speed and accuracy. Class-1 RFs (RF1, RF2 in bacteria, eRF1 in eukarya and aRF1 in archaea), have structural motifs that recognize stop codons in the decoding center (DC) and a universal GGQ motif for induction of ester bond hydrolysis in the peptidyl transfer center (PTC) 70 [A] away from the DC. The finding that free RF2 is compact with only 20 [A] between its codon reading and GGQ motifs came therefore as a surprise1. Cryo-electron microscopy (cryo-EM) then showed that ribosome-bound RF1 and RF2 have extended structures2,3, suggesting that bacterial RFs are compact when entering the ribosome and switch to the extended form in a stop signal-dependent manner3. FRET4, cryo-EM5,6 and X-ray crystallography7, along with a rapid kinetics study suggesting a pre-termination conformational change on the millisecond time-scale of ribosome-bound RF1 and RF28, have lent indirect support to this proposal. However, direct experimental evidence for such a short-lived compact conformation on the native pathway to RF-dependent termination is missing due to its transient nature. Here we use time-resolved cryo-EM9,10,11,12,13 to visualize compact and extended forms of RF1 and RF2 at 3.5 and 4 [A] resolution, respectively, in the codon-recognizing complex on the pathway to termination. About 25% of ribosomal complexes have RFs in the compact state at 24 ms reaction time after mixing RF and ribosomes, and within 60 ms virtually all ribosome-bound RFs are transformed to their extended forms.

biophysics↗

bioRxiv2018

Real-time structural dynamics of late steps in bacterial translation initiation visualized using time-resolved cryogenic electron microscopy

Bacterial translation initiation entails the tightly regulated joining of the 50S ribosomal subunit to an initiator transfer RNA (fMet-tRNAfMet)-containing 30S ribosomal initiation complex (IC) to form a 70S IC that subsequently matures into a 70S elongation-competent complex (70S EC). Rapid and accurate 70S IC formation is promoted by 30S IC-bound initiation factor (IF) 1 and the guanosine triphosphatase (GTPase) IF2, both of which must ultimately dissociate from the 70S IC before the resulting 70S EC can begin translation elongation1. Although comparison of 30S2-6 and 70S5,7-9 IC structures have revealed that the ribosome, IFs, and fMet-tRNAfMet can acquire different conformations in these complexes, the timing of conformational changes during 70S IC formation, structures of any intermediates formed during these rearrangements, and contributions that these dynamics might make to the mechanism and regulation of initiation remain unknown. Moreover, lack of an authentic 70S EC structure has precluded an understanding of ribosome, IF, and fMet-tRNAfMet rearrangements that occur upon maturation of a 70S IC into a 70S EC. Using time-resolved cryogenic electron microscopy (TR cryo-EM)10 we report the first, near-atomic-resolution view of how a time-ordered series of conformational changes drive and regulate subunit joining, IF dissociation, and fMet-tRNAfMet positioning during 70S EC formation. We have found that, within ~20-80 ms, rearrangements of the 30S subunit and IF2, uniquely captured in its GDP*Pi-bound state, stabilize fMet-tRNAfMet in its intermediate, 70S P/I, configuration7 and trigger dissociation of IF1 from the 70S IC. Within the next several hundreds of ms, dissociation of IF2 from the 70S IC is coupled to further remodeling of the ribosome that positions fMet-tRNAfMet into its final, P/P, configuration within the 70S EC. Our results demonstrate the power of TR cryo-EM to determine how a time-ordered series of conformational changes contribute to the mechanism and regulation of one of the most fundamental processes in biology.

molecular biology↗