Biology subjects

Shrestha, U.

Publications and source records attributed to Shrestha, U..

4 recordsLinked to original sources

bioRxiv2022

Identification and Characterization of Probiotics Isolates from Indigenous Chicken (Gallus domesticus) of Nepal

BackgroundExcessive and irrational use of antibiotics as growth promoters in poultry has been one of key factors contributing to increased emergence of antibiotics resistant bacteria. Drug resistant infections are becoming major concerns in poultry production impacting both human and poultry health. Several alternatives for antibiotic growth promoters are being sought, and the search for effective probiotics to be used as feed additives is amongst the promising ones. Our study aimed to isolate and test potential probiotics bacteria from cloacal swabs of various indigenous chicken (Gallus domesticus) breeds from rural outskirts of the Kathmandu valley (Nepal). MethodsSelective isolation of probiotics was conducted by micro-aerophilic enrichment of sample in MRS Broth at 37{degrees}C, followed by culturing on MRS agar supplemented with 5 g/L of CaCO3. Isolated bacterial colonies producing transparent halo were selected as potential lactic acid bacteria (LAB), and tested for their antibacterial activity, phenotypic and biochemical characteristics, acidic yield, and tolerance to acid and bile. ResultsA total of 90 potential LAB were isolated from cloacal samples collected from 41 free-ranging chickens of indigenous breeds. Of these, 52 LAB isolates (57%) showed variable antibacterial activity to at least one bacterial pathogen. Of 52 LAB, 46 isolates fulfilled phenotypic and biochemical criteria of Lactobacillus spp. Of these, 37 isolates produced varying percentage yields of lactic acid, 27 isolates showed survival at pH 3.0, and 17 isolates showed survival tolerances in the presence of 0.3% and 0.5% bile salts for 24 hours. Phylogenetic analysis of 16SrDNA sequencing of LAB isolates fulfilling in vitro probiotics properties showed that 3 isolates had genetic identity of 99.38% with Lactobacillus plantarum, while one isolate was genetically similar (99.85%) with the clade of L. reuteri, L. antri and L. panis. ConclusionsOur study identified four Lactobacillus spp. strains having potential probiotics properties. Further investigations are needed to evaluate these isolates to be used as poultry probiotics feed supplement.

microbiology↗

bioRxiv2021

Atomic-Resolution Prediction of Degrader-mediated Ternary Complex Structures by Combining Molecular Simulations with Hydrogen Deuterium Exchange

Targeted protein degradation (TPD) has emerged as a powerful approach in drug discovery for removing (rather than inhibiting) proteins implicated in diseases. A key step in this approach is the formation of an induced proximity complex, where a degrader molecule recruits an E3 ligase to the protein of interest (POI), facilitating the transfer of ubiquitin to the POI and initiating the proteasomal degradation process. Here, we address three critical aspects of the TPD process: 1) formation of the ternary complex induced by a degrader molecule, 2) conformational heterogeneity of the ternary complex, and 3) assessment of ubiquitination propensity via the full Cullin Ring Ligase (CRL) macromolecular assembly. The novel approach presented here combines experimental biophysical data--in this case hydrogen-deuterium exchange mass spectrometry (HDX-MS, which measures the solvent exposure of protein residues)--with all-atom explicit solvent molecular dynamics (MD) simulations aided by enhanced sampling techniques to predict structural ensembles of ternary complexes at atomic resolution. We present results demonstrating the efficiency, accuracy, and reliability of our approach to predict ternary structure ensembles using the bromodomain of SMARCA2 (SMARCA2BD) with the E3 ligase VHL as the system of interest. The simulations reproduce X-ray crystal structures - including prospective simulations validated on a new structure that we determined in this work (PDB ID: 7S4E) - with root mean square deviations (RMSD) of 1.1 to 1.6 [A]. The simulations also reveal a structural ensemble of low-energy conformations of the ternary complex within a broad energy basin. To further characterize the structural ensemble, we used snapshots from the aforementioned simulations as seeds for Hamiltonian replica exchange molecular dynamics (HREMD) simulations, and then perform 7.1 milliseconds of aggregate simulation time using Folding@home. The resulting free energy surface identifies the crystal structure conformation within a broad low-energy basin and the dynamic ensemble is consistent with solution-phase biophysical experimental data (HDX-MS and small-angle x-ray scattering, SAXS). Finally, we graft structures from the ternary complexes onto the full CRL and perform enhanced sampling simulations, where we find that differences in degradation efficiency can be explained by the proximity distribution of lysine residues on the POI relative to the E2-loaded ubiquitin. Several of the top predicted ubiquitinated lysine residues are validated prospectively through a ubiquitin mapping proteomics experiment.

biophysics↗

bioRxiv2021

Functional plasticity coupled with structural predispositions in auditory cortex shape successful music category learning

Categorizing sounds into meaningful groups helps listeners more efficiently process the auditory scene and is a foundational skill for speech perception and language development. Yet, how auditory categories develop in the brain through learning, particularly for nonspeech sounds, is not well understood. Here, we asked musically naive listeners to complete a brief ([~]20 min) training session where they learned to identify sounds from a nonspeech continuum (minor-major 3rd musical intervals). We used multichannel EEG to track behaviorally relevant neuroplastic changes in the auditory event-related potentials (ERPs) pre- to post-training. To rule out mere exposure-induced changes, neural effects were evaluated against a control group of 14 nonmusicians who did not undergo training. We also compared individual categorization performance with structural volumetrics of bilateral primary auditory cortex (PAC) from MRI to evaluate neuroanatomical substrates of learning. Behavioral performance revealed steeper (i.e., more categorical) identification functions in the posttest that correlated with better training accuracy. At the neural level, improvement in learners behavioral identification was characterized by smaller P2 amplitudes at posttest, particularly over right hemisphere. Critically, learning-related changes in the ERPs were not observed in control listeners, ruling out mere exposure effects. Learners also showed smaller and thinner PAC bilaterally, indicating superior categorization was associated with structural differences in primary auditory brain regions. Collectively, our data suggest successful auditory categorical learning of nonspeech sounds is characterized by short-term functional changes (i.e., greater post-training efficiency) in sensory coding processes superimposed on preexisting structural differences in bilateral auditory cortex.

neuroscience↗

bioRxiv2020

Inactivation of p21-Activated Kinase 2 (Pak2) Inhibits the Development of Nf2-Deficient Malignant Mesothelioma

Malignant mesotheliomas (MM) show frequent somatic loss of the NF2 tumor suppressor gene. The NF2 product, Merlin, is implicated in several tumor-related pathways, including p21-activated kinase (PAK) signaling. Merlin is both a phosphorylation target for PAK and a negative regulator of this oncogenic kinase. Merlin loss results in PAK activation, and PAK inhibitors hold promise for the treatment of NF2-deficient tumors. To test this possibility in an in vivo genetic system, Nf2f/f;Cdkn2af/f mice were crossed to mice with conditional knockout of Pak2, a highly expressed group I Pak member. Cohorts of these animals were injected in either the thoracic or peritoneal cavities with adeno-Cre virus to delete floxed alleles in the mesothelial lining. Loss of Pak2 resulted in a markedly decreased incidence and delayed onset and progression of pleural and peritoneal MMs in Nf2;Cdkn2a-deficient (NC) mice, as documented by Kaplan-Meier survival curves and in vivo bioluminescent imaging. RNA-seq revealed that MMs from NC;Pak2-/- mice showed downregulated expression of genes involved in several oncogenic pathways (Wnt, Akt) when compared to MMs from mice retaining Pak2. Kinome profiling showed that, as compared to NC MM cells, NC;Pak2-/- MM cells had multiple kinase changes indicative of an epithelial to mesenchymal transition. Collectively, these findings suggest that NC;Pak2-/- MMs adapt by reprogramming their kinome and gene signature profiles to bypass the need for PAK activity via the activation of other compensatory oncogenic kinase pathways. The identification of such secondary pathways offers opportunities for rational combination therapies to circumvent resistance to anti-PAK drugs.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cancer biology↗