Biology subjects

Shrestha, D.

Publications and source records attributed to Shrestha, D..

11 recordsLinked to original sources

bioRxiv2026

Industrialization restructures the domestic dog gut microbiome while preserving host specificity

Industrialization has reshaped human gut microbiomes, but its effects on other human-associated mammals remain poorly understood. Domestic dogs provide an informative comparative system because they have shared human environments and food systems for millennia yet retain distinct host biology. However, most canine microbiome studies have focused on industrialized companion animals, limiting our understanding of the ecological range of the domestic dog gut microbiome. We analyzed fecal 16S rRNA gene profiles from 261 dogs sampled across Nepal, Thailand, the United Arab Emirates, and the United States, spanning forager, agrarian, pastoralist, urban, and industrialized lifestyles; 257 dogs remained after excluding recent antibiotic exposure. Lifestyle was the strongest measured correlate of canine gut microbiome composition, and this structure persisted in restricted analyses of mature, non-shelter dogs sampled from temperate climate regions. Industrialized dogs differed from non-industrialized dogs through directional genus-level turnover, restructuring of VANISH- and BloSSUM-like microbial guilds, and shifts in predicted functional potential. Non-industrialized dogs were not microbiologically uniform: pastoralist dogs carried non-industrialized microbiome profiles but diverged from a simple forager-to-industrialized continuum. Cross-species comparisons with humans sampled across matched lifestyle categories showed parallel lifestyle-associated restructuring in both hosts, but host species remained the dominant axis of variation and the genera responding to industrialization were largely host-specific. These findings expand the ecological baseline for the domestic dog gut microbiome and identify industrialization as a major axis of microbiome restructuring in a long-term human-associated mammal. More broadly, they show that shared lifestyle transitions can impose parallel ecological pressures across host species without overriding host-specific community assembly.

microbiology↗

bioRxiv2026

Distinct Proteasomal Pathways Drive Oncogenic PPM1D Activation

PPM1D is a serine/threonine phosphatase and DNA damage response (DDR) regulator recurrently activated in cancer through amplification or C-terminal truncating mutations that increase its abundance. Here we show that truncating mutations fundamentally rewire PPM1D proteostasis, unmasking an oncogenic function of an alternative protein degradation pathway. While full-length PPM1D undergoes rapid ubiquitin-independent proteasomal degradation via a C-terminal degron, truncating mutations redirect degradation to a slower UBR5-mediated ubiquitin-dependent pathway. The resulting accumulation of PPM1D suppresses DDR signaling and enhances cellular fitness under genotoxic stress, which is further amplified by UBR5 loss. Consistent with selective pressure on this axis, cancers harboring PPM1D truncating mutations are enriched for UBR5 loss-of-function mutations. Together, these findings identify escape from ubiquitin-independent proteasomal degradation as a mechanism of oncogenic adaptation and establish proteostatic routing as a regulatory layer linking protein degradation, DDR signaling, and cancer evolution.

cell biology↗

bioRxiv2025

Exploring the Role of Kv1.3 and MAPK14 in Mediating Microglial Oxidative Stress and Neuroinflammation Following Organic Dust Exposure

BackgroundAgricultural contaminants, including inhaled organic dust (OD) and gases, are known to cause inflammation in the lungs and the brain. We investigated the role of the potassium channel Kv1.3 in organic dust (OD)-induced neuroinflammation models. Kv1.3 channels play a multifaceted role in microglial immune modulation, cancer, neurodegenerative diseases, and constitute a potential therapeutic target. MethodsWe used in vivo (C57BL/6 mice), in vitro (microglial cell line, primary microglia), and ex vivo (brain slice culture) models of OD-induced neuroinflammation. A sterile OD extract (ODE) was prepared, and mice were exposed to either normal saline or ODE intra-nasally for 5 weeks (5 days/week) to simulate an occupational exposure scenario. Primary microglia were isolated from neonatal mice for total RNA sequencing (RNA-seq). The ODE-induced expression of Kv1.3 was quantified using in vitro and ex vivo models with and without PAP-1 treatments. Exposure-induced changes in cytokines and reactive species markers were measured. Using western blot, we quantified phosphorylated p38 MAPK14 (p-p38 MAPK) and NOX2. We measured the currents through Kv1.3 channels using a microglial patch-clamp assay. ResultsExposure to ODE increased the expression of Kv1.3 and p-p38 MAPK in mouse microglia without affecting the Kv1.3 currents at the cell surface. Exposure increased the levels of inflammatory cytokines and NOX2. Kv1.3 inhibition with PAP-1 decreased inflammatory markers (TNF- and IL-6), levels of Kv1.3, p-p38 MAPK, NOX2, and nitrites. ConclusionOur study revealed that pharmacological inhibition of Kv1.3 potassium channels reduces ODE-induced neuroinflammation by decreasing inflammatory and oxidative stress markers.

pharmacology and toxicology↗

bioRxiv2022

Bacterial mobility and motility in porous media mimicked by microspheres

Bacterial motion in porous media are essential for their survival, proper functioning, and various applications. Here we investigated the motion of Escherichia coli bacteria in microsphere-mimicked porous media. We observed reduced bacterial velocity and enhanced directional changes of bacteria as the density of microspheres increased, while such changes happened mostly around the microspheres and due to the collisions with the microspheres. More importantly, we established and quantified the correlation between the bacterial trapping in porous media and the geometric confinement imposed by the microspheres. In addition, numerical simulations showed that the active Brownian motion model in the presence of microspheres resulted in bacterial motion that are consistent with the experimental observations. Our study suggested that it is important to distinguish the ability of bacteria to move easily - bacterial mobility - from the ability of bacteria to move independently - bacteria motility. Our results showed that bacterial motility remains similar in porous media, but bacterial mobility was significantly affected by the pore-scale confinement.

biophysics↗

bioRxiv2021

Parallel functional testing identifies enhancers active in early postnatal mouse brain

Enhancers are cis-regulatory elements that play critical regulatory roles in modulating developmental transcription programs and driving cell-type specific and context-dependent gene expression in the brain. The development of massively parallel reporter assays (MPRAs) has enabled high-throughput functional screening of candidate DNA sequences for enhancer activity. Tissue-specific screening of in vivo enhancer function at scale has the potential to greatly expand our understanding of the role of non-coding sequences in development, evolution, and disease. Here, we adapted a self-transcribing regulatory element MPRA strategy for delivery to early postnatal mouse brain via recombinant adeno-associated virus (rAAV). We identified and validated putative enhancers capable of driving reporter gene expression in mouse forebrain, including regulatory elements within an intronic CACNA1C linkage disequilibrium block associated with risk in neuropsychiatric disorder genetic studies. Paired screening and single enhancer in vivo functional testing, as we show here, represents a powerful approach towards characterizing regulatory activity of enhancers and understanding how enhancer sequences organize gene expression in normal and pathogenic brain development.

neuroscience↗

bioRxiv2020

Organic dust induced mitochondrial dysfunction could be targeted via cGAS-STING or mitochondrial NOX-2 inhibition

ABSTRACTOrganic dust (OD) exposure in animal production industries poses serious respiratory and other health risks. OD consists of microbial products and particulate matter and OD exposure induced respiratory inflammation is under intense investigation. However, the effect of OD exposure on brain largely remains unknown. Recently, we have shown that OD exposure of brain microglial cells induces an inflammatory phenotype with the release of mitochondrial DNA (mt-DNA). Therefore, we tested a hypothesis that OD-exposure induced secreted mt-DNA signaling drives the inflammation. OD samples were collected from commercial swine operations and a filter sterilized OD extract (ODE) was prepared. Mouse (C57BL/6) microglial cell line was treated with medium or ODE (5%) for 48 hours along with either PBS or mitoapocynin (MA, 10 μM, NOX-2 inhibitor). Microglia treated with control or anti-STING siRNA were exposed to medium or ODE. Next, mouse (C57BL/6) pups were euthanized under an approved protocol, organotypic brain slice cultures (BSCs) were prepared and exposed to medium or ODE with or without MA treatment daily for five days. Culture supernatant, cell pellets and mt-free cytosolic fractions were processed to quantify mt-superoxide, mt-DNA, cytochrome C, TFAM, mitochondrial stress markers and mt-DNA induced signaling via cGAS-STING and TLR9. Data were analyzed using one-way ANOVA and post-hoc tests. A p value of ≤ 0.05 was considered significant. ODE exposure increased the mt-superoxide formation andMA treatment decreased the ODE-induced mt-DNA release into cytosol. ODE exposure increased the cytochrome C and TFAM levels. ODE increased MFN1/2 and PINK1 but not DRP1 and MA treatment decreased the MFN2 expression. MA treatment decreased the ODE-exposure induced mt-DNA signaling via cGAS-STING and TLR9. Anti-STING siRNA decreased the ODE-induced increase in IRF3, IFN-β and Iba1 expression. In BSCs, MA-treatment decreased the ODE induced TNF-α, IL-6 and MFN1. Taken together, OD exposure induced mt-DNA signaling could be curtailed through mitochondrial NOX-2 inhibition or STING suppression to reduce neuroinflammation.Competing Interest StatementAGK has an equity interest in PK Biosciences Corporation located in Ames, IA. The terms of this arrangement have been reviewed and approved by Iowa State University per its conflict of interest policies. All other authors have declared no potential conflicts of interest.View Full Text

cell biology↗

bioRxiv2020

Organic Dust Exposure Induces Stress Response and Mitochondrial Dysfunction in Monocytic Cells

Exposure to airborne organic dust (OD), rich in microbial pathogen-associated molecular patterns, has been shown to induce inflammatory responses in the lung resulting in changes in airway structure and function. A common manifestation in lung inflammation is the occurrence of altered mitochondrial structure and bioenergetics, consequently regulating mitochondrial ROS (mROS) and creating a vicious cycle of mitochondrial dysfunction.The role of mitochondrial dysfunction in airway diseases such as COPD and asthma is well known. However, whether OD exposure induces mitochondrial dysfunction largely remains unknown. Therefore, in this study, we tested a hypothesis that OD exposure induces mitochondrial stress using a human monocytic cell line (THP-1). We examined the mechanisms of organic dust extract (ODE) exposure-induced mitochondrial structural and functional changes in THP-1 cells.In addition, the effect of co-exposure to ethyl pyruvate (EP), a known anti-inflammatory agent, or mitoapocynin (MA), a mitochondria targeting NOX2 inhibitor was examined. Transmission electron microscopy images showed significant changes in cellular and organelle morphology upon ODE exposure. ODE exposure with and without EP co-treatment increased the mtDNA leakage into the cytosol. Next, ODE exposure increased the PINK1 and Parkin expression, cytoplasmic cytochrome c levels and reduced mitochondrial mass and cell viability, indicating mitophagy. MA treatment was partially protective by decreasing Parkin expression, mtDNA and cytochrome c release and increasing cell viability.Competing Interest StatementAGK has an equity interest in PK Biosciences Corporation located in Ames, IA. The terms of this arrangement have been reviewed and approved by Iowa State University per its conflict of interest policies. All other authors have declared no potential conflicts of interest. AbbreviationsODOrganic DustODEOrgaic Dust ExtractEPEthyl PyruvateMAMitoapocyninLPSLipopolysaccharidePGNPeptidoglycanPAMPsPathogen Associated Molecular PatternsCOPDChronic Obstructive Pulmonary DiseaseAHRAirway hyperresponsivenessROSReactive Oxygen SpeciesRNSReactive Nitrogen SpeciesATPAdenosine TriphosphateOXPHOSOxidative PhosphorylationHMGB1High Mobility Group Box 1STATSignal Transducer and Activator of TranscriptionTPPTriphenylphosphoniumMPTP1-Methyl-4-Phenyl-1,2,3,6-TetrahydropyridineiNOSinducible Nitric Oxide SynthaseNOXNADPH OxidaseMTT3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromideTEMTransmission Electron MicroscopyDMSODimethyl SufoxidemtND1mitochondrial NADH dehydrogenase 1MFNMitofusinOPA1Optic Atrophy 1DRP1Dynamin-related protein 1EREndoplasmic ReticulumPINK1PTEN- induced kinase 1BNIP3Bcl-2 Homology 3 (BH3)-onlyMPTMitochondrial Permeability TransitionCOX4i2Cytochrome C Oxidase subunit 4 isoform 2ETCElectron Transport ChainSOD2Superoxide Dismutase 2mtDAMPsmitochondrial Damage Associated Molecular PattersmtTFAmitochondrial Transcription Factor AMGCMultinucleated Giant CellFBRForeign Body ReactionsOMMOuter Mitochondrial MembraneIMMInner Mitochondrial MembraneIMSIntermembrane SpaceILInterleukincGAScyclic GMP-AMP synthaseTLRToll-like receptorRAGEReceptor for advanced glycation end productsVDACVoltage-dependent anion channelView Full Text

cell biology↗

bioRxiv2019

High photon count rate super-resolution fluorescence fluctuation spectroscopy

Probing the diffusion of molecules has become a routine measurement across the life sciences, chemistry and physics. It provides valuable insights into reaction dynamics, oligomerisation, molecular (re-)organisation or cellular heterogeneities. Fluorescence correlation spectroscopy (FCS) is one of the widely applied techniques to determine diffusion dynamics in two and three dimensions. This technique relies on the temporal autocorrelation of intensity fluctuations but recording these fluctuations has thus far been limited by the detection electronics, which could not efficiently and accurately time-tag photons at high count rates. This has until now restricted the range of measurable dye concentrations, as well as the data quality of the FCS recordings, especially in combination with super-resolution stimulated emission depletion (STED) nanoscopy. Here, we investigate the applicability and reliability of (STED-)FCS at high photon count rates (average intensities of up to 40 MHz) using novel detection equipment, namely hybrid detectors and real-time gigahertz sampling of the photon streams implemented on a commercial microscope. By measuring the diffusion of fluorophores in solution and cytoplasm of live cells, as well as in model and cellular membranes, we show that accurate diffusion and concentration measurements are possible in these previously inaccessible high photon count regimes. Specifically, it offers much greater flexibility of experiments with biological samples with highly variable intensity, e.g. due to a wide range of expression levels of fluorescent proteins. In this context, we highlight the independence of diffusion properties of cytosolic GFP in a concentration range of approx. 0.01-1 M. We further show that higher photon count rates also allow for much shorter acquisition times, and improved data quality. Finally, this approach also pronouncedly increases the robustness of challenging live cell STED-FCS measurements of nanoscale diffusion dynamics, which we testify by confirming a free diffusion pattern for a fluorescent lipid analogue on the apical membrane of adherent cells.

biophysics↗

bioRxiv2018

Lipid composition but not curvature is a determinant of a low molecular mobility within HIV-1 lipid envelope

Human Immunodeficiency Virus type-1 (HIV-1) acquires its lipid membrane from the plasma membrane of the infected cell from where it buds out. Previous studies have shown that the HIV-1 envelope is a very low mobility environment with the diffusion of incorporated proteins two orders of magnitude slower than in plasma membrane. One of the reasons for this difference is thought to be due to HIV-1 membrane composition that is characterised by a high degree of rigidity and lipid packing. To further refine the model of the molecular mobility on HIV-1 surface, we here investigated the relative importance of membrane composition and curvature in Large Unilamellar Vesicles of different composition and size (0.2-1 m) by super-resolution STED microscopy-based fluorescence correlation spectroscopy (STED-FCS) analysis. We find that lipid composition and its rigidity but not membrane curvature play an important role in the decreased molecular mobility on vesicle surface thus confirming that this factor is an essential determinant of HIV-1 low surface mobility. Our results provide further insight into the dynamic properties of the surface of individual HIV-1 particles.

biophysics↗

bioRxiv2017

Complementary studies of lipid membrane dynamics using iSCAT and STED microscopy

Observation techniques with high spatial and temporal resolution, such as single-particle tracking (SPT) based on interferometric Scattering (iSCAT) microscopy, and fluorescence correlation spectroscopy applied on a super-resolution STED microscope (STED-FCS), have revealed new insights of the molecular organization of membranes. While delivering complementary information, there are still distinct differences between these techniques, most prominently the use of fluorescent dye-tagged probes for STED-FCS and a need for larger scattering gold nanoparticle tags for iSCAT. In this work we have used lipid analogues tagged with a hybrid fluorescent tag - gold nanoparticle construct, to directly compare the results from STED-FCS and iSCAT measurements of phospholipid diffusion on a homogeneous Supported Lipid Bilayer (SLB). These comparative measurements showed that while the mode of diffusion remained free, at least at the spatial (>40 nm) and temporal (50 [≤] t [≤] 100 ms) scales probed, the diffussion coefficient was reduced by 20- to 60-fold when tagging with 20 and 40 nm large gold particles as compared to when using dye-tagged lipid analogues. These FCS measurements of hybrid fluorescent tag - gold nanoparticle labeled lipids also revealed that commercially supplied streptavidin-coated gold nanoparticles contain large quantities of free streptavidin. Finally, the values of apparent diffusion coefficients obtained by STED-FCS and iSCAT differed by a factor of 2-3 across the techniques, while relative differences in mobility between different species of lipid analogues considered were identical in both approaches. In conclusion, our experiments reveal that large and potentially crosslinking scattering tags introduce a significant slow-down in diffusion on SLBs but no additional bias, and our labeling approach creates a new way of exploiting complementary information from STED-FCS and iSCAT measurements.

biophysics↗

bioRxiv2017

Advanced processing and analysis of conventional confocal microscopy generated scanning FCS data

Scanning Fluorescence Correlation Spectroscopy (scanning FCS) is a variant of conventional point FCS that allows molecular diffusion at multiple locations to be measured simultaneously. It enables disclosure of potential spatial heterogeneity in molecular diffusion dynamics and also the acquisition of a large amount of FCS data at the same time, providing large statistical accuracy. Here, we optimize the processing and analysis of these large-scale acquired sets of FCS data. On one hand we present FoCuS-scan, scanning FCS software that provides an end-to-end solution for processing and analysing scanning data acquired on commercial turnkey confocal systems. On the other hand, we provide a thorough characterisation of large-scale scanning FCS data over its intended time-scales and applications and propose a unique solution for the bias and variance observed when studying slowly diffusing species. Our manuscript enables researchers to straightforwardly utilise scanning FCS as a powerful technique for measuring diffusion across a broad range of physiologically relevant length scales without specialised hardware or expensive software.

biophysics↗