Biology subjects

Khadka, P.

Publications and source records attributed to Khadka, P..

6 recordsLinked to original sources

bioRxiv2025

The Arabidopsis UMAMIT30 transporter contributes to amino acid root exudation

ABSTRACTO_ST_ABSBackgroundC_ST_ABSRoot exudation is an important trait that enables plants to shape their interactions with soil-borne organisms. Amino acids present in root exudates play important roles in bacterial chemotaxis, bacterial metabolism, and root colonization, contributing to plant nutrition and health. Notwithstanding the importance of amino acids in shaping the rhizosphere microbiome, the identities of the plant amino acid transporters that mediate their root exudation have remained elusive. ResultsHere, we report that the Arabidopsis UMAMIT30 transporter, robustly expressed in root and shoot tissues, significantly contributes to amino acid root exudation. umamit30 loss-of-function mutants were compromised for amino acid root exudation as shown by the low concentration of amino acids, particularly glutamine, recovered from root exudates compared to wild-type plants. Amino acid quantification, as well as uptake and secretion assessments using radiolabelled glutamine, revealed that the shoots of umamit30 accumulate amino acids and have a reduced capacity to secrete glutamine, impacting root exudation. ConclusionsOur results identify UMAMIT30 as a broadly specific amino acid exporter strongly expressed in Arabidopsis vasculature. Loss-of-function mutants displayed reduced amino acid levels in root exudates, with significant drops in glutamine and asparagine among others, yet exhibited no detectable growth defects. UMAMIT30 disruption led to elevated shoot amino acid content and reduced glutamine efflux from shoots, suggesting a role in phloem uploading rather than root exudation alone. Despite decreased levels of root exuded amino acids, beneficial Pseudomonas interactions and plant-growth-promotion remained unaffected.

plant biology↗

bioRxiv2022

Elicitors of plant defense induce the accumulation of amino acids that suppress both bacterial virulence and growth

Plant immunity relies on the perception of Microbe-Associate Molecular Patterns (MAMPs) from invading microbes to induce defense responses that suppress attempted infections. It has been proposed that MAMP-triggered immunity (MTI) suppresses bacterial infections by suppressing the onset of bacterial virulence. However, the mechanisms by which plants exert this action are poorly understood. Here we uncover that MAMP-perception in Arabidopsis induces the accumulation of free amino acids (AA) in a salicylic acid (SA) dependent manner. When co-infiltrated with Glutamine and Serine, two of the MAMP-induced highly accumulating amino acids, Pseudomonas syringae pv. tomato DC3000 expressed low levels of virulence genes and failed to produce robust infections in otherwise susceptible plants. When applied exogenously, Glutamine and Serine suppressed bacterial virulence and bacterial growth directly without the need for MAMP-perception and SA-signaling, bypassing MAMP-elicited defense. In addition, an increased level of endogenous Glutamine in the leaf apoplast of a gain-of-function mutant of Glutamine Dumper-1 rescued the partially compromised bacterial virulence and growth suppressing phenotype of the SA-induced deficient-2 (sid2) mutant. Our data suggest that MTI suppresses bacterial infections by the direct suppressing effect of accumulating amino acids on the onset of bacterial virulence.

plant biology↗

bioRxiv2022

Single Cell Spatial Analysis Reveals the Topology of Immunomodulatory Purinergic Signaling in Glioblastoma

Glioblastoma develops an immunosuppressive microenvironment that fosters tumorigenesis and resistance to current therapeutic strategies. Here we use multiplexed tissue imaging and single-cell RNA-sequencing to characterize the composition, spatial organization, and clinical significance of extracellular purinergic signaling in glioblastoma. We show that glioblastoma exhibit strong expression of CD39 and CD73 ectoenzymes, correlating with increased adenosine levels. Microglia are the predominant source of CD39, while CD73 is principally expressed by tumor cells, particularly in tumors with amplification of EGFR and astrocyte-like differentiation. Spatially-resolved single-cell analyses demonstrate strong spatial correlation between tumor CD73 and microglial CD39, and that their spatial proximity is associated with poor clinical outcomes. Together, this data reveals that tumor CD73 expression correlates with tumor genotype, lineage differentiation, and functional states, and that core purine regulatory enzymes expressed by neoplastic and tumor-associated myeloid cells interact to promote a distinctive adenosine-rich signaling niche and immunosuppressive microenvironment potentially amenable to therapeutic targeting.

cancer biology↗

bioRxiv2020

HIV-specific T-cell responses reflect substantive in vivo interactions with infected cells despite long-term therapy

Antiretroviral therapies (ART) durably suppress HIV replication to undetectable levels - however, infection persists in the form of long-lived reservoirs of infected cells with integrated proviruses, that re-seed systemic replication if ART is interrupted. A central tenet of our current understanding of this persistence is that infected cells are shielded from immune recognition and elimination through a lack of antigen expression from proviruses. Efforts to cure HIV infection have therefore focused on reactivating latent proviruses to enable immune-mediated clearance, but these have yet to succeed in driving reductions in viral reservoirs. Here, we revisited the question of whether HIV reservoirs are predominately immunologically silent from a new angle, by querying the dynamics of HIV-specific T-cell responses over long-term ART for evidence of ongoing recognition of HIV-infected cells. We show that T-cell responses to autologous reservoir viruses persist over years, and that the maintenance of HIV-Nef-specific responses was uniquely associated with residual frequencies of infected cells. These responses disproportionately exhibited a cytotoxic, effector functional profile, indicative of recent in vivo recognition of HIV-infected cells. These results indicate substantial visibility of the HIV reservoir to T-cells on stable ART, presenting both opportunities and challenges for the development of therapeutic approaches to curing HIV infection.

immunology↗

bioRxiv2020

HIV Diversity Considerations in the Application of the Intact Proviral DNA Assay (IPDA)

Opening Paragraph (serves as abstract for submission) and BodyThe Intact Proviral DNA Assay (IPDA) was developed to address the critical need for a precise and scalable method for intact HIV reservoir quantification1. This duplexed droplet digital PCR (ddPCR) assay simultaneously targets the HIV Packaging Signal ({Psi}) and the Rev Responsive Element (RRE) within Envelope (env) to distinguish genomically intact proviruses against a large background of defective ones2. The IPDA requires less time, resources, and biological material than the gold standard for replication-competent HIV reservoir measurement, the Quantitative Viral Outgrowth Assay (QVOA)3, and is being adopted in research and clinical studies4-7. In our cohort of HIV-1 subtype B-infected individuals from North America however, the IPDA yielded a 28% failure rate due to HIV polymorphism. We further demonstrate that within-host HIV diversity can lead the IPDA to underestimate intact HIV reservoir size, which could negatively impact clinical trial results interpretation. While the IPDA represents an important methodological advance, HIV diversity should be addressed before its widespread adoption.

genetics↗

bioRxiv2019

Faster diffusive dynamics of histone-like nucleoid structuring proteins in live bacteria caused by silver ions

The antimicrobial activity and mechanism of silver ions (Ag+) have gained broad attention in recent years. However, dynamic studies are rare in this field. Here, we report our measurement of the effects of Ag+ ions on the dynamics of histone-like nucleoid structuring (H-NS) proteins in live bacteria using single-particle tracking photoactivated localization microscopy (sptPALM). It was found that treating the bacteria with Ag+ ions led to faster diffusive dynamics of H-NS proteins. Several techniques were used to understand the mechanism of the observed faster dynamics. Electrophoretic mobility shift assay on purified H-NS proteins indicated that Ag+ ions weaken the binding between H-NS proteins and DNA. Isothermal titration calorimetry confirmed that DNA and Ag+ ions interact directly. Our recently developed sensing method based on bent DNA suggested that Ag+ ions caused dehybridization of double-stranded DNA (i.e., dissociation into single strands). These evidences led us to a plausible mechanism for the observed faster dynamics of H-NS proteins in live bacteria when subjected to Ag+ ions: Ag+-induced DNA dehybridization weakens the binding between H-NS proteins and DNA. This work highlighted the importance of dynamic study of single proteins in the live cells for understanding the functions of antimicrobial agents to the bacteria.

biophysics↗