Biology subjects

Nguyen, K. D.

Publications and source records attributed to Nguyen, K. D..

7 recordsLinked to original sources

bioRxiv2024

Rapid Sterilization of Clinical Apheresis Blood Products using Ultra-High Dose Rate Radiation

BACKGROUND AND OBJECTIVESApheresis platelets products and plasma are essential for medical interventions, but both still have inherent risks associated with contamination and viral transmission. Platelet products are vulnerable to bacterial contamination due to storage conditions, while plasma requires extensive screening to minimize virus transmission risks. Here we investigate rapid irradiation to sterilizing doses for bacteria and viruses as an innovative pathogen reduction technology. MATERIALS AND METHODSWe configured a clinical linear accelerator to deliver ultra-high dose rate (6 kGy/min) irradiation to platelet and plasma blood components. Platelet aliquots spiked with 105 CFU of E.coli were irradiated with 0.1-20 kGy, followed by E.coli growth and platelet count assays. COVID Convalescent Plasma (CCP) aliquots were irradiated at a virus-sterilizing dose of 25 kGy and subsequently, RBD- specific antibody binding was assessed. RESULTS1 kGy irradiation of bacteria-spiked platelets reduced E.coli growth by 2.7- log without significant change of platelet count, and 5 kGy or higher produced complete growth suppression. The estimated sterilization (6-log bacterial reduction) dose was 2.3 kGy, corresponding to 31% platelet count reduction. A 25 kGy virus sterilizing dose to CCP produced a 9.2% average drop of RBD-specific IgG binding. CONCLUSIONThis study shows proof-of-concept of a novel rapid blood sterilization technique using a clinical linear accelerator. Promising platelet counts and CCP antibody binding were maintained at bacteria and virus sterilizing doses, respectively. This represents a potential point-of-care blood product sterilization solution. If additional studies corroborate these findings, this may be a practical method for ensuring blood products safety. HIGHLIGHTSO_LI1 kGy irradiation of bacteria-spiked platelets reduced E.coli growth by 2.7-log without significant change of platelet count, and 5 kGy or higher produced complete growth suppression. C_LIO_LIThe estimated sterilization (6-log bacterial reduction) dose was 2.3 kGy, corresponding to 31% platelet count reduction. C_LIO_LIA 25 kGy virus sterilizing dose to CCP produced a 9.2% average drop of RBD- specific IgG binding. C_LI

microbiology↗

bioRxiv2022

Chance and contingency in B cell evolution limit the similarity of antibody responses to infection across individuals

Antibodies result from the competition of B cell lineages evolving under selection for improved antigen recognition, a process known as affinity maturation. High-affinity antibodies to pathogens such as HIV, influenza, and SARS-CoV-2 are frequently reported to arise from B cells whose receptors, the precursors to antibodies, are encoded by particular immunoglobulin alleles. This raises the possibility that the presence of particular germline alleles in the B cell repertoire is a major determinant of the quality of the antibody response. Alternatively, initial differences in germline alleles propensities to form high-affinity receptors might be overcome by chance events during affinity maturation. We first investigate these scenarios in simulations: when germline-encoded fitness differences are large relative to the rate and effect size variation of somatic mutations, the same germline alleles persistently dominate the response of different individuals. In contrast, if germline-encoded advantages can be easily overcome by subsequent mutations, allele usage becomes increasingly divergent over time, a pattern we then observe in mice experimentally infected with influenza virus. We investigated whether affinity maturation might nonetheless strongly select for particular amino acid motifs across diverse genetic backgrounds, but we found no evidence of convergence to similar CDR3 sequences or amino acid substitutions. These results suggest that although germline-encoded specificities can lead to similar immune responses between individuals, diverse evolutionary routes to high affinity limit the genetic predictability of responses to infection and vaccination. Author SummaryAntibodies arise as B cell receptors encoded by the stochastic recombination of immunoglobulin genes. While those genes evolve over millions of years, the receptors themselves evolve within weeks as B cells compete under selection for improved antigen recognition. This competition shapes the response to infection and vaccination; how much the outcome depends on the initial choice of immunoglobulin genes versus subsequent receptor evolution is an open question that informs the predictability of the immune response and the long-term evolution of immunoglobulins. In simulations, we show that immunoglobulin genes with hardcoded specificity for the antigen can lead to either transient or persistent similarity in the response of different individuals. When the initial advantage is large relative to the effects of mutation, B cells using the same genes consistently dominate the response across individuals. Weaker initial advantages lead to similar responses early on but are later overcome by B cell evolution playing out differently in each individual due to chance events. We observe such increasingly divergent responses in mice infected with influenza virus. While long-term selection might hardcode specificities for particular pathogens on immunoglobulin genes, our results suggest diverse paths to potent antibodies can nonetheless limit the predictability of the response.

immunology↗

bioRxiv2021

Rheology of crossbridge ensembles

How skeletal muscle responds to mechanical perturbations, its rheology, is important for animal movement control. The molecular machinery of myosin II-actin crossbridge cycling is a crucial part of muscles rheological properties, and multiple models have been proposed for this mechanochemical process. But current understanding of the scale-connection from individual molecular motors to ensemble rheology is limited. Here we present computational and mathematical analyses of several different hypotheses of crossbridge dynamics, from 2-state to 5-state myosin II motor models, and show that an ensemble of actomyosin crossbridges exhibits surprisingly simple rheological behavior in all cases. The ensemble rheology is captured by a sum of at most three linear viscoelastic sub-processes, and as few as one for some crossbridge models. This simplification lends itself to computationally efficient phenomenological muscle models with experimentally measurable parameters, while still remaining grounded in crossbridge theory. However, the collapse of the ensemble behavior to few linear sub-processes identifies major limitations of crossbridge models that cannot be resolved by adding complexity to the crossbridge cycle and point to the roles of inter-crossbridge interactions and non-crossbridge elements.

biophysics↗

bioRxiv2020

Oligomerization of the Human Adenosine A2A Receptor Is Driven by the Intrinsically Disordered C-Terminus

G protein-coupled receptors (GPCRs) have long been shown to exist as oligomers with functional properties distinct from those of the monomeric counterparts, but the driving factors of GPCR oligomerization remain relatively unexplored. In this study, we focus on the human adenosine A2A receptor (A2AR), a model GPCR that forms oligomers both in vitro and in vivo. Combining experimental and computational approaches, we discover that the intrinsically disordered C-terminus of A2AR drives the homo-oligomerization of the receptor. The formation of A2AR oligomers declines progressively and systematically with the shortening of the C-terminus. Multiple interaction sites and types are responsible for A2AR oligomerization, including disulfide linkages, hydrogen bonds, electrostatic interactions, and hydrophobic interactions. These interactions are enhanced by depletion interactions along the C-terminus, forming a tunable network of bonds that allow A2AR oligomers to adopt multiple interfaces. This study uncovers the disordered C-terminus as a prominent driving factor for the oligomerization of a GPCR, offering important guidance for structure-function studies of A2AR and other GPCRs.

biophysics↗

bioRxiv2020

Proteogenomic analysis unveils the HLA Class I presented immunopeptidome in melanoma and EGFR mutant lung adenocarcinoma

Immune checkpoint inhibitor and adoptive lymphocyte transfer-based therapies have shown great therapeutic potential for cancers with high tumor mutation burden (TMB). Here, we employed mass spectrometry (MS)-based proteogenomic large-scale profiling to identify potential immunogenic human leukocyte antigen (HLA) Class I-presented peptides in both melanoma, a "hot tumor" with high TMB, and EGFR mutant lung adenocarcinoma, a "cold tumor" with low TMB. We identified several classes of neopeptides, including mutated neoantigens and more than 1000 post-translationally modified peptides representing 58 different PTMs. We constructed a cancer germline (CG) antigen database with 285 antigens and identified 42 Class I-presented CG antigens. Finally, we developed a non-canonical peptide discovery pipeline to identify 44 lncRNA-derived peptides and validated Class I binding for select neopeptides. We provide direct evidence of HLA Class I presentation of a large number of neopeptides for potential vaccine or adoptive cell therapy in melanoma and mutant EGFR lung cancer.

immunology↗

bioRxiv2020

Global proteome and phosphoproteome alterations in third generation EGFR TKI resistance reveal drug targets to circumvent resistance

Lung cancer is the leading cause of cancer mortality worldwide. The treatment of lung cancer patients harboring mutant EGFR with orally administered EGFR TKIs has been a paradigm shift. Osimertinib and rociletinib are 3rd generation irreversible EGFR TKIs targeting the EGFR T790M mutation. Osimertinib is the current standard care for patients with EGFR mutations due to increased efficacy, lower side effects, and enhanced brain penetrance. Unfortunately, all patients develop resistance. Genomic approaches have primarily been used to interrogate resistance mechanisms. Here, we have characterized the proteome and phosphoproteome of a series of isogenic EGFR mutant lung adenocarcinoma cell lines that are either sensitive or resistant to these drugs. To our knowledge, this is the most comprehensive proteomic dataset resource to date to investigate 3rd generation EGFR TKI resistance in lung adenocarcinoma. We have interrogated this unbiased global quantitative mass spectrometry dataset to uncover alterations in signaling pathways, reveal a proteomic signature of epithelial mesenchymal transition (EMT) and identify kinases and phosphatases with altered expression and phosphorylation in TKI resistant cells. Decreased tyrosine phosphorylation of key sites in the phosphatase SHP2 suggests its inhibition resulting in inhibition of RAS/MAPK and activation of PI3K/AKT pathways. Furthermore, we performed anticorrelation analyses of this phosphoproteomic dataset with the published drug-induced P100 phosphoproteomic datasets from the Library of Integrated Network-Based Cellular Signatures (LINCS) program to predict drugs with the potential to overcome EGFR TKI resistance. We identified dactolisib, a PI3K/mTOR inhibitor, which in combination with osimertinib overcomes resistance both in vitro and in vivo.One Sentence Summary Global quantitative proteome and phosphoproteome analyses to examine altered signaling pathways in isogenic 3rd generation EGFR TKI sensitive and resistant cells.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cancer biology↗

bioRxiv2018

Quantitative mass spectrometry to interrogate proteomic heterogeneity in metastatic lung adenocarcinoma and validate a novel somatic mutation CDK12-G879V

Lung cancer is the leading cause of cancer death both in men and women. Tumor heterogeneity is an impediment to targeted treatment of all cancers, including lung cancer. Here, we sought to characterize changes in tumor proteome and phosphoproteome by longitudinal, prospective collection of tumor tissue of an exceptional responder lung adenocarcinoma patient who survived with metastatic lung adenocarcinoma for more than seven years with HER2-directed therapy in combination with chemotherapy. We employed \"Super-SILAC\" and TMT labeling strategies to quantify the proteome and phosphoproteome of a lung metastatic site and ten different metastatic progressive lymph nodes collected across a span of seven years, including five lymph nodes procured at autopsy. We identified specific signaling networks enriched in lung compared to the lymph node metastatic sites. We correlated the changes in protein abundance with changes in copy number alteration (CNA) and transcript expression. To further interrogate the mass spectrometry data, patient-specific database was built incorporating all the somatic variants identified by whole genome sequencing (WGS) of genomic DNA from the lung, one lymph node metastatic site and blood. An extensive validation pipeline was built for confirmation of variant peptides. We validated 360 spectra corresponding to 55 germline and 6 somatic variant peptides. Targeted MRM assays demonstrated expression of two novel variant somatic peptides, CDK12-G879V and FASN-R1439Q, with expression in lung and lymph node metastatic sites, respectively. CDK12 G879V mutation likely results in a nonfunctional CDK12 kinase and chemotherapy susceptibility in lung metastatic sites. Knockdown of CDK12 in lung adenocarcinoma cells results in increased chemotherapy sensitivity, explaining the complete resolution of the lung metastatic sites in this patient.

cancer biology↗