Biology subjects

Kuiper, J.

Publications and source records attributed to Kuiper, J..

4 recordsLinked to original sources

bioRxiv2024

Enabling functionality and translation fidelity characterization of mRNA-based vaccines with a platform-based, antibody-free mass spectrometry detection approach

The success of mRNA-based therapeutics and vaccines can be attributed to their rapid development, adaptability to new disease variants, and scalable production. Modified ribonucleotides are often used in mRNA-based vaccines or therapeutics to enhance stability and reduce immunogenicity. However, substituting uridine with N1-methylpseudouridine has recently been shown to result in +1 ribosomal frameshifting that induces cellular immunity to the translated off-target protein. To accelerate vaccine development, it is critical to have analytical methods that can be rapidly brought online to assess the functionality and translation fidelity of mRNA constructs. Here, a platform-based, antibody-free method was developed using cell-free translation (CFT) and liquid chromatography-tandem mass spectrometry (MS) that can detect, characterize, and provide relative quantification of antigen proteins translated from mRNA vaccine drug substance. This workflow enabled the evaluation of mRNA subjected to thermal stress as well as bivalent (i.e., two mRNA encoding different antigen variants) drug substance. Additionally, the MS detection approach exhibited high sensitivity and specificity by accurately identifying all six translated proteins and their relative abundances in a dose-dependent manner following transfection of human cells with a hexavalent mRNA mixture encapsulated in lipid nanoparticles (LNPs), despite significant protein sequence homology. Expanding on these efforts, we show the utility of the CFT-MS approach in identifying the presence and junction of +1 ribosomal frameshifting resulting from N1-methylpseudouridation. Overall, this CFT-MS methodology offers a valuable analytical tool for the development and production of mRNA-based vaccines by facilitating the evaluation of mRNA quality and functionality while ensuring accurate translation of antigen proteins. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=91 SRC="FIGDIR/small/594137v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@e4c7a7org.highwire.dtl.DTLVardef@1efddd1org.highwire.dtl.DTLVardef@cbfcbaorg.highwire.dtl.DTLVardef@1ae1acc_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Female gene networks are expressed in myofibroblast-like smooth muscle cells in vulnerable atherosclerotic plaques.

Women presenting with coronary artery disease (CAD) more often present with fibrous atherosclerotic plaques, which are currently understudied. Phenotypically modulated smooth muscle cells (SMCs) contribute to atherosclerosis in women. How these phenotypically modulated SMCs shape female versus male plaques is unknown. Here, we show sex-stratified gene regulatory networks (GRNs) from human carotid atherosclerotic tissue. Prioritization of these networks identified two main SMC GRNs in late-stage atherosclerosis. Single-cell RNA-sequencing mapped these GRNs to two SMC phenotypes: a phenotypically modulated myofibroblast-like SMC network and a contractile SMC network. The myofibroblast-like GRN was mostly expressed in plaques that were vulnerable in females. Finally, mice orthologs of the female myofibroblast-like genes showed retained expression in advanced plaques from female mice but were downregulated in male mice during atherosclerosis progression. Female atherosclerosis is driven by GRNs that promote a fibrous vulnerable plaque rich in myofibroblast-like SMCs.

bioinformatics↗

bioRxiv2022

Enhanced photosynthetic efficiency for increased carbon assimilation and woody biomass production in hybrid poplar INRA 717-1B4

Increasing CO2 levels in the atmosphere and the resulting negative impacts on climate change have compelled global efforts to achieve carbon neutrality or negativity. Most such efforts focus on carbon sequestration through chemical or physical approaches. We aim to harness the power of synthetic biology to enhance plants natural ability to draw down and sequester carbon, thereby positively affecting climate change. Past decades of scientific progress have shed light on strategies to overcome the intrinsic limitations of carbon drawdown and fixation through photosynthesis, particularly in row crops in hopes of improving agricultural productivity for food security. Incorporating a photorespiration bypass in C3 plants has shown promising results of increased biomass and grain yield. Despite their globally dominant role in atmospheric carbon flux, the drawdown rates of most trees are currently limited by their C3 photosynthetic metabolism, and efforts to improve the photosynthetic capacity of trees, such as by reducing energy loss in photorespiration, are currently lacking. Here, we selected a photorespiration bypass pathway and tested its effectiveness on photosynthetic enhancement in hybrid poplar INRA717-IB4. The design includes a RNAi strategy to reduce the transportation of the photorespiration byproduct, glycolate, out of chloroplast and a shunt pathway to metabolize the retained glycolate back to CO2 for fixation through the Calvin-Benson cycle. Molecular and physiological data collected from two repeated growth experiments indicates that transgenic plants expressing genes in the photorespiration bypass pathway have increased photosynthetic efficiency, leading to faster plant growth and elevated biomass production. One lead transgenic event accumulated 53% more above-ground dry biomass over a five month growth period in a controlled environment. Pilot projects with photosynthesis-enhanced trees in the field are in progress. Our results provide a proof-of-concept for engineering trees to help combat climate change.

plant biology↗

bioRxiv2020

Common allotypes of ER aminopeptidase 1 have substrate- dependent and highly variable enzymatic properties

ObjectivePolymorphic variation of immune system proteins can drive variability of individual immune responses. ER aminopeptidase 1 (ERAP1) generates antigenic peptides for presentation by MHC class I molecules. Coding single nucleotide polymorphisms (SNPs) in ERAP1 have been associated with predisposition to inflammatory rheumatic disease and shown to affect functional properties of the enzyme, but the interplay between combinations of these SNPs as they exist in allotypes, has not been thoroughly explored. MethodsWe used phased genotype data to estimate ERAP1 allotype frequency in 2,504 individuals across five major human populations, generated highly pure recombinant enzymes corresponding to the 10 most common ERAP1 allotypes and systematically characterized their in vitro enzymatic properties. ResultsWe find that ERAP1 allotypes possess a wide range of enzymatic activities, up to 60-fold, whose ranking is substrate-dependent. Strikingly, allotype 10, previously associated with Behcets disease, is consistently a low-activity outlier, suggesting that a significant percentage of individuals carry a sub-active ERAP1 gene. Enzymatic analysis revealed that ERAP1 allotypes can differ in both catalytic efficiency and substrate affinity, differences that can change intermediate accumulation in multi-step trimming reactions. Alterations in efficacy of an allosteric inhibitor that targets the regulatory site suggest that allotypic variation influences the communication between the regulatory and the active site. ConclusionOur work defines the wide landscape of ERAP1 activity in human populations and demonstrates how common allotypes can induce substrate-dependent variability in antigen processing, thus contributing, in synergy with MHC haplotypes, to immune response variability and to predisposition to chronic inflammatory conditions.

biochemistry↗