Biology subjects

Reynolds, M.

Publications and source records attributed to Reynolds, M..

14 recordsLinked to original sources

bioRxiv2026

RUNX1 and YY1 modulate neuronal fate and energy metabolism in Alzheimer's disease

Loss of neuronal identity and metabolic dysfunction are features of Alzheimers disease (AD), yet the upstream-acting molecular drivers remain incompletely understood. By integrating multi-omics data from patient-derived induced neurons (iNs) and AD post-mortem human brains, we discovered that AD neurons express two master transcription factors (TFs), RUNX1 and YY1. While these TFs are primarily expressed during development where they play fundamental roles in cell fate determination and cellular bioenergetics, respectively, they can be reactivated in adult neurons in response to stress. To understand their functional role in AD neurons, we overexpressed RUNX1 or YY1 in aged iNs and found that the expression of each TF was sufficient to recapitulate two AD-associated features. Specifically, RUNX1 overexpression caused loss of neuronal fate, whereas YY1 overexpression regulated gene regulatory programs associated with metabolic dysfunction. Conversely, downregulation of either TF, in AD iNs, reinstated gene regulatory programs associated with a healthy mature neuronal phenotype. Together, these findings identify two transcriptional master regulators of the AD neuronal phenotype and establish a mechanistic foundation for further studying their role in the pathogenesis of AD and as putative therapeutical targets for the treatment of AD and age-associated neurodegeneration.

neuroscience↗

bioRxiv2026

Rethinking yield stability through phenotypic plasticity and its link to modern statistical methods

In the context of advocacy for yield stability, trade-offs between yield and yield stability, the frequent lack of definitions, and the variation in methods when they are explicit, we connect two perspectives: phenotypic plasticity and factor analytics. Phenotypic plasticity brings over a century of research in developmental biology, ecology and evolution, and is gaining traction in crop science. Factor analytics is an advanced linear mixed model of multi-environment data with factor analytic variance structures for the variety-by-environment interaction effect. We (1) review phenotypic plasticity, define agronomically adaptive plasticity where varieties (or practices) consistently return superior yield (or other traits) across environments with no trade-off, and describe percentile-plasticity plots to assess the agronomic value of plasticity; (2) outline factor analytic models, and (3) link plasticity and factor analytic models mathematically and empirically. We show that phenotypic plasticity of cereal yield correlates positively with overall performance obtained from factor analytic models when plasticity is adaptive and negatively when it is maladaptive. We conclude that phenotypic plasticity contributes biological meaning to opaque analytical approaches, and that biologically grounded statistics are needed to challenge weak agronomic narratives, such as advocacy for stability that might reflect decision biases rather than critical consideration of its benefits. HighlightUncritical advocacy for crop yield stability is common. Here we advance a biological-statistical synthesis of yield stability and test theoretical predictions with actual wheat and oat yield data.

plant biology↗

bioRxiv2024

The antimicrobial potential of traditional remedies of Indigenous Peoples from Canada against MRSA planktonic and biofilm bacteria in wound-infection mimetic conditions

Methicillin-resistant Staphylococcus aureus (MRSA) is the leading cause of wound infections, often progressing into serious invasive bloodstream infections. MRSA disproportionately affects Indigenous peoples in Canada with higher rates of skin and wound infections, an example of persistent gaps in health outcomes between Indigenous and non-Indigenous peoples precipitated by the legacy of colonialism. Conversely, Indigenous peoples have long used natural remedies for infections and other diseases; however, their knowledge was rarely considered for modern medicine. The stagnant antibiotic discovery pipeline and alarming rise of resistance to current antibiotics prompted us to turn to Indigenous medicine as an untapped source of antimicrobials. As such, we collected and prepared 85 extracts of medicinal plants of value to Indigenous Peoples spanning the Canadian Prairies. We explored the antimicrobial potential of these extracts against MRSA under wound infection-mimetic conditions compared to culture media typically used to study bacterial antibiotic responses and biofilms but not adequately representative of infection sites. We identified extracts with MRSA growth inhibitory [e.g., bergamot, dock, gaillardia, and dandelion extracts] and biofilm prevention and eradication [e.g., gumweed extracts] activities. Extracts, including those of chokecherry, hoary puccoon, and Northern bedstraw, were only active under wound infection-mimetic conditions, highlighting the relevance of antibiotic discovery under host-relevant conditions. Testing growth inhibitory extracts against an S. aureus cross-resistance platform suggested that they act through mechanisms likely distinct from known antibiotic classes. Together, through an interdisciplinary partnership leveraging Western approaches and traditional Indigenous knowledge, we identified plant extracts with promising antimicrobial potential for drug-resistant MRSA wound infections.

microbiology↗

bioRxiv2023

A SMART method for efficiently isolating monoclonal antibodies from individual rhesus macaque memory B cells

Characterizing antigen-specific B cells is a critical component of vaccine and infectious disease studies in rhesus macaques (RMs). However, it is challenging to capture immunoglobulin variable (IgV) genes from individual RM B cells using 5 multiplex (MTPX) primers in nested PCR reactions. In particular, the diversity within RM IgV gene leader sequences necessitates the use of large 5 MTPX primer sets to amplify IgV genes, decreasing PCR efficiency. To address this problem, we developed a switching mechanism at the 5 ends of the RNA transcript (SMART)-based method for amplifying IgV genes from single RM B cells, providing unbiased capture of Ig heavy and light chain pairs for cloning antibodies. We demonstrate this technique by isolating simian immunodeficiency virus (SIV) envelope-specific antibodies from single-sorted RM memory B cells. This approach has several advantages over existing methods for PCR cloning antibodies from RMs. First, optimized PCR conditions and SMART 5 and 3 rapid amplification of cDNA ends (RACE) reactions generate full-length cDNAs from individual B cells. Second, it appends synthetic primer binding sites to the 5 and 3 ends of cDNA during synthesis, allowing for PCR amplification of low-abundance antibody templates. Third, universal 5 primers are employed to amplify the IgV genes from cDNA, simplifying the primer mixes in the nested PCR reactions and improving the recovery of matched heavy and light chain pairs. We anticipate this method will enhance the isolation of antibodies from individual RM B cells, supporting the genetic and functional characterization of antigen-specific B cells.

immunology↗

bioRxiv2023

Analysis of intracellular transport dynamics using quantitative phase imaging and FRET-based calcium sensors

Understanding cellular responses to mechanical environmental stimuli is an essential goal of cellular mechanotransduction studies and remains a target for microscopy technique development. While fluorescence microscopy has been advanced for this purpose - its molecular sensitivity, the ability of quantitative phase imaging to visualize subcellular structure has yet to be widely applied, perhaps due to its limited specificity. Here we seek to combine Quantitative Phase Imaging (QPI) with a molecularly sensitive Forster resonance energy transfer (FRET) construct for cell mechanotransduction studies. The multimodal imaging instrument is applied to examine cellular response to hypo-osmotic stimulus by observing the influx of calcium ions using a FRET based sensor coupled with mapping of the redistribution of intracellular mass using QPI. The combined imaging modality enables discrimination of cell response by localized region and reveals distinct behavior for each. The analysis shows cell flattening and oscillatory mass transport in response to the stimulus. With the broad array of FRET sensors under development, the combination with QPI offers new avenues for studying cell response to environmental stimuli.

biophysics↗

bioRxiv2023

Regulatory network structure and environmental signals constrain transcription factor innovation

Evolutionary innovation of transcription factors frequently drives phenotypic diversification and adaptation to environmental change. Rewiring - that is gaining or losing connections to transcriptional target genes - is a key mechanism by which transcription factors evolve and innovate. However the frequency of functional adaptation varies between different regulators, even when they are closely related. To identify factors influencing propensity for rewiring, we utilise a Pseudomonas fluorescens SBW25 strain rendered incapable of flagellar mediated motility in soft-agar plates via deletion of the flagellar master regulator (fleQ). This bacterium can evolve to rescue flagellar motility via gene regulatory network rewiring of an alternative transcription factor to rescue activity of FleQ. Previously, we have identified two members (out of 22) of the RpoN-dependent enhancer binding protein (RpoN-EBP) family of transcription factors (NtrC and PFLU1132) that are capable of innovating in this way. These two transcription factors rewire repeatably and reliably in a strict hierarchy - with NtrC the only evolved rewiring route in a {Delta}fleQ background, and PFLU1132 the only evolved rewiring route in a {Delta}fleQ{Delta}ntrC background. However, why other members in the same transcription factor family have not been observed to rescue flagellar activity is unclear. Previous work shows that protein homology cannot fully explain this pattern, and mutations in rewired strains suggested high levels of transcription factor expression and activation drive rewiring. We predict that mutations that increase expression of the rewired transcription factor are vital to unlock rewiring potential. Here, we construct titratable expression mutant lines for 11 of the RpoN-EBPs in P. fluorescens. We show that in 5 additional RpoN-EBPs (HbcR, GcsR, DctD, AauR and PFLU2209), high expression levels result in different mutations conferring motility rescue, suggesting alternative rewiring pathways. Our results indicate that expression levels (and not protein homology) of RpoN-EBPs are a key constraining factor in determining rewiring potential. This suggests that transcription factors that can achieve high expression through few mutational changes, or transcription factors that are active in the selective environment, are more likely to innovate and contribute to adaptive gene regulatory network rewiring.

evolutionary biology↗

bioRxiv2022

ComBat Harmonization: Empirical Bayes versus Fully Bayes Approaches

Studying small effects or subtle neuroanatomical variation requires large-scale sample size data. As a result, combining neuroimaging data from multiple datasets is necessary. Variation in acquisition protocols, magnetic field strength, scanner build, and many other non-biologically related factors can introduce undesirable bias into studies. Hence, harmonization is required to remove the bias-inducing factors from the data. ComBat is one of the most common methods applied to features from structural images. ComBat models the data using a hierarchical Bayesian model and uses the empirical Bayes approach to infer the distribution of the unknown factors. The empirical Bayes harmonization method is computationally efficient and provides valid point estimates. However, it tends to underestimate uncertainty. This paper investigates a new approach, fully Bayesian ComBat, where Monte Carlo sampling is used for statistical inference. When comparing fully Bayesian and empirical Bayesian ComBat, we found Empirical Bayesian ComBat more effectively removed scanner strength information and was much more computationally efficient. Conversely, fully Bayesian ComBat better preserved biological disease and age-related information while performing more accurate harmonization on traveling subjects. The fully Bayesian approach generates a rich posterior distribution, which is useful for generating simulated imaging features for improving classifier performance in a limited data setting. We show the generative capacity of our model for augmenting and improving the detection of patients with Alzheimers disease. Posterior distributions for harmonized imaging measures can also be used for brain-wide uncertainty comparison and more principled downstream statistical analysis. Code for our new fully Bayesian ComBat extension is available at https://github.com/batmanlab/BayesComBat.

neuroscience↗

bioRxiv2021

Annotation of Putative Circadian Rhythm-Associated Genes in Diaphorina citri (Hemiptera : Liviidae)

The circadian rhythm is a process involving multiple genes that generates an internal molecular clock, allowing organisms to anticipate environmental conditions produced by the earths rotation on its axis. This report presents the results of the manual curation of twenty-seven genes likely associated with circadian rhythm in the genome of Diaphorina citri, the Asian citrus psyllid. This insect acts as the vector of the bacterial pathogen Candidatus Liberibacter asiaticus (CLas), the causal agent of citrus greening disease (Huanglongbing). This disease is the most severe detriment to citrus industries and has drastically decreased crop yields worldwide. Based on the genes identified in the psyllid genome, namely cry1 and cry2, D. citri likely possesses a circadian model similar to that of the lepidopteran butterfly, Danaus plexippus. Manual annotation of these genes will allow future molecular therapeutics to be developed that can disrupt the psyllid biology.

bioinformatics↗

bioRxiv2021

Cooperation between CYB5R3 and NOX4 via coenzyme Q mitigates endothelial inflammation

NADPH oxidase 4 (NOX4) regulates endothelial inflammation by producing reactive oxygen species. Since coenzyme Q (CoQ) mimics affect NOX4 activity, we hypothesize that cytochrome b5 reductase 3 (CYB5R3), a CoQ reductase abundant in vascular endothelial cells, modulates inflammatory activation. Mice lacking endothelial CYB5R3 (R3 KO), under lipopolysaccharides (LPS) challenge, showed exacerbated hypotension, decreased acetylcholine-induced vasodilation, and elevated vascular adhesion molecule 1 (Vcam-1) mRNA in aorta. In vitro, silencing Cyb5r3 enhanced LPS-induced VCAM-1 protein in a NOX4 dependent manner. APEX2- based electron microscopy and proximity biotinylation demonstrated CYB5R3s localization on the mitochondrial outer membrane and its interaction with NOX4, which was further confirmed by the proximity ligation assay. Notably, Cyb5r3 silenced HAECs had less total H2O2 but more mitochondrial O2*-. Using inactive or non-membrane bound active CYB5R3, we found CYB5R3 activity and membrane translocation were needed for optimal generation of H2O2 by NOX4. Lastly, CoQ deficient cells showed decreased NOX4-derived H2O2, indicating a requirement for endogenous CoQ in NOX4 activity. In conclusion, CYB5R3 mitigates endothelial inflammatory activation by assisting in NOX4-dependent H2O2 generation via CoQ. NOVELTY AND SIGNIFICANCEO_ST_ABSWhat Is Known?C_ST_ABSNADPH oxidase 4 (NOX4) reportedly produces primarily hydrogen peroxide (H2O2) and, to a lesser extent, superoxide (O2*-) and has been shown to have both beneficial and deleterious effects in the cardiovascular system. NOX4 activity can be affected by NAD(P)H quinone oxidoreductase 1 (NQO1), a CoQ reductase, and synthetic quinone compounds used to mimic CoQ. Cytochrome b5 reductase 3 (CYB5R3) is known to reduce CoQ and is highly expressed in endothelial cells. What New Information Does This Article Contribute?In vivo, the lack of endothelial CYB5R3 causes exacerbated lipopolysaccharides (LPS)-induced inflammatory signaling, endothelial dysfunction, and hypotension. Endothelial CYB5R3 mitigates inflammatory signaling by LPS and tumor necrosis factor (TNF-) in a NOX4 dependent manner. In endothelial cells, CYB5R3 and NOX4 reside in close proximity on the mitochondrial outer membrane. NOX4s ability to generate H2O2 depends on the membrane translocation and activity of CYB5R3 and the presence of endogenous CoQ. NONSTANDARD Abbreviations and Acronyms [Table 1] Protein names are abbreviated as capital letters (e.g., CYB5R3), while the corresponding gene names are annotated as in italic lower cases (e.g., Cyb5r3).

cell biology↗

bioRxiv2021

Heme stimulates platelet mitochondrial oxidant production via the activation of toll-like receptor 4 signaling to mediate targeted granule secretion

Hemolysis is a pathological component of many diseases and is associated with thrombosis and vascular dysfunction. Hemolytic products, including cell-free hemoglobin and free heme directly activate platelets. However, the effect of hemolysis on platelet degranulation, a central process in not only thrombosis, but also inflammatory and mitogenic signaling, remains less clear. Our group showed that hemoglobin-induced platelet activation involved the production of mitochondrial reactive oxygen species (mtROS). However, the molecular mechanism by which extracellular hemolysis induces platelet mtROS production, and whether the mtROS regulate platelet degranulation remains unknown. Here, we demonstrate using isolated human platelets that cell free heme is a more potent agonist for platelet activation than hemoglobin, and stimulates the release of a specific set of molecules from the -granule of platelets, including the glycoprotein thrombospondin-1 (TSP-1). We uncover the mechanism of heme-mediated platelet mtROS production which is dependent on the activation of platelet TLR4 signaling and leads to the downstream phosphorylation of complex-V by the serine kinase Akt. Notably, inhibition of platelet TLR4 or Akt, or scavenging mtROS prevents heme-induced granule release in vitro. Further, heme-dependent granule release is significantly attenuated in vivo in mice lacking TLR4 or those treated with the mtROS scavenger MitoTEMPO. These data elucidate a novel mechanism of TLR4-mediated mitochondrial regulation, establish the mechanistic link between hemolysis and platelet degranulation, and begin to define the heme and mtROS-dependent platelet secretome. These data have implications for hemolysis-induced thrombo-inflammatory signaling and for the consideration of platelet mitochondria as a therapeutic target in hemolytic disorders. Key pointsO_LIHeme induces platelet mtROS production by inhibiting complex-V activity via TLR4 signaling. C_LIO_LIHeme stimulated platelet granule secretion is regulated by mtROS. C_LI

cell biology↗

bioRxiv2021

Generation of SIV resistant T cells and Macrophages from Nonhuman Primate Induced Pluripotent Stem Cells with Edited CCR5 locus

Adoptive therapies with genetically modified somatic T cells rendered HIV resistant have shown promise for AIDS therapy. A renewable source of HIV resistant human T cells from induced pluripotent stem cells (iPSCs) would further facilitate and broaden the applicability of these therapies. Here, we report successful targeting of the CCR5 locus in iPSCs generated from peripheral blood T cells (T-iPSCs) or fibroblasts (fib-iPSCs) from Mauritian Cynomolgus macaques (MCM), using CRISPR/Cas9 technology. We found that CCR5 editing does not affect pluripotency or hematopoietic and T cell differentiation potentials of fib-iPSCs. However, deletion of CCR5 in T-iPSCs leads to selective loss of their T cell redifferentiation potential without affecting myeloid development. T cells and macrophages produced from CCR5-edited MCM- iPSCs did not support replication of the CCR5-tropic simian immunodeficiency viruses SIVmac239 (T-cell tropic) and SIVmac316 (macrophage-tropic). Overall, these studies provide a platform for further exploration of AIDS therapies based on gene-edited iPSCs in a nonhuman primate preclinical model.

cell biology↗

bioRxiv2020

Characterization of Wnt Signaling Genes in Diaphorina citri

The Asian citrus psyllid, Diaphorina citri, is an insect vector that transmits Candidatus Liberibacter asiaticus, the causal agent of the Huanglongbing (HLB) or citrus greening disease. This disease has devastated Floridas citrus industry and threatens Californias industry as well as other citrus producing regions around the world. To find novel solutions to the disease, a better understanding of the vector is needed. The D. citri genome has been used to identify and characterize genes involved in Wnt signaling pathways. Wnt signaling is utilized for many important biological processes in metazoans, such as patterning and tissue generation. Curation based on RNA sequencing data and sequence homology confirm twenty four Wnt signaling genes within the D. citri genome, including homologs for beta-catenin, Frizzled receptors, and seven Wnt-ligands. Through phylogenetic analysis, we classify D. citri Wnt-ligands as Wg/Wnt1, Wnt5, Wnt6, Wnt7, Wnt10, Wnt11, and WntA. The D. citri version 3.0 genome with chromosomal length scaffolds reveals a conserved Wnt1-Wnt6-Wnt10 gene cluster with gene configuration similar to that in Drosophila melanogaster. These findings provide a greater insight into the evolutionary history of D. citri and Wnt signaling in this important hemipteran vector. Manual annotation was essential for identifying high quality gene models. These gene models can further be used to develop molecular systems, such as CRISPR and RNAi, that target and control D. citri populations, to manage the spread of HLB. Manual annotation of Wnt signaling pathways was done as part of a collaborative community annotation project (https://citrusgreening.org/annotation/index).

genomics↗

bioRxiv2020

Genotypic differences in wheat yield determinants within a NAM population based on elite parents

Future grain yield (GY) improvements require the identification of beneficial traits within the context of high yield potential and not just based on the pleiotropic effect of traits such as crop height and heading date. We evaluated 1937 lines from Nested Association Mapping (NAM) population derived from 13 bi-parental varietal crosses under field conditions. We selected 493 lines with similar time to anthesis to that of the two checks used in the study (across and within each family) which reduced the range of plant height in the selected lines. Yield components were measured in these 493 lines from which 231 lines were selected by excluding lines with lowest number of grains so excluded low yielding lines. Later the subset of 231 lines were evaluated in two field experiments (2016-17, CS1 and 2017-18, CS2). Numerical and physiological components of grain yield were measured. The two-step selection maximised GY within an acceptable range of variation for height and anthesis. GY in 231 lines showed very high GxE interaction. Taking both seasons together, we selected lines from upper and lower quartile GY groups to identify stable beneficial trait combinations for improved GY. Differences in GY were explained by grain number driven by increased spike dry weight at anthesis (SDWa) and fruiting efficiency (FE). Increased GY was accompanied by sink limitation. The data points towards increases in grain number as the route towards future GY increases in wheat breeding.

physiology↗

bioRxiv2019

Chromosomal length reference assembly for Diaphorina citri using single-molecule sequencing and Hi-C proximity ligation with manually curated genes in developmental, structural and immune pathways

BackgroundDiaphorina citri is a vector of "Candidatus Liberibacter asiaticus" (CLas), the gram-negative bacterial pathogen associated with citrus greening disease. Control measures rely on pesticides with negative impacts on the environment, natural ecosystems and human and animal health. In contrast, gene-targeting methods have the potential to specifically target the vector species and/or reduce pathogen transmission. ResultsTo improve the genomic resources needed for targeted pest control, we assembled a D. citri genome based on PacBio long reads followed by proximity ligation-based scaffolding. The 474 Mb genome has 13 chromosomal-length scaffolds. 1,036 genes were manually curated as part of a community annotation project, composed primarily of undergraduate students. We also computationally identified a total of 1,015 putative transcription factors (TFs) and were able to infer motifs for 337 TFs (33 %). In addition, we produced a genome-independent transcriptome and genomes for D. citri endosymbionts. ConclusionsManual annotation provided more accurate gene models for use by researchers and also provided an excellent training opportunity for students from multiple institutions. All resources are available on CitrusGreening.org and NCBI. The chromosomal-length D. citri genome assembly serves as a blueprint for the development of collaborative genomics projects for other medically and agriculturally significant insect vectors.

genomics↗