Biology subjects

Reynolds, C. A.

Publications and source records attributed to Reynolds, C. A..

At least 19 recordsLinked to original sources

bioRxiv2026

AI-derived Protein Structures Validation: AlphaFold2 Models in the Twilight Zone

In any field, unquestioningly accepting artificial intelligence (AI) results should be considered bad practise. Here, we devised a comparative modelling-based strategy for validating protein structures that exploits the well-known observation that protein folds are far more conserved than protein sequences. We identify proteins with a similar fold to the AlphaFold-generated query protein and determine their structural alignment to the query. The hypothesis is that if the sequence alignment coincides with the structural alignment, then the structure is validated. The strategy is implemented on a helix-by-helix and strand-by-strand basis using a multi-template pairwise local profile alignment method that works well into the twilight zone. The method is illustrated by application to the transmembrane transporter PEPT1, for which the structure is known, and the S-deacylases ABHD13 and ABHD16A, for which only AI-generated models exist. ABHD16A is particularly challenging because a sequence alignment search with BLASTp does not reveal any structural homologues and therefore requires work with extremely remote homologues; however, both models are validated through this strategy and are stable during classical molecular dynamics simulations. The ability of the strategy to identify errors is assessed with reference to misaligned ABHD13 models and misfolded decoy proteins.

bioinformatics↗

bioRxiv2026

Deacylation of SNAP25 protein family isoforms reveals distinct substrate selectivities of α/β hydrolase domain (ABHD) deacylases

Reversible S-acylation controls protein trafficking, localisation and activity within cells, yet the limited understanding of the regulatory mechanisms of S-acylation/deacylation cycles represents one of the biggest obstacles in exploiting this prevalent post-translational modification for therapeutic purposes. Here, we identified ABHD13 and ABHD16A as novel deacylases regulating the SNAP25 family of SNARE proteins responsible for vesicular membrane fusion. We determined the mechanism and structural determinants for ABHD13-mediated deacylation and substrate selectivity towards SNAP25b, and identified a novel mechanism of acyl chain extraction for integral membrane deacylases. This is the first report of deacylase selectivity mapped to a single amino acid position in full-length proteins.

molecular biology↗

bioRxiv2025

Differential methylation clock ages across buffy coat (BC), peripheral blood mononuclear cells (PBMC), and saliva in individuals approaching midlife

Epigenetic aging prior to midlife is gaining interest as an intervenable period to address health and cognitive aging. Epigenetic changes may index DNA methylation aging rates, but methylation profiles may not be substitutable across tissues. We compared DNA methylation clocks and age acceleration in saliva, buffy coat (BC), and peripheral blood mononuclear cells (PBMC) collected in 91 individuals (7 unpaired, 20 siblings, 64 twins; 18 monozygotic (MZ), 14 dizygotic (DZ) pairs) from the Colorado Adoption/Twin Study of Lifespan behavioral development and cognitive aging (CATSLife1; Mean age=30.90 years [range=28.07-41.13]; 50.5% female). Across 15 DNA methylation clocks, chronological age and DNA methylation ages were moderately associated (mean Spearman correlations: r=0.37, Saliva; r=0.41, BC; r=0.38, PBMC). In mixed-effects models, saliva showed higher DNA methylation ages (B=3.83-16.46 years vs BC, p<0.001), whereas PBMC and BC were comparable (B=-0.06-0.39 years vs BC, p>0.447). The exception was the next-generation clock DunedinPace showing comparability (p=0.486). Similar patterns were observed for age acceleration estimates. Altogether, MZ pairs (meta-analytic r=0.49, 95%CI=0.32,0.66) and DZ pairs (meta-analytic r=0.38, 95%CI=0.25,0.52) were moderately correlated (Spearman), but MZ pairs showed heterogeneity across tissues (p<0.020): saliva was lower (mean r=0.33, SD=0.25) than BC (mean r=0.64, SD=0.10) and PBMC (mean r=0.49, SD=0.18). Next-generation PCGrimAge and DunedinPace clocks showed consistent zygosity correlations across tissues, while multi-tissue clocks (e.g., ZhangQ) showed comparable MZ-DZ correlations. While saliva-based DNA methylation is not a direct substitute for blood-based DNA methylation, BC and PBMC show comparability; nevertheless, all tissue types may be appropriate for DNA methylation aging studies when compared within tissues. Key Policy HighlightsO_LISaliva-based methylation is not a direct substitute for blood-based methylation. C_LIO_LISaliva-based DNA methylation clocks demonstrated relatively older methylation ages and faster age accelerations relative to blood-based DNA methylation clocks. C_LIO_LIBlood-based DNA methylation clocks were comparable across buffy coat and peripheral blood mononuclear cell tissues. C_LIO_LITwin similarity, particularly among identical twins, was higher for blood-based DNA methylation clocks and newer generation DNA methylation clocks. C_LIO_LIThree clocks (one first-generation multi-tissue and two next-generation) showed consistency and/or comparability across tissues and showed moderate to strong correlations among identical and fraternal twins, supporting the importance of tool development. C_LI

genetics↗

bioRxiv2024

A Multi-Strain, Biofilm-Forming Cocktail of Bacillus spp. and Pediococcus spp. Alters the Microbial Composition on Polyethylene Calf Housing Surfaces

Application of a beneficial microbial cocktail of Bacillus spp. and Pediococcus spp. was evaluated first for adherence to polyethylene calf hutch material, and second, to determine if application in situ to individual calf hutches post-cleaning influenced surface recolonization by enteric pathogens. Three treatments were utilized: 1) no application (NC), 2) chlorine-free, distilled water (DW), or 3) an application of a microbial inoculant containing Bacillus spp. and Pediococcus spp. at a concentration of 0.4 g/m2 of hutch space (LF). Thirty-six 15 x 15 cm pieces of naive, sterile polyethylene calf hutch material received either NC or LF, were incubated at 28{degrees}C, and bacterial growth was evaluated by total aerobic plate counts at 24, 48, and 72 h post-application. Thirty polyethylene calf hutches (n = 10/treatment) were randomized to either NC, DW, or LF 24 h after cleaning. Calves were placed in the hutches 24 h after treatment application and monitored daily for 28 d. In situ surface samples were randomized by time from five unique locations within the calf hutch interior: 24 h post-cleaning, then 24 h, 7 d, 14 d, and 21 d post-application. Total aerobic plate counts and culture-independent approaches RT-qPCR and 16S amplicon sequencing were used to detect and identify the composition of the bacterial community in situ. The bacteria in the inoculant were able to successfully colonize on polyethylene, and application to individual polyethylene calf housing in situ influenced microbial diversity and reduced the presence of some undesirable bacteria on high-contact interior surfaces. IMPORTANCEDue to its multifactorial nature, neonatal calf diarrhea can be difficult to manage on farms. Clean housing environments are a critical disease control point, especially for calves less than one month of age. Application of a beneficial biofilm-forming bacterial product after cleaning of neonatal calf housing may influence the microbial communities present on the surface, particularly those that may present disease risk to calves in early life.

microbiology↗

bioRxiv2024

In vivo functional profiling and structural characterisation of the human Glp1r A316T variant

Glucagon-like peptide-1 receptor agonists (GLP-1RAs) are effective therapies for type 2 diabetes (T2D) and obesity, yet patient responses are variable. Variation in the human Glp1r gene might be directly linked to therapeutic responses. A naturally occurring missense variant, A316T, protects against T2D and cardiovascular disease. Here, we have generated and characterised a human Glp1r A316T mouse model. Human Glp1rA316T/A316T mice displayed lower fasting blood glucose versus wildtype littermates, even under metabolic stress, and exhibited alterations in islet cytoarchitecture and /{beta}-cell identity under a high-fat, high-sucrose diet. This was however associated with blunted responses to GLP-1RAs in vivo. Further investigations in rodent and human {beta}-cell models demonstrated that human Glp1r A316T exhibits characteristics of constitutive activation but dampened GLP-1RA responses. Results are further supported by cryo-EM analyses and molecular dynamics simulations of GLP-1R A316T structure, collectively demonstrating that the A316T variant governs basal GLP-1R activity and pharmacological responses to GLP-1R-targeting therapies. TeaserThe Glp1r A316T missense variant displays improved glucose tolerance but impaired pharmacological incretin responses in vivo.

cell biology↗

bioRxiv2022

Hidden GPCR structural transitions addressed by multiple walker supervised molecular dynamics (mwSuMD)

The structural basis for the pharmacology of G protein-coupled receptors (GPCRs), the most abundant membrane proteins and the target of about 35% of approved drugs, is still a matter of intense study. What makes GPCRs challenging to study is the inherent flexibility and the metastable nature of interaction with extra-and intracellular partners that drive their effects. Here, we present a molecular dynamics (MD) adaptive sampling algorithm, namely multiple walker supervised molecular dynamics (mwSuMD), to address complex structural transitions involving GPCRs without energy input. We first report the binding and unbinding of the vasopressin peptide from its receptor V2. Successively, we present the complete transition of the glucagon-like peptide-1 receptor (GLP-1R) from inactive to active, agonist and Gs-bound state, and the GDP release from Gs. To our knowledge, this is the first time the whole sequence of events leading from an inactive GPCR to the GDP release is simulated without any energy bias. We demonstrate that mwSuMD can address complex binding processes intrinsically linked to protein dynamics out of reach of classic MD.

bioinformatics↗

bioRxiv2022

Is The Stalk of the SARS-CoV-2 Spike Protein Druggable?

The SARS-CoV-2 virus spike protein (SP) is the vector of the virus infectivity. The high propensity to mutate in key regions responsible for the recognition of the human angiotensinconverting enzyme 2 (hACE2) or the antibodies produced by the immune system following infection or vaccination makes subunit 1 of the SP a difficult to target and, to date, efforts have not delivered any ACE2 binding inhibitor yet. The inherent flexibility of the stalk region, within subunit S2, is key to SARS-CoV-2 high infectivity because it facilitates the receptor binding domain encounter with ACE2. Thus, it could be a valuable therapeutic target. By employing a fragment-based strategy, we computationally studied the druggability of the conserved part of the SP stalk by means of an integrated approach that combines molecular docking with high-throughput molecular dynamics simulations. Our results suggest that the druggability of the stalk is challenging and provide the structural basis for such difficulty.

bioinformatics↗

bioRxiv2022

The genetic and environmental etiology of blood-based biomarkers related to the risks of Alzheimer's Disease in a population-based sample of early old age men

The amyloid-tau-neurodegeneration (ATN) framework has led to an increased focus on Alzheimers disease (AD) biomarkers. The cost and invasiveness of obtaining biomarkers via cerebrospinal fluid has motivated efforts to develop sensitive blood-based biomarkers. Although AD is highly heritable, the biometric genetic and environmental etiology of blood-based biomarkers has never been explored. We therefore, analyzed plasma beta-amyloid (A{beta}40, A{beta}42, A{beta}42/40), total tautau (t-tautau), and neurofilament light (NFL) biomarkers in a sample of 1,050 men aged 60 to 73 years (m=68.2, SD=2.5) from the Vietnam Era Twin Study of Aging (VETSA). Unlike A{beta} and tautau, NFL does not define AD; however, as a biomarker of neurodegeneration it serves as the N component in the ATN framework. Univariate estimates suggest that familial aggregation in A{beta}42, A{beta}42/40, t-tau, and NFL is entirely explained by additive genetic influences accounting for 40%-58% of the total variance. All remaining variance is associated with unshared or unique environmental influences. For A{beta}40, a additive genetic (31%), shared environmental (44%), and unshared environmental (25%) influences contribute to the total variance. In the more powerful multivariate analysis of A{beta}42, A{beta}40, t-tau, and NFL, heritability estimates range from 32% to 58%. A{beta}40 and A{beta}42 are statistically genetically identical (rg = 1.00, 95%CI = 0.92,1.00) and are also moderately environmentally correlated (re = 0.66, 95%CI = 0.59, 0.73). All other genetic and environmental associations were non-significant or small. Our results suggest that plasma biomarkers are heritable and that A{beta}40 and A{beta}42 share the same genetic influences, whereas the genetic influences on plasma t-tau and NFL are mostly unique and uncorrelated with plasma A{beta} in early old-age men.

genetics↗

bioRxiv2022

Understanding The Role of Heparinoids on the SARS-CoV-2 Spike Protein through Molecular Dynamics Simulations

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to pose a threat, with an estimated number of deaths exceeding 5 million. SARS-CoV-2 entry into the cell is mediated by its transmembrane spike glycoprotein (S protein), and the angiotensin-converting enzyme 2 (ACE2) receptor on the human cell surface. The extracellular heparan sulphate (EcHS) enhances the S protein binding through a mechanism that is still unknown. Surprisingly, low molecular weight heparin (LMWH) and HS in the disaccharide form (dHS) hinder the S protein binding to ACE2, despite the similarity with EcHS. We investigated the molecular mechanism behind this inhibition through molecular dynamics (MD) simulations to understand the interaction pattern of the heparinoids with S protein and ACE2 receptor.

bioinformatics↗

bioRxiv2021

The genetic etiology of longitudinal measures of predicted brain ageing in a population-based sample of mid to late-age males

Magnetic resonance imaging data are being used in statistical models to predicted brain ageing (PBA) and as biomarkers for neurodegenerative diseases such as Alzheimers Disease. Despite their increasing application, the genetic and environmental etiology of global PBA indices is unknown. Likewise, the degree to which genetic influences in PBA are longitudinally stable and how PBA changes over time are also unknown. We analyzed data from 734 men from the Vietnam Era Twin Study of Aging with repeated MRI assessments between the ages 52 to 72 years. Biometrical genetic analyses twin models revealed significant and highly correlated estimates of additive genetic heritability ranging from 59% to 75%. Multivariate longitudinal modelling revealed that covariation between PBA at different timepoints could be explained by a single latent factor with 73% heritability. Our results suggest that genetic influences on PBA are detectable in midlife or earlier, are longitudinally very stable, and are largely explained by common genetic influences. HighlightsWe explored the genetic and environmental etiology of MRI-based predicted brain age (PBA) in a longitudinal sample of males starting in midlife. Genetic influences on PBA are detectable in midlife or earlier, are longitudinally very stable, and largely explained by common genetic influences.

genetics↗

bioRxiv2021

Determining the stability of genome-wide factors in BMI between ages 40 to 69 years.

Genome-wide association studies (GWAS) have successfully identified common variants associated with BMI. However, the stability of genetic variation influencing BMI from midlife and beyond is unknown. By analyzing BMI data collected from 165,717 men and 193,073 women from the UKBiobank, we performed BMI GWAS on six independent five-year age intervals between 40 and 73 years. We then applied genomic structural equation modeling (gSEM) to test competing hypotheses regarding the stability of genetic effects for BMI. LDSR genetic correlations between BMI assessed between ages 40 to 73 were all very high and ranged 0.89 to 1.00. Genomic structural equation modeling revealed that genetic variance in BMI at each age interval could not be explained by the accumulation of any age-specific genetic influences or autoregressive processes. Instead, a common set of stable genetic influences appears to underpin variation in BMI from middle to early old age in men and women alike.

genetics↗

bioRxiv2021

Dynamics of GLP-1R peptide agonist engagement are correlated with kinetics of G protein activation

The glucagon-like peptide-1 receptor (GLP-1R) has broad physiological roles and is a validated target for treatment of metabolic disorders. Despite recent advances in GLP-1R structure elucidation, detailed mechanistic understanding of how different peptides generate profound differences in G protein-mediated signalling is still lacking. Here we have combined cryo-electron microscopy, molecular dynamics simulations, receptor mutagenesis and pharmacological assays, to interrogate the mechanism and consequences of GLP-1R binding by four peptide agonists; glucagon-like peptide-1, oxyntomodulin, exendin-4 and exendin-P5. These data revealed that distinctions in peptide N-terminal interactions and dynamics with the GLP-1R transmembrane domain are reciprocally associated with differences in the allosteric coupling to G proteins. In particular, transient interactions with residues at the base of the binding cavity correlate with enhanced kinetics for G protein activation, providing a rationale for differences in G protein-mediated signalling efficacy from distinct agonists.

biochemistry↗

bioRxiv2021

Within-sibship GWAS improve estimates of direct genetic effects

Estimates from genome-wide association studies (GWAS) represent a combination of the effect of inherited genetic variation (direct effects), demography (population stratification, assortative mating) and genetic nurture from relatives (indirect genetic effects). GWAS using family-based designs can control for demography and indirect genetic effects, but large-scale family datasets have been lacking. We combined data on 159,701 siblings from 17 cohorts to generate population (between-family) and within-sibship (within-family) estimates of genome-wide genetic associations for 25 phenotypes. We demonstrate that existing GWAS associations for height, educational attainment, smoking, depressive symptoms, age at first birth and cognitive ability overestimate direct effects. We show that estimates of SNP-heritability, genetic correlations and Mendelian randomization involving these phenotypes substantially differ when calculated using within-sibship estimates. For example, genetic correlations between educational attainment and height largely disappear. In contrast, analyses of most clinical phenotypes (e.g. LDL-cholesterol) were generally consistent between population and within-sibship models. We also report compelling evidence of polygenic adaptation on taller human height using within-sibship data. Large-scale family datasets provide new opportunities to quantify direct effects of genetic variation on human traits and diseases.

genetics↗

bioRxiv2021

Partial agonism improves the anti-hyperglycaemic efficacy of an oxyntomodulin-derived GLP-1R/GCGR co-agonist

ObjectiveGlucagon-like peptide-1 and glucagon receptor (GLP-1R/GCGR) co-agonism can maximise weight loss and improve glycaemic control in type 2 diabetes and obesity. In this study we investigated the cellular and metabolic effects of modulating the balance between G protein activation and {beta}-arrestin-2 recruitment at GLP-1R and GCGR using oxyntomodulin (OXM)-derived co-agonists. This strategy has been previously shown to improve the duration of action of GLP-1R mono-agonists by reducing target desensitisation and downregulation. MethodsDipeptidyl dipeptidase-4 (DPP-4)-resistant OXM analogues were generated and assessed for a variety of cellular readouts. Molecular dynamic simulations were used to gain insights into the molecular interactions involved. In vivo studies were performed in mice to identify effects on glucose homeostasis and weight loss. ResultsLigand-specific reductions in {beta}-arrestin-2 recruitment led to reduced GLP-1R internalisation and prolonged glucose-lowering action in vivo. The putative benefits of GCGR agonism were retained, with equivalent weight loss compared to the GLP-1R mono-agonist liraglutide in spite of a lesser degree of food intake suppression. The compounds tested showed only a minor degree of biased agonism between G protein and {beta}-arrestin-2 recruitment at both receptors and were best classified as partial agonists for the two pathways measured. ConclusionsDiminishing {beta}-arrestin-2 recruitment may be an effective way to increase the therapeutic efficacy of GLP-1R/GCGR co-agonists. These benefits can be achieved by partial rather than biased agonism.

pharmacology and toxicology↗

bioRxiv2020

Deciphering the Agonist Binding Mechanism to the Adenosine A1 Receptor

Despite being amongst the most characterized G protein-coupled receptors (GPCRs), adenosine receptors (ARs) have always been a difficult target in drug design. To date, no agonist other than the natural effector and the diagnostic regadenoson has been approved for human use. Recently, the structure of the adenosine A1 receptor (A1R) was determined in the active, Gi protein complexed state; this has important repercussions for structure-based drug design. Here, we employed supervised molecular dynamics simulations and mutagenesis experiments to extend the structural knowledge of the binding of selective agonists to A1R. Our results identify new residues involved in the association and dissociation pathway, suggest the binding mode of N6-cyclopentyladenosine (CPA) related ligands, and highlight the dramatic effect that chemical modifications can have on the overall binding mechanism.

pharmacology and toxicology↗

bioRxiv2020

A Multisite Model of Allosterism for the Adenosine A1 Receptor

Despite being a target for about one-third of approved drugs, G protein-coupled receptors (GPCRs) still represent a tremendous reservoir for therapeutic strategies against countless diseases. For example, several cardiovascular and central nervous systems conditions could benefit from clinical agents that activate the adenosine 1 receptor (A1R), however, the pursuit of A1R agonists for clinical use are usually impeded by both on- and off-target side effects. One of the possible strategies to overcome this issue is the development of positive allosteric modulators (PAMs) capable of selectively enhancing the effect of a specific receptor subtype and triggering functional selectivity (a phenomenon also referred to as bias). Intriguingly, besides enforcing the effect of agonists upon binding to an allosteric site, most of the A1R PAMs display intrinsic partial agonism and orthosteric competition with antagonists. To rationalize this behaviour, we simulated the binding of the prototypical PAMs PD81723 and VCP171, the antagonist 13B, and the bitopic agonist VCP746. We propose that a single PAM can bind several A1R sites rather than a unique allosteric pocket, reconciling the structure-activity relationship and the mutagenesis results.

biophysics↗

bioRxiv2020

Non-opioid analgesia based on Gα signalling bias

The development of therapeutic agonists for G protein-coupled receptors (GPCRs) is hampered by the propensity of GPCRs to couple to multiple intracellular signalling pathways. This promiscuous coupling leads to numerous downstream cellular effects, some of which are therapeutically undesirable. This is especially the case for adenosine A1 receptors (A1Rs) whose clinical potential is undermined by the sedation and cardiorespiratory depression caused by conventional agonists. We have discovered that the A1R-selective agonist, BnOCPA, is a potent and powerful analgesic but does not cause sedation, bradycardia, hypotension or respiratory depression. This unprecedented discrimination between native A1Rs arises from BnOCPAs unique and exquisitely selective activation of Gob among the six Gi/o subtypes, and in the absence of {beta}-arrestin recruitment. BnOCPA thus demonstrates a highly-specific G-selective activation of the native A1R, sheds new light on GPCR signalling, and reveals new possibilities for the development of novel therapeutics based on the far-reaching concept of selective G agonism. Short summaryWe describe the selective activation of an adenosine A1 receptor-mediated intracellular pathway that provides potent analgesia in the absence of sedation or cardiorespiratory depression, paving the way for novel medicines based on the far-reaching concept of selective G agonism.

physiology↗

bioRxiv2019

A decade of epigenetic change in aging twins: genetic and environmental contributions to longitudinal DNA methylation

Summary/AbstractO_ST_ABSBackgroundC_ST_ABSEpigenetic changes may result from the interplay of environmental exposures and genetic influences and contribute to differences in age-related disease, disability and mortality risk. However, the etiologies contributing to stability and change in DNA methylation have rarely been examined longitudinally. MethodsWe considered DNA methylation in whole blood leukocyte DNA across a 10-year span in two samples of same-sex aging twins: (a) Swedish Adoption Twin Study of Aging (SATSA; N = 53 pairs, 53% female; 62.9 and 72.5 years, SD=7.2 years); (b) Longitudinal Study of Aging Danish Twins (LSADT; N = 43 pairs, 72% female, 76.2 and 86.1 years, SD=1.8 years). Joint biometrical analyses were conducted on 358,836 methylation probes in common. Bivariate twin models were fitted, adjusting for age, sex and country. ResultsOverall, results suggest genetic contributions to DNA methylation across 358,836 sites tended to be small and lessen across 10 years (broad heritability M=23.8% and 18.0%) but contributed to stability across time while person-specific factors explained emergent influences across the decade. Aging-specific sites identified from prior EWAS and methylation age clocks were more heritable than background sites. The 5,037 sites that showed the greatest heritable/familial-environmental influences (p<1E-07) were enriched for immune and inflammation pathways while 2,020 low stability sites showed enrichment in stress-related pathways. ConclusionsAcross time, stability in methylation is primarily due to genetic contributions, while novel experiences and exposures contribute to methylation differences. Elevated genetic contributions at age-related methylation sites suggest that adaptions to aging and senescence may be differentially impacted by genetic background.

genetics↗