Biology subjects

Gamage, K.

Publications and source records attributed to Gamage, K..

6 recordsLinked to original sources

bioRxiv2026

Genetic, intrinsic, and environmental determinants of innate immune cytokine responses in healthy four-year-old children

Abstract/SummaryInnate immune responses are crucial for host defence but vary markedly between individuals. Although determinants of this variation are well characterised in adults, data from healthy children remain scarce. We therefore profiled whole-blood cytokine responses to innate immune stimulation in 286 children aged approximately four years and examined genetic, host-intrinsic, and environmental correlates of response. Cytokine responses showed marked inter-individual heterogeneity and stimulus-specific patterns. The top 50 genetic variants explained a substantial proportion ([~]20-45%) of this variance across many stimulus conditions, including a biologically coherent association of the STING locus with cGAMP-induced cytokine production. In contrast, sex, age, adiposity, and perinatal variables showed limited or modest associations. Systemic inflammatory biomarkers of systemic inflammation (hsCRP, glycoprotein acetyls, granulocyte-to-lymphocyte ratio) were strongly positively associated with cytokine responses. Finally, seasonal population-level viral infection burden was positively associated with antiviral and inflammatory cytokine responses. Collectively, these findings advance our understanding of variation in early-life whole-blood cytokine responses, underscoring this developmental period as a critical window for understanding immune development trajectories relevant to long-term health.

immunology↗

bioRxiv2026

Measuring of the Quenching Rate of Atto-655 by Tryptophan

Intramolecular diffusion is an important, but often overlooked, property of intrinsically disordered proteins and plays an important role in folding, assembly and aggregation. Fluorescence resonance energy transfer (FRET) is used to observe reconfiguration over nanometer length scales while close range quenching over Angstrom length scales provides a complimentary view with different dynamics. There are several probe/quencher pairs that have been employed with varying levels of quantification of the quenching rate. Here we measure the electron transfer quenching parameters of the fluorophore Atto-655 by tryptophan using fluorescence correlation spectroscopy. Measurements with varying concentrations of quencher with low diffusion yield a distance dependent quenching rate. These parameters provide for a more quantitative analysis of measurements of intramolecular diffusion, particularly in crowded environments.

biophysics↗

bioRxiv2024

Post-translational Modification of α-Synuclein Modifies Monomer Dynamics and Aggregation Kinetics

The intrinsically disordered protein -Synuclein is identified as a major toxic aggregate in Parkinsons as well as several other neurodegenerative diseases. Recent work on this protein has focused on the effects of posttranslational modifications on aggregation kinetics. Among these, O-GlcNAcylation of -Synuclein has been observed to inhibit the aggregation propensity of the protein. Here we investigate the monomer dynamics of two O-GlcNAcylated -Synucleins, -Syn(gT72) and -Syn(gS87) and correlate them with the aggregation kinetics. We find that, compared to the unmodified protein, glycosylation at T72 makes the protein less compact and more diffusive while glycosylation at S87 makes the protein more compact and less diffusive. Based on a model of the earliest steps in aggregation, we predict that T72 should aggregate slower than unmodified protein, which is confirmed by ThT fluorescence measurements. In contrast, S87 should aggregate faster, which is not mirrored in ThT kinetics of later fibril formation but does not rule out a higher rate of formation of small oligomers. Together, these results show that posttranslational modifications do not uniformly affect aggregation propensity.

biophysics↗

bioRxiv2022

The Effect of Polymer Length in Phase Separation

Understanding the thermodynamics that drives liquid-liquid phase separation (LLPS) is quite important given the many numbers of diverse biomolecular systems undergoing this phenomenon. Regardless of the diversity, the processes underlying the formation of condensates exhibit physical similarities. Many studies have focused on condensates of long polymers, but very few systems of short polymer condensates have been observed and yet studied. Here we study a short polymer system of various lengths of poly-Adenine RNA and peptide formed by the RGRGG sequence repeats to understand the underlying thermodynamics of LLPS. We carried out MD simulations using the recently developed COCOMO coarse-grained (CG) model which revealed the possibility of condensates for lengths as short as 5-10 residues, which was then confirmed by experiment, making this one of the smallest LLPS systems yet observed. Condensation depends on polymer length and concentration, and phase boundaries were identified. A free energy model was also developed. Results show that the length dependent condensation is driven solely by entropy of confinement and identifies a negative free energy (-{Delta}G) of phase separation, indicating the stability of the condensates. The simplicity of this system will provide the basis for understanding more biologically realistic systems.

biophysics↗

bioRxiv2022

Characterizing Transient Protein-Protein Interactions by Trp-Cys Quenching and Computer Simulations

Transient protein-protein interactions occur frequently under the crowded conditions encountered in biological environments, yet they remain poorly understood. Here, tryptophan-cysteine quenching is introduced as an experimental approach that is ideally suited to characterize such interactions between proteins with minimal labeling due to its sensitivity to nano- to microsecond dynamics on sub-nanometer length scales. The experiments are paired with computational modeling at different resolutions including fully atomistic molecular dynamics simulations to provide interpretation of the experimental observables and add further insights at the molecular level. This approach is applied to model systems, villin variants and the drkN SH3 domain, in the presence of protein G crowders. It is demonstrated that Trp-Cys quenching experiments are able to not only distinguish between overall attractive and repulsive interactions between different proteins, but they can also discern variations in interaction preferences at different protein surface locations. The close integration between experiment and simulations also provides an opportunity to evaluate different molecular force fields for the simulation of concentrated protein solutions. Significance StatementBiological environments typically involve a variety of different proteins at very high concentrations where non-specific interactions are unavoidable. These interactions may go beyond simple crowding effects and involve transient contacts that may impact structure, dynamics, and ultimately function of proteins in vivo. While computer simulations have partially characterized such interactions, experimental data remain limited because established techniques are generally not well-suited to the characterization of dynamic processes on microsecond time and nanometer length scales. Tryptophan quenching by cysteine is introduced here as a new approach for studying transient protein encounters under concentrated conditions with the support of computational modeling. The study demonstrates that such experiments can resolve not just differences between different proteins but also residue-specific interaction preferences.

biophysics↗

bioRxiv2019

p75NTR and DR6 regulate distinct phases of axon degeneration demarcated by spheroid rupture.

The regressive events associated with trophic deprivation are critical for sculpting a functional nervous system. After nerve growth factor withdrawal, sympathetic axons maintain their structural integrity for roughly 18 hours (latent phase) followed by a rapid and near unison disassembly of axons over the next 3 hours (catastrophic phase). Here we examine the molecular basis by which axons transition from latent to catastrophic phases of degeneration following trophic withdrawal. Prior to catastrophic degeneration, we observed an increase in intra-axonal calcium. This calcium flux is accompanied by p75 neurotrophic factor receptor (NTR)-Rho-actin dependent expansion of calcium rich axonal spheroids that eventually rupture, releasing their contents to the extracellular space. Conditioned media derived from degenerating axons is capable of hastening transition into the catastrophic phase of degeneration. We also found that death receptor 6 (DR6) but not p75NTR is required for transition into the catastrophic phase in response to conditioned media but not for the intra-axonal calcium flux, spheroid formation, or rupture that occurs toward the end of latency. Our results support the existence of an inter-axonal degenerative signal that promotes catastrophic degeneration among trophically deprived axons.

neuroscience↗