Biology subjects

Silver, J.

Publications and source records attributed to Silver, J..

8 recordsLinked to original sources

bioRxiv2026

Type II topoisomerase substrate geometry revealed through combined experiment and computation.

Type II topoisomerases (topo IIs) are essential enzymes that regulate DNA topology through a strand-passage mechanism in which a duplex DNA (transfer-segment) is passed through a transiently cleaved second duplex DNA (gate-segment). Biochemical and structural approaches have revealed critical details of the binding and cleavage of the gate-segment DNA. However, capture of the transfer-segment DNA has proven more difficult to resolve due to the transient nature of the interaction. Nonetheless, selection of the transfer segment with a specific conformation or orientation relative to the gate segment is predicted to govern aspects of topo II activity, including chiral discrimination and the ability to reduce topological complexity below equilibrium. To determine the conformation and orientation of the transfer-segment relative to the gate segment, we combined experimental single-molecule measurements of topo II unlinking a single DNA crossing with Brownian dynamics simulations of the DNA crossing. By correlating the unlinking rate with the geometric features of the DNA crossing, we obtain the complete three-dimensional preferred crossing geometry for strand passage. Strikingly, the preferred crossing geometries for Escherichia coli topoisomerase IV and Methanosarcina mazei topoisomerase VI are distinct and provide structural models of the DNA synapse selected for strand passage along with a mechanistic basis for their differing activities and biological functions. The approach we develop is generalizable, providing unique insights into the kinetic selection of DNA synapse structure.

biophysics↗

bioRxiv2024

Mitochondria isolated from male skeletal muscle contain a distinct 1 population of miRNA that are differentially expressed following acute exercise

Initially thought to localise at the cytosol and nucleus only, emerging evidence indicates that miRNAs also localise within the mitochondria where they could regulate diverse pathological and physiological processes. Therefore, the aim of the current study was to use small RNA sequencing to profile and compare the entire population of miRNAs in human skeletal muscle of healthy males in whole-tissue and in isolated mitochondria at rest and in response to acute endurance exercise. Twelve healthy males (age 26 {+/-} 4 years, mean {+/-} SD) cycled for 60 min at 70% VO2peak and muscle biopsies were collected at rest, immediately after and 3 h after exercise. The mitochondria were isolated by immunoprecipitation, further purified, then the resident RNA was sequenced to assess the mitochondrial transcriptome. Small RNA sequencing revealed that mitochondria isolated from male skeletal muscle tissue contain a small and distinct population of miRNAs. Of the approximately 110 mature miRNAs detected in skeletal muscle mitochondria at each time-point, the canonical myo-miRs miR-1, miR-133 and miR-206 families constituted on average 45% of total mitochondria miRNA reads. However, none of these canonical myo-miRs were differentially expressed in mitochondria following endurance exercise. One miRNA, hsa-miR-146b-3p, was differentially expressed in both whole muscle tissue and mitochondria when adjusted for multiple testing (FDR <0.05). Future research is now required to investigate miRNA-mRNA interactions in the mitochondria of skeletal muscle tissue. KEY POINTS SUMMARYO_LIEmerging evidence suggests microRNA are localised in the mitochondria of skeletal muscle cells and may play a role in regulating mitochondrial function. C_LIO_LIWe recently optimised an approach to isolate RNA from mitochondria of human skeletal muscle that is free from contaminating cytosolic RNA and suitable for RNA sequencing. C_LIO_LIIn this study we examined the microRNA population from male skeletal muscle mitochondria before and in the hours following 60 minutes of moderate intensity cycling exercise. C_LIO_LIWe detected around 110 microRNAs in skeletal muscle mitochondria, with the muscle enriched myo-miR such as miR-1, miR-133 and miR-206 families constituting almost half of the reads. However, only one microRNA, hsa-miR-146b-3p, was differentially expressed whereby it increased [~] 10-fold following exercise. C_LIO_LIThe results provide new knowledge into how mitochondria might be regulated at the subcellular level and in response to physiological stressors such as exercise. C_LI

cell biology↗

bioRxiv2024

Collagen I is a critical organizer of scarring and CNS regeneration failure

Although axotomized neurons retain the ability to initiate the formation of growth cones and attempt to regenerate after spinal cord injury, the scar area formed as a result of the lesion in most adult mammals contains a variety of reactive cells that elaborate multiple extracellular matrix and enzyme components that are not suitable for regrowth1,2. Newly migrating axons in the vicinity of the scar utilize upregulated LAR family receptor protein tyrosine phosphatases, such as PTP{sigma}, to associate with extracellular chondroitin sulphate proteoglycans (CSPGs), which have been discovered to tightly entrap the regrowing axon tip and transform it into a dystrophic non-growing endball. The scar is comprised of two compartments, one in the lesion penumbra, the glial scar, composed of reactive microglia, astrocytes and OPCs; and the other in the lesion epicenter, the fibrotic scar, which is made up of fibroblasts, pericytes, endothelial cells and inflammatory cells. While the fibrotic scar is known to be strongly inhibitory, even more so than the glial scar, the molecular determinants that curtail axon elongation through the injury core are largely uncharacterized. Here, we show that one sole member of the entire family of collagens, collagen I, creates an especially potent inducer of endball formation and regeneration failure. The inhibitory signaling is mediated by mechanosensitive ion channels and RhoA activation. Staggered systemic administration of two blood-brain barrier permeable-FDA approved drugs, aspirin and pirfenidone, reduced fibroblast incursion into the complete lesion and dramatically decreased collagen I, as well as CSPG deposition which were accompanied by axonal growth and considerable functional recovery. The anatomical substrate for robust axonal regeneration was provided by laminin producing GFAP+ and NG2+ bridging cells that spanned the wound. Our results reveal a collagen I-mechanotransduction axis that regulates axonal regrowth in spinal cord injury and raise a promising strategy for rapid clinical application.

neuroscience↗

bioRxiv2022

Recovery of forearm and fine digit function after chronic spinal cord injury by simultaneous blockade of inhibitory matrix CSPG production and the receptor PTPσ.

Spinal cord injuries, for which there are limited effective clinical treatments, result in enduring paralysis and hypoesthesia due, in part, to the inhibitory microenvironment that develops and limits regeneration/sprouting, especially during chronic stages. Recently, we discovered that targeted enzymatic modulation of the potently inhibitory chondroitin sulfate proteoglycan (CSPG) component of the extracellular and perineuronal net (PNN) matrix via Chondroitinase ABC (ChABC) can rapidly restore robust respiratory function to the previously paralyzed hemi-diaphragm after remarkably long times post-injury (up to 1.5 years) following a cervical level 2 lateral hemi-transection. Importantly, ChABC treatment at cervical level 4 in this chronic model also elicited rapid, albeit modest, improvements in upper arm function. In the present study, we sought to further optimize and elucidate the capacity for nerve sprouting and/or regeneration to restore gross as well as fine motor control of the forearm and digits at lengthy chronic stages post injury. However, instead of using ChABC, we utilized a novel and more clinically relevant systemic, non-invasive combinatorial treatment strategy designed to both reduce and overcome inhibitory CSPGs simultaneously and spatially extensively. Following a three-month upper cervical spinal hemi-lesion using adult female Sprague Dawley rats, we show that the combined treatment has a profound effect on functional recovery of the chronically paralyzed forelimb and paw, specifically during walking as well as precision movements of the digits. Our exciting pre-clinical findings will begin to enhance our understanding of the basic mechanisms underlying functionally beneficial regenerative events occurring at chronic injury stages for clinically relevant translational benefits. Significance statementOvercoming the persistent axon inhibitory environment following a functionally debilitating incomplete spinal cord lesion has long proven to be an elusive dilemma, especially months to years after the initial spinal injury. Current therapeutic and rehabilitative techniques for patients suffering from chronic cervical spinal insults minimally, if at all, address this structural hindrance and support limited return of crucial behaviors such as voluntary use of the arms and hands. Our investigation into the behavioral and anatomical consequences of systemically perturbing the high-affinity binding interaction between the receptor PTP{sigma} and the extracellular chondroitin sulfate proteoglycans highlight an underlying barrier to the restoration of forelimb/paw walking and eating behavior 12-weeks after a cervical spinal hemi-transection.

neuroscience↗

bioRxiv2022

Purification of mitochondria from skeletal muscle tissue for transcriptomic analyses reveals localisation of nuclear-encoded non-coding RNAs

Mitochondria are central to cellular function, particularly in metabolically active tissues such as skeletal muscle. Nuclear-encoded RNAs typically localise within the nucleus and cytosol but a small population may also translocate to subcellular compartments such as mitochondria. We aimed to investigate the nuclear-encoded RNAs that localise within the mitochondria of skeletal muscle cells and tissue. Intact mitochondria were isolated via immunoprecipitation (IP) followed by enzymatic treatments (RNase-A and proteinase-K) optimised to remove transcripts located exterior to mitochondria, making it amenable for high-throughput transcriptomic sequencing. Small-RNA sequencing libraries were successfully constructed from as little as 1.8 ng mitochondrial RNA input. Small-RNA sequencing of mitochondria from rat myoblasts revealed the enrichment of over 200 miRNAs. Whole-transcriptome RNA sequencing of enzymatically-purified mitochondria isolated by IP from skeletal muscle tissue showed a striking similarity in the degree of purity compared to mitoplast preparations which lack an outer mitochondrial membrane. In summary, we describe a novel, powerful sequencing approach applicable to animal and human tissues and cells that can facilitate the discovery of nuclear-encoded RNA transcripts localised within skeletal muscle mitochondria.

cell biology↗

bioRxiv2022

Cholesterol in the cargo membrane reduces kinesin-1 binding to microtubules in the presence of tau

Intracellular cargos are often membrane-bound and transported by microtubule-based motors in the presence of microtubule-associated proteins (MAPs). Whereas increasing evidence reveals how MAPs impact the interactions between motors and microtubules, critical questions remain about the impact of the cargo membrane on transport. Here we combined in vitro optical trapping with theoretical approaches to determine the effect of a lipid cargo membrane on kinesin-based transport in the presence of MAP tau. Attaching kinesins to a fluid lipid membrane decreases the inhibitory effect of tau in comparison to membrane-free cargos. Adding cholesterol, which reduces kinesin diffusion in the cargo membrane, amplifies the inhibitory effect of tau on kinesin in a dosage-dependent manner. Our findings can be understood in a framework in which kinesin diffusion in the cargo membrane counteracts binding-site occlusion by tau. Our study establishes a direct link between the physical properties of cargo membrane and MAP-based regulation of kinesin-1.

biophysics↗

bioRxiv2021

Chronic kidney disease alters Pin1 phosphorylation and parathyroid hormone mRNA binding proteins leading to secondary hyperparathyroidism

Parathyroid hormone (PTH) regulates calcium metabolism and bone strength. Chronic kidney disease leads to secondary hyperparathyroidism (SHP) which increases morbidity and mortality. In experimental SHP, the increased PTH gene expression is due to enhanced PTH mRNA stability, mediated by changes in its interaction with stabilizing AUF1 and destabilizing KSRP. Pin1 isomerizes target proteins and leads to KSRP dephosphorylation. In SHP, Pin1 isomerase activity is decreased and phosphorylated KSRP fails to bind PTH mRNA, resulting in high PTH mRNA stability and levels. However, the up- and down-stream mechanisms by which kidney failure stimulates the parathyroid remain elusive. We now reveal a pathway where kidney failure induces parathyroid Pin1 phosphorylation, linking Pin1, KSRP and PTH mRNA stability as key players for the high PTH expression in SHP. We identified by mass-spectrometry, changes in rat parathyroid proteome and phosphoproteome profiles induced by impaired renal function, including KSRP phosphorylation at Pin1 target sites. Furthermore, both acute and chronic kidney failure led to parathyroid-specific Pin1 Ser16 and Ser71 phosphorylation, which disrupts Pin1 activity. Accordingly, pharmacologic Pin1 inhibition, that mimics the decreased Pin1 activity in SHP, increased PTH expression ex-vivo in parathyroid organ cultures and in transfected cells, through the PTH mRNA protein-interacting element and KSRP phosphorylation at potential Pin1-binding motifs. Therefore, kidney failure leads to loss of parathyroid Pin1 activity by inducing Pin1 phosphorylation. This predisposes parathyroids to increase PTH production through impaired PTH mRNA decay that is dependent on KSRP phosphorylation. Pin1 and KSRP phosphorylation and the Pin1-KSRP-PTH mRNA axis thus drive SHP.

molecular biology↗

bioRxiv2021

IL-33 promotes innate lymphoid cell-dependent IFN-γ production required for innate immunity to Toxoplasma gondii

IL-33 is an alarmin required for resistance to the parasite Toxoplasma gondii, but its role in innate resistance to this infection is unclear. T. gondii infection promotes increased stromal cell expression of IL-33 and levels of parasite replication correlate with IL-33 release. In response to infection, a subset of innate lymphoid cells (ILC) emerges composed of IL-33R+ NK cells and ILC1s. In Rag-/- mice, where NK cells and ILC1 provide an innate mechanism of resistance to T. gondii, the loss of IL-33R reduced ILC responses and increased parasite replication. Furthermore, administration of IL-33 to Rag-/- mice resulted in a marked decrease in parasite burden, increased production of IFN-{gamma} and the recruitment and expansion of inflammatory monocytes associated with parasite control. These protective effects of exogenous IL-33 were dependent on endogenous IL-12p40 and the ability of IL-33 to enhance ILC production of IFN-{gamma}. These results highlight that IL-33 synergizes with IL-12 to promote ILC-mediated resistance to T. gondii.

immunology↗