Biology subjects

Gordon, J.

Publications and source records attributed to Gordon, J..

33 records · Page 2Linked to original sources

bioRxiv2022

A cell-based, spike protein binding assay highlights differences in antibody neutralising capacity for SARS-CoV-2 variants

The engagement of the SARS-CoV-2 spike protein with ACE2 is a critical step for viral entry to human cells and accordingly blocking this interaction is a major determinant of the efficacy of monoclonal antibody therapeutics and vaccine-elicited serum antibodies. The emergence of SARS-CoV-2 variants necessitates the development of adaptable assays that can be applied to assess the effectiveness of therapeutics. Through testing of a range of recombinant spike proteins, we have developed a cell based, ACE2/spike protein binding assay that characterises monoclonal anti-spike protein antibodies and neutralising antibodies in donor serum. The assay uses high-content imaging to quantify cell bound spike protein fluorescence. Using spike proteins from the original Wuhan SARS-CoV-2 virus, as well as the delta and omicron variants, we identify differential blocking activity of three monoclonal antibodies directed against the spike receptor binding domain. Importantly, biological activity in the spike binding assay translated to efficacy in a SARS-CoV-2 infection assay. Hence, the spike binding assay has utility to monitor anti-spike antibodies against the major known SARS-CoV-2 variants and is readily adaptable to quantify impact of antibodies against new and emerging SARS-CoV-2 variants.

cell biology↗

bioRxiv2022

Skeletal muscle-derived extracellular vesicles are altered with chronic contractile activity

Extracellular vesicles (EVs) are small lipid bilayer-delimited particles that are secreted by all cells, playing a central role in cellular communication. EVs are released from skeletal muscle during exercise, but the effects of contractile activity on skeletal muscle-derived EVs (Skm-EVs) are poorly understood due to the challenges in distinguishing Skm-EVs derived from exercising muscle in vivo. Using tunable resistive pulse sensing (TRPS), we previously demonstrated that chronic contractile activity (CCA) increased the secretion of Skm-EVs from C2C12 myotubes, while their size and zeta potential remained unchanged. Here, we aimed to determine whether similar results could be obtained using an alternative method of EV characterization, dynamic light scattering (DLS). C2C12 myoblasts were differentiated into myotubes, and electrically paced (3h/day x 4days @14V, C-PACE EM, IonOptix) to mimic chronic exercise in vitro. EVs were isolated from conditioned media of control and stimulated myotubes using differential ultracentrifugation, and characterized based on size and zeta potential. The mean size of EVs from chronically stimulated myotubes (CCA-EVs, 132 nm) was 26% smaller than control (CON-EVs, 178 nm). Size distribution analysis revealed that CCA-EVs were enriched in small EVs (100-150 nm), while CON-EVs were largely composed of 200-250 nm sized vesicles. Additionally, zeta potential was 27% lower in CCA-EVs compared to CON-EVs. Our data indicate that the effect of CCA on facilitating the release of smaller, more stable EVs, is a robust finding, reproducible by multiple methods of EV characterization. Future studies investigating the mechanisms by which CCA influences Skm-EV biogenesis and secretion are warranted.

cell biology↗

bioRxiv2022

Differential Impact of Doxorubicin Dose on Cell Death and Autophagy Pathways during Acute Cardiotoxicity.

Doxorubicin (DOX) is an effective anthracycline used in chemotherapeutic regimens for a variety of haematological and solid tumors. However, its utility remains limited by its well-described, but poorly understood cardiotoxicity. Despite numerous studies describing various forms of regulated cell death and their involvement in DOX-mediated cardiotoxicity, the predominate form of cell death remains unclear. Part of this inconsistency lies in a lack of standardization of in vivo and in vitro model design. To this end, the objective of this study was to characterize acute low- and high-dose DOX exposure on cardiac structure and function in C57BL/6N mice, and evaluate regulated cell death pathways and autophagy both in vivo and in cardiomyocyte culture models. Acute low-dose DOX had little impact on cardiac structure or function; however, acute high-dose DOX elicited substantial cardiac necrosis resulting in diminished cardiac mass and volume, with a corresponding reduced cardiac output, and without impacting ejection fraction or fibrosis. Low-dose DOX consistently activated caspase-signaling with evidence of mitochondrial permeability transition. However, acute high-dose DOX had only modest impact on common necrotic signaling pathways, but instead led to an inhibition in autophagic flux. Intriguingly, when autophagy was inhibited in cultured cardiomyoblasts, DOX-induced necrosis was enhanced. Collectively, these observations implicate inhibition of autophagy flux as an important component of the acute necrotic response to DOX, but also suggest that acute high-dose DOX exposure does not recapitulate the disease phenotype observed in human cardiotoxicity.

physiology↗

bioRxiv2021

Discovery of imidazole-based inhibitors of P. falciparum cGMP-dependent protein kinase

The discovery of new targets for treatment of malaria and in particular those aimed at the pre-erythrocytic stage in the life cycle, advanced with the demonstration that orally administered inhibitors of Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) could clear infection in a murine model. This enthusiasm was tempered by unsatisfactory safety and/or pharmacokinetic issues found with these chemotypes. To address the urgent need for new scaffolds, this manuscript presents initial structure-activity relationships in an imidazole scaffold at four positions, representative in vitro ADME, hERG characterization and cell-based anti-parasitic activity. This series of PfPKG inhibitors has good in vitro PfPKG potency, low hERG activity and cell-based anti-parasitic activity against multiple Plasmodium species that appears to correlate with in vitro potency.

pharmacology and toxicology↗

bioRxiv2021

Interaction between sperm whales (Physeter macrocephalus) and northern bottlenose whales (Hyperoodon ampullatus) and deep-water trawlers in the Northwest Atlantic

Commercial fisheries have increased in all the worlds oceans with diverse unintended impacts on marine ecosystems. As a result of resource overlap, interactions between cetaceans and fisheries are a common occurrence and, in many cases, these give rise to significant conservation issues. Research on the distribution and types of such interactions is important for efficient management. In this study, we describe the behaviors of two whale species: sperm whales (Physeter macrocephalus) and northern bottlenose whales (Hyperoodon ampullatus), interacting with benthic trawlers fishing off the eastern Grand Banks of the western North Atlantic in 2007. Whale interactions were only observed when vessels were targeting Greenland halibut (Reinnhardtius hippoglossoides) in deep-water fishing areas and were most common during net hauling. Sperm whales and northern bottlenose whales appeared to engage in feeding behavior close to the surface during hauling, especially during the latter stages, suggesting they targeted fish escapees rather than discards. Using photo-identification methods, seven individual sperm whales were identified with multiple resights of six individuals being recorded over an almost two month period. The maximum distance between two resights was 234 km, suggesting individual sperm whales were repeatedly targeting and even following fishing vessels over multiple days and between fishing areas. By contrast, there were no photographic resights of individual northern bottlenose whales within this study, or with substantial photo-identification catalogues from other adjacent high density areas, suggesting that individuals of this species may be less likely to follow vessels or move between areas. This study documents the earliest confirmed records of northern bottlenose whales in this remote region. These interactions and high encounter rates may indicate that adjacent populations are recovering from the previous century of commercial whaling. Our study provides new insights and details on whale-fisheries interactions, which can inform future research and help managers understand the real and perceived impacts on fisheries and whales.

animal behavior and cognition↗

bioRxiv2021

Acidity and sulfur oxidation intermediate concentrations controlled by O2-driven partitioning of sulfur oxidizing bacteria in a mine tailings impoundment

Acidification of freshwater in mining impacted areas is a major global environmental problem catalyzed by sulfur-oxidizing bacteria (SOB). To date, little is known about the active bacteria in mine tailings impoundments and their environmental niches. Here, biological sulfur oxidation was investigated over four years in a mine tailings impoundment, integrating sulfur geochemistry, genome-resolved metagenomics and metatranscriptomics. We demonstrated oxygen driven niche partitioning of SOB and their metabolic pathways that explain acidity generation and thiosulfate persistence. Four chemolithoautotrophic SOB, Halothiobacillus, Thiobacillus, Sulfuricurvum and Sediminibacterium comprised 37% to 73% of the analyzed communities. The impoundment waters alternated between the dominance of Halothiobacillus versus a Thiobacillus, Halothiobacillus, Sulfuricurvum and Sediminibacterium consortia. Halothiobacillus dominance was associated with lower pH values ([~]4.3), higher [H+]/[SO42-] and lower [S2O32-], collectively indicative of extensive sulfur oxidation. Halothiobacillus, which couple sulfur oxidation via the Sox pathway to aerobic respiration or NO2- reduction, were present throughout the depth profile, yet their expression of sox genes occurred only in upper highly oxygenated waters. Conversely, when consortia of Thiobacillus, Halothiobacillus, Sulfuricurvum and Sediminibacterium dominated, recycling/disproportionating reactions were more prevalent. Thiobacillus, which dominated deeper micro-oxic/anoxic waters, oxidized sulfur primarily through the rDSR pathway, coupled to NO3-/NO2- reduction, resulting in lower [H+]/[SO42-] and higher [S2O32-] relative to upper waters. These field results mirror the Sox/rDSR-geochemical patterns of experimental SOB enrichments and reveal opportunities for biological treatments of recalcitrant reduced sulfur compounds, as well as gene-based monitoring and in situ RNA detection to predict the onset of problematic geochemistry.

microbiology↗

bioRxiv2021

Discovery of novel potent, cell-permeable inhibitors of P. falciparum cGMP-dependent protein kinase.

The discovery of new targets for treatment of malaria advanced with the demonstration that orally administered inhibitors of Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) could clear infection in a murine model. This enthusiasm was tempered by unsatisfactory safety and/or pharmacokinetic issues found with these chemotypes. To address the urgent need for new scaffolds, we recently reported the discovery and optimization of novel, potent isoxazole-based PfPKG inhibitors that lacked any obvious safety warnings. This manuscript presents representative in vitro ADME, hERG characterization and cell-based antiparasitic activity of these PfPKG inhibitors. We also report the discovery and structure-activity relationships of a new series with good potency, low hERG activity and cell-based anti-parasitic activity comparable to a literature standard.

pharmacology and toxicology↗

bioRxiv2021

The breast pre-cancer atlas illustrates the molecular and micro-environmental diversity of ductal carcinoma in situ

Micro-environmental and molecular factors mediating the progression of Breast Ductal Carcinoma In Situ (DCIS) are not well understood, impeding the development of prevention strategies and the safe testing of treatment de-escalation. We addressed methodological barriers and characterized the mutational, transcriptional, histological and microenvironmental landscape across 85 multiple micro-dissected regions from 39 cases. Most somatic alterations, including whole genome duplications, were clonal, but genetic divergence increased with physical distance. Phenotypic and subtype heterogeneity frequently associated with underlying genetic heterogeneity and regions with low-risk features preceded those with high-risk features according to the inferred phylogeny. B- and T-lymphocytes spatial analysis identified 3 immune states, including an epithelial excluded state located preferentially at DCIS regions, and characterized by histological and molecular features of immune escape, independently from molecular subtypes. Such breast pre-cancer atlas with uniquely integrated observations will help scope future expansion studies and build finer models of outcomes and progression risk.

genomics↗

bioRxiv2020

Trunk Control during Gait: Walking with Wide and Narrow Step Widths Present Distinct Challenges

The active control of the trunk plays an important role in frontal plane gait stability. We characterized trunk control in response to different step widths using a novel feedback system and examined the different effects of wide and narrow step widths as they each present unique task demands. Twenty healthy young adults walked on a treadmill at 1.25 m/s at five prescribed step widths: 0.33, 1.67, 1, 1.33, 1.67 times preferred step width. Motion capture was used to record trunk kinematics, and surface electromyography was used to record longissimus muscle activation bilaterally. Vector coding was used to analyze coordination between pelvis and thorax segments of the trunk. Results showed that while center of mass only varied across step width in the mediolateral direction, trunk kinematics in all three planes were affected by changes in step width. Angular excursions of the trunk segments increased only with wider widths in the transverse plane. Thorax-pelvis kinematic coordination was affected more by wider widths in transverse plane and by narrower widths in the frontal plane. Peak longissimus activation and bilateral co-activation increased as step widths became narrower. As a control task, walking with varied step widths is not simply a continuum of adjustments from narrow to wide. Rather, narrowing step width and widening step width from the preferred width represent distinct control challenges that are managed in different ways. This study provides foundation for future investigations on the trunk during gait in different populations.

neuroscience↗

bioRxiv2020

Cryo-EM Structures of the SARS-CoV-2 Endoribonuclease Nsp15

New therapeutics are urgently needed to inhibit SARS-CoV-2, the virus responsible for the on-going Covid-19 pandemic. Nsp15, a uridine-specific endoribonuclease found in all coronaviruses, processes viral RNA to evade detection by RNA-activated host defense systems, making it a promising drug target. Previous work with SARS-CoV-1 established that Nsp15 is active as a hexamer, yet how Nsp15 recognizes and processes viral RNA remains unknown. Here we report a series of cryo-EM reconstructions of SARS-CoV-2 Nsp15. The UTP-bound cryo-EM reconstruction at 3.36 [A] resolution provides molecular details into how critical residues within the Nsp15 active site recognize uridine and facilitate catalysis of the phosphodiester bond, whereas the apo-states reveal active site conformational heterogeneity. We further demonstrate the specificity and mechanism of nuclease activity by analyzing Nsp15 products using mass spectrometry. Collectively, these findings advance understanding of how Nsp15 processes viral RNA and provide a structural framework for the development of new therapeutics.

biochemistry↗

bioRxiv2020

Comparative Modeling of CDK9 Inhibitors to Explore Selectivity and Structure-Activity Relationships

Cyclin-dependent kinase 9 (CDK9) plays a key role in transcription elongation, and more recently it was also identified as the molecular target of a series of diaminothiazole compounds that reverse epigenetic silencing in a phenotypic assay. To better understand the structural basis underlying these compounds activity and selectivity, we developed a comparative modeling approach that we describe herein. Briefly, this approach draws upon the strong structural conservation across the active conformation of all protein kinases, and their shared pattern of interactions with Type I inhibitors. Because of this, we hypothesized that the large collection of inhibitor/kinase structures available in the Protein Data Bank (PDB) would enable accurate modeling of this diaminothiazole series in complex with each CDK family member. We apply this new comparative modeling pipeline to build each of these structural models, and then demonstrate that these models provide retrospective rationale for the structure-activity relationships that ultimately guided optimization to the lead diaminothiazole compound MC180295 (14e). We then solved the crystal structure of the 14e/CDK9 complex, and found the resulting structure to be in excellent agreement with our corresponding comparative model. Finally, inspired by these models, we demonstrate how structural changes to 14e can be used to rationally tune this compounds selectivity profile. With the emergence of CDK9 as a promising target for therapeutic intervention in cancer, we anticipate that comparative modeling can provide a valuable tool to guide optimization of potency and selectivity of new inhibitors targeting this kinase.

cancer biology↗

bioRxiv2020

A versatile vector system for the fast generation of knock-in cell lines with CRISPR

Until recent advancements in genome editing via CRISPR/Cas9 technology, understanding protein function typically involved artificially overexpressing proteins of interest. Despite that CRISPR/Cas9 has ushered in a new era of possibilities for modifying endogenous genes with labeling tags (knock-in) to more accurately study proteins under physiological conditions, the technique is largely underutilized due to its tedious, multi-step process. Here we outline a homologous recombination system (FAST-HDR) to be used in combination with CRISPR/Cas9 that significantly simplifies and accelerates this process while introducing multiplexing to allow live-cell studies of 3 endogenous proteins within the same cell line. Furthermore, the recombination vectors are assembled in a single reaction that is enhanced for eliminating false positives and reduces the overall creation time for the knockin cell line from ~8 weeks to <15 days. Finally, the system utilizes a modular construction to allow for seamlessly swapping labeling tags to ensure flexibility according to the area under study. We validated this new methodology by developing advanced cell lines with 3 fluorescent-labeled endogenous proteins that support high-content phenotypic drug screening without using antibodies or exogenous staining. Therefore, Fast-HDR cell lines provide a robust alternative for studying multiple proteins of interest in live cells without artificially overexpressing labeled proteins.

molecular biology↗

bioRxiv2019

Nix induced mitochondrial fission, mitophagy, and myocyte insulin resistance are abrogated by PKA phosphorylation.

Lipotoxicity is a form of cellular stress caused by the accumulation of lipids resulting in mitochondrial dysfunction and insulin resistance in muscle. Previously, we demonstrated that the mitophagy receptor Nix is responsive to lipotoxicity and accumulates in response to diacylglycerols induced by high-fat (HF) feeding. In addition, previous studies have implicated autophagy and mitophagy in muscle insulin sensitivity. To provide a better understanding of these observations, we undertook gene expression array and shot-gun metabolomics studies in soleus muscle from rodents on an HF diet. Interestingly, we observed a modest reduction in several autophagy-related genes including Beclin-1, ATG3, and -5. Moreover, we observed alterations in the fatty acyl composition of cardiolipins and phosphatidic acids. Given the previously reported roles of these phospholipids and Nix in mitochondrial dynamics, we investigated aberrant mitochondrial fission and turn-over as a mechanism of myocyte insulin resistance. In a series of gain-of-function and loss-of-function experiments in rodent and human myotubes, we demonstrate that Nix accumulation triggers mitochondrial depolarization, fragmentation, calcium-dependent activation of DRP1, and mitophagy. In addition, Nix-induced mitochondrial fission leads to myotube insulin resistance through activation of mTOR-p70S6 kinase inhibition of IRS1, which is contingent on phosphatidic acids and Rheb. Finally, we demonstrate that Nix-induced mitophagy and insulin resistance can be reversed by direct phosphorylation of Nix by PKA, leading to the translocation of Nix from the mitochondria and sarcoplasmic reticulum to the cytosol. These findings provide insight into the role of Nix-induced mitophagy and myocyte insulin resistance during an overfed state when overall autophagy-related gene expression is reduced. Furthermore, our data suggests a mechanism by which exercise or pharmacological activation of PKA may overcome myocyte insulin resistance. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/825828v2_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@15d0a4org.highwire.dtl.DTLVardef@34f358org.highwire.dtl.DTLVardef@5427f3org.highwire.dtl.DTLVardef@10ae48a_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2019

Nol9 is a Spatial Regulator for the Human ITS2 pre-rRNA Processing Complex

The ribosome plays a universal role in translating the cellular proteome. Defects in the ribosome assembly factor Las1L are associated with congenital lethal motor neuron disease and X-linked intellectual disability disorders, yet its role in processing precursor ribosomal RNA (pre-rRNA) is largely unclear. The Las1L endoribonuclease associates with the Nol9 polynucleotide kinase to form the internal transcribed spacer 2 (ITS2) pre-rRNA processing machinery. Together, Las1L-Nol9 catalyzes RNA cleavage and phosphorylation to mark the ITS2 for degradation. While ITS2 processing is critical for the production of functional ribosomes, the regulation of mammalian Las1L-Nol9 remains obscure. Here we characterize the human Las1L-Nol9 complex and identify critical molecular features that regulate its assembly and spatial organization. We establish that Las1L and Nol9 form a higher-order complex and identify the regions responsible for orchestrating this intricate architecture. Structural analysis by high-resolution imaging defines the intricate spatial pattern of Las1L-Nol9 within the nucleolar sub-structure linked with late pre-rRNA processing events. Furthermore, we uncover a Nol9 encoded nucleolar localization sequence that is responsible for nucleolar transport of the assembled LasL-Nol9 complex. Together, these data provide a mechanism for the assembly and nucleolar localization of the human ITS2 pre-rRNA processing complex.

molecular biology↗

bioRxiv2019

NOTCH1 signaling establishes the medullary thymic epithelial cell progenitor pool during mouse fetal development

The cortical and medullary thymic epithelial cell (cTEC and mTEC) lineages are essential for inducing T cell lineage commitment, T cell positive selection and the establishment of self-tolerance, but the mechanisms controlling their fetal specification and differentiation are poorly understood. Here, we show that Notch signaling is required to specify and expand the mTEC lineage. Notch1 is expressed by and active in TEC progenitors. Deletion of Notch1 in TECs resulted in depletion of mTEC progenitors and dramatic reductions in mTECs during fetal stages, consistent with defects in mTEC specification and progenitor expansion. Conversely, forced Notch signaling in all TEC resulted in widespread expression of mTEC progenitor markers and profound defects in TEC differentiation. In addition, lineage-tracing analysis indicated that all mTECs have a history of receiving a Notch signal, consistent with Notch signaling occurring in mTEC progenitors. Interestingly, this lineage analysis also showed that cTECs are divided between Notch lineage-positive and lineage-negative populations, identifying a previously unknown complexity in the cTEC lineage.

developmental biology↗