Biology subjects

Williams, R.

Publications and source records attributed to Williams, R..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

A multistate capture-recapture model to estimate reproduction of North Atlantic right whales

The recent steep decline of critically endangered North Atlantic right whales (Eubalaena glacialis) can be attributed to high mortality combined with low reproduction. While the former is a clear result of anthro-pogenic activity, the latter involves more complexity. Evidence suggests that both short-term fluctuations in prey availability and long-term decline in health are responsible for depressed right whale calving rates. To facilitate an assessment of extinction risk, we developed a multistate capture-recapture model that estimated the probability of calving using extensive sightings data from 1990-2019. The model estimated sub-lethal effects of severe injury on calving probability, and modeled temporal variability in calving as related to indices of prey availability (Calanus spp. biomass) and an apparent regime shift. The average annual probability of calving for known-breeding females, given average prey conditions, decreased from 0.217 [0.162, 0.281] to 0.142 [0.067, 0.252] after the 2010 regime shift. The model indicated strong evidence of a relationship between calving probability and the prey index from the eastern Gulf of Maine, though this relationship effectively disappeared after 2010; moderate evidence for a relationship with prey from the southwest Gulf of St. Lawrence remained. Weak evidence of reduced calving probability due to severe injury resulted from low sample sizes, given increased mortality for individuals observed with severe injuries. The regime effect is hypothesized to be capturing a long-term decline in health due to a combination of decreasing habitat quality resulting from climate change and potentially chronic sublethal injuries (e.g., entanglements). Our reproduction model provides demographic parameter estimates that can be used in population projections for North Atlantic right whales, though uncertainty remains in the mechanisms responsible for recent declines in calving.

ecology↗

bioRxiv2024

The extracellular heparan sulfatase SULF2 limits myeloid IFNbeta signaling and Th17 responses in inflammatory arthritis.

Heparan sulfate (HS) proteoglycans are important regulators of cellular responses to soluble mediators such as chemokines, cytokines and growth factors. We profiled changes in expression of genes encoding HS core proteins, biosynthesis enzymes and modifiers during macrophage polarisation, and found that the most highly regulated gene was Sulf2, an extracellular HS 6-O-sulfatase that was markedly downregulated in response to pro-inflammatory stimuli. We then generated Sulf2+/- bone marrow chimeric mice and examined inflammatory responses in antigen-induced arthritis, as a model of rheumatoid arthritis. Resolution of inflammation was impaired in myeloid Sulf2+/- chimeras, with elevated joint swelling and increased abundance of pro-arthritic Th17 cells in synovial tissue. Transcriptomic and in vitro analyses indicated that Sulf2 deficiency increased type I interferon signaling in bone marrow-derived macrophages, leading to elevated expression of the Th17-inducing cytokine IL-6. This establishes that dynamic remodeling of HS by Sulf2 limits type I interferon signaling in macrophages, and so protects against Th17-driven pathology.

immunology↗

bioRxiv2024

Determining susceptibility loci in triple negative breast cancer using a novel pre-clinical model

Breast cancer (BC) is the most common cancer and the second cause of death in US women. Our lack of understanding of how genetic variants affect molecular mechanisms that mediate BC aggression poses a substantial obstacle to advancements in cancer diagnosis and therapy. To examine genetic variants on BC traits, a novel murine model was created with robust phenotypic and genomic variation. The FVB C3(1)-T-antigen ("C3Tag") mouse develops spontaneous tumors in the mammary glands of female mice with a mean latency of 4-5 months of age. This genetically engineered mouse model (GEMM) is well established to resemble human basal-like TNBC. TNBC is an aggressive subtype with few clinical approaches and poor patient outcomes. Thus, to model human heterogeneity in BC outcomes, we systematically crossed the C3Tag GEMM into the BXD recombinant inbred family - the largest and best characterized genetic reference population. The new model is termed "BXD-BC" and F1 hybrids of the cross have isogenic genomes that are reproducible. BXD-BCs are a potent tool to determine the impact of genetic modifiers on BC tumor traits. We hypothesized that examination of BXD-BC GEMMs will enable the identification of susceptibility loci, candidate genes, and molecular networks that underlie variation of multiple BC phenotypes. Using N=29 BXD-BC strains, we demonstrated significant heritable variations in the severity of TNBC characteristics such as tumor latency, multiplicity, and survival. Interestingly, 2 BXD-BC strains never developed tumors out to 1 year of age. Thus, BXD-BC strains demonstrate variance in cancer susceptibility and progression compared to the parent C3Tag GEMM, indicating the presence of genetic modifiers. Through an unbiased systematic quantification of breast cancer severity across BXD-BC hybrids, we identified several significant quantitative trait loci (QTL) and candidate genes for specific tumor traits. In combination with public human GWAS datasets, we defined syntenic regions, candidate genes, and underlying networks through cross-species systems genetics analyses to demonstrate the translational validity of conserved, biologically relevant, and targetable candidates. Our findings suggest conserved candidates predicting TNBC patient survival. In sum, the BXD-BC resource is an innovative, reliable, and robust preclinical model that reflects robust genetic heterogeneity. Using cutting edge systems genetics, we have identified genetic modifiers of BC phenotypic variation that could be targeted to advance therapeutic limitations or as biomarkers of risk or response to therapy.

genetics↗

bioRxiv2023

Reliability of dynamic causal modelling of resting state magnetoencephalography

This study assesses the reliability of resting-state dynamic causal modelling (DCM) of magneto-electroencephalography under conductance-based canonical microcircuit models, in terms of both posterior parameter estimates and model evidence. We use resting state magneto-electroencephalography (MEG) data from two sessions, acquired two weeks apart, from a cohort with high between-subject variance arising from Alzheimers disease. Our focus is not on the effect of disease, but on the predictive validity of the methods implicit in their reliability, which is crucial for future studies of disease progression and drug intervention. To assess the predictive validity of first-level DCMs, we compare model evidence associated with the covariance among subject-specific free energies (i.e., the quality of the models) with vs. without interclass correlations. We then used parametric empirical Bayes (PEB) to investigate the predictive validity of DCM parameters at the between subject level. Specifically, we examined the evidence for or against parameter differences (i) within-subject, within-session, between-epochs; (ii) within-subject between-session and (iii) within-site between-subjects, accommodating the conditional dependency among parameter estimates. We show that for data acquired close in time, and under similar circumstances, more than 95% of inferred DCM parameters are unlikely to differ, speaking to mutual predictability over sessions. Using PEB, we show a reciprocal relationship between a conventional definition of reliability and the conditional dependency among inferred model parameters. Our analyses confirm the predictive validity and reliability of the conductance-based DCMs for resting-state neurophysiological data. In this respect, the implicit generative modelling is suitable for interventional and longitudinal studies of neurological and psychiatric disorders. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/562379v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@135c964org.highwire.dtl.DTLVardef@38e4a7org.highwire.dtl.DTLVardef@12bc358org.highwire.dtl.DTLVardef@25f110_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

A multistate capture-recapture model to estimate cause-specific injury and mortality of North Atlantic right whales

Understanding the causes of mortality for a declining species is essential for developing effective conservation and management strategies, particularly when anthropogenic activities are the primary threat. Using a competing hazards framework allows for robust estimation of the cause-specific variation that may exist across multiple dimensions, such as time and individual. Here, we estimated cause-specific rates of severe injury and mortality for North Atlantic right whales (Eubalaena glacialis), a critically endangered species that is currently in peril due to human-caused interactions. We developed a multistate capture-recapture model that leveraged 30 years of intensive survey effort yielding sightings of individuals with injury assessments and necropsies of carcass recoveries. We examined variation in the hazard rates of severe injury and mortality due to entanglements in fishing gear and vessel strikes as explained by year and the age and reproductive status of the individual. We found strong evidence for increased rates of severe entanglement injuries after 2013 and for females with calves, with consequently higher marginal mortality. The model results also suggested that despite vessel strikes causing a lower average rate of severe injuries, the higher mortality rate conditional on injury results in significant total mortality risk, particularly for females resting from a recent calving event. Large uncertainty in the estimation of carcass recovery rate for vessel strike deaths permeated into the apportionment of mortality causes. The increased rates of North Atlantic right whale mortality in the last decade, particularly for reproducing females, puts the species at risk of severe decline. By apportioning the human-caused threats using a quantitative approach with estimation of relevant uncertainty, this work can guide development of conservation and management strategies to facilitate species recovery.

ecology↗

bioRxiv2023

Peripherally derived LGI1-reactive monoclonal antibodies cause epileptic seizures in vivo

One striking clinical hallmark in patients with autoantibodies to leucine-rich glioma inactivated 1 (LGI1) is the very frequent focal seizure semiologies, including faciobrachial dystonic seizures (FBDS), in addition to the amnesia. Polyclonal serum IgGs have successfully modelled the cognitive changes in vivo but not seizures. Hence, it remains unclear whether LGI1-autoantibodies are sufficient to cause seizures. We tested this with the molecularly precise monoclonal antibodies directed against LGI1 (LGI1-mAbs), derived from patient circulating B cells. These were directed towards both major domains of LGI1, LRR (n=5) and EPTP (n=5) and infused intracerebroventricularly over 7 days into juvenile male Wistar rats using osmotic pumps. Continuous wireless EEG was recorded from a depth electrode placed in hippocampal CA3 plus behavioural tests for memory and hyperexcitability were performed. Following infusion completion (Day 9), post-mortem brain slices were studied using electrophysiology and immunostaining. By comparison to control-mAb injected rats (n=6), video-EEG analysis over 9 days revealed convulsive and non-convulsive seizure activity in rats infused with LGI1-mAbs, with a significant number of ictal events (245{+/-}83 vs. 7.8{+/-}7.8 in controls; p=0.002). Memory was not impaired in the novel object recognition test. Local field potential recordings from postmortem brain slices showed spontaneous ictal-like spike activity in the CA3 region (p=0.03). The LGI1-mAbs bound most strongly in the hippocampal CA3 region and induced a significant reduction in Kv1.1 cluster number in this subfield (6 controls; 7 LGI1-mAbs; p=0.01) Peripherally-derived human LGI1-mAbs infused into rodent CSF provide strong evidence of direct in vivo epileptogenesis with molecular correlations. These findings fulfill criteria for LGI1-antibodies in seizure causation.

neuroscience↗

bioRxiv2023

Pathogenic entero- and salivatypes harbour changes in microbiome virulence and antimicrobial resistance genes with increasing chronic liver disease severity

Background & AimsLife-threatening complications of cirrhosis are triggered by bacterial infections, with the ever-increasing threat of antimicrobial resistance (AMR). Alterations in the gut microbiome in decompensated cirrhosis (DC) and acute-on-chronic liver failure (ACLF) are recognised to influence clinical outcomes, whilst the role of the oral microbiome is still being explored. Our aims were to simultaneously interrogate the gut and oral micro- and mycobiome in cirrhotic patients, and assess microbial community structure overlap in relation to clinical outcomes, as well as alterations in virulence factors and AMR genes. Methods18 healthy controls (HC), 20 stable cirrhotics (SC), 50 DC, 18 ACLF and 15 with non-liver sepsis (NLS) i.e. severe infection but without cirrhosis, were recruited at a tertiary liver centre. Shotgun metagenomic sequencing was undertaken from saliva (S) and faecal (F) samples (paired where possible). Salivatypes and enterotypes based on clustering of genera were calculated and compared in relation to cirrhosis severity and in relation to specific clinical parameters. Virulence and antimicrobial resistance genes (ARGs) were evaluated in both oral and gut niches, and distinct resistotypes identified. ResultsSpecific saliva- and enterotypes revealed a greater proportion of pathobionts with concomitant reduction in autochthonous genera with increasing cirrhosis severity, and in those with hyperammonemia. Overlap between oral and gut microbiome communities was observed and was significantly higher in DC and ACLF vs SC and HCs, independent of antimicrobial, beta-blocker and acid suppressant use. Two distinct gut microbiome clusters [ENT2/ENT3] harboured genes encoding for the phosphoenolpyruvate:sugar phosphotransferase system (PTS) system and other virulence factors in patients with DC and ACLF. Substantial numbers of ARGs (oral: 1,218 and gut: 672) were detected with 575 ARGs common to both sites. The cirrhosis resistome was significantly different to HCs, with three and four resistotypes identified for the oral and gut microbiome, respectively. DiscussionOral and gut microbiome profiles differ significantly with increasing severity of cirrhosis, with progressive dominance of pathobionts and loss of commensals. DC and ACLF have significantly worse microbial diversity than NLS, despite similar antimicrobial exposure, supporting the additive patho-biological effect of cirrhosis. The degree of microbial community overlap between sites, frequency of virulence factors and presence of ARGs, all increment significantly with hepatic decompensation. These alterations may predispose to higher infection risk, poorer response to antimicrobial therapy and worsening outcomes, and provide the rationale for developing non-antibiotic-dependent microbiome-modulating therapies.

physiology↗

bioRxiv2023

Sale of critically endangered sharks in the United States

Shark meat is widely available in the United States in grocery stores and seafood markets. The meat is often mislabeled or generically labeled as "shark". The ambiguity of these generic labels makes it challenging to assess the conservation implications of this practice and for consumers to avoid species with high mercury concentrations. For this study we purchased and DNA barcoded 30 shark products purchased in the United States to determine their species identity and conservation status. These samples consisted of 19 filets sold in grocery stores, seafood markets, and Asian specialty markets (mostly in North Carolina) and 11 ordered online as "jerky". 70% of samples were "soft mislabeled" (i.e., labeled generically as shark but not as a specific species). Of the nine samples labeled to species, eight were mislabeled (e.g., spinner shark labeled as mako shark). Only one sample was correctly labeled. All 30 samples were identified as shark and came from 11 different species, including three species listed by the IUCN as Critically Endangered: great hammerhead, scalloped hammerhead, and tope. The first two species have been found to contain very high levels of mercury, illustrating the implications of seafood mislabeling for human health. The widespread availability of shark meat in U.S. grocery stores is surprising given the dramatic decline of shark populations globally. Moreover, the fact that nearly all shark meat is either mislabeled or not labeled to species amplifies the problem. Accurate, verified product labels for shark meat would benefit consumers and shark conservation efforts, and should be a priority for the seafood industry.

genetics↗

bioRxiv2023

Optical nanosensor passivation enables highly sensitive detection of the inflammatory cytokine IL-6

Interleukin-6 (IL-6) is known to a play critical role in the progression of inflammatory diseases such as cardiovascular disease, cancer, sepsis, viral infection, neurological disease, and autoimmune diseases. Emerging diagnostic and prognostic tools, such as optical nanosensors, experience challenges in successful clinical application in part due to protein corona formation dampening their selectivity and sensitivity. To address this problem, we explored the rational screening of several classes of biomolecules to be employed as agents in non-covalent surface passivation as a strategy to screen interference from non-specific proteins. Findings from this screening were applied to the detection of IL-6 by a fluorescent antibody-conjugated single-walled carbon nanotube (SWCNT)-based nanosensor. The IL-6 nanosensor exhibited highly sensitive and specific detection after passivation with a polymer, poly-L-lysine, as demonstrated by IL-6 detection in human serum within a clinically relevant range of 25 pg/mL to 25,000 pg/mL, exhibiting a limit of detection over three orders of magnitude lower than prior antibody-conjugated SWCNT sensors. This work holds the potential for rapid and highly sensitive detection of IL-6 in clinical settings with future application to other cytokines or disease-specific biomarkers.

bioengineering↗

bioRxiv2022

LAT1 enables T cell activation under inflammatory conditions: a new therapeutic target for rheumatoid arthritis

ObjectiveTo assess the L-type amino acid transporter-1 (LAT1) as a possible therapeutic target for rheumatoid arthritis (RA). MethodsSynovial LAT1 expression was monitored by immunohistochemistry and transcriptomic datasets. The contribution of LAT1 to gene expression and immune synapse formation was assessed by RNA-sequencing and total internal reflection fluorescent (TIRF) microscopy, respectively. Mouse models of RA were used to assess the impact of therapeutic targeting of LAT1. ResultsLAT1 was strongly expressed by CD4+ T cells in the synovial membrane of patients with active RA and the level of expression correlated with levels of ESR and CRP as well as DAS-28 scores. Deletion of LAT1 in murine CD4+ T cells inhibited the development of experimental arthritis and prevented the differentiation of CD4+ T cells expressing IFN-{gamma} and TNF-, without affecting regulatory T cells. LAT1 deficient CD4+ T cells demonstrated reduced transcription of genes associated with TCR/CD28 signalling, including Akt1, Akt2, Nfatc2, Nfkb1 and Nfkb2. Functional studies using TIRF microscopy revealed a significant impairment of immune synapse formation with reduced recruitment of CD3{zeta} and phospho-tyrosine signalling molecules in LAT1 deficient CD4+ T cells from the inflamed joints but not the draining lymph nodes of arthritic mice. Finally, it was shown that a small molecule LAT1 inhibitor, currently undergoing clinical trials in man, was highly effective in treating experimental arthritis in mice. ConclusionsIt was concluded that LAT1 plays a critical role in activation of pathogenic T cell subsets under inflammatory conditions and represents a promising new therapeutic target for RA. Key MessagesO_ST_ABSWhat is already known about this subject?C_ST_ABSO_LILAT1 is an amino acid transporter that has previously been shown to play a role in T cell activation. C_LI What does this study add?O_LILAT1 is expressed by synovial T cells in human rheumatoid arthritis and the level of expression correlates with disease severity. C_LIO_LILAT1 expression by T cells is necessary for development of severe arthritis in animal models. C_LIO_LILAT1 is required for immune synapse formation and activation of pathogenic CD4+ T cell subsets in the inflamed joint, but not the lymph nodes. C_LIO_LIA small molecular weight LAT1 inhibitor, currently in clinical trials for cancer, is highly effective in animal models of rheumatoid arthritis. C_LI How might this impact on clinical practice of future developments?O_LIThe context-specific nature of LAT1 involvement in T cell activation positions it as an ideal therapeutic target to distinguish between pathogenic and protective T cell responses and this study provides the scientific rationale for clinical evaluation of LAT1 inhibitors in the treatment of rheumatoid arthritis. C_LI

immunology↗

bioRxiv2022

Chromatin remodeller Chd7 is developmentally regulated in the neural crest by tissue-specific transcription factors

Neurocristopathies such as CHARGE syndrome result from aberrant neural crest development. A large proportion of CHARGE cases are attributed to mutations in the gene encoding CHD7, chromodomain helicase DNA binding protein 7, which remodels chromatin. While the role for CHD7 in neural crest development is well documented, it remains elusive how this seemingly ubiquitous factor is specifically upregulated in neural crest cells. Here, we use epigenomic profiling of chick neural crest to identify a cohort of enhancers regulating Chd7 expression in neural crest cells and other tissues. We functionally validate upstream transcription factor binding at candidate enhancers, revealing novel epistatic relationships between neural crest master regulators and Chd7. To our knowledge, this is the first report of tissue-specific regulation of a chromatin remodeller. Furthermore, we find conserved enhancer features in human embryonic epigenomic data and validate the activity of the human equivalent CHD7 enhancers in the chick embryo. Collectively our findings embed Chd7 in the neural crest gene regulatory network and offer potentially clinically relevant elements for interpreting CHARGE syndrome cases without causative allocation.

developmental biology↗

bioRxiv2022

ROCK2 Knockout Improves Proliferation Rate in a Cellular Model of Down Syndrome

In a cellular model of Down Syndrome, hTERT immortalized RPE-1 (human retinal pigment epithelial-1) cells carrying an extra copy of chromosome 21 exhibit reduced fitness, in part, as an increase in doubling time (or a reduction in cell proliferation rate) in culture. ROCK2 (Rho associated coiled-coil containing kinase 2) was identified in a whole genome CRISPR knockout (KO) screen designed to identify genes and pathways that could be therapeutically targeted to improve cell proliferation (Replogle JM, et al. manuscript in preparation). ROCK2 KO cell lines of both RPE-1 euploid and trisomy 21 aneuploid cells were created using CRISPR. Trisomy 21 ROCK2 KO cell lines showed a modest increase in cell proliferation rate compared to the parental aneuploid cells, similar to the relative effect that ROCK2 knockout had in the whole genome CRISPR screen. Euploid ROCK2 KO cell lines showed no difference in growth rate vs their ROCK2 expressing counterparts. Changes in doubling time in response to two pharmaceutical ROCK inhibitors, Fasudil and Y27632, also showed the same modest increase in cell proliferation rate in the trisomy 21 cells. The actin cytoskeleton, a target of ROCK2 regulation, exhibited long stress fibers that aligned across multiple contiguous cells in confluent trisomy 21 ROCK2 KO cells compared to the disorganized stress fibers of the parental trisomy 21 cells with normal ROCK2 expression.

cell biology↗

bioRxiv2022

Within-host diversity improves phylogenetic and transmission reconstruction of SARS-CoV-2 outbreaks

Accurate inference of who infected whom in an infectious disease outbreak is critical for the delivery of effective infection prevention and control. The increased resolution of pathogen whole-genome sequencing has significantly improved our ability to infer transmission events. Despite this, transmission inference often remains limited by the lack of genomic variation between the source case and infected contacts. Although within-host genetic diversity is common among a wide variety of pathogens, conventional whole-genome sequencing phylogenetic approaches to reconstruct outbreaks exclusively use consensus sequences, which consider only the most prevalent nucleotide at each position and therefore fail to capture low frequency variation within samples. We hypothesized that including within-sample variation in a phylogenetic model would help to identify who infected whom in instances in which this was previously impossible. Using whole-genome sequences from SARS-CoV-2 multi-institutional outbreaks as an example, we show how within-sample diversity is stable among repeated serial samples from the same host, is transmitted between those cases with known epidemiological links, and how this improves phylogenetic inference and our understanding of who infected whom. Our technique is applicable to other infectious diseases and has immediate clinical utility in infection prevention and control.

evolutionary biology↗

bioRxiv2022

Considering decoupled phenotypic diversification between ontogenetic phases in macroevolution: An example using Triggerfishes (Balistidae)

Across the Tree of Life, most studies of phenotypic disparity and diversification have been restricted to adult organisms. However, many lineages have distinct ontogenetic phases that do not reflect the same traits as their adult forms. Non-adult disparity patterns are particularly important to consider for coastal ray-finned fishes, which often have juvenile phases with distinct phenotypes. These juvenile forms are often associated with sheltered nursery environments, with phenotypic shifts between adults and juvenile stages that are readily apparent in locomotor morphology. However, whether this ontogenetic variation in locomotor morphology reflects a decoupling of diversification dynamics between life stages remains unknown. Here we investigate the evolutionary dynamics of locomotor morphology between adult and juvenile triggerfishes. Integrating a time-calibrated phylogenetic framework with geometric morphometric approaches and measurement data of fin aspect ratio and incidence, we reveal a mismatch between morphospace occupancy, the evolution of morphological disparity, and the tempo of trait evolution between life stages. Collectively, our results illuminate how the heterogeneity of morpho-functional adaptations can decouple the mode and tempo of morphological diversification between ontogenetic stages.

evolutionary biology↗

bioRxiv2021

Simultaneous pathway engagement of translation in astrocytes, circadian rhythm in GABAergic neurons and cytoskeleton in glutamatergic neurons precede electroencephalographic changes in neurodegenerative prion disease

Selective vulnerability is an enigmatic feature of neurodegenerative diseases (NDs), whereby a widely expressed protein causes lesions in specific brain regions and cell types. This selectivity may arise from cells possessing varying capacities to regain proteostasis when stressed by cytotoxic protein conformers. Using the RiboTag method in mice, translational responses of five neural subtypes to acquired prion disease (PrD) were measured. Pre-onset and disease onset timepoints were chosen based on longitudinal electroencephalography (EEG) that revealed a gradual increase in theta power between 10- and 18-weeks after prion injection, resembling a clinical feature of human PrD. At disease onset, marked by significantly increased theta power and histopathological lesions, mice had pronounced translatome changes in all five cell types despite having a normal outward appearance. Remarkably, at a pre-onset stage, prior to EEG and neuropathological changes, we found that 1) translatomes of astrocytes indicated a sharply reduced synthesis of ribosomal and mitochondrial components, 2) excitatory neurons showed increased expression of cytoskeletal genes, and 3) inhibitory neurons revealed reduced expression of circadian rhythm network genes. Further assessment for the role of circadian rhythms using a jet lag paradigm modestly exacerbated disease. These data demonstrate that early translatome responses to neurodegeneration emerge prior to other signs of disease and are unique to different cell types. Therapeutic strategies may need to target multiple pathways, each in specific populations of cells, early in the disease process.

neuroscience↗

bioRxiv2021

Pathway profiling of a novel SRC inhibitor, AZD0424, in combination with MEK inhibitors.

A more comprehensive understanding of how cells respond to drug intervention, the likely immediate signalling responses and how resistance may develop within different microenvironments allows us anticipate how cells adapt to targeted therapy enabling more informed prediction of rational drug combinations. The non-receptor tyrosine kinase SRC regulates many cellular signalling processes and pharmacological inhibition has long been a target of drug discovery projects for the treatment of cancer. Here we describe the in vitro and in vivo characterisation of the small molecule SRC inhibitor, AZD0424. We show that AZD0424 potently inhibits the phosphorylation of tyrosine-416 of SRC (IC50 [~] 100 nM) in many cancer cell lines; however inhibition of cell viability, via a G1 cell cycle arrest, was observed only in a sub-set of cancer cell lines in the low (on target) micromolar range. We profiled the changes in intracellular pathway signalling in cancer cells following exposure to AZD0424 and other targeted therapies using Reverse Phase Protein Array analysis. We demonstrate that SRC is activated in response to MEK inhibitor (trametinib or AZD6244)-treatment of KRAS mutant colorectal cell lines (HCT116 and DLD1) and that AZD0424 abrogates this. Cell lines treated with trametinib or AZD6244 in combination with AZD0424 revealed reduction of EGFR, FAK and SRC compensatory activation, and, synergistically inhibits cell viability in vitro. In vivo, trametinib-treatment of mice bearing HCT116 tumours increased phosphorylation of SRC on Tyr416, and when combined with AZD0424, inhibition of tumour growth is greater than trametinib alone. We also demonstrate that drug-induced resistance to trametinib is not re-sensitised by AZD0424 treatment in vitro, likely as a result of multiple compensatory signalling mechanisms; however inhibition of SRC remains an effective way to block invasion of trametinib resistant tumour cells. These data imply that inhibiting SRC may offer a useful addition to MEK inhibitor combination strategies.

cancer biology↗

bioRxiv2021

Segregation of neural crest specific lineage trajectories from a heterogeneous neural plate border territory only emerges at neurulation

The epiblast of vertebrate embryos is comprised of neural and non-neural ectoderm, with the border territory at their intersection harbouring neural crest and cranial placode progenitors. Here we profile avian epiblast cells as a function of time using single-cell RNA-seq to define transcriptional changes in the emerging neural plate border. The results reveal gradual establishment of heterogeneous neural plate border signatures, including novel genes that we validate by fluorescent in situ hybridisation. Developmental trajectory analysis shows that segregation of neural plate border lineages only commences at early neurulation, rather than at gastrulation as previously predicted. We find that cells expressing the prospective neural crest marker Pax7 contribute to multiple lineages, and a subset of premigratory neural crest cells shares a transcriptional signature with their border precursors. Together, our results suggest that cells at the neural plate border remain heterogeneous until early neurulation, at which time progenitors become progressively allocated toward defined lineages.

developmental biology↗

bioRxiv2021

Understanding SARS-CoV-2 budding through molecular dynamics simulations of M and E protein complexes

SARS-CoV-2 and other coronaviruses pose major threats to global health, yet computational efforts to understand them have largely overlooked the process of budding, a key part of the coronavirus life cycle. When expressed together, coronavirus M and E proteins are sufficient to facilitate budding into the ER-Golgi intermediate compartment (ERGIC). To help elucidate budding, we ran atomistic molecular dynamics (MD) simulations using the Feig laboratorys refined structural models of the SARS-CoV-2 M protein dimer and E protein pentamer. Our MD simulations consisted of M protein dimers and E protein pentamers in patches of membrane. By examining where these proteins induced membrane curvature in silico, we obtained insights around how the budding process may occur. Multiple M protein dimers acted together to induce global membrane curvature through protein-lipid interactions while E protein pentamers kept the membrane planar. These results could eventually help guide development of antiviral therapeutics which inhibit coronavirus budding.

biophysics↗