Biology subjects

Lewis, A. J.

Publications and source records attributed to Lewis, A. J..

18 recordsLinked to original sources

bioRxiv2026

Rank-based integration identifies convergent disease mechanisms across omics

The rapid growth of omics studies offers new opportunities to uncover disease mechanisms, yet findings from individual studies or single omics may be influenced by cohort-, platform- and layer-specific variation. Integrating evidence across studies and omics layers can reveal robust biological signals that are reproducible across molecular layers, but differences in assay platforms, sample sizes and feature coverage complicate direct combination of summary statistics. Here we present the Omics Rank-Based Integration Tool (ORBIT), a direction-aware framework for prioritizing concordant signals across omics layers using only within-dataset feature ranks and effect directions. ORBIT tests directional rank consistency across omics layers against a closed-form variance-gamma null distribution while accounting for inter-dataset correlation and incomplete feature overlap. Simulations show that ORBIT maintains well-calibrated false-positive rates under inter-omics correlation, increasing numbers of omics layers and missingness, while detecting concordant signals with increasing power as the number of layers grows. We applied ORBIT to tubulointerstitial transcriptomic data from patients with chronic kidney disease in the C-PROBE cohort and proteomic data from the Kidney Precision Medicine Project, identifying a concordant injury programme marked by inflammatory and fibrotic remodeling together with impaired energy metabolism. We also used ORBIT to integrate ten transcriptomic, four proteomic and one translatomic datasets in dilated cardiomyopathy heart tissue, identifying extracellular matrix remodeling and mitochondrial energy failure as the dominant concordant biological programmes. In both diseases, ORBIT prioritized concordant genes and identified pathways that were not significant in any single omics layer alone. These results show that directional rank integration can be used for integrating heterogeneous omics summary data and prioritizing robust molecular mechanisms.

bioinformatics↗

bioRxiv2026

Cryo-ET and Sub-Volume Analysis of Fibrillar Pathology in the Neurodegenerative Human Brain

Structural studies of amyloid fibrils extracted from brain tissue have identified disease-specific fibril polymorphs. However, the mechanisms driving distinct polymorphs remain unclear because no method currently links the cellular context, composition and ultrastructure of individual aggregates to their constituent fibril polymorphs. Here, we present a workflow for in situ cryo-electron tomography (cryo-ET) to study amyloids within individual aggregates in the human neurodegenerative postmortem brain. Using chemically fixed tissue, we studied in situ tau fibrils in the hippocampus of an Alzheimer's disease (AD) donor and -synuclein fibrils within a glial nuclear inclusion in the cingulate gyrus of a multiple systems atrophy (MSA) donor. Subtomogram averaging and helical reconstruction of fibril subvolumes yielded low-resolution (~30-35A) density maps, which were compared to existing ex vivo structures. Preserved ultrastructure enabled detailed observations of pathology, including an alpha-synuclein fibril penetrating the nuclear envelope, providing mechanistic insight into intranuclear aggregate formation. Combined with existing high-resolution fibril structures lacking spatial context, cryo-ET offers a powerful approach to understanding amyloid polymorphism in neurodegenerative diseases, advancing anti-amyloid therapies and diagnostic tools.

neuroscience↗

bioRxiv2026

Ultrastructural remodelling of tau fibrils during ghost tangle formation in Alzheimer's disease brain

Tau aggregation into intracellular neurofibrillary tangles (NFTs) is one of the major hallmarks of Alzheimers disease (AD). Based on neuropathological studies, NFTs have been classified into pre-tangles, mature tangles, and ghost tangles, however the ultrastructural transitions between these stages remain poorly understood. Here, we used correlative light and electron microscopy (CLEM) to structurally characterize tau tangle maturity states in post-mortem human AD brain tissue. Pre-tangles showed no consistent fibrillar ultrastructure. Mature tangles contained densely packed, highly aligned paired helical filaments (PHF) and straight filaments (SF), often organized in spatially distinct bundles within the neuronal soma. Ghost tangles lacked cellular organelles and were composed predominantly of thin fibrils compartmentalized by membranous structures, with fibril morphology differing between compartmentalized and non-compartmentalized regions. Electron tomography and fibril segmentation demonstrated that these fibrils were significantly thinner than PHFs and SFs while immunogold labeling using the 2E9 tau marker confirmed the presence of tau within both mature and ghost tangle fibrils. GFAP-positive astrocytic processes infiltrated fibril-rich compartments within ghost tangles, linking astrocytic engagement with the emergence of this distinct ultrastructural organization. Together, our findings show that ghost-tangles contain a structurally distinct population of tau fibrils, suggesting that tau aggregates undergo astrocytic-mediated structural remodeling at late stages of pathology.

pathology↗

bioRxiv2026

In vivo evidence for bleb-induced survival signaling in metastatic melanoma

Bleb signaling is a cellular process in which pressure-driven plasma membrane protrusions generate localized micron-scale membrane curvature that recruits cytosolic septin complexes, assembling signaling hubs that promote cell survival1. Our prior work showed that this morphology-encoded signaling pathway is necessary to sustain anchorage-independent survival of BRAF and NRAS mutant melanoma cells in vitro. However, whether bleb-induced signaling occurs in vivo and contributes to cancer progression is unclear. Here, we develop complementary in vivo and ex vivo assays spanning human patient samples and mouse xenograft models to mechanistically interrogate bleb signaling directly in physiologic contexts. We observe that bleb-associated septin hubs are exclusively formed in poorly adherent amoeboid tumor cells at the invasive margin, within malignant effusions, and at distant metastatic sites, while well-adhered cells in the tumor interior show no signs of septin hub formation. Accordingly, pharmacological pathway disruption specifically kills disseminated tumor cells within these low-adhesion microenvironments while having no appreciable effect on adhered cells in the tumor interior, resulting in reduced metastatic burden and delayed disease recurrence in vivo. This work confirms septin-mediated bleb signaling as a previously unrecognized vulnerability of disseminated cancer cells and demonstrates that this morphology-encoded pathway operates in vivo to support disease progression, preserving cancer cell viability under conditions in which cell survival signals from the environment are muted. These findings suggest novel opportunities to target survival signaling in micrometastatic disseminated cancer cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/697601v1_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@1770384org.highwire.dtl.DTLVardef@87e567org.highwire.dtl.DTLVardef@1e5d6b2org.highwire.dtl.DTLVardef@724c5f_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2025

Cryo-EM structure of alpha-Synuclein Fibrils Harboring the Dementia with Lewy Bodies - Associated E83Q Mutation

Aggregation of -synuclein (-Syn) into amyloid fibrils underlies the pathology of synucleinopathies. Rare familial mutations can modulate -Syn aggregation and may give rise to distinct fibril conformations linked to disease heterogeneity. The E83Q mutation, identified in a Dementia with Lewy Body disease (DLB) patient with atypical Lewy body distribution, has been shown to accelerate -Syn aggregation and enhance neuronal seeding, yet its structural basis remains unclear. Here, we determined the cryo-electron microscopy structure at 3.4 [A] resolution of recombinant full-length human -Syn fibrils harboring the E83Q mutation. The fibrils adopt a double-protofilament architecture with a conserved Greek-key-like core spanning residues 36-99, stabilized by intra- and inter-filament salt bridges. Despite this conserved fold, the E83Q mutation induces a distinct local rearrangement within the hydrophobic region: substitution of Glu83 with Gln reorients residue 83 inward, abolishing solvent exposure and altering interactions with the N-terminal region. This structural shift brings the hydrophobic region closer to the N-terminus, shielding residues implicated in post-translational modification and ligand binding. Our findings reveal how a single charge-neutralizing mutation reshapes -Syn fibril architecture, providing a structural framework to understand the enhanced aggregation and pathogenic properties associated with the E83Q variant.

neuroscience↗

bioRxiv2025

Correlative Ultrastructural Mapping of Lewy Pathology Reveals Regional Diversity in Parkinsons and Dementia with Lewy bodies

Lewy body diseases, including Parkinsons disease (PD) and dementia with Lewy bodies (DLB), are defined by neuronal accumulation of misfolded -synuclein (-Syn), yet the ultrastructural diversity of these inclusions across brain regions and disease contexts remains unclear. Here, we applied large-scale correlative light and electron microscopy (CLEM) to map -Syn pathology across cortical regions (entorhinal cortex, ENT; anterior cingulate cortex, AC; hippocampal CA2 region) and substantia nigra (SN) in clinically and pathologically confirmed PD and DLB donors. We identified pronounced regional heterogeneity in Lewy pathology, with cortical inclusions showing diverse maturation stages at the ultrastructural level, ranging from low-density fibrils interspersed with organelles to highly compact fibrillar inclusions. In the SN of DLB donors, we observed the full range of classical nigral LB morphologies previously described in PD. We additionally characterized diverse neuritic -Syn pathologies in DLB and identified a distinct population of electron-dense, degenerating, -Syn-positive cortical neurons not previously reported. Importantly, we found no significant difference in LB ultrastructure between PD and DLB in either cortical or nigral pathology. In contrast, quantitative analysis of >10,000 mitochondria revealed disease- and region-specific signatures of altered mitochondrial homeostasis. PD showed increased mitochondrial density and enlargement in the SN, whereas DLB showed increased mitochondrial density only in the ENT. Mitochondrial enlargement was exclusive to PD. These findings indicate that LB ultrastructure alone does not distinguish PD from DLB; instead, region-specific mitochondrial phenotypes may better reflect disease identity and regional susceptibility. Overall, we provide a high-resolution framework for human Lewy pathology in PD and DLB, revealing that ultrastructural responses to -Syn pathology are driven primarily by neuronal identity and regional vulnerability. Our results highlight the need for disease- and region-specific models that capture human phenotypes to advance mechanistic understanding and therapeutic targeting of synucleinopathies.

pathology↗

bioRxiv2025

Developmental and transcriptional programs that define the initiation of the forelimb

The vertebrate forelimb initiates as a localized swelling in the somatic lateral plate mesoderm (somatic LPM) in response to TBX5-dependent transcription. The molecular pathways driving limb morphogenesis have been extensively studied but the steps directly preceding limb bud formation remain poorly characterized. To address this, we defined the temporal onset of forelimb initiation in mouse embryos using sequencing based high-throughput approaches (RNA-seq, scRNA-seq, ChIP-seq, and Ribo-ITP) benchmarked to known features in forelimb development, identifying four distinct stages. Using scRNA-seq at the onset of forelimb-specific transcription, we determined the transcriptional profile of the somatic LPM and identified signature genes that distinguish the nascent forelimb from other cell types. This group includes multiple genes involved in neural projection as well as cell adhesion. Interestingly, these genes are highly enriched for TBX5 binding sites, suggesting they are candidate early transcriptional targets of TBX5. As TBX5 is essential for forelimb outgrowth, the identification of these genes suggests new mechanistic models for TBX5-driven limb initiation.

developmental biology↗

bioRxiv2025

Multiscale analysis of myelin alterations in skin biopsies from synucleinopathies.

Loss of myelin and demyelination play a role in the pathophysiology of Parkinsons disease (PD) and related neurodegenerative diseases, but little is known about the ultrastructure of the myelin-axon unit in the peripheral nervous system of subjects diseased with synucleinopathies. We here present an analysis of the myelin ultrastructure and the myelin protein abundance that characterize myelinated axons of nerve fiber bundles in cervical skin biopsies of 45 pathologically confirmed PD, DLB, MSA and non-neurological control donors. We calculated a myelin damage score and classified over 1100 myelin sheaths by looking at myelin fragmentation and swellings with correlative light and electron microscopy. We found a higher load of myelin damage in the PD compared to MSA and control groups. Quantification with ELISA did not reveal any differences in myelin protein zero (MPZ) concentrations in skin tissue homogenates between synucleinopathies. The observed structural abnormalities in the myelin sheaths may help to discriminate among subjects with synucleinopathies and control subjects and to understand the involvement of the peripheral innervation in the diseases. Our multiscale analysis of peripheral nerves highlights their potential as future biomarkers for the detection and differentiation of synuclein diseases.

neuroscience↗

bioRxiv2025

Immunomodulation by AZD1656 reverses cardiac dysfunction, metabolic remodelling and reduces infarct size in type 2 diabetic cardiomyopathy

Type 2 diabetes (T2D) precipitates diabetic cardiomyopathy (dbCM), a condition characterized by chronic inflammation, metabolic dysregulation and impaired cardiac performance. Here we show that the glucokinase activator AZD1656, originally developed for glycaemic control but later identified to have immunomodulatory effects, reverses cardiac dysfunction and metabolic remodelling in dbCM. In obese, hyperglycaemic db/db mice with diastolic dysfunction, six weeks of AZD1656 treatment improved myocardial performance, reduced infarct size and enhanced post-ischaemic recovery. Integrated metabolic, functional and histological analyses revealed restoration of mitochondrial metabolism and attenuation of fibrosis. Mechanistically, AZD1656 remodelled the cardiac immune landscape by promoting regulatory T-cell infiltration. These findings demonstrate a link between cardiac inflammation and metabolic remodelling in dbCM and highlight that modulation of immune cells and metabolism can protect the diabetic heart. Targeting immunometabolic pathways may therefore offer a therapeutic strategy to alleviate cardiac dysfunction and reduce infarct vulnerability in T2D

physiology↗

bioRxiv2024

1.94 Angstrom structure of synthetic alpha-synuclein fibrils seeding MSA neuropathology

Summary ParagraphMultiple system atrophy (MSA) is a rapidly progressive neurodegenerative disease of unknown cause, typically affecting individuals aged 50-60 and leading to death within a decade1-3. It is characterized by glial cytoplasmic inclusions (GCIs) composed of fibrillar alpha-synuclein (aSyn)4-8, whose formation shows parallels with prion propagation9,10. While fibrils extracted from MSA brains have been structurally characterized11, their ability to replicate in a "protein-only" manner has been questioned12, and their capacity to induce GCIs in vivo remains unexplored. By contrast, the synthetic fibril strain 1B13,14, assembled from recombinant human aSyn, self-replicates in vitro and induces GCIs in mice15 - suggesting direct relevance to MSA - but awaited scrutiny at an atomic scale. Here, we report high-resolution structural analyses of 1B fibrils and of fibrils extracted from diseased mice injected with 1B that developed GCIs (1BP). We show in vivo that conformational templating enables fibril strain replication, resulting in MSA-like inclusion pathology. Remarkably, the structures of 1B and 1BP are highly similar and mimic the fold of aSyn observed in one protofilament of fibrils isolated from MSA patients11. Moreover, reinjection of crude mouse brain homogenates containing 1BP into new mice reproduces the same MSA-like pathology induced by the parent synthetic seed 1B. Our findings identify 1B as a synthetic pathogen capable of self-replication in vivo and reveal structural features of 1B/1BP that may underlie MSA pathology, offering insights for therapeutic strategies.

neuroscience↗

bioRxiv2024

Modulating Cardiac Energetics in Cardio-Metabolic Syndromes: A mechanistic, hyperpolarized MR Trial of Ninerafaxstat Treatment

BackgroundType 2 diabetes (T2D) and obesity are key contributors for heart failure (HF)- development, especially for HF with a preserved ejection fraction (HFpEF). On a molecular basis, excessive use of fatty acids (FA) induces lipotoxicity which in turn promotes inflammation, reduces mitochondrial pyruvate dehydrogenase (PDH) activity and impairs myocardial energetics and -function. Harnessing in-vivo, real time measurement of cellular metabolism via hyperpolarized pyruvate MR, we aimed to assess the effects of ninerafaxstat, a selective FA oxidation inhibitor, on cardiac energetics, metabolism & diastolic function in patients with cardio-metabolic syndromes. MethodsIMPROVE-DiCE was an open-label, mechanistic phase 2a trial. 21 participants received 200mg ninerafaxstat twice daily for four (n=5) or eight weeks (n=16). Myocardial energetics (phosphocreatine to adenosine triphosphate ratio, PCr/ATP), metabolism and function were assessed pre-& post-treatment using magnetic resonance imaging (MRI), 31P- and 1H-MR spectroscopy (MRS). We utilised hyperpolarized [1-13C]pyruvate MRS to assess in-vivo PDH-flux (n=9) and plasma metabolomics and proteomics to assess whole body metabolism. ResultsPatients presented with impaired PCr/ATP, (median 1.6 [IQR 1.4, 2.1]), myocardial steatosis (2.2 % [IQR 1.5, 3.2]) and LV diastolic dysfunction (peak circumferential diastolic strain rate 0.86/s [IQR 0.82, 1.06]) at baseline. Ninerafaxstat treatment improved myocardial energetics by 32% (p<0.01), reduced myocardial triglyceride content by 34% (p=0.03) and showed a trend towards improved PDH-flux (mean 45% increase, p=0.08). Diastolic function was significantly improved post-treatment (peak diastolic strain rate by 10%, peak LV filling rate by 11%, both p<0.05). ConclusionsMetabolic modulation with ninerafaxstat significantly improved myocardial energetics, reduced myocardial steatosis and improved LV diastolic filling. Combining hyperpolarized MRS and metabolomics, is a powerful approach to examine the mechanism of action of novel metabolic modulators. REGISTRATIONURL: https://clinicaltrials.gov; Unique identifier: NCT04826159 O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/591019v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@86fae1org.highwire.dtl.DTLVardef@1c1037eorg.highwire.dtl.DTLVardef@cc5ca9org.highwire.dtl.DTLVardef@d789d1_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

Plant Phenolics Inhibit Focal Adhesion Kinase and Suppress Host Cell Invasion by Uropathogenic Escherichia coli

Traditional folk treatments for the prevention and management of urinary tract infections (UTIs) and other infectious diseases often include plants and plant extracts that are rich in phenolic and polyphenolic compounds. These have been ascribed a variety of activities, including inhibition of bacterial interactions with host cells. Here we tested a panel of four well-studied phenolic compounds - caffeic acid phenethyl ester (CAPE), resveratrol, catechin, and epigallocatechin gallate - for effects on host cell adherence and invasion by uropathogenic Escherichia coli (UPEC). These bacteria, which are the leading cause of UTIs, can bind and subsequently invade bladder epithelial cells via an actin-dependent process. Intracellular UPEC reservoirs within the bladder are often protected from antibiotics and host defenses, and likely contribute to the development of chronic and recurrent infections. Using cell culture-based assays, we found that only resveratrol had a notable negative effect on UPEC adherence to bladder cells. However, both CAPE and resveratrol significantly inhibited UPEC entry into the host cells, coordinate with attenuated phosphorylation of the host actin regulator Focal Adhesion Kinase (FAK, or PTK2) and marked increases in the numbers of focal adhesion structures. We further show that the intravesical delivery of resveratrol inhibits UPEC infiltration of the bladder mucosa in a murine UTI model, and that resveratrol and CAPE can disrupt the ability of other invasive pathogens to enter host cells. Together, these results highlight the therapeutic potential of molecules like CAPE and resveratrol, which could be used to augment antibiotic treatments by restricting pathogen access to protective intracellular niches. IMPORTANCEUrinary tract infections (UTIs) are exceptionally common and increasingly difficult to treat due to the ongoing rise and spread of antibiotic resistant pathogens. Furthermore, the primary cause of UTIs, uropathogenic Escherichia coli (UPEC), can avoid antibiotic exposure and many host defenses by invading the epithelial cells that line the bladder surface. Here we identified two plant-derived phenolic compounds that disrupt activation of the host machinery needed for UPEC entry into bladder cells. One of these compounds (resveratrol) effectively inhibited UPEC invasion of the bladder mucosa in a mouse UTI model, and both phenolic compounds significantly reduced host cell entry by other invasive pathogens. These findings suggest that select phenolic compounds can be used to supplement existing antibacterial therapeutics by denying uropathogens shelter within host cells and tissues, and help explain some of the benefits attributed to traditional plant-based medicines.

microbiology↗

bioRxiv2022

Rapid iPSC inclusionopathy models shed light on formation, consequence and molecular subtype of alpha-synuclein inclusions

Intracellular inclusions accompanying neurodegeneration are histopathologically and ultrastructurally heterogeneous but the significance of this heterogeneity is unclear. iPSC models, while promising for disease modeling, do not form inclusions in a reasonable timeframe and suffer from limited tractability. Here, we developed an iPSC toolbox utilizing piggyBac-based or targeted transgenes to rapidly induce CNS cells with concomitant expression of aggregation-prone proteins. This system is amenable to screening and longitudinal tracking at single-cell and single-inclusion resolution. For proof-of-principle, cortical neuron -synuclein "inclusionopathy" models were engineered to form inclusions through exogenous seeding or -synuclein mutation. These models recapitulated known fibril- and lipid-rich inclusion subtypes, uncovering dynamic interactions between them, and refined the classification of inclusions in postmortem brain. Genetic-modifier and protein-interaction screens pinpointed proteins like RhoA whose sequestration into specific inclusion subtypes is likely to be toxic. This iPSC platform should enhance our understanding of proteinaceous pathologies in neurodegeneration and facilitate therapeutics development.

neuroscience↗

bioRxiv2021

Balanced Input from the tRNA Prenyltransferase MiaA Controls the Stress Resistance and Virulence Potential of Extraintestinal Pathogenic Escherichia coli

An ability to adapt to rapidly changing and often hostile environments is key to the success of many bacterial pathogens. In Escherichia coli, the highly conserved enzymes MiaA and MiaB mediate the sequential prenylation and methylthiolation of adenosine-37 within tRNAs that decode UNN codons. Here, we show that MiaA, but not MiaB, is critical to the fitness and virulence of extraintestinal pathogenic E. coli (ExPEC), a major cause of urinary tract and bloodstream infections. Deletion of miaA has pleiotropic effects, rendering ExPEC especially sensitive to stressors like nitrogen and oxygen radicals and osmotic shock. We find that stress can stimulate striking changes in miaA expression, which in turn can increase translational frameshifting and markedly alter the bacterial proteome. Cumulatively, these data indicate that ExPEC, and likely other organisms, can vary MiaA levels as a means to fine-tune translation and the spectrum of expressed proteins in response to changing environmental challenges.

microbiology↗

bioRxiv2020

De novo coding variants in the AGO1 gene cause a neurodevelopmental disorder with intellectual disability

High-impact pathogenic variants in more than 1,000 protein-coding genes cause Mendelian forms of neurodevelopmental disorders (NDD), including the newly reported AGO2 gene. This study describes the molecular and clinical characterization of 28 probands with NDD harboring heterozygous AGO1 coding variants. De novo status was always confirmed when parents were available (26/28). A total of 15 unique variants leading to amino acid changes or deletions were identified: 12 missense variants, two in-frame deletions of one codon, and one canonical splice variant leading to a deletion of two amino acid residues. Some variants were recurrently identified in several unrelated individuals: p.(Phe180del), p.(Leu190Pro), p.(Leu190Arg), p.(Gly199Ser), p.(Val254Ile) and p.(Glu376del). AGO1 encodes the Argonaute 1 protein, which functions in gene-silencing pathways mediated by small non-coding RNAs. Three-dimensional protein structure predictions suggest that these variants might alter the flexibility of the AGO1 linkers domains, which likely would impair its function in mRNA processing. Affected individuals present with intellectual disability of varying severity, as well as speech and motor delay, autistic behavior and additional behavioral manifestations. Our study establishes that de novo coding variants in AGO1 are involved in a novel monogenic form of NDD, highly similar to AGO2 phenotype.

genetics↗

bioRxiv2020

A Genome-wide CRISPR-Cas9 Screen Identifies Host Factors Essential for Optimal Plasmodium Liver Stage Development.

Prior to initiating symptomatic malaria, a single Plasmodium sporozoite infects a hepatocyte and develops into thousands of merozoites, in part by scavenging host resources. We show that host microtubules dynamically reorganize around the developing liver stage (LS) parasite. Using a genome-wide CRISPR-Cas9 screen, we identified host regulators of cytoskeleton organization, vesicle trafficking, ER/Golgi stress and lipid biogenesis that regulate Plasmodium LS development. These novel regulators of infection, including Centromere Protein J (CENPJ), led us to interrogate how microtubule organizing centers (MTOCs) are regulated during infection. Foci of {gamma}-tubulin localized to the parasite periphery; depletion of CENPJ exacerbated this re-localization and increased infection. Further, we show that the Golgi acts as a non-centrosomal MTOC by organizing {gamma}-tubulin and stimulating microtubule nucleation at the parasite periphery. Collectively, we show that the Plasmodium LS recruits the host Golgi to form MT mediated conduits along which host organelles are recruited to the PVM, to support liver stage development. Our findings suggest many host-targeted pharmacological inhibitors may inhibit LS infection.

cell biology↗

bioRxiv2019

A CRISPR Knockout Screen Identifies Foxf1 as a Suppressor of Colorectal Cancer Metastasis That Acts Through Reduced mTOR Signalling

IntroductionA greater understanding of molecular mechanisms underlying metastasis is necessary for development of new strategies to prevent and treat cancer.\n\nMethodsWe performed a genome-wide CRISPR/Cas9 knockout screen in MC38 colorectal cancer (CRC) cells transplanted orthotopically into mice to identify genes that promote metastasis. We undertook focussed molecular analyses to identify mechanisms underlying metastasis.\n\nResultsThe screen identified several gene knockouts over-represented in lung metastases, including Dptor (mTOR signalling) and Foxf1 (gastrointestinal tumour predisposition). We validate that loss of Foxf1 promotes metastasis, increased Foxf1 expression restrained cellular migration in-vitro and human CRC metastases express lower Foxf1 than paired primary tumours. Analysis of gene expression changes downstream of Foxf1 identified increased mTOR signalling as a possible mechanism of metastasis caused by Foxf1 loss, consistent with Dptor identification. We confirmed this mechanism demonstrating that mTOR inhibitor sirolimus reduced lung metastasis burden in xenografts.\n\nConclusionMesenchymal Foxf1 plays a major role in intestinal development. We have shown for the first time, through an unbiased genetic screen, that reduced epithelial Foxf1 results in raised mTOR signalling and metastasis.\n\nAuthorship statementLennard Lee-study concept and design, acquisition of data, analysis, interpretation of data, drafting of the manuscript, statistical analysis and obtained funding. Connor Woolley-acquisition of data, analysis and interpretation of data. Thomas Starkey-acquisition of data, analysis, interpretation of data, drafting of the manuscript. Luke Freeman-Mills-interpretation of data. Andrew Bassett-technical and material support. Fanny Fanchini-technical support. Lai Mun Wang-acquisition of data and study supervision. Annabelle Lewis-study supervision. Roland Arnold-analysis, interpretation of data, statistical analysis. Ian Tomlinson-study supervision and critical revision of the manuscript.\n\nConflict of InterestThe authors whose names are listed above declare that they have no conflict of interest.

cancer biology↗

bioRxiv2018

Assessing the effect of hypoxia on cardiac metabolism using hyperpolarized 13C magnetic resonance spectroscopy

Abstract summaryHypoxia plays a role in many diseases and can have a wide range of effects on cardiac metabolism depending on the extent of the hypoxic insult. Non-invasive imaging methods could shed valuable light on the metabolic effects of hypoxia on the heart in vivo. Hyperpolarized carbon-13 magnetic resonance spectroscopy (HP 13C MRS) in particular is an exciting technique for imaging metabolism that could provide such information.\n\nThe aim of our work was, therefore, to establish whether hyperpolarized 13C MRS can be used to assess the in vivo response of cardiac metabolism to systemic acute and chronic hypoxic exposure. Groups of healthy male Wistar rats were exposed to either acute (30 minutes), one week or three weeks of hypoxia. In vivo MRS of hyperpolarized [1-13C]pyruvate was carried out along with assessments of physiological parameters and ejection fraction. No significant changes in heart rate, respiration rate, or ejection fraction were observed at any timepoint. Haematocrit was elevated after one week and three weeks of hypoxia.\n\nThirty minutes of hypoxia resulted in a significant reduction in pyruvate dehydrogenase (PDH) flux, whereas one or three weeks of hypoxia resulted in a PDH flux that was not different to normoxic animals. Conversion of hyperpolarized [1-13C]pyruvate into [1-13C]lactate was elevated following acute hypoxia, suggestive of enhanced anaerobic glycolysis. Elevated HP pyruvate to lactate conversion was also seen at the one-week timepoint, in concert with an increase in lactate dehydrogenase (LDH) expression. Following three weeks of hypoxic exposure, cardiac metabolism was comparable to that observed in normoxia.\n\nWe have successfully visualized of the effects of systemic hypoxia on cardiac metabolism using hyperpolarized 13C MRS, with differences observed following 30 minutes and 1 week of hypoxia. This demonstrates the potential of in vivo hyperpolarized 13C MRS data for assessing the cardiometabolic effects of hypoxia in disease.

physiology↗