Biology subjects

McDonald, S.

Publications and source records attributed to McDonald, S..

13 recordsLinked to original sources

bioRxiv2026

Pro-inflammatory microglia drive escalated alcohol consumption during early abstinence

Despite growing evidence that neuroimmune dysfunction contributes to Alcohol Use Disorder (AUD) pathology, the underlying neuroinflammatory mechanisms that may promote alcohol consumption are not as clear. We recently report that specific knockdown of interferon regulatory factor 7 (IRF7) in the anterior insula (aIC) mitigates escalation in ethanol self-administration in rats. In addition, we find pro-inflammatory activation of microglia contributes to other AUD-related behavioral impairments. Here, we sought to determine if pro-inflammatory activation of microglia from ethanol contributes to elevations in IRF7 and ethanol self-administration in rats. Male Wistar rats were trained under our ethanol self-administration paradigm (15% v/v; FR2 vs inactive lever) followed by 1-4 cycles of chronic intermittent ethanol vapor exposure (CIE). To inhibit microglia, rats were treated with minocycline (30mg/kg, i.p.) before and after each ethanol vapor session. Escalation in self-administration and biochemical markers were assessed 72 hours into abstinence. We found CIE increased ethanol self-administration, which was positively correlated with aIC IRF7 levels. Minocycline treatment blunted IRF7 expression and alleviated ethanol self-administration following CIE. LC-MS/MS proteomics of primary microglia from the aIC of rats following CIE with and without minocycline treatment indicate minocycline promotes metabolic and ribosomal re-wiring of microglia during early abstinence. These data suggest a role for microglia in driving both IRF7 levels and escalation in ethanol self-administration in early abstinence.

neuroscience↗

bioRxiv2026

A multi-modal flow phantom for quantitative PET/Spectral CT

PurposeIn this work, we aimed to establish a flow phantom for multi-modal PET and spectral CT imaging to improve blood flow quantification. MethodsA modular flow phantom was built with materials compatible with both PET and spectral CT. A peristaltic pump was used to allow for recirculation. Pores were installed through the aorta to allow for tissue exchange between the blood and tissue compartments, and valves were placed in line with the aorta to control the pressure gradient between compartments. We characterized the system using saline bolus experiments, dynamic PET imaging, and iodine-based spectral CT acquisitions. A blood flow (K1) of 1.0 mL/min/mL with a pressure range of approximately 1.0-3.0 psi was targeted. Using compartmental modeling, we estimate K1 across phantom configurations and evaluate the consistency of perfusion-related parameters derived from saline, PET, and spectral CT measurements. ResultsWith the four pore, two valve configuration, target K1 of 1.0 mL/min/mL was achieved with a physiologic pressure range (2.2-3.5 psi) and a pump speed of 150 rpm. Further, the flow phantom was also able to recapitulate K1 across a range of values through adjustable modifications to the phantom configuration. ConclusionsWe present a modular multimodal flow phantom with a tissue-mimicking compartment, vascular tubing with an adjustable number of pores and valves, and 3D-printed components to support tunable exchange between blood-pool and tissue compartments and controlled dynamic perfusion imaging with same-session PET and spectral CT. Such a setup will enable the development of multi-modal approaches for evaluating tissue perfusion.

bioengineering↗

bioRxiv2026

Microtubule Stabilization and Biomaterial Guidance Synergize to Enhance CST Regeneration and Motor Recovery After Chronic SCI.

Spinal cord injury (SCI) results in loss of sensory and motor function below the level of damage, with chronic injuries presenting unique challenges for regenerative therapies. While multichannel biomaterial interventions have shown promise in promoting axonal regeneration, circuit restoration, and motor recovery in acute SCI, achieving similar outcomes in chronic injury models remains challenging due to a combination of intrinsic and extrinsic factors. These include the reduced capacity of the neuronal cell body to sustain a growth-activated state and the formation of a physical and chemical barrier at the injury site, preventing axonal growth. To address these challenges and promote motor recovery after chronic injury, we investigated the combinatorial effect of two regenerative approaches: 1) the implantation of poly (lactide-co-glycolide) (PLG) biomaterial bridge to guide axonal growth through the injury site, and 2) the delivery of Epothilone B (EpoB), a microtubule stabilizer that strengthens axons to promote regrowth. We used a transgenic mouse model that selectively expresses a red fluorescent protein variant (tdTomato) reporter throughout the corticospinal tract (CST) under control of the Crym promoter (Crym-tdTomato). We demonstrated that the combination of bridge implantation 60 days after surgical hemisection at C5 with EpoB improved locomotor function. At 12 weeks post-bridge implantation, immunohistology revealed axon regeneration in mice receiving implantation, but not EpoB or no-implant controls. The addition of EpoB significantly increased the volume of both total and CST axons regenerating through the biomaterial channels. Diffusion tensor magnetic resonance imaging (DTI) analysis identified enhanced fractional anisotropy (FA), axial diffusivity (AD), and mean diffusivity (MD) in the bridge region in the combination treatment group, consistent with new intact axons. Furthermore, EpoB enhanced the myelination of regenerated axons in the bridge. Finally, we investigated the proteomic profile of corticospinal neurons ipsilateral and contralateral to the SCI lesion and bridge, comparing the effect of EpoB treatment. Mass spectrometry-based analysis of laser-captured cells in this paradigm identified activation of a regeneration program by corticospinal neurons. These findings present a novel approach to enhance regenerative neural repair and locomotor recovery in chronic SCI.

neuroscience↗

bioRxiv2025

Inorganic pyrophosphate disrupts amorphous hydrated bone mineral interfaces in hypophosphatasia

Bone mineral molecular architecture is tightly regulated by the kinetics of calcium phosphate phase transformations. In the rare skeletal disease hypophosphatasia (HPP), caused by inactivating mutations in the ALPL gene encoding tissue-nonspecific alkaline phosphatase (TNSALP), accumulation of inorganic pyrophosphate (PPi) alters these phase dynamics. Using solid-state nuclear magnetic resonance spectroscopy and high-resolution electron microscopy, in patient samples we show that bone mineral from compound heterozygous HPP patients exhibits a loss of hydrated amorphous interfacial phases and instead contains highly crystalline hydroxyapatite (HAP), correlating with abnormally high bone mineral density and brittle atypical femoral fractures. Synthetic and cellular models demonstrate that elevated PPi impedes normal phase transitions from amorphous calcium phosphate precursors, bypassing intermediate states and driving ordered HAP nucleation. These findings link disrupted mineral phase kinetics to pathological bone mineral molecular structure, emphasising the critical importance of the hydrated amorphous shell around bone mineral for its material properties and redefining HPP as a molecular mineralization disorder. Our findings also illustrate how biochemical cues regulate non-equilibrium crystallization pathways in biomineralization.

biophysics↗

bioRxiv2024

Hedgehog signaling controls cytotoxic T cell migration in the tumour microenvironment.

Cytotoxic T lymphocytes effectively eliminate cancer cells. Their abundance in the tumour microenvironment is one of the strongest pan-cancer predictors of clinical response. Here, we show that Hedgehog (Hh) signaling regulates T cell migration into tumours. Using conditional knockout mouse models of central Hh signaling components Ihh, Smo and Gli1 in CD8 T cells, we show that Smo deletion greatly impairs the anti-tumour response in vivo due to diminished CD8 T cell migration into the tumour microenvironment. The migration defect is mediated exclusively by Smo, both in in vivo cancer models and in vitro migration assays. This effect is independent of the canonical Hh pathway and relies on the GPCR function of Smo to regulate the migration of murine and human CD8 T cells via RhoA. Hh signaling is critical during embryonic development and adult stem cell homeostasis, but is also amplified in multiple cancer types. Hh inhibitors targeting SMO have been clinically-approved and shown efficacy in the treatment of Hh-driven basal cell carcinoma and medulloblastoma but have failed in clinical trials in other solid cancers with upregulated Hh signaling. We demonstrate that SMO inhibitors specifically decrease CD8 T cell migration into the tumour microenvironment, both in murine cancer models and resected BCCs from patients treated with the SMO inhibitor vismodegib, providing the first mechanistic explanation as to why Hh inhibitors have failed in solid cancers. Our data establishes a novel link between Hh inhibition in vivo and the anti-tumour immune response and reveals a fundamental mechanism controlling T cell migration. The work provides the basis for improved Hh targeting approaches in the clinic and new entry points into enhancing migration in T cell therapies.

immunology↗

bioRxiv2023

Concerted epithelial and stromal changes during progression of Barrett's Esophagus to invasive adenocarcinoma exposed by multi-scale, multi-omics analysis

Esophageal adenocarcinoma arises from Barretts esophagus, a precancerous metaplastic replacement of squamous by columnar epithelium in response to chronic inflammation. Multi-omics profiling, integrating single-cell transcriptomics, extracellular matrix proteomics, tissue-mechanics and spatial proteomics of 64 samples from 12 patients paths of progression from squamous epithelium through metaplasia, dysplasia to adenocarcinoma, revealed shared and patient-specific progression characteristics. The classic metaplastic replacement of epithelial cells was paralleled by metaplastic changes in stromal cells, ECM and tissue stiffness. Strikingly, this change in tissue state at metaplasia was already accompanied by appearance of fibroblasts with characteristics of carcinoma-associated fibroblasts and of an NK cell-associated immunosuppressive microenvironment. Thus, Barretts esophagus progresses as a coordinated multi-component system, supporting treatment paradigms that go beyond targeting cancerous cells to incorporating stromal reprogramming. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=187 SRC="FIGDIR/small/544265v1_ufig1.gif" ALT="Figure 1"> View larger version (62K): org.highwire.dtl.DTLVardef@64c375org.highwire.dtl.DTLVardef@15c6ef2org.highwire.dtl.DTLVardef@177dee0org.highwire.dtl.DTLVardef@c945e7_HPS_FORMAT_FIGEXP M_FIG Graphical AbstractTo obtain a comprehensive picture of the coordinated changes in epithelial, stromal and immune compartments during development of Barretts-associated esophageal adenocarcinoma, patient-matched samples corresponding to various phases of disease progression were collected from 12 patients, each of which had at a given time point lesions at multiple stages progression (matched-normal, metaplasia, dysplasia, and carcinoma). Matched "normal" gastric tissues were also collected. These sample were analyzed by single cell RNA-sequencing (scRNAseq) for single-cell resolution transcriptomics and Copy Number Variant (CNV), by proteomics for extracellular matrix (ECM) proteins, by Atomic Force Microscopy (AFM for tissue stiffness and by CODEX spatial proteomics imaging The integrative multi-omics analysis exposed drastic alterations in cell type composition and shifts in cell states in all three compartments. A large subpopulation of fibroblasts absent in the normal esophagus and characteristic of dysplasia and adenocarcinoma sample, that based on markers would indeed be considered cancer associated fibroblasts (CAF), appeared already in the metaplastic phase. This fibroblast subpopulation had transcriptomes virtually indistinguishable with fibroblasts of the cancer free gastric epithelium in these patients C_FIG

cancer biology↗

bioRxiv2021

The Food Additive Xanthan Gum Drives Adaptation of the Human Gut Microbiota

The diets of industrialized countries reflect the increasing use of processed foods, often with the introduction of novel food additives. Xanthan gum is a complex polysaccharide with unique rheological properties that have established its use as a widespread stabilizer and thickening agent1. However, little is known about its direct interaction with the gut microbiota, which plays a central role in digestion of other, chemically-distinct dietary fiber polysaccharides. Here, we show that the ability to digest xanthan gum is surprisingly common in industrialized human gut microbiomes and appears to be contingent on the activity of a single bacterium that is a member of an uncultured bacterial genus in the family Ruminococcaceae. We used a combination of enrichment culture, multi-omics, and recombinant enzyme studies to identify and characterize a complete pathway in this uncultured bacterium for the degradation of xanthan gum. Our data reveal that this keystone degrader cleaves the xanthan gum backbone with a novel glycoside hydrolase family 5 (GH5) enzyme before processing the released oligosaccharides using additional enzymes. Surprisingly, some individuals harbor a Bacteroides species that is capable of consuming oligosaccharide products generated by the keystone Ruminococcaceae or a purified form of the GH5 enzyme. This Bacteroides symbiont is equipped with its own distinct enzymatic pathway to cross-feed on xanthan gum breakdown products, which still harbor the native linkage complexity in xanthan gum, but it cannot directly degrade the high molecular weight polymer. Thus, the introduction of a common food additive into the human diet in the past 50 years has promoted the establishment of a food chain involving at least two members of different phyla of gut bacteria.

microbiology↗

bioRxiv2021

Inactivation of the Hippo Tumor Suppressor Pathway Promotes Melanoma

Human melanomas are commonly driven by activating mutations in BRAF, which promote melanocyte proliferation through constitutive stimulation of the MAPK pathway. However, oncogenic BRAF alone is insufficient to promote melanoma; instead, its expression merely induces a transient burst of proliferation that ultimately ceases with the development of benign nevi (i.e. moles) comprised of growth-arrested melanocytes. The tumor suppressive mechanisms that induce this melanocytic growth arrest remain poorly understood. Recent modeling studies have suggested that the growth arrest of nevus melanocytes is not solely due to oncogene activation in individual cells, but rather due to cells sensing and responding to their collective overgrowth, similar to what occurs in normal tissues. This cell growth arrest is reminiscent of the arrest induced by activation of the Hippo tumor suppressor pathway, which is an evolutionarily conserved pathway known to regulate organ size. Herein, we demonstrate that oncogenic BRAF signaling activates the Hippo pathway in vitro, which leads to inhibition of the pro-growth transcriptional co-activators YAP and TAZ, ultimately promoting the growth arrest of melanocytes. We also provide evidence that the Hippo tumor suppressor pathway is activated in growth-arrested nevus melanocytes in vivo, both from single-cell sequencing of mouse models of nevogenesis and human tissue samples. Mechanistically, we observe that oncogenic BRAF promotes both ERK-dependent alterations in the actin cytoskeleton and whole-genome-doubling events, and that these two effects independently promote Hippo pathway activation. Lastly, we demonstrate that abrogation of the Hippo pathway, via melanocyte-specific deletion of the Hippo kinases Lats1/2, enables oncogenic BRAF-expressing melanocytes to bypass nevus formation, thus leading to the rapid onset of melanoma with 100% penetrance. This model is clinically relevant, as co-heterozygous loss of LATS1/2 is observed in [~]15% of human melanomas. Collectively, our data reveal that the Hippo pathway enforces the stable growth arrest of nevus melanocytes and therefore represents a critical and previously unappreciated barrier to melanoma development.

cancer biology↗

bioRxiv2020

Volatiles from Serratia marcescens, S. proteamaculans, and Bacillus subtilis Inhibit Growth of Rhizopus stolonifer and Other Fungi

The common soil bacteria Serratia marcescens, Serratia proteamaculans, and Bacillus subtilis produce small molecular weight volatile compounds that are fungi-static against multiple species, including the zygomycete mold Rhizopus stolonifer (Mucoromycota) and the model filamentous mold Neurospora crassa (Ascomycota). The compounds or the bacteria can be exploited in development of biological controls to prevent establishment of fungi on food and surfaces. Here, we quantified and identified bacteria-produced volatiles using headspace sampling and gas chromatography-mass spectrometry. We found that each bacterial species in culture has a unique volatile profile consisting of dozens of compounds. Using multivariate statistical approaches, we identified compounds in common or unique to each species. Our analysis suggested that three compounds, dimethyl trisulfide, anisole, and 2-undecanone, are characteristic of the volatiles emitted by these antagonistic bacteria. We developed bioassays for testing inhibition of each compound and found dimethyl trisulfide and anisole were the most potent. This work establishes a pipeline for translating volatile profiles of cultured bacteria into high quality candidate fungistatic compounds which may be useful in combination as antifungal control products. IMPORTANCEBacteria may benefit by producing fungistatic volatiles that limit fungal growth providing a mechanism to exclude competitors for resources. Volatile production is potentially mediating long distance biological control and competitive in-teractions among microbes, but the specific bioactive compounds are poorly characterized. This work provides evidence that fungistatic compounds in complex blends can be identified using machine-learning and multivariate approaches. This is the first step in identifying pathways responsible for fungistatic volatile production in order to phenotype and select natural strains for biocontrol ability, or engineer bacteria with relevant pathways.

microbiology↗

bioRxiv2020

Validation of a Single-step, Single-tube Reverse Transcription-Loop-Mediated Isothermal Amplification Assay for Rapid Detection of SARS-CoV-2 RNA

2.IntroductionThe SARS-CoV-2 pandemic of 2020 has resulted in unparalleled requirements for RNA extraction kits and enzymes required for virus detection, leading to global shortages. This has necessitated the exploration of alternative diagnostic options to alleviate supply chain issues. AimTo establish and validate a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of SARS-CoV-2 from nasopharyngeal swabs. MethodologyWe used a commercial RT-LAMP mastermix from OptiGene Ltd in combination with a primer set designed to detect the CDC N1 region of the SARS-CoV-2 nucleocapsid (N) gene. A single-tube, single-step fluorescence assay was implemented whereby as little as 1 L of universal transport medium (UTM) directly from a nasopharyngeal swab could be used as template, bypassing the requirement for RNA purification. Amplification and detection could be conducted in any thermocycler capable of holding 65{degrees}C for 30 minutes and measure fluorescence in the FAM channel at one-minute intervals. ResultsAssay evaluation by assessment of 157 clinical specimens previously screened by E-gene RT-qPCR revealed assay sensitivity and specificity of 87% and 100%, respectively. Results were fast, with an average time-to-positive (Tp) for 93 clinical samples of 14 minutes (SD {+/-}7 minutes). Using dilutions of SARS-CoV-2 virus spiked into UTM, we also evaluated assay performance against FDA guidelines for implementation of emergency-use diagnostics and established a limit-of-detection of 54 Tissue Culture Infectious Dose 50 per ml (TCID50 mL-1), with satisfactory assay sensitivity and specificity. A comparison of 20 clinical specimens between four laboratories showed excellent interlaboratory concordance; performing equally well on three different, commonly used thermocyclers, pointing to the robustness of the assay. ConclusionWith a simplified workflow, N1-STOP-LAMP is a powerful, scalable option for specific and rapid detection of SARS-CoV-2 and an additional resource in the diagnostic armamentarium against COVID-19. 3. Data summaryThe authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

microbiology↗

bioRxiv2020

Stabilising selection causes grossly altered but stable karyotypes in metastatic colorectal cancer

Aneuploidy, the loss and gain of whole and part chromosomes, is near-ubiquitous in cancer genomes and likely defines cancer cell biology. However, the temporal evolutionary dynamics that select for aneuploidy remain uncharacterised. Here we perform longitudinal genomic analysis of 755 samples from a total of 167 patients with colorectal-derived neoplastic lesions that represent distinct stages of tumour evolution through metastasis and treatment. Adenomas typically had few copy number alterations (CNAs) and most were subclonal, whereas cancers had many clonal CNAs, suggesting that progression goes through a CNA bottleneck. Individual CRC glands from the same tumour typically had very similar karyotypes, despite evidence of ongoing instability at the cell level in patient tumours, cell lines and organoids. CNAs in metastatic lesions sampled from liver and other organs, after chemotherapy or targeted therapies, and in late recurrences were typically similar to the primary tumour. Mathematical modelling and statistical inference indicated that these data are consistent with the action of negative selection on CNAs that traps cancer cell genomes on a fitness peak defined by the specific pattern of chromosomal aberrations. These data suggest that the initial progression of colorectal cancer requires the traversal of a rugged fitness landscape and subsequent CNA evolution, including metastatic dissemination and therapeutic resistance, is constrained by negative selection.

cancer biology↗

bioRxiv2020

Quality control of HLA-DR molecules by the lysosomal aspartyl protease, cathepsin D

Major histocompatibility complex class II (MHCII) molecules display peptides on antigen-presenting cells (APCs) for inspection by CD4+ T cells. MHCII surface levels and life span are regulated post-translationally by peptide loading and ubiquitin-dependent lysosomal targeting, but proteases responsible for MHCII protein degradation remain unidentified. Here, we examined the role of aspartyl proteases in MHCII protein degradation and characterised the form of MHCII that is degraded. Exposure of immature monocyte-derived dendritic cells (MoDCs) and KG-1 acute myeloid leukaemia cells to the aspartyl protease inhibitor, pepstatin A (PepA), caused accumulation of human leukocyte antigen (HLA)-DR molecules in intracellular vesicles. Statistically significant PepA effects on MHCII protein expression were also observed in murine APCs. In KG-1 cells, cathepsin D (CatD) was the sole expressed aspartyl protease, and shRNA-mediated knockdown ablated the PepA effect, providing formal proof of CatD involvement. In vitro, CatD initiated specific cleavage of recombinant DR at F54, a site flanking the peptide-binding groove. Immunochemical characteristics of PepA-rescued DR molecules in KG-1 cells were consistent with selective CatD attack on HLA-DR molecules that lack association with the MHCII chaperone, invariant chain, or with stably bound peptide. We propose that CatD has a critical role in the selective lysosomal disposal of mature HLA-DR molecules that have lost, or never acquired, bound peptide, explaining how MHCII protein life span is coupled to peptide loading.

biochemistry↗

bioRxiv2019

Green, orange, red, and far-red optogenetic tools derived from cyanobacteriochromes

Existing optogenetic tools for controlling protein-protein interactions are available in a limited number of wavelengths thereby limiting opportunities for multiplexing. The cyanobacteriochrome (CBCR) family of photoreceptors responds to an extraordinary range of colors, but light-dependent binding partners for CBCR domains are not currently known. We used a phage-display based approach to develop small (~50-residue) monomeric binders selective for the green absorbing state (Pg), or for the red absorbing state (Pr) of the CBCR Am1_c0023g2 with a phycocyanobilin chromophore and also for the far-red absorbing state (Pfr) of Am1_c0023g2 with a biliverdin chromophore. These bind in a 1:1 mole ratio with KDs for the target state from 0.2 to 2 M and selectivities from 10 to 500-fold. We demonstrate green, orange, red, and far-red light-dependent control of protein-protein interactions in vitro and also in vivo where these multicolor optogenetic tools are used to control transcription in yeast.

synthetic biology↗