Biology subjects

Brown, M.

Publications and source records attributed to Brown, M..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Super-resolution microscopy reveals distinct epigenetic states regulated by estrogen receptor activity

Changes in gene expression regulated by ligand-dependent transcription factors such as estrogen receptor- (ER) involves the recruitment of coactivators including p300 that acetylates histone H3 at lysine 27 (H3K27ac). While H3K27ac marks active enhancers, the detailed chromatin architecture of enhancers remains unclear. Using super-resolution microscopy, we reveal distinct structural states of H3K27ac modified chromatin in response to ER activation. In estradiol (E2)-treated cells, H3K27ac modified chromatin adopts open, elongated structures, while ER inhibition induces compact, spherical H3K27ac modified chromatin conformations. Using MED1, a core subunit of the Mediator complex that bridges transcription factors with RNA polymerase II (Pol II), we demonstrate that larger H3K27ac structures are preferentially associated with active enhancers, whereas more compact structures show reduced MED1 association, consistent with a less active or inactive state. A constitutively active ER mutation linked to endocrine therapy resistance in breast cancer maintains open chromatin states independent of ligand, suggesting sustained transcriptional activity. Our findings provide the first direct visualization of H3K27ac associated chromatin structural dynamics, challenging the assumption that H3K27ac modification alone is sufficient to lead to enhancer activation. By demonstrating that H3K27ac architecture is dynamically regulated by ER, we establish a new paradigm for understanding epigenetic regulation and highlight potential therapeutic targets for endocrine therapy resistant cancers.

cancer biology↗

bioRxiv2025

Desiccation-induced DNA damage facilitates drug resistance in Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) is an obligate human pathogen that depends on its ability to spread from host-to-host to survive as a species. Yet, knowledge of transmission-specific traits remains lacking. Here, we report the discovery of a specific adaptive response to desiccation, a stress intrinsically linked to the generation of the aerosol droplets within which Mtb transmits. We show that desiccation inflicts oxidative damage and activates Mtbs DNA repair responses but that this repair is imperfect and results in mutations. We further show that activation of these DNA repair responses is accompanied by increased expression of the transcription-coupled repair factor, mfd, but that this expression serves to buffer the fitness cost of specific resistance-conferring mutations in rpoB, the target of the frontline drug rifampin, rather than to facilitate transcription-coupled DNA repair. Silencing mfd during aerosolization impairs survival of strains harboring the rifampin resistance allele S450L. This function is further supported by whole genome sequence data from over 50,000 clinically circulating strains. These studies indicate that Mtb has evolved transmission-specific stress responses that have enabled it to leverage desiccation-induced DNA damage as a potential source of genetic diversification and drug resistance.

microbiology↗

bioRxiv2025

Lack of synergy between AR targeted therapies and PARP inhibitors in homologous recombination-proficient prostate cancer

Recent clinical trials have explored the combination of androgen receptor (AR) pathway inhibitors and poly (ADP-ribose) polymerase (PARP) inhibitors as a potential treatment for castration-resistant prostate cancer. This combination treatment is based on the premise that AR directly regulates expression of DNA repair genes, leading to synergy between PARP and AR inhibition. Despite some promising preclinical evidence, this combination therapy has shown limited efficacy in patients with homologous recombination (HR)-proficient tumors. To investigate this discrepancy between preclinical and clinical results, we profiled the effects of PARP inhibition in prostate cancer models in the presence or absence of AR inhibition. Surprisingly, AR inhibition impaired response to PARP inhibitors in castration-sensitive cells and had no effect on response in castration-resistant cells. AR inhibition also did not regulate DNA repair in either the castration-resistant or castration-sensitive setting. Instead, we find that cell cycle progression is required for response to PARP inhibition in homologous-recombination proficient prostate cancer. STATEMENT OF SIGNIFICANCEAndrogen deprivation does not inhibit DNA repair and does not synergize with PARP inhibition in prostate cancer with intact homologous recombination repair.

cancer biology↗

bioRxiv2025

Intratumoral cDC1-T Cell Clusters Serve as Sites of Local Costimulation to Enhance CTL-Mediated Tumor Rejection

T cells are essential for anti-tumor immunity, but their ability to eliminate tumors depends on coordinated interactions with type 1 conventional dendritic cells (cDC1s). While cDC1s are known for cross-presenting tumor-derived antigens in lymph nodes to prime CD8+ T cells, their role within the tumor itself remains less well understood. Here, we use the Skin Tumor Array by Micro-Poration (STAMP) model to investigate how cDC1-T cell interactions shape immune responses and influence tumor fate. Our data reveal that it is the spatial distribution of both cDC1s and T cells that determines whether a tumor can be rejected. We defined three primary immunotypes based on the spatial distribution of T cells and cDC1s: T cell-inflamed/dendritic cell-inflamed (TC-In/DC-In) tumors, where T cells and cDC1s co-infiltrate the tumor; T cell-inflamed/dendritic cell-excluded (TC-In/DC-Ex) tumors, where T cells infiltrate but cDC1s remain at the periphery; and T cell-excluded/dendritic cell-excluded (TC-Ex/DC-Ex) tumors, which lack both cDC1 and T cell infiltration. Notably, TC-In/DC-In tumors are more likely to undergo rejection, whereas TC-In/DC-Ex tumors persist despite T cell infiltration. Within TC-In/DC-In tumors, cDC1s engage in direct interactions with T cells, upregulate co-stimulatory molecules, and sustain effector T cell responses, while cDC1s in TC-In/DC-Ex tumors express higher migration-associated genes, suggesting a propensity to exit the tumor. We further show that chemokine modulation, particularly through CXCL9, CCL5, and XCL1, can reshape immune infiltration patterns to promote intra-tumoral cDC1-T cell clustering and improve tumor rejection. These findings underscore the unexpectedly important role of cDC1 positioning and function in sustaining effective anti-tumor immunity and highlight spatially organized cDC1-T cell clusters as critical hubs for local T cell activation.

immunology↗

bioRxiv2025

Pathogenic Viruses, Genome Integrations, and Viral::Human Chimeric Transcripts Detected by VirusIntegrationFinder Across >30k Human Tumor and Normal Samples

Viruses are a leading cause of human morbidity and mortality. Certain viruses, including human papillomaviruses (HPVs), play a significant role in the etiology of cancer. Detection of viral DNA insertions in the human genome from next generation sequencing data defines viral associations with cancer and other diseases, identifies impacted organs and tissues, provides insights into disease mechanisms and has the potential to enhance clinical evaluations. In this study, we developed VirusIntegrationFinder (CTAT-VIF), a tool for surveying human genome insertions of various human viruses using both DNA and RNA sequencing data. We applied CTAT-VIF to analyze a dataset of over 30,000 tumor and normal samples, as well as more than 1,000 cancer cell lines. This effort resulted in the compilation of a catalog of over 30,0000 virus-human DNA or RNA junctions at more than 20,000 insertion loci and reassessed viral cancer-insertion hotspots across the human genome. Furthermore, we characterized the functional impacts of insertions with respect to human copy number alterations, effects on the expression of flanking human genes, and the identification of potentially oncogenic chimeric human and human/virus fusion transcripts at insertion loci. In addition to confirming known viral associations with specific tumor types, our study revealed both shared and virus-specific insertion hotspots in addition to variable functional impacts based on virus type. Besides some rare events of interest, we also found evidence for sequencing contamination, which underscores the need for vigilance when studying viral content or genome integrations.

cancer biology↗

bioRxiv2025

Lowering CD40L expression in Murine Lupus Results in an Increase in Disease Indicators in Female but not Male B6 mice

Our lab has previously described a mouse model (CD40L{Delta}5) that produces only 60% of WT CD40L due to a targeted deletion in an RNA binding site within the CD40L message. The CD40L{Delta}5 mutation, which destabilizes CD40L mRNA during T cell activation, causes disrupted germinal center (GC) formation leading to reduced levels of memory B cells and switched antibodies. In this study, we used our model of limited CD40L expression to investigate its effect on Systemic Lupus Erythematosus (SLE or lupus). Two mouse models of SLE were assessed: The first, termed PIL, used pristane to induce disease over a six-month period and the second utilized a chronic graft-versus-host disease (bm12-cGVHD) that resembles lupus based on multiple parameters and allowed us to monitor the early events in disease development. Importantly, we found that in both model systems female mice expressing the CD40L{Delta}5 mutation showed a consistent increase in elevated antibody secreting cells and autoantibody titers. Also, PIL female CD40L{Delta}5 mice had higher levels of immunocomplex deposition in the kidney compared to all other cohorts. In the bm12-cGVHD model, cellular increases in GC cells along with an altered cytokine profile of donor CD4+ T cells and host dendritic cells (DCs) reflected a significant skewing of CD40L{Delta}5 female CD4 T cells towards a Th2 phenotype. Overall, our results support a more nuanced role for CD40L in lupus than previously described and suggest a sex-determined threshold of CD40L-CD40 signaling that demarcates an interface between protection and exacerbation at the very early steps of disease progression.

immunology↗

bioRxiv2025

Coordinated macrophage and T cell interactions mediate response to checkpoint blockade in colorectal cancer.

Mismatch repair deficiency (MMRd), either due to inherited or somatic mutation, is prevalent in colorectal cancer (CRC) and other cancers. While anti-PD-1 therapy is utilized in both local and advanced disease, up to 50% of MMRd CRC fail to respond. Using animal and human models of MMRd, we determined that interactions between MHC+ C1Q+ CXCL9+ macrophages and TCF+ BHLHE40+ PRF1+ T cell subsets are associated with control of MMRd tumor growth, during anti-PD-1 treatment. In contrast, resistance is associated with upregulation of TIM3, LAG3, TIGIT, and PD-1 expression on T cells, and infiltration of the tumor with immunosuppressive TREM2+ macrophages and monocytes. By combining anti-PD-1 with anti-LAG3/CTLA4/TREM2, up to 100% tumor eradication was achieved in MMRd CRC and remarkably, in >70% in MMRp CRC. This study identifies key T cell and macrophage subsets mediating the efficacy of immunotherapy in overcoming immune escape in both MMRd and MMRp CRC settings. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/637954v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@1778bd6org.highwire.dtl.DTLVardef@17da4e5org.highwire.dtl.DTLVardef@1d4d1d2org.highwire.dtl.DTLVardef@11b58e3_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIAnti-PD-1 therapy leads to the accumulation and colocalization of MHCI/II+ C1Q+ CXCL9+ macrophages and DCs with TCF+ CCL5+ T cells that have high TCR diversity. C_LIO_LIResistance to anti-PD-1 therapy involves multiple T cell checkpoints, TREM2+ macrophages, IL1B+ TREM1+ monocytes and neutrophils, and IFITM+ tumor cells. C_LIO_LISimultaneous blockade of PD-1, LAG3, CTLA-4 and TREM2 dramatically prevents progression of both MMRd and MMRp tumors. C_LIO_LICombination therapy completely eliminates tumors by leveraging MHC+ macrophage, CD4+ and CD8+ T cell interactions, facilitating durable anti-tumor effects. C_LI

immunology↗

bioRxiv2024

The probability and duration of immigration in microbial communities

Immigration is a fundamental feature of microbial communities. We propose a method to determine the probability (Pi) that a number of immigrants (i) can attain an abundance N using the ratio of the probabilities of death q and division or "birth" p and the gamblers ruin equation: Pi= (1-(q/p)i/(1-(q/p)N). We estimate the probability of successful bioaugmentation or transplantation, the fate of a mutation infection and extinction. For example, an inoculum of 108 bacteria with a q/p of 1.00000001 has a 10-43 chance of attaining an abundance of 1010. The immigration parameter used in neutral models, m is 1/(1-q/p). We calculated the long-term average value of m and q/p in a wastewater treatment plant. The value of m varies by >5 orders of magnitude, with a curious bimodal distribution. However, all the values of q/p are very close to, but greater than, 1. We expect the long-term average value of q/p to be [~]1 in all stable microbial communities. In the absence of migration, bacterial populations with a q/p [≥]1 will go extinct with probability 1. The link between q/p and infectious dose is known and we demonstrate that, in principle, the gamblers ruin equation can estimate the infectious dose in naturally occurring infections, using Vibrio cholerae "carriers" to illustrate the point. We use the ratio q/p to estimate the time (measured in events or solar time) for a given change in abundance to happen. When q/p=1, extinction in even a small microbial population will take thousands of years.

microbiology↗

bioRxiv2024

Mobile Human Brain Imaging using Functional Ultrasound

Imagine being able to study the human brain in real-world scenarios while the subject displays natural behaviors such as locomotion, social interaction, or spatial navigation. The advent of ultrafast ultrasound imaging brings us closer to this goal with functional Ultrasound imaging (fUSi), a new mobile neuroimaging technique. Here, we present real-time fUSi monitoring of brain activity during walking in a subject with a clinically approved sonolucent skull implant. Our approach utilizes personalized 3D-printed fUSi-helmets for stability, optical tracking for cross-modal validation with fMRI, advanced signal processing to estimate hemodynamic responses and facial tracking of a lick licking paradigm. These combined efforts allowed us to show consistent fUSi signals over 20 months, even during high-motion activities like walking. These results demonstrate the feasibility of fUSi for monitoring brain activity in real-world contexts, marking an important milestone for fUSi-based insights in clinical and neuroscientific research.

neuroscience↗

bioRxiv2024

Epigenetic investigation of multifocal small intestinal neuroendocrine tumours reveals accelerated ageing of tumours and epigenetic alteration of metabolic genes

BackgroundSmall intestinal neuroendocrine tumours (SI-NETs) are the most common malignancy of the small intestine and around 50% of patients present in clinic with multifocal disease. Recent investigations into the genomic architecture of multifocal SI-NETs have found evidence that these synchronous primary tumours evolve independently of each other. They also have extremely low mutational burden and few known driver genes, suggesting that epigenetic dysregulation may be driving tumorigenesis. Very little is known about epigenetic gene regulation, metabolism and ageing in these tumours, and how these traits differ across multiple tumours within individual patients. MethodsIn this study, we performed the first investigation of genome-wide DNA methylation in multifocal SI-NETs, assessing multiple primary tumours within each patient (n=79 primary tumours from 14 patients) alongside matched metastatic tumours (n=12) and normal intestinal epithelial tissue (n=9). We assessed multifocal SI-NET differential methylation using a novel method, comparing primary tumours with matched normal epithelial tissue and an enterochromaffin-enriched cell line to enrich for tumour-specific effects. This method reduced the identification of false positive methylation differences driven by cell composition differences between tumour and normal epithelial tissue. We also assessed tumour ageing using epigenetic clocks and applied metabolic predictors in the dataset to assess methylation variation across key metabolic genes. ResultsWe have identified 12,392 tumour-specific differentially methylated positions (Bonferroni corrected p<0.05) which were enriched for neural pathways. The expression levels of the genes associated with top sites were also found to be significantly altered in SI-NETs. Age acceleration was observed across SI-NETs and a variability in epigenetic age of tumours within each patient, which we believe is reflecting the order in which tumours have developed. This is supported by the correlation of age acceleration with somatic mutational count in the tumours. We have identified SI-NET associated alterations to the methylation patterns in key metabolic genes compared to matched normal tissue, which is more pronounced in metastatic tumours and tumours harbouring chromosome 18 loss of heterozygosity, indicating metabolic differences in these tumour subtypes. ConclusionsWe have identified accelerated ageing and changes to regulation of metabolic genes, alongside an epigenetic signature of multifocal SI-NETs. These findings add to our understanding of multifocal SI-NET biology and their molecular differences which may be instrumental in the development of these elusive tumours.

cancer biology↗

bioRxiv2024

Sugar phosphate-mediated inhibition of peptidoglycan precursor synthesis

Antibiotic tolerance, the widespread ability of diverse pathogenic bacteria to sustain viability in the presence of typically bactericidal antibiotics for extended time periods, is an understudied steppingstone towards antibiotic resistance. The Gram-negative pathogen Vibrio cholerae, the causative agent of cholera, is highly tolerant to {beta}-lactam antibiotics. We previously found that the disruption of glycolysis, via deletion of pgi (vc0374, glucose-6-phosphate isomerase), resulted in significant cell wall damage and increased sensitivity towards {beta}-lactam antibiotics. Here, we uncover the mechanism of this resulting damage. We find that glucose causes growth inhibition, partial lysis, and a damaged cell envelope in {Delta}pgi. Supplementation with N-acetylglucosamine, but not other carbon sources (either from upper glycolysis, TCA cycle intermediates, or cell wall precursors) restored growth, re-established antibiotic resistance towards {beta}-lactams, and recovered cellular morphology of a pgi mutant exposed to glucose. Targeted metabolomics revealed the cell wall precursor synthetase enzyme GlmU (vc2762, coding for the bifunctional enzyme that converts glucosamine-1P to UDP-GlcNAc) as a critical bottleneck and mediator of glucose toxicity in {Delta}pgi. In vitro assays of GlmU revealed that sugar phosphates (primarily glucose-1-phosphate) inhibit the acetyltransferase activity of GlmU (likely competitively), resulting in compromised PG and LPS biosynthesis. These findings identify GlmU as a critical branchpoint enzyme between central metabolism and cell envelope integrity and reveal the molecular mechanism of {Delta}pgi glucose toxicity in Vibrio cholerae. ImportanceSugar-phosphate toxicity is a well characterized phenomenon that is seen within diverse bacterial species, and yet the molecular underpinnings remain elusive. We previously discovered that disrupting Vibrio choleraes ability to eat glucose (by disrupting the pgi gene), also resulted in a damaged cell envelope. Upon deletion of pgi, glucose-phosphate levels rapidly build and inhibit the enzymatic activity of GlmU, a key step of bacterial peptidoglycan precursor synthesis. GlmU inhibition causes enhanced killing by antibiotics and a pronounced cell envelope defect. Thus, GlmU serves as a prime target for novel drug development. This research opens new routes through which central metabolism and sugar-phosphate toxicity modulate antibiotic susceptibility.

microbiology↗

bioRxiv2024

Random subsamples of animal populations can reveal intrinsic differences in sociality with key implications in ecology, conservation and disease transmission

Animal populations are under mounting stress from the dual threats of climate change and rapid global human population growth, raising significant concerns about declining wildlife and the rising risk of zoonotic diseases. In many species, social interactions can be a highly plastic suite of behaviours that are responsive to these disturbances and are consequential to other processes like disease transmission and population dynamics. Studying social interactions can be challenging in that researchers often rely on wildlife population subsamples due to practical constraints and costs, which can introduce biases in the reliability of social network metrics. We investigated the extent to which subsamples can depict intrinsic characteristics of wildlife populations using data from three distinct species: peri-urban fallow deer, Alpine ibex and Angolan giraffe. We showed that random subsamples of these populations could still reveal differences in their social behaviour, indicating that, as long as researchers have a reliable estimate of population size, subsampling animal populations can be an effective and precise method to infer their sociality and offer valuable empirical data for management, conservation and zoonotic disease ecology. Furthermore, we demonstrate that non-random sampling, influenced for instance by animal personality and related trappability, can introduce significant biases in social network estimates. These findings underscore the importance of accounting for sampling biases in social network analysis and offer a robust framework for using partial networks in ecological studies and conservation management.

ecology↗

bioRxiv2024

Oatk: a de novo assembly tool for complex plant organelle genomes

Plant organelle genomes, particularly the large mitochondrial genomes with intricate repetitive structures, present significant challenges for assembly. The advent of long-read sequencing technologies provides a transformative opportunity to generate complete genomes, but problems of resolving alternative structures remain. Here we introduce a novel tool for plant organelle genome assembly from high-accuracy long reads. Our method employs a k-mer based assembler for rapid assembly graph construction, integrates a profile HMM gene database for robust organelle sequence annotation, and leverages a new search method to find the best supported path through the assembly graph. We describe high-quality organelle assemblies for 195 plant species and demonstrate improvements over other methods. The assembled genomes provide multiple insights into structural complexity, heteroplasmy, and DNA exchange between organelles.

bioinformatics↗

bioRxiv2024

T cell epitope mapping reveals immunodominance of evolutionarily conserved regions within SARS-CoV-2 proteome.

As SARS-CoV-2 variants continue to emerge capable of evading neutralizing antibodies, it has become increasingly important to fully understand the breadth and functional profile of T cell responses to determine their impact on the immune surveillance of variant strains. Here, sampling healthy individuals, we profiled the kinetics and polyfunctionality of T cell immunity elicited by mRNA vaccination. Modeling of anti-spike T cell responses against ancestral and variant strains of SARS-CoV-2 suggested that epitope immunodominance and cross-reactivity are major predictive determinants of T cell immunity. To identify immunodominant epitopes across the viral proteome, we generated a comprehensive map of CD4+ and CD8+ T cell epitopes within non-spike proteins that induced polyfunctional T cell responses in convalescent patients. We found that immunodominant epitopes mainly resided within regions that were minimally disrupted by mutations in emerging variants. Conservation analysis across historical human coronaviruses combined with in silico alanine scanning mutagenesis of non-spike proteins underscored the functional importance of mutationally-constrained immunodominant regions. Collectively, these findings identify immunodominant T cell epitopes across the mutationally-constrained SARS-CoV-2 proteome, potentially providing immune surveillance against emerging variants, and inform the design of next-generation vaccines targeting antigens throughout SARS-CoV-2 proteome for broader and more durable protection. One Sentence SummaryPolyfunctional CD8+ and CD4+ T cells directed against SARS-CoV-2 target mutationally constrained regions of the viral proteome.

immunology↗

bioRxiv2024

Exploiting the Achilles' Heel of Viral RNA Processing to Develop Novel Antivirals

Viruses continue to pose a significant health burden to the human population, and recent history has shown a concerning surge in viral threats. Treatment options for viral infections are limited, and viruses have proven adept at evolving resistance to many existing therapies, highlighting a significant vulnerability in our defenses. In response to this challenge, we explored the modulation of cellular RNA metabolic processes as an alternative paradigm to antiviral development. Many viruses depend on the host cells RNA splicing machinery, and small alterations to this host process results in catastrophic changes in viral protein production, ultimately inhibiting virus replication. Previously, the small molecule 5342191 was identified as a potent inhibitor of HIV-1 replication by altering viral RNA accumulation at doses that minimally affect host gene expression. In this report, we document 5342191 as a potent inhibitor of adenovirus, coronavirus, and influenza replication. In each case, 5342191-mediated reduction in virus replication was associated with altered viral RNA accumulation and loss of viral structural protein expression. Interestingly, while resistant viruses were rapidly isolated for compounds targeting either virus-encoded proteases or polymerases, we have not yet isolated 534219-resistant variants of coronavirus or influenza. As with HIV-1, 5342191s inhibition of coronaviruses and influenza is mediated through the activation of specific cell signaling networks, including GPCR and/or MAPK signaling pathways that ultimately affect SR kinase expression. Together, these studies highlight the therapeutic potential of compounds that target cellular processes essential for the replication of multiple viruses. Not only do these compounds hold promise as broad-spectrum antivirals, but they also offer the potential of greater resilience in combating viral infections.

microbiology↗

bioRxiv2024

New imaging tools reveal live cellular collagen secretion, fibril dynamics and network organisation.

Although light microscopy has been used to examine the early trafficking of collagen within the cell, much of our understanding of the detailed organisation of cell deposited collagen is from static electron microscopy studies. To understand the dynamics of live cell collagen deposition and fibril organisation, we generated a bright photostable mNGCol12 fusion protein and employed a range of microscopy techniques to follow its intracellular transport and elucidate extracellular fibril formation. Our findings reveal the dynamics of fibril growth and the dynamic nature of collagen network interactions at the cellular level. Notably we observed molecular events that build network organisation, including fibril bundling, bifurcation, directionality along existing fibrils, and looping/intertwining behaviours. Strikingly, mNGCol12 fluorescence intensity maxima can mark a fibril before another growing collagen fibril intersects at this location. Our study shows that N-terminal protease site is not an absolute requirement for collagen fibril incorporation and is, to our knowledge, the first time that cell-directed collagen fibrillogenesis has been visualised at high resolution in real time. The approach paves the way for assessing the dynamic organisation and assembly of collagen into the extracellular matrix in skin models and other tissues during health, ageing and disease.

cell biology↗

bioRxiv2024

Rapamycin increases murine lifespan but does not reduce mineral volume in the Matrix GLA Protein (MGP) knockout mouse model of medial arterial calcification.

Peripheral artery disease (PAD) is the narrowing of the arteries that carry blood to the lower extremities. PAD has been traditionally associated with atherosclerosis. However, recent studies have found that thrombotic events triggered by medial arterial calcification (MAC) is the primary cause of chronic limb ischemia below the knee. MAC is localized around the elastic fibers surrounding smooth muscle cells (SMCs) in arteries. Matrix GLA protein (MGP) binds circulating calcium and prevents hydroxyapatite mineral deposition, while also modulating proosteogenic signaling by attenuating BMP-2-mediated activation of Runx2 gene expression. Mgp-/- mice develop severe MAC and die around 8 weeks after birth due to aortic rupture or heart failure. We previously discovered a rare genetic disease Arterial Calcification due to Deficiency of CD73 (ACDC), in which patients present with extensive MAC in their lower extremity arteries. Using a patient-specific induced pluripotent stem cell model, we found that rapamycin inhibited calcification. Here we investigated whether rapamycin could reduce MAC in vivo using the Mgp-/- murine model. Mgp+/+ and Mgp-/- mice received 5mg/kg rapamycin or vehicle. Calcification content was assessed via microCT, and vascular morphology and extracellular matrix content were assessed histologically. Immunostaining and western blot analysis were used to examine SMC phenotype and extracellular matrix content. Rapamycin prolonged Mgp-/- mice lifespan, decreased mineral density in the arteries, maintained SMC contractile phenotype, and improved vessel structure, however, calcification volume was unchanged. Mgp-/- mice with SMC-specific deletion of Raptor or Rictor did not recapitulate treatment with rapamycin. These findings suggest rapamycin promotes beneficial vascular remodeling in vessels with MAC. NEWS AND NOTEWORTHYPeripheral artery disease (PAD) is associated with medial arterial calcification (MAC), which involves calcification of arterial elastic fibers and smooth muscle cells (SMCs). Matrix GLA protein (MGP) inhibits vascular calcification, and Mgp-/- mice develop severe MAC. Using this model, we found rapamycin prolonged lifespan, reduced arterial mineral density, maintained SMC contractile phenotype, and improved vessel structure, though calcification volume remained unchanged. Findings highlight rapamycins potential for vascular remodeling in MAC.

physiology↗

bioRxiv2024

Obtaining super-resolved images at the mesoscale through Super-Resolution Radial Fluctuations

Super-resolution microscopy overcomes the diffraction limit of light to achieve higher spatial resolutions than are typically available when using light microscopy techniques. However, these methods are usually restricted to imaging a very small field of view (FOV). Here, we have applied one of these super-resolution techniques, Super-Resolution Radial Fluctuations (SRRF) in conjunction with the Mesolens, which has the unusual combination of a low-magnification and high numerical aperture, to obtain super-resolved images over a FOV of 4.4 mm x 3.0 mm. We assessed the accuracy of these SRRF images through error maps calculated using a secondary analysis method, Super-resolution Quantitative Image Rating and Reporting of Error Locations (SQUIRREL). We demonstrate it is possible to achieve images with a resolution of 446.3 {+/-} 10.9 nm, providing a [~]1.6-fold improvement in spatial resolution over a uniquely large field, with consistent structural agreement between raw data and SRRF processed images. MotivationCurrent super-resolution imaging techniques allow for a greater understanding of cellular structures however they are often complex or only have the ability to image a few cells at once. This small field of view may not represent the behaviour across the entire sample and the manual selection of which restricted ROI to use may introduce bias. Currently, this is often circumvented by stitching and tiling methods which stitch many small ROI together, however this can result in artefacts across an image which poses an issue when analysing data. To combat this, we have used the Mesolens alongside Super-Resolution Radial Fluctuations analysis, to obtain super-resolved images over a field of view of 4.4 mm x 3.0 mm with minimal error.

biophysics↗