Biology subjects

Day, A.

Publications and source records attributed to Day, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Riverine input and eddy edge effects on microeukaryotic biodiversity in the Northern Gulf

Marine microorganisms drive the biogeochemical processes that sustain ocean ecosystems, such as primary production, nutrient cycling, and the transfer of carbon and energy to higher trophic levels. Single-celled eukaryotic organisms (microbial eukaryotes or protists) represent a multifaceted group that contribute to food web dynamics as primary producers, consumers, parasites, and nutrient remineralizers. The Northern Gulf of Mexico is a productive, river-influenced, semi-enclosed ecosystem with strong economic ties. To gain detailed insight into Gulf-based microbial communities, we present an 18S rRNA gene metabarcoding survey across 12 stations from the Louisiana coast to offshore Northern Gulf encompassing the surface to over 2,000 m. Together, water mass, the ratio of dissolved inorganic carbon to total alkalinity, distance to the coast, and depth structured protistan species composition; a secondary signal was associated with the edge of a Loop Current eddy. At the Mississippi River-Gulf interface, diatoms dominated the upper water column depths, with dinoflagellates, parasitic Syndiniales, and rhizaria making up the majority of the offshore communities throughout the entire water column. Shifts in species composition with Northern Gulf environmental gradients reflect varied trophic strategies and have implications for carbon transfer efficiency and food web structure. These results establish a baseline characterization of microeukaryotic biodiversity across coastal-to-offshore and surface to deep-sea gradients that provide critical context for future assessments of Northern Gulf ecosystem resilience.

ecology↗

bioRxiv2026

A Direct DNA Extraction Workflow for Metabarcoding Fungal Bioaerosols from Adhesive Samplers

Monitoring fungal bioaerosols is essential for understanding their impacts on human health and crop productivity. However, current monitoring approaches rely on culture-dependent techniques, which underestimate the diversity of fungal exposures. Adhesive-coated films can effectively capture airborne particles but have been regarded as unsuitable for DNA downstream analysis, owing to the lack of standardised DNA extraction methods for the adhesive capture surface. Here, we present an optimised DNA extraction protocol for adhesive film, which we developed and validated to enable direct and culture-independent ITS2-based metabarcoding of fungal bioaerosols collected on a 96 well plate sealing film ( Sticky sampler). This validated approach was applied to field samples collected from four agricultural farms and from a domestic garden to evaluate seasonal and spatial changes in fungal bioaerosols. Our analysis uncovered pronounced site-specific contrasts linked to geography and land management, as well as clear seasonal shifts in garden fungal communities. This study demonstrates that adhesive samplers, combined with optimised DNA extraction, provide a practical and inexpensive tool for fungal bioaerosol research by resolving ecologically meaningful spatiotemporal dynamics of airborne fungi. This protocol opens new opportunities for highly scalable surveillance of environmental bioaerosols.

microbiology↗

bioRxiv2025

HDAC inhibition unlocks tumor plasticity and enhances immunotherapy response in Myc- Driven Small Cell Lung Cancer

Small Cell Lung Cancer (SCLC) is a highly aggressive malignancy, accounting for approximately 15% of all lung cancer cases. Characterized by low immunogenicity, SCLC may utilize epigenetic mechanisms to evade immune detection. Here, we demonstrate that entinostat, a class I histone deacetylase inhibitor (HDACi) upregulates immune-related genes in human SCLC cells. In vivo, we confirmed entinostat treatment increased expression of immunecheckpoint ligands and antigen presentation machinery in Myc-driven tumors in a Rb1/Trp53/MycT58A (RPM) SCLC mouse model, while shifting tumors from a neuroendocrine(NE)-high to a NE-low phenotype. Notably, combining entinostat with anti-PD-1 immunotherapy significantly enhances T-cell infiltration, suppresses tumor growth, and prolongs survival in RPM allograft models. These findings underscore the potential of entinostat to reprogram the immunological landscape and NE status of SCLC, enhance immune checkpoint blockade efficacy, and improve therapeutic outcomes.

cancer biology↗

bioRxiv2025

Proteomic profiling of extracellular vesicles distinguishes prostate cancer molecular subtypes

Prostate cancer is the most common non-cutaneous cancer among men in the United States. Most prostate cancers are driven by androgen receptor (AR) signaling, but there are an increasing number of cases that lose AR and gain neuroendocrine (NE) features (AR-/NE+) or lack both (AR-/NE-). These latter subtypes are particularly aggressive and lethal. Extracellular vesicles (EVs) have shown great potential as biomarkers for noninvasive liquid biopsy assays, as EVs contain biomolecules from their cells of origin. Here, we used a shotgun proteomics approach with mass spectrometry to interrogate the global proteome of EVs isolated from prostate cancer cell lines reflecting diverse clinical subtypes, including AR-/NE+ and AR-/NE-models. We identified 3,952 EV proteins, which clustered largely by tumor subtype and provided enough proteomic coverage to derive classic gene signatures of AR or NE identity that are of high relevance for prostate cancer prognostication. EVs isolated from AR+ cells displayed high levels of proteins regulated by AR and mTOR signaling. EVs isolated from AR-/NE+ cells contained known NE markers such as SYP and CHGA, whereas EVs from AR-/NE-models were enriched in basal cell markers and proteins that regulate epithelial-to-mesenchymal transition (EMT). We integrated our cell line data with recently published EV proteomics data from 27 advanced prostate cancer patients and found 2,733 overlapping proteins including cell surface markers relevant to prostate cancer, AR activity indicators, and proteins enriched in specific subtypes (AR+, AR-/NE-, AR-/NE+). This approach is especially promising for rare cancer subtypes, such as prostate cancers that lose AR- related features and gain NE features, so as to optimize the use of these liquid biopsy samples for clinical decision making.

cancer biology↗

bioRxiv2025

Candida albicans Colonization Modulates Murine Ethanol Consumption and Behavioral Responses Through Elevation of Serum Prostaglandin E2 and Impact on the Striatal Dopamine System

Candida albicans is a commensal yeast that is a common component of the gastrointestinal (GI) microbiome of humans. C. albicans has been shown to bloom in the GI tract of individuals with alcohol use disorder (AUD) and can promote and increase the severity of alcoholic liver disease (ALD). However, the effects of C. albicans blooms on the host in the context of AUD or AUD-related phenotypes, such as ethanol preference, have been unstudied. In this work, we report a reduction in ethanol consumption and preference in mice colonized with C. albicans. C. albicans-colonized mice exhibited elevated levels of serum PGE2 and reduced ethanol preference was reversed by injection with antagonists of PGE2 receptors. Further, injection of mice with a PGE2 derivative decreased their ethanol preference. These results show that PGE2 acting on its receptors EP1 and EP2 drives reduced ethanol preference in C. albicans-colonized mice. We also showed altered transcription of dopamine receptors in the dorsal striatum of C. albicans-colonized mice and more rapid acquisition of ethanol conditioned taste aversion, suggesting alterations to reinforcement or aversion learning. Finally, C. albicans-colonized mice were more susceptible to ethanol-induced motor coordination impairment showing significant alterations to the behavioral effects of ethanol. This study identifies a member of the fungal microbiome that alters ethanol preference and demonstrates a role for PGE2 signaling in these phenotypes. ImportanceCandida albicans is a commensal yeast that is found in the gut of most individuals. C. albicans has been shown to contribute to alcoholic liver disease. Outside of this, the impact of intestinal fungi on alcohol-use disorder (AUD) had been unstudied. As AUD is a complex disorder characterized by high relapse rates, and there are only 3 FDA-approved therapies for the maintenance of abstinence, it is important to study novel AUD contributors to find new therapeutic targets. Here we show that an intestinal fungus, C. albicans, can alter mammalian ethanol consumption through an immune modulator, prostaglandin E2. The results highlight novel contributors to AUD-related phenotypes and further implicate the gut-brain axis in AUD. Future studies could lead to new therapeutic avenues for the treatment of AUD.

microbiology↗

bioRxiv2024

Mapping herpesvirus-driven impacts on the cellular milieu and transcriptional profile of Kaposi sarcoma in patient-derived mouse models

Kaposi sarcoma (KS) is defined by aberrant angiogenesis driven by Kaposi sarcoma herpesvirus (KSHV)-infected spindle cells with endothelial characteristics. KS research is hindered by rapid loss of KSHV infection upon explant culture of tumor cells. Here, we establish patient-derived KS xenografts (PDXs) upon orthotopic implantation of cutaneous KS biopsies in immunodeficient mice. KS tumors were maintained in 27/28 PDX until experimental endpoint, up to 272 days in the first passage of recipient mice. KSHV latency associated nuclear antigen (LANA)+ endothelial cell density increased by a mean 4.3-fold in 14/15 PDX analyzed by IHC at passage 1 compared to respective input biopsies, regardless of implantation variables and clinical features of patients. The Ki-67 proliferation marker colocalized with LANA more frequently in PDXs. Spatial transcriptome analysis revealed increased expression of viral transcripts from latent and lytic gene classes in the PDX. The expanded KSHV+ regions of the PDX maintained signature gene expression of KS tumors, with enrichment in pathways associated with angiogenesis and endothelium development. Cells with characteristics of tumor-associated fibroblasts derived from PDX were propagated for 15 passages. These fibroblast-like cells were permissive for de novo KSHV infection, and one lineage produced CXCL12, a cancer-promoting chemokine. Spatial analysis revealed that fibroblasts are a likely source of CXCL12 signaling to CXCR4 that was upregulated in KS regions. The reproducible expansion of KSHV-infected endothelial cells in PDX from multiple donors and recapitulation of a KS tumor gene signature supports the application of patient-derived KS mouse models for studies of pathogenesis and novel therapies. One Sentence SummaryTumor virus-driven expansion of endothelial cells with a transcriptional signature of Kaposi sarcoma in a large cohort of patient-derived xenografts provides a platform to discover cell communications within the tumor microenvironment.

microbiology↗

bioRxiv2024

Stress contingent changes in Hog1 pathway architecture and regulation in Candida albicans

The Hog1 stress-activated protein kinase (SAPK) is a key mediator of stress resistance and virulence in Candida albicans. Hog1 activation via phosphorylation of the canonical TGY motif is mediated by the Pbs2 MAPKK, which itself is activated by the Ssk2 MAPKKK. Although this three-tiered SAPK signalling module is well characterised, it is unclear how Hog1 activation is regulated in response to different stresses. Functioning upstream of the Ssk2 MAPKKK is a two-component related signal transduction system comprising three sensor histidine kinases, a phosphotransfer protein Ypd1, and a response regulator Ssk1. Here, we report that Ssk1 is a master regulator of the Hog1 SAPK that promotes stress resistance and Hog1 phosphorylation in response to diverse stresses, except high osmotic stress. Notably, we find Ssk1 regulates Hog1 in a two-component independent manner by functioning as a scaffolding protein to promote interactions between the Ssk2 and Pbs2 kinases. We propose this scaffolding function is important to maintain a basal level of Hog1 phosphorylation which is necessary for oxidative stress, but not osmotic stress, mediated Hog1 activation. We find that osmotic stress triggers robust Pbs2 phosphorylation which drives its dissociation from Ssk2. In contrast, Pbs2 is not robustly phosphorylated following oxidative stress and the Ssk1-mediated Ssk2-Pbs2 interaction remains intact. Instead, oxidative stress-stimulated increases in phosphorylated Hog1 is dependent on the inhibition of protein tyrosine phosphatases that negatively regulate Hog1 coupled with the Ssk1-mediated promotion of basal Hog1 activity. Furthermore, we find that inhibition of protein tyrosine phosphatases is linked to the hydrogen peroxide induced oxidation of these negative regulators in a mechanism that is dependent thioredoxin. Taken together these data reveal stress contingent changes in Hog1 pathway architecture and regulation and uncover a novel mode of action of the Ssk1 response regulator in SAPK regulation. Author summaryAs a core stress regulator, the Hog1 stress-activated protein kinase (SAPK), is a key virulence determinant in many fungal pathogens. Despite this, little is known regarding the mechanisms by which different stresses trigger the phosphorylation and activation of Hog1. Here we present three novel findings regarding Hog1 regulation in the human fungal pathogen C. albicans. Firstly, we find that the response regulator protein, Ssk1, is a master regulator of Hog1 that forms a scaffold for the upstream Hog1-activating kinases, Ssk2 and Pbs2. Secondly, this scaffolding role maintains a basal level of Hog1 phosphorylation, which is important for responses to stresses, such as oxidative stress, that do not stimulate activation of the upstream Ssk2 and Pbs2 kinases. Instead, oxidative stress induced Hog1 phosphorylation is mediated through the oxidation and inactivation of protein tyrosine phosphatases that negatively regulate Hog1. Finally, we show that high osmotic stress induces the robust phosphorylation and activation of the upstream kinase Pbs2, which drives its dissociation from the Ssk1-mediated scaffold. These new insights into the regulation of the C. albicans Hog1 SAPK pathway offer new strategies to therapeutically target this core virulence determinant.

microbiology↗

bioRxiv2024

Critical analysis of polycyclic tetramate macrolactam biosynthetic cluster phylogeny and functional diversity

Polycyclic tetramate macrolactams (PTMs) are bioactive natural products commonly associated with certain actinobacterial and proteobacterial lineages. These molecules have been the subject of numerous structure-activity investigations since the 1970s. New members continue to be pursued in wild and engineered bacterial strains, and advances in PTM biosynthesis suggests their outwardly simplistic biosynthetic gene clusters (BGCs) belie unexpected product complexity. Towards addressing the origins of this complexity and understanding its influence on PTM discovery, we engaged in a combination of bioinformatics to systematically classify PTM BGCs, and PTM-targeted metabolomics to compare the products of select BGC types. By comparing groups of producers and BGC mutants, we exposed knowledge gaps that complicate bioinformatics-driven product predictions. In sum, we provide new insights into the evolution of PTM BGCs while systematically accounting for the PTMs discovered thus far. The combined computational and metabologenomic findings presented here should prove useful for guiding future discovery. IMPORTANCEPolycyclic tetramate macrolactam (PTM) pathways are frequently found within the genomes of biotechnologically-important bacteria, including Streptomyces and Lysobacter spp. Their molecular products are typically bioactive, having substantial agricultural and therapeutic interest. Leveraging bacterial genomics for the discovery of new related molecules is thus desirable, but drawing accurate structural predictions from bioinformatics alone remains challenging. This difficulty stems from a combination of previously underappreciated biosynthetic complexity and remaining knowledge gaps, compounded by a stream of yet-uncharacterized PTM biosynthetic loci gleaned from recently sequenced bacterial genomes. We engaged in the following study to create a useful framework for cataloging historic PTM clusters, identifying new cluster variations, and tracing evolutionary paths for these molecules. Our data suggests new PTM chemistry remains discoverable in nature. However, our metabolomic and mutational analyses emphasize practical limitations to genomics-based discovery by exposing hidden complexity.

microbiology↗

bioRxiv2023

Selection of Ethanol Tolerant Strains of Candida albicans by Repeated Ethanol Exposure Results in Strains with Reduced Susceptibility to Fluconazole

Candida albicans is a commensal yeast that has important impacts on host metabolism and immune function, and can establish life-threatening infections in immunocompromised individuals. Previously, C. albicans colonization has been shown to contribute to the progression and severity of alcoholic liver disease. However, relatively little is known about how C. albicans responds to changing environmental conditions in the GI tract of individuals with alcohol use disorder, namely repeated exposure to ethanol. In this study, we repeatedly exposed C. albicans to high concentrations (10% vol/vol) of ethanol--a concentration that can be observed in the upper GI tract of humans following consumption of alcohol. Following this repeated exposure protocol, ethanol small colony (Esc) variants of C. albicans isolated from these populations exhibited increased ethanol tolerance, altered transcriptional responses to ethanol, and cross-resistance/tolerance to the frontline antifungal fluconazole. These Esc strains exhibited chromosomal copy number variations and carried polymorphisms in genes previously associated with the acquisition of fluconazole resistance during human infection. This study identifies a selective pressure that can result in evolution of fluconazole tolerance and resistance without previous exposure to the drug.

microbiology↗

bioRxiv2023

Unraveling the Global Proteome and Phosphoproteome of Prostate Cancer Patient-Derived Xenografts

Resistance to androgen deprivation therapies leads to metastatic castration-resistant prostate cancer (mCRPC) of adenocarcinoma (AdCa) origin that can transform to emergent aggressive variant prostate cancer (AVPC) which has neuroendocrine (NE)-like features. To this end, we used LuCaP patient-derived xenograft (PDX) tumors, clinically relevant models that reflects and retains key features of the tumor from advanced prostate cancer patients. Here we performed proteome and phosphoproteome characterization of 48 LuCaP PDX tumors and identified over 94,000 peptides and 9,700 phosphopeptides corresponding to 7,738 proteins. When we compared 15 NE versus 33 AdCa PDX samples, we identified 309 unique proteins and 476 unique phosphopeptides that were significantly altered and corresponded to proteins that are known to distinguish these two phenotypes. Assessment of protein and RNA concordance from these tumors revealed increased dissonance in transcriptionally regulated proteins in NE and metabolite interconversion enzymes in AdCa.

cancer biology↗

bioRxiv2023

Saracatinib synergizes with enzalutamide to downregulate androgen receptor activity in castration resistant prostate cancer

Prostate cancer (PCa) remains the most diagnosed non-skin cancer amongst the American male population. Treatment for localized prostate cancer consists of androgen deprivation therapies (ADTs), which typically inhibit androgen production and the androgen receptor (AR). Though initially effective, a subset of patients will develop resistance to ADTs and the tumors will transition to castration-resistant prostate cancer (CRPC). Second generation hormonal therapies such as abiraterone acetate and enzalutamide are typically given to men with CRPC. However, these treatments are not curative and typically prolong survival only by a few months. Several resistance mechanisms contribute to this lack of efficacy such as the emergence of AR mutations, AR amplification, lineage plasticity, AR splice variants (AR-Vs) and increased kinase signaling. Having identified SRC kinase as a key tyrosine kinase enriched in CRPC patient tumors from our previous work, we evaluated whether inhibition of SRC kinase synergizes with enzalutamide or chemotherapy in several prostate cancer cell lines expressing variable AR isoforms. We observed robust synergy between the SRC kinase inhibitor, saracatinib, and enzalutamide, in the AR-FL+/AR-V+ CRPC cell lines, LNCaP95 and 22Rv1. We also observed that saracatinib significantly decreases AR Y534 phosphorylation, a key SRC kinase substrate residue, on AR-FL and AR-Vs, along with the AR regulome, supporting key mechanisms of synergy with enzalutamide. Lastly, we also found that the saracatinib-enzalutamide combination reduced DNA replication compared to the saracatinib-docetaxel combination, resulting in marked increased apoptosis. By elucidating this combination strategy, we provide pre-clinical data that suggests combining SRC kinase inhibitors with enzalutamide in select patients that express both AR-FL and AR-Vs.

cancer biology↗

bioRxiv2022

Differentiation of human induced pluripotent stem cells into cortical neural stem cells

Efficient and effective methods for converting human induced pluripotent stem cells (iPSC) into differentiated derivatives are critical for performing robust, large-scale studies of development and disease modelling, and for providing a source of cells for regenerative medicine. Here, we describe a 14-day neural differentiation protocol which allows for the scalable, simultaneous differentiation of multiple iPSC lines into cortical neural stem cells (NSCs). We currently employ this protocol to differentiate and compare sets of engineered iPSC lines carrying loss of function alleles in developmental disorder associated genes, alongside isogenic wildtype controls. Using RNA sequencing (RNA-Seq), we can examine the changes in gene expression brought about by each disease gene knockout, to determine its impact on neural development and explore mechanisms of disease. The 10-day Neural Induction period uses the well established dual-SMAD inhibition approach combined with Wnt/{beta}-Catenin inhibition to selectively induce formation of cortical NSCs. This is followed by a 4-day Neural Maintenance period facilitating NSC expansion and rosette formation, and NSC cryopreservation. We also describe methods for thawing and passaging the cryopreserved NSCs, which are useful in confirming their viability for further culture. Routine implementation of ICC Quality Control confirms the presence of PAX6-positive and/or FOXG1-positive NSCs and the absence of OCT4-positive iPSCs after differentiation. RNA-Seq, flow cytometry, immunocytochemistry (ICC) and RT-qPCR provide additional confirmation of robust presence of NSC markers in the differentiated cells. The broader utility and application of our protocol is demonstrated by the successful differentiation of wildtype iPSC lines from five additional independent donors. This paper thereby describes an efficient method for the production of large numbers of high purity cortical NSCs, which are widely applicable for downstream research into developmental mechanisms, further differentiation into postmitotic cortical neurons, or other applications such as large-scale drug screening experiments.

developmental biology↗

bioRxiv2022

Mouse and cellular models of KPTN-related disorder implicate mTOR signalling in cognitive and progressive overgrowth phenotypes

KPTN-related disorder (KRD) is an autosomal recessive disorder associated with germline variants in KPTN (kaptin), a component of the mTOR regulatory complex KICSTOR. To gain further insights into the pathogenesis of KRD, we analysed mouse knockout and human stem cell KPTN loss-of-function models. Kptn-/- mice display many of the key KRD phenotypes, including brain overgrowth, behavioural abnormalities, and cognitive deficits. Assessment of affected individuals has identified concordant selectivity of cognitive deficits, postnatal onset of brain overgrowth, and a previously unrecognised KPTN dosage-sensitivity, resulting in increased head circumference in their heterozygous parents. Molecular and structural analysis of Kptn-/- mice revealed pathological changes, including differences in brain size, shape, and cell numbers primarily due to abnormal postnatal brain development. Both the mouse and differentiated iPSC models of the disorder display transcriptional and biochemical evidence for altered mTOR pathway signalling, supporting the role of KPTN in regulating mTORC1. Increased mTOR signalling downstream of KPTN is rapamycin sensitive, highlighting possible therapeutic avenues with currently available mTOR inhibitors. These findings place KRD in the broader group of mTORC1 related disorders affecting brain structure, cognitive function, and network integrity.

genetics↗

bioRxiv2021

High-throughput RNA isoform sequencing using programmable cDNA concatenation

Alternative splicing is a core biological process that enables profound and essential diversification of gene function. Short-read RNA sequencing approaches fail to resolve RNA isoforms and therefore primarily enable gene expression measurements - an isoform unaware representation of the transcriptome. Conversely, full-length RNA sequencing using long-read technologies are able to capture complete transcript isoforms, but their utility is deeply constrained due to throughput limitations. Here, we introduce MAS-ISO-seq, a technique for programmably concatenating cDNAs into single molecules optimal for long-read sequencing, boosting the throughput >15 fold to nearly 40 million cDNA reads per run on the Sequel IIe sequencer. We validated unambiguous isoform assignment with MAS-ISO-seq using a synthetic RNA isoform library and applied this approach to single-cell RNA sequencing of tumor-infiltrating T cells. Results demonstrated a >30 fold boosted discovery of differentially spliced genes and robust cell clustering, as well as canonical PTPRC splicing patterns across T cell subpopulations and the concerted expression of the associated hnRNPLL splicing factor. Methods such as MAS-ISO-seq will drive discovery of novel isoforms and the transition from gene expression to transcript isoform expression analyses.

genetics↗

bioRxiv2021

Scalable Clustering with Supervised Linkage Methods

Data clustering plays a significant role in biomedical sciences, particularly in single-cell data analysis. Researchers use clustering algorithms to group individual cells into populations that can be evaluated across different levels of disease progression, drug response, and other clinical statuses. In many cases, multiple sets of clusters must be generated to assess varying levels of cluster specificity. For example, there are many subtypes of leukocytes (e.g. T cells), whose individual preponderance and phenotype must be assessed for statistical/functional significance. In this report, we introduce a novel hierarchical density clustering algorithm (HAL-x) that uses supervised linkage methods to build a cluster hierarchy on raw single-cell data. With this new approach, HAL-x can quickly predict multiple sets of labels for immense datasets, achieving a considerable improvement in computational efficiency on large datasets compared to existing methods. We also show that cell clusters generated by HAL-x yield near-perfect F1-scores when classifying different clinical statuses based on single-cell profiles. Our hierarchical density clustering algorithm achieves high accuracy in single cell classification in a scalable, tunable and rapid manner. We make HAL-x publicly available at: https://pypi.org/project/hal-x/

bioinformatics↗

bioRxiv2021

Characterizing Human Mesenchymal Stromal Cells Immune Modulatory Potency Using Targeted Lipidomic Profiling of Sphingolipids

Cell therapies are expected to increase over the next decade due to increasing demand for clinical applications. Mesenchymal stromal cells (MSCs) have been explored to treat a number of diseases, with some successes in early clinical trials. Despite early successes, poor MSC characterization results in lessened therapeutic capacity once in vivo. Here, we characterized bone-marrow (BM), adipose derived and umbilical cord tissue MSCs sphingolipids (SLs), a class of bioactive lipids, using liquid chromatography - tandem mass spectrometry. We found ceramide levels differed based upon donors sex in BM-MSCs. We detected fatty acyl chain variants in MSCs from all 3 sources. Linear discriminant analysis revealed that MSCs separated based on tissue source. Principal component analysis showed IFN-{gamma} primed and unstimulated MSCs separated according to their SL signature. Lastly, we detected higher ceramide levels in low IDO MSCs, indicating sphingomeylinase or ceramidase enzymatic activity may be involved in their immune potency.

bioengineering↗

bioRxiv2021

Increased complement activation is a distinctive feature of severe SARS-CoV-2 infection.

Complement activation has been implicated in the pathogenesis of severe SARS-CoV-2 infection. However, it remains to be determined whether increased complement activation is a broad indicator of critical illness (and thus, no different in COVID-19). It is also unclear which pathways are contributing to complement activation in COVID-19, and, if complement activation is associated with certain features of severe SARS-CoV-2 infection, such as endothelial injury and hypercoagulability. To address these questions, we investigated complement activation in the plasma from patients with COVID-19 prospectively enrolled at two tertiary care centers. We compared our patients to two non-COVID cohorts: (a) patients hospitalized with influenza, and (b) patients admitted to the intensive care unit (ICU) with acute respiratory failure requiring invasive mechanical ventilation (IMV). We demonstrate that circulating markers of complement activation (i.e., sC5b-9) are elevated in patients with COVID-19 compared to those with influenza and to patients with non-COVID-19 respiratory failure. Further, the results facilitate distinguishing those who are at higher risk of worse outcomes such as requiring ICU admission, or IMV. Moreover, the results indicate enhanced activation of the alternative complement pathway is most prevalent in patients with severe COVID-19 and is associated with markers of endothelial injury (i.e., Ang2) as well as hypercoagulability (i.e., thrombomodulin and von Willebrand factor). Our findings identify complement activation to be a distinctive feature of COVID-19, and provide specific targets that may be utilized for risk prognostication, drug discovery and personalized clinical trials. SUMMMARYComplement has been implicated in COVID-19. However, whether this is distinctive of COVID-19 remains unanswered. Ma et al report increased complement activation in COVID-19 compared to influenza and non-COVID respiratory failure, and demonstrate alternative pathway activation as a key marker of multiorgan failure and death.

immunology↗

bioRxiv2020

Structure of a dominant negative mutant reveals a stalled intermediate in anthrax protective antigen pore maturation

Anthrax is a severe bacterial infection caused by Bacillus anthracis, which produces a tripartite toxin that includes protective antigen (PA), lethal factor (LF) and edema factor (EF). A series of dominant-negative mutations have been previously identified that prevent the heptameric PA prepore from forming the pH-induced, membrane spanning beta-barrel pore that is required for translocation of EF and LF to the cytoplasm of the infected cell. Here we show that the dominant negative D425A mutation stalls the formation of the pore at a reversible intermediate maturation state, which exhibits many of the structural aspects of the pore but fails to form the phi({phi})-clamp and beta-barrel structure needed for full pore maturation. Overall, this structure reveals that {phi}-clamp and beta-barrel pore formation are later steps in the pathway to pore formation, thereby providing a regulatory mechanism to prevent premature translocation of EF and LF.

biophysics↗