Biology subjects

Thomas, D.

Publications and source records attributed to Thomas, D..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

A next-generation, histological atlas of the human brain and its application to automated brain MRI segmentation

Magnetic resonance imaging (MRI) is the standard tool to image the human brain in vivo. In this domain, digital brain atlases are essential for subject-specific segmentation of anatomical regions of interest (ROIs) and spatial comparison of neuroanatomy from different subjects in a common coordinate frame. High-resolution, digital atlases derived from histology (e.g., Allen atlas [7], BigBrain [13], Julich [15]), are currently the state of the art and provide exquisite 3D cytoarchitectural maps, but lack probabilistic labels throughout the whole brain. Here we present NextBrain, a next - generation probabilistic atlas of human brain anatomy built from serial 3D histology and corresponding highly granular delineations of five whole brain hemispheres. We developed AI techniques to align and reconstruct [~]10,000 histological sections into coherent 3D volumes with joint geometric constraints (no overlap or gaps between sections), as well as to semi-automatically trace the boundaries of 333 distinct anatomical ROIs on all these sections. Comprehensive delineation on multiple cases enabled us to build the first probabilistic histological atlas of the whole human brain. Further, we created a companion Bayesian tool for automated segmentation of the 333 ROIs in any in vivo or ex vivo brain MRI scan using the NextBrain atlas. We showcase two applications of the atlas: automated segmentation of ultra-high-resolution ex vivo MRI and volumetric analysis of Alzheimers disease and healthy brain ageing based on [~]4,000 publicly available in vivo MRI scans. We publicly release: the raw and aligned data (including an online visualisation tool); the probabilistic atlas; the segmentation tool; and ground truth delineations for a 100 m isotropic ex vivo hemisphere (that we use for quantitative evaluation of our segmentation method in this paper). By enabling researchers worldwide to analyse brain MRI scans at a superior level of granularity without manual effort or highly specific neuroanatomical knowledge, NextBrain holds promise to increase the specificity of MRI findings and ultimately accelerate our quest to understand the human brain in health and disease.

neuroscience↗

bioRxiv2023

Tumor- and Nerve-Derived Axon Guidance Molecule Promotes Pancreatic Ductal Adenocarcinoma Progression and Metastasis through Macrophage Reprogramming

Axon guidance molecules were found to be the gene family most frequently altered in pancreatic ductal adenocarcinoma (PDA) through mutations and copy number changes. However, the exact molecular mechanism regarding PDA development remained unclear. Using genetically engineered mouse models to examine one of the axon guidance molecules, semaphorin 3D (SEMA3D), we found a dual role for tumor-derived SEMA3D in malignant transformation of pancreatic epithelial cells and a role for nerve-derived SEMA3D in PDA development. This was demonstrated by the pancreatic-specific knockout of the SEMA3D gene from the KRASG12D and TP53R172H mutation knock-in, PDX1-Cre (KPC) mouse model which demonstrated a delayed tumor initiation and growth comparing to the original KPC mouse model. Our results showed that SEMA3D knockout skews the macrophages in the pancreas away from M2 polarization, providing a potential mechanistic role of tumor-derived SEMA3D in PDA development. The KPC mice with the SEMA3D knockout remained metastasis-free, however, died from primary tumor growth. We then tested the hypothesis that a potential compensation mechanism could result from SEMA3D which is naturally expressed by the intratumoral nerves. Our study further revealed that nerve-derived SEMA3D does not reprogram macrophages directly, but reprograms macrophages indirectly through ARF6 signaling and lactate production in PDA tumor cells. SEMA3D increases tumor-secreted lactate which is sensed by GPCR132 on macrophages and subsequently stimulates pro-tumorigenic M2 polarization in vivo. Tumor intrinsic- and extrinsic-SEMA3D induced ARF6 signaling through its receptor Plexin D1 in a mutant KRAS-dependent manner. Consistently, RNA sequencing database analysis revealed an association of higher KRASMUT expression with an increase in SEMA3D and ARF6 expression in human PDAs. Moreover, multiplex immunohistochemistry analysis showed an increased number of M2-polarized macrophages proximal to nerves in human PDA tissue expressing SEMA3D. Thus, this study suggests altered expression of SEMA3D in tumor cells lead to acquisition of cancer-promoting functions and the axon guidance signaling originating from nerves is "hijacked" by tumor cells to support their growth. Other axon guidance and neuronal development molecules may play a similar dual role which is worth further investigation. One sentence summaryTumor- and nerve-derived SEMA3D promotes tumor progression and metastasis through macrophage reprogramming in the tumor microenvironment. STATEMENT OF SIGNIFICANCEThis study established the dual role of axon guidance molecule, SEMA3D, in the malignant transformation of pancreatic epithelial cells and of nerve-derived SEMA3D in PDA progression and metastasis. It revealed macrophage reprogramming as the mechanism underlying bothroles. Together, this research elucidated how inflammatory responses promote invasive PDA progression and metastasis through an oncogenic process.

cancer biology↗

bioRxiv2023

Mitochondrial translocation of TFEB regulates complex I and inflammation

TFEB is a master regulator of autophagy, lysosome biogenesis and mitochondrial metabolism that works, and immunity, primarily through transcription controlled by cytosol-to-nuclear translocation. Emerging data indicate additional regulatory interactions at the surface of organelles such as lysosomes. Here we show that TFEB has a non-transcriptional role in mitochondria, regulating the electron transport chain complex I to down-modulate inflammation. Proteomic analysis revealed extensive TFEB co-precipitation with several mitochondrial proteins, whose interactions are disrupted upon infection with S. Typhimurium. Localization of TFEB in the mitochondrial matrix was confirmed by high resolution confocal microscopy and biochemistry with translocation dependent on a conserved N-terminal TOMM20-binding motif enhanced by mTOR inhibition. Within the mitochondria, TFEB and protease LONP1 antagonistically co-regulate complex I, reactive oxygen species and the inflammatory response. Consequently, during infection, lack of TFEB specifically in the mitochondria exacerbates the expression of pro-inflammatory cytokines, contributing to innate immune pathogenesis.

cell biology↗

bioRxiv2022

Macromolecular crowding is surprisingly unable to deform the structure of a model biomolecular condensate

The crowded interior of a living cell makes experiments on simpler in vitro systems attractive. Although these reveal interesting phenomena, their biological relevance can be questionable. A topical example is the phase separation of intrinsically-disordered proteins into biomolecular condensates, which is proposed to underlie the membraneless compartmentalisation of many cellular functions. How a cell reliably controls biochemical reactions in compartments open to the compositionally-varying cytoplasm is an important question for understanding cellular homeostasis. Computer simulations are often used to study the phase behaviour of model biomolecular condensates, but the number of relevant parameters explodes as the number of protein components increases. It is unfeasible to exhaustively simulate such models for all parameter combinations, although interesting phenomena are almost certainly hidden in the jungle of their high-dimensional parameter space. Here we have studied the phase behaviour of a model biomolecular condensate in the presence of a polymeric crowding agent. We used a novel compute framework to execute dozens of simultaneous simulations spanning the protein/crowder concentration space. We then combined the results into a graphical representation for human interpretation, which provided an efficient way to search the models high-dimensional parameter space. We found that steric repulsion from the crowder drives a near-critical system across the phase boundary, but the molecular arrangement within the resulting biomolecular condensate is rather insensitive to the crowder concentration and molecular weight. We propose that a cell may use the local cytoplasmic concentration to assist formation of biomolecular condensates, while relying on the dense phase reliably providing a stable, structured, fluid milieu for cellular biochemistry despite being open to its changing environment.

biophysics↗

bioRxiv2022

Sequence-to-expression approach to identify etiological non-coding DNA variations in P53 and cMYC-driven diseases

Disease risk prediction based on DNA sequence and transcriptional profile can improve disease screening, prevention, and potential therapeutic approaches by revealing contributing genetic factors and altered networks. Despite identifying many disease-associated DNA variants through genome-wide association studies, distinguishing deleterious non-coding DNA variations remains poor for most common diseases. We previously reported that non-coding variations disrupting cis-overlapping motifs (CisOMs) of opposing transcription factors significantly affect enhancer activity. Analyzing publicly available ChIP-seq data for P53 and cMYC in human embryonic stem cells and mouse embryonic cells showed that [~]344-366 genomic regions are co-occupied by P53 and cMYC. We identified, on average, two CisOMs per region, suggesting that co-occupancy is evolutionarily conserved in vertebrates. Therefore, we designed in vitro experiments to uncover the significance of the co-occupancy and competitive binding and inhibition between P53 and cMYC on target gene expression. We found that treating U2OS cells with doxorubicin increased P53 protein level while reducing cMYC level. In contrast, no change in protein levels was observed in Raji cells. ChIP-seq analysis showed that 16-922 genomic regions were co-occupied by P53 and cMYC before and after treatment, and substitutions of cMYC signals by P53 were detected after doxorubicin treatment in U2OS. Around 187 expressed genes near co-occupied regions were altered at mRNA level according to RNA-seq data. We utilized a computational motif-matching approach to determine that changes in predicted P53 binding affinity by DNA variations in CisOMs of co-occupied elements significantly correlate with alterations in reporter gene expression. We performed a similar analysis using SNPs mapped in CisOMs for P53 and cMYC from ChIP-seq data in U2OS and Raji, and expression of target genes from the GTEx portal. We found a significant correlation between change in motif-predicted cMYC binding affinity by SNPs in CisOMs and gene expression. In conclusion, our study suggests a generally applicable approach to filter etiological non-coding variations associated with P53 and cMYC-dependent diseases. Author SummaryMost DNA variants associated with common complex diseases fall outside the protein-coding regions of the genome, making them hard to detect and relate to a function. Although many computational tools are available for prioritizing functional disease risk variants outside the protein-coding regions of the genome, the precision of prediction of these tools is mostly unreliable and hence not close to cancer risk prediction. This study brings to light a novel way to improve prediction accuracy of publicly available tools by integrating the impact of cis-overlapping binding sites of opposing cancer proteins, such as P53 and cMYC, in their analysis to filter out deleterious DNA variants outside the protein-coding regions of the human genome. Using a biology-based statistical approach, DNA variants within cis-overlapping motifs impacting the binding affinity of opposing transcription factors can significantly alter the expression of target genes and regulatory networks. This study brings us closer to developing a generally applicable approach capable of filtering etiological non-coding variations in co-occupied genomic regions of P53 and cMYC family members to improve disease risk assessment.

genetics↗

bioRxiv2022

THE RELATIONSHIP BETWEEN TERROIR AND THE PHENOLOGY OF BAROSSA SHIRAZ

Background and AimsVine phenology results from the interaction between the genotype, environment and management, with implications for fruit, and wine composition. The impact of weather, site and management practices, underlying elements of terroir, impacting the timing of key phenological stages were explored across the Barossa Zone (GI). Methods and ResultsVine phenology was assessed in three zones of 24 vineyards over three vintages using the E-L scale before veraison, and total soluble sugars (TSS) in berries during ripening. We explored the associations between weather, plant traits and viticultural variables, and development in four periods: pre-budburst, budburst-flowering, flowering-veraison and veraison-maturity. The spatial structure of the timing of phenological events suggested three main groups of vineyards. This structure followed gradients in topography and soils across the landscape, and were maintained despite the effect of the season (vintage). On average, differences between early and late groups of vineyards were 13 days at budburst, 20 days at flowering and 24 days at TSS = 24 {degrees}Brix. Phenology responded mainly to temperature until flowering, and to temperature and canopy size from flowering to maturity. The strength of the relationship between the duration of the period and temperature ranked pre-budburst (r2 = 0.94) > budburst-flowering (r2 = 0.40) > veraison-maturity (r2 = 0.17). Duration of pre-budburst and budburst-flowering periods was shortened at 6 d {degrees}C-1, compared to 2 d {degrees}C-1 for veraison-maturity. The duration from veraison to maturity increased with yield (r2 = 0.29, Pa < 0.0001). ConclusionsThe spatial variation in development was maintained despite vintage effects and management practices. Variation in temperature due to topography and elevation were the major drivers of vine phenological development until flowering. During ripening, development was driven by temperature and carbon capture and partitioning. Significance of the StudyThis is the first attempt to show spatial variability on phenology across the Barossa Valley GI. The observed switch on drivers on phenology during development from temperature-driven processed before flowering to resource-dominated processes during ripening have implications for modelling and vineyard management.

plant biology↗

bioRxiv2022

Genetically modified IL-2 bone marrow-derived myeloid cells reprogram the glioma immunosuppressive tumormicroenvironment

Gliomas are the most prevalent type of brain tumors and one of the leading causes of cancer-related death in the adolescent and young adult population (AYA). Two-thirds of glioma AYA patients are affected by low-grade gliomas (LGGs), but there are no specific treatments. Therefore, a percentage of LGG patients experience tumor relapse and malignant progression to high-grade glioma which leads to fatal outcomes. In part, malignant progression is potentiated by the immunosuppressive stromal component of the tumor microenvironment (TME) underscored by M2-macrophages and a paucity of cytotoxic T cells. As a result, first-line immunotherapies have failed to improve outcomes for patients with progressive high-grade gliomas. Here, we report the efficacy of an in vivo approach that demonstrates the potential for a novel cell-mediated innate immunotherapy designed to abrogate immunosuppressive mechanisms within the glioma TME and enhance the recruitment of activated effector T cells. A single dose of engineered bone marrow-derived myeloid cells that release Interleukin-2 (GEMys-IL2) was used systemically to treat mice with LGG tumors systemically. Our results demonstrate that GEMys-IL2 efficiently crossed the blood brain barrier (BBB), infiltrated the glioma microenvironment, and reprogrammed the infiltrating immune cell composition and transcriptome. In addition, GEMys-IL2 impaired tumor progression and extended survival in a LGG immunocompetent mouse model. In conclusion, we demonstrated that GEMys-IL2 have a therapeutic effect in vivo, thus supporting its potential application as a novel immunotherapy that warrants further investigation.

cancer biology↗

bioRxiv2022

Small molecule mediated stabilization of PP2A modulates the Homologous Recombination pathway and potentiates DNA damage-induced cell death

High-Grade Serous Carcinoma (HGSC) is the most common and lethal ovarian cancer subtype. PARP-inhibitors (PARPi) have become the mainstay of HGSC targeted therapy, given that these tumors are driven by a high degree of genomic instability and Homologous Recombination (HR) defects. Nonetheless, only [~]30% of patients initially respond to treatment, ultimately relapsing with resistant disease. Thus, despite recent advances in drug development and increased understanding of genetic alterations driving HGSC progression, mortality has not declined, highlighting the need for novel therapies. Using a Small Molecule Activator of Protein Phosphatase 2A (PP2A) (SMAP-061), we investigated the mechanism by which PP2A stabilization induces apoptosis in Patient-Derived HGSC cells and Xenograft (PDX) models alone or in combination with PARPi. We uncovered that PP2A genes essential for transformation (B56,B56{gamma} and PR72) and basal phosphatase activity (PP2A-A and -C) are heterozygously lost in the majority of HGSC. Moreover, loss of these PP2A genes correlates with worse overall patient survival. We show that SMAP-061 stabilization of PP2A inhibits the HR output by targeting RAD51, leading to chronic accumulation of DNA damage and ultimately apoptosis. Furthermore, combination of SMAP-061 and PARPi leads to enhanced apoptosis in both HR-proficient and -deficient cells and in patient-derived xenograft models. Our studies identify PP2A as novel regulator of HR and introduces PP2A activators as a potential treatment for HGSC tumors. Our studies further emphasize the potential of PP2A modulators to overcome PARPi insensitivity, given that targeting RAD51 has presented benefits in overcoming PARPi-resistance driven by BRCA1/2 mutation reversions.

cancer biology↗

bioRxiv2022

Genetic architecture of spatially complex color patterning in hybrid Mimulus

Coloration in living organisms varies in hue, saturation, and pattern. Here we use genetic mapping to investigate all three elements in monkeyflowers Mimulus luteus var. variegatus and M. cupreus, whose flower petals differ in both yellow carotenoid and magenta anthocyanin pigmentation and whose hybrids exhibit anthocyanin pattern variation not seen in either parent. We report two QTLs associated with carotenoid intensity, and show that lighter yellow petals accumulate relatively high proportions of beta-carotene at the expense of downstream carotenoids. We propose that the derived loss of carotenoid saturation in M. l. variegatus is due to a function-reducing mutation in a Beta-Carotene Hydroxylase candidate gene, coupled with a reduction in the availability of carotenoid-storing chromoplasts influenced by the ORANGE candidate gene. We next identify five QTLs associated with the spatial patterning of anthocyanin pigment. Two QTLs, 9b and 23, each contain genes in the anthocyanin-activating subgroup of the MYB family. We hypothesize that MYB5a/NEGAN, at QTL-9b, activates spot formation in hybrids as part of a Turing-type reaction-diffusion system, while a MYB at QTL-23 activates solid, unpatterned anthocyanin pigment and may also be able to activate the variegatus allele of MYB5a/NEGAN. In addition to the apparently stochastic placement of most spots (consistent with a Turing mechanism), some pattern aspects - notably spots at the petal tips - appear to conform to a positional specification model in which spot location is predetermined. Collectively, this work begins to identify how combinations of yellow and red hues, shifts in carotenoid intensity, and two cryptic patterning systems all contribute to the complexity of color divergence between two close relatives.

genetics↗

bioRxiv2022

Macrocycle-stabilization of its interaction with 14-3-3 increases plasma membrane localization and activity of CFTR

Impaired activity of the chloride channel CFTR is the cause of cystic fibrosis. 14-3-3 proteins have been shown to stabilize CFTR and increase its biogenesis and activity. Here, we report the identification and mechanism of action of a macrocycle stabilizing the 14-3-3/CFTR complex, a first-in-class molecular glue. This molecule rescues plasma membrane localization and chloride transport of F508del-CFTR and works additively with the CFTR pharmacological chaperone corrector lumacaftor (VX-809).

biochemistry↗

bioRxiv2021

Differences between intrinsic and acquired nucleoside analogue resistance in acute myeloid leukaemia cells

BackgroundSAMHD1 mediates resistance to anti-cancer nucleoside analogues, including cytarabine, decitabine, and nelarabine that are commonly used for the treatment of leukaemia, through cleavage of their triphosphorylated forms. Hence, SAMHD1 inhibitors are promising candidates for the sensitisation of leukaemia cells to nucleoside analogue-based therapy. Here, we investigated the effects of the cytosine analogue CNDAC, which has been proposed to be a SAMHD1 substrate, in the context of SAMHD1. MethodsCNDAC was tested in 13 acute myeloid leukaemia (AML cell lines), in 26 acute lymphoblastic leukaemia cell lines, ten AML sublines adapted to various antileukaemic drugs, 24 single cell-derived clonal AML sublines, and primary leukaemic blasts from 24 AML patients. Moreover, 24 CNDAC-resistant sublines of the AML cell lines HL-60 and PL-21 were established. The SAMHD1 gene was disrupted using CRISPR/Cas9 and SAMHD1 depleted using RNAi, and the viral Vpx protein. Forced DCK expression was achieved by lentiviral transduction. SAMHD1 promoter methylation was determined by PCR after treatment of genomic DNA with the methylation-sensitive HpaII endonuclease. Nucleoside (analogue) triphosphate levels were determined by LC-MS/MS. CNDAC interaction with SAMHD1 was analysed by an enzymatic assay and by crystallisation. ResultsAlthough the cytosine analogue CNDAC was anticipated to inhibit SAMHD1, SAMHD1 mediated intrinsic CNDAC resistance in leukaemia cells. Accordingly, SAMHD1 depletion increased CNDAC triphosphate (CNDAC-TP) levels and CNDAC toxicity. Enzymatic assays and crystallisation studies confirmed CNDAC-TP to be a SAMHD1 substrate. In 24 CNDAC-adapted acute myeloid leukaemia (AML) sublines, resistance was driven by DCK (catalyses initial nucleoside phosphorylation) loss. CNDAC-adapted sublines displayed cross-resistance only to other DCK substrates (e.g. cytarabine, decitabine). Cell lines adapted to drugs not affected by DCK or SAMHD1 remained CNDAC sensitive. In cytarabine-adapted AML cells, increased SAMHD1 and reduced DCK levels contributed to cytarabine and CNDAC resistance. ConclusionIntrinsic and acquired resistance to CNDAC and related nucleoside analogues are driven by different mechanisms. The lack of cross-resistance between SAMHD1/ DCK substrates and non-substrates provides scope for next-line therapies after treatment failure.

pharmacology and toxicology↗

bioRxiv2021

A multimodal iPSC platform for cystic fibrosis drug testing

Cystic fibrosis (CF) is a monogenic lung disease caused by dysfunction of the cystic fibrosis transmembrane regulator (CFTR) anion channel, resulting in significant morbidity and mortality. The progress in elucidating the role of the CFTR channel using established animal and cell-based models led to the recent discovery of effective CFTR modulators for most individuals with CF. However, a subset of individuals with CF do not respond to these modulators and there is an urgent need to develop novel therapeutic strategies. In this study, we assembled a panel of iPSCs derived from individuals with common or rare variants representative of three distinct classes of CFTR dysfunction. To measure CFTR function in patient-specific iPSCs we adapted two established in vitro assays of CFTR function to iPSC-derived airway cells. In both a 3-D spheroid assay using forskolin-induced swelling as well as planar cultures composed of polarized mucociliary airway epithelial cells, we quantified CFTR baseline function and response to CFTR modulators and detected genotype-specific differences. Our results demonstrate the potential of the human iPSC platform as a research tool to study cystic fibrosis and in particular accelerate therapeutic development for CF caused by rare mutations.

developmental biology↗

bioRxiv2021

Identification of Leucinostatins from Ophiocordyceps sp. as Antiparasitic Agents Against Trypanosoma cruzi

Safe and effective treatments for Chagas disease, a potentially fatal parasitic infection associated with cardiac and gastrointestinal pathology and caused by the kinetoplastid parasite Trypanosoma cruzi, have yet to be developed. Benznidazole and nifurtimox, which are currently the only available drugs against T. cruzi, are associated with severe adverse effects and questionable efficacy in the late stage of the disease. Natural products have proven to be a rich source of new chemotypes for other infectious agents. We utilized a microscopy-based high-throughput phenotypic screen to identify inhibitors of T. cruzi from a library of natural product samples obtained from fungi procured through a Citizen Science Soil Collection Program (https://whatsinyourbackyard.org/), and the Great Lakes (USA) benthic environment. We identified five leucinostatins (A, B, F, NPDG C and NPDG D) as potent inhibitors of the intracellular amastigote form of T. cruzi. Leucinostatin B also showed in vivo efficacy in a mouse model of Chagas disease. Given prior reports that leucinostatins A and B have antiparasitic activity against the related kinetoplastid T. brucei, our findings suggest a potential cross-trypanocidal compound class and provide a platform for further chemical derivatization of a potent chemical scaffold against T. cruzi.

microbiology↗

bioRxiv2021

Mild CF Lung Disease is Associated with Bacterial Community Stability

Microbial communities in the airways of persons with CF (pwCF) are variable, may include genera that are not typically associated with CF, and their composition can be difficult to correlate with long-term disease outcomes. Leveraging two large datasets characterizing sputum communities of 167 pwCF and associated metadata, we identify five bacterial community types. These communities explain 24% of the variability in lung function in this cohort, far more than single factors like Simpson diversity, which explains only 4%. Subjects with Pseudomonas-dominated communities tended to be older and have reduced percent predicted FEV1 (ppFEV1) than subjects with Streptococcus-dominated communities, consistent with previous findings. To assess the predictive power of these five communities in a longitudinal setting, we used random forests to classify 346 additional samples from 24 subjects observed 8 years on average in a range of clinical states. Subjects with mild disease were more likely to be observed at baseline, that is, not in the context of a pulmonary exacerbation, and community structure in these subjects was more self-similar over time, as measured by Bray-Curtis distance. Interestingly, we found that subjects with mild disease were more likely to remain in a mixed Pseudomonas community, providing some support for the climax-attack model of the CF airway. In contrast, patients with worse outcomes were more likely to show shifts among community types. Our results suggest that bacterial community instability may be a risk factor for lung function decline and indicates the need to better understand factors that drive shifts in community composition.

microbiology↗

bioRxiv2020

The Virucidal Efficacy of Oral Rinse Components Against SARS-CoV-2 In Vitro

The ability of widely-available mouthwashes to inactivate SARS-CoV-2 in vitro was tested using a protocol capable of detecting a 5-log10 reduction in infectivity, under conditions mimicking the naso/oropharynx. During a 30 second exposure, two rinses containing cetylpyridinium-chloride and a third with ethanol/ethyl lauroyl arginate eliminated live virus to EN14476 standards (>4-log10 reduction), while others with ethanol/essential oils and povidone-iodine (PVP-I) eliminated virus by 2-3-log10. Chlorhexidine or ethanol alone displayed little or no ability to inactivate virus. Studies are warranted to determine whether these formulations can inactivate virus in the human oropharynx in vivo, and whether this might impact transmission risk.

microbiology↗

bioRxiv2020

Cardiac glycosides cause selective cytotoxicity in human macrophages and ameliorate white adipose tissue homeostasis

Cardiac glycosides (CGs) inhibit the Na+,K+-ATPase and are widely prescribed medicines for chronic heart failure and cardiac arrhythmias. Recently, CGs have been described to induce inflammasome activation in human macrophages, suggesting a cytotoxicity that remains to be elucidated in tissues. Here we show that human monocyte-derived macrophages (hMDMs) undergo cell death following incubation with nanomolar concentrations of CGs, and in particular with ouabain (IC50=50 nM). The ouabain-induced cell death is more efficient in hMDMs compared to non-adherent PBMC populations and is through on-target inhibition of Na,K-ATPAse activity, as it causes an intracellular depletion of K+, while inducing accumulation of Na+ and Ca2+ levels. Consistently, the cell-death caused by these ion imbalances can be rescued by addition of potassium chloride in hMDMs. Strikingly, white adipose tissue (WAT) explants from morbidly obese patients cultured with nanomolar concentrations of ouabain causes depletion of macrophages, decreases type VI collagen levels, and ameliorates insulin-sensitivity ex vivo. These results suggest that the usage of nanomolar concentration of CGs can be an attractive therapeutic avenue in metabolic syndrome characterised by pathogenic infiltration and activation of macrophages.

immunology↗

bioRxiv2020

Quantifying fluorescent glycan uptake to elucidate strain-level variability in foraging behaviors of rumen bacteria

Gut microbiomes have vast catabolic potential and are essential to host health and nutrition. An in-depth understanding of the metabolic pathways in these ecosystems will enable us to design treatments (i.e. prebiotics) that influence microbiome structure and enhance host physiology. Currently, the investigation of metabolic pathways relies on inferences derived from metagenomics or in vitro cultivations, however, novel approaches targeting specific cell physiologies can illuminate the functional potential encoded within microbial (meta)genomes to accurately assess metabolic abilities. Here, we present a multi-faceted study using complimentary next-generation physiology and omics approaches to characterize the microbial adaptation to a prebiotic in the rumen ecosystem. Using fluorescently labeled polysaccharides, we identified bacteria that actively metabolize a glycan prebiotic in the rumen microbiome ex vivo. Subsequently, we characterized strain-level variability in carbohydrate utilization systems and predict metabolic strategies of isolated bovine-adapted strains of Bacteroides thetaiotaomicron using comparative whole genome sequencing, RNA-Seq, and carbohydrate-active enzyme fingerprinting.

microbiology↗

bioRxiv2020

Alterations of multiple alveolar macrophage states in chronic obstructive pulmonary disease

Despite the epidemics of chronic obstructive pulmonary disease (COPD), the cellular and molecular mechanisms of this disease are far from being understood. Here, we characterize and classify the cellular composition within the alveolar space and peripheral blood of COPD patients and control donors using a clinically applicable single-cell RNA-seq technology corroborated by advanced computational approaches for: machine learning-based cell-type classification, identification of differentially expressed genes, prediction of metabolic changes, and modeling of cellular trajectories within a patient cohort. These high-resolution approaches revealed: massive transcriptional plasticity of macrophages in the alveolar space with increased levels of invading and proliferating cells, loss of MHC expression, reduced cellular motility, altered lipid metabolism, and a metabolic shift reminiscent of mitochondrial dysfunction in COPD patients. Collectively, single-cell omics of multi-tissue samples was used to build the first cellular and molecular framework for COPD pathophysiology as a prerequisite to develop molecular biomarkers and causal therapies against this deadly disease.

systems biology↗