Biology subjects

Webb, A.

Publications and source records attributed to Webb, A..

31 records · Page 2Linked to original sources

bioRxiv2021

Caspase-4/11 exacerbates disease severity in SARS-CoV-2 infection by promoting inflammation and thrombosis

SARS-CoV-2 is a worldwide health concern, and new treatment strategies are needed 1. Targeting inflammatory innate immunity pathways holds therapeutic promise, but effective molecular targets remain elusive. Here, we show that human caspase-4 (CASP4), and its mouse homologue, caspase-11 (CASP11), are upregulated in SARS-CoV-2 infections, and that CASP4 expression correlates with severity of SARS-CoV-2 infection in humans. SARS-CoV-2-infected Casp11-/- mice were protected from severe weight loss and lung pathology, including blood vessel damage, compared to wild-type (WT) and gasdermin-D knock out (Gsdmd-/-) mice. GSDMD is a downstream effector of CASP11 and CASP1. Notably, viral titers were similar in the three genotypes. Global transcriptomics of SARS-CoV-2-infected WT, Casp11-/- and Gsdmd-/- lungs identified restrained expression of inflammatory molecules and altered neutrophil gene signatures in Casp11-/- mice. We confirmed that protein levels of inflammatory mediators IL-1{beta}, IL6, and CXCL1, and neutrophil functions, were reduced in Casp11-/- lungs. Additionally, Casp11-/- lungs accumulated less von Willebrand factor, a marker for endothelial damage, but expressed more Kruppel-Like Factor 2, a transcription factor that maintains vascular integrity. Overall, our results demonstrate that CASP4/11, promotes detrimental SARS-CoV-2-associated inflammation and coagulopathy, largely independently of GSDMD, identifying CASP4/11 as a promising drug target for treatment and prevention of severe COVID-19.

immunology↗

bioRxiv2021

Selective Modulator of Nuclear Receptor PPARy with Reduced Adipogenic Potential Ameliorates Experimental Nephrotic Syndrome

BackgroundGlomerular disease, often manifesting as nephrotic syndrome (NS) with high proteinuria, can be refractory to standard treatment and is typically associated with hypoalbuminemia, hypercholesterolemia and hypercoagulopathy. We hypothesized that the nuclear receptor PPAR{gamma} can be selectively modulated using a novel partial agonist, GQ-16, to gain therapeutic advantage over traditional PPAR{gamma} agonists (e.g. thiazolidinediones) for the treatment of glomerular disease. MethodsNephropathy was induced with puromycin amino-nucleoside (PAN) in Wistar rats and treated with Pioglitazone (Pio) or GQ-16. Plasma, serum, and urine chemistries were performed, and kidneys, glomeruli, liver, and white adipose tissue (WAT) were harvested. Lipid accumulation and adipogenic gene expression were measured in adipocytes. ResultsPAN-induced proteinuria was significantly reduced with Pio to 64% of PAN-value. It was reduced robustly with GQ-16 to 81% of PAN, which was comparable to controls. While both GQ-16 and Pio restored glomerular Nphs1 and hepatic Pcsk9 expression and reduced hypercholesterolemia, GQ-16 also restored glomerular Nrf2, and reduced hypoalbuminemia and hypercoagulopathy. Furthermore, RNA-seq analysis identified both common and distinct restored glomerular genes downstream of Pio and GQ-16. Pio but not GQ-16 significantly induced aP2 (fatty acid binding protein) in adipocytes and in WAT. Pio induced more lipid accumulation than GQ-16 in differentiated adipocytes. Both, Pio and GQ-16 induced insulin sensitizing adipokines in WAT with varying degrees. ConclusionsSelective modulation of PPAR{gamma} by a partial agonist, GQ-16, is more advantageous than pioglitazone in reducing proteinuria and NS associated co-morbidities, while reducing the adipogenic side-effects conferred by traditional PPAR{gamma} full agonists. Translational StatementThe authors have previously reported that type-II diabetes drugs, thiazolidinediones (PPAR{gamma} agonists), also provide beneficial effects in reducing podocyte and glomerular injury. However, these drugs are associated with adverse effects such as weight gain, and their effects on glomerular disease-associated features are largely unexplored. Their current findings demonstrate that PPAR{gamma} can be selectively modulated by its partial agonist, GQ-16, which reduces proteinuria and improves nephrotic syndrome (NS) with reduced side-effects typically conferred by thiazolidinediones. These findings not only deepen our molecular understanding of the role of PPAR{gamma} in glomerular disease and underscore the potential for partial agonists of PPAR{gamma}, such as GQ-16 as a treatment modality for NS, but also lend the possibility of its potential benefits in diabetic nephropathy.

molecular biology↗

bioRxiv2021

Fetal-like reversion in the regenerating intestine is regulated by mesenchymal Asporin

Epithelial tissues undergo fetal-like cellular reprogramming to regenerate after damage1,2. Although the mesenchyme and the extracellular matrix (ECM) play critical roles in tissue homeostasis and regeneration2-5, their role in repurposing developmental programs in epithelium is unknown. To model epithelial regeneration, we culture intestinal epithelium on decellularized small intestinal scaffold (iECM), and identify Asporin (Aspn), an ECM bound proteoglycan, as a critical mediator of cellular reprogramming. Aspn is produced by the mesenchyme, and we show that its effect on epithelial Tgf{beta}-signalling via CD44 is critical for fetal-like conversion. Furthermore, we demonstrate that Aspn is transiently increased upon chemotherapy-induced damage and pivotal for a timely induction of the fetal-like state and tissue regeneration. In summary, we establish a platform for modelling epithelial injury responses ex vivo, and show that the mesenchymal Aspn-producing niche controls tissue repair by regulating epithelial fetal-like reprogramming.

developmental biology↗

bioRxiv2021

Transcriptional milestones in Dictyostelium development

Development of the social amoeba Dictyostelium discoideum begins by starvation of single cells and ends in multicellular fruiting bodies 24 hours later. These major morphological changes are accompanied by sweeping gene expression changes, encompassing nearly half of the 13,000 genes in the genome. To explore the relationships between the transcriptome and developmental morphogenesis, we performed time-series RNA-sequencing analysis of the wild type and 20 mutant strains with altered morphogenesis. These strains exhibit arrest at different developmental stages, accelerated development, or terminal morphologies that are not typically seen in the wild type. Considering eight major morphological transitions, we identified 1,371 milestone genes whose expression changes sharply between two consecutive transitions. We also identified 1,099 genes as members of 21 regulons, which are groups of genes that remain coordinately regulated despite the genetic, temporal, and developmental perturbations in the dataset. The gene annotations in these milestones and regulons validate known transitions and reveal several new physiological and functional transitions during development. For example, we found that DNA replication genes are co-regulated with cell division genes, so they are co-expressed in mid-development even though chromosomal DNA is not replicated at that time. Altogether, the dataset includes 486 transcriptional profiles, across developmental and genetic conditions, that can be used to identify new relationships between gene expression and developmental processes and to improve gene annotations. We demonstrate the utility of this resource by showing that the cycles of aggregation and disaggregation observed in allorecognition-defective mutants involve a dedifferentiation process. We also show unexpected variability and sensitivity to genetic background and developmental conditions in two commonly used genes, act6 and act15, and robustness of the coaA gene. Finally, we propose that gpdA should be used as a standard for mRNA quantitation because it is less sensitive to genetic background and developmental conditions than commonly used standards. The dataset is available for democratized exploration without the need for programming skills through the web application dictyExpress and the data mining environment Orange.

genomics↗

bioRxiv2021

Off-resonance saturation as an MRI method to quantify ferritin-bound iron in the post-mortem brain

PurposeTo employ an Off-Resonance Saturation (ORS) method to measure the ferritin-bound iron pool, which is an endogenous contrast agent which can give information on cellular iron status. MethodsAn ORS acquisition protocol was implemented on a 7T preclinical scanner and the contrast maps were fitted to an established analytical model. The method was validated by correlation and Bland-Altman analysis on a ferritin-containing phantom. Ferritin-iron maps were obtained from post-mortem tissue of patients with neurological diseases characterized by brain iron accumulation, i. e. Alzheimers disease, Huntingtons disease and aceruloplasminemia, and validated with histology. Transverse relaxation rate and magnetic susceptibility values were also obtained for comparison. ResultsIn post-mortem tissue, the ferritin-iron contrast strongly co-localizes with histological iron staining, in all the cases. Quantitative iron values obtained via the ORS method are in agreement with literature. ConclusionsOff-resonance saturation is an effective way to detect iron in grey matter structures, while mitigating for the presence of myelin. If a reference region with little iron is available in the tissue, the method can produce quantitative iron maps. This method is applicable in the study of brain diseases characterized by brain iron accumulation and complement existing iron-sensitive parametric methods.

biophysics↗

bioRxiv2020

Effects of Alzheimer's disease and formalin fixation on the different mineralised-iron forms in the human brain

Iron accumulation in the brain is a phenomenon common to many neurodegenerative diseases, perhaps most notably Alzheimers disease (AD). We present here magnetic analyses of post-mortem brain tissue of patients who had severe Alzheimers disease, and compare the results with those from healthy controls. Isothermal remanent magnetization experiments were performed to assess the extent to which different magnetic carriers are affected by AD pathology and formalin fixation. While Alzheimers brain material did not show higher levels of magnetite/maghemite nanoparticles than corresponding controls, the ferrihydrite mineral, known to be found within the core of ferritin proteins and hemosiderin aggregates, almost doubled in concentration in patients with Alzheimers pathology, strengthening the conclusions of our previous studies. As part of this study, we also investigated the effects of sample preparation, by performing experiments on frozen tissue as well as tissue which had been fixed in formalin for a period of five months. Our results showed that the two different preparations did not critically affect the concentration of magnetic carriers in brain tissue, as observable by SQUID magnetometry.

biophysics↗

bioRxiv2020

A draft genome of grass pea (Lathyrus sativus), a resilient diploid legume

We have sequenced the genome of grass pea (Lathyrus sativus), a resilient diploid (2n=14) legume closely related to pea (Pisum sativum). We determined the genome size of the sequenced European accession (LS007) as 6.3 Gbp. We generated two assemblies of this genome, i) EIv1 using Illumina PCR-free paired-end sequencing and assembly followed by long-mate-pair scaffolding and ii) Rbp using Oxford Nanopore Technologies long-read sequencing and assembly followed by polishing with Illumina paired-end data. EIv1 has a total length of 8.12 Gbp (including 1.9 billion Ns) and scaffold N50 59,7 kbp. Annotation has identified 33,819 high confidence genes in the assembly. Rbp has a total length of 6.2 Gbp (with no Ns) and a contig N50 of 155.7 kbp. Gene space assessment using the eukaryote BUSCO database showed completeness scores of 82.8 % and 89.8%, respectively.

genomics↗

bioRxiv2020

Iron and lipocalin-2 modulate cellular responses in the tumor micro-environment of pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic disease with poor outcomes. Iron is known to signal cellular responses, and its levels are regulated by lipocalin-2 (LCN2) expression, a PDAC pro-tumorigenic molecule. However, how iron and LCN2 function in PDAC is unclear. Here we demonstrate that iron levels regulate PDAC cell proliferation, invasion, expression of epithelial to mesenchymal tumor markers, and pro-inflammatory cytokines. Iron chelation increased the expression of the LCN2 receptor SLC22A17 in pancreatic stellate cells and the anti-metastatic gene NDRG1 in PDAC cells. Deletion of Lcn2 in mouse tumor cells modulated the expression of genes involved in extracellular matrix deposition and cell migration. Moreover, cellular iron responses were dependent on the Kras mutation status of cells, and LCN2 expression levels. Deletion of Lcn2 expression in PDAC suggests a protective role against metastasis. Thus, iron modulation and LCN2 blockade could serve as potential therapeutic approaches against PDAC.

cancer biology↗

bioRxiv2019

The Pop-gen Pipeline Platform: A Software Platform for Facilitating Population Genomic Analyses

Here we present the Pop-Gen Pipeline Platform (PPP), a software platform with the goal of reducing the computational expertise required for conducting population genomic analyses. The PPP was designed as a collection of scripts that facilitate common population genomic workflows in a consistent and standardized Python environment. Functions were developed to encompass entire workflows, including: input preparation, file format conversion, various population genomic analyses, output generation, and visualization. By facilitating entire workflows, the PPP offers several benefits to prospective end users - it reduces the need of redundant in-house software and scripts that would require development time and may be error-prone, or incorrect. The platform has also been developed with reproducibility and extensibility of analyses in mind. The PPP is an open-source package that is available for download and use at https://ppp.readthedocs.io/en/latest/PPP_pages/install.html

bioinformatics↗

bioRxiv2019

UVA and UVB elicit distinct mutational signatures in melanoma

BRAF-mutant melanomas are more likely than NRAS-mutant melanomas to arise in anatomical locations protected from chronic sun damage. We hypothesized that this discrepancy in tumor location is a consequence of the differential sensitivity of BRAF and NRAS-mutant melanocytes to ultraviolet light (UV)-mediated carcinogenesis. We tested this hypothesis by comparing the mutagenic consequences of a single, narrow band ultraviolet-A (UVA; 320-400nm) or ultraviolet-B (UVB; 280-320nm) exposure in mouse models predisposed to Braf- or Nras-mutant melanoma. Exposures approximated the amount of UVA or UVB energy contained in ~40 minutes of summer sunlight. Tumor onset was accelerated in all UVB-, but only half of UVA- irradiated mice as compared to unirradiated controls. Melanomas from both mouse models, harbored recurrent mutations affecting the RING domain of MAP3K1 and Actin-binding domain of Filamin A irrespective of UV status. Melanomas from UVB-irradiated, Braf-mutant mice averaged twice as many SNVs (1,025 vs. 435) and five times as many dipyrimidine variants (33.3 vs. 5.7) than tumors from similarly irradiated Nras-mutant mice. We identified a mutational signature enriched in UVB-accelerated tumors which mirrored COSMIC signatures associated with human skin cancer. Notably, this signature was enriched to a greater extent in Braf- than Nras-mutant murine melanomas. These data suggest that oncogenic BRAF may enhance UVB carcinogenesis to promote melanoma formation at anatomic sites with low or intermittent sun exposure.

cancer biology↗

bioRxiv2019

Genome-wide association analyses identify variants in IRF4 associated with Acute Myeloid Leukemia and Myelodysplastic Syndrome susceptibility

The role of common genetic variation in susceptibility to acute myeloid leukemia (AML), and myelodysplastic syndrome (MDS), a group of rare clonal hematologic disorders characterized by dysplastic hematopoiesis and high mortality, remains unclear. We performed AML and MDS genome-wide association studies (GWAS) in the DISCOVeRY-BMT cohorts (2309 cases and 2814 controls). Association analysis based on subsets (ASSET) was used to conduct a summary statistics SNP-based analysis of MDS and AML subtypes. For each AML and MDS case and control we used PrediXcan to estimate the component of gene expression determined by their genetic profile and correlate this imputed gene expression level with risk of developing disease in a transcriptome-wide association study (TWAS). ASSET identified an increased risk for de novo AML and MDS (OR=1.38, 95% CI, 1.26-1.51, Pmeta=2.8x10-12) in patients carrying the T allele at rs12203592 in Interferon Regulatory Factor 4 (IRF4), a transcription factor which regulates myeloid and lymphoid hematopoietic differentiation. Our TWAS analyses showed increased IRF4 gene expression is associated with increased risk of de novo AML and MDS (OR=3.90, 95% CI, 2.36-6.44, Pmeta =1.0x10-7). The identification of IRF4 by both GWAS and TWAS contributes valuable insight on the role of genetic variation in AML and MDS susceptibility.

genomics↗

bioRxiv2018

Identification of a divergent cytochrome c oxidase complex in the mitochondrial proteome of Toxoplasma gondii

The mitochondrion of apicomplexan parasites is critical for parasite survival, although the full complement of proteins that localize to this organelle has not been defined. Here we undertake two independent approaches to elucidate the mitochondrial proteome of the apicomplexan Toxoplasma gondii. We identify 421 mitochondrial proteins, many of which lack homologs in the animals that these parasites infect, and most of which are important for parasite growth. We demonstrate that one such protein, termed TgApiCox25, is an important component of the parasite cytochrome c oxidase (COX) complex. We identify numerous other apicomplexan-specific components of COX, and conclude that apicomplexan COX, and apicomplexan mitochondria more generally, differ substantially in their protein composition from the hosts they infect. Our study highlights the diversity that exists in mitochondrial proteomes across the eukaryotic domain of life, and provides a foundation for defining unique aspects of mitochondrial biology in an important phylum of parasites.

microbiology↗

bioRxiv2018

BAX requires VDAC2 to mediate apoptosis and to limit tumor development

Intrinsic apoptosis is critical for normal physiology including the prevention of tumor formation. BAX and BAK are essential for mediating this process and for the cytotoxic action of many anticancer drugs. BAX and BAK are thought to act in a functionally redundant manner and are considered to be regulated similarly. From an unbiased genome-wide CRISPR/Cas9 screen, we identified VDAC2 (voltage-dependent anion channel 2) as essential for BAX, but not BAK, to function. The genetic deletion of VDAC2 abrogated the association of BAX and BAK with mitochondrial complexes that contain VDAC1, VDAC2 and VDAC3. By disrupting its localization to mitochondria, BAX is rendered completely ineffective. Moreover, we defined an interface unique to VDAC2 that is required to drive BAX activity. Consequently, interfering with this interaction or deleting VDAC2 phenocopied the loss of BAX, including impairing the killing of tumor cells by anti-cancer agents such as the BCL-2 inhibitor venetoclax. Furthermore, the ability of BAX to prevent tumor formation was attenuated in the absence of VDAC2. Taken together, our studies show for the first time that BAX-mediated apoptosis, but not BAK-mediated apoptosis, is critically dependent on VDAC2, hence revealing the differential regulation of BAX and BAK.

cell biology↗