Biology subjects

Cooper, L.

Publications and source records attributed to Cooper, L..

27 records · Page 2Linked to original sources

bioRxiv2021

Expression of CD49f defines subsets of human regulatory T cells with divergent transcriptional landscape and function that correlate with ulcerative colitis disease activity

ObjectiveAdoptive regulatory T cell (Treg) therapy is being trialled for treatment of different autoimmune disorders, including inflammatory bowel diseases (IBD). In-depth understanding of the biological variability of Treg in the human blood may be required to improve IBD immune monitoring and treatment strategies. MethodsThrough a combination of quantitative proteomic, multiparametric flow cytometry, RNA-sequencing data analysis and functional assays on Treg enriched from the blood of ulcerative colitis (UC) patients and healthy controls, we investigated the association between CD49f expression, Treg phenotype and function, and UC disease activity. ResultsHigh-dimensional analysis and filtering defined two distinct subsets of human Treg based on the presence or absence of CD49f with divergent transcriptional landscape and functional activities. CD49f negative Treg are enriched for functional Treg markers and present significantly increased suppressive capacity. In contrast, CD49f high Treg display a pro-inflammatory Th17-like phenotype and accumulate in the blood of UC patients. Dysregulation on CD49f Treg subsets in UC patients correlate with disease activity. ConclusionOverall our findings uncover the importance of CD49f expression on Treg in physiological immunity and in pathological autoimmunity.

immunology↗

bioRxiv2021

Potent, Novel SARS-CoV-2 PLpro Inhibitors Block Viral Replication in Monkey and Human Cell Cultures

Antiviral agents blocking SARS-CoV-2 viral replication are desperately needed to complement vaccination to end the COVID-19 pandemic. Viral replication and assembly are entirely dependent on two viral cysteine proteases: 3C-like protease (3CLpro) and the papain-like protease (PLpro). PLpro also has deubiquitinase (DUB) activity, removing ubiquitin (Ub) and Ub-like modifications from host proteins, disrupting the host immune response. 3CLpro is inhibited by many known cysteine protease inhibitors, whereas PLpro is a relatively unusual cysteine protease, being resistant to blockade by such inhibitors. A high-throughput screen of biased and unbiased libraries gave a low hit rate, identifying only CPI-169 and the positive control, GRL0617, as inhibitors with good potency (IC50 < 10 {micro}M). Analogues of both inhibitors were designed to develop structure-activity relationships; however, without a co-crystal structure of the CPI-169 series, we focused on GRL0617 as a starting point for structure-based drug design, obtaining several co-crystal structures to guide optimization. A series of novel 2-phenylthiophene-based non-covalent SARS-CoV-2 PLpro inhibitors were obtained, culminating in low nanomolar potency. The high potency and slow inhibitor off-rate were rationalized by newly identified ligand interactions with a "BL2 groove" that is distal from the active site cysteine. Trapping of the conformationally flexible BL2 loop by these inhibitors blocks binding of viral and host protein substrates; however, until now it has not been demonstrated that this mechanism can induce potent and efficacious antiviral activity. In this study, we report that novel PLpro inhibitors have excellent antiviral efficacy and potency against infectious SARS-CoV-2 replication in cell cultures. Together, our data provide structural insights into the design of potent PLpro inhibitors and the first validation that non-covalent inhibitors of SARS-CoV-2 PLpro can block infection of human cells with low micromolar potency.

microbiology↗

bioRxiv2020

Novel ACE2-IgG1 fusions with increased activity against SARS-CoV-2

SARS-CoV2, the etiologic agent of COVID-19, uses ACE2 as a cell entry receptor. Soluble ACE2 has been shown to have neutralizing antiviral activity but has a short half-life and no active transport mechanism from the circulation into the alveolar spaces of the lung. To overcome this, we constructed an ACE2-human IgG1 fusion protein with mutations in the catalytic domain of ACE2. This fusion protein contained a LALA mutation that abrogates Fcr{gamma} binding, but retains FcRN binding to prolong the half-life, as well as achieve therapeutic concentrations in the lung lavage. Interestingly, a mutation in the catalytic domain of ACE2, MDR504, completely abrogated catalytic activity, but significantly increased binding to SARS-CoV2 spike protein in vitro. This feature correlated with more potent viral neutralization in a plaque assay. Parental administration of the protein showed stable serum concentrations with a serum half-life of [~] 145 hours with excellent bioavailability in the epithelial lining fluid of the lung. Prophylactic administration of MDR504 significantly attenuated SARS-CoV2 infection in a murine model. These data support that the MDR504 hACE2-Fc is an excellent candidate for pre or post-exposure prophylaxis or treatment of COVID-19.

immunology↗

bioRxiv2020

In vivo high-content screening in zebrafish for developmental nephrotoxicity of approved drugs

Despite widespread drug exposure, for example during gestation or in prematurely born children, organ-specific developmental toxicity of most drugs is poorly understood. Developmental and functional abnormalities are a major cause of kidney diseases during childhood; however, the potential causal relationship to exposure with nephrotoxic drugs during nephrogenesis is widely unknown. To identify developmental nephrotoxic drugs in a large scale, we established and performed an automated high-content screen to score for phenotypic renal alterations in the Tg(wt1b:EGFP) zebrafish line. During early nephrogenesis, embryos were exposed to a compound library of approved drugs. After treatment, embryos were aligned within microtiter plates using 3D-printed orientation tools enabling the robust acquisition of consistent dorsal views of pronephric kidneys by automated microscopy. To qualitatively and quantitatively score and visualize phenotypes, we developed software tools for the semi-automated analysis, processing and visualization of this large image-based dataset. Using this scoring scheme, we were able to categorize compounds based on their potential developmental nephrotoxic effects. About 10% of tested drugs induced pronephric phenotypes including glomerular and tubular malformations, or overall changes in kidney morphology. Major chemical compound groups identified to cause glomerular and tubular alterations included dihydropyridine derivatives, HMG CoA reductase inhibitors, fibrates, imidazole, benzimidazole and triazole derivatives, corticosteroids, glucocorticoids, acetic acid derivatives and propionic acid derivatives. In conclusion, the presented study demonstrates the large-scale screening of kidney-specific toxicity of approved drugs in a live vertebrate embryo. The associated technology and tool-sets can be easily adapted for other organ systems providing a unique platform for in vivo large-scale assessment of organ-specific developmental toxicity or other biomedical applications. Ultimately, the presented data and associated visualization and browsing tools provide a resource for potentially nephrotoxic drugs and for further investigations.

developmental biology↗

bioRxiv2020

Low coverage genomic data resolve the population divergence and gene flow history of an Australian rain forest fig wasp

Population divergence and gene flow are key processes in evolution and ecology. Model-based analysis of genome-wide datasets allows discrimination between alternative scenarios for these processes even in non-model taxa. We used two complementary approaches (one based on the blockwise site frequency spectrum (bSFS), the second on the Pairwise Sequentially Markovian Coalescent (PSMC)) to infer the divergence history of a fig wasp, Pleistodontes nigriventris. Pleistodontes nigriventris and its fig tree mutualist Ficus watkinsiana are restricted to rain forest patches along the eastern coast of Australia, and are separated into northern and southern populations by two dry forest corridors (the Burdekin and St. Lawrence Gaps). We generated whole genome sequence data for two haploid males per population and used the bSFS approach to infer the timing of divergence between northern and southern populations of P. nigriventris, and to discriminate between alternative isolation with migration (IM) and instantaneous admixture (ADM) models of post divergence gene flow. Pleistodontes nigriventris has low genetic diversity ({pi} = 0.0008), to our knowledge one of the lowest estimates reported for a sexually reproducing arthropod. We find strongest support for an ADM model in which the two populations diverged ca. 196kya in the late Pleistocene, with almost 25% of northern lineages introduced from the south during an admixture event ca. 57kya. This divergence history is highly concordant with individual population demographies inferred from each pair of haploid males using PSMC. Our analysis illustrates the inferences possible with genome-level data for small population samples of tiny, non-model organisms and adds to a growing body of knowledge on the population structure of Australian rain forest taxa.

evolutionary biology↗

bioRxiv2019

A glycosaminoglycan extract from Portunus pelagicus inhibits BACE1, the β secretase implicated in Alzheimer’s disease

Therapeutic options for Alzheimers disease, the most common form of dementia, are currently restricted to palliative treatments. The glycosaminoglycan heparin, widely used as a clinical anticoagulant, has previously been shown to inhibit the Alzheimers disease-relevant {beta}-secretase 1 (BACE1). Despite this, the deployment of pharmaceutical heparin for the treatment of Alzheimers disease is largely precluded by its potent anticoagulant activity. Furthermore, ongoing concerns regarding the use of mammalian sourced heparins, primarily due to prion diseases and religious beliefs, hinder the deployment of alternative heparin based therapeutics. A marine-derived, heparan sulphate-containing glycosaminoglycan extract isolated from the crab Portunus pelagicus, was identified to inhibit human BACE1 with comparable bioactivity to that of mammalian heparin (IC50 = 1.85 g.mL-1 (R2 = 0.94) and 2.43 g.mL-1 (R2 = 0.93), respectively) possessing highly attenuated anticoagulant activities. The results from several structural techniques suggest that the interactions between BACE1 and the extract from P. pelagicus are complex and distinct from those of heparin.

biochemistry↗

bioRxiv2019

STATegra: a comprehensive multi-omics dataset of B-cell differentiation in mouse

Multi-omics approaches use a diversity of high-throughput technologies to profile the different molecular layers of living cells. Ideally, the integration of this information should result in comprehensive systems models of cellular physiology and regulation. However, most multi-omics projects still include a limited number of molecular assays and there have been very few multi-omic studies that evaluate dynamic processes such as cellular growth, development and adaptation. Hence, we lack formal analysis methods and comprehensive multi-omics datasets that can be leveraged to develop true multi-layered models for dynamic cellular systems. Here we present the STATegra multi-omics dataset that combines measurements from up to 10 different omics technologies applied to the same biological system, namely the well-studied mouse pre-B-cell differentiation. STATegra includes high-throughput measurements of chromatin structure, gene expression, proteomics and metabolomics, and it is complemented with single-cell data. To our knowledge, the STATegra collection is the most diverse multi-omics dataset describing a dynamic biological system.

bioinformatics↗

bioRxiv2018

A cell cycle-coordinated nuclear compartment for Polymerase II transcription encompasses the earliest gene expression before global genome activation

Most metazoan embryos commence development with rapid cleavages without zygotic gene expression and their genome activation is delayed until the mid-blastula transition (MBT). However, a set of genes escape global repression during the extremely fast cell cycles, which lack gap phases and their transcription is activated before the MBT. Here we describe the formation and the spatio-temporal dynamics of a distinct transcription compartment, which encompasses the earliest detectable transcription during the first wave of genome activation. Simultaneous 4D imaging of expression of pri-miR430 and zinc finger genes by a novel, native transcription imaging approach reveals a pair of shared transcription compartments regulated by homolog chromosome organisation. These nuclear compartments carry the majority of nascent RNAs and transcriptionally active Polymerase II, are depleted of compact chromatin and represent the main sites for detectable transcription before MBT. We demonstrate that transcription occurs in the S-phase of the cleavage cycles and that the gradual slowing of these cell cycles are permissive to transcription before global genome activation. We propose that the demonstrated transcription compartment is part of the regulatory architecture of nucleus organisation, and provides a transcriptionally competent, supporting environment to facilitate early escape from the general nuclear repression before global genome activation.

developmental biology↗

bioRxiv2017

Determining the Specificity of Cascade Binding, Interference, and Priming in vivo

In CRISPR-Cas immunity systems, short CRISPR RNAs (crRNAs) are bound by CRISPR-associated (Cas) proteins, and these complexes target invading nucleic acid molecules for degradation in a process known as interference. In type I CRISPR-Cas systems, the Cas protein complex that binds DNA is known as Cascade. Association of Cascade with target DNA can also lead to acquisition of new immunity elements in a process known as primed adaptation. Here, we assess the specificity determinants for Cascade-DNA interaction, interference, and primed adaptation in vivo, for the type I-E system of Escherichia coli. Remarkably, as few as 5 bp of crRNA-DNA are sufficient for association of Cascade with a DNA target. Consequently, a single crRNA promotes Cascade association with numerous off-target sites, and the endogenous E. coli crRNAs direct Cascade binding to >100 chromosomal sites. In contrast to the low specificity of Cascade-DNA interactions, >18 bp are required for both interference and primed adaptation. Hence, Cascade binding to sub-optimal, off-target sites is inert. Our data support a model in which initial Cascade association with DNA targets requires only limited sequence complementarity at the crRNA 5{square} end, whereas recruitment and/or activation of the Cas3 nuclease, a prerequisite for interference and primed adaptation, requires extensive base-pairing.\n\nIMPORTANCEMany bacterial and archaeal species encode CRISPR-Cas immunity systems that protect against invasion by foreign DNA. In the Escherichia coli CRISPR-Cas system, a protein complex, Cascade, binds 61 nt CRISPR RNAs (crRNAs). The Cascade complex is directed to invading DNA molecules through base-pairing between the crRNA and target DNA. This leads to recruitment of the Cas3 nuclease that destroys the invading DNA molecule and promotes acquisition of new immunity elements. We make the first in vivo measurements of Cascade binding to DNA targets. Thus, we show that Cascade binding to DNA is highly promiscuous; endogenous E. coli crRNAs can direct Cascade binding to >100 chromosomal locations. By contrast, we show that target degradation and acquisition of new immunity elements requires highly specific association of Cascade with DNA, limiting CRISPR-Cas function to the appropriate targets.

microbiology↗