Biology subjects

Spencer, J.

Publications and source records attributed to Spencer, J..

35 records · Page 2Linked to original sources

bioRxiv2023

Unanticipated interacting features of human gut-associated lymphoid tissues link microbiota, intestinal immunity and autoimmunity

Gut-associated lymphoid tissue (GALT) is organised lymphoid tissue that is chronically activated by the intestinal microbiota. It generates the IgA response that is critical for intestinal homeostasis. By iterative application of multiplexed technologies, we identify enrichment of double-negative 2 (DN2:CD27-IgD-CD21loCD11chi) B cells in GALT, where they comprise the majority of intraepithelial and subepithelial B cells. We show that DN2 B cells in GALT interact with DC in the sub-epithelial dome that express DNASE1L3 and microbicides. Unlike in mice, DNASE1L3 in humans does not associate with apoptotic debris, but is located between sampled bacteria and host tissue where it is co-expressed with C1Q, consistent with management of bacterial debris. Thus we demonstrate that DN2 B cells that are otherwise associated with lupus nephritis, and DNASE1L3 and C1q that are lupus autoantigens, are microbiota-associated, interacting components of normal intestinal immunity.

immunology↗

bioRxiv2023

The Crystal Structure of the Hsp90-LA1011 Complex and the Mechanism by which LA1011 may Improve the Prognosis of Alzheimer's Disease.

Chaperone systems play a major role in the decline of cognition and contribute to neurological pathologies such as Alzheimers disease (AD). While such a decline may occur naturally with age or with stress or trauma, the mechanisms involved have remained elusive. The current models suggest that amyloid-{beta} (A{beta}) plaque formation leads to the hyperphosphorylation of tau by a Hsp90 dependent process that triggers tau neurofibrillary tangle formation and neurotoxicity. Several co-chaperones of Hsp90 can influence the phosphorylation of tau, including FKBP51, FKBP52 and PP5. In particular elevated levels of FKBP51 occur with age and stress and are further elevated in AD. Recently, the dihydropyridine, LA1011 was shown to reduce tau pathology and amyloid plaque formation in transgenic AD mice, probably through its interaction with Hsp90 although the precise mode of action is currently unknown. Here, we present a co-crystal structure of LA1011 in complex with a fragment of Hsp90. We show that LA1011 can disrupt binding of FKBP51, which might help to rebalance the Hsp90-FKBP51 chaperone machinery and provide a favourable prognosis towards AD. Clinically, this is highly significant, as AD is generally a disease affecting older patients, where slowing of disease progression could result in AD no longer being life limiting.

molecular biology↗

bioRxiv2023

Inhibition mechanism and antiviral activity of an α-ketoamide based SARS-CoV-2 main protease inhibitor

SARS-CoV-2 has demonstrated extraordinary ability to evade antibody immunity by antigenic drift. Small molecule drugs may provide effective therapy while being part of a solution to circumvent SARS-CoV-2 immune escape. In this study we report an -ketoamide based peptidomimetic inhibitor of SARS-CoV-2 main protease (Mpro), RAY1216. Enzyme inhibition kinetic analysis established that RAY1216 is a slow-tight inhibitor with a Ki of 8.6 nM; RAY1216 has a drug-target residence time of 104 min compared to 9 min of PF-07321332 (nirmatrelvir), the antiviral component in Paxlovid, suggesting that RAY1216 is approximately 12 times slower to dissociate from the protease-inhibitor complex compared to PF-07321332. Crystal structure of SARS-CoV-2 Mpro:RAY1216 complex demonstrates that RAY1216 is covalently attached to the catalytic Cys145 through the -ketoamide warhead; more extensive interactions are identified between bound RAY1216 and Mpro active site compared to PF-07321332, consistent with a more stable acyl-enzyme inhibition complex for RAY1216. In cell culture and human ACE2 transgenic mouse models, RAY1216 demonstrates comparable antiviral activities towards different SARS-CoV-2 virus variants compared to PF-07321332. Improvement in pharmacokinetics has been observed for RAY1216 over PF-07321332 in various animal models, which may allow RAY1216 to be used without ritonavir. RAY1216 is currently undergoing phase III clinical trials (https://clinicaltrials.gov/ct2/show/NCT05620160) to test real-world therapeutic efficacy against COVID-19.

biochemistry↗

bioRxiv2022

Tautomer-specific deacylation and {Omega}-loop flexibility explain carbapenem-hydrolyzing, broad-spectrum activity of the KPC-2 β-lactamase

KPC-2 (Klebsiella pneumoniae carbapenemase-2) is a globally disseminated serine-{beta}-lactamase (SBL) responsible for extensive {beta}-lactam antibiotic resistance in Gram-negative pathogens. SBLs inactivate {beta}-lactams via a mechanism involving a hydrolytically labile covalent acyl-enzyme intermediate. Carbapenems, the most potent {beta}-lactams, evade activity of many SBLs by forming long-lived inhibitory acyl-enzymes; however, carbapenemases such as KPC-2 efficiently catalyze deacylation of carbapenem-derived acyl-enzymes. We present high-resolution (1.25-1.4 [A]) crystal structures of KPC-2 acyl-enzymes with representative penicillins (ampicillin), cephalosporins (cefalothin) and carbapenems (imipenem, meropenem and ertapenem), obtained utilizing an isosteric deacylation-deficient mutant (E166Q). Mobility of the {Omega}-loop (residues 165-170) negatively correlates with antibiotic turnover rates (kcat), highlighting the role of this region in positioning catalytic residues for efficient hydrolysis of different {beta}-lactams. Carbapenem-derived acyl-enzyme structures reveal predominance of the {Delta}1-(2R) imine tautomer, except for the imipenem acyl-enzyme, which is present in dual occupancy in both {Delta}1-(2R) and (2S) configurations. Quantum mechanics/molecular mechanics (QM/MM) molecular dynamics simulations of deacylation of the KPC-2:meropenem acyl-enzyme, using an adaptive string method (ASM), show that the {Delta}1-(2R) isomer has a 7 kcal/mol higher barrier for the (rate-determining) formation of the tetrahedral deacylation intermediate than the {Delta}2 tautomer. The simulations identify tautomer-specific differences in hydrogen bonding networks involving the carbapenem C-3 carboxylate and the deacylating water, that, together with stabilization by protonated N-4 of accumulating negative charge during oxyanion formation, accelerate deacylation of the {Delta}2-enamine acyl-enzyme compared to the {Delta}1-imine. Taken together, our data show how the flexible {Omega}-loop helps confer broad spectrum activity upon KPC-2, while carbapenemase activity stems from efficient deacylation of the {Delta}2-enamine acyl-enzyme tautomer. Differentiation of the barriers associated with deacylation of these subtly different {beta}-lactam isomers further identifies ASM as a sensitive method for calculation of reaction energetics that can accurately model turnover and, potentially, predict the impact of substrate modifications or point mutations upon activity.

biochemistry↗

bioRxiv2022

An Azobenzene G-quadruplex Ligand Exhibits Promising Antibacterial Activity against Escherichia coli

There is great need for novel strategies to tackle antimicrobial resistance, in particular in Gram-negative species such as Escherichia coli that cause opportunistic infections of already compromised patients. Here we demonstrate, following a screen of G-quadruplex (G4) ligand candidates, that a novel pyridinium-functionalized azobenzene L9 shows promising antibacterial activity (MIC values [≤] 4 g/mL) against multi-drug resistant E. coli. Tandem Mass Tag (TMT) proteomics of E. coli treated with sub-lethal concentrations of L9, identified that, consistent with its superior antibacterial activity, L9 treatment influences expression levels of more G4-associated proteins than the analogous ligands L5 (stiff-stilbene) or pyridostatin (PDS), and upregulates multiple essential proteins involved in translation. Biophysical analysis showed L9 binds potential target G4-containing sequences, identified from proteomic experiments and by bioinformatics, with variable affinity, in contrast to the two comparator G4 ligands (L5, PDS) that better stabilize G4 structures but have lower antimicrobial activity. Fluorescence microscopy-based Bacterial Cytological Profiling (BCP) suggests that the L9 mechanism of action is distinct from other antibiotic classes. These findings support strategies discovering potential G4 ligands as antibacterial candidates for priority targets such as multi-drug resistant E. coli, warranting their further exploration as potential novel therapeutic leads with G4-mediated modes of action. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/506212v2_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@18197a0org.highwire.dtl.DTLVardef@109b120org.highwire.dtl.DTLVardef@14be8eeorg.highwire.dtl.DTLVardef@a97048_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2022

Rarity: Discovering rare cell populations from single-cell imaging data

BackgroundCell type identification plays an important role in the analysis and interpretation of single-cell data and can be carried out via supervised or unsupervised clustering approaches. Supervised methods are best suited where we can list all cell types and their respective marker genes a priori. While unsupervised clustering algorithms look for groups of cells with similar expression properties. This property permits the identification of both known and unknown cell populations, making unsupervised methods suitable for discovery.. Success is dependent on the relative strength of the expression signature of each group as well as the number of cells. Rare cell types therefore present a particular challenge that are magnified when they are defined by differentially expressing a small number of genes. Typical unsupervised approaches fail to identify such rare subpopulations, and these cells tend to be absorbed into more prevalent cell types. ResultsIn order to balance these competing demands, we have developed a novel statistical framework for unsupervised clustering, named Rarity, that enables the discovery process for rare cell types to be more robust, consistent and interpretable. We achieve this by devising a novel clustering method based on a Bayesian latent variable model in which we assign cells to inferred latent binary on/off expression profiles. This lets us achieve increased sensitivity to rare cell populations while also allowing us to control and interpret potential false positive discoveries. ConclusionsWe systematically study the challenges associated with rare cell type identification and demonstrate the utility of Rarity on various IMC data sets.

bioinformatics↗

bioRxiv2021

Disrupted Peyer's patch microanatomy in COVID-19 including germinal centre atrophy independent of local virus

Confirmed SARS-coronavirus-2 infection with gastrointestinal symptoms and changes in microbiota associated with coronavirus disease 2019 (COVID-19) severity have been previously reported, but the disease impact on the architecture and cellularity of ileal Peyers patches (PP) remains unknown. Here we analysed post-mortem tissues from throughout the gastrointestinal (GI) tract of patients who died with COVID-19. When virus was detected by PCR in the GI tract, immunohistochemistry identified virus in epithelium and lamina propria macrophages, but not in lymphoid tissues. Immunohistochemistry and imaging mass cytometry (IMC) analysis of ileal PP revealed depletion of germinal centres (GC), disruption of B cell/T cell zonation and decreased potential B and T cell interaction and lower nuclear density in COVID-19 patients. This occurred independent of the local viral levels. The changes in PP demonstrate that the ability to mount an intestinal immune response is compromised in severe COVID-19, which could contribute to observed dysbiosis.

immunology↗

bioRxiv2021

ChAdOx1 nCoV-19 (AZD1222) vaccine elicits monoclonal antibodies with potent cross-neutralizing activity against SARS-CoV-2 viral variants

Although the antibody response to COVID-19 vaccination has been studied extensively at the polyclonal level using immune sera, little has been reported on the antibody response at the monoclonal level. Here we isolate a panel of 44 anti-SARS-CoV-2 monoclonal antibodies (mAbs) from an individual who received two doses of the ChAdOx1 nCoV-19 (AZD1222) vaccine at a 12-week interval. We show that despite a relatively low serum neutralization titre, mAbs with potent neutralizing activity against the current SARS-CoV-2 variants of concern (B.1.1.7, P.1, B.1.351 and B.1.617.2) were obtained. The vaccine elicited neutralizing mAbs form 8 distinct competition groups and bind epitopes overlapping with neutralizing mAbs elicited following SARS-CoV-2 infection. AZD1222 elicited mAbs are more mutated than mAbs isolated from convalescent donors 1-2 months post infection. Spike reactive IgG+ B cells were still detectable 9-months post boost. These findings give molecular insights into AZD1222 elicited antibody response.

immunology↗

bioRxiv2021

A SIMPLI (Single-cell Identification from MultiPLexed Images) approach for spatially resolved tissue phenotypingat single-cell resolution.

Multiplexed imaging technologies enable the study of biological tissues at single-cell resolution while preserving spatial information. Currently, high-dimension imaging data analysis is technology-specific and requires multiple tools, restricting analytical scalability and result reproducibility. Here we present SIMPLI (Single-cell Identification from MultiPlexed Images), a novel, flexible and technology-agnostic software that unifies all steps of multiplexed imaging data analysis. After raw image processing, SIMPLI performs a spatially resolved, single-cell analysis of the tissue slide as wells as cell-independent quantifications of marker expression to investigate features undetectable at the cell level. SIMPLI is highly customisable and can run on desktop computers as well as high-performance computing environments, enabling workflow parallelisation for large datasets. SIMPLI produces multiple tabular and graphical outputs at each step of the analysis. Its containerised implementation and minimum configuration requirements make SIMPLI a portable and reproducible solution for multiplexed imaging data analysis. SIMPLI is available at: https://github.com/ciccalab/SIMPLI.

bioinformatics↗

bioRxiv2021

Green Fluorescent Carbon Dots as Targeting Probes for LED-Dependent Bacterial Killing

The emergence of antimicrobial resistance represents a significant health and economic challenge worldwide. The slow pace of antibacterial discovery necessitates strategies for optimal use of existing agents, including effective diagnostics able to drive informed prescribing; and development of alternative therapeutic strategies that go beyond traditional small-molecule approaches. Thus, the development of novel probes able to target bacteria for detection and killing, and that can pave the way to effective theranostic strategies, is of great importance. Here we demonstrate that metal-free green-emitting fluorescent carbon dots (FCDs) synthesized from glucosamine HCl and m-phenylenediamine, and featuring 2,5-deoxyfructosazine on a robust amorphous core, can label both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa) bacterial pathogens within 10 minutes of exposure. Moreover, effective killing of Gram-positive and -negative bacteria can be induced by combining FCD treatment with irradiation by LED light in the visible range. Cell-based, electron microscopy and Tandem Mass Tag (TMT) proteomic experiments indicate that FCD administration in combination with LED exposure gives rise to local heating, ROS production, and membrane- and DNA-damage, suggesting multiple routes to FCD-mediated bacterial killing. Our data identify FCDs as materials that combine facile synthesis from low-cost precursors with labelling and light-dependent killing of clinically important bacterial species, and that thus warrant further exploration as the potential bases for novel theranostics.

microbiology↗

bioRxiv2021

Two subsets of human marginal zone B cells resolved by global analysis of lymphoid tissues and blood

B cells generate antibodies that are essential for immune protection. Major events driving B cell responses occur in lymphoid tissues, which guide antigen acquisition and support cellular interactions, yet complexities of B cell subsets in human lymphoid tissues are poorly understood. Here we perform undirected, global profiling of B cells in matched human lymphoid tissues from deceased transplant organ donors and tracked dissemination of B cell clones. In addition to identifying unanticipated features of tissue-based B cell differentiation, we resolve two clonally independent subsets of marginal zone B cells that differ in cell surface and transcriptomic profiles, tendency to disseminate, distribution bias within splenic marginal zone microenvironment and immunoglobulin repertoire diversity and hypermutation frequency. Each subset is represented in spleen, gut-associated lymphoid tissue, mesenteric lymph node, and also blood. Thus, we provide clarity and diffuse controversy surrounding human MZB - the elephant in the room of human B cell biology.

immunology↗

bioRxiv2021

Allosteric communication in Class A β-lactamases occurs via Cooperative Coupling of Loop Dynamics

Allosteric effects control protein (e.g. enzyme) activity in ways that are not fully understood. Better understanding of allosteric effects, and tools to identify them, would offer promising alternative strategies to inhibitor development. Through a combination of equilibrium and nonequilibrium molecular dynamics simulations, we identify allosteric effects and communication pathways from two distant ligand binding sites to important active site structural elements that control enzymatic activity in two prototypical class A {beta}-lactamases, TEM-1 and KPC-2. Both of these enzymes are important determinants of antibiotic resistance in widespread bacterial pathogens. The simulations show that the allosteric sites are connected to the active site in both enzymes, (e.g. affecting the conformation of the {Omega}-loop) highlighting how allosteric inhibitors may exert their effects. Nonequilibrium simulations reveal pathways of communication operating over distances of 30 [A] or more. In these identified signaling pathways, the propagation of the signal occurs through cooperative coupling of loop dynamics. Notably, 50% or more clinically relevant amino acid substitutions in each enzyme map onto the identified signal transduction pathways. This suggests that clinically important variation may affect, or be driven by, differences in allosteric behavior, providing a mechanism by which amino acid substitutions may affect the relationship between spectrum of activity, catalytic turnover and potential allosteric behavior in this clinically important enzyme family. Simulations of the type presented here will help in identifying and analyzing such differences.

biophysics↗

bioRxiv2020

Immunogenomic profile of colorectal cancer response to immune checkpoint blockade

Colorectal cancers (CRCs) show variable response to immune checkpoint blockade, which can only partially be explained by the variability of tumour mutational burden. To dissect the cellular and molecular determinants of response we performed a multi-omic screen of 721 cancer regions from patients treated with Pembrolizumab (KEYNOTE 177 clinical trial) or Nivolumab. Multi-regional whole exome, RNA and T-cell receptor sequencing show that, within hypermutated CRCs, response to both anti-PD1 agents is not positively associated with tumour mutational burden but with high clonality of immunogenic mutations, expanded T cells, low activation of the WNT pathway and active immune escape mechanisms. Coupling high-dimensional imaging mass cytometry with multiplexed immunofluorescence and computational spatial analysis, we observe that responsive hypermutated CRCs are rich in cytotoxic and proliferating PD1-expressing CD8 cells interacting with high-density clusters of PDL1-expressing antigen presenting macrophages. We propose that anti-PD1 agents release the PD1-PDL1 interaction between CD8 T cells and macrophages thus promoting cytotoxic anti-tumour activity.

cancer biology↗

bioRxiv2020

Human marginal zone B cell development from early T2 progenitors

B cells emerge from the bone marrow as transitional (TS) B cells that differentiate through T1, T2 and T3 stages to become naive B cells. We have identified a bifurcation of human B cell maturation from the T1 stage forming IgMhi and IgMlo developmental trajectories. IgMhi T2 cells have higher expression of 4{beta}7 integrin and lower expression of IL4 receptor (IL4R) compared to the IgMlo branch and are selectively recruited into gut-associated lymphoid tissue. IgMhi T2 cells also share transcriptomic features with marginal zone B cells (MZB). Lineage progression from T1 cells to MZB via an IgMhi trajectory is identified by pseudotime analysis of scRNA-sequencing data. Reduced frequency of IgMhi gut homing T2 cells is observed in severe SLE and is associated with reduction of MZB and their putative IgMhi precursors. The collapse of the gut-associated MZB maturational axis in severe SLE affirms its existence and importance for maintaining health.

cell biology↗

bioRxiv2020

Acetyl-Leucine slows disease progression in lysosomal storage disorders

Acetyl-DL-leucine (ADLL) is a derivative of the branched chain amino acid leucine. In observational clinical studies ADLL improved symptoms of ataxia, in particular in patients with the lysosomal storage disorder (LSD), Niemann-Pick disease type C 1 (NPC1). Here, we investigated ADLL and its enantiomers acetyl-L-leucine (ALL) and acetyl-D-leucine (ADL) in symptomatic Npc1-/- mice and observed an improvement in ataxia with both enantiomers and ADLL. When ADLL and ALL were administered pre-symptomatically to Npc1-/- mice, both treatments delayed disease progression and extended life span, whereas ADL did not. These data are consistent with ALL being the neuroprotective enantiomer. Altered glucose and antioxidant metabolism were found to be implicated as one of the potential mechanisms of action of the L enantiomer in Npc1-/- mice. When miglustat and ADLL were used in combination significant synergy resulted. In agreement with these pre-clinical data, when NPC1 patients were evaluated after 12 months of ADLL treatment, rates of disease progression were slowed, with stabilisation or improvement in multiple neurological domains. A beneficial effect of ADLL on gait was also observed in this study in a mouse model of GM2 gangliosidosis (Sandhoff disease) and in Tay-Sachs and Sandhoff disease patients in individual-cases of off-label-use. Taken together, we have identified an unanticipated neuroprotective effect of acetyl-L-leucine and underlying mechanisms of action in LSDs, supporting its further evaluation in clinical trials in lysosomal disorders.

neuroscience↗

bioRxiv2020

High sensitivity detection of SARS-CoV-2 using multiplex PCR and a multiplex-PCR-based metagenomic method

Many detection methods have been used or reported for the diagnosis and/or surveillance of COVID-19. Among them, reverse transcription polymerase chain reaction (RT-PCR) is the most commonly used because of its high sensitivity, typically claiming detection of about 5 copies of viruses. However, it has been reported that only 47-59% of the positive cases were identified by some RT-PCR methods, probably due to low viral load, timing of sampling, degradation of virus RNA in the sampling process, or possible mutations spanning the primer binding sites. Therefore, alternative and highly sensitive methods are imperative. With the goal of improving sensitivity and accommodating various application settings, we developed a multiplex-PCR-based method comprised of 343 pairs of specific primers, and demonstrated its efficiency to detect SARS-CoV-2 at low copy numbers. The assay produced clean characteristic target peaks of defined sizes, which allowed for direct identification of positives by electrophoresis. We further amplified the entire SARS-CoV-2 genome from 8 to half a million viral copies purified from 13 COVID-19 positive specimens, and detected mutations through next generation sequencing. Finally, we developed a multiplex-PCR-based metagenomic method in parallel, that required modest sequencing depth for uncovering SARS-CoV-2 mutational diversity and potentially novel or emerging isolates.

genomics↗

bioRxiv2017

Fast and Accurate Genomic Analyses using Genome Graphs

The human reference genome serves as the foundation for genomics by providing a scaffold for alignment of sequencing reads, but currently only reflects a single consensus haplotype, which impairs read alignment and downstream analysis accuracy. Reference genome structures incorporating known genetic variation have been shown to improve the accuracy of genomic analyses, but have so far remained computationally prohibitive for routine large-scale use. Here we present a graph genome implementation that enables read alignment across 2,800 diploid genomes encompassing 12.6 million SNPs and 4.0 million indels. Our Graph Genome Pipeline requires 6.5 hours to process a 30x coverage WGS sample on a system with 36 CPU cores compared with 11 hours required by the GATK Best Practices pipeline. Using complementary benchmarking experiments based on real and simulated data, we show that using a graph genome reference improves read mapping sensitivity and produces a 0.5% increase in variant calling recall, or about 20,000 additional variants being detected per sample, while variant calling specificity is unaffected. Structural variations (SVs) incorporated into a graph genome can be genotyped accurately under a unified framework. Finally, we show that iterative augmentation of graph genomes yields incremental gains in variant calling accuracy. Our implementation is a significant advance towards fulfilling the promise of graph genomes to radically enhance the scalability and accuracy of genomic analyses.

bioinformatics↗