Biology subjects

Richardson, D.

Publications and source records attributed to Richardson, D..

13 recordsLinked to original sources

bioRxiv2026

TARGETING THE CAPSULE OF KLEBSIELLA PNEUMONIAE WITH A CATIONIC CR3-BINDING PROTEIN ENHANCES PHAGOCYTOSIS AND PROMOTES BACTERIAL CLEARANCE AND SURVIVAL IN A MOUSE SEPSIS MODEL

Platelet Factor 4 (PF4), a cationic antimicrobial peptide, serves as a ligand for the myeloid-specific phagocytic receptor CR3 (Mac-1, CD11b/CD18). We previously demonstrated that recombinant dimeric PF4 (rdPF4) functions as a bacterial opsonin, enhancing phagocytosis of Gram-positive Staphylococcus aureus and facilitating clearance of both antibiotic-susceptible and methicillin-resistant S. aureus in a mouse model of infectious peritonitis. In this study, we examined whether rdPF4 is pathogen-agnostic by assessing its effect on phagocytosis of Gram-negative encapsulated Klebsiella pneumoniae, a WHO Bacterial Priority Pathogen. We demonstrate that rdPF4 enhances CR3-mediated phagocytosis of both live and heat-inactivated high-virulence K2 and low-virulence K3 strains of K. pneumoniae by various mouse and human macrophage cell lines, as well as primary neutrophils and macrophages. It also increased phagocytosis of carbapenem-resistant K. pneumoniae. rdPF4 did not directly kill bacteria but acted as an opsonin binding to the negatively charged bacterial capsule and creating recognition sites for CR3 on leukocytes. In a mouse sepsis model, a single dose of rdPF4 significantly enhanced bacterial clearance from the lungs, liver, and peritoneum and reduced bacteremia. Histological analyses showed that rdPF4 provided substantial protection to lung and liver tissues against K. pneumoniae-induced damage. Consistent with these findings, rdPF4 treatment increased the survival rates of infected mice. These results show that rdPF4 effectively targets the capsule, a key virulence factor of K. pneumoniae, thereby reducing the bacteriums ability to evade the host immune response. Overall, the data suggest a common mechanism in which cationic rdPF4, by binding to the negatively charged surfaces of both Gram-negative and Gram-positive bacteria, diminishes their antiphagocytic properties.

microbiology↗

bioRxiv2026

Inbreeding depression by polygenic load following a severe population bottleneck

It is poorly understood how populations survive extreme bottlenecks despite severe inbreeding. We investigate the genomic architecture of inbreeding in the once critically endangered Seychelles warbler (Acrocephalus sechellensis) using 37 years of individual-based monitoring and 1.8 million SNPs. Linkage disequilibrium patterns reveal a historical effective population size of [~]270 that plummeted to [~]13 coinciding with human colonisation events of the Seychelles archipelago. Contemporary genomes are over one-third inbred from recent inbreeding measured by runs of homozygosity (ROH) formed since human colonisation of the Seychelles, 50 generations ago (mean FROH < 50 generations ago = 0.38), and we identify significant inbreeding depression across cross key fitness traits. On average, a 17.9% reduction in lifespan and a 15.1% reduction in lifetime offspring production were associated with each 10% increase in individual inbreeding. Genome-wide scans reveal this depression is caused by a polygenic load. Our results suggest that a history of small population size could have facilitated the selective purging of severe genetic load, while mildly deleterious alleles escaped selection via drift. A partitioned genetic load architecture potentially enabled species recovery despite the inbreeding depression that followed near-extinction.

evolutionary biology↗

bioRxiv2026

A Seychelles warbler genomic toolkit

Understanding evolutionary processes is greatly facilitated by high-quality data on genetic variation. We report the development of a genomic toolkit for a recently bottlenecked, long-term studied species, the Seychelles warbler (Ptimerl dezil; Acrocephalus sechellensis). This toolkit comprises a reference genome assembled into 31 chromosomes, together with functional annotations and reference-panel-free imputation of whole-genome sequences from 1,935 individuals. The genomic data have been used to assign the sequenced individuals into a genetic pedigree. Individual genomic data are associated with a suite of phenotypic metadata, amassed from three decades of fieldwork in this closed, long-term monitored population. We compared sex and parentage assigned using the genomic data with the previously recorded sex and parentage metadata to identify and correct 41 sample DNA samples labelled with the wrong identity. This population resource enables a wide range of analyses, that include, but are not limited to phylogenetics, metabarcoding, recombination rates, linkage patterns, adaptation, heritability, demographic history, selection, and inbreeding estimates. We wish to encourage interest from researchers seeking to collaborate on future analyses and data collection. Overall, our methods demonstrate the potential of next generation sequencing and statistical tools to provide dense genomic datasets at large sample sizes for wild populations.

genomics↗

bioRxiv2025

Transplantation of GABAergic Interneuron Progenitors Restores Cortical Circuit Function in an Alzheimer's Disease Mouse Model

In addition to dementia, Alzheimers patients suffer from sleep impairments and aberrations in sleep-dependent brain rhythms. Deficits in inhibitory GABAergic interneuron function disrupt one of those rhythms, slow oscillation in particular, and actively contribute to Alzheimers progression. We tested the degree to which transplantation of healthy donor interneuron progenitors would restore slow oscillation rhythm in young APP mice. We harvested medial ganglionic eminence (MGE) progenitors from mouse embryos and transplanted them into host APP mutant cortices. 3D light-sheet and structured illumination microscopy revealed that transplanted MGE progenitors survived and matured into healthy interneurons. In vivo multiphoton calcium imaging and voltage-sensitive dye imaging showed functional integration and slow oscillation rescue in absence or presence of optogenetic stimulation. Our work provides proof-of-concept evidence that stem cell therapy may serve as a viable strategy to rescue functional impairments in cortical circuits of APP mice and potentially those of Alzheimers patients.

neuroscience↗

bioRxiv2025

Genome-wide cline analysis identifies new locus contributing to a barrier to gene flow across an Antirrhinum hybrid zone

Identification of the genomic regions that contribute to reproductive isolation and how they interact is a major goal of evolutionary genetics. Much effort has focused on locating candidate genes and potential barrier loci by scanning genomes for regions of excess differentiation (FST). An alternative, and perhaps more robust approach, would be to scan for genomic regions exhibiting steep clines in allele frequency across a hybrid zone. Here we develop this approach and apply it to genomic data from across a hybrid zone between varieties of Antirrhinum majus subspecies majus with different flower colour patterns (A. m. m var. pseudomajus and A. m. m var. striatum). Most steep clines are clustered in seven genomic regions, only four of which were present from FST scans between all pair-wise comparisons. Six of these regions carry previously identified loci that influence flower colour in the hybrid zone. The seventh region harbours a novel locus, RUBIA, modifying magenta intensity. Clines at RUBIA approached towards fixation on magenta side of the hybrid zone, whilst remaining polymorphic on the yellow side. This polymorphism on the yellow side may reflect a smaller phenotypic effect of RUBIA in yellow compared to magenta genetic backgrounds. Our findings illustrate how whole-genome cline scans in hybrid zones can robustly detect genomic regions contributing to phenotypic differences and highlight how different reproductive barrier loci interact across the genome.

evolutionary biology↗

bioRxiv2023

PLATELET FACTOR 4 (PF4) IMPROVES SURVIVAL IN A MURINE MODEL OF ANTIBIOTIC-SUSCEPTIBLE AND METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS PERITONITIS

The complement receptor CR3, also known as integrin Mac-1 (CD11b/CD18), is one of the major phagocytic receptors on the surface of neutrophils and macrophages. We previously demonstrated that in its protein ligands, Mac-1 binds sequences enriched in basic and hydrophobic residues and strongly disfavors negatively charged sequences. The avoidance by Mac-1 of negatively charged surfaces suggests that the bacterial wall and bacterial capsule possessing net negative electrostatic charge may repel Mac-1 and that the cationic Mac-1 ligands can overcome this evasion by acting as opsonins. Indeed, we previously showed that opsonization of Gram-negative Escherichia coli with several cationic peptides, including PF4 (Platelet Factor 4), strongly augmented phagocytosis by macrophages. Here, we investigated the effect of recombinant PF4 (rPF4) on phagocytosis of Gram-positive Staphylococcus aureus in vitro and examined its impact in a mouse model of S. aureus peritonitis. Characterization of the interaction of rPF4 with nonencapsulated and encapsulated S. aureus showed that rPF4 localizes on the bacterial surface, thus making it available for Mac-1. Furthermore, rPF4 did not have direct bactericidal and bacteriostatic activity and was not toxic to host cells. rPF4 enhanced phagocytosis of S. aureus bioparticles by various primary and cultured Mac-1-expressing leukocytes by several folds. It also increased phagocytosis of live nonencapsulated and encapsulated bacteria. Notably, the augmentation of phagocytosis by rPF4 did not compromise the intracellular killing of S. aureus by macrophages. Using a murine S. aureus peritonitis model, we showed that treatment of infected mice with rPF4 caused a significant increase in the clearance of antibiotic-susceptible S. aureus and its methicillin-resistant (MRSA) variant and markedly improved survival. These findings indicate that rPF4 binding to the bacterial surface circumvents its antiphagocytic properties, improving host defense against antibiotic-susceptible and antibiotic-resistant bacteria.

cell biology↗

bioRxiv2022

Improvements on Sample Preparation and Peptide Separation for Reduced Peptide Mapping Based Multi-Attribute Method analysis of Therapeutic Monoclonal Antibodies Using Lys-C Digestion

The mass spectrometry based multi-attribute method (MAM) has gained popularity in the field of biopharmaceutical analysis as it promises a single method for comprehensive monitoring of multiple product quality attributes (PQAs) and product purity. Sample preparation for protein digestion and peptide separation are critical considerations for a reduced peptide mapping-based MAM. To avoid desalting steps required in tryptic protein digestion and in order to improve peptide separation for hydrophilic peptides, we developed an improved robust sample preparation using Lys-C protease for high-throughput MAM testing. Additionally, this method optimizes the peptide retention and separation of a stability-indicating VSNK peptide using a HSS T3 column for comprehensive PQA monitoring. A fully automated sample preparation had similar assay variations for PQAs monitoring compared to manual sample preparation. To the best of our knowledge, this is the first report of a high-resolution MS-based MAM using Lys-C digestion with enhanced PQA monitoring for hydrophilic peptides. The improved, robust MAM workflow for protein digestion and peptide separation will pave the way for broader MAM qualification and its applications for the characterization and quality control of therapeutic monoclonal antibodies.

biochemistry↗

bioRxiv2021

The plant-specific SCL30a SR protein regulates ABA-dependent seed traits and salt stress tolerance during germination

SR (serine/arginine-rich) proteins are conserved RNA-binding proteins best known as key regulators of splicing, which have also been implicated in other steps of gene expression. Despite mounting evidence for their role in plant development and stress responses, the molecular pathways underlying SR protein regulation of these processes remain elusive. Here we show that the plant-specific SCL30a SR protein negatively regulates abscisic acid (ABA) signaling to control important seed traits and salt stress responses during germination in Arabidopsis. The SCL30a gene is upregulated during seed imbibition and germination, and its loss of function results in smaller seeds displaying enhanced dormancy and elevated expression of ABA-responsive genes as well as of genes repressed during the germination process. Moreover, the knockout mutant is hypersensitive to ABA and high salinity, while transgenic plants overexpressing SCL30a exhibit reduced ABA sensitivity and enhanced tolerance to salt stress during seed germination. An ABA biosynthesis inhibitor rescues the mutants enhanced sensitivity to stress, and epistatic analyses confirm that this hypersensitivity requires a functional ABA pathway. Finally, seed ABA levels are unchanged by altered SCL30a expression, indicating that the SR protein positively regulates stress tolerance during seed germination by reducing sensitivity to the phytohormone. Our results reveal a new key player in ABA-mediated control of early development and stress response, and underscore the role of plant SR proteins as important regulators of the ABA signaling pathway. Author SummarySeed germination is a critical step in plant development determining the transition to aerial growth and exposure to a more challenging environment. As such, seeds have evolved mechanisms that prevent germination under adverse conditions, thereby increasing the chances of plant survival. As a general regulator of plant development and a key mediator of stress responses, the hormone abscisic acid (ABA) promotes a prolonged non-germinating state called dormancy, influences seed size and represses germination under environmental stress. Here, we show that an RNA-binding protein, SCL30a, controls seed size, dormancy, germination and tolerance to high salinity in the model plant Arabidopsis thaliana. Loss of SCL30a gene function results in smaller and more dormant seeds with reduced ability to germinate in a high-salt environment; by contrast, SCL30a overexpression produces larger seeds that germinate faster under salt stress. Using a large-scale gene expression analysis, we identify the ABA hormonal pathway as a putative target of SCL30a. We then use genetic and pharmacological tools to unequivocally demonstrate that the uncovered biological functions of SCL30a are achieved through modulation of the ABA pathway. Our study reveals a novel regulator of key seed traits and has biotechnological implications for crop improvement under adverse environments.

plant biology↗

bioRxiv2020

Profiling Active Enzymes for Polysorbate Degradation in Biotherapeutics by Activity-Based Protein Profiling

Polysorbate is widely used to maintain stability of biotherapeutic proteins in formulation development. Degradation of polysorbate can lead to particle formation in drug products, which is a major quality concern and potential patient risk factor. Enzymatic activity from residual enzymes such as lipases and esterases can cause polysorbate degradation. Their high activity, even at low concentration, constitutes a major analytical challenge. In this study, we evaluated and optimized the activity-based protein profiling (ABPP) approach to identify active enzymes responsible for polysorbate degradation. Using chemical probes to enrich active serine hydrolases, more than 80 proteins were identified in harvested cell culture fluid (HCCF) from monoclonal antibodies (mAb) production. A total of 8 known lipases were identified by ABPP, while only 5 lipases were identified by a traditional abundance-based proteomics (TABP) approach. Interestingly, phospholipase B-like 2 (PLBL2), a well-known problematic HCP was not found to be active in process-intermediates from two different mAbs. In a proof-of-concept study, phospholipase A2 group VII (PLA2G7) and sialic acid acetylesterase (SIAE) were identified by ABPP as possible root causes of polysorbate-80 degradation. The established ABBP approach can fill the gap between lipase abundance and activity, which enables more meaningful polysorbate degradation investigations for biotherapeutic development.

bioengineering↗

bioRxiv2019

Mouse retinal cell behaviour in space and time using light sheet fluorescence microscopy

As the general population ages and the incidence of diabetes increases epidemically, more people are affected by eye diseases, such as retinopathies. It is therefore critical to improve imaging of eye disease mouse models. Here, we demonstrate that 1) rapid, quantitative 3D and 4D (time lapse) imaging of cellular and subcellular processes in the murine eye is feasible, with and without tissue clearing, using light-sheet fluorescent microscopy (LSFM) and 2) LSFM readily reveals new features of even well studied eye disease mouse models, such as the Oxygen-Induced Retinopathy (OIR) model. Through correlative LSFM-Confocal imaging we find that flat-mounting retinas for confocal microscopy significantly distorts tissue morphology. The minimized distortion with LSFM dramatically improved analysis of pathological vascular tufts in the OIR model revealing \"knotted\" morphologies, leading to a proposed new tuft nomenclature. Furthermore, live-imaging of OIR tuft formation revealed abnormal cell motility and altered filopodia dynamics. We conclude that quantitative 3D/4D LSFM imaging and analysis has the potential to advance our understanding of pathological processes in the eye, in particular neuro-vascular degenerative processes.

developmental biology↗

bioRxiv2019

Variation in PU.1 binding and chromatin looping at neutrophil enhancers influences autoimmune disease susceptibility

Neutrophils play fundamental roles in innate inflammatory response, shape adaptive immunity1, and have been identified as a potentially causal cell type underpinning genetic associations with immune system traits and diseases2,3 The majority of these variants are non-coding and the underlying mechanisms are not fully understood. Here, we profiled the binding of one of the principal myeloid transcriptional regulators, PU.1, in primary neutrophils across nearly a hundred volunteers, and elucidate the coordinated genetic effects of PU.1 binding variation, local chromatin state, promoter-enhancer interactions and gene expression. We show that PU.1 binding and the associated chain of molecular changes underlie genetically-driven differences in cell count and autoimmune disease susceptibility. Our results advance interpretation for genetic loci associated with neutrophil biology and immune disease.

genetics↗

bioRxiv2017

Comparative analysis of neutrophil and monocyte epigenomes

Neutrophils and monocytes provide a first line of defense against infections as part of the innate immune system. Here we report the integrated analysis of transcriptomic and epigenetic landscapes for circulating monocytes and neutrophils with the aim to enable downstream interpretation and functional validation of key regulatory elements in health and disease. We collected RNA-seq data, ChIP-seq of six histone modifications and of DNA methylation by bisulfite sequencing at base pair resolution from up to 6 individuals per cell type. Chromatin segmentation analyses suggested that monocytes have a higher number of cell-specific enhancer regions (4-fold) compared to neutrophils. This highly plastic epigenome is likely indicative of the greater differentiation potential of monocytes into macrophages, dendritic cells and osteoclasts. In contrast, most of the neutrophil-specific features tend to be characterized by repressed chromatin, reflective of their status as terminally differentiated cells. Enhancers were the regions where most of differences in DNA methylation between cells were observed, with monocyte-specific enhancers being generally hypomethylated. Monocytes show a substantially higher gene expression levels than neutrophils, in line with epigenomic analysis revealing that gene more active elements in monocytes. Our analyses suggest that the overexpression of c-Myc in monocytes and its binding to monocyte-specific enhancers could be an important contributor to these differences. Altogether, our study provides a comprehensive epigenetic chart of chromatin states in primary human neutrophils and monocytes, thus providing a valuable resource for studying the regulation of the human innate immune system.

genomics↗

bioRxiv2017

Spin coated chitin films for biosensors and its analysis are depended on chitin-surface interactions

Chitin, abundant in nature, is a renewable resource with many possible applications in bioengineering. Biosensors, capable of label-free and in-line evaluation, play an important role in the investigation of chitin synthesis, degradation and interaction with other materials. This work presents a comparative study of the usefulness of a chitin surface preparation, either on gold (Au) or on polystyrene (PS). In both cases the most common method to dissolve chitin was used, followed by a simple spin-coating procedure. Multi-parametric surface plasmon resonance (MP-SPR), modeling of the optical properties of the chitin layers, scanning electron microscopy, and contact angle goniometry were used to confirm: the thickness of the layers in air and buffer, the refractive indices of the chitin layers in air and buffer, the hydrophobicity, the binding properties of the chitin binding domain (CBD) of Bacillus circulans, and the split-intein capture process. Binding of the CBD differed between chitin on Au versus chitin on PS in terms of binding strength and binding specificity due to a less homogenous structured chitin-surface on Au in comparison to chitin on PS, despite a similar thickness of both chitin layers in air and after running buffer over the surfaces. The use of the simple method to reproduce chitin films on a thin polystyrene layer to study chitin as a biosensor and for chitin binding studies was obvious from the SPR studies and the binding studies of CBD as moiety of chitinases or as protein fusion partner. In conclusion, stable chitin layers for SPR studies can be made from chitin in a solution of dimethylacetamide (DMA) and lithium chloride (LiCl) followed by spin-coating if the gold surface is protected with PS.

biophysics↗