Biology subjects

Collins, E.

Publications and source records attributed to Collins, E..

10 recordsLinked to original sources

bioRxiv2026

Interrogating the Mechanisms of Cas9-mediated Allele Conversion

Allele conversion describes a process where a heterozygous variant is made homozygous. Recently, it has been shown that allele conversion can be triggered by DNA damage at the heterozygous site. This process has the potential to repair pathogenic heterozygous mutations; however, the efficiency is low. Here, we endeavoured to understand the mechanism underlying allele conversion, ultimately to raise allele conversion efficiency to functionally relevant levels. To test this, we developed a Compound Heterozygous Allele Conversion Reporter (CHACR) cell line. This line comprises knocked-in fluorescent protein encoding genes, with heterozygous inactivating mutations resulting in different fluorescence profiles from each allele. These mutations create protospacer adjacent motifs (PAM) for Cas9 recognition, where allele-specific gRNAs (AS-gRNAs) target the heterozygous mutations. We showed that applying these AS-gRNAs with either Cas9 nuclease or Cas9(D10A) nickase can recover mCherry fluorescence. Sorting and sequencing these fluorescent cells revealed wild-type sequences, suggesting allele conversion repaired the mutation using the homologous allele as a template. Allele conversion can also be triggered using an adenine base editor with an AS-gRNA, and this allele conversion mechanism can be manipulated by inhibiting DNA-PKcs or overexpressing RAD51. This work introduces a model for measuring allele conversion, and modifiers of this mechanism.

molecular biology↗

bioRxiv2025

The First Chromosome-Scale Genome Assembly for Dermacentor reticulatus: A Key Vector of Tick-Borne Pathogens of Public and Veterinary Health Importance in Europe

BackgroundDermacentor reticulatus is a key tick species across Europe and an established vector of multiple pathogens that affect both human and animal health. Despite its significant role in disease transmission, genomic data for this species remain limited. Here, we present the first chromosome-scale genome assembly of D. reticulatus, constructed using Oxford Nanopore long-read sequencing data. MethodsHigh molecular weight DNA was extracted from a female Dermacentor reticulatus collected in Devon, UK, and sequenced using Oxford Nanopore long-read technology. Genome assemblies were generated using both guided and unguided approaches, followed by repeat masking, gene prediction, and functional annotation. Genome completeness was assessed using BUSCO, and comparative, phylogenetic, and functional analyses were performed against other Dermacentor and tick species. ResultsThis chromosome-scale genome assembly revealed a repeat-rich genome, with approximately 61.9% of the total sequence consisting of repetitive elements. Benchmarking universal single-copy ortholog (BUSCO) analysis demonstrated strong genome completeness, with guided assembly (chromosome) achieving a score of 97.1%, closely matching those of related Dermacentor species. For comparison, unguided assembly (scaffold) yielded a BUSCO score of 96.7%. Gene annotation following repeat masking resulted in BUSCO completeness scores of 95.1% (guided) and 95.0% (unguided). Functional characterisation included Pfam domain assignment and Gene Ontology analysis. Additionally, we assembled the mitochondrial genome (15,103 bp), comprising 38 genes, providing further insight into D. reticulatus phylogenetic placement. ConclusionsThis genomic resource establishes a foundation for functional genomics, evolutionary studies and supports future research in vector biology and the control of tick-borne diseases.

genomics↗

bioRxiv2025

CD11c+ Tbet+ B cells constrain obesity- and vaccination-induced germinal center B cells and T helper cells

Obesity is a rapidly growing public health crisis associated simultaneously with increased metabolic disease and humoral immune suppression to vaccination or infection. Inflammatory CD11c+T-bet+ B cells increase in spleen and adipose tissue during obesity and exacerbate metabolic dysfunction via antibodies. We now find that during obesity Tbet+ B cells also expand in the liver but not omentum or mesenteric fat. Obese mice also develop increased splenic CXCR5+ TFH and hepatic CXCR5-TPH cells which serve as likely partners for antigen-experienced MHC-II+ CD11c+ Tbet+ B cells. We also observed that antibodies in obese mice, previously found to contribute to metabolic disease, largely circulate as inflammatory autoantigen-bound immune complexes. Obese mice lacking T-bet in B cells also develop increased autoantibody titers and expanded splenic germinal center (GC) B and T helper cells. T-bet+ B cell-deficient mice make a similarly enhanced GC, TFH, TPH response to haptenated-protein vaccination with a corresponding increase in antibody affinity, although there is no additive effect of obesity. These results are consistent with GC inhibition by expanded CD11c+ B cells demonstrated by others to occur during autoimmunity, suggesting a broadly universal mechanism which may explain reduced humoral immunity and poor clinical outcomes following infection in patients with obesity and other forms of chronic inflammation.

immunology↗

bioRxiv2025

DRP1/DMNL-1-mediated mitochondrial fission augments Rickettsia parkeri replication in macrophages

Pathogenic Spotted Fever Group (SFG) Rickettsia species, including Rickettsia parkeri replicate in endothelial cells, monocytes, and macrophages in vitro and during infections in murine models of disease. We demonstrated that R. parkeri survives and proliferates within phagocytes and avoids intracellular killing within lysosomal compartments. We found that infection of human macrophage-like cells with a related SFG Rickettsia, R. conorii, resulted in a significant increase in mitochondria-associated proteins, suggesting that mitochondrial functions are involved in Rickettsia pathogenesis. Several intracellular bacterial pathogens manipulate host cell mitochondrial networks and stimulate mitochondrial fission mediated by a GTP-binding regulatory protein, DRP1/DMNL1, to promote intracellular replication. Here, we investigated the contribution of DRP1 in the growth of R. parkeri in macrophages. Murine immortalized bone marrow derived macrophages (iBMDMs) and primary human monocyte derived macrophages were infected with R. parkeri and mitochondrial dynamics (fission and network) were assessed by immunofluorescence microscopy. R. parkeri proliferated in macrophages, which coincided with a significant increase in mitochondria content and fission compared to uninfected cells. R. parkeri infection led to increases in host cell ATP production primarily due to mitochondrial respiration and bacteria were often found co-localized with mitochondrial fragments. Importantly, R. parkeri growth was significantly impacted in DRP1 deficient macrophages. These results suggest that the modulation of mitochondria content and dynamics are essential for replication and survival of pathogenic SFG Rickettsia species in macrophages and suggest that the metabolic requirements for obligate intracellular pathogens may differ from other pathogenic Gram-negative intracellular bacteria.

microbiology↗

bioRxiv2024

Long-term hybridization in a karst window reveals the genetic basis of eye loss in cavefish

Eye loss is a hallmark trait of animals inhabiting perpetual darkness, yet the precise genetic variants underlying this evolutionary change remain largely unknown. The Mexican tetra (Astyanax mexicanus) provides a powerful model for dissecting the genetic basis of eye degeneration, as sighted surface fish and multiple independently evolved blind cave populations remain interfertile; yet despite decades of research and numerous QTL studies, the genetic basis of eye loss has remained unresolved at the level of specific variants. Here, we exploit a rare natural experiment in the Caballo Moro cave, where the collapse of a karst window created a partially illuminated pool inhabited by both fully eyed and completely eyeless cavefish of closely related genetic background. Whole-genome sequencing reveals a long-standing hybrid population between cave and surface lineages, enabling a dramatic refinement of the genetic architecture of eye degeneration to 203 candidate SNPs across 41 genes. Among these, we identified a nonsynonymous mutation in the lens gap-junction protein Connexin-50 (Cx50). CRISPR-based disruption of cx50 induces early eye loss in surface fish, and F2 laboratory crosses confirm genetic linkage between cx50 variants and eye size. Additional Cx50 mutations are present in independent cavefish populations and correlate with reduced eye size. Notably, variants in conserved regions of Cx50 also occur in other cave-dwelling fish and subterranean mammals, suggesting repeated evolutionary targeting of this gene. Introduction of the Caballo Moro mutation into mice causes cataracts and reduced eye and lens size, confirming its functional impact. Together, these findings identify the first SNP directly implicated in cavefish eye loss, demonstrate the power of natural hybrid populations to resolve the genetic basis of complex traits, and reveal Cx50 as a case of molecular convergence in vertebrate eye degeneration.

evolutionary biology↗

bioRxiv2024

Megabouts: a flexible pipeline for zebrafish locomotion analysis

Accurate quantification of animal behavior is crucial for advancing neuroscience and for defining reliable physiological markers. We introduce Megabouts (megabouts.ai), a software package standardizing zebrafish larvae locomotion analysis across experimental setups. Its flexibility, achieved with a Transformer neural network, allows the classification of actions regardless of tracking methods or frame rates. We demonstrate Megabouts ability to quantify sensorimotor transformations and enhance sensitivity to drug-induced phenotypes through high-throughput, high-resolution behavioral analysis.

animal behavior and cognition↗

bioRxiv2024

Uncovering the genetic diversity in Aedes aegypti insecticide resistance genes through global comparative genomics

Insecticides are essential to control the transmission of vector-borne diseases to humans and animals, but their efficacy is being threatened by the spread of resistance across multiple medically important mosquito species. An example of this is Aedes aegypti - a major vector of arboviruses, including Zika, dengue, yellow fever, West Nile, and Chikungunya, with widespread insecticide resistance reported in the Americas and Asia, while data from Africa is more limited. Here we investigate the global genetic diversity in four insecticide resistance associated genes: ace-1, GSTe2, rdl and vgsc. Apart from vgsc, the other genes have been less investigated in Ae. aegypti, and limited genetic diversity information is available. We explore a large whole-genome sequencing dataset of 729 Ae. aegypti across 15 countries including nine in Africa. Among the four genes, we identified 1,829 genetic variants including 474 non-synonymous substitutions, as well as putative copy number variations in GSTe2 and vgsc. Among these are many previously documented insecticide resistance mutations which were present at different frequencies and combinations depending on origin of samples. Global insecticide resistance phenotypic data demonstrated variable resistance in geographic areas with resistant genotypes. These warrant further investigation to assess their functional contribution to insecticide resistant phenotypes and their potential development into genetic panels for operational surveillance. Overall, our work provides the first global catalogue and geographic distribution of known and new amino-acid mutations and duplications that can be used to guide the identification of resistance drivers in Ae. aegypti and thereby support monitoring efforts and strategies for vector control.

genomics↗

bioRxiv2022

Towards environmental detection, quantification, and molecular characterization of Anopheles stephensi and Aedes aegypti from larval breeding sites

The invasion and establishment of An. stephensi mosquitoes in the Horn of Africa represents a significant regional threat, which may jeopardise malaria control, particularly in urban areas which were formally free from disease transmission. Novel vector surveillance methods are urgently needed, both agnostic to mosquito larval morphology, and simple to implement at the sampling stage. Using new multiplex TaqMan assays, specifically targeting An. stephensi and Ae. aegypti, we validated the use of environmental DNA (eDNA) for simultaneous vector detection in shared artificial breeding sites. Study findings demonstrated that An. stephensi and Ae. aegypti eDNA deposited by as few as one second instar larva in 1L of water was detectable. Characterization of molecular insecticide resistance mechanisms, using novel amplicon-sequencing panels for both vector species, was possible from eDNA shed by as few as 32 second instar larvae in 50ml of water. An. stephensi eDNA, derived from emergent pupae for 24 hours, was remarkably stable, and still detectable ~2 weeks later. eDNA surveillance has the potential to be implemented in local endemic communities and points of country entry, to monitor the spread of invasive vector species. Further studies are required to validate the feasibility of this technique under field conditions.

molecular biology↗

bioRxiv2020

FastSK: Fast Sequence Analysis with Gapped String Kernels

Gapped k-mer kernels with Support Vector Machines (gkm-SVMs) have achieved strong predictive performance on regulatory DNA sequences on modestly-sized training sets. However, existing gkm-SVM algorithms suffer from slow kernel computation time, as they depend exponentially on the sub-sequence feature-length, number of mismatch positions, and the tasks alphabet size. In this work, we introduce a fast and scalable algorithm for calculating gapped k-mer string kernels. Our method, named FastSK, uses a simplified kernel formulation that decomposes the kernel calculation into a set of independent counting operations over the possible mismatch positions. This simplified decomposition allows us to devise a fast Monte Carlo approximation that rapidly converges. FastSK can scale to much greater feature lengths, allows us to consider more mismatches, and is performant on a variety of sequence analysis tasks. On 10 DNA transcription factor binding site (TFBS) prediction datasets, FastSK consistently matches or outperforms the state-of-the-art gkmSVM-2.0 algorithms in AUC, while achieving average speedups in kernel computation of [~]100x and speedups of [~]800x for large feature lengths. We further show that FastSK outperforms character-level recurrent and convolutional neural networks across all 10 TFBS tasks. We then extend FastSK to 7 English-language medical named entity recognition datasets and 10 protein remote homology detection datasets. FastSK consistently matches or outperforms these baselines. Our algorithm is available as a Python package and as C++ source code1.

bioinformatics↗

bioRxiv2018

Investigating the relationship between insecticide resistance, underlying molecular mechanisms and malaria prevalence in Anopheles gambiae s.l. from Guinea

The threat of insecticide resistance across sub-Saharan Africa is anticipated to severely impact the continued effectiveness of malaria vector control. We investigated the effect of carbamate and pyrethroid resistance on Anopheles gambiae s.l age, Plasmodium falciparum infection and characterized molecular resistance mechanisms in Guinea. Pyrethroid resistance was intense, with survivors of ten times the insecticidal concentration required to kill susceptible individuals. The L1014F kdr allele was significantly associated with mosquito survival following deltamethrin or permethrin treatment (p=0.003 and p=0.04, respectively). N1575Y and I1527T mutations were identified in 13% and 10% of individuals, respectively, but neither conferred increased pyrethroid tolerance. Partial restoration of pyrethroid susceptibility following synergist pre-exposure suggest a role for mixed-function oxidases. Carbamate resistance was lower and significantly associated with the G119S Ace-1 mutation (p=0.001). Oocyst rates were 6.8% and 4.2% among resistant and susceptible mosquitoes, respectively; survivors of bendiocarb exposure were significantly more likely to be infected (p=0.03). Resistant mosquitoes had significantly lower parity rates; however, a subset of intensely pyrethroid-resistant vectors were more likely to be parous (p=0.042 and p=0.045, for survivors of five and ten times the diagnostic dose of insecticides, respectively). Our findings emphasize the need for additional studies directly assessing the influence of insecticide resistance on mosquito fitness.

genetics↗