Biology subjects

Watkins, C.

Publications and source records attributed to Watkins, C..

16 recordsLinked to original sources

bioRxiv2026

Aβ Aggregates Bind the U1 Spliceosomal Ribonucleoprotein in Alzheimer Disease Brain

Missplicing due to U1 small nuclear ribonucleoprotein (U1 snRNP) insolubilization and dysfunction has been identified in Alzheimer disease (AD) brain. Cytoplasmic aggregation and mislocalization of the U1 snRNP partly co-localizes with tau neurofibrillary tangles, and some evidence for tau-U1 binding has been identified. However, tau-U1 co-localization by immunohistochemistry is only partial, and insoluble U1 small nuclear RNA (snRNA) binding proteins correlate better with A{beta} than with tau in unbiased proteomics. While investigating the sedimentation characteristics of A{beta} aggregates capable of diffusing out of AD brain tissue, we unexpectedly found that some were bound to small RNA. A{beta} immunoprecipitation and deep sequencing revealed the U1 snRNA, with specific binding confirmed through reverse immunoprecipitation and oligonucleotide hybridization. Double immunoelectron microscopy revealed decoration of A{beta} fibrils, more than tau fibrils, with the U1-70k protein, a component of the U1 snRNP. Immunofluorescence of unfixed cryostat sections but not formalin fixed paraffin embedded sections of AD brain revealed labeling of a subset of amyloid plaques with anti-U1-70k antibodies, confirmed by RNA in situ hybridization of the U1 snRNA. We conclude that a subset of AD brain A{beta} aggregates are bound to the U1 snRNP at the edges of some amyloid plaques, explaining prior proteomics findings. These data provide a potential link between A{beta} aggregation and spliceosome dysfunction and unite A{beta} with other fibril-forming proteins across the neurodegenerative diseases whose aggregation is affected by RNA binding.

neuroscience↗

bioRxiv2026

Ancestry-specific rewiring of BCR-MAPK signaling in sarcoidosis B cells

Sarcoidosis is a heterogenous disease of unknown etiology characterized by non-caseating granulomas. Disease prevalence and presentation vary significantly by ancestry and ranges from acute, self-resolving disease to severe, chronic disease. Following previous reports suggesting B cells in the development and pathogenesis of sarcoidosis, we present here results of single-cell RNA sequencing, supporting B cell involvement in sarcoidosis through altered immediate early response, rewiring of MAPK signaling, and ancestry-specific preferential expansion of B cell receptors. Peripheral blood mononuclear cells were obtained from individuals of African or European Ancestry (AA and EA, respectively) including 48 healthy controls, 59 sarcoidosis patients, and 28 systemic lupus erythematosus (SLE) patients. SLE samples were used as a disease control. Differential expression analysis highlighted many differentially expressed genes (DEGs) with almost 5x more in the AA sarcoidosis versus AA control group compared to the EA sarcoidosis versus EA control group. B cells had the most DEGs of all cell types and expression patterns were similar between ancestries, however, sarcoidosis had an opposite transcription pattern than SLE, demonstrating an alternative immune response to acute activation than that seen in a prototypical autoinflammatory disease. This trend was maintained when examining specialized B cell subsets, with the most pronounced effect in the AA sarcoidosis versus AA control comparison. Our results strongly support further investigation of the role of humoral immune response in sarcoidosis and the potential to highlight patient groups likely to benefit from existing B cell therapies.

immunology↗

bioRxiv2025

Comparative Landscape of Small RNAs in Tissue and Liquid Biopsies for Liver Transplant Outcomes

BackgroundIschemia-reperfusion injury (IRI) is an inevitable consequence of liver transplantation, arising during donor organ procurement and reoxygenation. Severe IRI is a leading contributor to early allograft dysfunction (EAD), a post-transplant complication associated with reduced graft survival. Current postoperative biomarkers provide limited time for intervention, highlighting a need to identify preoperative biomarkers of IRI. Meanwhile, tRNA fragments (tRFs) have emerged as novel biomarkers in various diseases but remain unexplored in the context of liver transplant. ResultsWe performed small RNA sequencing on 96 paired donor liver biopsies from 48 patients to investigate IRI-associated transcript changes. In parallel, 161 donor liver perfusates were analyzed as a non-invasive surrogate for tissue. Across samples, microRNAs (miRNAs) and tRFs were the most abundant. Perfusate expression strongly correlated with biopsies, supporting their value as a non-invasive source of small RNAs. Comparison between post-reperfusion and pre-implantation biopsies revealed that IRI reprogrammed tRF expression. Stratification by clinical outcome showed that patients who developed EAD exhibited specific small RNA signatures in both biopsy and perfusate. Receiver operating characteristic (ROC) analysis revealed a miRNA-based model that achieved an AUC of 0.772, outperforming donor risk index alone (AUC = 0.665), representing a 10.7% increase in discriminative capacity. ConclusionsThese results are the first to establish tRFs as IRI-responsive biomolecules abundant in both donor liver tissue and non-invasive perfusate. In particular, various small RNAs emerged as promising candidate biomarkers for early detection of EAD. These results lay the foundation to further investigate the prognostic utility of tRFs/miRNAs in liver transplantation.

genomics↗

bioRxiv2025

High-quality genome assembly for the genetically improved Abbassa Nile tilapia enables the reconstruction of X and Y haplotypes

The success of the Nile tilapia (Oreochromis niloticus) as an aquaculture species is partly the result of continuous selective breeding leading to high performing strains. These elite strains have been derived from breeding populations of diverse origins and crosses with other Oreochromis species. Owing to the complex and unique evolutionary histories of each strain, existing reference genomes of wild populations are unsuitable to implement genomic selection for beneficial traits such as growth or environmental resilience in aquaculture programmes. Here we generated a high-quality genome assembly and annotation of the WorldFish Genetically Improved Abbassa Nile tilapia (GIANT) elite strain using a combination of PacBio HiFi, and Omni-C Illumina sequencing. As a male Abbassa Nile tilapia was used for the generation of the genome assembly, we reconstructed both X and Y haplotypes, identifying both amhY and amh{Delta}y on LG23 indicating that Abbassa likely shares the same sex determination system as GIFT, and thereby differs from the existing reference genome, whose sex determination loci are located on LG1.

genomics↗

bioRxiv2025

Whole genome sequencing reveals how plasticity and genetic differentiation underlie sympatric morphs of Arctic charr

Salmonids have a remarkable ability to form sympatric morphs after postglacial colonization of freshwater lakes. These morphs often exhibit differences in morphology, feeding, and spawning behaviour. Here we explore the genetics of morph differentiation by establishing a high-quality, annotated reference genome for the Arctic charr and use this as a resource for population genomic analysis of morphs from two Norwegian and two Icelandic lakes. The four lakes represent the spectrum of genetic differentiation between morphs from one lake with no genetic differentiation between morphs, implying phenotypic plasticity only, to two lakes with locus-specific genetic differentiation, implying incomplete reproductive isolation, and one lake with strong genome-wide divergence consistent with complete reproductive isolation. As many as 12 putative inversions ranging from 0.45 to 3.25 Mbp in size segregated among the four morphs present in one lake, Thingvallavatn, and these contributed significantly to the genetic differentiation among morphs. None of the putative inversions was found in any of the other lakes, but there were cases of partial haplotype sharing in similar morph contrasts in other lakes. The results are consistent with a highly polygenic basis of morph differentiation with limited genetic parallelism between lakes. The results support a model where morph differentiation is usually first established due to phenotypic plasticity that results in niche expansion and separation, followed by gradual development of reproductive isolation and locus-specific differentiation and eventually complete reproductive isolation and genome-wide divergence. A major explanation for salmonids ability to diversify into multiple sympatric morphs is likely the genome complexity caused by their ancient whole genome duplication that enhances evolvability.

evolutionary biology↗

bioRxiv2024

Chromosome-scale genome assembly and de novo annotation of Alopecurus aequalis.

Alopecurus aequalis is a winter annual or short-lived perennial bunchgrass which has in recent years emerged as the dominant agricultural weed of barley and wheat in certain regions of China and Japan, causing significant yield losses. Its robust tillering capacity and high fecundity, combined with the development of both target and non-target-site resistance to herbicides means it is a formidable challenge to food security. Here we report on a chromosome-scale assembly of A. aequalis with a genome size of 2.83 Gb. The genome contained 33,758 high-confidence protein-coding genes with functional annotation. Comparative genomics revealed that the genome structure of A. aequalis is more similar to Hordeum vulgare rather than the more closely related Alopecurus myosuroides and has undergone an expansion of cytochrome P450 genes, a gene family involved in non-target-site herbicide resistance.

plant biology↗

bioRxiv2024

O-glycosylation contributes to mammalian glycoRNA biogenesis

There is an increasing appreciation for the role of cell surface glycans in modulating interactions with extracellular ligands and participating in intercellular communication. We recently reported the existence of sialoglycoRNAs, where mammalian small RNAs are covalently linked to N-glycans through the modified base acp3U and trafficked to the cell surface. However, little is currently known about the role for O-glycosylation, another major class of carbohydrate polymer modifications. Here, we use parallel genetic, enzymatic, and mass spectrometry approaches to demonstrate that O-linked glycan biosynthesis is responsible for the majority of sialoglycoRNA levels. By examining the O-glycans associated with RNA from cell lines and colon organoids we find known and previously unreported O-linked glycan structures. Further, we find that O-linked glycans released from small RNA from organoids derived from ulcerative colitis patients exhibit higher levels of sialylation than glycans from healthy organoids. Together, our work provides flexible tools to interrogate O-linked glycoRNAs (O-glycoRNA) and suggests that they may be modulated in human disease.

molecular biology↗

bioRxiv2024

Most human DNA replication initiation is dispersed throughout the genome with only a minority within previously identified initiation zones

BackgroundThe identification of sites of DNA replication initiation in mammalian cells has been challenging. Here, we present unbiased detection of replication initiation events in human cells using BrdU incorporation and single-molecule nanopore sequencing. ResultsIncreases in BrdU incorporation allow us to measure DNA replication dynamics, including identification of replication initiation, fork direction and termination on individual nanopore sequencing reads. Importantly, initiation and termination events are identified on single-molecules with high resolution, throughout S-phase, genome-wide and at high coverage at specific loci using targeted enrichment. We find a significant enrichment of initiation sites within the broad initiation zones identified by population level studies. However, these focused initiation sites only account for [~]20% of all identified replication initiation events. Most initiation events are dispersed throughout the genome and are missed by cell population approaches. This indicates that most initiation occurs at sites that, individually, are rarely used. These dispersed initiation sites contrast with the focused sites identified by population studies, in that they do not show a strong relationship to transcription or a particular epigenetic signature. ConclusionsWe show here that single-molecule sequencing enables unbiased detection and characterisation of DNA replication initiation events, including the numerous dispersed initiation events that replicate most of the human genome.

genomics↗

bioRxiv2024

The genome sequence of the Violet Carpenter Bee, Xylocopa violacea (Linnaeus, 1785): a hymenopteran species undergoing range expansion.

We present a reference genome assembly from an individual male Violet Carpenter Bee (Xylocopa violacea, Linnaeus, 1758). The assembly is 1.02 gigabases in span. 48% of the assembly is scaffolded into 17 pseudo-chromosomal units. The mitochondrial genome has also been assembled and is 21.8 kilobases in length. The genome is highly repetitive, likely representing a highly heterochromatic architecture expected of bees from the genus Xylocopa. We also use an evidence-based methodology to annotate 10,152 high confidence coding genes. This genome was sequenced as part of the pilot project of the European Reference Genome Atlas (ERGA) and represents an important addition to the genomic resources available for Hymenoptera.

genomics↗

bioRxiv2023

Exogenous mitochondrial transfer increases energy expenditure and attenuates adiposity gains in mice with diet-induced obesity

Obesity is associated with chronic multi-system bioenergetic stress that may be improved by increasing the number of healthy mitochondria available across organ systems. However, treatments capable of increasing mitochondrial content are generally limited to endurance exercise training paradigms, which are not always sustainable long-term, let alone feasible for many patients with obesity. Recent studies have shown that local transfer of exogenous mitochondria from healthy donor tissues can improve bioenergetic outcomes and alleviate the effects of tissue injury in recipients with organ specific disease. Thus, the aim of this project was to determine the feasibility of systemic mitochondrial transfer for improving energy balance regulation in the setting of diet-induced obesity. We found that transplantation of mitochondria from lean mice into mice with diet-induced obesity attenuated adiposity gains by increasing energy expenditure and promoting the mobilization and oxidation of lipids. Additionally, mice that received exogenous mitochondria demonstrated improved glucose uptake, greater insulin responsiveness, and complete reversal of hepatic steatosis. These changes were, in part, driven by adaptations occurring in white adipose tissue. Together, these findings are proof-of-principle that mitochondrial transplantation is an effective therapeutic strategy for limiting the deleterious metabolic effects of diet-induced obesity in mice.

physiology↗

bioRxiv2023

The modified RNA base acp3U is an attachment site for N-glycans in glycoRNA

We recently identified glycoRNA--a previously undescribed glycoconjugate--which consists of RNAs modified with secretory N-glycans and presented on the cell surface. While previous work supported a covalent linkage between RNA and glycans, the direct chemical nature of the RNA-glycan connection was not described. Here we develop a sensitive and scalable protocol to detect and characterize native glycoRNAs. Leveraging periodate oxidation and aldehyde ligation (rPAL) and Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS), we identified the modified RNA base 3-(3-amino-3-carboxypropyl)uridine (acp3U) as a site of attachment of N-glycans in glycoRNA. The sensitivity and robustness of rPAL provided the first evidence of a direct glycan-RNA linkage, and its flexibility will enable further characterization of glycoRNA biology.

molecular biology↗

bioRxiv2023

Unveiling APOL1 Haplotypes: A Novel Classification Through Probe-Independent Quantitative Real-Time PCR

IntroductionApolipoprotein-L1 (APOL1) is a primate-specific protein component of high- density lipoprotein (HDL). Two variants of APOL1 (G1 and G2), provide resistance to parasitic infections in African Americans but are also implicated in kidney-related diseases and transplant outcomes in recipients. This study aims to identify these risk variants using a novel probe- independent quantitative real-time PCR method in a high African American recipient cohort. Additionally, it aims to develop a new stratification approach based on haplotype-centric model. MethodsGenomic DNA was extracted from recipient PBMCs using SDS lysis buffer and proteinase K. Quantitative PCR assay with modified forward primers and a common reverse primer enabled us to identify single nucleotide polymorphisms (SNPs) and the 6-bp deletion quantitatively. Additionally, we used sanger sequencing to verify our QPCR findings. ResultsOur novel probe-independent qPCR effectively distinguished homozygous wild-type, heterozygous SNPs/deletion, and homozygous SNPs/deletion, with at least 4-fold differences. High prevalence of APOL1 variants was observed (18% two-risk alleles, 34% one-risk allele) in our recipient cohort. Intriguingly, up to 12-month follow-up revealed no significant impact of recipient APOL1 variants on transplant outcomes. Ongoing research will encompass more time points and a larger patient cohort, allowing a comprehensive evaluation of G1/G2 variant subgroups categorized by new haplotype scores, enriching our understanding. ConclusionsOur cost-effective and rapid qPCR technique facilitates APOL1 genotyping within hours. Prospective and retrospective studies will enable comparisons with long-term allograft rejection, potentially predicting early/late-stage transplant outcomes based on haplotype evaluation in this diverse group of kidney transplant recipients.

genetics↗

bioRxiv2023

Interleukin-27-induced HIV-resistant dendritic cells suppress reveres transcription following virus entry in an SPTBN1, Autophagy, and YB-1 independent manner

Interleukin (IL)-27, a member of the IL-12 family of cytokines, induces human immunodeficiency virus (HIV)-resistant monocyte-derived macrophages and T cells. This resistance is mediated via the downregulation of spectrin beta, non-erythrocytic 1 (SPTBN1), induction of autophagy, or suppression of the acetylation of Y-box binding protein-1 (YB-1); however, the role of IL-27 administration during the induction of immature monocyte-derived dendritic cells (iDC) is poorly investigated. In the current study, we investigated the function of IL-27-induced iDC (27DC) on HIV infection. 27DC inhibited HIV infection by 95 {+/-} 3 % without significant changes in the expression of CD4, CCR5, and SPTBN1 expression, autophagy induction and acetylation of YB-1 compared to iDC. An HIV proviral DNA copy number assay displayed that 27DC suppressed reverse transcriptase (RT) reaction without influencing the virus entry. A DNA microarray analysis was performed to identify the differentially expressed genes between 27DC and iDC. Compared to iDC, 51 genes were differentially expressed in 27DC, with more than 3-fold changes in four independent donors. Cross-reference analysis with the reported 2,214 HIV regulatory host genes identified nine genes as potential interests: Ankyrin repeat domain 22, Guanylate binding protein (GBP)-1, -2, -4, -5, Stabilin 1, Serpin family G member 1 (SERPING1), Interferon alpha inducible protein 6, and Interferon-induced protein with tetratricopeptide repeats 3. A knock-down study using si-RNA failed to determine a key factor associated with the anti-HIV activity due to the induction of robust amounts of off-target effects. Overexpression of each protein in cells had no impact on HIV infection. Thus, we could not define the mechanism of the anti-HIV effect in 27DC. However, our findings indicated that IL-27 differentiates monocytes into HIV-resistant DC, and the inhibitory mechanism differs from IL-27-induced HIV-resistant macrophages and T cells.

immunology↗

bioRxiv2022

Chromosome-level genome sequence of the Genetically Improved Farmed Tilapia (GIFT, Oreochromis niloticus) highlights regions of introgression with O. mossambicus

BackgroundThe Nile tilapia (Oreochromis niloticus) is the third most important freshwater fish for aquaculture. Its success is directly linked to continuous breeding efforts focusing on production traits such as growth rate and weight. Among those elite strains, the Genetically Improved Farmed Tilapia (GIFT) programme initiated by WorldFish is now distributed worldwide. To accelerate the development of the GIFT strain through genomic selection, a high-quality reference genome is necessary. FindingsUsing a combination of short (10X Genomics) and long read (PacBio HiFi, PacBio CLR) sequencing and a genetic map for the GIFT strain, we generated a chromosome level genome assembly for the GIFT. Using genomes of two closely related species (O. mossambicus, O. aureus), we characterised the extent of introgression between these species and O. niloticus that has occurred during the breeding process. Over 11Mb of O. mossambicus genomic material could be identified within the GIFT genome, including genes associated with immunity but also with traits of interest such as growth rate. ConclusionBecause of the breeding history of elite strains, current reference genomes might not be the most suitable to support further studies into the GIFT strain. We generated a chromosome level assembly of the GIFT strain, characterising its mixed origins, and the potential contributions of introgressed regions to selected traits.

genomics↗

bioRxiv2021

Transcript accumulation rates in the early C. elegans embryo

Dynamic changes in transcription are widespread in developing embryos, where cell cycles are rapid and cell fate decisions need to be made quickly, often before the next cell division. Some fate decisions in the early Caenorhabditis elegans embryo overcome these constraints through the rapid production of high absolute levels of transcription factor mRNAs. Single cell accumulation rates for a small subset of developmental genes are known, but genome-scale measurements are lacking. Furthermore, how different aspects of transcription kinetics are fine-tuned for different genes to achieve the appropriate RNA levels is still being worked out. We describe a novel strategy to analyze single cell RNA sequencing data from the early C. elegans embryo. We estimate the absolute accumulation rates of zygotic genes up to the 16-cell stage and calibrate predicted rates with single molecule transcript imaging. We show that rapid transcript accumulation is common across different cell types and lineages and rates are the highest soon after zygotic transcription begins. High-rate transcription is a characteristic of genes encoding transcription factors with functions in cell fate specification. These genes share common genomic features and are more likely to have undergone recent duplication. We identify core promoter motifs that might drive high absolute RNA accumulation rates. We measured the contributions of core promoter elements to accumulation rate for one rapidly accumulating gene, ceh-51, which is required for mesoderm development. We find that mutating individual motifs modestly decreases the accumulation rate of ceh-51 mRNA, suggesting multifactorial control of transcript accumulation rates. These results are a step towards estimating absolute transcription kinetics during embryonic fate specification and understanding how transcript dosage drives developmental decisions.

developmental biology↗

bioRxiv2020

An accessible, efficient and global approach for the large-scale sequencing of bacterial genomes

We have developed an efficient and inexpensive pipeline for streamlining large-scale collection and genome sequencing of bacterial isolates. Evaluation of this method involved a worldwide research collaboration focused on the model organism Salmonella enterica, the 10KSG consortium. By optimising a logistics pipeline that collected isolates as thermolysates, permitting shipment in ambient conditions, the project assembled a diverse collection of 10,419 clinical and environmental isolates from low- and middle-income countries in less than one year. The bacteria were obtained from 51 countries/territories dating from 1949 to 2017, with a focus on Africa and Latin-America. All isolates were collected in barcoded tubes and genome sequenced using an optimised DNA extraction method and the LITE pipeline for library construction. After Illumina sequencing, the total reagent cost was less than USD$10 per genome. Our method can be applied to genome-sequence other large bacterial collections at a relatively low cost, within a limited timeframe, to support global collaborations.

microbiology↗