Biology subjects

Williams, B.

Publications and source records attributed to Williams, B..

At least 55 records · Page 3Linked to original sources

bioRxiv2023

Systematic assessment of long-read RNA-seq methods for transcript identification and quantification

AbstractThe Long-read RNA-Seq Genome Annotation Assessment Project (LRGASP) Consortium was formed to evaluate the effectiveness of long-read approaches for transcriptome analysis. The consortium generated over 427 million long-read sequences from cDNA and direct RNA datasets, encompassing human, mouse, and manatee species, using different protocols and sequencing platforms. These data were utilized by developers to address challenges in transcript isoform detection and quantification, as well as de novo transcript isoform identification. The study revealed that libraries with longer, more accurate sequences produce more accurate transcripts than those with increased read depth, whereas greater read depth improved quantification accuracy. In well-annotated genomes, tools based on reference sequences demonstrated the best performance. When aiming to detect rare and novel transcripts or when using reference-free approaches, incorporating additional orthogonal data and replicate samples are advised. This collaborative study offers a benchmark for current practices and provides direction for future method development in transcriptome analysis.

genomics↗

bioRxiv2023

Dichotomy of neutralizing antibody, B cell and T cell responses to SARS-CoV-2 vaccination and protection in healthy adults

Heterogeneity in SARS-CoV-2 vaccine responses is not understood. Here, we identify four patterns of live-virus neutralizing antibody responses: individuals with hybrid immunity (with confirmed prior infection); rare individuals with low responses (paucity of S1-binding antibodies); and surprisingly, two further groups with distinct serological repertoires. One group - broad responders - neutralize a range of SARS-CoV-2 variants, whereas the other - narrow responders - neutralize fewer, less divergent variants. This heterogeneity does not correlate with Ancestral S1-binding antibody, rather the quality of the serological response. Furthermore, IgDlowCD27-CD137+ B cells and CCR6+ CD4+ T cells are enriched in broad responders before dose 3. Notably, broad responders have significantly longer infection-free time after their third dose. Understanding the control and persistence of these serological profiles could allow personalized approaches to enhance serological breadth after vaccination.

immunology↗

bioRxiv2023

The ENCODE4 long-read RNA-seq collection reveals distinct classes of transcript structure diversity

The majority of mammalian genes encode multiple transcript isoforms that result from differential promoter use, changes in exonic splicing, and alternative 3 end choice. Detecting and quantifying transcript isoforms across tissues, cell types, and species has been extremely challenging because transcripts are much longer than the short reads normally used for RNA-seq. By contrast, long-read RNA-seq (LR-RNA-seq) gives the complete structure of most transcripts. We sequenced 264 LR-RNA-seq PacBio libraries totaling over 1 billion circular consensus reads (CCS) for 81 unique human and mouse samples. We detect at least one full-length transcript from 87.7% of annotated human protein coding genes and a total of 200,000 full-length transcripts, 40% of which have novel exon junction chains. To capture and compute on the three sources of transcript structure diversity, we introduce a gene and transcript annotation framework that uses triplets representing the transcript start site, exon junction chain, and transcript end site of each transcript. Using triplets in a simplex representation demonstrates how promoter selection, splice pattern, and 3 processing are deployed across human tissues, with nearly half of multitranscript protein coding genes showing a clear bias toward one of the three diversity mechanisms. Evaluated across samples, the predominantly expressed transcript changes for 74% of protein coding genes. In evolution, the human and mouse transcriptomes are globally similar in types of transcript structure diversity, yet among individual orthologous gene pairs, more than half (57.8%) show substantial differences in mechanism of diversification in matching tissues. This initial large-scale survey of human and mouse long-read transcriptomes provides a foundation for further analyses of alternative transcript usage, and is complemented by short-read and microRNA data on the same samples and by epigenome data elsewhere in the ENCODE4 collection.

genomics↗

bioRxiv2023

D614G and Omicron SARS-CoV-2 variant spike proteins differ in the effects of N-glycan modifications on spike expression, virus infectivity, and neutralization by some therapeutic antibodies

The SARS-CoV-2 spike glycoprotein has 22 potential N-linked glycosylation sites per monomer that are highly conserved among diverse variants, but how individual glycans affect virus entry and neutralization of Omicron variants has not been extensively characterized. Here we compared the effects of specific glycan deletions or modifications in the Omicron BA.1 and D614G spikes on spike expression, processing, and incorporation into pseudoviruses, as well as on virus infectivity and neutralization by therapeutic antibodies. We found that loss of potential glycans at spike residues N717 and N801 each conferred a loss of pseudovirus infectivity for Omicron but not for D614G or Delta variants. This decrease in infectivity correlated with decreased spike processing and incorporation into Omicron pseudoviruses. Oligomannose-enriched Omicron pseudoviruses generated in GnTI- cells or in the presence of kifunensine were non-infectious, whereas D614G or Delta pseudoviruses generated under similar conditions remained infectious. Similarly, authentic SARS-CoV-2 grown in the presence of kifunensine decreased titers more for the BA.1.1 variant than Delta or D614G variants relative to their respective, untreated controls. Finally, we found that loss of some N-glycans, including N343 and N234, increased the maximum percent neutralization by the class 3 S309 monoclonal antibody against D614G but not BA.1 variants, while these glycan deletions altered the neutralization potency of the class 1 COV2-2196 and Etesevimab monoclonal antibodies without affecting maximum percent neutralization. The maximum neutralization by some antibodies also varied with the glycan composition, with oligomannose-enriched pseudoviruses conferring the highest percent neutralization. These results highlight differences in the interactions between spike glycans and residues among SARS-CoV-2 variants that can affect spike expression, virus infectivity, and susceptibility of variants to antibody neutralization. Author summary (non-technical)The SARS-CoV-2 spike surface protein is covered in glycans that may affect its function and ability to evade antibodies. Omicron variants have over 30 mutations compared to the D614G variant, yet all 22 potential N-glycosylation sites are highly conserved. Here we compared the impact of glycan changes in the spikes of the Omicron and D614G variants on virus infectivity and neutralization. We found that loss of specific glycans in the transmembrane subunit of spike greatly reduced Omicron, but not D614G, spike expression and incorporation into pseudoviruses. Changes in the overall glycan composition also reduced the infectivity of Omicron pseudovirus and authentic virus more than D614G pseudoviruses and authentic viruses. We further show that changes in specific glycans directly or indirectly affected susceptibility of pseudoviruses to therapeutic antibodies, but the effects differed among the variants. These findings highlight differences in the interplay between spike glycans and amino acid residues among SARS-CoV-2 variants that can contribute to spike plasticity and modify spike expression, function, and immune evasion properties.

microbiology↗

bioRxiv2023

Knockdown of Atg1 or Atg18 in adult adipocytes impairs lipophagy and reduces lifespan of fruit flies (Drosophila melanogaster)

Autophagy, a lysosome-based eukaryotic cellular degradation system, has previously been implicated in lifespan regulation in different animal models. In this report, we show that expression of the RNAi transgenes targeting the transcripts of the key autophagy genes such as Atg1 or Atg18 in adult fly muscle or glia does not affect the overall levels of autophagosomes in those tissues and does not change the lifespan of the tested flies, but lifespan reduction phenotype has become apparent when Atg1 RNAi or Atg18 RNAi is expressed in a non-tissue-specific manner through a Tub-Gal4 in adult flies or after lipophagy is eradicated through the knockdown of Atg1 or Atg18 in adult fly adipocytes. Lifespan reduction was also observed when Atg1 or Atg18 was knocked down in adult fly enteroblasts and middle gut stem cells. Over-expression of wildtype Atg1 in adult fly muscle or adipocytes reduces lifespan. High levels of ubiquitinated protein aggregates could be the culprit of the reduced lifespan of Atg1 over-expression flies. Our research data presented here have highlighted the important functions of the key autophagy genes in adult fly adipocytes, enteroblasts, and midgut stem cells for lifespan regulation and their undetermined functions in adult fly muscle and glia. SummaryIn this research, we have demonstrated that the key autophagy genes play important roles in fly lifespan regulation through adult adipose tissues, enteroblasts, and middle gut stem cells.

genetics↗

bioRxiv2023

C. elegans SMOC-1 interacts with both BMP and glypican to regulate BMP signaling

ABSTRACTSSecreted modular calcium binding (SMOC) proteins are conserved matricellular proteins found in organisms from C. elegans to humans. SMOC homologs characteristically contain one or two extracellular calcium (EC) binding domain(s) and one or two thyroglobulin type-1 (TY) domain(s). SMOC proteins in Drosophila and Xenopus have been found to interact with cell surface heparan sulfate protein glycans (HSPGs) to exert both positive and negative influences on the conserved bone morphogenetic protein (BMP) signaling pathway. In this study, we used a combination of biochemical, structural modeling, and molecular genetic approaches to dissect the functions of the sole SMOC protein in C. elegans. We showed that SMOC-1 binds LON-2/glypican, as well as the mature domain of DBL-1/BMP. Moreover, SMOC-1 can simultaneously bind LON-2/glypican and DBL-1/BMP. The interaction between SMOC-1 and LON-2/glypican is mediated by the EC domain of SMOC-1, while the interaction between SMOC-1 and DBL-1/BMP involves full-length SMOC-1. We further showed that while SMOC-1(EC) is sufficient to promote BMP signaling when overexpressed, both the EC and TY domains are required for SMOC-1 function at the endogenous locus. Finally, when overexpressed, SMOC-1 can promote BMP signaling in the absence of LON-2/glypican. Taken together, our findings led to a model where SMOC-1 functions both negatively in a LON-2-dependent manner and positively in a LON-2-independent manner to regulate BMP signaling. Our work provides a mechanistic basis for how the evolutionarily conserved SMOC proteins regulate BMP signaling.

developmental biology↗

bioRxiv2022

In situ structure of a dimeric hibernating ribosome from a eukaryotic intracellular pathogen

Translational control is an essential process for the cell to adapt to varying physiological or environmental conditions. To survive adverse conditions such as low nutrient levels, translation can be shut down almost entirely by inhibiting ribosomal function. Here we investigated eukaryotic hibernating ribosomes from the microsporidian parasite Spraguea lophii in situ by a combination of cryo-electron tomography (cryoET) and single particle cryoEM. We show that microsporidian spores contain ribosomes primed for host cell invasion and thus shed new light on the infection mechanism of this important pathogen. Prior to host infection, virtually all ribosomes are locked in the 100 S dimeric state, which appears to be formed by a unique dimerization mechanism that is distinct from its bacterial counterparts. Within the dimer, the hibernation factor MDF1 is bound within the E site, locking the L1 stalk in a closed conformation, and thus preventing the translation of mRNAs to polypeptides.

molecular biology↗

bioRxiv2022

Emergence of new subgenomic mRNAs in SARS-CoV-2

Two mutations occurred in SARS-CoV-2 early during the COVID-19 pandemic that have come to define circulating virus lineages1: first a change in the spike protein (D614G) that defines the B.1 lineage and second, a double substitution in the nucleocapsid protein (R203K, G204R) that defines the B.1.1 lineage, which has subsequently given rise to three Variants of Concern: Alpha, Gamma and Omicron. While the latter mutations appear unremarkable at the protein level, there are dramatic implications at the nucleotide level: the GGG[->]AAC substitution generates a new Transcription Regulatory Sequence (TRS) motif, driving SARS-CoV-2 to express a novel subgenomic mRNA (sgmRNA) encoding a truncated C-terminal portion of nucleocapsid (N.iORF3), which is an inhibitor of type I interferon production. We find that N.iORF3 also emerged independently within the Iota variant, and further show that additional TRS motifs have convergently evolved to express novel sgmRNAs; notably upstream of Spike within the nsp16 coding region of ORF1b, which is expressed during human infection. Our findings demonstrate that SARS-CoV-2 is undergoing evolutionary changes at the functional RNA level in addition to the amino acid level, reminiscent of eukaryotic evolution. Greater attention to this aspect in the assessment of emerging strains of SARS-CoV-2 is warranted.

microbiology↗

bioRxiv2022

A comparison of feature selection methodologies and learning algorithms in the development of a DNA methylation-based telomere length estimator

The field of epigenomics holds great promise in understanding and treating disease with advances in machine learning (ML) and artificial intelligence being vitally important in this pursuit. Increasingly, research now utilises DNA methylation measures at cytosine-guanine dinucleotides (CpG) to detect disease and estimate biological traits such as aging. Given the high dimensionality of DNA methylation data, feature-selection techniques are commonly employed to reduce dimensionality and identify the most important subset of features. In this study, we test and compare a range of feature-selection methods and ML algorithms in the development of a novel DNA methylation-based telomere length (TL) estimator. We found that principal component analysis in advance of elastic net regression led to the overall best performing estimator when evaluated using a nested cross-validation analysis and two independent test cohorts. In contrast, the baseline model of elastic net regression with no prior feature reduction stage performed worst - suggesting a prior feature-selection stage may have important utility. The variance in performance across tested approaches shows that estimators are sensitive to data set heterogeneity and the development of an optimal DNA methylation-based estimator should benefit from the robust methodological approach used in this study. Additionally, we observed that different DNA methylation-based TL estimators, which have few common CpGs, are associated with many of the same biological entities. Moreover, our methodology which utilises a range of feature-selection approaches and ML algorithms could be applied to other biological markers and disease phenotypes, to examine their relationship with DNA methylation and predictive value.

bioinformatics↗

bioRxiv2021

Dissociable roles for the striatal cholinergic system in different flexibility contexts.

The production of behavioural flexibility requires the coordination and integration of information from across the brain, by the dorsal striatum. In particular, the striatal cholinergic system is thought to be important for the modulation of striatal activity. Research from animal literature has shown that chemical inactivation of the dorsal striatum leads to impairments in reversal learning. Furthermore, proton magnetic resonance spectroscopy work has shown that the striatal cholinergic system is also important for reversal learning in humans. Here, we aim to assess whether the state of the dorsal striatal cholinergic system at rest is related to flexible behaviour in reversal learning. We provide preliminary results showing that variability in choline in the dorsal striatum is significantly related to both the number perseverative and regressive errors that participants make, and their rate of learning from positive and negative prediction errors. These findings, in line with previous work, suggest the resting state of dorsal striatal cholinergic system has important implications for producing flexible behaviour. However, these results also suggest the system may have heterogeneous functionality across different types of tasks measuring behavioural flexibility. These findings provide a starting point for further interrogation into understanding the functional role of the striatal cholinergic system in flexibility.

neuroscience↗

bioRxiv2021

Cortical and thalamic influences on striatal involvement in instructed, serial reversal learning; implications for the organisation of flexible behaviour.

Cognitive flexibility is essential for enabling an individual to respond adaptively to changes in their environment. Evidence from human and animal research suggests that the control of cognitive flexibility is dependent on an array of neural architecture. Cortico-basal ganglia circuits have long been implicated in cognitive flexibility. In particular, the role of the striatum is pivotal, acting as an integrative hub for inputs from the prefrontal cortex and thalamus, and modulation by dopamine and acetylcholine. Striatal cholinergic modulation has been implicated in the flexible control of behaviour, driven by input from the centromedian-parafascicular nuclei of the thalamus. However, the role of this system in humans is not clearly defined as much of the current literature is based on animal work. Here, we aim to investigate the roles corticostriatal and thalamostriatal connectivity in serial reversal learning. Functional connectivity between the left centromedian-parafascicular nuclei and the associative dorsal striatum was significantly increased for negative feedback compared to positive feedback. Similar differences in functional connectivity were observed for the right lateral orbitofrontal cortex, but these were localised to when participants switched to using an alternate response strategy following reversal. These findings suggest that connectivity between the centromedian-parafascicular nuclei and the striatum may be used to generally identify potential changes in context based on negative outcomes, and the effect of this signal on striatal output may be influenced by connectivity between the lateral orbitofrontal cortex and the striatum.

neuroscience↗

bioRxiv2021

The ENDS of assumptions; an online tool for the Epistemic Nonparametric Drug-response Scoring

MotivationThe drugs sensitivity analysis is often elucidated from drug dose-response curves. These curves capture the degree of cell viability (or inhibition) over a range of induced drugs, often with parametric assumptions that are rarely validated. ResultsWe present a class of nonparametric models for the curve fitting and scoring of drug dose-responses. To allow a more objective representation of the drug sensitivity, these epistemic models devoid of any parametric assumptions attached to the linear fit, allow the parallel indexing such as IC50 and AUC. Specifically, three nonparametric models including Spline, Monotonic, and Bayesian (npS, npM, npB) and the parametric Logistic (pL) are implemented. Other indices including Maximum Effective Dose (MED) and Drug-response Span Gradient (DSG) pertinent to the npS are also provided to facilitate the interpretation of the fit. The collection of these models are implemented in an online app, standing as useful resource for drug dose-response curve fitting and analysis. AvailabilityThe ENDS is freely available online at https://irscope.shinyapps.io/ENDS/ and source codes can be obtained from https://github.com/AmiryousefiLab/ENDS. Supplementary informationSupplementary data are available at Bioinformatics and https://irscope.shinyapps.io/ENDS/ Contactali.amiryousefi@helisnki.fi; jing.tang@helisnki.fi. ContributionsAA conceived the study and developed the models, AA and BW adopted and implemented the methods, JT provided the funding, AA, BW, MJ, and JT wrote the paper.

pharmacology and toxicology↗

bioRxiv2021

Qualification of ELISA and neutralization methodologies to measure SARS-CoV-2 humoral immunity using human clinical samples

In response to the SARS-CoV-2 pandemic many vaccines have been developed and evaluated in human clinical trials. The humoral immune response magnitude, composition and efficacy of neutralizing SARS-CoV-2 are essential endpoints for these trials. Robust assays that are reproducibly precise, linear, and specific for SARS-CoV-2 antigens would be beneficial for the vaccine pipeline. In this work we describe the methodologies and clinical qualification of three SARS-CoV-2 endpoint assays. We developed and qualified Endpoint titer ELISAs for total IgG, IgG1, IgG3, IgG4, IgM and IgA to evaluate the magnitude of specific responses to the trimeric spike (S) antigen and total IgG specific to the spike receptor binding domain (RBD) of SARS-CoV-2. We also qualified a pseudovirus neutralization assay which evaluates functional antibody titers capable of inhibiting the entry and replication of a lentivirus containing the Spike antigen of SARS-CoV-2. To complete the suite of assays we qualified a plaque reduction neutralization test (PRNT) methodology using the 2019-nCoV/USA-WA1/2020 isolate of SARS-CoV-2 to assess neutralizing titers of antibodies in plasma from normal healthy donors and convalescent COVID-19 individuals.

immunology↗

bioRxiv2021

Desiccation-driven senescence and its repression in Xerophyta schlechteri are regulated at extremely low water contents

O_LIVegetative desiccation tolerance, the ability to survive loss of over 90% of cellular water, is an extremely rare trait in Angiosperms. Xerophyta schlechteri survives such extreme water deficit by entering prolonged quiescence and suppressing drought-induced senescence in most of the leaf area, except the apical tip. Information on the molecular regulation of senescence in such plants is scarce and this is the first study to investigate such regulation in senescing and non-senescing tissues of the same leaf. C_LIO_LIGenome-wide RNA sequencing enabled comparison of senescent and non-senescent tissues during desiccation and early rehydration, establishment of the water content range in which senescence is initiated and identification of molecular mechanisms employed to bring about cellular death. C_LIO_LISenescence-associated genes (XsSAG) specific to this species were identified and two potential regulatory sites were enriched in regions upstream to these XsSAGs, allowing us to create a model of senescence regulation in X. schlechteri based on homology with known Arabidopsis senescence regulators. C_LIO_LIWe hypothesise that desiccation-driven senescence occurs as a result of a convergence of signals around MAPK6 to trigger WRKY-mediated ethylene synthesis and XsSAG expression, not unlike aging and stress-related senescence in Arabidopsis, but at remarkably lower water contents (<35% RWC). C_LI

plant biology↗

bioRxiv2020

Deep learning and automated Cell Painting reveal Parkinson's disease-specific signatures in primary patient fibroblasts

Drug discovery for diseases such as Parkinsons disease are impeded by the lack of screenable cellular phenotypes. We present an unbiased phenotypic profiling platform that combines automated cell culture, high-content imaging, Cell Painting, and deep learning. We applied this platform to primary fibroblasts from 91 Parkinsons disease patients and matched healthy controls, creating the largest publicly available Cell Painting image dataset to date at 48 terabytes. We use fixed weights from a convolutional deep neural network trained on ImageNet to generate deep embeddings from each image and train machine learning models to detect morphological disease phenotypes. Our platforms robustness and sensitivity allow the detection of individual-specific variation with high fidelity across batches and plate layouts. Lastly, our models confidently separate LRRK2 and sporadic Parkinsons disease lines from healthy controls (receiver operating characteristic area under curve 0.79 (0.08 standard deviation)), supporting the capacity of this platform for complex disease modeling and drug screening applications.

cell biology↗

bioRxiv2020

Spatial modulation of dark versus bright stimulus responses in mouse visual cortex

A fundamental task of the visual system is to respond to luminance increments and decrements. In primary visual cortex (V1) of cats and primates, luminance decrements elicit stronger, faster, and more salient neural activity (OFF responses) than luminance increments (ON responses). However, studies of V1 in ferrets and mice show that ON responses may be stronger. These discrepancies may arise from differences in species, experimental conditions, or from measuring responses in single neurons versus populations. Here, we examined OFF versus ON responses across different regions of visual space in both single neurons and populations of mouse V1. We used high-density silicon probes and whole-cell patch-clamp recordings to assess OFF versus ON dominance in local field potential (LFP), single neuron, and membrane potential responses. Across these levels, we found that OFF responses clearly dominated in the central visual field, whereas ON responses were more evident in the periphery. These observations were clearest in LFP and subthreshold membrane potential. Our findings consolidate and resolve prior conflicting results and reveal that retinotopy may provide a common organizing principle for spatially biasing OFF versus ON processing in mammalian visual systems.

neuroscience↗

bioRxiv2020

Liver x receptor alpha drives chemoresistance in response to side-chain hydroxycholesterols in triple negative breast cancer

Triple negative breast cancer (TNBC) is challenging to treat successfully because targeted therapies do not exist. Instead, systemic therapy is typically restricted to cytotoxic chemotherapy, which fails more often in patients with elevated circulating cholesterol. Liver x receptors are ligand-dependent transcription factors that are homeostatic regulators of cholesterol, and are linked to regulation of broad-affinity xenobiotic transporter activity in non-tumor tissues. We show that LXR ligands confer chemotherapy resistance in TNBC cell lines and xenografts, and that LXRalpha is necessary and sufficient to mediate this resistance. Furthermore, in TNBC patients who had cancer recurrences, LXRalpha and ligands were independent markers of poor prognosis and correlated with P-glycoprotein expression. However, in patients who survived their disease, LXRalpha signaling and P-glycoprotein were decoupled. These data reveal a novel chemotherapy resistance mechanism in this poor prognosis subtype of breast cancer. We conclude that systemic chemotherapy failure in some TNBC patients is caused by co-opting the LXRalpha:P-glycoprotein axis, a pathway highly targetable by therapies that are already used for prevention and treatment of other diseases.

cancer biology↗

bioRxiv2020

Laser-activated drug implant for controlled release to the posterior segment of the eye

The current standard of care for posterior segment eye diseases, such as age-related macular degeneration and diabetic macular edema, is frequent intravitreal injections or sustained-release drug implants. Drug implants have side effects due to the burst release of the drugs, and their release cannot be easily controlled after implantation. Present study attempts to develop a dosage-controllable drug delivery implant which consists of a nanoporous biodegradable PLGA capsule and light-activated liposomes. Controllable drug release from the implant was achieved by using pulsed near-infrared (NIR) laser both in vitro and in vivo. The in vitro drug release kinetics from two different initial dose implants, 1000 g and 500 g, was analyzed by fitting zero order and first order kinetics, as well as the Korsmeyer-Peppas and Higuchi models. The 1000 g and 500 g implants fit the first-order and zero-order kinetics model, respectively, the best. The multiple drug releases in the vitreous was determined by in vivo fluorimeter, which was consistent with the in vitro data. The dose released was also clinically relevant. Histology and optical and ultrasound imaging data showed no abnormality in the eyes received implant treatment suggesting that the drug delivery system was safe to the retina. This on-demand dose-controllable drug delivery system could be potentially used for long-term posterior eye disease treatment.

bioengineering↗