Biology subjects

Acman, M.

Publications and source records attributed to Acman, M..

9 recordsLinked to original sources

bioRxiv2026

RNA isoform-resolved multiplexed sequencing with bioorthogonal barcoding

RNA isoform dysregulation drives disease pathogenesis and is the target of FDA-approved splice-switching therapeutics. However, multiplexed sequencing methods discard splice junction information because only 3' termini are barcoded and counted. Here, we repurpose acylation and click chemistries to conjugate bioorthogonal barcodes (bobcodes) directly onto multiple internal positions along cellular RNAs. Bobcoded RNAs from multiple samples are pooled for multiplexed cDNA synthesis, during which reverse transcriptase switches from each RNA template onto its tethered bobcode with greater than 99% accuracy in species mixing experiments. Bobcode attachment intervals set cDNA insert sizes without a library fragmentation step, and priming with poly(dT) or random hexamers selects between 3'-end counting and full-length isoform capture. A bioorthogonal barcode-sequencing (BOB-seq v0.1) drug screen identifies transcriptome-wide on- and off-target RNA splicing effects and outperforms existing multiplexing RNA sequencing methods in workflow simplicity, sample-to-sample variability, and barcoding accuracy. Bobcodes add isoform resolution to scalable multiplexed RNA sequencing.

genomics↗

bioRxiv2025

Localization of KANK2 to Focal or Fibrillar Adhesions Influences Migration in Melanoma Cell Lines

BackgroundIntegrins form focal adhesions (FAs) at the cell edge and fibrillar adhesions (FBs) located centrally. Talin 1 is essential to FAs, while talin2 is found in FAs and FBs. KANK (kidney ankyrin repeat-containing) family proteins regulate adhesion dynamics and are recruited to adhesions through interaction with talins. Previously, we showed in MDA-MB-435S melanoma cells that KANK2 is part of integrin V{beta}5 FAs, that its interaction with talin2 regulates actin-microtubule (MT) crosstalk and that KANK2 knockdown mimics the effect of integrin V or {beta}5 knockdown by reducing cell migration. Here, in another melanoma cell line RPMI-7951 we observed that KANK2 is part of FAs and FBs and that KANK2 knockdown increases cell migration. Therefore, we analyse integrin adhesion complexes in RPMI-7951 cells, explore the localization and role of KANK2 in FAs and FBs. MethodsKnockdown in human melanoma RPMI-7951 cells was achieved by transfection with gene-specific siRNAs. Integrin adhesion complexes were isolated and analysed by mass spectrometry. Immunofluorescence analysis, live cell imaging and the proximity ligation assays were done using confocal microscopy. Cell migration was assessed using Transwell Cell Culture Inserts. MTT assays were performed to determine cell sensitivity to paclitaxel. Data were statistically evaluated using one-way or two-way ANOVA or unpaired Students t-tests in GraphPad Software. ResultsWe demonstrate that RPMI-7951 melanoma cells use integrin V{beta}5 FAs and integrin 5{beta}1 FBs for adhesion, and that KANK2 is part of both structures. KANK2 is predominantly in proximity to talin1 at the cell edge (FAs) and in proximity to talin2 in the cell center (FBs). KANK2 in FAs functionally interacts with talin1 to maintain FAs, and with talin2 to regulate their dynamics. KANK2 is a component of FBs, and its knockdown mimics integrin 5 knockdown by increasing MT-dependent cell migration. ConclusionsOur study reveals the distinct roles of KANK2 in FAs and FBs. We show that KANK2 is a component of FBs, linking them to MTs and promoting their stabilisation. Loss of integrin 5 or KANK2 from FBs increases cell migration, a process that relies on the MT cytoskeleton. Plain English summaryCells adhere to their surroundings using specialized structures called adhesions, which are formed by different types of integrins and have different cell localisations: focal adhesions at the cell edge and fibrillar adhesions in the cell center. Talin1 is essential for focal adhesions, while talin2 is present in focal and fibrillar adhesions. KANK (kidney ankyrin repeat-containing) family proteins regulate adhesion dynamics by connecting talins to the microtubule cytoskeleton. Our study explored the different roles of KANK2 in focal or fibrillar adhesions, by focusing on its interactions with integrins and talins. We show that in RPMI-7951 cells, which form V{beta}5 focal and 5{beta}1 fibrillar adhesions, KANK2 is present in both of these adhesions. KANK2 supports V{beta}5 focal adhesion maintenance via talin1 and regulates their dynamics via talin2. KANK2 is also a component of 5{beta}1 fibrillar adhesions, and its depletion mimics integrin 5 knockdown and enhances cell migration, a process dependent on the microtubule cytoskeleton. In conclusion, our study demonstrates that KANK2 plays distinct roles in different adhesion structures, contributing to both focal and fibrillar adhesion function.

cell biology↗

bioRxiv2021

A phylogeny-based metric for estimating changes in transmissibility from recurrent mutations in SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in late 2019 and spread globally to cause the COVID-19 pandemic. Despite the constant accumulation of genetic variation in the SARS-CoV-2 population, there was little evidence for the emergence of significantly more transmissible lineages in the first half of 2020. Starting around November 2020, several more contagious and possibly more virulent Variants of Concern (VoCs) were reported in various regions of the world. These VoCs share some mutations and deletions that haven arisen recurrently in distinct genetic backgrounds. Here, we build on our previous work modelling the association of mutations to SARS-CoV-2 transmissibility and characterise the contribution of individual recurrent mutations and deletions to estimated viral transmissibility. We then assess how patterns of estimated transmissibility in all SARS-CoV-2 clades have varied over the course of the COVID-19 pandemic by summing transmissibility estimates for all individual mutations carried by any sequenced genome analysed. Such an approach recovers the Delta variant (21A) as the most transmissible clade currently in circulation, followed by the Alpha variant (20I). By assessing transmissibility over the time of sampling, we observe a tendency for estimated transmissibility within clades to slightly decrease over time in most clades. Although subtle, this pattern is consistent with the expectation of a decay in transmissibility in mainly non-recombining lineages caused by the accumulation of weakly deleterious mutations. SARS-CoV-2 remains a highly transmissible pathogen, though such a trend could conceivably play a role in the turnover of different global viral clades observed over the pandemic so far. CaveatsO_LIThis work is not about the severity of disease. We do not analyse the severity of disease. We do not present any evidence that SARS-CoV-2 has decreased in severity. C_LIO_LILineage replacement dynamics are affected by many factors. The trend we recover for a decrease in inferred transmissibility of a clade over time is a small effect. We caution against over-interpretation. This result would not affect the management of the SARS-CoV-2 pandemic: for example, we make no claims about any impact on the efficacy of particular non-pharmaceutical interventions (NPIs). C_LIO_LIOur phylogeny-based method to infer changes in estimated transmissibility due to recurrent mutations and deletions makes a number of simplifying assumptions. These may not all be valid. The consistent trend for the slight decrease we report might be due to an as-yet-unidentified systematic bias. C_LI

genomics↗

bioRxiv2021

Role of the mobilome in the global dissemination of the carbapenem resistance gene blaNDM

The mobile resistance gene blaNDM encodes the NDM enzyme which hydrolyses carbapenems, a class of antibiotics used to treat some of the most severe bacterial infections. blaNDM is globally distributed across a variety of Gram-negative bacteria on multiple plasmids, typically located within a highly recombining and transposon-rich genomic region. This complexity means the dynamics underlying the dissemination of blaNDM remain poorly resolved. In this work, we compile a dataset of over 6000 bacterial genomes harbouring the blaNDM gene including 104 newly generated PacBio hybrid assemblies from clinical and livestock associated isolates across China. We develop a novel computational approach to track structural variants surrounding blaNDM in bacterial genomes. This allows us to identify the prevalent genomic contexts of blaNDM and reconstruct the key mobile genetic elements and events in its global spread. We estimate that blaNDM emerged on a Tn125 transposon before 1985 but only reached a global prevalence around a decade after its first recorded observation in 2005. We find that the Tn125 transposon played an important role in early plasmid-mediated jumps of blaNDM but was overtaken by other elements in recent years including IS26-flanked pseudo-composite transposons and Tn3000. Lastly, we observe a notable correlation between plasmid backbones bearing blaNDM and the sampling location of isolates. This observation suggests that the dissemination of resistance genes is mainly driven by successive between-plasmid transposon jumps, with plasmid exchange much more restricted due to the adaptation of plasmids to specific bacterial hosts.

microbiology↗

bioRxiv2020

Detection of a bedaquiline / clofazimine resistance reservoir in Mycobacterium tuberculosis predating the antibiotic era

Drug resistance in tuberculosis (TB) poses a major ongoing challenge to public health. The recent inclusion of bedaquiline into TB drug regimens has improved treatment outcomes, but this advance is threatened by the emergence of strains of Mycobacterium tuberculosis (Mtb) resistant to bedaquiline. Clinical bedaquiline resistance is most frequently conferred by off-target resistance-associated variants (RAVs) in the mmpR5 gene (Rv0678), the regulator of an efflux pump, which can also confer cross-resistance to clofazimine, another TB drug. We compiled a dataset of 3,682 Mtb genomes, including 150 carrying variants in mmpR5 that have been associated to borderline (henceforth intermediate) or confirmed resistance to bedaquiline. We identified eight cases where RAVs were present in the genomes of strains collected prior to the use of bedaquiline in TB treatment regimes. Phylogenetic reconstruction points to multiple emergence events and circulation of RAVs in mmpR5, some estimated to predate the introduction of bedaquiline. However, epistatic interactions can complicate bedaquiline drug-susceptibility prediction from genetic sequence data. Indeed, in one clade of isolates where the RAV Ile67fs is estimated to have emerged prior to the antibiotic era, co-occurrence of mutations in mmpL5 are found to neutralise bedaquiline resistance. The presence of a pre-existing reservoir of Mtb strains carrying bedaquiline RAVs prior to its clinical use augments the need for rapid drug susceptibility testing and individualised regimen selection to safeguard the use of bedaquiline in TB care and control.

genomics↗

bioRxiv2020

No evidence for increased transmissibility from recurrent mutations in SARS-CoV-2

The COVID-19 pandemic is caused by the coronavirus SARS-CoV-2, which jumped into the human population in late 2019 from a currently uncharacterised animal reservoir. Due to this extremely recent association with humans, SARS-CoV-2 may not yet be fully adapted to its human host. This has led to speculations that some lineages of SARS-CoV-2 may be evolving towards higher transmissibility. The most plausible candidate mutations under putative natural selection are those which have emerged repeatedly and independently (homoplasies). Here, we formally test whether any of the recurrent mutations that have been observed in SARS-CoV-2 are significantly associated with increased viral transmission. To do so, we develop a phylogenetic index to quantify the relative number of descendants in sister clades with and without a specific allele. We apply this index to a carefully curated set of recurrent mutations identified within a dataset of 46,723 SARS-CoV-2 genomes isolated from patients worldwide. We do not identify a single recurrent mutation in this set convincingly associated with increased viral transmission. Instead, recurrent SARS-CoV-2 mutations currently in circulation appear to be evolutionary neutral. Recurrent mutations also seem primarily induced by the human immune system via host RNA editing, rather than being signatures of adaptation to the novel human host. In conclusion, we find no evidence at this stage for the emergence of significantly more transmissible lineages of SARS-CoV-2 due to recurrent mutations.

genomics↗

bioRxiv2020

Metagenomic evidence for a polymicrobial signature of sepsis

Our understanding of the host component of sepsis has made significant progress. However, detailed study of the microorganisms causing sepsis, either as single pathogens or microbial assemblages, has received far less attention. Metagenomic data offer opportunities to characterise the microbial communities found in septic and healthy individuals. In this study we apply gradient-boosted tree classifiers and a novel computational decontamination technique built upon SHapley Additive exPlanations (SHAP) to identify microbial hallmarks which discriminate blood metagenomic samples of septic patients from that of healthy individuals. Classifiers had high performance when using the read assignments to microbial genera (AUROC = 0.995), including after removal of species confirmed as the cause of sepsis through clinical testing (AUROC = 0.915). Models trained on single genera were inferior to those employing a polymicrobial model and we identified multiple co-occurring bacterial genera absent from healthy controls. ImportanceWhile prevailing diagnostic paradigms seek to identify single pathogens, our results point to the involvement of a polymicrobial community in sepsis. We demonstrate the importance of the microbial component in characterising sepsis, which may offer new biological insights into the aetiology of sepsis and allow the development of clinical diagnostic or even prognostic tools.

bioinformatics↗

bioRxiv2019

Large-scale network analysis captures biological features of bacterial plasmids

Many bacteria can exchange genetic material through horizontal gene transfer (HGT) mediated by plasmids and plasmid-borne transposable elements. Here, we study the population structure and dynamics of over 10,000 bacterial plasmids, by quantifying their genetic similarities and reconstructing a network based on their shared k-mer content. We use a community detection algorithm to assign plasmids into cliques, which correlate with plasmid gene content, bacterial host range, GC content, and existing classifications based on replicon and mobility (MOB) types. Further analysis of plasmid population structure allows us to uncover candidates for yet undescribed replicon genes and to identify transposable elements as the main drivers of HGT at broad phylogenetic scales. Our work illustrates the potential of network-based analyses of the bacterial mobilome and opens up the prospect of a natural, exhaustive classification framework for bacterial plasmids.

genomics↗

bioRxiv2019

Emergence of metabolic landscapes in yeast monolayer colonies

Microbial colonies are fascinating structures in which growth and internal organization reflect complex morphogenetic processes. Here, we generated a microfluidics device with arrays of long monolayer yeast colonies to further global understanding of how intercellular metabolic interactions affect the internal structure of colonies within defined boundary conditions. We observed the emergence of stable glucose gradients using fluorescently labelled hexose transporters and quantified the spatial correlations with intracolony growth rates and expression of other genes regulated by glucose availability. These landscapes depended on the external glucose concentration as well as secondary gradients, e.g., amino acid availability. This work demonstrates the regulatory genetic networks governing cellular physiological adaptation are the key to internal structuration of cellular assemblies. This approach could be used in the future to decipher the interplay between long-range metabolic interactions, cellular development and morphogenesis in more complex systems.

biophysics↗