Biology subjects

Blocker, A.

Publications and source records attributed to Blocker, A..

4 recordsLinked to original sources

bioRxiv2025

X-Atlas/Orion: Genome-wide Perturb-seq Datasets via a Scalable Fix-Cryopreserve Platform for Training Dose-Dependent Biological Foundation Models

The rapid expansion of massively parallel sequencing technologies has enabled the development of foundation models to uncover novel biological findings. While these have the potential to significantly accelerate scientific discoveries by creating AI-driven virtual cell models, their progress has been greatly limited by the lack of large-scale high-quality perturbation data, which remains constrained due to scalability bottlenecks and assay variability. Here, we introduce "Fix-Cryopreserve-ScRNAseq" (FiCS) Perturb-seq, an industrialized platform for scalable Perturb-seq data generation. We demonstrate that FiCS Perturb-seq exhibits high sensitivity and low batch effects, effectively capturing perturbation-induced transcriptomic changes and recapitulating known biological pathways and protein complexes. In addition, we release X-Atlas: Orion edition (X-Atlas/Orion), the largest publicly available Perturb-seq atlas. This atlas, generated from two genome-wide FiCS Perturb-seq experiments targeting all human protein-coding genes, comprises eight million cells deeply sequenced to over 16,000 unique molecular identifiers (UMIs) per cell. Furthermore, we show that single guide RNA (sgRNA) abundance can serve as a proxy for gene knockdown (KD) efficacy. Leveraging the deep sequencing and substantial cell numbers per perturbation, we also show that stratification by sgRNA expression can reveal dose-dependent genetic effects. Taken together, we demonstrate that FiCS Perturb-seq is an efficient and scalable platform for high-throughput Perturb-seq screens. Through the release of X-Atlas/Orion, we highlight the potential of FiCS Perturb-seq to address current scalability and variability challenges in data generation, advance foundation model development that incorporates gene-dosage effects, and accelerate biological discoveries.

genomics↗

bioRxiv2024

Maternal and Neonatal Colonization with Multidrug Resistant and Extended Spectrum beta-Lactamase Producing Escherichia coli and Klebsiella pneumoniae in a Cameroonian Labour Ward

BackgroundEscherichia coli and Klebsiella pneumoniae rank among the primary bacterial culprits in neonatal infections and fatalities in sub-Saharan Africa. This study sought to characterize the phenotypic and genotypic features of Escherichia coli and Klebsiella pneumoniae in a labour ward in Yaounde, Cameroon. MethodsA prospective and cross-sectional study spanning five months, from February 21 to June 30, 2022. Recto-vaginal swabs were obtained from expectant mothers, and nasopharyngeal swabs were collected from their babies. The samples were cultured on eosin methylene blue agar and isolates identified using the Enterosystem 18R kit. Extended-spectrum {beta}-lactamase (ESBL) production was assessed using CHROMAgar ESBL and the double disc synergy test. Antibiotic susceptibility was determined by the Kirby-Bauer disk diffusion method. Polymerase chain reaction (PCR) was employed to detect {beta}-lactamase genes blaSHV, blaCTX-M and blaTEM. ERIC-PCR was used to assess the clonal relatedness of isolates. ResultsE. coli was predominantly found in pregnant women (81%) and neonates (55%) while K. pneumoniae predominated in healthcare workers. Almost all pregnant women (90%) were colonized by one or more multi-drug resistant (MDR) isolates with 52% being concomitantly ESBL producers. Altogether, 22 neonates were positive for E. coli and/or K. pneumoniae and 19 (91%) were colonized by a MDR isolate. The blaCTX-M (75%) was the leading {beta}-lactamase gene detected. ConclusionOur study suggests that MDR- and ESBL-E. coli and K. pneumoniae are circulating at high prevalence in labour Yaounde. It emphasizes the necessity for strict infection prevention and control measures in conjunction with effective antimicrobial stewardship in the country.

molecular biology↗

bioRxiv2020

Bacterial flagella disrupt host cell membranes and interact with cytoskeletal components

Bacterial flagella have many established roles beyond swimming motility. Despite clear evidence of flagella-dependent adherence, the specificity of ligands and mechanisms of binding are still debated. In this study, the molecular basis of E. coli O157:H7 and S. Typhimurium flagella binding to epithelial cell cultures was investigated. Flagella interactions with host cell surfaces were intimate and crossed cellular boundaries as demarcated by actin and membrane labelling. SEM revealed flagella disappearing into cellular surfaces and TEM of S. Typhiumurium indicated host membrane deformation and disruption in proximity to flagella. Motor mutants of E. coli O157:H7 and S. Typhimurium caused reduced haemolysis compared to wild-type, indicating that membrane disruption was in part due to flagella rotation. Flagella from E. coli O157 (H7), EPEC O127 (H6), and S. Typhimurium (P1 & P2 flagella) were shown to bind to purified intracellular components of the actin cytoskeleton and directly increase in vitro actin polymerisation rates.

microbiology↗

bioRxiv2019

The AAA+ ATPase ClpX Is Critical for Growth and Development of Chlamydia trachomatis

Abstract:Chlamydia trachomatis (Ctr) is an obligate intracellular bacterium that undergoes a complex developmental cycle in which the bacterium differentiates between two functionally and morphologically distinct forms, the EB and RB, each of which expresses its own specialized repertoire of proteins. Both primary (EB to RB) and secondary (RB to EB) differentiation require protein turnover, and we hypothesize that proteases are critical for mediating differentiation. The Clp protease system is well conserved in bacteria and important for protein turnover. Minimally, the system relies on a serine protease subunit, ClpP, and a AAA+ ATPase, such as ClpX, that recognizes and unfolds substrates for ClpP degradation. In Chlamydia, ClpX is encoded within an operon 3 to clpP2. We present evidence that the chlamydial ClpX and ClpP2 orthologs are essential to organism viability and development. We demonstrate here that chlamydial ClpX is a functional ATPase and forms the expected homohexamer in vitro. Overexpression of a ClpX mutant lacking ATPase activity had a limited impact on DNA replication or secondary differentiation but, nonetheless, reduced EB viability with observable defects in EB morphology noted. Conversely, the overexpression of a catalytically inactive ClpP2 mutant significantly impacted developmental cycle progression by reducing the overall number of organisms. Blocking clpP2X transcription using CRISPR interference led to a decrease in bacterial growth, and this effect was complemented in trans by a plasmid copy of clpP2. Taken together, our data indicate that ClpX and the associated ClpP2 serve distinct functions in chlamydial developmental cycle progression and differentiation. Importance Chlamydia trachomatis is the leading cause of infectious blindness globally and the most reported bacterial sexually transmitted infection both domestically and internationally. Given the economic burden, the lack of an approved vaccine, and the use of broad-spectrum antibiotics for treatment of infections, an understanding of chlamydial growth and development is critical for the advancement of novel, targeted antibiotics. The Clp proteins comprise an important and conserved protease system in bacteria. Our work highlights the importance of the chlamydial Clp proteins to this clinically important bacterium. Additionally, our study implicates the Clp system playing an integral role in chlamydial developmental cycle progression, which may help establish models of how Chlamydia spp. and other bacteria progress through their respective developmental cycles. Our work also contributes to a growing body of Clp-specific research that underscores the importance and versatility of this system throughout bacterial evolution and further validates Clp proteins as drug targets.

microbiology↗