Biology subjects

Abraham, S.

Publications and source records attributed to Abraham, S..

At least 19 recordsLinked to original sources

bioRxiv2026

The frequency of resistant Escherichia coli isolation from seagulls is positively related to human population density

Antimicrobial resistance (AMR) is a major global public health threat. Wild birds, including seagulls, are increasingly recognised as potential reservoirs and disseminators of resistant bacteria linked to human activity. The objective of the study was to assess the association between human population density and the occurrence of Escherichia coli resistant to critically important antimicrobials in Australian seagulls. Faecal samples were collected from seagull populations in coastal regions across Australia representing differing human population densities. Resistant E. coli isolates were identified and characterised using multilocus sequence typing and plasmid incompatibility group analysis to determine relatedness to human associated lineages. The frequency of resistant E. coli isolation increased with human population density. The predominant sequence types ST10, ST131 and ST354 comprised 24.5% of isolates and belong to globally distributed human associated lineages linked to extraintestinal pathogenic E. coli. Many isolates carried IncF and IncI plasmids, which are key vectors of blaCTX-M extended spectrum beta lactamase genes and plasmid mediated quinolone resistance determinants commonly reported in human clinical strains. IMPORTANCEThese findings support the contention that seagulls primarily acquire resistant bacteria through contact with anthropogenic activities. Once acquired, these bacteria may be disseminated to other seagulls, birds and animals, including being transmitted to humans.

microbiology↗

bioRxiv2026

Natural variation in male frequency fails to predict inbreeding responses in Caenorhabditis elegans

In androdioecious species like Caenorhabditis elegans, where the primary mode of reproduction is self-fertilization, the evolutionary role of males has long puzzled biologists. One proposed benefit of males is the potential to escape inbreeding depression. We tested this by enforcing seven generations of inbreeding across nine C. elegans strains differing in baseline male frequency and measuring competitive relative fitness before and after inbreeding. We then relaxed inbreeding for four generations to assess recovery. We predicted that strains with higher male frequency, and greater opportunity for outcrossing, would exhibit faster recovery once inbreeding was relaxed. Strains varied substantially in their responses with most showing significant fitness declines and partial recovery but neither the magnitude of inbreeding depression nor the extent of recovery correlated with male frequency. These results show that male frequency is a poor predictor of inbreeding responses and does not reliably reflect realized outcrossing or its fitness consequences.

evolutionary biology↗

bioRxiv2025

Cell State Chaos Underpins the Evolution of SMARCA4-Deficient Dedifferentiated Endometrial Cancer

Dedifferentiated endometrial carcinoma (DDEC) is a histologically unique cancer type, wherein well-differentiated regions lie adjacent to morphologically distinct, high-grade lesions that are histologically undifferentiated. Previous studies have determined that in nearly half of the cases dedifferentiation is associated with the genomic inactivation of SMARCA4, a catalytic subunit belonging to the SWI/SNF chromatin remodelling complex (SWI/SNF CRC), suggesting that SMARCA4 loss causes dedifferentiation. Herein, using gene editing, we reveal that when serially passaged in mice, SMARCA4-deficient endometrial cancer cells repeatably and predictably generate heterogeneous admixtures of differentiated and undifferentiated cells, resembling human DDEC. Surprisingly, despite this metamorphosis, SMARCA4 loss does not induce lineage plasticity nor reprogramming to a less differentiated fate. Rather, single-cell sequencing combined with barcoding demonstrated that SMARCA4 loss induces a dysregulated epigenome that allows cells to randomly move through cellular states that are otherwise shared with SMARCA4-expressing well-differentiated cancer cells. This finding was validated using a cohort of patient samples, such that epithelial fate markers (E-CADHERIN) can be detected in morphologically undifferentiated cells. Collectively, this work constitutes the first repeatable model of human dedifferentiated cancer and suggests that histological dedifferentiation is not due to the acquisition of a stem cell-like fate. Rather, undifferentiated tissue emerges due to epigenomic dysfunction concomitant with the arbitrary movement of cancer cells between cellular states.

cancer biology↗

bioRxiv2025

NF-kB-Dependent Transcriptional Regulation of Piezo1 Mediates Bacterial Clearance on Stiffened Lung

Respiratory pathogens, such as Pseudomonas aeruginosa damage the alveolar-capillary barrier leading to lung injury and stiffness. Lung stiffness is a key macrophage signal for bacterial clearance, but it remains unknown how stiffness-sensing mechanosensitive ion channels in macrophages are regulated during pneumonia. Macrophage Piezo1 is critical to bacterial clearance in experimental pneumonia in vivo; however, identification of putative matrix-derived signals and the mechanism of their effects remain to be determined. To our knowledge, our work is the first to show that during pneumonia, transcription of the mechanosensitive ion channel Piezo1 is increased in macrophages by the NF-{kappa}B transcription factor, p65, through its signaling adaptor protein, MyD88, leading to increased Piezo1 Ca2+ channel activity. Piezo1 mRNA abundance is increased in association with open chromatin at the Piezo1 promoter in macrophages. The enhanced level of Piezo1 increases the abundance of transcription factor EB (Tfeb) resulting in lysosome biogenesis and stiffness-dependent phagolysosome maturation, a critical step for macrophage bacterial clearance. Our data support the mechanism whereby transcription of macrophage Piezo1 is enhanced by p65 to augment bacterial clearance on an injured, stiffened lung matrix during pneumonia. Therefore, Piezo1 is a future therapeutic target against pneumonia-induced lung injury.

immunology↗

bioRxiv2024

Rules of engagement for condensins and cohesins guide mitotic chromosome formation

During mitosis, interphase chromatin is rapidly converted into rod-shaped mitotic chromosomes. Using Hi-C, imaging, proteomics and polymer modeling, we determine how the activity and interplay between loop-extruding SMC motors accomplishes this dramatic transition. Our work reveals rules of engagement for SMC complexes that are critical for allowing cells to refold interphase chromatin into mitotic chromosomes. We find that condensin disassembles interphase chromatin loop organization by evicting or displacing extrusive cohesin. In contrast, condensin bypasses cohesive cohesins, thereby maintaining sister chromatid cohesion while separating the sisters. Studies of mitotic chromosomes formed by cohesin, condensin II and condensin I alone or in combination allow us to develop new models of mitotic chromosome conformation. In these models, loops are consecutive and not overlapping, implying that condensins do not freely pass one another but stall upon encountering each other. The dynamics of Hi-C interactions and chromosome morphology reveal that during prophase loops are extruded in vivo at [~]1-3 kb/sec by condensins as they form a disordered discontinuous helical scaffold within individual chromatids.

molecular biology↗

bioRxiv2024

Oxytocin signaling regulates maternally-directed behavior during early life

Oxytocin is essential in shaping social behavior across the lifespan. While the role of oxytocin signaling in parental care has been widely investigated, little is known about its function in social behavior during early life. This is partly due to the lack of precise technologies for studying the developing brain. Here, we studied the role of oxytocin in pup social behavior under acute separation from and reunion with the mother. We show that the activity of oxytocin neurons was increased by acute maternal separation and returned to baseline after reunion. Behaviorally, maternally-separated pups emitted more ultrasonic vocalizations upon reunion, which were further modulated by nipple attachment behavior. These effects were attenuated by blocking the oxytocin receptor during maternal separation. To investigate the role of oxytocin neurons with higher precision, we established a method for transcranial optogenetic silencing of neuronal activity in untethered and freely behaving pups. Using this approach, we found that silencing of oxytocin neurons during maternal separation disrupted vocal behavior during separation and reunion in a sex-specific manner. Our findings reveal an important role of oxytocin in context-dependent vocal communication in pups, offering new insights into the mechanisms of social behavior during early life.

neuroscience↗

bioRxiv2023

Unique territorial and sub-chromosomal organization revealed in the holocentric moth Bombyx mori

Hallmarks of multicellular eukaryotic genome organization are chromosome territories, compartments, and loop-extrusion-mediated structures, including TADs. However, these are mainly observed in model organisms, and most eukaryotes remain unexplored. Using Hi-C in the silkworm Bombyx mori we discover a novel chromatin folding structure, compartment S, which is "secluded" from the rest of the chromosome. This compartment exhibits loop extrusion features and a unique genetic and epigenetic landscape, and it localizes towards the periphery of chromosome territories. While euchromatin and heterochromatin display preferential compartmental contacts, S domains are remarkably devoid of contacts with other regions, including with other S domains. Polymer simulations show that this contact pattern can only be explained by high loop-extrusion activity within compartment S, combined with low extrusion elsewhere through the genome. This unique, targeted extrusion represents a novel phenomenon and underscores how evolutionarily conserved mechanisms--compartmentalization and loop extrusion--can be repurposed to create new 3D genome architectures.

genomics↗

bioRxiv2023

An Ionic Lock Mediates Interdomain Interactions in G protein αi subunits and Determines Gαi Subtype Signaling Specificity

Highly homologous members of the Gi family, Gi1-3, have distinct tissue distributions and physiological functions, yet the functional properties of these proteins with respect to GDP/GTP binding and regulation of adenylate cyclase are very similar. We recently identified PDZ-RhoGEF (PRG) as a novel Gi1 effector, however, it is poorly activated by Gi2. Here, in a proteomic proximity labeling screen we observed a strong preference for Gi1 relative to Gi2 with respect to engagement of a broad range of potential targets. We investigated the mechanistic basis for this selectivity using PRG as a representative target. Substitution of either the helical domain (HD) from Gi1 into Gi2 or substitution of a single amino acid, A230 in Gi2 to the corresponding D in Gi1, largely rescues PRG activation and interactions with other Gi targets. Molecular dynamics simulations combined with Bayesian network models revealed that in the GTP bound state, dynamic separation at the HD-Ras-like domain (RLD) interface is prevalent in Gi2 relative to Gi1 and that mutation of A230s4h3.3 to D in Gi2 stabilizes HD-RLD interactions through formation of an ionic interaction with R145HD.11 in the HD. These interactions in turn modify the conformation of Switch III. These data support a model where D229s4h3.3 in Gi1 interacts with R144HD.11 stabilizes a network of interactions between HD and RLD to promote protein target recognition. The corresponding A230 in Gi2 is unable to form the "ionic lock" to stabilize this network leading to an overall lower efficacy with respect to target interactions. This study reveals distinct mechanistic properties that could underly differential biological and physiological consequences of activation of Gi1 or Gi2 by GPCRs.

pharmacology and toxicology↗

bioRxiv2023

Escherichia coli isolated from commercial layer hens in Australia remain susceptible to critically important antimicrobials

Controlling the use of the most critically important antimicrobials (CIAs) in food animals has been identified as one of the key measures required to curb the transmission of antimicrobial resistant bacteria from animals to humans. Expanding the evidence demonstrating the effectiveness of restricting CIA usage for preventing the emergence of resistance to key drugs amongst commensal organisms in animal production would do much to strengthen international efforts to control antimicrobial resistance (AMR). As Australia has strict controls on antimicrobial use in layer hens, and internationally comparatively low levels of poultry disease due to strict national biosecurity measures, we investigated whether these circumstances have resulted in curtailing development of critical forms of AMR. The work comprised a cross-sectional national survey of 62 commercial layer farms with each assessed for AMR in Escherichia coli isolates recovered from faeces. Minimum inhibitory concentration analysis using a panel of 13 antimicrobials was performed on 296 isolates with those exhibiting phenotypic resistance to fluoroquinolones (a CIA) or multi-class resistance (MCR) subjected to whole genome sequencing. Overall, 52.0% of the isolates were susceptible to all antimicrobials tested, and all isolates were susceptible to ceftiofur, chloramphenicol and colistin. Resistance was observed to ampicillin (16.2%), cefoxitin (1.4%), ciprofloxacin (2.7%), florfenicol (2.4%), gentamicin (1.0%), streptomycin (4.7%), tetracycline (37.8%) and trimethoprim/sulfamethoxazole (10.5%). Multi-class resistance was observed in 23 isolates (7.7%), with one isolate (ST746) exhibiting resistance to five antimicrobial classes. Whole genome sequencing found that ciprofloxacin-resistant (fluoroquinolone) isolates were devoid of both known chromosomal mutations in the quinolone resistance determinant regions and plasmid-mediated quinolone resistance genes (qnr) - other than in one isolate (ST155) which carried the qnrS gene. Two MCR E. coli isolates with ciprofloxacin-resistance were found to be carrying known resistance genes including aadA1, dfrA1, strA, strB, sul1, sul2, tet(A), blaTEM-1B, qnrS1 and tet(A). Overall, this study found that E. coli from layer hens in Australia have low rates of AMR, likely due to strict control on antimicrobial usage achieved by the sum of regulation and voluntary measures.

microbiology↗

bioRxiv2022

Cooltools: enabling high-resolution Hi-C analysis in Python

Chromosome conformation capture (3C) technologies reveal the incredible complexity of genome organization. Maps of increasing size, depth, and resolution are now used to probe genome architecture across cell states, types, and organisms. Larger datasets add challenges at each step of computational analysis, from storage and memory constraints to researchers time; however, analysis tools that meet these increased resource demands have not kept pace. Furthermore, existing tools offer limited support for customizing analysis for specific use cases or new biology. Here we introduce cooltools (https://github.com/open2c/cooltools), a suite of computational tools that enables flexible, scalable, and reproducible analysis of high-resolution contact frequency data. Cooltools leverages the widely-adopted cooler format which handles storage and access for high-resolution datasets. Cooltools provides a paired command line interface (CLI) and Python application programming interface (API), which respectively facilitate workflows on high-performance computing clusters and in interactive analysis environments. In short, cooltools enables the effective use of the latest and largest genome folding datasets.

bioinformatics↗

bioRxiv2022

Antimicrobial Resistance, Genomic, and Public Health Insights into Enterococcus spp. from Australian Cattle

Enterococci are opportunistic, potentially life-threatening pathogens of humans that are difficult to manage due to antimicrobial resistance. Historically, enterococci entering the food-chain through livestock have been viewed as a likely source of antimicrobial resistance in humans. Australian human-derived clinical enterococci have a propensity to be resistant to multiple classes of antimicrobials including vancomycin. Recent Australian studies involving pigs and chicken have virtually excluded these species as reservoirs of infection for resistant enterococci in humans. However, the Australian bovine population has not been similarly assessed. This study investigates the antimicrobial resistance profiles of enterococci from Australian cattle and the phylogenetic relationship between E. faecium isolated from cattle and human sepsis cases. Minimum inhibitory concentration assays were performed for E. faecium (n=343), E. faecalis (n=92), and E. hirae (n=284) against a panel of 15 antimicrobials. The majority of isolates were sensitive to all tested antimicrobials. Erythromycin resistance was most prevalent for E. faecium isolates (18.7%), daptomycin for E. faecalis (12.1%) and tetracycline for E. hirae (13.3%). Phenotypically, 1 E. faecalis was resistant to vancomycin and 9 were resistant to linezolid (E. faecium n=4, E. faecalis n=2, E. hirae n=3) but this was not confirmed with any know genotype. A subset of 67 E. faecium isolates selected for comparative phylogenetic analysis revealed that bovine isolates clustered with other livestock-derived and van-negative human isolates. In conclusion, there is a low prevalence of antimicrobial resistance amongst enterococci from Australian cattle which are unlikely to be precursor strains to vancomycin-resistant strains currently circulating in Australian hospitals. ImportanceEnterococci resistant to critically important antimicrobials such as vancomycin and linezolid are difficult to manage in health care settings. Historically, there has been the belief that livestock can act as a reservoir of resistance for human infections. Previous studies in poultry and pork have demonstrated that isolates derived from these livestock are unlikely to be precursor strains for currently circulating vancomycin resistant-Enterococci causing infection in humans. To date, there has been no study looking at the genetic similarity of bovine derived Enterococci and the relationship to human pathogenic strains. In this study we performed phenotypic and genotypic characterization of bovine derived Enterococci along with comparative phylogenetic analysis with other livestock derived and human sepsis derived E. faecium isolates. We found that while non-vancomycin resistant strain sharing occurs between hosts, cattle are unlikely to be precursor strains for vancomycin resistant human E. faecium infections.

microbiology↗

bioRxiv2022

Improving and evaluating deep learning models of cellular organization

MotivationCells contain dozens of major organelles and thousands of other structures, many of which vary extensively in their number, size, shape and spatial distribution. This complexity and variation dramatically complicates the use of both traditional and deep learning methods to build accurate models of cell organization. Most cellular organelles are distinct objects with defined boundaries that do not overlap, while the pixel resolution of most imaging methods is not sufficient to resolve these boundaries. Thus while cell organization is conceptually object-based, most current methods are pixel-based. Using extensive image collections in which particular organelles were fluorescently-labeled, deep learning methods can be used to build conditional autoencoder models for particular organelles. A major advance occurred with the use of a U-net approach to make multiple models all conditional upon a common reference, unlabeled image, allowing the relationships between different organelles to be at least partially inferred. ResultsWe have developed improved GAN-based approaches for learning these models and have also developed novel criteria for evaluating how well synthetic cell images reflect the properties of real images. The first set of criteria measure how well models preserve the expected property that organelles do not overlap. We also developed a modified loss function that allows retraining of the models to minimize that overlap. The second set of criteria uses object-based modeling to compare object shape and spatial distribution between synthetic and real images. Our work provides the first demonstration that, at least for some organelles, deep learning models can capture object-level properties of cell images. AvailabilityA Reproducible Research Archive containing all source code, generated images and analysis results will be made available at http://murphylab.cbd.cmu.edu/Software upon publication. Contactmurphy@cmu.edu Supplementary informationSupplementary data are available at Bioinformatics online.

bioinformatics↗

bioRxiv2021

Proximity to human settlement is directly related to carriage of critically important antimicrobial-resistant Escherichia coli and Klebsiella pneumoniae in Silver Gulls

Human population and activities play an important role in dissemination of antimicrobial resistant bacteria. This study investigated the relationship between carriage rates of critically important antimicrobial-resistant (CIA-R) Escherichia coli and Klebsiella pneumoniae by Silver Gulls and their proximity to human populations. Faecal swabs (n=229) were collected from Silver Gulls across 10 southern coastline locations in Western Australia (WA). The sampling locations included main town centres and remote areas. Fluoroquinolone and extended-spectrum cephalosporin-resistant E. coli and K. pneumoniae were isolated and tested for antimicrobial sensitivity. Genome sequencing was performed to validate phenotypic resistance profiles and determine the molecular characteristics of strains. CIA-R E. coli and K. pneumoniae were detected in 69 (30.1%) and 20 (8.73%) of the faecal swabs respectively. Two large urban locations tested positive for CIA-R E. coli (frequency ranging from 34.3%-84.3%), and/or for CIA-R K. pneumoniae (frequency ranging from 12.5%-50.0%). A small number of CIA-R E. coli (3/31, 9.7%) were identified at a small tourist town, but no CIA-R bacteria were recovered from gulls at remote sites. Commonly detected E. coli sequence types (STs) included ST131 (12.5%) and ST1193 (10.0%), and five K. pneumoniae STs were found. Resistance genes including blaCTX-M-3, blaCTX-M-15 and blaCTX-M-27 were identified in both bacterial species. High-level colonisation of CIA-R E. coli and K. pneumoniae in Silver Gulls in and around urban areas compared to remote locations substantiates that anthropogenic activities are strongly associated with acquisition of resistant bacteria by gulls. ImportanceHumans play an important role in dissemination of antimicrobial resistant bacteria. This study investigated the relationship between carriage rates of resistant bacterial pathogens (Escherichia coli and Klebsiella pneumoniae) among Silver Gulls and their proximity to human populations. The frequency of resistant E. coli carriage was high (ranging from 34.3 - 84.3%) in the samples collected from areas with high human population density while resistant K. pneumoniae frequencies at these sites varied from 0 to 50%. However, resistant E. coli and K. pneumoniae were not recovered from any of the remote sites that did not have a permanent human population. This study, conducted across a large stretch of the southwestern Australian coastline, indicated that seagulls act as vectors in carrying and disseminating antimicrobial resistant bacteria, including clinically significant strains. High-level colonisation of resistant E. coli and K. pneumoniae in Silver Gulls in and around urban areas compared to remote locations substantiates that human activities are strongly associated with acquisition of resistant bacteria by Silver gulls.

microbiology↗

bioRxiv2021

Identification of G Protein αi Signaling Partners by Proximity Labeling Reveals a Network of Interactions that Includes PDZ-RhoGEF

G protein-coupled receptors (GPCRs) that couple to the Gi family of G proteins are key regulators of cell and tissue physiology. Our recent work has discovered novel roles for Gi in migration of neutrophils and fibrosarcoma cells downstream of activated chemoattractant receptors, but the molecular target(s) of Gi in these processes remain to be identified. We adopted an intact cell proximity-based labeling approach using BioID2 coupled to tandem mass tag (TMT)-based quantitative proteomics to identify proteins that selectively interact with the GTP-bound form of Gi1. Multiple targets were identified and validated for selective biotinylation by active BioID2-Gi1(Q204L), suggesting a previously unappreciated network of interactions for activated Gi proteins in intact cells. Extensive characterization of one candidate protein, PDZ-RhoGEF (PRG), revealed that active-Gi1 strongly activates PRG. Strikingly, large differences in the ability of Gi1, Gi2, and Gi3 isoforms to activate PRG were observed despite over 85% sequence identity. We also demonstrate the functional relevance of the interaction between active Gi and PRG ex vivo in primary human neutrophils. Identification and characterization of new targets regulated by Gi both individually and in networks provide insights that will aid not only in investigation of diverse functional roles of Gi-coupled GPCRs in biology but also in the development of novel therapeutic approaches. SummaryProximity-based labeling approach was used to identify signaling networks and signaling mechanisms downstream of Gi-coupled receptors.

pharmacology and toxicology↗

bioRxiv2021

Validation of selective agars for detection and quantification of Escherichia coli resistant to critically important antimicrobials

Success in the global fight against antimicrobial resistance (AMR) is likely to improve if surveillance can be performed more rapidly, affordably and on a larger scale. An approach based on robotics and agars incorporated with antimicrobials has enormous potential to achieve this. However, there is a need to identify the combinations of selective agars and key antimicrobials yielding the most accurate counts of susceptible and resistant organisms. A series of designed experiments involving 1,202 plates identified the best candidate-combinations from six commercially available agars and five antimicrobials using 18 Escherichia coli strains as either pure cultures or inoculums within faeces. The effect of various design factors on colony counts were analysed in generalised linear models. Without antimicrobials, Brilliance E. coli (Brilliance) and CHROMagar ECC (CHROMagar) agars yielded 28.9% and 23.5% more colonies than MacConkey agar. The order of superiority of agars remained unchanged when faecal samples with and without spiking of resistant E. coli were inoculated onto agars with or without specific antimicrobials. When incorporating antimicrobials at varying concentrations, it was revealed that ampicillin, tetracycline and ciprofloxacin are suitable for incorporation into Brilliance and CHROMagar agars at all defined concentrations. Gentamicin was only suitable for incorporation at 8 and 16 g/mL while ceftiofur was only suitable at 1 g/mL. CHROMagar ESBL agar supported growth of a wider diversity of extended-spectrum cephalosporin-resistant E. coli. The findings demonstrate the potential for combining robotics with agars to deliver AMR surveillance on a vast scale with greater sensitivity of detection and strategic relevance. IMPORTANCEEstablished models of surveillance for AMR in livestock typically have a low sampling intensity which creates a tremendous barrier to understanding the variation of resistance amongst animal and food enterprises. However, developments in laboratory robotics now make it possible to rapidly and affordably process high volumes of samples. Combined with modern selective agars incorporating antimicrobials, this forms the basis of a novel surveillance process for identifying resistant bacteria by chromogenic reaction including accurately detecting and quantifying their presence even when present at low concentration. As Escherichia coli is a widely preferred indicator bacterium for AMR surveillance, this study identifies the optimal selective agar for quantifying resistant E. coli by assessing the growth performance on agars with antimicrobials. The findings are the first step towards exploiting laboratory robotics in an up-scaled approach to AMR surveillance in livestock with wider adaptations in food, clinical microbiology and public health.

microbiology↗

bioRxiv2021

Robotic Antimicrobial Susceptibility Platform (RASP): A Next Generation Approach to One-Health Surveillance of Antimicrobial Resistance.

BackgroundSurveillance of antimicrobial resistance (AMR) is critical to reducing its wide-reaching impact. Its reliance on sample size invites solutions to longstanding constraints regarding scalability. A robotic platform (RASP) was developed for high-throughput AMR surveillance in accordance with internationally recognised standards (CLSI and ISO 20776-1:2019) and validated through a series of experiments. MethodsExperiment A compared RASPs ability to achieve consistent MICs to that of a human technician across eight replicates for four E. coli isolates. Experiment B assessed RASPs agreement with human performed MICs across 91 E. coli isolates with a diverse range of AMR profiles. Additionally, to demonstrate its real-world applicability, the RASP workflow was then applied to five faecal samples where a minimum of 47 E. coli per animal (239 total) were evaluated using an AMR indexing framework. ResultsFor each drug-rater-isolate combination in experiment A, there was a clear consensus of the MIC and deviation from the consensus remained within one doubling-dilution (the exception being gentamicin at two dilutions). Experiment B revealed a concordance correlation coefficient of 0.9670 (95%CI: 0.9670 - 0.9670) between the robot and human performed MICs. RASPs application to the five faecal samples highlighted the intra-animal diversity of gut commensal E. coli, identifying between five and nine unique isolate AMR phenotypes per sample. ConclusionsWhile adhering to internationally accepted guidelines, RASP was superior in throughput, cost and data resolution when compared to an experienced human technician. Integration of robotics platforms in the microbiology laboratory is a necessary advancement for future One-Health AMR endeavours.

microbiology↗

bioRxiv2020

Systematic evaluation of chromosome conformation capture assays

Chromosome conformation capture (3C)-based assays are used to map chromatin interactions genome-wide. Quantitative analyses of chromatin interaction maps can lead to insights into the spatial organization of chromosomes and the mechanisms by which they fold. A number of protocols such as in situ Hi-C and Micro-C are now widely used and these differ in key experimental parameters including cross-linking chemistry and chromatin fragmentation strategy. To understand how the choice of experimental protocol determines the ability to detect and quantify aspects of chromosome folding we have performed a systematic evaluation of experimental parameters of 3C-based protocols. We find that different protocols capture different 3D genome features with different efficiencies. First, the use of cross-linkers such as DSG in addition to formaldehyde improves signal-to-noise allowing detection of thousands of additional loops and strengthens the compartment signal. Second, fragmenting chromatin to the level of nucleosomes using MNase allows detection of more loops. On the other hand, protocols that generate larger multi-kb fragments produce stronger compartmentalization signals. We confirmed our results for multiple cell types and cell cycle stages. We find that cell type-specific quantitative differences in chromosome folding are not detected or underestimated by some protocols. Based on these insights we developed Hi-C 3.0, a single protocol that can be used to both efficiently detect chromatin loops and to quantify compartmentalization. Finally, this study produced ultra-deeply sequenced reference interaction maps using conventional Hi-C, Micro-C and Hi-C 3.0 for commonly used cell lines in the 4D Nucleome Project.

genomics↗

bioRxiv2020

BRD4-mediated repression of p53 is a target for combination therapy in AML

Acute Myeloid Leukemia (AML) is a typically-lethal molecularly heterogeneous disease, with few broad-spectrum therapeutic targets. Unusually, most AML retain wild-type TP53, encoding the pro-apoptotic tumor suppressor p53. MDM2 inhibitors (MDM2i), which activate wild-type p53, and BET inhibitors (BETi), targeting the BET-family co-activator BRD4, both show encouraging pre-clinical activity, but limited clinical activity as single agents. Here, we report synergistic toxicity of combined MDM2i and BETi towards AML cell lines, primary human blasts and mouse models, resulting from BETis ability to evict an unexpected repressive form of BRD4 from p53 target genes, and hence potentiate MDM2i-induced p53 activation. These results indicate that wild-type TP53 and a transcriptional repressor function of BRD4 together represent a potential broad-spectrum synthetic therapeutic vulnerability for AML.

cell biology↗