Biology subjects

Hughes, D.

Publications and source records attributed to Hughes, D..

At least 19 recordsLinked to original sources

bioRxiv2026

Spaghettification: How extreme replichore imbalance impacts bacterial replication and morphology

Bacterial chromosomes are typically organized into two similarly sized replichores to synchronize bidirectional replication and fusion within a Tus/ter-defined termination zone. Previous studies have suggested that replichore balance in both Salmonella and E. coli is maintained by selection for efficient and properly coordinated replication termination and chromosome segregation. However, there is a lack of understanding of the causal relationship between replichore asymmetry, cellular fitness, and replication-fork convergence, as well as how the termination machinery behaves when the two replichores differ substantially in length. Here we dissect the impact of replichore asymmetry by engineering 41 isogenic Salmonella inversion mutants spanning progressive imbalances, complemented by duplication-based asymmetries. Our constructed strains show that even cells with severely imbalanced chromosomes, where one replichore is 11-fold longer in size than the other (0.38 Mb vs. 4.48 Mb) are viable. However, relative fitness declined linearly with replichore size difference at approximately -0.17 per Mb. Additionally, highly imbalanced strains exhibited substantial division defects with severe cell elongation and lysis. Replication profiling showed that, even under extreme replichore asymmetry, forks consistently converge at the innermost ter sites of the shorter replichore and that two sequential Tus/ter barriers were sufficient for complete replication fork arrest. Together, these results establish a quantitative rule linking replichore asymmetry to fitness, define the termination position under imbalance, and reveal a dual Tus/ter barrier that safeguards termination when one replichore reaches the terminus region ahead of the other. Collectively these results connect chromosome architecture to replication mechanics and cell-division outcomes.

microbiology↗

bioRxiv2025

Mitochondrial stress in Fabry disease

Fabry disease (FD) is clinically heterogeneous. As some GLA variants attain similar levels of residual activity but result in a range of phenotypes, our aim is to understand factors influencing phenotypic variability. The mitochondrial unfolded protein response (mtUPR) is a stress response mechanism activated by multiple forms of mitochondrial dysfunction including accumulation of misfolded protein. As mitochondrial dysfunction has been reported, we investigated intracellular levels of heat shock protein 60 (Hsp60) by western blotting in 27 FD patients: 11 N215S (7 males) and 16 non-N215S (7 males) vs 4 heathy controls (HC, 3 males and 1 female). Serum Fibroblast Growth Factor-21 (FGF21), and Growth Differentiation Factor-15 (GDF-15) were also measured. Clinical outcomes explored included the Mainz Severity Score Index (MSSI), the Age-Adjusting Severity Scores (AASS), estimated glomerular filtration rate (eGFR) and left ventricular mass indexed to height (LVMI). Globotriaosylsphingosine (lyso-Gb3) data was available for a subset of participants. Hsp60 showed no significant differences between groups (males FD: 0.29 vs HC: 0.11, females HC: 0.26 vs FD: 0.19 Hsp60/LC), however, differences among FD patients were noted. While some had over 2-fold that of HCs, others had less than half of HCs despite genotype and gender. When analysed in terms of severity scores, the N215S group with higher levels of Hsp60 corresponded with a milder phenotype. LVMI and eGFR also seemed to improve with higher levels of Hsp60 only for this group. In terms of FGF-21 and GDF-15, lower levels showed a trend with higher Hsp60 levels and LVMI in the N215S group. To conclude, our findings suggest a potential role for mtUPR activation, as evidenced by intracellular Hsp60 levels, in modulating cardiac and renal manifestations in Fabry disease. These preliminary associations highlight the need for longitudinal studies to validate Hsp60 and mitokines as biomarkers of disease progression, aiming to inform personalized approaches that improve outcomes across Fabry disease phenotypes.

pathology↗

bioRxiv2025

Sex differences in alpha galactosidase protein processing and its impact on disease severity in Fabry disease

Fabry disease (FD) is an X-linked disorder due to mutations in the -galactosidase A (GLA) gene. The condition is characterized by low GLA activity and accumulation of toxic sphingolipids. Some patients present full disease symptoms whereas others have one system affected, generally the heart or kidney. This suggests that a mutation in the GLA gene is necessary to cause FD, but other factors may contribute to its clinical expression. To investigates the impact of GLA mutant protein processing on GLA activity and disease expression, 26 individuals with FD (14 males) were studied. Clinical outcomes explored included the Mainz Severity Score Index (MSSI), the Age-Adjusting Severity Scores (AASS), glomerular filtration rate (GFR), and left ventricular mass index (LVMI). Globotriaosylsphingosine (lyso-Gb3) data was available for a subset of participants. Whole cell lysates were employed to assess GLA activity and GLA protein levels. Additionally, endoglycosidase H digestion was performed on cell lysates to quantify each GLA protein form: immature 50 kDa endoplasmic reticulum form and mature 46 kDa lysosomal form. The latter was employed to calculate lysosomal GLA activity and its relationship with clinical outcomes was studied. Fabry participants exhibited more of the immature form of GLA protein (0.23 vs 0.66, p= 0.04). Female patients exhibited higher total GLA activity (19.0 vs 5.1 nmol/hr/mg), total GLA protein levels 0.13 vs 0.50 GLA/LC, p= 0.003), and 46 kDa mature lysosomal form levels than male patients (0.53 vs 0.10, p= 0.001). Additionally, females showed a significant correlation between the GLA mature form and GLA activity (r2= 0.59, p= 0.04). Consistently, lysosomal GLA activity exhibited significant associations with MSSI (r2= -0.66, p= 0.02) and GFR (r2= 0.59, p= 0.04) only in this sex group. These results suggest that total GLA protein levels are linked to the severity of FD manifestations, particularly in females via enzyme activity.

pathology↗

bioRxiv2025

Development of a bioassay guided genome mining approach for antifungal natural product discovery from pseudomonads

Zymoseptoria tritici causes Septoria Leaf Blotch disease of wheat and has evolved to overcome most chemical and genetic control methods. As such, new tools are required for future disease control. We identified Pseudomonas isolates that antagonise Z. tritici through the production of secreted secondary metabolites, using a novel in vitro Z. tritici antagonism assay. In addition to high-throughput qualitative assessment of Pseudomonas antagonism of Z. tritici, a quantitative assessment identified variation in the sensitivity of Z. tritici isolates to antagonism by a subset of Pseudomonas isolates. Genome assemblies of 3 strongly antagonistic Pseudomonas isolates were found to contain a predicted Biosynthetic Gene Cluster (BGC) with high sequence similarity to a reference BGC encoding the biosynthesis of the known antifungal compound 2,4-diacetylphloroglucinol (2,4-DAPG). Mutagenesis of the core biosynthetic gene phlD resulted in a loss of 2,4-DAPG production in Pseudomonas isolate Roth82, and a loss of Z. tritici inhibition in the antagonism assay. These results demonstrate that the described in vitro antagonism assay can be used to identify, quantify and mechanistically characterise bacterial antagonism of Z. tritici through the production of secondary metabolites. This is the first study to find significant differences in the response of genetically diverse isolates of Z. tritici isolates to bacterial antagonists, suggesting sensitivity to bacterial antagonism exists as a quantitative trait within natural Z. tritici populations. Our approach can be used to identify and characterise putatively novel BGCs that encode natural products with antifungal activity against Z. tritici.

microbiology↗

bioRxiv2025

Uncovering functional deterioration in the rhizosphere microbiome associated with wheat dwarfing

BackgroundOne of the biggest developments of wheat domestication was the development of semi-dwarf cultivars that respond well to fertilisers and produce higher yields without lodging. Consequently, this change has also impacted the wheat microbiome, often resulting in reduced selection of taxa and a loss of network complexity in the rhizospheres of semi-dwarf cultivars. Given the importance of rhizosphere microbiomes for plant health and performance, it is imperative that we understand if and how these changes have affected their function. Here, we use shotgun metagenomics to classify the functional potential of prokaryote communities from the rhizospheres of tall and semi-dwarf cultivars to compare the impact of wheat dwarfing on rhizosphere microbiome functions. ResultsWe found distinct taxonomic and functional differences between tall and semi-dwarf wheat rhizosphere communities and identified that semi-dwarf wheat microbiomes were less distinct from bulk soil communities. Of the 113 functional genes that were differentially abundant between tall and semi-dwarf cultivars, 95 % were depleted in semi-dwarf cultivars and 65 % of differentially abundant reads best mapped to genes involved in staurosporine biosynthesis (antibiotic product), plant cell wall degradation (microbial mediation of plant root architecture, overwintering energy source for microbes) and sphingolipid metabolism (signal bioactive molecules). ConclusionsOverall, our findings indicate that green revolution breeding has developed wheat cultivars with a reduced rhizosphere effect. The consequences of this are likely detrimental to the development of microbiome-assisted agriculture which will require a strong rhizosphere selective environment for the establishment of a beneficial plant root microbiome. We believe our results are of striking importance and highlight that implementation of microbiome facilitated agriculture as part of a sustainable crop production strategy will require an overhaul of wheat breeding programmes to consider plant-microbe interactions, especially in the root environment.

microbiology↗

bioRxiv2025

Single-cell profiling of human bone marrow reveals multiple myeloma progression is accompanied by an increase in CD56bright bone marrow resident NK cells.

Natural killer (NK) cells play a key role in the innate immune response against tumour progression. While the immune microenvironment in multiple myeloma (MM) becomes increasingly dysfunctional during disease evolution, little is known about changes in the NK cell compartment. Using primary samples from clinical trial patients, we performed detailed phenotypic analyses of bone marrow mononuclear cells from MGUS, SMM, and newly diagnosed MM patients. We found that disease progression is associated with an increase in CD56bright NK cells with a dynamic positive association between this NK cell subset and local tumour burden. We generated a large single-cell RNA sequencing dataset of >100,000 NK cells from healthy donor individuals and plasma cell disorder patients and identify a bone marrow specific CD56bright-like NK cell population (BM-NK) that is enriched in the marrow of MM patients. These findings highlight the evolution of the NK-cell compartment in MM and suggest a role for BM-resident CD56bright NK cells with impaired cytotoxicity in promoting immune evasion.

cancer biology↗

bioRxiv2025

Lithography-free Water Stable Conductive Polymer Nanowires

Free-standing nanowires can gain intracellular access without causing cellular stress or apoptosis. Current approaches to generate nanowires focus on lithographic patterning and inorganic materials (Si, GaAs, Al2O3, etc.) while organic materials are less explored. Use of organic conductive polymers allows for creation of soft mixed ion-electron conducting nanowires. Processing conductive polymers into nanowires is challenging due to the harsh chemicals and processing conditions used. Here, we demonstrate a lithography-free and scalable method to generate all-organic water-stable nanowires composed of conductive polymers. A nanoporous membrane is filled with conductive polymer in solution followed by a cross-linking step to make the polymer water stable. The surface of the membrane is anisotropically etched using a reactive ion etcher to reveal the polymer inside the pores, which extend from the membrane as nanowires. We interface the nanowires with model algal cells and human primary hematopoietic stem and progenitor cells. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/631660v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@bf44b3org.highwire.dtl.DTLVardef@13782baorg.highwire.dtl.DTLVardef@1679366org.highwire.dtl.DTLVardef@fe1677_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

Apramycin efficacy in the treatment of carbapenem-resistant Enterobacterales in murine blood stream infection models

BackgroundThe aminoglycoside apramycin has been proposed as a drug candidate for the treatment of critical Gram-negative systemic infections. However, its potential in the treatment of drug-resistant bloodstream infections (BSIs) has yet to be assessed. MethodsThe resistance gene annotations of 26 493 blood culture isolates were analyzed. In vitro profiling of apramycin comprised cell-free translation assays, broth microdilution, and frequency of resistance determination. The efficacy of apramycin was studied in a mouse peritonitis model for nine E. coli and K. pneumoniae isolates. ResultsGenotypic aminoglycoside resistance was identified in 87.8% of all 6973 carbapenem-resistant Enterobacterales blood-culture isolates, in comparison to 46.4% of colistin and 2.1% of apramycin resistance. Apramycin activity against methylated ribosomes was > 100-fold higher than other aminoglycosides. Frequencies of resistance were < 10-9 at 8 x MIC. Tentative epidemiological cutoffs (ECOFFs) were determined as 8 g/mL for E. coli and 4 g/mL for K. pneumoniae. A single dose of 5 to 13 mg/kg resulted in a 1-log CFU reduction in the blood and peritoneum. Two doses of 80 mg/kg, resulting in an exposure that resembles the AUC observed for a single 30 mg/kg dose in humans, resulted in complete eradication of carbapenem- and aminoglycoside-resistant bacteremias. ConclusionEncouraging coverage and potent in-vivo efficacy against a selection of highly drug-resistant Enterobacterales isolates in the mouse peritonitis model warrants further consideration of apramycin as a drug candidate for the treatment and prophylaxis of BSI.

microbiology↗

bioRxiv2023

Reduction of a-synuclein aggregates by PIKfyve inhibition via TFEB-mediated lysosomal biogenesis in a Parkinson disease model

Parkinson disease is a neurodegenerative disorder characterised by impairment of motor function, and is associated with a progressive accumulation of insoluble aggregates of misfolded alpha-synuclein. In the present study, we exploited the SH-SY5Y cell model overexpressing a pro-aggregation form of alpha-synuclein to investigate the efficacy of PIKfyve-mediated lysosomal biogenesis, through TFEB, as potential target for Parkinson therapy. To investigate this, we exploited high-content imaging along with enzymatic assays to follow the progression of lysosomal biogenesis, lysosomal function and alpha-synuclein accumulation. The cellular model exploited in this study recapitulated important elements of the biochemical phenotype observed in Parkinson patient-derived neurons, including synuclein aggregates and impaired glucocerebrosidase (GCase) function. PIKfyve inhibition by YM201636 resulted in a lysosomal-dependant reduction of alpha-synuclein aggregates as early as 24 hours post-treatment. The mechanism of action of YM201636 was shown to be TFEB-mediated, with an increase in TFEB in the nuclei which subsequently resulted in increased lysosomal markers LAMP1 and GCase. PIKfyve inhibtion efficacy was also tested in differentiated SH-SY5Y cells, exhibiting a neuron-like morphology. In these cells, YM201636 also significantly reduced alpha-synuclein aggregates and increased TFEB nuclear presence. These findings suggest that PIKfyve inhibition could be exploited as therapeutic target for Parkinson disease.

neuroscience↗

bioRxiv2023

A new antibiotic from an uncultured bacterium binds to an immutable target

Antimicrobial resistance is a leading mortality factor worldwide. Here we report the discovery of clovibactin, a new antibiotic, isolated from uncultured soil bacteria. Clovibactin efficiently kills drug-resistant bacterial pathogens without detectable resistance. Using biochemical assays, solid-state NMR, and atomic force microscopy, we dissect its mode of action. Clovibactin blocks cell wall synthesis by targeting pyrophosphate of multiple essential peptidoglycan precursors (C55PP, Lipid II, LipidWTA). Clovibactin uses an unusual hydrophobic interface to tightly wrap around pyrophosphate, but bypasses the variable structural elements of precursors, accounting for the lack of resistance. Selective and efficient target binding is achieved by the irreversible sequestration of precursors into supramolecular fibrils that only form on bacterial membranes that contain lipid-anchored pyrophosphate groups. Uncultured bacteria offer a rich reservoir of antibiotics with new mechanisms of action that could replenish the antimicrobial discovery pipeline.

microbiology↗

bioRxiv2023

An Aging-Susceptible Circadian Rhythm Controls Cutaneous Antiviral Immunity

Aged skin is prone to viral infections, but the mechanisms responsible for this immunosenescent immune risk are unclear. We observed that aged murine and human skin expressed reduced antiviral proteins (AVPs) and circadian regulators including Bmal1 and Clock. Bmal1 and Clock were found to control rhythmic AVP expression in skin and such circadian-control of AVPs was diminished by disruption of immune cell interleukin 27 signaling and deletion of Bmal1/Clock genes in mouse skins, as well as siRNA-mediated knockdown of CLOCK in human primary keratinocytes. We found that treatment of circadian enhancing agents, nobiletin and SR8278, reduced infection of herpes simplex virus 1 (HSV1) in epidermal explants and human keratinocytes in a Bmal1/Clock-dependent manner. Circadian enhancing treatment also reversed susceptibility of aging murine skin and human primary keratinocytes to viral infection. These findings reveal an evolutionarily conserved and age-sensitive circadian regulation of cutaneous antiviral immunity, underscoring circadian restoration as an antiviral strategy in aging populations.

immunology↗

bioRxiv2022

ASO targeting temperature-controlled RBM3 poison exon splicing prevents neurodegeneration in vivo

Neurodegenerative diseases are increasingly prevalent in the aging population, yet currently no disease-modifying treatments are available. Increasing the expression of the cold-shock protein, RBM3, through therapeutic hypothermia is remarkably neuroprotective, but cooling poses a health risk itself, strongly limiting its clinical application. Selective upregulation of RBM3 at normothermia thus holds immense therapeutic potential. Here we identify a poison exon within the RBM3 gene that is solely responsible for cold-induced RBM3 expression. Genetic removal or ASO-mediated manipulation of this exon yields high RBM3 levels independent of cooling. Notably, a single administration of ASO to exclude the poison exon, using FDA-approved chemistry, results in long-lasting increase of RBM3 expression in mouse brains. In prion-diseased mice, this treatment leads to remarkable neuroprotection, with prevention of neuronal loss and spongiosis despite high levels of prion protein. RBM3-inducing ASOs could thus broadly deliver protection in humans in conditions ranging from acute brain injury to Alzheimers disease. One sentence summaryInducing cold shock protein RBM3 by modulating its alternative splicing at normothermia is neuroprotective in vivo

molecular biology↗

bioRxiv2022

Weakly supervised Unet: an image classifier which learns to explain itself

BackgroundExplainability is a major limitation of current convolutional neural network (CNN) image classifiers. A CNN is required which supports its image-level prediction with a voxel-level segmentation. MethodsA weakly-supervised Unet architecture (WSUnet) is proposed to model voxel classes, by training with image-level supervision. WSUnet computes the image-level class prediction from the maximal voxel class prediction. Thus, voxel-level predictions provide a causally verifiable saliency map for the image-level decision. WSUnet is applied to explainable lung cancer detection in CT images. For comparison, current model explanation approaches are also applied to a standard CNN. Methods are compared using voxel-level discrimination metrics and a clinician preference survey. ResultsIn test data from two external institutions, WSUnet localised the tumour precisely at voxel-level (Precision: 0.93 [0.93-0.94]), achieving superior voxel-level discrimination to the best comparator (AUPR: 0.55 [0.54-0.55] vs. 0.36 [0.35-0.36]). Clinicians preferred WSUnet predictions in most test instances (Clinician Preference Rate: 0.72 [0.68-0.77]). ConclusionsWSUnet is a simple extension of the Unet, which facilitates voxel-level modelling from image-level labels. As WSUnet supports its image-level prediction with a causative voxel-level segmentation, it functions as a self-explaining image classifier. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=194 SRC="FIGDIR/small/507144v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@52e3ccorg.highwire.dtl.DTLVardef@1e981f0org.highwire.dtl.DTLVardef@151f31eorg.highwire.dtl.DTLVardef@13046a2_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract The weakly-supervised Unet converts voxel-level predictions to image-level predictions using a global max-pooling layer. Thus, loss is computed at image-level. Following training with image-level labels, voxel-level predictions are extracted from the voxel-level output layer. C_FIG FundingAuthors acknowledge funding support from the UK Research & Innovation London Medical Imaging and Artificial Intelligence Centre; Wellcome/Engineering and Physical Sciences Research Council Centre for Medical Engineering at Kings College London [WT 203148/Z/16/Z]; National Institute for Health Research Biomedical Research Centre at Guys & St Thomas Hospitals and Kings College London; National Institute for Health Research Biomedical Research Centre at Guys & St Thomas Hospitals and Kings College London; Cancer Research UK National Cancer Imaging Translational Accelerator [C1519/A28682]. For the purpose of open access, authors have applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission. HIGHLIGHTSO_LIWSUnet is a weakly supervised Unet architecture which can learn semantic segmentation from data labelled only at image-level. C_LIO_LIWSUnet is a convolutional neural network image classifier which provides a causally verifiable voxel-level explanation to support its image-level prediction. C_LIO_LIIn application to explainable lung cancer detection, WSUnets voxel-level output localises tumours precisely, outperforming current model explanation methods. C_LIO_LIWSUnet is a simple extension of the standard Unet architecture, requiring only the addition of a global max-pooling layer to the output. C_LI

bioinformatics↗

bioRxiv2022

High Quality Phasing Using Linked-Read Whole Genome Sequencing of Patient Cohorts Informs Genetic Understanding of Complex Traits

Phasing of heterozygous alleles is critical for interpretation of cis-effects of disease-relevant variation. For population studies, phase is often inferred from external data but read-based phasing approaches that span long genomic distances would be more accurate because they enable both genotype and phase to be obtained from a single dataset. To demonstrate how read-based phasing can provide functional insights, we sequenced 477 individuals with Cystic Fibrosis (CF) using linked-read sequencing. We benchmark read-based phasing with different short- and long-read sequencing technologies, prioritize linked-read technology as the most informative and produce a benchmark phase call set from reference sample HG002 for the community. The 477 samples display an average phase block N50 of 4.39 Mb. We use these samples to construct a graph representation of CFTR haplotypes, which facilitates understanding of complex CF alleles. Fine-mapping and phasing of the chr7q35 trypsinogen locus associated with CF meconium ileus demonstrates a 20 kb deletion and a PRSS2 missense variant p.Thr8Ile (rs62473563) independently contribute to meconium ileus risk (p=0.0028, p=0.011, respectively) and are PRSS2 pancreas eQTLs (p=9.5e-7 and p=1.4e-4, respectively), explaining the mechanism by which these polymorphisms contribute to CF. Phase enables access to haplotypes that can be used for genome graph or reference panel construction, identification of cis-effects, and for understanding disease associated loci. The phase information from linked-reads provides a causal explanation for variation at a CF-relevant locus which also has implications for the genetic basis of non-CF pancreatitis to which this locus has been reported to contribute.

genetics↗

bioRxiv2021

Train-the-Trainer as an Effective Approach to Building Global Networks of Experts in Genomic Surveillance of AMR

Advanced genomics and sequencing technologies are increasingly becoming critical for global health applications such as pathogen and antimicrobial resistance (AMR) surveillance. Limited resources challenge capacity development in low- and middle-income countries (LMICs), with few countries having genomics facilities and adequately trained staff. Training research and public health experts who are directly involved in the establishment of such facilities offers an effective, but limited, solution to a growing need. Instead, training them to impart their knowledge and skills to others provides a sustainable model for scaling up the much needed capacity and capability for genomic sequencing and analysis locally with global impact. We designed and developed a Train-the-Trainer course integrating pedagogical aspects with genomic and bioinformatics activities. The course was delivered to 18 participants from 12 countries in Africa, Asia, and Latin America. A combination of teaching strategies culminating in a group project created a foundation for continued development at home institutions. Upon follow-up after 6 months, at least 40% of trainees had initiated training programs and collaborations to build capacity at local, national, and regional level. This work provides a framework for implementing a training and capacity building program for the application of genomics tools and resources in AMR surveillance. 40-word summaryThis work provides a framework for implementing a training and capacity building program for the application of genomics tools and resources in AMR surveillance. We outline a Train-the-Trainer course integrating pedagogical aspects with genomic and bioinformatics activities.

scientific communication and education↗

bioRxiv2020

TET2 drives 5hmc marking of GATA6 and epigenetically defines pancreatic ductal adenocarcinoma transcriptional subtypes

Background and AimsPancreatic ductal adenocarcinoma (PDAC) is characterised by advanced disease stage at presentation, aggressive disease biology and resistance to therapy resulting in extremely poor five-year survival <10%. PDAC is classified into transcriptional subtypes with distinct survival characteristics, although how these arise is not known. Epigenetic deregulation, rather than genetics, has been proposed to underpin progression but exactly why is unclear and hindered by analysis of clinical samples. MethodsGenome-wide epigenetic mapping of DNA modifications 5-hydroxymethylcytosine (5mc) and 5-hydroxymethylcytosine (5hmc) using oxidative bisulphite sequencing (oxBS). Bioinformatics using iCluster and mutational profiling to identify overlap with transcriptional signatures in FFPE from resected patients and confirmation in vivo. ResultsWe find that more aggressive squamous-like PDAC subtypes result from epigenetic inactivation of loci including GATA6 that promote differentiated classical-pancreatic subtypes. We show that squamous-like PDAC transcriptional subtypes are associated with greater loss of 5hmc due to reduced expression of the 5mc-hydroxylase TET2. Furthermore, we find that SMAD4 directly supports TET2 levels in the pancreas and classical-pancreatic tumors and loss of SMAD4 expression is associated reduced 5hmc, GATA6 and squamous-like tumors. Importantly, enhancing TET2 stability using Metformin and VitaminC/ascorbic acid (AA) restores 5hmc and GATA6 levels, reverting squamous-like tumor phenotypes and WNT-dependence in vitro and in vivo. ConclusionsWe identify epigenetic deregulation of pancreatic differentiation as an underpinning event behind the emergence of transcriptomic subtypes in PDAC. Our data shows that restoring epigenetic control increases biomarkers of classical-pancreatic tumors and raises the possibility that combination of Vitamin C and Metformin may prolong survival in patients with squamous-like pancreatic cancer.

cancer biology↗

bioRxiv2020

Joint antibiotic and phage therapy: addressing the limitations of a seemingly ideal phage for treating Staphylococcus aureus infections

In response to increasing frequencies of antibiotic-resistant pathogens, there has been a resurrection of interest in the use of bacteriophage to treat bacterial infections: phage therapy. Here we explore the potential of a seemingly ideal phage, PYOSa, for combination phage and antibiotic treatment of Staphylococcus aureus infections. (i) This K-like phage has a broad host range; all 83 tested clinical isolates of S.aureus tested were susceptible to PYOSa. (ii) Because of the mode of action of PYOSa S. aureus is unlikely to generate classical receptor-site mutants resistant to PYOSa; none were observed in the 13 clinical isolates tested. (iii) PYOSa kills S. aureus at high rates. On the downside, the results of our experiments and tests of the joint action of PYOSa and antibiotics raise issues that must be addressed before PYOSa is employed clinically. Despite the maintenance of the phage, PYOSa does not clear the populations of S. aureus. Due to the ascent of a phenotypically diverse array of small colony variants following an initial demise, the bacterial populations return to densities similar to that of phage-free controls. Using a combination of mathematical modeling and in vitro experiments, we postulate and present evidence for a mechanism to account for the demise-resurrection dynamics of PYOSa and S. aureus. Critically for phage therapy, our experimental results suggest that treatment with PYOSa followed by bactericidal antibiotics can clear populations of S. aureus more effectively than the antibiotics alone. Significance StatementThe increasing frequency of antibiotic-resistant pathogens has fostered a quest for alternative means to treat bacterial infections. Prominent in this quest is a therapy that predates antibiotics: bacteriophage. This study explores the potential of a phage, PYOSa, for treating Staphylococcus aureus infections in combination with antibiotics. On first consideration, this phage, isolated from a commercial therapeutic cocktail, seems ideal for this purpose. The results of this population dynamic and genomic analysis study identify a potential liability of using PYOSa for therapy. Due to the production of potentially pathogenic atypical small colony variants, PYOSa alone cannot eliminate S. aureus populations. However, we demonstrate that by following the administration of PYOSa with bactericidal antibiotics, this limitation and potential liability can be addressed.

microbiology↗

bioRxiv2020

Primate phylogenomics uncovers multiple rapid radiations and ancient interspecific introgression

Our understanding of the evolutionary history of primates is undergoing continual revision due to ongoing genome sequencing efforts. Bolstered by growing fossil evidence, these data have led to increased acceptance of once controversial hypotheses regarding phylogenetic relationships, hybridization and introgression, and the biogeographical history of primate groups. Among these findings is a pattern of recent introgression between species within all major primate groups examined to date, though little is known about introgression deeper in time. To address this and other phylogenetic questions, here we present new reference genome assemblies for three Old World Monkey species: Colobus angolensis ssp. palliatus (the black and white colobus), Macaca nemestrina (southern pig-tailed macaque), and Mandrillus leucophaeus (the drill). We combine these data with 23 additional primate genomes to estimate both the species tree and individual gene trees using thousands of loci. While our species tree is largely consistent with previous phylogenetic hypotheses, the gene trees reveal high levels of genealogical discordance associated with multiple primate radiations. We use strongly asymmetric patterns of gene tree discordance around specific branches to identify multiple instances of introgression between ancestral primate lineages. In addition, we exploit recent fossil evidence to perform fossil-calibrated molecular dating analyses across the tree. Taken together, our genome-wide data help to resolve multiple contentious sets of relationships among primates, while also providing insight into the biological processes and technical artifacts that led to the disagreements in the first place.

evolutionary biology↗