Biology subjects

Graham, J.

Publications and source records attributed to Graham, J..

At least 19 recordsLinked to original sources

bioRxiv2026

Lipopolysaccharide truncation and restoration drives a trade-off in resistance to two phages in Pseudomonas aeruginosa

Phage therapy is a promising treatment for multidrug resistant bacterial infections, and for patients no longer able to tolerate antibiotic treatments. A major challenge for phage therapy is emergent phage resistance, which target bacteria acquire by structurally modifying or masking phage receptors to prevent adsorption. Functionally diverse phage cocktails that target a broad range of receptors are less prone to resistance as there is a higher fitness cost associated with modifying multiple receptors. Expanding phage libraries with well-characterised phages that target a broad range of receptors would aid in timely and strategic design of functionally diverse phage cocktails. Here, we aimed to isolate phages targeting novel receptors by enriching wastewater samples on a Pseudomonas aeruginosa PAO1 {Delta}pilA {Delta}galU unmarked deletion mutant lacking O-antigen, outer core lipopolysaccharide (LPS) and type IV pili (T4P) - the three most common Pseudomonas phage receptors. This led to the isolation of a novel phage, named Vale. Vale was predicted to bind the LPS inner core as it could only infect strains with truncated LPS, suggesting that the outer core LPS blocks Vale from accessing the inner core. We identified a trade-oM in resistance to Vale and another phage, Tor, that targets the LPS outer core, mediated by host-derived LPS modifications. The PAO1 host evolved resistance to Tor by 100-200kb genomic deletions, which resulted in LPS truncation and sensitivity to Vale. Complete LPS restoration in the {Delta}pilA {Delta}galU mutant conferred resistance to Vale and sensitivity to Tor in two out of three replicates. Combined treatment with Tor and Vale delayed the emergence of resistance in PAO1 for at least three times longer than individual phage treatments. This study provides an example of how using phage receptors to strategically design phage cocktails can minimise the likelihood of emergent phage resistance. Graphical abstractA summary of the LPS modifications, genomic mutations and phage susceptibilities of Tor and Vale resistant mutants. "Parent strain" refers to PAO1 {Delta}hsdR. {Delta}pilA {Delta}galU refers to PAO1 {Delta}hsdR {Delta}pilA {Delta}galU. The parent strain gains resistance to Tor via LPS truncation associated with 100-200 kb genomic deletions, resulting in sensitivity to Vale. {Delta}pilA {Delta}galU gains resistance to Vale by restoring its LPS, conferring sensitivity to Tor in two out of three repeats. As resistance to one phage sensitises bacteria to the other, combined treatment with both phages suppresses phage resistance for longer than individual treatments. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/700494v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1efee0forg.highwire.dtl.DTLVardef@f64bfeorg.highwire.dtl.DTLVardef@1f71130org.highwire.dtl.DTLVardef@1897bd6_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

A phylogenetic contribution to understanding the panzootic spread of African swine fever: from the global to the local scale

African Swine Fever virus has become a primary concern for veterinarian health agencies and pig producers worldwide. The current panzootic of the virus genotype II is having a devastating impact on pig production in Africa, Europe, Asia, Oceania and Hispaniola (Caribbean). Due to its high persistence and mortality rate, disease control policies require enhanced passive surveillance activities, wild boar depopulation, containment and other costly interventions, as a safe and effective vaccine is not currently available. Since 2007, several disease clusters have emerged far from both its original range, in South-Eastern Africa, and far from other affected suid populations. These transmissions were likely caused by anthropogenic movement of contaminated material, facilitated by the persistence of the virus in the environment and on contaminated material. The objective of this research is to understand the spatio-temporal dynamic of the African Swine Fever virus panzootic, with a specific focus on clusters from mainland Italy. We mapped and analysed the virus spread using 228 whole-genome sequences available from online repositories and from the Italian cases/outbreaks, combined with their metadata. We inferred the pathogen phylogenies using a Bayesian phylodynamic model, with which we obtained a time-scaled and spatially explicit maximum clade credibility tree. Our results showed a non-negligible number of long-distance transmissions across regions or continents, coupled with dense local dynamics, particularly in areas where the disease affected a naive population. The distribution of spatial distances inferred along the trees branches further highlighted these trends, and revealed how previously observed survival times in pork products could allow the virus to traverse distances up to 900km. Finally, from the available sequences, we identified at least seven separate introduction events in Europe, of which at least three caused new clusters on mainland Italy. This study provides important insights on the African Swine Fever virus introduction into many affected areas worldwide and highlights the crucial role of genomic surveillance to correctly track the pathogen spread, and to monitor the virus potential evolution.

molecular biology↗

bioRxiv2025

Development of a Flex-Seq SNP panel for raspberry (Rubus idaeus L.) and validation through linkage map construction and identification of QTL for several traits of agronomic importance to raspberry breeding

High-throughput and reproducible genotyping platforms are critical for advancing genetic research and breeding in horticultural crops. Here, the development and validation of a custom single nucleotide polymorphism (SNP) panel using the Flex-Seq genotyping platform for red raspberry (Rubus idaeus L.) is described. SNPs were derived from existing linkage maps and RNA-seq data, resulting in a panel of 5,639 high-confidence, bi-allelic markers distributed across the seven chromosomes of the R. idaeus Malling Jewel reference genome. The panel was used to genotype 457 red raspberry accessions including 161 individuals from a bi-parental mapping population (Parisx486), enabling the construction of high-density linkage maps and the identification of quantitative trait loci (QTL) for fruit size, leaf colour, plant vigour, and thorn density. Genome-wide association studies (GWAS) identified a major QTL for thornlessness on chromosome 4, co-locating with a candidate HOX3 gene, and multiple QTLs associated with anthocyanin biosynthesis genes for leaf colour. The SNP panel demonstrated utility for linkage mapping and trait association analyses, offering a powerful resource for marker-assisted selection and genetic improvement in red raspberry.

genomics↗

bioRxiv2025

Development of an eco-friendly RNAi yeast attractive targeted sugar bait that silences the Shaker gene in spotted-wing drosophila, Drosophila suzukii

BackgroundDrosophila suzukii, or spotted-wing drosophila (SWD), (Diptera: Drosophilidae), are invasive vinegar flies of East Asian origin that have wreaked havoc on the small fruit and berry industry. In locations where SWD are well established, weekly chemical insecticide applications are necessary, resulting in increased economic costs, unwanted environmental impacts ensuing from loss of non-targeted organisms, and the eventual emergence of populations that are resistant to these insecticides. It is therefore critical that new classes of biorational pesticides and cost-effective technologies for controlling SWD are identified. ResultsHere, we used the attractive properties of Saccharomyces cerevisiae, bakers yeast, which was designed to express an RNA interference (RNAi) pesticide that specifically targets the SWD Shaker (Sh) gene, to lure and kill flies that feed on the yeast, which was delivered in a feeder as a component of an attractive targeted sugar bait (ATSB). The yeast, which was heat killed prior to preparation of the ATSB, silenced the Sh gene, resulting in severe neural defects and 96{+/-}9% fly mortality in laboratory trials. The RNAi yeast was successfully fed to the flies in an easily assembled soda bottle feeder that continuously rewetted the yeast with soda, which lured and killed the flies in simulated field trials. Despite this toxicity observed in SWD, consumption of the yeast had no impact on the survival of other dipteran insects. ConclusionThis promising ATSB technology, which was prepared with a new class of RNAi yeast insecticides, could one day be an effective component in integrated SWD control programs.

genetics↗

bioRxiv2024

Sympathetic innervation of interscapular brown adipose tissue is not a predominant mediator of oxytocin-elicited reductions of body weight and adiposity in male diet-induced obese mice

Previous studies indicate that CNS administration of oxytocin (OT) reduces body weight in high fat diet-induced obese (DIO) rodents by reducing food intake and increasing energy expenditure (EE). We recently demonstrated that hindbrain (fourth ventricular [4V]) administration of OT elicits weight loss and elevates interscapular brown adipose tissue temperature (TIBAT, a surrogate measure of increased EE) in DIO mice. What remains unclear is whether OT-elicited weight loss requires increased sympathetic nervous system (SNS) outflow to IBAT. We hypothesized that OT-induced stimulation of SNS outflow to IBAT contributes to its ability to activate BAT and elicit weight loss in DIO mice. To test this hypothesis, we determined the effect of disrupting SNS activation of IBAT on the ability of 4V OT administration to increase TIBAT and elicit weight loss in DIO mice. We first determined whether bilateral surgical SNS denervation to IBAT was successful as noted by [≥] 60% reduction in IBAT norepinephrine (NE) content in DIO mice. NE content was selectively reduced in IBAT at 1-, 6- and 7-weeks post-denervation by 95.9{+/-}2.0, 77.4{+/-}12.7 and 93.6{+/-}4.6% (P<0.05), respectively and was unchanged in inguinal white adipose tissue, pancreas or liver. We subsequently measured the effects of acute 4V OT (1, 5 {micro}g {approx} 0.99, 4.96 nmol) on TIBAT in DIO mice following sham or bilateral surgical SNS denervation to IBAT. We found that the high dose of 4V OT (5 {micro}g {approx} 4.96 nmol) elevated TIBAT similarly in sham mice as in denervated mice. We subsequently measured the effects of chronic 4V OT (16 nmol/day over 29 days) or vehicle infusions on body weight, adiposity and food intake in DIO mice following sham or bilateral surgical denervation of IBAT. Chronic 4V OT reduced body weight by 5.7{+/-}2.23% and 6.6{+/-}1.4% in sham and denervated mice (P<0.05), respectively, and this effect was similar between groups (P=NS). OT produced corresponding reductions in whole body fat mass (P<0.05). Together, these findings support the hypothesis that sympathetic innervation of IBAT is not necessary for OT-elicited increases in BAT thermogenesis and reductions of body weight and adiposity in male DIO mice.

pharmacology and toxicology↗

bioRxiv2024

Silencing of a raspberry homologue of VRN1 is associated with disruption of dormancy induction and misregulation of subsets of dormancy-associated genes.

Winter dormancy is a key process in the phenology of temperate perennials. The changing climate is severely impacting its course leading to economic losses in agriculture. A better understanding of the underlying mechanisms, as well as the genetic basis of the different responses, are necessary for the development of climate-resilient cultivars. This study aims to provide an insight into winter dormancy in red raspberry (Rubus idaeus L). We report the transcriptomic profiles during dormancy in two raspberry cultivars with contrasting responses. The cultivar Glen Ample showed a typical perennial phenology, whereas Glen Dee registered consistent dormancy dysregulation, exhibiting active growth and flowering out of season. RNA-seq combined with weighted gene co-expression network analysis (WGCNA) highlighted gene clusters in both genotypes that exhibited time-dependent expression profiles, Functional analysis of Glen Ample gene clusters highlighted the significance of the cell and structural development prior to dormancy entry as well the role of genetic and epigenetic processes such as RNAi and DNA methylation in regulating gene expression. On the contrary, dormancy release in Glen Ample was associated with upregulation of transcripts associated with the resumption of metabolism, nucleic acid biogenesis and processing signal response pathways. Many of the processes occurring in Glen Ample were dysregulated in Glen Dee and twenty-eight transcripts exhibiting time-dependent expression in Ample that also had an Arabidopsis homologue were not found in all samples from Glen Dee. These included a gene with homology to Arabidopsis VRN1 (RiVRN1.1) that exhibited a sharp decline in expression following dormancy induction in Glen Ample. Characterisation of the gene region in the Glen Dee genome revealed two large insertions upstream of the ATG start codon. We propose that non-expression of a specific VRN1 homologue in Glen Dee causes dormancy misregulation as a result of inappropriate expression of a subset of genes that are directly or indirectly regulated by RiVRN1.1. HIGHLIGHTThe raspberry cultivar Glen Dee exhibits aberrant winter dormancy status associated with insertions in the upstream promoter region of a VRN1 (RiVRN1.1) homologue that silence expression, allowing the identification of dormancy-associated genetic modules that are regulated by RiVRN1.1.

plant biology↗

bioRxiv2023

Statistics to prioritize rare variants in family-based sequencing studies with disease subtypes

Family-based sequencing studies are increasingly used to find rare genetic variants of high risk for disease traits with familial clustering. In some studies, families with multiple disease subtypes are collected and the exomes of affected relatives are sequenced for shared rare variants. Since different families can harbor different causal variants and each family harbors many rare variants, tests to detect causal variants can have low power in this study design. Our goal is rather to prioritize shared variants for further investigation by, e.g., pathway analyses or functional studies. The transmission-disequilibrium test prioritizes variants based on departures from Mendelian transmission in parent-child trios. Extending this idea to families, we propose methods to prioritize rare variants shared in affected relatives with two disease subtypes, with one subtype more heritable than the other. Global approaches condition on a variant being observed in the study and assume a known probability of carrying a causal variant. In contrast, local approaches condition on a variant being observed in specific families to eliminate the carrier probability. Our simulation results indicate that global approaches are robust to mis-specification of the carrier probability and prioritize more effectively than local approaches even when the carrier probability is misspecified.

genetics↗

bioRxiv2023

Single cell transcriptomics of the human parasite Schistosoma mansoni first intra-molluscan stage reveals tentative tegumental and stem cell regulators.

BackgroundSchistosomiasis is a major Neglected Tropical Disease, caused by the infection with blood flukes in the genus Schistosoma. To complete the life cycle, the parasite undergoes asexual and sexual reproduction within an intermediate snail host and a definitive mammalian host, respectively. The intra-molluscan phase provides a critical amplification step that ensures a successful transmission. However, the cellular and molecular mechanisms underlying the development of the intra-molluscan stages remain poorly understood. MethodsS. mansoni mother sporocysts were dissociated into single cell suspensions, and live cells enriched and sequenced using the single cell 10X Genomics Chromium platform. We defined somatic and stem/germinal cell clusters, identified cell type-enriched Gene Ontology (GO) terms, and predicted transcription factor binding sites for key marker genes. ResultsSix cell clusters comprising stem/germinal, two tegument, muscle, neuron, and parenchyma were identified and validated by Fluorescence in situ Hybridisation (FISH). GO term analysis predicted key biological processes for each of the clusters. Using the Self-Assembling Manifold (SAM) algorithm, three sub-clusters were identified within the stem/germinal cell population. Furthermore, transcription factor binding sites and putative transcription factors were predicted for stem/germinal and tegument clusters. ConclusionsWe report a spatially validated single cell transcriptomic analysis of the first intra-molluscan stage of S. mansoni. Key cell regulators were identified, paving the way for future analyses to unveil their role during the parasite development and interaction with its intermediate host.

developmental biology↗

bioRxiv2023

A single-cell atlas of the free-living miracidium larva of Schistosoma mansoni

Schistosomes are parasitic flatworms that cause the water-borne disease schistosomiasis, affecting millions of people worldwide. The miracidium larva of schistosomes represents the first post-embryonic stage of development and is critical to transmission. After hatching, a miracidium infects a freshwater snail and transforms into a mother sporocyst, where its stem cells generate daughter sporocysts that give rise to many human-infective cercariae larvae. To understand this important life cycle stage at the cellular and molecular levels, we have used single-cell RNA sequencing, in situ hybridisation and image analysis to create a whole-body cell atlas of the miracidium larva of Schistosoma mansoni. Our atlas shows that each miracidium is composed of [~]365 cells and 19 transcriptionally distinct cell types. We show that 93% of miracidium cells are somatic (57% neural, 19% muscle, 13% epidermal (tegument), 2% parenchyma, 2% protonephridia), and the remaining 7% are stem cells. Cellular diversity within tissue types is revealed, and is highest in neurons. There are two stem cell populations, and they show different activation and potency states. Trajectory analysis indicates that one stem cell population is the origin of the tegument lineage and the other likely contains pluripotent cells. Furthermore, each of these stem populations is transcriptionally distinct based on sex-linked gene expression in male and female larvae. Through single cell transcriptomics and in-situ hybridisation we identified every cell in the whole organism revealing the organisation of the miracidium. This single cell atlas provides the foundation to understand the development and interaction of cell types and tissues as they change over a life cycle that is characterised by complex morphological changes.

genomics↗

bioRxiv2023

Raspberry plant stress detection using hyperspectral imaging

Monitoring plant responses to stress is an ongoing challenge for crop breeders, growers and agronomists. The measurement of below ground stress is particularly challenging as plants do not always show visible signs of stress in the above ground organs, particularly at early stages. Hyperspectral imaging is a technique that could be used to overcome this challenge if associations between plant spectral data and specific stresses can be determined. In this study, three genotypes of red raspberry plants grown under controlled conditions in a glasshouse were subjected to below ground biotic stresses (root pathogen Phytophthora rubi and root herbivore Otiorhynchus sulcatus) or abiotic stress (soil water availability) and regularly imaged using hyperspectral cameras over this period. Significant differences were observed in plant biophysical traits (canopy height and leaf dry mass) and canopy reflectance spectrum between the three genotypes and the imposed stress treatments. The ratio of reflectance at 469nm and 523nm showed a significant genotype-by-treatment interaction driven by differential genotypic responses to the Phytophthora rubi treatment. This indicates that spectral imaging can be used to identify variable plant stress responses in raspberry plants.

plant biology↗

bioRxiv2022

Inference of gene-environment interaction from heterogeneous case-parent trios

In genetic epidemiology, log-linear models of population risk may be used to study the effect of genotypes and exposures on the relative risk of a disease. Such models may also include gene-environment interaction terms that allow the genotypes to modify the effect of the exposure, or equivalently, the exposure to modify the effect of genotypes on the relative risk. When a measured test locus is in linkage disequilibrium with an unmeasured causal locus, exposure-related genetic structure in the population can lead to spurious gene-environment interaction; that is, to apparent gene-environment interaction at the test locus in the absence of true gene-environment interaction at the causal locus. Exposure-related genetic structure occurs when the distributions of exposures and of haplotypes at the test and causal locus both differ across population strata. A case-parent trio design can protect inference of genetic main effects from confounding bias due to genetic structure in the population. Unfortunately, when the genetic structure is exposure-related, the protection against confounding bias for the genetic main effect does not extend to the gene-environment interaction term. We show that current methods to reduce the bias in estimated gene-environment interactions from case-parent trio data can only account for simple population structure involving two strata. To fill this gap, we propose to directly accommodate multiple population strata by adjusting for genetic principal components. We evaluate our approach through simulation and illustrate it on data from a study of genetic modifiers of cleft palate.

genetics↗

bioRxiv2022

Identification of genetic loci in lettuce mediating quantitative resistance to fungal pathogens

Lactuca sativa L. (lettuce) is an important leafy vegetable crop grown and consumed globally. Chemicals are routinely used to control major pathogens, including the causal agents of grey mould (Botrytis cinerea) and lettuce drop (Sclerotinia sclerotiorum). With increasing prevalence of pathogen resistance to fungicides and environmental concerns, there is an urgent need to identify sources of genetic resistance to B. cinerea and S. sclerotiorum in lettuce. We demonstrated genetic variation for quantitative resistance to B. cinerea and S. sclerotiorum in a set of 97 diverse lettuce and wild relative accessions, and between the parents of lettuce mapping populations. Transcriptome profiling across multiple lettuce accessions enabled us to identify genes with expression correlated with resistance, predicting the importance of post-transcriptional gene regulation in the lettuce defence response. We identified five genetic loci influencing quantitative resistance in a F10 mapping population derived from a Lactuca serriola (wild relative) x lettuce cross, which each explained 5-10% of the variation. Differential gene expression analysis between the parent lines, and integration of data on correlation of gene expression and resistance in the diversity set, highlighted potential causal genes underlying the quantitative trait loci. Key MessageWe demonstrate genetic variation for quantitative resistance against important fungal pathogens in lettuce and its wild relatives, map loci conferring resistance and predict key molecular mechanisms using transcriptome profiling.

plant biology↗

bioRxiv2022

A Longitudinal Study of Dominant E. coli Lineages and Antimicrobial Resistance in the Gut of Children Living in an Upper Middle-income Country

The gastrointestinal tract constitutes a complex and diverse ecosystem. Escherichia coli is one of the most frequently studied and characterized species in the gut ecosystem, nevertheless, there has been little research to determine their diversity and population dynamics in the intestines of children over time. In this prospective study, a fresh fecal sample was obtained from children longitudinally over one year (30 fecal samples at sampling period 1 and 22 fecal samples at sampling periods 2 and 3). From each stool sample, five E. coli colonies were randomly selected (n = 405 E. coli isolates total) in order to characterize the genotype and phenotypic antimicrobial resistance patterns. We found that all numerically dominant E. coli lineages in childrens intestines were transient colonizers, and antimicrobial resistance phenotypes of these strains varied significantly over time without any apparent selective force. Whole-genome sequencing of 3 isolates belonging to ST131 found in one child during the sampling period I and II indicated that isolates were three different ST 131 clones that carried extended-spectrum {beta}-lactamase (ESBL) genes. IMPORTANCEThe length of residency and numeric dominance of antimicrobial-resistant E. coli may affect the extent to which an isolate contributes to the dissemination of antimicrobial resistance in a community. We studied the persistence of numerically dominant and antimicrobial-resistant lineages of E. coli in the human intestine. We found that E. coli lineages in the children's gut change considerably and rapidly over time. This study suggests that some phenotypic resistance patterns may result from the random distribution of genes in E. coli populations over time and may not be associated with differential exposure to antimicrobials.

microbiology↗

bioRxiv2021

An exploration of linkage fine-mapping on sequences from case-control studies

Linkage analysis maps genetic loci for a heritable trait by identifying genomic regions with excess relatedness among individuals with similar trait values. Analysis may be conducted on related individuals from families, or on samples of unrelated individuals from a population. For allelically heterogeneous traits, population-based linkage analysis can be more powerful than genotypic-association analysis. Here, we focus on linkage analysis in a population sample, but use sequences rather than individuals as our unit of observation. Earlier investigations of sequence-based linkage mapping relied on known sequence relatedness, whereas we infer relatedness from the sequence data. We propose two ways to associate similarity in relatedness of sequences with similarity in their trait values and compare the resulting linkage methods to two genotypic-association methods. We also introduce a procedure to label case sequences as potential carriers or non-carriers of causal variants after an association has been found. This post-hoc labeling of case sequences is based on inferred relatedness to other case sequences. Our simulation results indicate that methods based on sequence-relatedness improve localization and perform as well as genotypic-association methods for detecting rare causal variants. Sequence-based linkage analysis therefore has potential to fine-map allelically heterogeneous disease traits.

genetics↗

bioRxiv2021

Genomic epidemiology of Mycobacterium bovis infection in sympatric badger and cattle populations in Northern Ireland.

BackgroundBovine tuberculosis (bTB) is a costly, epidemiologically complex, multi-host, endemic disease. Pathogen whole genome sequencing can improve the resolution of epidemiological tracing. We genome sequenced an exceptional data set of 619 Mycobacterium bovis isolates from badgers and cattle in a 100km2 bTB hotspot. Historical molecular subtyping data permitted the targeting of an endemic pathogen lineage, whose long-term persistence provided an opportunity to study genome epidemiology in detail. To assess whether badger population genetic structure was associated with the spatial distribution of pathogen genetic diversity, we microsatellite genotyped hair samples from 769 badgers trapped in this area. ResultsEight lineages of M. bovis were circulating in the study area, seven of which were likely non-endemic, and imported by animal movement. The endemic lineage exhibited low genetic diversity with an average inter-isolate genetic distance of 7.6 SNPs (s.d. {+/-} 4.0), consistent with contemporary transmission. Bayesian phylogenetic methods determined an evolutionary rate of 0.30 substitutions per genome per year for this lineage, estimating its emergence 40-50 years before present, while Bayesian Skyline analysis identified significant population expansion of the endemic lineage in the 1990s and again in 2011-2012. The phylogeny revealed distinct sub-lineages, all of which contained isolates from both cattle and badger hosts, indicative of the sharing of closely related strains and inter-species transmission. However, the presence of significant badger population genetic structure was not associated with the spatial distribution of M. bovis genetic diversity. ConclusionsOur data provided unparalleled detail on the evolutionary history of an endemic M. bovis lineage. Findings are consistent with ongoing interspecies transmission in the study area but suggest that badger intra-species transmission may not be a major driver of persistence in this area. In addition, the data collected permitted the tracking of incursions of novel pathogen lineages into the study area and means to determine if they were involved in disease transmission.

evolutionary biology↗

bioRxiv2020

Analytical validation and performance characteristics of a 48-gene next-generation sequencing panel for detecting potentially actionable genomic alterations in myeloid neoplasms

Identification of genomic mutations by molecular testing plays an important role in diagnosis, prognosis, and treatment of myeloid neoplasms. Next-generation sequencing (NGS) is an efficient method for simultaneous detection of clinically significant genomic mutations with high sensitivity. However, due to lack of standard NGS protocols, the application of NGS for hematologic malignancies into clinical settings remains limited. We report development and validation of a 48-gene NGS panel for molecular profiling of myeloid neoplasms including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and myeloproliferative neoplasms (MPN). Target regions were captured by hybridization with complementary biotinylated DNA baits, and NGS was performed on an Illumina NextSeq500 instrument. A bioinformatics pipeline that was developed in-house was used to detect single nucleotide variations (SNVs), insertions/deletions (indels), and FLT3 internal tandem duplications (FLT3-ITD). An analytical validation study was performed on 184 unique specimens for variants with allele frequencies [≥]5%. Variants identified by the 48-gene panel were compared to those identified by a 35-gene hematologic neoplasms panel using an additional 137 unique specimens. The developed assay was applied to a large cohort (n=2,053) of patients with suspected myeloid neoplasms. Analytical validation yielded 99.6% sensitivity (95% CI: 98.9-99.9%) and 100% specificity (95% CI: 100%). Concordance of variants detected by the 2 tested panels was 100%. Among patients with suspected myeloid neoplasms (n=2,053), 54.5% patients harbored at least one clinically significant mutation: 77% in AML patients, 48% in MDS, and 45% in MPN. Together, these findings demonstrate that the assay can identify mutations associated with diagnosis, prognosis, and treatment options of myeloid neoplasms.

cancer biology↗

bioRxiv2020

A longitudinal study of E. coli lineages and antimicrobial resistance in Ecuadorian children

The gastrointestinal tract (GIT) constitutes a complex and diverse ecosystem. Escherichia coli is one of the most frequently studied and characterized species in the gut ecosystem. Nevertheless, there has been little research to determine their diversity and population dynamics in the intestines of children over time. Many intestinal E. coli lineages carry antimicrobial resistance and virulence genes, which have implications in disease and public health. In this one-year prospective study, a fresh fecal sample was obtained from 30 children longitudinally for one year (n = 82 fecal samples). From each stool sample, five Escherichia coli colonies were randomly selected to characterize their genotype and phenotypic antimicrobial resistance pattern (n = 405 E. coli isolates). We found that the most numerically dominant E. coli lineages in childrens intestines were transient colonizers, and phenotypic antimicrobial resistance varied significantly over time, however, ST131 a multi-drug resistant pathogen, and 3 additional STs persisted in a childs intestine for 3 months or more. IMPORTANCEThe length of residency and numeric dominance of antimicrobial-resistant E. coli may affect the extent to which an isolate contributes to the dissemination of antimicrobial resistance. We studied the persistence of numerically dominant and antimicrobial-resistant lineages of E. coli in the human intestine and found that E. coli lineages in the gut of children change rapidly over time.

microbiology↗

bioRxiv2020

Baseline T cell immune phenotypes predict virologic and disease control upon SARS-CoV infection

The COVID-19 pandemic has revealed that infection with SARS-CoV-2 can result in a wide range of clinical outcomes in humans, from asymptomatic or mild disease to severe disease that can require mechanical ventilation. An incomplete understanding of immune correlates of protection represents a major barrier to the design of vaccines and therapeutic approaches to prevent infection or limit disease. This deficit is largely due to the lack of prospectively collected, pre-infection samples from indiviuals that go on to become infected with SARS-CoV-2. Here, we utilized data from a screen of genetically diverse mice from the Collaborative Cross (CC) infected with SARS-CoV to determine whether circulating baseline T cell signatures are associated with a lack of viral control and severe disease upon infection. SARS-CoV infection of CC mice results in a variety of viral load trajectories and disease outcomes. Further, early control of virus in the lung correlates with an increased abundance of activated CD4 and CD8 T cells and regulatory T cells prior to infections across strains. A basal propensity of T cells to express IFNg and IL17 over TNFa also correlated with early viral control. Overall, a dysregulated, pro-inflammatory signature of circulating T cells at baseline was associated with severe disease upon infection. While future studies of human samples prior to infection with SARS-CoV-2 are required, our studies in mice with SARS-CoV serve as proof of concept that circulating T cell signatures at baseline can predict clinical and virologic outcomes upon SARS-CoV infection. Identification of basal immune predictors in humans could allow for identification of individuals at highest risk of severe clinical and virologic outcomes upon infection, who may thus most benefit from available clinical interventions to restrict infection and disease. SummaryWe used a screen of genetically diverse mice from the Collaborative Cross infected with mouse-adapted SARS-CoV in combination with comprehensive pre-infection immunophenotyping to identify baseline circulating immune correlates of severe virologic and clinical outcomes upon SARS-CoV infection.

immunology↗