Biology subjects

Robinson, E.

Publications and source records attributed to Robinson, E..

48 records · Page 3Linked to original sources

bioRxiv2019

M. tuberculosis microvariation is common and is associated with transmission: analysis of three years prospective universal sequencing in England

BackgroundThe prevalence, association with disease status, and public health impact of infection with mixtures of M. tuberculosis strains is unclear, in part due to limitations of existing methods for detecting mixed infections.\n\nMethodsWe developed an algorithm to identify mixtures of M. tuberculosis strains using next generation sequencing data, assessing performance using simulated sequences. We identified mixed M. tuberculosis strains when there was at least one mixed nucleotide position, and where both the mixtures components were present in similar isolates from other individuals. We determined risk factors for mixed infection among isolations of M. tuberculosis in England using logistic regression. We used survival analyses to assess the association between mixed infection and putative transmission.\n\nFindings6,560 isolations of TB were successfully sequenced in England 2016-2018. Of 3,691 (56%) specimens for which similar sequences had been isolated from at least two other individuals, 341 (9.2%) were mixed. Infection with lineages other than Lineage 4 were associated with mixed infection. Among the 1,823 individuals with pulmonary infection with Lineage 4 M. tuberculosis, mixed infection was associated with significantly increased risk of subsequent isolation of closely related organisms from a different individual (HR 1.43, 95% CI 1.05,1.94), indicative of transmission.\n\nInterpretationMixtures of transmissible strains occur in at least 5% of tuberculosis infections in England; when present in pulmonary disease, such mixtures are associated with an increased risk of tuberculosis transmission.\n\nFundingPublic Health England; NIHR Health Protection Research Unit Oxford; European Union.\n\nResearch in Context\n\nEvidence Before This StudyWe searched Pubmed using the search terms tuberculosis and mixed or mixture for English Language articles published up to 1 April 2019. Studies, most performed without the benefit of genomic sequencing, report mixed TB infection from a range of medium and high prevalence areas and show it to be associated with delayed treatment response. Modelling suggests detection and treatment of mixed TB infection is an important goal for TB eradication campaigns. Although routine DNA sequencing of M. tuberculosis isolates is becoming widespread, efficient methods for detecting mixed infection from such data are underdeveloped, and the true prevalence of mixed infection and its association with transmission is unclear.\n\nAdded Value of This StudyThis study investigated a large series of TB isolations obtained as part of a routine Mycobacterial sequencing program by two reference laboratories, in a low incidence area, England. We developed an efficient generalisable approach to identify transmitted mixed M. tuberculosis infection; our approach is capable of sensitive and specific detection of a single mixed nucleotide position. We identified mixed infection of similar strains ( microvariation) in about 9.2% of the M. tuberculosis samples which we were able to assess, and found evidence of increased transmission from individuals with mixed infection.\n\nImplications of All the Available EvidenceTB microvariation is a risk factor for TB transmission, even in the low incidence area studied. Although an efficient and highly specific technique identifying microvariation exists, it relies on comparison with similar sequences isolated from other patients. Sharing of sequence data from the many TB sequencing programs being deployed globally will increase the sensitivity of microvariation detection, and may assist targeted public health interventions.

microbiology↗

bioRxiv2019

Ultrasound- and microbubble-mediated targeted delivery of therapeutic microRNA-loaded nanocarriers to deep liver and kidney tissues in pigs

In this study, we designed and validated a platform for ultrasound (US) and microbubble (MB)-mediated delivery of FDA-approved pegylated poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs) loaded with anticancer microRNAs (miRNAs) to deep tissues in a pig model. Small RNAs have the ability to reprogram tumor cells and sensitize them to clinically used chemotherapy. However, to overcome their short intravascular circulation half-life and achieve controlled and sustained release into tumor cells, anticancer miRNAs need to be encapsulated into NPs. Moreover, focused US combined with gas-filled MBs provides a safe and noninvasive way to improve the permeability of tumor vasculature and increase the delivery efficiency of drug-loaded nanocarriers. A single handheld, curvilinear US array was used in this study for image-guidance and therapy with clinical-grade SonoVue MBs. First, we validated the platform on phantoms to optimize the MB cavitation dose based on acoustic parameters, including peak negative pressure, pulse length, and pulse repetition frequency. We then tested the system in vivo by delivering PLGA-NPs co-loaded with antisense-miRNA-21 and antisense-miRNA-10b in pig liver and kidney. Enhanced miRNA delivery was observed (1.9- to 3.7-fold increase) as a result of the US-MB treatment compared to untreated control regions. Additionally, we used highly fluorescent semiconducting polymer nanoparticles (SPNs) co-delivered with miRNA-loaded PLGA-NPs to visually assess NP delivery. Fluorescent microscopy of SPNs confirmed NP extravasation and showed the presence of particles in the extravascular compartment. Hematoxylin and eosin staining of treated tissues did not reveal tissue damage. The results presented in this manuscript suggest that enhanced delivery of miRNA-loaded NPs to target regions in deep organs is feasible in large animal models using the proposed platform.

bioengineering↗

bioRxiv2019

Portion size does not influence food consumption in rats

In studies of eating behavior that have been conducted in humans, the tendency to consume more when given larger portions of food, known as the portion size effect (PSE), is one of the most robust and widely replicated findings. Despite this, the mechanisms that underpin it are still unknown. In particular, it is unclear whether the PSE arises from higher-order social and cognitive processes that are unique to humans or, instead, reflects more fundamental processes that drive feeding, such as conditioned food-seeking. Importantly, studies in rodents and other animals have yet to show convincing evidence of a PSE. In this series of studies, we used several methods to test for a PSE in adult male Sprague-Dawley rats. Our approaches included using visually identifiable portions of a palatable food; training on a plate cleaning procedure; providing portion sizes of food pellets that were signaled by auditory and visual food-predictive cues; providing food with amorphous shape properties; and providing standard chow diet portions in home cages. In none of these manipulations did larger portions increase food intake. In summary, our data indicate that a PSE does not appear to be present in rats or, if it is, it is more nuanced, dependent on experimental procedure and/or smaller in size than it is in humans. In turn, these findings suggest that the widely-replicated PSE in humans may be more likely to reflect higher-order cognitive and social processes than fundamental conditioned behaviors.\n\nHighlightsO_LIPortion size effect (PSE) refers to increased food intake induced by large portions.\nC_LIO_LIAlthough widely replicated in human feeding studies, it may not exist animals.\nC_LIO_LIPresence of a PSE in animals would shed light on mechanisms, which are not known.\nC_LIO_LIHere, we find no evidence of PSE in rats under a number of experimental conditions.\nC_LIO_LIThis suggests that the human PSE is more likely due to socio-cognitive processes.\nC_LI

animal behavior and cognition↗

bioRxiv2018

MinION Nanopore Sequencing of Multiple Displacement Amplified Mycobacteria DNA Direct from Sputum

Sequencing of pathogen DNA directly from clinical samples offers the possibilities of rapid diagnosis, faster antimicrobial resistance prediction and enhanced outbreak investigation. The approach is especially advantageous for infections caused by species which grow very slowly in culture, such as Mycobacteria tuberculosis. Since the pathogen of interest may represent as little as 0.01% of the total DNA, enrichment of the input material for target sequences by specific amplification and, or depletion of non-target DNA (human, other bacteria) is essential for success. Here, we investigated the potential of isothermal multiple displacement amplification by Phi29 polymerase. We directed the amplification reaction towards Mycobacteria DNA in sputum samples by exploiting in our oligonucleotide primer design, their high GC content (approximately 65%) relative to human DNA. Amplified DNA was then sequenced using the Oxford Nanopore Technology MinION. In addition, a model system comprising standardised mock clinical samples was designed. Pooled infection negative human sputum samples were spiked with enumerated Mycobacterium bovis (BCG) Pasteur strain at concentrations spanning the typical range at which Mycobacterium tuberculosis is found in human sputum samples (106 - 101 BCG cells/ml). To assess the amount of BCG sequence enrichment achieved, sample DNA was sequenced both before, and after amplification. Reads from amplified samples, which mapped to a BCG reference genome, comprised short repeated sequences - apparently transcribed multiple times from the same fragment of BCG DNA. Therefore post-amplification, the samples were enriched for BCG sequences relative to unamplified sequences (8,101 BCG reference mapped reads, increasing to 28,617 at 106 BCG cells/ml sample), but BCG genome coverage declined markedly (for example 89.4% to 4.1%). In summary, the use of standardised mock clinical samples allowed direct comparison of data from different Mycobacteria enrichment experiments and sequencing runs. However, optimal conditions for multiple displacement amplification of minority Mycobacteria DNAs, remain to be identified.

microbiology↗

bioRxiv2018

Identifying mixed Mycobacterium tuberculosis infection and laboratory cross-contamination during Mycobacterial sequencing programs

IntroductionDetecting laboratory cross-contamination and mixed tuberculosis infection are important goals of clinical Mycobacteriology laboratories.\n\nObjectivesTo develop a method detecting mixtures of different M. tuberculosis lineages in laboratories performing Mycobacterial next generation sequencing (NGS).\n\nSettingPublic Health England National Mycobacteriology Laboratory Birmingham, which performs Illumina sequencing on DNA extracted from positive Mycobacterial Growth Indicator tubes.\n\nMethodsWe analysed 4,156 samples yielding M. tuberculosis from 663 MiSeq runs, obtained during development and production use of a diagnostic process using NGS. Counts of the most common (major) variant, and all other variants (non-major variants) were determined from reads mapping to positions defining M. tuberculosis lineages. Expected variation was estimated during process development.\n\nResultsFor each sample we determined the non-major variant proportions at 55 sets of lineage defining positions. The non-major variant proportion in the two most mixed lineage defining sets (F2 metric) was compared with that in the 47 least mixed lineage defining sets (F47 metric). Three patterns were observed: (i) not mixed by either metric, (ii) high F47 metric suggesting mixtures of multiple lineages, and (iii) samples compatible with mixtures of two lineages, detected by differential F2 metric elevation relative to F47. Pattern (ii) was observed in batches, with similar patterns in the H37Rv control present in each run, and is likely to reflect cross-contamination. During production, the proportions of samples in each pattern were 97%, 2.8%, and 0.001%, respectively.\n\nConclusionThe F2 and F47 metrics described could be used for laboratory process control in laboratories sequencing M. tuberculosis.

microbiology↗

bioRxiv2018

Amazon Mechanical Turk as a platform for borderline personality disorder research

Researchers investigating the psychological processes underlying specific mental health problems often have difficulties achieving large enough samples for adequately powered studies. This can be particularly problematic when studying psychopathology with low base rates in typical samples (i.e., undergraduate and community). A relatively new approach to recruitment and testing employs online crowdsourcing to rapidly measure the characteristics and behavior of large numbers of people. We tested the feasibility of researching borderline personality disorder (BPD) in this manner using one large crowdsourcing site, Amazon Mechanical Turk (MTurk). Specifically, we examined prevalence rates of psychopathology in a large MTurk sample, as well as the demographic, psychosocial, and psychiatric characteristics of individuals who met criteria for BPD. These characteristics were compared across three groups: those who met criteria for BPD currently, those who met criteria for remitted BPD, and those who had never met criteria for BPD. The results suggest that MTurk may be ideally suited for studying individuals with a wide range of pathology, from healthy to intensely symptomatic to remitted.

animal behavior and cognition↗

bioRxiv2018

Whole genome sequencing for predicting Mycobacterium abscessus drug susceptibility

Mycobacterium abscessus is emerging as an important pathogen in chronic lung diseases with concern regarding patient to patient transmission. The recent introduction of routine whole genome sequencing (WGS) as a replacement for existing reference techniques in England provides an opportunity to characterise the genetic determinants of resistance. We conducted a systematic review to catalogue all known resistance determining mutations. This knowledge was used to construct a predictive algorithm based on mutations in the erm(41) and rrl genes which was tested on a collection of 203 sequentially acquired clinical isolates for which there was paired genotype/phenotype data. A search for novel resistance determining mutations was conducted using an heuristic algorithm.\n\nThe sensitivity of existing knowledge for predicting resistance in clarithromycin was 95% (95% CI 89 - 98%) and the specificity was 66% (95% CI 54 - 76%). Subspecies alone was a poor predictor of resistance to clarithromycin. Eight potential new resistance conferring SNPs were identified. WGS demonstrates probable resistance determining SNPs in regions the NTM-DR line probe cannot detect. These mutations are potentially clinically important as they all occurred in samples predicted to be inducibly resistant, and for which a macrolide would therefore currently be indicated. We were unable to explain all resistance, raising the possibility of the involvement of other as yet unidentified genes.

microbiology↗

bioRxiv2018

A quantitative evaluation of MIRU-VNTR typing against whole-genome sequencing for identifying Mycobacterium tuberculosis transmission: A prospective observational cohort study

SummaryO_ST_ABSBackgroundC_ST_ABSMycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat (MIRU-VNTR) typing is widely used in high-income countries for Mycobacterium tuberculosis typing. Whole-genome sequencing (WGS) is known to deliver greater specificity, but no quantitative prospective comparison has yet been undertaken.\n\nMethodsWe studied isolates from the English Midlands, sampled consecutively between 1 January 2012 and 31 December 2015. In addition to routinely performed MIRU-VNTR typing, DNA was extracted from liquid cultures and sequenced using Illumina technology. Demographic and epidemiological data were extracted from the Enhanced Tuberculosis Surveillance system maintained by Public Health England. Closely related samples, defined using a threshold of five single nucleotide variants (SNVs), were compared to samples with identical MIRU-VNTR profiles, with shared epidemiological risk factors, and to those with both characteristics.\n\nFindings1,999 patients were identified for whom at least one M. tuberculosis isolate had been MIRU-VNTR typed and sequenced. Comparing epidemiological risk factors with close genetic relatedness, only coresidence had a positive predictive value of over 5%. Excluding co-resident individuals, 18.6% of patients with identical MIRU-VNTR profiles were within 5 SNVs. Where patients also shared social risk factors and ethnic group, this rose to 48%. Only 8% of MIRU-VNTR linked pairs in lineage 1 were within 5 SNV, compared to 31% in lineage 4.\n\nInterpretationIn the setting studied, MIRU-VNTR typing and epidemiological risk factors are poorly predictive of close genomic relatedness, assessed by SNV. MIRU-VNTR performance varies markedly by lineage.\n\nFundingPublic Health England, National Institute of Health Research Oxford Biomedical Research Centre.

microbiology↗

bioRxiv2018

Control of artefactual variation in reported inter-sample relatedness during clinical use of a Mycobacterium tuberculosis sequencing pipeline

Contact tracing requires reliable identification of closely related bacterial isolates. When we noticed the reporting of artefactual variation between M. tuberculosis isolates during routine next generation sequencing of Mycobacterium spp, we investigated its basis in 2,018 consecutive M. tuberculosis isolates. In the routine process used, clinical samples were decontaminated and inoculated into broth cultures; from positive broth cultures DNA was extracted, sequenced, reads mapped, and consensus sequences determined. We investigated the process of consensus sequence determination, which selects the most common nucleotide at each position. Having determined the high-quality read depth and depth of minor variants across 8,006 M. tuberculosis genomic regions, we quantified the relationship between the minor variant depth and the amount of non-Mycobacterial bacterial DNA, which originates from commensal microbes killed during sample decontamination. In the presence of non-Mycobacterial bacterial DNA, we found significant increases in minor variant frequencies of more than 1.5 fold in 242 regions covering 5.1% of the M. tuberculosis genome. Included within these were four high variation regions strongly influenced by the amount of non-Mycobacterial bacterial DNA. Excluding these four regions from pairwise distance comparisons reduced biologically implausible variation from 5.2% to 0% in an independent validation set derived from 226 individuals. Thus, we have demonstrated an approach identifying critical genomic regions contributing to clinically relevant artefactual variation in bacterial similarity searches. The approach described monitors the outputs of the complex multi-step laboratory and bioinformatics process, allows periodic process adjustments, and will have application to quality control of routine bacterial genomics.

microbiology↗

bioRxiv2017

Automated detection of Mycobacterial growth on 96-well plates for rapid and accurate Tuberculosis drug susceptibility testing

M. tuberculosis grows slowly and is challenging to work with experimentally compared with many other bacteria. Although microtitre plates have the potential to enable high-throughput phenotypic testing of M. tuberculosis, they can be difficult to read and interpret. Here we present a software package, the Automated Mycobacterial Growth Detection Algorithm (AMyGDA), that measures how much M. tuberculosis is growing in each well of a 96-well microtitre plate. The plate used here has serial dilutions of 14 anti-tuberculosis drugs, thereby permitting the minimum inhibitory concentrations (MICs) to be elucidated. The two participating laboratories each inoculated ten 96-well plates with the standard H37Rv reference strain and, after two weeks incubation, measured the MICs for all 14 drugs on each plate and took a photograph. By analysing the images, we demonstrate that AMyGDA is reproducible, and that the MICs measured are comparable to those measured by a laboratory scientist. AMyGDA software will be used by the Comprehensive Resistance Prediction for Tuberculosis: an International Consortium (CRyPTIC) to measure the drug susceptibility profile of a large number (> 30,000) of samples of M. tuberculosis from patients over the next few years.

microbiology↗

bioRxiv2017

A genetic investigation of sex bias in the prevalence of attention deficit hyperactivity disorder

Attention-deficit/hyperactivity disorder (ADHD) shows substantial heritability and is 2-7 times more common in males than females. We examined two putative genetic mechanisms underlying this sex bias: sex-specific heterogeneity and higher burden of risk in female cases. We analyzed genome-wide common variants from the Psychiatric Genomics Consortium and iPSYCH Project (20,183 cases, 35,191 controls) and Swedish population-registry data (N=77,905 cases, N=1,874,637 population controls). We find strong genetic correlation for ADHD across sex and no mean difference in polygenic burden across sex. In contrast, siblings of female probands are at an increased risk of ADHD, compared to siblings of male probands. The results also suggest that females with ADHD are at especially high risk of comorbid developmental conditions. Overall, this study supports a greater familial burden of risk in females with ADHD and some clinical and etiological heterogeneity. However, autosomal common variants largely do not explain the sex bias in ADHD prevalence.

genetics↗

bioRxiv2017

Back-Translating A Rodent Measure Of Negative Bias Into Humans: The Impact Of Induced Anxiety And Unmedicated Mood And Anxiety Disorders

BackgroundMood and anxiety disorders are ubiquitous but current treatment options are ineffective for large numbers of sufferers. Moreover, recent years have seen a number of promising pre-clinical interventions fail to translate into clinical efficacy in humans. Improved treatments are unlikely without better animal-human translational pipelines. Here, we directly adapt-i.e. back-translate - a rodent measure of negative affective bias into humans, and explore its relationship with a)pathological mood and anxiety symptoms (study one) and b)transient induced anxiety (study two).\n\nMethodParticipants who met criteria for mood or anxiety disorder symptomatology according to a face-to-face neuropsychiatric interview were included in the symptomatic group. N = 77(47 asymptomatic; Female = 21; 30 symptomatic; Female = 25) participants completed study one and N = 47 asymptomatic participants (25 female) completed study two. Outcome measures were choice ratios, reaction times and parameters recovered from a computational model of reaction time; the drift diffusion model (DDM).\n\nResultsSymptomatic individuals demonstrated increased negative affective bias relative to asymptomatic individuals (proportion high reward = 0.42(SD = 0.14), and 0.53(SD = 0.17), respectively) as well as reduced DDM drift rate (p = 0.004). No significant effects were observed for the within-subjects anxiety-induction in study 2.\n\nConclusionHumans with pathological anxiety symptoms directly mimic rodents undergoing anxiogenic manipulation. The lack of sensitivity to transient anxiety suggests the paradigm may, moreover, be primarily sensitive to clinically relevant symptoms. Our results establish a direct translational pipeline (and candidate therapeutics screen) from negative affective bias in rodents to pathological mood and anxiety symptoms in humans, and link it to a computational model of reaction time.

animal behavior and cognition↗