Characterisation of the mode of action of Aurodox, a Type III secretion system inhibitor from Streptomyces goldiniensis

Recent work has demonstrated that the polyketide natural product Aurodox, from Streptomyces goldiniensis is able to block the pathogenesis of the murine pathogen Citrobacter rodentium. In this work we aimed to aimed gain a better understanding of the mechanism of action of the compound. We show that Aurodox downregulates the expression of the Type Three Secretion Systems of enteropathogenic and enterohaemorhagic Escherichia coli. Furthermore, we have used transcriptomic analysis to show that Aurodox inhibits the expression at the transcriptional level by repressing the master regulator, ler. Our data support a model in which Aurodox acts upstream of ler and not directly on the secretion system itself. Finally, we have shown that Aurodox, unlike some traditional antibiotics, does not induce expression of RecA, which is essential for the production of Shiga toxin. We propose that these properties nominate Aurodox as a promising anti-virulence therapy for the treatment of these infections

The antimicrobial peptide cathelicidin drives development of experimental autoimmune encephalomyelitis in mice by affecting Th17 differentiation

Multiple sclerosis (MS) is a highly prevalent demyelinating autoimmune condition; the mechanisms regulating its severity and progression are unclear. The IL-17-producing Th17 subset of T cells has been widely implicated in MS and in the mouse model, experimental autoimmune encephalomyelitis (EAE). However, the differentiation and regulation of Th17 cells during EAE remain incompletely understood. Although evidence is mounting that the antimicrobial peptide cathelicidin profoundly affects early T cell differentiation, no studies have looked at its role in longer-term T cell responses. Now, we report that cathelicidin drives severe EAE disease. It is released from neutrophils, microglia, and endothelial cells throughout disease; its interaction with T cells potentiates Th17 differentiation in lymph nodes and Th17 to exTh17 plasticity and IFN-γ production in the spinal cord. As a consequence, mice lacking cathelicidin are protected from severe EAE. In addition, we show that cathelicidin is produced by the same cell types in the active brain lesions in human MS disease. We propose that cathelicidin exposure results in highly activated, cytokine-producing T cells, which drive autoimmunity; this is a mechanism through which neutrophils amplify inflammation in the central nervous system

Inter- and intra-reproducibility of genotypes from sheep technical replicates on Illumina and Affymetrix platforms

peer-reviewedBackground Accurate genomic analyses are predicated upon access to accurate genotype input data. The objective of this study was to quantify the reproducibility of genotype data that are generated from the same genotype platform and from different genotyping platforms. Methods Genotypes based on 51,121 single nucleotide polymorphisms (SNPs) for 84 animals that were each genotyped on Illumina and Affymetrix platforms and for another 25 animals that were each genotyped twice on the same Illumina platform were compared. Genotypes based on 11,323 SNPs for an additional 21 animals that were genotyped on two different Illumina platforms by two different service providers were also compared. Reproducibility of the results was measured as the correlation between allele counts and as genotype and allele concordance rates. Results A mean within-animal correlation of 0.9996 was found between allele counts in the 25 duplicate samples that were genotyped on the same Illumina platform and varied from 0.9963 to 1.0000 per animal. The mean (minimum, maximum) genotype and allele concordance rates per animal between the 25 duplicate samples were equal to 0.9996 (0.9968, 1.0000) and 0.9993 (0.9937, 1.0000), respectively. The concordance rate between the two different Illumina platforms was also near 1. A mean within-animal correlation of 0.9738 was found between genotypes that were generated on the Illumina and Affymetrix platforms and varied from 0.9505 to 0.9812 per animal. The mean (minimum, maximum) within-animal genotype and allele concordance rates between the Illumina and Affymetrix platforms were equal to 0.9711 (0.9418, 0.9798) and 0.9845 (0.9695, 0.9889), respectively. The genotype concordance rate across all genotypes increased from 0.9711 to 0.9949 when the SNPs used were restricted to those with three high-resolution genotype clusters which represented 75.2% of the called genotypes. Conclusions and implications Our results suggest that, regardless of the genotype platform or service provider, high genotype concordance rates are achieved especially if they are restricted to high-quality extracted DNA and SNPs that result in high-quality genotypes.Financial support from the Irish Department of Agriculture, Stimulus Research Fund project OVIGEN is gratefully acknowledged. The contribution from the International Sheep Genomics Consortium in the development of the low-density custom genotyping platform is also acknowledged

Development of an improved blood-stage malaria vaccine targeting the essential RH5-CyRPA-RIPR invasion complex

Reticulocyte-binding protein homologue 5 (RH5), a leading blood-stage Plasmodium falciparum malaria vaccine target, interacts with cysteine-rich protective antigen (CyRPA) and RH5-interacting protein (RIPR) to form an essential heterotrimeric “RCR-complex”. We investigate whether RCR-complex vaccination can improve upon RH5 alone. Using monoclonal antibodies (mAbs) we show that parasite growth-inhibitory epitopes on each antigen are surface-exposed on the RCR-complex and that mAb pairs targeting different antigens can function additively or synergistically. However, immunisation of female rats with the RCR-complex fails to outperform RH5 alone due to immuno-dominance of RIPR coupled with inferior potency of anti-RIPR polyclonal IgG. We identify that all growth-inhibitory antibody epitopes of RIPR cluster within the C-terminal EGF-like domains and that a fusion of these domains to CyRPA, called “R78C”, combined with RH5, improves the level of in vitro parasite growth inhibition compared to RH5 alone. These preclinical data justify the advancement of the RH5.1 + R78C/Matrix-M™ vaccine candidate to Phase 1 clinical trial

Phylogenetic organization of bacterial activity.

Phylogeny is an ecologically meaningful way to classify plants and animals, as closely related taxa frequently have similar ecological characteristics, functional traits and effects on ecosystem processes. For bacteria, however, phylogeny has been argued to be an unreliable indicator of an organism\u27s ecology owing to evolutionary processes more common to microbes such as gene loss and lateral gene transfer, as well as convergent evolution. Here we use advanced stable isotope probing with (13)C and (18)O to show that evolutionary history has ecological significance for in situ bacterial activity. Phylogenetic organization in the activity of bacteria sets the stage for characterizing the functional attributes of bacterial taxonomic groups. Connecting identity with function in this way will allow scientists to begin building a mechanistic understanding of how bacterial community composition regulates critical ecosystem functions.The ISME Journal advance online publication, 4 March 2016; doi:10.1038/ismej.2016.28

Interstate migration of the US poverty population: Immigration “pushes” and welfare magnet “pulls”

This study evaluates the social and demographic structure of poverty migration during the 1985–90 period based on an analysis of recent census data. Particular attention is given to the roles of two policy-relevant factors that are proposed to be linked to poverty migration. The first of these is the role of immigration from abroad and its effect on the net out-migration of longer-term residents with below-poverty incomes, from States receiving the highest volume of immigrants. Such a response, it is argued, could result from job competition or other economic and social costs associated with immigration. The second involves the poverty population “magnet” effect associated with State welfare benefits (AFDC and Food Stamp payments) which has come under renewed scrutiny in light of the impending reform of the federal welfare program. The impact of both of these factors on interstate poverty migration is evaluated in a broader context that takes cognizance of other sociodemographic subgroups, and State-level attributes that are known to be relevant in explaining internal migration. This research employs an exceptionally rich data base of aggregate migration flows, specially tabulated from the full migration sample of the 1990 US census (based on the “residence 5 years ago” question). It also employs an analysis technique, the nested logit model, which identifies separately the “push” and “pull” effects of immigration, welfare benefits, and other State attributes on the migration process. Our findings are fairly clear. The high volume of immigration to selected US States does affect a selective out-migration of the poverty population, which is stronger for whites, Blacks and other non-Asian minorities as well as the least-educated. These results are consistent with arguments that internal migrants are responding to labor market competition from similarly educated immigrants. Moreover, we found that the impact of immigration occurs primarily as a “push” rather than a reduced “pull.” In contrast, State welfare benefits exert only minimal effects on the interstate migration of the poverty population—either as “pulls” or “pushes,” although some demographic segments of that population are more prone to respond than others. In addition to these findings, our results reveal the strong impact that a State's racial and ethnic composition exerts in both retaining and attracting migrants of like race and ethnic groups. This suggests the potential for a greater cross-state division in the US poverty population, by race and ethnic status.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/43484/1/11111_2005_Article_BF02208337.pd

Development of an amplicon-based sequencing approach in response to the global emergence of mpox

The 2022 multicountry mpox outbreak concurrent with the ongoing Coronavirus Disease 2019 (COVID-19) pandemic further highlighted the need for genomic surveillance and rapid pathogen whole-genome sequencing. While metagenomic sequencing approaches have been used to sequence many of the early mpox infections, these methods are resource intensive and require samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there was an urgent need for a more sensitive and broadly applicable sequencing approach. Highly multiplexed amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Here, we used PrimalScheme to develop a primer scheme for human monkeypox virus that can be used with many sequencing and bioinformatics pipelines implemented in public health laboratories during the COVID-19 pandemic. We sequenced clinical specimens that tested presumptively positive for human monkeypox virus with amplicon-based and metagenomic sequencing approaches. We found notably higher genome coverage across the virus genome, with minimal amplicon drop-outs, in using the amplicon-based sequencing approach, particularly in higher PCR cycle threshold (Ct) (lower DNA titer) samples. Further testing demonstrated that Ct value correlated with the number of sequencing reads and influenced the percent genome coverage. To maximize genome coverage when resources are limited, we recommend selecting samples with a PCR Ct below 31 Ct and generating 1 million sequencing reads per sample. To support national and international public health genomic surveillance efforts, we sent out primer pool aliquots to 10 laboratories across the United States, United Kingdom, Brazil, and Portugal. These public health laboratories successfully implemented the human monkeypox virus primer scheme in various amplicon sequencing workflows and with different sample types across a range of Ct values. Thus, we show that amplicon-based sequencing can provide a rapidly deployable, cost-effective, and flexible approach to pathogen whole-genome sequencing in response to newly emerging pathogens. Importantly, through the implementation of our primer scheme into existing SARS-CoV-2 workflows and across a range of sample types and sequencing platforms, we further demonstrate the potential of this approach for rapid outbreak response.This publication was made possible by CTSA Grant Number UL1 TR001863 from the National Center for Advancing Translational Science (NCATS), a component of the National Institutes of Health (NIH) awarded to CBFV. INSA was partially funded by the HERA project (Grant/ 2021/PHF/23776) supported by the European Commission through the European Centre for Disease Control (to VB).info:eu-repo/semantics/publishedVersio

Oral abstracts 3: RA Treatment and outcomesO13. Validation of jadas in all subtypes of juvenile idiopathic arthritis in a clinical setting

Background: Juvenile Arthritis Disease Activity Score (JADAS) is a 4 variable composite disease activity (DA) score for JIA (including active 10, 27 or 71 joint count (AJC), physician global (PGA), parent/child global (PGE) and ESR). The validity of JADAS for all ILAR subtypes in the routine clinical setting is unknown. We investigated the construct validity of JADAS in the clinical setting in all subtypes of JIA through application to a prospective inception cohort of UK children presenting with new onset inflammatory arthritis. Methods: JADAS 10, 27 and 71 were determined for all children in the Childhood Arthritis Prospective Study (CAPS) with complete data available at baseline. Correlation of JADAS 10, 27 and 71 with single DA markers was determined for all subtypes. All correlations were calculated using Spearman's rank statistic. Results: 262/1238 visits had sufficient data for calculation of JADAS (1028 (83%) AJC, 744 (60%) PGA, 843 (68%) PGE and 459 (37%) ESR). Median age at disease onset was 6.0 years (IQR 2.6-10.4) and 64% were female. Correlation between JADAS 10, 27 and 71 approached 1 for all subtypes. Median JADAS 71 was 5.3 (IQR 2.2-10.1) with a significant difference between median JADAS scores between subtypes (p < 0.01). Correlation of JADAS 71 with each single marker of DA was moderate to high in the total cohort (see Table 1). Overall, correlation with AJC, PGA and PGE was moderate to high and correlation with ESR, limited JC, parental pain and CHAQ was low to moderate in the individual subtypes. Correlation coefficients in the extended oligoarticular, rheumatoid factor negative and enthesitis related subtypes were interpreted with caution in view of low numbers. Conclusions: This study adds to the body of evidence supporting the construct validity of JADAS. JADAS correlates with other measures of DA in all ILAR subtypes in the routine clinical setting. Given the high frequency of missing ESR data, it would be useful to assess the validity of JADAS without inclusion of the ESR. Disclosure statement: All authors have declared no conflicts of interest. Table 1Spearman's correlation between JADAS 71 and single markers DA by ILAR subtype ILAR Subtype Systemic onset JIA Persistent oligo JIA Extended oligo JIA Rheumatoid factor neg JIA Rheumatoid factor pos JIA Enthesitis related JIA Psoriatic JIA Undifferentiated JIA Unknown subtype Total cohort Number of children 23 111 12 57 7 9 19 7 17 262 AJC 0.54 0.67 0.53 0.75 0.53 0.34 0.59 0.81 0.37 0.59 PGA 0.63 0.69 0.25 0.73 0.14 0.05 0.50 0.83 0.56 0.64 PGE 0.51 0.68 0.83 0.61 0.41 0.69 0.71 0.9 0.48 0.61 ESR 0.28 0.31 0.35 0.4 0.6 0.85 0.43 0.7 0.5 0.53 Limited 71 JC 0.29 0.51 0.23 0.37 0.14 -0.12 0.4 0.81 0.45 0.41 Parental pain 0.23 0.62 0.03 0.57 0.41 0.69 0.7 0.79 0.42 0.53 Childhood health assessment questionnaire 0.25 0.57 -0.07 0.36 -0.47 0.84 0.37 0.8 0.66 0.4

Genetic determinants of telomere length from 109,122 ancestrally diverse whole-genome sequences in TOPMed

Genetic studies on telomere length are important for understanding age-related diseases. Prior GWAS for leukocyte TL have been limited to European and Asian populations. Here, we report the first sequencing-based association study for TL across ancestrally-diverse individuals (European, African, Asian and Hispanic/Latino) from the NHLBI Trans-Omics for Precision Medicine (TOPMed) program. We used whole genome sequencing (WGS) of whole blood for variant genotype calling and the bioinformatic estimation of telomere length in n=109,122 individuals. We identified 59 sentinel variants (p-value OBFC1indicated the independent signals colocalized with cell-type specific eQTLs for OBFC1 (STN1). Using a multi-variant gene-based approach, we identified two genes newly implicated in telomere length, DCLRE1B (SNM1B) and PARN. In PheWAS, we demonstrated our TL polygenic trait scores (PTS) were associated with increased risk of cancer-related phenotypes