Emergence of novel human pathogens pose significant challenges to human health as highlighted by the SARS-CoV-2 pandemic. Wastewater based epidemiology (WBE) has previously been employed to identify ...viral pathogens and outbreaks by testing samples from regional wastewater treatment plants. Near source tracking (NST) allows for more targeted WBE by analysing samples from individual buildings such as schools or even individual floors such as in multi-floor office buildings. Despite the public health advantages of WBE, few strategies exist for optimising NST sampling methodologies. Therefore, we developed a protocol to evaluate virus detection in NST sampling using Pepper Mild Mottle Virus (PMMoV) as a proxy for RNA viruses. PMMoV is the most abundant enteric human associated RNA virus and is present in peppers/pepper-containing foods. Two bespoke TaqMan RT-PCR assays were developed to detect a PMMoV genomic 5' region and a capsid associated gene. To evaluate the protocol against field samples, pepper homogenates were flushed down an in-use toilet (Liverpool School of Tropical Medicine, UK) to spike wastewater with PMMoV on multiple days, and samples collected from two sewage access points to validate NST samplers. These wastewater samples were assessed for PMMoV based on Ct values and results compared to pepper and Tabasco derived PMMoV positive controls. Positive detection of PMMoV was comparable and consistent in ten independent samples across two NST samplers regardless of pepper homogenate spiking. We have developed two novel one step TaqMan assays that amplify both PMMoV targets in viral RNA extractions from peppers, Tabasco, and wastewater samples with cDNA synthesis through to RT-PCR results taking approximately 30 minutes. Pepper homogenate flushing was not required to detect PMMoV in our wastewater samples, however this strategy of flushing PMMoV containing materials outlined here could be valuable in assessing and validating NST in buildings with no previous or current sewage flow.
The human microbiome is affected by multiple factors, including the environment and host genetics. In this study, we analyzed the salivary microbiomes of an extended family of Ashkenazi Jewish ...individuals living in several cities and investigated associations with both shared household and host genetic similarities. We found that environmental effects dominated over genetic effects. While there was weak evidence of geographical structuring at the level of cities, we observed a large and significant effect of shared household on microbiome composition, supporting the role of the immediate shared environment in dictating the presence or absence of taxa. This effect was also seen when including adults who had grown up in the same household but moved out prior to the time of sampling, suggesting that the establishment of the salivary microbiome earlier in life may affect its long-term composition. We found weak associations between host genetic relatedness and microbiome dissimilarity when using family pedigrees as proxies for genetic similarity. However, this association disappeared when using more-accurate measures of kinship based on genome-wide genetic markers, indicating that the environment rather than host genetics is the dominant factor affecting the composition of the salivary microbiome in closely related individuals. Our results support the concept that there is a consistent core microbiome conserved across global scales but that small-scale effects due to a shared living environment significantly affect microbial community composition.
Previous research shows that the salivary microbiomes of relatives are more similar than those of nonrelatives, but it remains difficult to distinguish the effects of relatedness and shared household environment. Furthermore, pedigree measures may not accurately measure host genetic similarity. In this study, we include genetic relatedness based on genome-wide single nucleotide polymorphisms (SNPs) (rather than pedigree measures) and shared environment in the same analysis. We quantify the relative importance of these factors by studying the salivary microbiomes in members of a large extended Ashkenazi Jewish family living in different locations. We find that host genetics plays no significant role and that the dominant factor is the shared environment at the household level. We also find that this effect appears to persist in individuals who have moved out of the parental household, suggesting that aspects of salivary microbiome composition established during upbringing can persist over a time scale of years.
Recent studies have provided a detailed census of genes that are mutated in acute myeloid leukemia (AML). Our next challenge is to understand how this genetic diversity defines the pathophysiology of ...AML and informs clinical practice.
We enrolled a total of 1540 patients in three prospective trials of intensive therapy. Combining driver mutations in 111 cancer genes with cytogenetic and clinical data, we defined AML genomic subgroups and their relevance to clinical outcomes.
We identified 5234 driver mutations across 76 genes or genomic regions, with 2 or more drivers identified in 86% of the patients. Patterns of co-mutation compartmentalized the cohort into 11 classes, each with distinct diagnostic features and clinical outcomes. In addition to currently defined AML subgroups, three heterogeneous genomic categories emerged: AML with mutations in genes encoding chromatin, RNA-splicing regulators, or both (in 18% of patients); AML with TP53 mutations, chromosomal aneuploidies, or both (in 13%); and, provisionally, AML with IDH2(R172) mutations (in 1%). Patients with chromatin-spliceosome and TP53-aneuploidy AML had poor outcomes, with the various class-defining mutations contributing independently and additively to the outcome. In addition to class-defining lesions, other co-occurring driver mutations also had a substantial effect on overall survival. The prognostic effects of individual mutations were often significantly altered by the presence or absence of other driver mutations. Such gene-gene interactions were especially pronounced for NPM1-mutated AML, in which patterns of co-mutation identified groups with a favorable or adverse prognosis. These predictions require validation in prospective clinical trials.
The driver landscape in AML reveals distinct molecular subgroups that reflect discrete paths in the evolution of AML, informing disease classification and prognostic stratification. (Funded by the Wellcome Trust and others; ClinicalTrials.gov number, NCT00146120.).
Antimicrobial resistance (AMR) in clinically relevant bacteria is a growing threat to public health globally. In these bacteria, antimicrobial resistance genes are often associated with mobile ...genetic elements (MGEs), which promote their mobility, enabling them to rapidly spread throughout a bacterial community.
The tool MobileElementFinder was developed to enable rapid detection of MGEs and their genetic context in assembled sequence data. MGEs are detected based on sequence similarity to a database of 4452 known elements augmented with annotation of resistance genes, virulence factors and detection of plasmids.
MobileElementFinder was applied to analyse the mobilome of 1725 sequenced Salmonella enterica isolates of animal origin from Denmark, Germany and the USA. We found that the MGEs were seemingly conserved according to multilocus ST and not restricted to either the host or the country of origin. Moreover, we identified putative translocatable units for specific aminoglycoside, sulphonamide and tetracycline genes. Several putative composite transposons were predicted that could mobilize, among others, AMR, metal resistance and phosphodiesterase genes associated with macrophage survivability. This is, to our knowledge, the first time the phosphodiesterase-like pdeL has been found to be potentially mobilized into S. enterica.
MobileElementFinder is a powerful tool to study the epidemiology of MGEs in a large number of genome sequences and to determine the potential for genomic plasticity of bacteria. This web service provides a convenient method of detecting MGEs in assembled sequence data. MobileElementFinder can be accessed at https://cge.cbs.dtu.dk/services/MobileElementFinder/.
Abstract
Antibiotic-resistant Gram-positive bacteria are responsible for morbidity and mortality in healthcare environments. Enterococcus faecium, Enterococcus faecalis, Staphylococcus aureus and ...Streptococcus pneumoniae can all exhibit clinically relevant multidrug resistance phenotypes due to acquired resistance genes on mobile genetic elements. It is possible that clinically relevant multidrug-resistant Clostridium difficile strains will appear in the future, as the organism is adept at acquiring mobile genetic elements (plasmids and transposons). Conjugative transposons of the Tn916/Tn1545 family, which carry major antibiotic resistance determinants, are transmissible between these different bacteria by a conjugative mechanism during which the elements are excised by a staggered cut from donor cells, converted to a circular form, transferred by cell−cell contact and inserted into recipient cells by a site-specific recombinase. The ability of these conjugative transposons to acquire additional, clinically relevant antibiotic resistance genes importantly contributes to the emergence of multidrug resistance.
In this review we explore the diversity of the Tn916 / Tn1545 family of mobile genetic elements in four major Gram positive pathogens and show that these elements are central to the dissemination of antibiotic resistance among these pathogens.
Chimeric antigen receptors (CARs) are synthetic receptors that reprogram T cells to kill cancer. The success of CAR-T cell therapies highlights the promise of programmed immunity and suggests that ...applying CAR strategies to other immune cell lineages may be beneficial. Here, we engineered a family of Chimeric Antigen Receptors for Phagocytosis (CAR-Ps) that direct macrophages to engulf specific targets, including cancer cells. CAR-Ps consist of an extracellular antibody fragment, which can be modified to direct CAR-P activity towards specific antigens. By screening a panel of engulfment receptor intracellular domains, we found that the cytosolic domains from Megf10 and FcRɣ robustly triggered engulfment independently of their native extracellular domain. We show that CAR-Ps drive specific engulfment of antigen-coated synthetic particles and whole human cancer cells. Addition of a tandem PI3K recruitment domain increased cancer cell engulfment. Finally, we show that CAR-P expressing murine macrophages reduce cancer cell number in co-culture by over 40%.
Solvent organization is a key but underexploited contributor to the thermodynamics of protein–ligand recognition, with implications for ligand discovery, drug resistance, and protein engineering. ...Here, we explore the contribution of solvent to ligand binding in the Haemophilus influenzae virulence protein SiaP. By introducing a single mutation without direct ligand contacts, we observed a >1000-fold change in sialic acid binding affinity. Crystallographic and calorimetric data of wild-type and mutant SiaP showed that this change results from an enthalpically unfavorable perturbation of the solvent network. This disruption is reflected by changes in the normalized atomic displacement parameters of crystallographic water molecules. In SiaP’s enclosed cavity, relative differences in water-network dynamics serve as a simple predictor of changes in the free energy of binding upon changing protein, ligand, or both. This suggests that solvent structure is an evolutionary constraint on protein sequence that contributes to ligand affinity and selectivity.
The Tn916 family is a group of mobile genetic elements that are widespread among many commensal and pathogenic bacteria. These elements are found primarily, but not exclusively, in the Firmicutes. ...They are integrated into the bacterial genome and are capable of conjugative transfer to a new host and, often, intracellular transposition to a different genomic site - hence their name: 'conjugative transposons', or 'integrative conjugative elements'. An increasing variety of Tn916 relatives are being reported from different bacteria, harbouring genes coding for resistance to various antibiotics and the potential to encode other functions, such as lantibiotic immunity. This family of mobile genetic elements has an extraordinary ability to acquire accessory genes, making them important vectors in the dissemination of various traits among environmental, commensal and clinical bacteria. These elements are also responsible for genome rearrangements, providing considerable raw material on which natural selection can act. Therefore, the study of this family of mobile genetic elements is essential for a better understanding and control of the current rise of antibiotic resistance among pathogenic bacteria.
(Inter)nationalising the antibiotic research and development pipeline Singer, Andrew C; Kirchhelle, Claas; Roberts, Adam P
Lancet. Infectious diseases/The Lancet. Infectious diseases,
February 2020, 2020-Feb, 2020-02-00, 20200201, 2020-02, Letnik:
20, Številka:
2
Journal Article
Recenzirano
Odprti dostop
In this Personal View, we critically examine the wider context of international efforts to stimulate commercial antibiotic research and development via public–private initiatives. Despite these ...efforts, antibiotics remain a global resource without an international support structure that is commensurate to the risks from antibiotic-resistant infections and the long-term nature of required solutions. To protect this resource, we propose a two-pronged antibiotic research and development strategy based on a short-term strengthening of incentives (such as market entry rewards) to maximise the delivery of existing opportunities in the pipeline, and on a concurrent medium-term to long-term establishment of a global, publicly funded antibiotic research and development institute. Designed sustainably to deliver novel and first-in-class antibiotics targeting key human health gaps, the institute and its staff would become a global resource that, unlike the private pharmaceutical sector, would be managed as an open science platform. Our model of internationalised public research and development would maximise scientific synergy and cross-fertilisation, minimise replication of effort, acquire and preserve existing know-how, and ensure equitable and sustainable access to novel and effective antibiotics. Its genuinely global focus would also help counteract tendencies to equate donor with global health priorities. Our proposal is not radical. Historical precedent and developments in other research areas show that sustained international funding of publicly owned research can hasten the delivery of critically needed drugs and lower barriers to access.
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