A Mycobacterial Enzyme Essential for Cell Division Synergizes with Resuscitation-Promoting Factor

The final stage of bacterial cell division requires the activity of one or more enzymes capable of degrading the layers of peptidoglycan connecting two recently developed daughter cells. Although this is a key step in cell division and is required by all peptidoglycan-containing bacteria, little is known about how these potentially lethal enzymes are regulated. It is likely that regulation is mediated, at least partly, through protein–protein interactions. Two lytic transglycosylases of mycobacteria, known as resuscitation-promoting factor B and E (RpfB and RpfE), have previously been shown to interact with the peptidoglycan-hydrolyzing endopeptidase, Rpf-interacting protein A (RipA). These proteins may form a complex at the septum of dividing bacteria. To investigate the function of this potential complex, we generated depletion strains in M. smegmatis. Here we show that, while depletion of rpfB has no effect on viability or morphology, ripA depletion results in a marked decrease in growth and formation of long, branched chains. These growth and morphological defects could be functionally complemented by the M. tuberculosis ripA orthologue (rv1477), but not by another ripA-like orthologue (rv1478). Depletion of ripA also resulted in increased susceptibility to the cell wall–targeting β-lactams. Furthermore, we demonstrate that RipA has hydrolytic activity towards several cell wall substrates and synergizes with RpfB. These data reveal the unusual essentiality of a peptidoglycan hydrolase and suggest a novel protein–protein interaction as one way of regulating its activity

The Monarch Initiative in 2019: an integrative data and analytic platform connecting phenotypes to genotypes across species.

In biology and biomedicine, relating phenotypic outcomes with genetic variation and environmental factors remains a challenge: patient phenotypes may not match known diseases, candidate variants may be in genes that haven\u27t been characterized, research organisms may not recapitulate human or veterinary diseases, environmental factors affecting disease outcomes are unknown or undocumented, and many resources must be queried to find potentially significant phenotypic associations. The Monarch Initiative (https://monarchinitiative.org) integrates information on genes, variants, genotypes, phenotypes and diseases in a variety of species, and allows powerful ontology-based search. We develop many widely adopted ontologies that together enable sophisticated computational analysis, mechanistic discovery and diagnostics of Mendelian diseases. Our algorithms and tools are widely used to identify animal models of human disease through phenotypic similarity, for differential diagnostics and to facilitate translational research. Launched in 2015, Monarch has grown with regards to data (new organisms, more sources, better modeling); new API and standards; ontologies (new Mondo unified disease ontology, improvements to ontologies such as HPO and uPheno); user interface (a redesigned website); and community development. Monarch data, algorithms and tools are being used and extended by resources such as GA4GH and NCATS Translator, among others, to aid mechanistic discovery and diagnostics

Uncovering Ecosystem Service Bundles through Social Preferences

Ecosystem service assessments have increasingly been used to support environmental management policies, mainly based on biophysical and economic indicators. However, few studies have coped with the social-cultural dimension of ecosystem services, despite being considered a research priority. We examined how ecosystem service bundles and trade-offs emerge from diverging social preferences toward ecosystem services delivered by various types of ecosystems in Spain. We conducted 3,379 direct face-to-face questionnaires in eight different case study sites from 2007 to 2011. Overall, 90.5% of the sampled population recognized the ecosystem’s capacity to deliver services. Formal studies, environmental behavior, and gender variables influenced the probability of people recognizing the ecosystem’s capacity to provide services. The ecosystem services most frequently perceived by people were regulating services; of those, air purification held the greatest importance. However, statistical analysis showed that socio-cultural factors and the conservation management strategy of ecosystems (i.e., National Park, Natural Park, or a non-protected area) have an effect on social preferences toward ecosystem services. Ecosystem service trade-offs and bundles were identified by analyzing social preferences through multivariate analysis (redundancy analysis and hierarchical cluster analysis). We found a clear trade-off among provisioning services (and recreational hunting) versus regulating services and almost all cultural services. We identified three ecosystem service bundles associated with the conservation management strategy and the rural-urban gradient. We conclude that socio-cultural preferences toward ecosystem services can serve as a tool to identify relevant services for people, the factors underlying these social preferences, and emerging ecosystem service bundles and trade-offs

REQUITE: A prospective multicentre cohort study of patients undergoing radiotherapy for breast, lung or prostate cancer

Purpose: REQUITE aimed to establish a resource for multi-national validation of models and biomarkers that predict risk of late toxicity following radiotherapy. The purpose of this article is to provide summary descriptive data. Methods: An international, prospective cohort study recruited cancer patients in 26 hospitals in eight countries between April 2014 and March 2017. Target recruitment was 5300 patients. Eligible patients had breast, prostate or lung cancer and planned potentially curable radiotherapy. Radiotherapy was prescribed according to local regimens, but centres used standardised data collection forms. Pre-treatment blood samples were collected. Patients were followed for a minimum of 12 (lung) or 24 (breast/prostate) months and summary descriptive statistics were generated. Results: The study recruited 2069 breast (99% of target), 1808 prostate (86%) and 561 lung (51%) cancer patients. The centralised, accessible database includes: physician-(47,025 forms) and patient-(54,901) reported outcomes; 11,563 breast photos; 17,107 DICOMs and 12,684 DVHs. Imputed genotype data are available for 4223 patients with European ancestry (1948 breast, 1728 prostate, 547 lung). Radiation-induced lymphocyte apoptosis (RILA) assay data are available for 1319 patients. DNA (n = 4409) and PAXgene tubes (n = 3039) are stored in the centralised biobank. Example prevalences of 2-year (1-year for lung) grade >= 2 CTCAE toxicities are 13% atrophy (breast), 3% rectal bleeding (prostate) and 27% dyspnoea (lung). Conclusion: The comprehensive centralised database and linked biobank is a valuable resource for the radiotherapy community for validating predictive models and biomarkers. Patient summary: Up to half of cancer patients undergo radiation therapy and irradiation of surrounding healthy tissue is unavoidable. Damage to healthy tissue can affect short-and long-term quality-of-life. Not all patients are equally sensitive to radiation "damage" but it is not possible at the moment to identify those who are. REQUITE was established with the aim of trying to understand more about how we could predict radiation sensitivity. The purpose of this paper is to provide an overview and summary of the data and material available. In the REQUITE study 4400 breast, prostate and lung cancer patients filled out questionnaires and donated blood. A large amount of data was collected in the same way. With all these data and samples a database and biobank were created that showed it is possible to collect this kind of information in a standardised way across countries. In the future, our database and linked biobank will be a resource for research and validation of clinical predictors and models of radiation sensitivity. REQUITE will also enable a better understanding of how many people suffer with radiotherapy toxicity

Evaluating ethanol-based sample preservation to facilitate use of DNA barcoding in routine freshwater biomonitoring programs using benthic macroinvertebrates.

Molecular methods, such as DNA barcoding, have the potential to enhance biomonitoring programs worldwide. Altering routinely used sample preservation methods to protect DNA from degradation may pose a potential impediment to application of DNA barcoding and metagenomics for biomonitoring using benthic macroinvertebrates. Using higher volumes or concentrations of ethanol, requirements for shorter holding times, or the need to include additional filtering may increase cost and logistical constraints to existing biomonitoring programs. To address this issue we evaluated the efficacy of various ethanol-based sample preservation methods at maintaining DNA integrity. We evaluated a series of methods that were minimally modified from typical field protocols in order to identify an approach that can be readily incorporated into existing monitoring programs. Benthic macroinvertebrates were collected from a minimally disturbed stream in southern California, USA and subjected to one of six preservation treatments. Ten individuals from five taxa were selected from each treatment and processed to produce DNA barcodes from the mitochondrial gene cytochrome c oxidase I (COI). On average, we obtained successful COI sequences (i.e. either full or partial barcodes) for between 93-99% of all specimens across all six treatments. As long as samples were initially preserved in 95% ethanol, successful sequencing of COI barcodes was not affected by a low dilution ratio of 2∶1, transfer to 70% ethanol, presence of abundant organic matter, or holding times of up to six months. Barcoding success varied by taxa, with Leptohyphidae (Ephemeroptera) producing the lowest barcode success rate, most likely due to poor PCR primer efficiency. Differential barcoding success rates have the potential to introduce spurious results. However, routine preservation methods can largely be used without adverse effects on DNA integrity

Developing Indicators of Nutrient Pollution in Streams Using 16S rRNA Gene Metabarcoding of Periphyton-Associated Bacteria

Indicators based on nutrient-biota relationships in streams can inform water quality restoration and protection programs. Bacterial assemblages could be particularly useful indicators of nutrient effects because they are species-rich, important contributors to ecosystem processes in streams, and responsive to rapidly changing conditions. Here, we sampled 25 streams weekly (12–14 times each) and used 16S rRNA gene metabarcoding of periphyton-associated bacteria to quantify the effects of total phosphorus (TP) and total nitrogen (TN). Threshold indicator taxa analysis identified assemblage-level changes and amplicon sequence variants (ASVs) that increased or decreased with increasing TP and TN concentrations (i.e., low P, high P, low N, and high N ASVs). Boosted regression trees confirmed that relative abundances of gene sequence reads for these four indicator groups were associated with nutrient concentrations. Gradient forest analysis complemented these results by using multiple predictors and random forest models for each ASV to identify portions of TP and TN gradients at which the greatest changes in assemblage structure occurred. Synthesized statistical results showed bacterial assemblage structure began changing at 24 µg TP/L with the greatest changes occurring from 110 to 195 µg/L. Changes in the bacterial assemblages associated with TN gradually occurred from 275 to 855 µg/L. Taxonomic and phylogenetic analyses showed that low nutrient ASVs were commonly Firmicutes, Verrucomicrobiota, Flavobacteriales, and Caulobacterales, Pseudomonadales, and Rhodobacterales of Proteobacteria, whereas other groups, such as Chitinophagales of Bacteroidota, and Burkholderiales, Rhizobiales, Sphingomonadales, and Steroidobacterales of Proteobacteria comprised the high nutrient ASVs. Overall, the responses of bacterial ASV indicators in this study highlight the utility of metabarcoding periphyton-associated bacteria for quantifying biotic responses to nutrient inputs in streams

Cumulative frequency graphs of sequence length (base pairs) by treatment.

<p>Circles represent the points at which additional sequences are accumulated. Dashed horizontal lines represent the cutoff between partial barcodes (to the left) and full barcodes (to the right).</p

Higher taxa (i.e. Class/Order), lower taxon (i.e. Family/Genus), and individuals per replicate.

<p>Higher taxa (i.e. Class/Order), lower taxon (i.e. Family/Genus), and individuals per replicate.</p

Cumulative frequency graphs of sequence length (base pairs) by lower taxa.

<p>Circles represent the points at which additional sequences are accumulated. Dashed horizontal lines represent the cutoff between partial barcodes (to the left) and full barcodes (to the right).</p

Primer pairs used (forward+reverse) and number of sequence reactions each pair was used.

<p>Primer pairs used (forward+reverse) and number of sequence reactions each pair was used.</p