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Abstract X-ray grating interferometry allows for the simultaneous acquisition of attenuation, differential-phase contrast, and dark-field images, resulting from X-ray attenuation, refraction, and small-angle scattering, respectively. The modulated phase grating (MPG) interferometer is a recently developed grating interferometry system capable of generating a directly resolvable interference pattern using a relatively large period grating envelope function that is sampled at a pitch that is small enough that X-ray spatial coherence can be achieved by using a microfocus X-ray source or G0 grating. We present the theory of the MPG interferometry system for a 2-dimensional staggered grating, derived using Fourier optics, and we compare the theoretical predictions with experiments we have performed with a microfocus X-ray system at Pennington Biomedical Research Center, LSU. The theoretical and experimental fringe visibility is evaluated as a function of grating-to-detector distance. Additionally, quantitative experiments are performed with porous carbon and alumina compounds, and the mean normalized dark-field signal is compared with independent porosimetry measurements. Qualitative analysis of attenuation and dark-field images of a dried anchovy are shown.

Abstract The rise of drug resistance has become a global crisis, with >1 million deaths due to resistant bacterial infections each year. Pseudomonas aeruginosa, in particular, remains a serious problem with limited solutions due to complex resistance mechanisms that now lead to more than 32,000 multidrug-resistant (MDR) infections and over 2000 deaths in the U.S. annually. While the emergence of resistant bacteria has become ominously common, identification of useful new drug classes has been limited over the past over 40 years. We found that a potential novel therapeutic, the peptide-mimetic TM5, is effective at killing P. aeruginosa and displays sufficiently low toxicity in mammalian cells to allow for use in treatment of infections. Interestingly, TM5 kills P. aeruginosa more rapidly than traditional antibiotics, within 30–60 min in vitro, and is effective against a range of clinical isolates, including extensively drug resistant strains. In vivo, TM5 significantly reduced bacterial load in the lungs within 24 h compared to untreated mice and demonstrated few adverse effects. Taken together, these observations suggest that TM5 shows promise as an alternative therapy for MDR P. aeruginosa respiratory infections.

Abstract Background Proteus syndrome is an ultra-rare mosaic overgrowth disorder. Individuals with Proteus syndrome can develop emphysematous and cystic changes of the lung that may lead to progressive respiratory symptoms and require surgical intervention. This retrospective study seeks to quantify the radiographic features of Proteus syndrome-associated lung disease using computed tomography (CT) of the chest. The first method derives a Cystic Lung Score (CLS) by using a computer-aided diagnostic tool to quantify the fraction of cystic involvement of the lung. The second method yields a Clinician Visual Score (CVS), an observer reported scale of severity based on multiple radiographic features. The aim of this study was to determine if these measurements are associated with clinical symptoms, pulmonary function test (PFT) measurements, and if they may be used to assess progression of pulmonary disease. Results One hundred and thirteen imaging studies from 44 individuals with Proteus syndrome were included. Dyspnea and oxygen use were each associated with higher CLS (p = 0.001 and

Integration of the Internet of Things (IoT) in the automotive industry has brought benefits as well as security challenges. Significant benefits include enhanced passenger safety and more comprehensive vehicle performance diagnostics. However, current onboard and remote vehicle diagnostics do not include the ability to detect counterfeit parts. A method is needed to verify authentic parts along the automotive supply chain from manufacture through installation and to coordinate part authentication with a secure database. In this study, we develop an architecture for anti-counterfeiting in automotive supply chains. The core of the architecture consists of a cyber-physical trust anchor and authentication mechanisms connected to blockchain-based tracking processes with cloud storage. The key parameters for linking a cyber-physical trust anchor in embedded IoT include identifiers (i.e., serial numbers, special features, hashes), authentication algorithms, blockchain, and sensors. A use case was provided by a two-year long implementation of simple trust anchors and tracking for a coffee supply chain which suggests a low-cost part authentication strategy could be successfully applied to vehicles. The challenge is authenticating parts not normally connected to main vehicle communication networks. Therefore, we advance the coffee bean model with an acoustical sensor to differentiate between authentic and counterfeit tires onboard the vehicle. The workload of secure supply chain development can be shared with the development of the connected autonomous vehicle networks, as the fleet performance is degraded by vehicles with questionable replacement parts of uncertain reliability.

The functional and causal relationships among the inferotemporal face patches remain elusive. Here, using fMRI and pharmacological inactivation, the authors discover an antero-posterior bidirectional and fundal-lateral parallel organization of these face patches.

Face stimuli that are perceived as emotionally expressive rather than neutral are associated with specific neural responses in V1. Here the authors show that valence information perceived from facial expressions is computed in the amygdala and fed back to V1 via direct anatomical projections.

Heskett et al. describe several members of a class of long non-coding RNAs, known as ASARs, which show distinct epigenetic regulation between subclonal lineages and are essential for normal DNA replication timing and stability of human autosomes.

Abstract The indoor environment is the primary location for the transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), largely driven by respiratory particle accumulation in the air and increased connectivity between the individuals occupying indoor spaces. In this study, we aimed to track a cohort of subjects as they occupied a COVID-19 isolation dormitory to better understand the impact of subject and environmental viral load over time, symptoms, and room ventilation on the detectable viral load within a single room. We find that subject samples demonstrate a decrease in overall viral load over time, symptoms significantly impact environmental viral load, and we provide the first real-world evidence for decreased aerosol SARS-CoV-2 load with increasing ventilation, both from mechanical and window sources. These results may guide environmental viral surveillance strategies and be used to better control the spread of SARS-CoV-2 within built environments and better protect those caring for individuals with COVID-19.

Abstract Background Clinical outcome assessments are important tools for measuring the natural history of disease and efficacy of an intervention. The heterogenous phenotype and difficult to quantity features of Proteus syndrome present challenges to measuring clinical outcomes. To address these, we designed a global clinical assessment for Proteus syndrome, a rare mosaic overgrowth disorder. The Clinical Gestalt Assessment (CGA) aims to evaluate change over time in this phenotypically diverse disorder. Results We gathered paired serial photographs and radiographs obtained at 12-to-36-month intervals from our natural history study of Proteus syndrome. The chronologic order of each set was blinded and presented to clinicians familiar with overgrowth disorders. They were asked to determine the chronologic order and, based on that response, rate global clinical change using a seven-point scale (Much Worse, Worse, Minimally Worse, No Change, Minimally Improved, Improved, Much Improved). Following a pilot, we tested the inter-rater reliability of the CGA using eight cases rated by eight clinicians. Raters identified the correct chronologic order in 53 of 64 (83%) of responses. There was low inter-rater variance and poor to moderate reliability with an intraclass correlation coefficient of 0.46 (95% CI 0.24–0.75). The overall estimate of global change was Minimally Worse over time, which is an accurate reflection of the natural history of Proteus syndrome. Conclusions The CGA is a tool to evaluate clinical change over time in Proteus syndrome and may be a useful adjunct to measure clinical outcomes in prospective therapeutic trials.

Wheat yields have plateaued in the UK over the last 25 years, during which time most arable land has been annually cropped continuously with short rotations dominated by cereals. Arable intensification has depleted soil organic matter and biology, including mycorrhizas, which are affected by tillage, herbicides, and crop genotype. Here, we test whether winter wheat yields, mycorrhization, and shoot health can be improved simply by adopting less intensive tillage and adding commercial mycorrhizal inoculum to long-term arable fields, or if 3-year grass-clover leys followed direct drilling is more effective for biological regeneration of soil with reduced N fertiliser. We report a trial of mycorrhization, ear pathology, and yield performance of the parents and four double haploid lines from the Avalon x Cadenza winter wheat population in a long-term arable field that is divided into replicated treatment plots. These plots comprised wheat lines grown using ploughing or disc cultivation for 3 years, half of which received annual additions of commercial arbuscular mycorrhizal (AM) inoculum, compared to 3-year mown grass-clover ley plots treated with glyphosate and direct-drilled. All plots annually received 35 kg of N ha−1 fertiliser without fungicides. The wheat lines did not differ in mycorrhization, which averaged only 34% and 40% of root length colonised (RLC) in the ploughed and disc-cultivated plots, respectively, and decreased with inoculation. In the ley, RLC increased to 52%. Two wheat lines were very susceptible to a sooty ear mould, which was lowest in the ley, and highest with disc cultivation. AM inoculation reduced ear infections by >50% in the susceptible lines. In the ley, yields ranged from 7.2 to 8.3 t ha−1, achieving 92 to 106% of UK average wheat yield in 2018 (7.8 t ha−1) but using only 25% of average N fertiliser. Yields with ploughing and disc cultivation averaged only 3.9 and 3.4 t ha−1, respectively, with AM inoculum reducing yields from 4.3 to 3.5 t ha−1 in ploughed plots, with no effect of disc cultivation. The findings reveal multiple benefits of reintegrating legume-rich leys into arable rotations as part of a strategy to regenerate soil quality and wheat crop health, reduce dependence on nitrogen fertilisers, enhance mycorrhization, and achieve good yields.

Background: Cardiovascular disease (CVD) is the number one cause of death in the United States with risk factors including hypertension, hyperlipidemia, diabetes, obesity, smoking, physical inactivity, age, genetics, and unhealthy diets. A university-based workplace wellness program (WWP) consisting of an annual biometric screening assessment with targeted, individualized health coaching was implemented in an effort to reduce these risk factors while encouraging and nurturing ideal cardiovascular health. Objective: The purpose of this study was to examine and describe the prevalence of single and combined, or multiple, CVD risk factors within a workplace wellness dataset. Methods: Cluster analysis was used to determine CVD risk factors within biometric screening data (BMI, waist circumference, LDL, total cholesterol, HDL, triglycerides, blood glucose age, ethnicity, and gender) collected during WWP interventions. Results: The cluster analysis provided visualizations of the distributions of participants having specific CVD risk factors. Of the 8,802 participants, 1,967 (22.4%) had no CVD risk factor, 1,497 (17%) had a single risk factor, and 5,529 (60.5%) had two or more risk factors. The majority of sample members are described as having more than one CVD risk factor with 78% having multiple. Conclusion: Cluster analysis demonstrated utility and efficacy in categorizing participant data based on their CVD risk factors. A baseline analysis of data was captured and provided understanding and awareness into employee health and CVD risk. This process and analysis facilitated WWP planning to target and focus on education to promote ideal cardiovascular health.

Halting biodiversity loss on land (Sustainable Development Goal [SDG] 15) is an unfolding problem, and as such, requires novel solutions. Citizen science (CS) promises large quantities of data, but introduces the challenge of ensuring these are valuable to conservation research and can inform meaningful action. This paper contributes to this endeavour, examining the impact of systematic as opposed to unstructured fieldwork on the biodiversity monitoring value of data from the Second Southern African Bird Atlas Project (SABAP2). SABAP2 “atlasers” work within a fine-scale grid system to generate avian species checklists that are comprehensive at the time of fieldwork. Though valuable, unstructured fieldwork efforts paint an incomplete picture; effective conservation action requires monitoring—keeping a finger on the pulse of local biodiversity through consistent and systematic data collection. Systematic collection allows for the detection of nuanced biological patterns such as seasonal population trends and movements, rapidly alerting scientists to anomalies and galvanizing swift response. It is, however, a demanding protocol, and implementation requires careful consideration of participant impact and motivations. Here, we used a newly developed approach for measuring temporal data quality to examine the systematic atlasing efforts of a CS community in the Hessequa Atlasing Area, South Africa, assessing the biodiversity monitoring value of structured data collection versus opportunistic checklists. We found that structured data collection increased the temporal resolution of atlas data, and thus its monitoring quality. We discuss challenges in maintaining achievable fieldwork goals for participants, and examine Hessequa’s project structure and participant motivations to provide recommendations for future project management.

Abstract Background The widespread use of next-generation sequencing has identified an important role for somatic mosaicism in many diseases. However, detecting low-level mosaic variants from next-generation sequencing data remains challenging. Results Here, we present a method for Position-Based Variant Identification (PBVI) that uses empirically-derived distributions of alternate nucleotides from a control dataset. We modeled this approach on 11 segmental overgrowth genes. We show that this method improves detection of single nucleotide mosaic variants of 0.01–0.05 variant allele fraction compared to other low-level variant callers. At depths of 600 × and 1200 ×, we observed > 85% and > 95% sensitivity, respectively. In a cohort of 26 individuals with somatic overgrowth disorders PBVI showed improved signal to noise, identifying pathogenic variants in 17 individuals. Conclusion PBVI can facilitate identification of low-level mosaic variants thus increasing the utility of next-generation sequencing data for research and diagnostic purposes.

Abstract Background Somatic single nucleotide variants have gained increased attention because of their role in cancer development and the widespread use of high-throughput sequencing techniques. The necessity to accurately identify these variants in sequencing data has led to a proliferation of somatic variant calling tools. Additionally, the use of simulated data to assess the performance of these tools has become common practice, as there is no gold standard dataset for benchmarking performance. However, many existing somatic variant simulation tools are limited because they rely on generating entirely synthetic reads derived from a reference genome or because they do not allow for the precise customizability that would enable a more focused understanding of single nucleotide variant calling performance. Results SomatoSim is a tool that lets users simulate somatic single nucleotide variants in sequence alignment map (SAM/BAM) files with full control of the specific variant positions, number of variants, variant allele fractions, depth of coverage, read quality, and base quality, among other parameters. SomatoSim accomplishes this through a three-stage process: variant selection, where candidate positions are selected for simulation, variant simulation, where reads are selected and mutated, and variant evaluation, where SomatoSim summarizes the simulation results. Conclusions SomatoSim is a user-friendly tool that offers a high level of customizability for simulating somatic single nucleotide variants. SomatoSim is available at https://github.com/BieseckerLab/SomatoSim .

Abstract In this work, we sought to delineate the prevalence of cardiothoracic imaging findings of Proteus syndrome in a large cohort at our institution. Of 53 individuals with a confirmed diagnosis of Proteus syndrome at our institution from 10/2001 to 10/2019, 38 individuals (men, n = 23; average age = 24 years) underwent cardiothoracic imaging (routine chest CT, CT pulmonary angiography and/or cardiac MRI). All studies were retrospectively and independently reviewed by two fellowship-trained cardiothoracic readers. Disagreements were resolved by consensus. Differences between variables were analyzed via parametric and nonparametric tests based on the normality of the distribution. The cardiothoracic findings of Proteus syndrome were diverse, but several were much more common and included: scoliosis from bony overgrowth (94%), pulmonary venous dilation (62%), band-like areas of lung scarring (56%), and hyperlucent lung parenchyma (50%). In addition, of 20 individuals who underwent cardiac MRI, 9/20 (45%) had intramyocardial fat, mostly involving the endocardial surface of the left ventricular septal wall. There was no statistically significant difference among the functional cardiac parameters between individuals with and without intramyocardial fat. Only one individual with intramyocardial fat had mildly decreased function (LVEF = 53%), while all others had normal ejection fraction.

Purpose: To investigate the incidence, seasonal variation, and differences among age, sex, and race for rhegmatogenous retinal detachment (RRD) repair, retinal break (RB) treatment, and posterior vitreous detachment (PVD) in the Intelligent Research in Sight (IRIS) Registry. Design: Retrospective database study. Participants: Patients in the IRIS Registry who underwent RRD repair, RB treatment, or cataract surgery (CS) based on Current Procedural Terminology codes and PVD diagnosis based on International Classification of Diseases, Ninth and Tenth Revision, codes. Methods: Daily incidence rates were defined as the ratio of patients who underwent RRD repair or RB treatment and patients with a diagnosis of PVD to the total number of patients followed on a given day within the IRIS Registry. The CS group was included as a comparison for seasonal variation. Rates were stratified by decade of life, sex, and race. Main Outcome Measures: Time series trends for incidence rates of RRD, RB, and PVD. Results: A total of 7 115 774 patients received a diagnosis of incident PVD, 237 646 patients underwent RRD repair, and 359 022 patients underwent RB treatment. Also included were 5 940 448 patients who underwent CS. The mean daily incidence for RRD repair, RB treatment, PVD diagnosis, and CS were 0.46 per 100 000 patients, 0.70 per 100 000 patients, 13.90 per 100 000 patients, and 11.80 per 100 000 patients, respectively. Men showed higher incidence of RRD repair and RB treatment than women, whereas women showed higher incidence of PVD diagnosis. Rhegmatogenous retinal detachment incidence was higher in White people compared with other races. Seasonal decreases in PVD, RB treatment, RRD repair, and CS corresponded to national holidays, with larger decreases in winter months. Kaplan-Meier estimates showed that RRD repair and RB treatment typically occurred within 60 days of PVD diagnosis. Conclusions: Within the IRIS Registry, the highest incidence of RRD was in the 6th and 7th decade of life. There was a higher incidence of RRD repair and RB treatment in men compared with women. The seasonal variation associated with national holidays was less pronounced for RRD repair and RB treatment.

AbstractHumans process faces by using a network of face-selective regions distributed across the brain. Neuropsychological patient studies demonstrate that focal damage to nodes in this network can impair face recognition, but such patients are rare. We approximated the effects of damage to the face network in neurologically normal human participants by using theta burst transcranial magnetic stimulation (TBS). Multi-echo functional magnetic resonance imaging (fMRI) resting-state data were collected pre- and post-TBS delivery over the face-selective right superior temporal sulcus (rpSTS), or a control site in the right motor cortex. Results showed that TBS delivered over the rpSTS reduced resting-state connectivity across the extended face processing network. This connectivity reduction was observed not only between the rpSTS and other face-selective areas, but also between nonstimulated face-selective areas across the ventral, medial, and lateral brain surfaces (e.g., between the right amygdala and bilateral fusiform face areas and occipital face areas). TBS delivered over the motor cortex did not produce significant changes in resting-state connectivity across the face processing network. These results demonstrate that, even without task-induced fMRI signal changes, disrupting a single node in a brain network can decrease the functional connectivity between nodes in that network that have not been directly stimulated.

Abstract Spatial reasoning is a critical skill in many everyday tasks and in science, technology, engineering, and mathematics disciplines. The current study examined how training on mental rotation (a spatial reasoning task) impacts the completeness of an encoded representation and the ability to rotate the representation. We used a multisession, multimethod design with an active control group to determine how mental rotation ability impacts performance for a trained stimulus category and an untrained stimulus category. Participants in the experimental group (n = 18) showed greater improvement than the active control group (n = 18) on the mental rotation tasks. The number of saccades between objects decreased and saccade amplitude increased after training, suggesting that participants in the experimental group encoded more of the object and possibly had more complete mental representations after training. Functional magnetic resonance imaging data revealed distinct neural activation associated with mental rotation, notably in the right motor cortex and right lateral occipital cortex. These brain areas are often associated with rotation and encoding complete representations, respectively. Furthermore, logistic regression revealed that activation in these brain regions during the post-training scan significantly predicted training group assignment. Overall, the current study suggests that effective mental rotation training protocols should aim to improve the encoding and manipulation of mental representations.

The sustainable intensification of agriculture involves providing sufficient food and other ecosystem services without going beyond the limits of the earth’s system. Here a project management approach is suggested to help guide agricultural policy to deliver these objectives. The first step is to agree measurable outcomes, integrating formal policy goals with the often much less formal and much more diverse goals of individual farmers. The second step is to assess current performance. Ideally, this will involve the use of farm-scale metrics that can feed into process models that address social and environmental domains as well as production issues that can be benchmarked and upscaled to landscape and country. Some policy goals can be delivered by supporting ad hoc interventions, while others require the redesign of the farming system. A pipeline of research, knowledge and capacity building is needed to ensure the continuous increase in farm performance. System models can help prioritise policy interventions. Formal optimization of land use is only appropriate if the policy goals are clear, and the constraints understood. In practice, the best approach may depend on the scale of action that is required, and on the amount of resource and infrastructure available to generate, implement and manage policy.

Plain Language Summary Through living with conditions and/or engaging with health and social care services patients, public and service users become experts by experience. In Canada and the UK, the active involvement of experts by experience in ophthalmology research (as well as in other specialties) positively benefits all stages of the research cycle; improves the experience and outcomes for patients taking part in research; drives better engagement between researchers, the public and other key stakeholders; and benefits these expert’s own sense of wellbeing and achievement. At the moment, the extent to which experts by experience are active in ophthalmology research around the world is unclear, but likely to be minimal. To enable more research to benefit from the contribution of experts by experience, global efforts to improve the continuity and quality of reporting and evidence of impact are needed.

ABSTRACT Social and political policy, human activities, and environmental change affect the ways in which microbial communities assemble and interact with people. These factors determine how different social groups are exposed to beneficial and/or harmful microorganisms, meaning microbial exposure has an important socioecological justice context. Therefore, greater consideration of microbial exposure and social equity in research, planning, and policy is imperative. Here, we identify 20 research questions considered fundamentally important to promoting equitable exposure to beneficial microorganisms, along with safeguarding resilient societies and ecosystems. The 20 research questions we identified span seven broad themes, including the following: (i) sociocultural interactions; (ii) Indigenous community health and well-being; (iii) humans, urban ecosystems, and environmental processes; (iv) human psychology and mental health; (v) microbiomes and infectious diseases; (vi) human health and food security; and (vii) microbiome-related planning, policy, and outreach. Our goal was to summarize this growing field and to stimulate impactful research avenues while providing focus for funders and policymakers.

Solid-state Friction Stir Additive Manufacturing has recently gained attention as a result of its capacity to fabricate large-scale parts while preserving the mechanical properties of the feedstock material. However, the correlation between the quality of layer-by-layer bonding of the deposited metal and processing parameters has remained unknown. Neutron imaging techniques, with 90% total transmission per cm, are employed for Al6061 parts fabricated by MELD® Technology as a non-destructive evaluation approach for the first time to investigate the layer-by-layer structure of a stadium-shaped ingot in different sections. The post-processed results show the fabricated parts with an optimized set of processing parameters are void-free. However, the hydrocarbon-based feedstock lubricant segregates between the layers, which consequently may lead to non-uniform weaker mechanical properties along the build direction and stimulate crack initiation during mechanical loading. The tensile test results show 14% lower strain-to-failure values in alleged contaminated areas in transmission imaging results. Additionally, layer bonding is significantly impacted by hot-on-hot and hot-on-cold layer deposition schemes, especially for larger layer thicknesses.

Laser powder bed fusion (LPBF)-based additive manufacturing (AM) has the flexibility in fabricating parts with complex geometries. However, using non-optimized processing parameters or using certain feedstock powders, internal defects (pores, cracks, etc.) may occur inside the parts. Having a thorough and statistical understanding of these defects can help researchers find the correlations between processing parameters/feedstock materials and possible internal defects. To establish a tool that can automatically detect defects in AM parts, in this research, X-ray CT images of Inconel 939 samples fabricated by LPBF are analyzed using U-Net architecture with different sets of hyperparameters. The hyperparameters of the network are tuned in such a way that yields maximum segmentation accuracy with reasonable computational cost. The trained network is able to segment the unbalanced classes of pores and cracks with a mean intersection over union (mIoU) value of 82% on the test set, and has reduced the characterization time from a few weeks to less than a day compared to conventional manual methods. It is shown that the major bottleneck in improving the accuracy is uncertainty in labeled data and the necessity for adopting a semi-supervised approach, which needs to be addressed first in future research.

ABSTRACT Noncoding small RNAs (sRNAs) are crucial for the posttranscriptional regulation of gene expression in all organisms and are known to be involved in the regulation of bacterial virulence. In the human pathogen Bordetella pertussis, which causes whooping cough, virulence is controlled primarily by the master two-component system BvgA (response regulator)/BvgS (sensor kinase). In this system, BvgA is phosphorylated (Bvg+ mode) or nonphosphorylated (Bvg− mode), with global transcriptional differences between the two. B. pertussis also carries the bacterial sRNA chaperone Hfq, which has previously been shown to be required for virulence. Here, we conducted transcriptomic analyses to identify possible B. pertussis sRNAs and to determine their BvgAS dependence using transcriptome sequencing (RNA-seq) and the prokaryotic sRNA prediction program ANNOgesic. We identified 143 possible candidates (25 Bvg+ mode specific and 53 Bvg− mode specific), of which 90 were previously unreported. Northern blot analyses confirmed all of the 10 ANNOgesic candidates that we tested. Homology searches demonstrated that 9 of the confirmed sRNAs are highly conserved among B. pertussis, Bordetella parapertussis, and Bordetella bronchiseptica, with one that also has homologues in other species of the Alcaligenaceae family. Using coimmunoprecipitation with a B. pertussis FLAG-tagged Hfq, we demonstrated that 3 of the sRNAs interact directly with Hfq, which is the first identification of sRNA binding to B. pertussis Hfq. Our study demonstrates that ANNOgesic is a highly useful tool for the identification of sRNAs in this system and that its combination with molecular techniques is a successful way to identify various BvgAS-dependent and Hfq-binding sRNAs. IMPORTANCE Noncoding small RNAs (sRNAs) are crucial for posttranscriptional regulation of gene expression in all organisms and are known to be involved in the regulation of bacterial virulence. We have investigated the presence of sRNAs in the obligate human pathogen B. pertussis, using transcriptome sequencing (RNA-seq) and the recently developed prokaryotic sRNA search program ANNOgesic. This analysis has identified 143 sRNA candidates (90 previously unreported). We have classified their dependence on the B. pertussis two-component system required for virulence, namely, BvgAS, based on their expression in the presence/absence of the phosphorylated response regulator BvgA, confirmed several by Northern analyses, and demonstrated that 3 bind directly to B. pertussis Hfq, the RNA chaperone involved in mediating sRNA effects. Our study demonstrates the utility of combining RNA-seq, ANNOgesic, and molecular techniques to identify various BvgAS-dependent and Hfq-binding sRNAs, which may unveil the roles of sRNAs in pertussis pathogenesis.

ABSTRACT Humans are inextricably linked to each other and our natural world, and microorganisms lie at the nexus of those interactions. Microorganisms form genetically flexible, taxonomically diverse, and biochemically rich communities, i.e., microbiomes that are integral to the health and development of macroorganisms, societies, and ecosystems. Yet engagement with beneficial microbiomes is dictated by access to public resources, such as nutritious food, clean water and air, safe shelter, social interactions, and effective medicine. In this way, microbiomes have sociopolitical contexts that must be considered. The Microbes and Social Equity (MSE) Working Group connects microbiology with social equity research, education, policy, and practice to understand the interplay of microorganisms, individuals, societies, and ecosystems. Here, we outline opportunities for integrating microbiology and social equity work through broadening education and training; diversifying research topics, methods, and perspectives; and advocating for evidence-based public policy that supports sustainable, equitable, and microbial wealth for all.

Hydrogenotrophic methanogens are ubiquitous chemoautotrophic archaea inhabiting globally distributed deep-sea hydrothermal vent ecosystems and associated subseafloor niches within the rocky subseafloor, yet little is known about how they adapt and diversify in these habitats. To determine genomic variation and selection pressure within methanogenic populations at vents, we examined five Methanothermococcus single cell amplified genomes (SAGs) in conjunction with 15 metagenomes and 10 metatranscriptomes from venting fluids at two geochemically distinct hydrothermal vent fields on the Mid-Cayman Rise in the Caribbean Sea. We observed that some Methanothermococcus lineages and their transcripts were more abundant than others in individual vent sites, indicating differential fitness among lineages. The relative abundances of lineages represented by SAGs in each of the samples matched phylogenetic relationships based on single-copy universal genes, and genes related to nitrogen fixation and the CRISPR/Cas immune system were among those differentiating the clades. Lineages possessing these genes were less abundant than those missing that genomic region. Overall, patterns in nucleotide variation indicated that the population dynamics of Methanothermococcus were not governed by clonal expansions or selective sweeps, at least in the habitats and sampling times included in this study. Together, our results show that although specific lineages of Methanothermococcus co-exist in these habitats, some outcompete others, and possession of accessory metabolic functions does not necessarily provide a fitness advantage in these habitats in all conditions. This work highlights the power of combining single-cell, metagenomic, and metatranscriptomic datasets to determine how evolution shapes microbial abundance and diversity in hydrothermal vent ecosystems.

Abstract Background The American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP) clinical variant interpretation guidelines established criteria for different types of evidence. This includes the strong evidence codes PS3 and BS3 for “well-established” functional assays demonstrating a variant has abnormal or normal gene/protein function, respectively. However, they did not provide detailed guidance on how functional evidence should be evaluated, and differences in the application of the PS3/BS3 codes are a contributor to variant interpretation discordance between laboratories. This recommendation seeks to provide a more structured approach to the assessment of functional assays for variant interpretation and guidance on the use of various levels of strength based on assay validation. Methods The Clinical Genome Resource (ClinGen) Sequence Variant Interpretation (SVI) Working Group used curated functional evidence from ClinGen Variant Curation Expert Panel-developed rule specifications and expert opinions to refine the PS3/BS3 criteria over multiple in-person and virtual meetings. We estimated the odds of pathogenicity for assays using various numbers of variant controls to determine the minimum controls required to reach moderate level evidence. Feedback from the ClinGen Steering Committee and outside experts were incorporated into the recommendations at multiple stages of development. Results The SVI Working Group developed recommendations for evaluators regarding the assessment of the clinical validity of functional data and a four-step provisional framework to determine the appropriate strength of evidence that can be applied in clinical variant interpretation. These steps are as follows: (1) define the disease mechanism, (2) evaluate the applicability of general classes of assays used in the field, (3) evaluate the validity of specific instances of assays, and (4) apply evidence to individual variant interpretation. We found that a minimum of 11 total pathogenic and benign variant controls are required to reach moderate-level evidence in the absence of rigorous statistical analysis. Conclusions The recommendations and approach to functional evidence evaluation described here should help clarify the clinical variant interpretation process for functional assays. Further, we hope that these recommendations will help develop productive partnerships with basic scientists who have developed functional assays that are useful for interrogating the function of a variety of genes.