GeNLP: a web tool for NLP-based exploration and prediction of microbial gene function.

SUMMARY: GeNLP is a web application that enables exploring microbial gene "semantics" and predictions of uncharacterized gene families based on their genomic context. It utilizes a pre-trained language model to uncover gene relationships and allows users to access and utilize the data as well as make their own predictions through an interactive interface. AVAILABILITY AND IMPLEMENTATION: The web application is accessible from all browsers at: http://gnlp.bursteinlab.org/. All source codes are freely available from GitHub under the MIT license here: https://github.com/burstein-lab/genomic-nlp-server.

WarA, a remote homolog of NpmA and KamB from Nocardia wallacei, confers broad spectrum aminoglycoside resistance in Nocardia and Mycobacteria.

OBJECTIVES: Aminoglycoside resistance in bacteria is typically conferred by specific drug-modifying enzymes. Infrequently, such resistance is achieved through 16S ribosomal RNA methyltransferases, such as NpmA and KamB encoded by Escherichia coli and Streptoalloteichus tenebrarius, respectively. These enzymes are not widespread and have not been described in Nocardia species to date. METHODS: We report the genomic mining of 18 Nocardia wallacei isolates that were found to be specifically and substantially resistant to amikacin. RESULTS: We identified a gene coding for a protein with very distant homology to NpmA and KamB. However, 3-D modeling revealed that the tertiary structure of these three proteins was highly similar. Cloning and expressing this gene in two susceptible bacteria Nocardia asteroides, and Mycobacterium smegmatis (another Actinobacterium) led to high-level, pan-aminoglycoside resistance in both cases. We named this gene warA (Wallacei Amikacin Resistance A). CONCLUSIONS: This is the first description and experimental characterization of a gene of this family in Nocardia, and the first demonstration that such activity could lead to pan-aminoglycoside resistance in Mycobacteria as well. The discovery of this novel gene has important biotechnology and clinical implications.

Using big sequencing data to identify chronic SARS-Coronavirus-2 infections.

The evolution of SARS-Coronavirus-2 (SARS-CoV-2) has been characterized by the periodic emergence of highly divergent variants. One leading hypothesis suggests these variants may have emerged during chronic infections of immunocompromised individuals, but limited data from these cases hinders comprehensive analyses. Here, we harnessed millions of SARS-CoV-2 genomes to identify potential chronic infections and used language models (LM) to infer chronic-associated mutations. First, we mined the SARS-CoV-2 phylogeny and identified chronic-like clades with identical metadata (location, age, and sex) spanning over 21 days, suggesting a prolonged infection. We inferred 271 chronic-like clades, which exhibited characteristics similar to confirmed chronic infections. Chronic-associated mutations were often high-fitness immune-evasive mutations located in the spike receptor-binding domain (RBD), yet a minority were unique to chronic infections and absent in global settings. The probability of observing high-fitness RBD mutations was 10-20 times higher in chronic infections than in global transmission chains. The majority of RBD mutations in BA.1/BA.2 chronic-like clades bore predictive value, i.e., went on to display global success. Finally, we used our LM to infer hundreds of additional chronic-like clades in the absence of metadata. Our approach allows mining extensive sequencing data and providing insights into future evolutionary patterns of SARS-CoV-2.

Phenotypic and genotypic analysis of antimicrobial resistance in Nocardia species.

BACKGROUND: Antimicrobial resistance is common in Nocardia species but data regarding the molecular mechanisms beyond their resistance traits are limited. Our study aimed to determine the species distribution, the antimicrobial susceptibility profiles, and investigate the associations between the resistance traits and their genotypic determinants. METHODS: The study included 138 clinical strains of Nocardia from nine Israeli microbiology laboratories. MIC values of 12 antimicrobial agents were determined using broth microdilution. WGS was performed on 129 isolates of the eight predominant species. Bioinformatic analysis included phylogeny and determination of antimicrobial resistance genes and mutations. RESULTS: Among the isolates, Nocardia cyriacigeorgica was the most common species (36%), followed by Nocardia farcinica (16%), Nocardia wallacei (13%), Nocardia abscessus (9%) and Nocardia brasiliensis (8%). Linezolid was active against all isolates, followed by trimethoprim/sulfamethoxazole (93%) and amikacin (91%). Resistance to other antibiotics was species-specific, often associated with the presence of resistance genes or mutations: (1) aph(2″) in N. farcinica and N. wallacei (resistance to tobramycin); (ii) blaAST-1 in N. cyriacigeorgica and Nocardia neocaledoniensis (resistance to amoxicillin/clavulanate); (iii) blaFAR-1 in N. farcinica (resistance to ceftriaxone); (iv) Ser83Ala substitution in the gyrA gene in four species (resistance to ciprofloxacin); and (v) the 16S rRNA m1A1408 methyltransferase in N. wallacei isolates (correlating with amikacin resistance). CONCLUSIONS: Our study provides a comprehensive understanding of Nocardia species diversity, antibiotic resistance patterns, and the molecular basis of antimicrobial resistance. Resistance appears to follow species-related patterns, suggesting a lesser role for de novo evolution or transmission of antimicrobial resistance.

Genome-wide analysis of a model-derived binge eating disorder phenotype identifies risk loci and implicates iron metabolism.

Binge eating disorder (BED) is the most common eating disorder, yet its genetic architecture remains largely unknown. Studying BED is challenging because it is often comorbid with obesity, a common and highly polygenic trait, and it is underdiagnosed in biobank data sets. To address this limitation, we apply a supervised machine-learning approach (using 822 cases of individuals diagnosed with BED) to estimate the probability of each individual having BED based on electronic medical records from the Million Veteran Program. We perform a genome-wide association study of individuals of African (n = 77,574) and European (n = 285,138) ancestry while controlling for body mass index to identify three independent loci near the HFE, MCHR2 and LRP11 genes and suggest APOE as a risk gene for BED. We identify shared heritability between BED and several neuropsychiatric traits, and implicate iron metabolism in the pathophysiology of BED. Overall, our findings provide insights into the genetics underlying BED and suggest directions for future translational research.

CRISPR-based genetic diagnostics in microgravity.

Monitoring astronauts' health during space missions poses many challenges, including rapid assessment of crew health conditions. Sensitive genetic diagnostics are crucial for examining crew members and the spacecraft environment. CRISPR-Cas12a, coupled with isothermal amplification, has proven to be a promising biosensing system for rapid, on-site detection of genomic targets. However, the efficiency and sensitivity of CRISPR-based diagnostics have never been tested in microgravity. We tested the use of recombinase polymerase amplification (RPA) coupled with the collateral cleavage activity of Cas12a for genetic diagnostics onboard the International Space Station. We explored the detection sensitivity of amplified and unamplified target DNA. By coupling RPA with Cas12a, we identified targets in attomolar concentrations. We further assessed the reactions' stability following long-term storage. Our results demonstrate that CRISPR-based detection is a powerful tool for on-site genetic diagnostics in microgravity, and can be further utilized for long-term space endeavors to improve astronauts' health and well-being.

Detecting and Adjusting for Hidden Biases due to Phenotype Misclassification in Genome-Wide Association Studies.

With the advent of healthcare-based genotyped biobanks, genome-wide association studies (GWAS) leverage larger sample sizes, incorporate patients with diverse ancestries and introduce noisier phenotypic definitions. Yet the extent and impact of phenotypic misclassification on large-scale datasets is not currently well understood due to a lack of statistical methods to estimate relevant parameters from empirical data. Here, we develop a statistical method and scalable software, PheMED, Phenotypic Measurement of Effective Dilution, to quantify phenotypic misclassification across GWAS using only summary statistics. We illustrate how the parameters estimated by PheMED relate to the negative and positive predictive value of the labeled phenotype, compared to ground truth, and how misclassification of the phenotype yields diluted effect-sizes of variant-phenotype associations. Furthermore, we apply our methodology to detect multiple instances of statistically significant dilution in real-world data. We demonstrate how effective dilution biases downstream GWAS replication and heritability analyses despite utilizing current best practices, and provide a dilution-aware meta-analysis approach that outperforms existing methods. Consequently, we anticipate that PheMED will be a valuable tool for researchers to address phenotypic data quality issues both within and across cohorts.

Internal Medicine Residents' Experience Performing Routine Assessment of What Matters Most to Patients Upon Hospital Admission.

PROBLEM: Failure to elicit patients' values, goals, and priorities can result in missed opportunities to provide patient-centered care. Little is known about resident physicians' direct experience of eliciting patients' values, goals, and priorities and integrating them into routine hospital care. INTERVENTION: In 2017, we asked resident physicians on general internal medicine wards rotations to elicit and document a "Personal History" from patients upon hospital admission, in addition to a traditional social history. We defined a Personal History as documenting "what matters most to the patient and why." The purpose of the Personal History was to understand and consider patients' values, goals, and priorities. We then conducted qualitative interviews of the resident physicians to understand their experiences eliciting and integrating patients' values, goals, and priorities in routine hospital care. CONTEXT: We performed this exploratory intervention at a large high-volume urban hospital. Two teams from general medicine wards participated in the Personal History intervention. We conducted voluntary interviews of eligible residents (n = 14/15; 93%) about their experience after they completed their general wards rotations. Using the coproduction model, our aim was to explore how patients' self-expertise can be combined with physicians' medical expertise to achieve patient-centered care. IMPACT: Four major themes were identified: 1) Taking a Personal History had value, and eliciting patients' self-expertise had the potential to change medical decision making, 2) Situational and relational factors created barriers to obtaining a Personal History, 3) Variability in buy-in with the proposed intervention affected effort, and 4) Meaningful Personal History taking could be an adaptive and longitudinal process. Perceived benefits included improved rapport with patients, helpful for patients with complex medical history, and improved physician-patient communication. Barriers included patient distress, lack of rapport, and responses from patients which did not add new insights. Accountability from attending physicians affected resident effort. Suggested future applications were for patients with serious illness, integration into electronic health records, and skills taught in medical education. LESSONS LEARNED: Resident physicians had generally positive views of eliciting a Personal History from patients upon admission to the hospital. Overall, many residents conveyed the perceived ability to elicit and consider patient's values, goals, and priorities in certain situations (e.g., patient not in distress, adequate rapport, lack of competing priorities such as medical emergencies or overwhelming workloads). External factors, such as electronic health record design and accountability from attending physicians, may further promote residents' efforts to routinely incorporate patients' values, goals, and priorities in clinical care. Increasing familiarity among both resident physicians and patients in routinely discussing patients' values, goals, and priorities may facilitate patient-centered practice.

Rapid and sensitive on-site genetic diagnostics of pest fruit flies using CRISPR-Cas12a.

BACKGROUND: Bactrocera zonata, a major fruit pest species, is gradually spreading west from its native habitat in East Asia. In recent years it has become a significant threat to the Mediterranean area, with the potential of invading Europe, the Americas, and Australia. To prevent it spreading, monitoring efforts in cultivation sites and border controls are carried out. Despite these efforts, and due to morphological similarities between B. zonata and other pests in relevant developmental stages, the monitoring process is challenging, time-consuming, and requires external assistance from professional laboratories. CRISPR-Cas12a genetic diagnostics has been rapidly developing in recent years and provides an efficient tool for the genetic identification of pathogens, viruses, and other genetic targets. Here we design a CRISPR-Cas12a detection assay that differentially detects two major pest species, B. zonata and Ceratitis capitata. RESULTS: We demonstrate the specificity and high sensitivity of this method. Identification of target pests was done using specific and universal primers on pooled samples, enabling differentiation of pests with high certainty. We also demonstrate reaction stability over time for future on-site applications. DISCUSSION: Our easy-to-use and affordable assay employs a simple DNA extraction technique together with isothermal amplification and Cas12a-based detection. This method is highly modular, and the presented target design method can be applied to a wide array of pests. This approach can be easily adapted to fit local threats and requires minimal training of operators in border controls and other relevant locations, reshaping pest control and making state-of-the-art technologies available worldwide, including in developing countries. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Penetrance and Pleiotropy of Polygenic Risk Scores for Schizophrenia, Bipolar Disorder, and Depression Among Adults in the US Veterans Affairs Health Care System.

Importance: Serious mental illnesses, including schizophrenia, bipolar disorder, and depression, are heritable, highly multifactorial disorders and major causes of disability worldwide. Objective: To benchmark the penetrance of current neuropsychiatric polygenic risk scores (PRSs) in the Veterans Health Administration health care system and to explore associations between PRS and broad categories of human disease via phenome-wide association studies. Design, Setting, and Participants: Extensive Veterans Health Administration's electronic health records were assessed from October 1999 to January 2021, and an embedded cohort of 9378 individuals with confirmed diagnoses of schizophrenia or bipolar 1 disorder were found. The performance of schizophrenia, bipolar disorder, and major depression PRSs were compared in participants of African or European ancestry in the Million Veteran Program (approximately 400 000 individuals), and associations between PRSs and 1650 disease categories based on ICD-9/10 billing codes were explored. Last, genomic structural equation modeling was applied to derive novel PRSs indexing common and disorder-specific genetic factors. Analysis took place from January 2021 to January 2022. Main Outcomes and Measures: Diagnoses based on in-person structured clinical interviews were compared with ICD-9/10 billing codes. PRSs were constructed using summary statistics from genome-wide association studies of schizophrenia, bipolar disorder, and major depression. Results: Of 707 299 enrolled study participants, 459 667 were genotyped at the time of writing; 84 806 were of broadly African ancestry (mean [SD] age, 58 [12.1] years) and 314 909 were of broadly European ancestry (mean [SD] age, 66.4 [13.5] years). Among 9378 individuals with confirmed diagnoses of schizophrenia or bipolar 1 disorder, 8962 (95.6%) were correctly identified using ICD-9/10 codes (2 or more). Among those of European ancestry, PRSs were robustly associated with having received a diagnosis of schizophrenia (odds ratio [OR], 1.81 [95% CI, 1.76-1.87]; P < 10-257) or bipolar disorder (OR, 1.42 [95% CI, 1.39-1.44]; P < 10-295). Corresponding effect sizes in participants of African ancestry were considerably smaller for schizophrenia (OR, 1.35 [95% CI, 1.29-1.42]; P < 10-38) and bipolar disorder (OR, 1.16 [95% CI, 1.11-1.12]; P < 10-10). Neuropsychiatric PRSs were associated with increased risk for a range of psychiatric and physical health problems. Conclusions and Relevance: Using diagnoses confirmed by in-person structured clinical interviews and current neuropsychiatric PRSs, the validity of an electronic health records-based phenotyping approach in US veterans was demonstrated, highlighting the potential of PRSs for disentangling biological and mediated pleiotropy.

Deciphering microbial gene function using natural language processing.

Revealing the function of uncharacterized genes is a fundamental challenge in an era of ever-increasing volumes of sequencing data. Here, we present a concept for tackling this challenge using deep learning methodologies adopted from natural language processing (NLP). We repurpose NLP algorithms to model "gene semantics" based on a biological corpus of more than 360 million microbial genes within their genomic context. We use the language models to predict functional categories for 56,617 genes and find that out of 1369 genes associated with recently discovered defense systems, 98% are inferred correctly. We then systematically evaluate the "discovery potential" of different functional categories, pinpointing those with the most genes yet to be characterized. Finally, we demonstrate our method's ability to discover systems associated with microbial interaction and defense. Our results highlight that combining microbial genomics and language models is a promising avenue for revealing gene functions in microbes.

A Scoping Review on the Concept of Physician Caring.

BACKGROUND: Physicians' interest in the health and well-being of their patients is a tenet of medical practice. Physicians' ability to act upon this interest by caring for and about their patients is central to high-quality clinical medicine and may affect burnout. To date, a strong theoretical and empirical understanding of physician caring does not exist. To establish a practical, evidence-based approach to improve health care delivery and potentially address physician burnout, we sought to identify and synthesize existing conceptual models, frameworks, and definitions of physician caring. METHODS: We performed a scoping review on physician caring. In November 2019 and September 2020, we searched PubMed MEDLINE, Embase, PsycINFO, CINAHL, and CENTRAL Register of Controlled Trials to identify conceptual models, frameworks, and definitions of physician caring. Eligible articles involved discussion or study of care or caring among medical practitioners. We created a content summary and performed thematic analysis of extracted data. RESULTS: Of 11,776 articles, we reviewed the full text of 297 articles; 61 articles met inclusion criteria. Commonly identified concepts referenced Peabody's "secret of care" and the ethics of care. In bioethics, caring is described as a virtue. Contradictions exist among concepts of caring, such as whether caring is an attitude, emotion, or behavior, and the role of relationship development. Thematic analysis of all concepts and definitions identified six aspects of physician caring: (1) relational aspects, (2) technical aspects, (3) physician attitudes and characteristics, (4) agency, (5) reciprocity, and (6) physician self-care. DISCUSSION: Caring is instrumental to clinical medicine. However, scientific understanding of what constitutes caring from physicians is limited by contradictions across concepts. A unifying concept of physician caring does not yet exist. This review proposes six aspects of physician caring which can be used to develop evidence-based approaches to improve health care delivery and potentially mitigate physician burnout.

Chimeric CRISPR-CasX enzymes and guide RNAs for improved genome editing activity.

A compact protein with a size of <1,000 amino acids, the CRISPR-associated protein CasX is a fundamentally distinct RNA-guided nuclease when compared to Cas9 and Cas12a. Although it can induce RNA-guided genome editing in mammalian cells, the activity of CasX is less robust than that of the widely used S. pyogenes Cas9. Here, we show that structural features of two CasX homologs and their guide RNAs affect the R-loop complex assembly and DNA cleavage activity. Cryo-EM-based structural engineering of either the CasX protein or the guide RNA produced two new CasX genome editors (DpbCasX-R3-v2 and PlmCasX-R1-v2) with significantly improved DNA manipulation efficacy. These results advance both the mechanistic understanding of CasX and its application as a genome-editing tool.

Association of Primary Care Physician Compensation Incentives and Quality of Care in the United States, 2012-2016.

BACKGROUND: Physician compensation incentives may have positive or negative effects on clinical quality. OBJECTIVE: To assess the association between various physician compensation incentives on technical indicators of primary care quality. DESIGN: Cross-sectional, nationally representative retrospective analysis. PARTICIPANTS: Visits by adults to primary care physicians in the National Ambulatory Medical Care Survey from 2012-2016. We analyzed 49,580 sampled visits, representing 1.45 billion primary care visits. MAIN MEASURES: We assessed the association between 5 compensation incentives - quality measure performance, patient experience scores, individual productivity, practice financial performance, or practice efficiency - and 10 high-value and 7 low-value care measures as well as high-value and low-value care composites. KEY RESULTS: Quality measure performance was an incentive in 22% of visits; patient experience scores, 17%; individual productivity, 57%; practice financial performance, 63%; and practice efficiency, 12%. In adjusted models, none of the compensation incentives were consistently associated with individual high- and low-value measures. None of the compensation incentives were associated with high- or low-value care composites. For example, quality measure performance compensation was not significantly associated with high-value care (visits with quality incentive, 47% of eligible measures met; without quality incentive, 43%; adjusted odds ratio [aOR], 1.02; 95% confidence interval [CI], 0.91 to 1.15) or low-value care (aOR, 0.99; 95% CI, 0.82-1.19). Physician compensation incentives that might be expected to increase low-value care did not: patient experience (aOR for low-value care composite, 0.83; 95% CI, 0.65-1.05), individual productivity (aOR, 1.03; 95% CI, 0.88-1.22), and practice financial performance (aOR, 1.05; 95% CI, 0.81-1.36). CONCLUSION: In this retrospective, cross-sectional, nationally representative analysis of care in the United States, physician compensation incentives were not generally associated with more or less high- or low-value care.

Engineered B cells expressing an anti-HIV antibody enable memory retention, isotype switching and clonal expansion.

HIV viremia can be controlled by chronic antiretroviral therapy. As a potentially single-shot alternative, B cells engineered by CRISPR/Cas9 to express anti-HIV broadly neutralizing antibodies (bNAbs) are capable of secreting high antibody titers. Here, we show that, upon immunization of mice, adoptively transferred engineered B cells home to germinal centers (GC) where they predominate over the endogenous response and differentiate into memory and plasma cells while undergoing class switch recombination (CSR). Immunization with a high affinity antigen increases accumulation in GCs and CSR rates. Boost immunization increases the rate of engineered B cells in GCs and antibody secretion, indicating memory retention. Finally, antibody sequences of engineered B cells in the spleen show patterns of clonal selection. Therefore, B cells can be engineered into what could be a living and evolving drug.

A scoutRNA Is Required for Some Type V CRISPR-Cas Systems.

CRISPR-Cas12c/d proteins share limited homology with Cas12a and Cas9 bacterial CRISPR RNA (crRNA)-guided nucleases used widely for genome editing and DNA detection. However, Cas12c (C2c3)- and Cas12d (CasY)-catalyzed DNA cleavage and genome editing activities have not been directly observed. We show here that a short-complementarity untranslated RNA (scoutRNA), together with crRNA, is required for Cas12d-catalyzed DNA cutting. The scoutRNA differs in secondary structure from previously described tracrRNAs used by CRISPR-Cas9 and some Cas12 enzymes, and in Cas12d-containing systems, scoutRNA includes a conserved five-nucleotide sequence that is essential for activity. In addition to supporting crRNA-directed DNA recognition, biochemical and cell-based experiments establish scoutRNA as an essential cofactor for Cas12c-catalyzed pre-crRNA maturation. These results define scoutRNA as a third type of transcript encoded by a subset of CRISPR-Cas genomic loci and explain how Cas12c/d systems avoid requirements for host factors including ribonuclease III for bacterial RNA-mediated adaptive immunity.

The distinction of CPR bacteria from other bacteria based on protein family content.

Candidate phyla radiation (CPR) bacteria separate phylogenetically from other bacteria, but the organismal distribution of their protein families remains unclear. Here, we leveraged sequences from thousands of uncultivated organisms and identified protein families that co-occur in genomes, thus are likely foundational for lineage capacities. Protein family presence/absence patterns cluster CPR bacteria together, and away from all other bacteria and archaea, partly due to proteins without recognizable homology to proteins in other bacteria. Some are likely involved in cell-cell interactions and potentially important for episymbiotic lifestyles. The diversity of protein family combinations in CPR may exceed that of all other bacteria. Over the bacterial tree, protein family presence/absence patterns broadly recapitulate phylogenetic structure, suggesting persistence of core sets of proteins since lineage divergence. The CPR could have arisen in an episode of dramatic but heterogeneous genome reduction or from a protogenote community and co-evolved with other bacteria.

Tiny Hidden Genes within Our Microbiome.

Exploration of tiny protein-coding sequences within the human microbiome reveals thousands of conserved gene families that have been overlooked by traditional analyses. These small proteins may play key roles in the crosstalk among bacteria within the microbiome and in interactions with their human hosts.

Mediterranean grassland soil C-N compound turnover is dependent on rainfall and depth, and is mediated by genomically divergent microorganisms.

Soil microbial activity drives the carbon and nitrogen cycles and is an important determinant of atmospheric trace gas turnover, yet most soils are dominated by microorganisms with unknown metabolic capacities. Even Acidobacteria, among the most abundant bacteria in soil, remain poorly characterized, and functions across groups such as Verrucomicrobia, Gemmatimonadetes, Chloroflexi and Rokubacteria are understudied. Here, we have resolved 60 metagenomic and 20 proteomic data sets from a Mediterranean grassland soil ecosystem and recovered 793 near-complete microbial genomes from 18 phyla, representing around one-third of all microorganisms detected. Importantly, this enabled extensive genomics-based metabolic predictions for these communities. Acidobacteria from multiple previously unstudied classes have genomes that encode large enzyme complements for complex carbohydrate degradation. Alternatively, most microorganisms encode carbohydrate esterases that strip readily accessible methyl and acetyl groups from polymers like pectin and xylan, forming methanol and acetate, the availability of which could explain the high prevalence of C1 metabolism and acetate utilization in genomes. Microorganism abundances among samples collected at three soil depths and under natural and amended rainfall regimes indicate statistically higher associations of inorganic nitrogen metabolism and carbon degradation in deep and shallow soils, respectively. This partitioning decreased in samples under extended spring rainfall, indicating that long-term climate alteration can affect both carbon and nitrogen cycling. Overall, by leveraging natural and experimental gradients with genome-resolved metabolic profiles, we link microorganisms lacking prior genomic characterization to specific roles in complex carbon, C1, nitrate and ammonia transformations, and constrain factors that impact their distributions in soil.

Author Correction: Hydrogen-based metabolism as an ancestral trait in lineages sibling to the Cyanobacteria.

The original version of this Article contained errors in Fig. 4. In panel a, the labels 'F420-reducing NiFe hydrogenase (group 3a)' and 'Group 2 NiFe hydrogenase' were misplaced. These errors have been corrected in both the PDF and HTML versions of the Article.