OpenFold: retraining AlphaFold2 yields new insights into its learning mechanisms and capacity for generalization.

AlphaFold2 revolutionized structural biology with the ability to predict protein structures with exceptionally high accuracy. Its implementation, however, lacks the code and data required to train new models. These are necessary to (1) tackle new tasks, like protein-ligand complex structure prediction, (2) investigate the process by which the model learns and (3) assess the model's capacity to generalize to unseen regions of fold space. Here we report OpenFold, a fast, memory efficient and trainable implementation of AlphaFold2. We train OpenFold from scratch, matching the accuracy of AlphaFold2. Having established parity, we find that OpenFold is remarkably robust at generalizing even when the size and diversity of its training set is deliberately limited, including near-complete elisions of classes of secondary structure elements. By analyzing intermediate structures produced during training, we also gain insights into the hierarchical manner in which OpenFold learns to fold. In sum, our studies demonstrate the power and utility of OpenFold, which we believe will prove to be a crucial resource for the protein modeling community.

Implementation of a lifestyle and life-skills intervention to prevent weight-gain and cardiometabolic abnormalities in young people with first-episode psychosis as part of routine care: The Keeping the Body in Mind program.

OBJECTIVES: In 2013, a cluster-controlled pilot study found the 12-week Keeping the Body in Mind (KBIM) lifestyle and life skills intervention was able to prevent weight gain in a small sample of youth experiencing first-episode psychosis (FEP) with fewer than 4 weeks of antipsychotic exposure. This study aims to evaluate the effectiveness of KBIM as routine care on anthropometry and metabolic biochemistry in a larger sample of youth with FEP across three community mental health services. METHOD: This retrospective chart audit was conducted on youth with FEP, prescribed a therapeutic dose of antipsychotic medication, and who engaged with KBIM between 2015 and 2019. Primary outcomes were weight and waist circumference. Secondary outcomes were blood pressure, blood glucose, and blood lipids. Outcomes were collected in at baseline and at 12 weeks. Data on program engagement were obtained from the participant's medical file. RESULTS: One-hundred and eighty-two people met inclusion criteria, and up to 134 people had baseline and 12-week data on one or more outcome. Mean number of sessions attended was 11.1 (SD = 7.3). Increases in weight and waist circumference were limited to 1.5 kg (SD = 5.3, t(133) = 3.2, p = .002) and 0.7 cm (SD = 5.8, t(109) = 1.2, p = .23) respectively. Eighty-one percent of participants did not experience clinically significant weight gain (>7% of baseline weight). There were no significant changes in blood pressure or metabolic biochemistry. CONCLUSION: The prevention of substantial gains in weight and waist circumference observed in the initial pilot study was maintained with implementation of KBIM as part of routine clinical care for youth with FEP.

Erratum: Pharmacist interventions for appropriate COVID-19 antiviral therapy in long-term care facilities: A public health initiative - ERRATUM.

[This corrects the article DOI: 10.1017/ash.2023.246.].

The Relationship Between Early Term Birth and the Risk of Later Childhood Mental Disorders Within a Pregnancy Cohort.

This study examines whether gestational age, birth weight, and early term birth is associated with childhood mental disorders in 342 pregnant women recruited at less than 20 weeks gestation and were then followed up until 4 years postpartum, including 93 children born at early term. Women were assessed at recruitment using the Structured Clinical Interview for DSM. At 4 years of age their children were assessed using the Preschool Age Psychiatric Assessment (PAPA) and the Child Behavior Checklist (CBCL). This study found earlier birth predicted an increased risk for anxiety disorders and demonstrated a significant interaction between gestational age and lower birthweight. The risk for ADHD increased with lower gestational age independent of birthweight. In contrast, gestational age was not associated with Oppositional Defiant Disorder, Conduct Disorder, internalizing or externalizing symptoms. These findings highlight the important differences in the association of early term birth and vulnerability for specific mental disorders.

In-depth assessment of critical access hospital stewardship program adherence to the CDC Core Elements in Iowa and Nebraska.

In 21 antimicrobial stewardship programs in critical-access hospitals in Nebraska and Iowa that self-reported nonadherence to a CDC Core Element or Elements, in-depth program assessment and feedback revealed that accountability and education most needed improvement. Recommendations included providing physician and pharmacist training, tracking interventions, and providing education. Program barriers included lack of time and/or personnel and antimicrobial stewardship and/or infectious diseases expertise.

Local structural flexibility drives oligomorphism in computationally designed protein assemblies.

Many naturally occurring protein assemblies have dynamic structures that allow them to perform specialized functions. For example, clathrin coats adopt a wide variety of architectures to adapt to vesicular cargos of various sizes. Although computational methods for designing novel self-assembling proteins have advanced substantially over the past decade, most existing methods focus on designing static structures with high accuracy. Here we characterize the structures of three distinct computationally designed protein assemblies that each form multiple unanticipated architectures, and identify flexibility in specific regions of the subunits of each assembly as the source of structural diversity. Cryo-EM single-particle reconstructions and native mass spectrometry showed that only two distinct architectures were observed in two of the three cases, while we obtained six cryo-EM reconstructions that likely represent a subset of the architectures present in solution in the third case. Structural modeling and molecular dynamics simulations indicated that the surprising observation of a defined range of architectures, instead of non-specific aggregation, can be explained by constrained flexibility within the building blocks. Our results suggest that deliberate use of structural flexibility as a design principle will allow exploration of previously inaccessible structural and functional space in designed protein assemblies.

OpenProteinSet: Training data for structural biology at scale.

Multiple sequence alignments (MSAs) of proteins encode rich biological information and have been workhorses in bioinformatic methods for tasks like protein design and protein structure prediction for decades. Recent breakthroughs like AlphaFold2 that use transformers to attend directly over large quantities of raw MSAs have reaffirmed their importance. Generation of MSAs is highly computationally intensive, however, and no datasets comparable to those used to train AlphaFold2 have been made available to the research community, hindering progress in machine learning for proteins. To remedy this problem, we introduce OpenProteinSet, an open-source corpus of more than 16 million MSAs, associated structural homologs from the Protein Data Bank, and AlphaFold2 protein structure predictions. We have previously demonstrated the utility of OpenProteinSet by successfully retraining AlphaFold2 on it. We expect OpenProteinSet to be broadly useful as training and validation data for 1) diverse tasks focused on protein structure, function, and design and 2) large-scale multimodal machine learning research.

Use of a beta-lactam graded challenge process for inpatients with self-reported penicillin allergies at an academic medical center.

Background: The Antimicrobial Stewardship Program (ASP) at Nebraska Medicine collaborated with a board-certified allergist to develop a penicillin allergy guidance document for treating inpatients with self-reported allergy. This guidance contains an algorithm for evaluating and safely challenging penicillin-allergic patients with beta-lactams without inpatient allergy consults being available. Methods: Following multi-disciplinary review, an order set for beta-lactam graded challenges (GC) was implemented in 2018. This contains recommended monitoring and detailed medication orders to challenge patients with various beta-lactam agents. Inpatient orders for GC from 3/2018-6/2022 were retrospectively reviewed to evaluate ordering characteristics, outcomes of the challenge, and whether documentation of the allergy history was updated. All beta-lactam challenges administered to inpatients were included, and descriptive statistics were performed. Results: Overall, 157 GC were administered; 13 with oral amoxicillin and 144 with intravenous (IV) beta-lactams. Ceftriaxone accounted for the most challenges (43%). All oral challenges were recommended by an Infectious Diseases consult service, as were a majority of IV challenges (60%). Less than one in five were administered in an ICU (19%). Almost all (n = 150, 96%) were tolerated without any adverse event. There was one reaction (1%) of hives and six (4%) involving a rash, none of which had persistent effects. Allergy information was updated in the electronic health record after 92% of the challenges. Conclusion: Both intravenous and oral beta-lactam graded challenges were implemented successfully in a hospital without a regular inpatient allergy consult service. They were well-tolerated, administered primarily in non-ICU settings, and were often ordered by non-specialist services. In patients with a self-reported penicillin allergy, these results demonstrate the utility and safety of a broadly adopted beta-lactam GC process.

Implementation of a Rapid Multiplex Polymerase Chain Reaction Pneumonia Panel and Subsequent Antibiotic De-escalation.

Background: Net effects of implementation of a multiplex polymerase chain reaction (PCR) pneumonia panel (PNP) on antimicrobial stewardship are thus far unknown. This retrospective study evaluated the real-world impact of the PNP on time to antibiotic de-escalation in critically ill patients treated for pneumonia at an academic medical center. Methods: This retrospective, quasi-experimental study included adult intensive care unit (ICU) patients with respiratory culture results from 1 May to 15 August 2019 (pre-PNP group) and adult ICU patients with PNP results from 1 May to 15 August 2020 (PNP group) at Nebraska Medical Center. Patients were excluded for the following reasons: any preceding positive coronavirus disease 2019 PCR test, lack of antibiotic receipt, or non-respiratory tract infection indications for antibiotics. The primary outcome was time to discontinuation of anti-methicillin-resistant Staphylococcus aureus (MRSA) therapy. Secondary outcomes included time to discontinuation of antipseudomonal therapy, frequency of early discontinuation for atypical coverage, and overall duration (in days) of antibiotic therapy for pneumonia. Results: Sixty-six patients in the pre-PNP group and 58 in the PNP group were included. There were significant differences in patient characteristics between groups. The median time to anti-MRSA agent discontinuation was 49.1 hours in the pre-PNP and 41.8 hours in the PNP group (P = .28). The median time to discontinuation of antipseudomonal agents was 134.4 hours in the pre-PNP versus 98.1 hours in the PNP group (P = .47). Other outcomes were numerically but not significantly improved in our sample. Conclusions: This early look at implementation of a multiplex PNP did not demonstrate a statistically significant difference in antibiotic use but lays the groundwork to further evaluate a significant real-world impact on antibiotic de-escalation in ICU patients treated for pneumonia.

Nurse-Led Physical Health Interventions for People with Mental Illness: A Scoping Review of International Literature.

People with mental illness have a higher prevalence of co-occurring physical health conditions and poor health behaviors, leading a mortality gap of up to 16 years, compared with the general population. Nurses working in mental health settings play an important role in addressing factors influencing sub-optimal physical health. Therefore, this scoping review aimed to identify nurse-led physical health interventions and align interventions to eight recognized physical healthcare priority areas (i.e. Equally Well in Victoria Framework). A systematic search strategy was used to identify relevant literature. Data extraction included alignment to the Equally Well priority areas, research design, and indication of co-design (meaningful and collaborative involvement of consumers and significant others) and recovery-oriented practice (focusing on needs and goals of a consumer's recovery journey). All included papers (n = 74) were aligned to at least one of eight Equally Well priority areas. Papers were predominately quantitative (n = 64, 86%), with the remainder mixed methods (n = 9, 9%) or qualitative (n = 4, 5%). Most papers were aligned to improving metabolic health and support to quit smoking. One study focused on nurse-led intervention designed to reduce falls. Recovery-oriented practice was evident in six papers. No paper described evidence of co-design. A research gap was identified for nurse-led intervention to reduce falls and improve dental/oral care. Relative to mental healthcare policy, there is a need for future nurse-led physical health research to be co-designed and include recovery-oriented practice. Evaluation and description of future nurse-led physical interventions should seek to report perspectives of key stakeholders as these remain relatively unknown.

Methanogen activity and microbial diversity in Gulf of Cádiz mud volcano sediments.

The Gulf of Cádiz is a tectonically active continental margin with over sixty mud volcanoes (MV) documented, some associated with active methane (CH4) seepage. However, the role of prokaryotes in influencing this CH4 release is largely unknown. In two expeditions (MSM1-3 and JC10) seven Gulf of Cádiz MVs (Porto, Bonjardim, Carlos Ribeiro, Captain Arutyunov, Darwin, Meknes, and Mercator) were analyzed for microbial diversity, geochemistry, and methanogenic activity, plus substrate amended slurries also measured potential methanogenesis and anaerobic oxidation of methane (AOM). Prokaryotic populations and activities were variable in these MV sediments reflecting the geochemical heterogeneity within and between them. There were also marked differences between many MV and their reference sites. Overall direct cell numbers below the SMTZ (0.2-0.5 mbsf) were much lower than the general global depth distribution and equivalent to cell numbers from below 100 mbsf. Methanogenesis from methyl compounds, especially methylamine, were much higher than the usually dominant substrates H2/CO2 or acetate. Also, CH4 production occurred in 50% of methylated substrate slurries and only methylotrophic CH4 production occurred at all seven MV sites. These slurries were dominated by Methanococcoides methanogens (resulting in pure cultures), and prokaryotes found in other MV sediments. AOM occurred in some slurries, particularly, those from Captain Arutyunov, Mercator and Carlos Ribeiro MVs. Archaeal diversity at MV sites showed the presence of both methanogens and ANME (Methanosarcinales, Methanococcoides, and ANME-1) related sequences, and bacterial diversity was higher than archaeal diversity, dominated by members of the Atribacterota, Chloroflexota, Pseudomonadota, Planctomycetota, Bacillota, and Ca. "Aminicenantes." Further work is essential to determine the full contribution of Gulf of Cádiz mud volcanoes to the global methane and carbon cycles.

Macrocyclic ß-Sheets Stabilized by Hydrogen Bond Surrogates.

Mimics of protein secondary and tertiary structure offer rationally-designed inhibitors of biomolecular interactions. ß-Sheet mimics have a storied history in bioorganic chemistry and are typically designed with synthetic or natural turn segments. We hypothesized that replacement of terminal inter-ß-strand hydrogen bonds with hydrogen bond surrogates (HBS) may lead to conformationally-defined macrocyclic ß-sheets without the requirement for natural or synthetic ß-turns, thereby providing a minimal mimic of a protein ß-sheet. To access turn-less antiparallel ß-sheet mimics, we developed a facile solid phase synthesis protocol. We surveyed a dataset of protein ß-sheets for naturally observed interstrand side chain interactions. This bioinformatics survey highlighted an over-abundance of aromatic-aromatic, cation-π and ionic interactions in ß-sheets. In correspondence with natural ß-sheets, we find that minimal HBS mimics show robust ß-sheet formation when specific amino acid residue pairings are incorporated. In isolated ß-sheets, aromatic interactions endow superior conformational stability over ionic or cation-π interactions. Circular dichroism and NMR spectroscopies, along with high-resolution X-ray crystallography, support our design principles.

Cryo-EM-guided engineering of T-box-tRNA modules with enhanced selectivity and sensitivity in translational regulation.

Riboswitches are non-coding RNA elements that play vital roles in regulating gene expression. Their specific ligand-dependent structural reorganization facilitates their use as templates for design of engineered RNA switches for therapeutics, nanotechnology and synthetic biology. T-box riboswitches bind tRNAs to sense aminoacylation and control gene expression via transcription attenuation or translation inhibition. Here we determine the cryo-EM structure of the wild-type Mycobacterium smegmatis ileS T-box in complex with its cognate tRNA Ile . This structure shows a very flexible antisequestrator region that tolerates both 3'-OH and 2',3'-cyclic phosphate modification at the 3' end of tRNA Ile . Elongation of one helical turn (11-base pair) in both the tRNA acceptor arm and T-box Stem III maintains T-box-tRNA complex formation and increases the selectivity for tRNA 3' end modification. Moreover, elongation of Stem III results in ∼6-fold tighter binding to tRNA, which leads to increased sensitivity of downstream translational regulation indicated by precedent translation. Our results demonstrate that cryo-EM can guide RNA engineering to design improved riboswitch modules for translational regulation, and potentially a variety of additional functions.

Community science designed ribosomes with beneficial phenotypes.

Functional design of ribosomes with mutant ribosomal RNA (rRNA) can expand opportunities for understanding molecular translation, building cells from the bottom-up, and engineering ribosomes with altered capabilities. However, such efforts are hampered by cell viability constraints, an enormous combinatorial sequence space, and limitations on large-scale, 3D design of RNA structures and functions. To address these challenges, we develop an integrated community science and experimental screening approach for rational design of ribosomes. This approach couples Eterna, an online video game that crowdsources RNA sequence design to community scientists in the form of puzzles, with in vitro ribosome synthesis, assembly, and translation in multiple design-build-test-learn cycles. We apply our framework to discover mutant rRNA sequences that improve protein synthesis in vitro and cell growth in vivo, relative to wild type ribosomes, under diverse environmental conditions. This work provides insights into rRNA sequence-function relationships and has implications for synthetic biology.

RNA 3D Modeling with FARFAR2, Online.

Understanding the three-dimensional structure of an RNA molecule is often essential to understanding its function. Sampling algorithms and energy functions for RNA structure prediction are improving, due to the increasing diversity of structural data available for training statistical potentials and testing structural data, along with a steady supply of blind challenges through the RNA-Puzzles initiative. The recent FARFAR2 algorithm enables near-native structure predictions on fairly complex RNA structures, including automated selection of final candidate models and estimation of model accuracy. Here, we describe the use of a publicly available webserver for RNA modeling for realistic scenarios using FARFAR2, available at https://rosie.rosettacommons.org/farfar2 . We walk through two cases in some detail: a simple model pseudoknot from the frameshifting element of beet western yellows virus modeled using the "basic interface" to the webserver and a replication of RNA-Puzzle 20, a metagenomic twister sister ribozyme, using the "advanced interface." We also describe example runs of FARFAR2 modeling including two kinds of experimental data: a c-di-GMP riboswitch modeled with low-resolution restraints from MOHCA-seq experiments and a tandem GA motif modeled with 1H NMR chemical shifts.

Tackling change in mental health service delivery: A qualitative evaluation of a lifestyle program targeting mental health staff - Keeping our Staff in Mind (KoSiM).

ISSUES ADDRESSED: People with severe mental illness have adverse health outcomes compared to the general population. Lifestyle interventions are effective in improving health outcomes in this population. Current cultural processes in mental health services do not generally incorporate physical health care practices. Innovative education is required to improve knowledge and confidence of staff in the delivery of preventative health measures. METHODS: The Keeping our Staff in Mind (KoSiM) program delivered a brief lifestyle intervention to mental health staff. A qualitative analysis following the Standards for Reporting Qualitative Research was undertaken. Semi-structured interviews designed to elicit information about the acceptability of the program and the impact of the intervention on participants' personal and professional lives. The interviews were analysed using thematic analysis, with coding independently developed and reviewed by three authors. RESULTS: Of the 103 eligible participants, 75 were interviewed. Responses revealed four main themes: (i) positive changes in clinician's approach to physical health care, (ii) improvements in attitudes to self-care and family wellbeing, (iii) positive changes in workplace culture associated with physical health care delivery and (iv) high levels of acceptability of the program. CONCLUSION: The KoSiM model may be useful in other settings as a means of changing the culture of mental health services to better integrate physical health care as a core part of mental health service provision. SO WHAT?: A novel approach using staff focussed lifestyle interventions model may cut through the resistance that is encountered when implementing proven methods of clinical intervention where cultural barriers exist.

Computationally-guided design and selection of high performing ribosomal active site mutants.

Understanding how modifications to the ribosome affect function has implications for studying ribosome biogenesis, building minimal cells, and repurposing ribosomes for synthetic biology. However, efforts to design sequence-modified ribosomes have been limited because point mutations in the ribosomal RNA (rRNA), especially in the catalytic active site (peptidyl transferase center; PTC), are often functionally detrimental. Moreover, methods for directed evolution of rRNA are constrained by practical considerations (e.g. library size). Here, to address these limitations, we developed a computational rRNA design approach for screening guided libraries of mutant ribosomes. Our method includes in silico library design and selection using a Rosetta stepwise Monte Carlo method (SWM), library construction and in vitro testing of combined ribosomal assembly and translation activity, and functional characterization in vivo. As a model, we apply our method to making modified ribosomes with mutant PTCs. We engineer ribosomes with as many as 30 mutations in their PTCs, highlighting previously unidentified epistatic interactions, and show that SWM helps identify sequences with beneficial phenotypes as compared to random library sequences. We further demonstrate that some variants improve cell growth in vivo, relative to wild type ribosomes. We anticipate that SWM design and selection may serve as a powerful tool for rRNA engineering.

Deep learning models for predicting RNA degradation via dual crowdsourcing.

Medicines based on messenger RNA (mRNA) hold immense potential, as evidenced by their rapid deployment as COVID-19 vaccines. However, worldwide distribution of mRNA molecules has been limited by their thermostability, which is fundamentally limited by the intrinsic instability of RNA molecules to a chemical degradation reaction called in-line hydrolysis. Predicting the degradation of an RNA molecule is a key task in designing more stable RNA-based therapeutics. Here, we describe a crowdsourced machine learning competition ('Stanford OpenVaccine') on Kaggle, involving single-nucleotide resolution measurements on 6,043 diverse 102-130-nucleotide RNA constructs that were themselves solicited through crowdsourcing on the RNA design platform Eterna. The entire experiment was completed in less than 6 months, and 41% of nucleotide-level predictions from the winning model were within experimental error of the ground truth measurement. Furthermore, these models generalized to blindly predicting orthogonal degradation data on much longer mRNA molecules (504-1,588 nucleotides) with improved accuracy compared with previously published models. These results indicate that such models can represent in-line hydrolysis with excellent accuracy, supporting their use for designing stabilized messenger RNAs. The integration of two crowdsourcing platforms, one for dataset creation and another for machine learning, may be fruitful for other urgent problems that demand scientific discovery on rapid timescales.

Enhancing the Australian Gridded Climate Dataset rainfall analysis using satellite data.

Rainfall estimation over large areas is important for a thorough understanding of water availability, influencing societal decision-making, as well as being an input for scientific models. Traditionally, Australia utilizes a gauge-based analysis for rainfall estimation, but its performance can be severely limited over regions with low gauge density such as central parts of the continent. At the Australian Bureau of Meteorology, the current operational monthly rainfall component of the Australian Gridded Climate Dataset (AGCD) makes use of statistical interpolation (SI), also known as optimal interpolation (OI) to form an analysis from a background field of station climatology. In this study, satellite observations of rainfall were used as the background field instead of station climatology to produce improved monthly rainfall analyses. The performance of these monthly datasets was evaluated over the Australian domain from 2001 to 2020. Evaluated over the entire national domain, the satellite-based SI datasets had similar to slightly better performance than the station climatology-based SI datasets with some individual months being more realistically represented by the satellite-SI datasets. However, over gauge-sparse regions, there was a clear increase in performance. For a representative sub-domain, the Kling-Gupta Efficiency (KGE) value increased by + 8% (+ 12%) during the dry (wet) season. This study is an important step in enhancing operational rainfall analysis over Australia.