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.

Three-dimensional structure-guided evolution of a ribosome with tethered subunits.

RNA-based macromolecular machines, such as the ribosome, have functional parts reliant on structural interactions spanning sequence-distant regions. These features limit evolutionary exploration of mutant libraries and confound three-dimensional structure-guided design. To address these challenges, we describe Evolink (evolution and linkage), a method that enables high-throughput evolution of sequence-distant regions in large macromolecular machines, and library design guided by computational RNA modeling to enable exploration of structurally stable designs. Using Evolink, we evolved a tethered ribosome with a 58% increased activity in orthogonal protein translation and a 97% improvement in doubling times in SQ171 cells compared to a previously developed tethered ribosome, and reveal new permissible sequences in a pair of ribosomal helices with previously explored biological function. The Evolink approach may enable enhanced engineering of macromolecular machines for new and improved functions for synthetic biology.

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.

Computationally designed peptide macrocycle inhibitors of New Delhi metallo-ß-lactamase 1.

The rise of antibiotic resistance calls for new therapeutics targeting resistance factors such as the New Delhi metallo-ß-lactamase 1 (NDM-1), a bacterial enzyme that degrades ß-lactam antibiotics. We present structure-guided computational methods for designing peptide macrocycles built from mixtures of l- and d-amino acids that are able to bind to and inhibit targets of therapeutic interest. Our methods explicitly consider the propensity of a peptide to favor a binding-competent conformation, which we found to predict rank order of experimentally observed IC50 values across seven designed NDM-1- inhibiting peptides. We were able to determine X-ray crystal structures of three of the designed inhibitors in complex with NDM-1, and in all three the conformation of the peptide is very close to the computationally designed model. In two of the three structures, the binding mode with NDM-1 is also very similar to the design model, while in the third, we observed an alternative binding mode likely arising from internal symmetry in the shape of the design combined with flexibility of the target. Although challenges remain in robustly predicting target backbone changes, binding mode, and the effects of mutations on binding affinity, our methods for designing ordered, binding-competent macrocycles should have broad applicability to a wide range of therapeutic targets.

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.

Accelerated cryo-EM-guided determination of three-dimensional RNA-only structures.

The discovery and design of biologically important RNA molecules is outpacing three-dimensional structural characterization. Here, we demonstrate that cryo-electron microscopy can routinely resolve maps of RNA-only systems and that these maps enable subnanometer-resolution coordinate estimation when complemented with multidimensional chemical mapping and Rosetta DRRAFTER computational modeling. This hybrid 'Ribosolve' pipeline detects and falsifies homologies and conformational rearrangements in 11 previously unknown 119- to 338-nucleotide protein-free RNA structures: full-length Tetrahymena ribozyme, hc16 ligase with and without substrate, full-length Vibrio cholerae and Fusobacterium nucleatum glycine riboswitch aptamers with and without glycine, Mycobacterium SAM-IV riboswitch with and without S-adenosylmethionine, and the computer-designed ATP-TTR-3 aptamer with and without AMP. Simulation benchmarks, blind challenges, compensatory mutagenesis, cross-RNA homologies and internal controls demonstrate that Ribosolve can accurately resolve the global architectures of RNA molecules but does not resolve atomic details. These tests offer guidelines for making inferences in future RNA structural studies with similarly accelerated throughput.

De novo 3D models of SARS-CoV-2 RNA elements from consensus experimental secondary structures.

The rapid spread of COVID-19 is motivating development of antivirals targeting conserved SARS-CoV-2 molecular machinery. The SARS-CoV-2 genome includes conserved RNA elements that offer potential small-molecule drug targets, but most of their 3D structures have not been experimentally characterized. Here, we provide a compilation of chemical mapping data from our and other labs, secondary structure models, and 3D model ensembles based on Rosetta's FARFAR2 algorithm for SARS-CoV-2 RNA regions including the individual stems SL1-8 in the extended 5' UTR; the reverse complement of the 5' UTR SL1-4; the frameshift stimulating element (FSE); and the extended pseudoknot, hypervariable region, and s2m of the 3' UTR. For eleven of these elements (the stems in SL1-8, reverse complement of SL1-4, FSE, s2m and 3' UTR pseudoknot), modeling convergence supports the accuracy of predicted low energy states; subsequent cryo-EM characterization of the FSE confirms modeling accuracy. To aid efforts to discover small molecule RNA binders guided by computational models, we provide a second set of similarly prepared models for RNA riboswitches that bind small molecules. Both datasets ('FARFAR2-SARS-CoV-2', https://github.com/DasLab/FARFAR2-SARS-CoV-2; and 'FARFAR2-Apo-Riboswitch', at https://github.com/DasLab/FARFAR2-Apo-Riboswitch') include up to 400 models for each RNA element, which may facilitate drug discovery approaches targeting dynamic ensembles of RNA molecules.

Theoretical basis for stabilizing messenger RNA through secondary structure design.

RNA hydrolysis presents problems in manufacturing, long-term storage, world-wide delivery and in vivo stability of messenger RNA (mRNA)-based vaccines and therapeutics. A largely unexplored strategy to reduce mRNA hydrolysis is to redesign RNAs to form double-stranded regions, which are protected from in-line cleavage and enzymatic degradation, while coding for the same proteins. The amount of stabilization that this strategy can deliver and the most effective algorithmic approach to achieve stabilization remain poorly understood. Here, we present simple calculations for estimating RNA stability against hydrolysis, and a model that links the average unpaired probability of an mRNA, or AUP, to its overall hydrolysis rate. To characterize the stabilization achievable through structure design, we compare AUP optimization by conventional mRNA design methods to results from more computationally sophisticated algorithms and crowdsourcing through the OpenVaccine challenge on the Eterna platform. We find that rational design on Eterna and the more sophisticated algorithms lead to constructs with low AUP, which we term 'superfolder' mRNAs. These designs exhibit a wide diversity of sequence and structure features that may be desirable for translation, biophysical size, and immunogenicity. Furthermore, their folding is robust to temperature, computer modeling method, choice of flanking untranslated regions, and changes in target protein sequence, as illustrated by rapid redesign of superfolder mRNAs for B.1.351, P.1 and B.1.1.7 variants of the prefusion-stabilized SARS-CoV-2 spike protein. Increases in in vitro mRNA half-life by at least two-fold appear immediately achievable.

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.

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.

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.

RNA-Puzzles Round IV: 3D structure predictions of four ribozymes and two aptamers.

RNA-Puzzles is a collective endeavor dedicated to the advancement and improvement of RNA 3D structure prediction. With agreement from crystallographers, the RNA structures are predicted by various groups before the publication of the crystal structures. We now report the prediction of 3D structures for six RNA sequences: four nucleolytic ribozymes and two riboswitches. Systematic protocols for comparing models and crystal structures are described and analyzed. In these six puzzles, we discuss (i) the comparison between the automated web servers and human experts; (ii) the prediction of coaxial stacking; (iii) the prediction of structural details and ligand binding; (iv) the development of novel prediction methods; and (v) the potential improvements to be made. We show that correct prediction of coaxial stacking and tertiary contacts is essential for the prediction of RNA architecture, while ligand binding modes can only be predicted with low resolution and simultaneous prediction of RNA structure with accurate ligand binding still remains out of reach. All the predicted models are available for the future development of force field parameters and the improvement of comparison and assessment tools.

Accurate de novo design of hyperstable constrained peptides.

Naturally occurring, pharmacologically active peptides constrained with covalent crosslinks generally have shapes that have evolved to fit precisely into binding pockets on their targets. Such peptides can have excellent pharmaceutical properties, combining the stability and tissue penetration of small-molecule drugs with the specificity of much larger protein therapeutics. The ability to design constrained peptides with precisely specified tertiary structures would enable the design of shape-complementary inhibitors of arbitrary targets. Here we describe the development of computational methods for accurate de novo design of conformationally restricted peptides, and the use of these methods to design 18-47 residue, disulfide-crosslinked peptides, a subset of which are heterochiral and/or N-C backbone-cyclized. Both genetically encodable and non-canonical peptides are exceptionally stable to thermal and chemical denaturation, and 12 experimentally determined X-ray and NMR structures are nearly identical to the computational design models. The computational design methods and stable scaffolds presented here provide the basis for development of a new generation of peptide-based drugs.

Better together: Elements of successful scientific software development in a distributed collaborative community.

Many scientific disciplines rely on computational methods for data analysis, model generation, and prediction. Implementing these methods is often accomplished by researchers with domain expertise but without formal training in software engineering or computer science. This arrangement has led to underappreciation of sustainability and maintainability of scientific software tools developed in academic environments. Some software tools have avoided this fate, including the scientific library Rosetta. We use this software and its community as a case study to show how modern software development can be accomplished successfully, irrespective of subject area. Rosetta is one of the largest software suites for macromolecular modeling, with 3.1 million lines of code and many state-of-the-art applications. Since the mid 1990s, the software has been developed collaboratively by the RosettaCommons, a community of academics from over 60 institutions worldwide with diverse backgrounds including chemistry, biology, physiology, physics, engineering, mathematics, and computer science. Developing this software suite has provided us with more than two decades of experience in how to effectively develop advanced scientific software in a global community with hundreds of contributors. Here we illustrate the functioning of this development community by addressing technical aspects (like version control, testing, and maintenance), community-building strategies, diversity efforts, software dissemination, and user support. We demonstrate how modern computational research can thrive in a distributed collaborative community. The practices described here are independent of subject area and can be readily adopted by other software development communities.

Structure and ligand binding of the glutamine-II riboswitch.

We have determined the structure of the glutamine-II riboswitch ligand binding domain using X-ray crystallography. The structure was solved using a novel combination of homology modeling and molecular replacement. The structure comprises three coaxial helical domains, the central one of which is a pseudoknot with partial triplex character. The major groove of this helix provides the binding site for L-glutamine, which is extensively hydrogen bonded to the RNA. Atomic mutation of the RNA at the ligand binding site leads to loss of binding shown by isothermal titration calorimetry, explaining the specificity of the riboswitch. A metal ion also plays an important role in ligand binding. This is directly bonded to a glutamine carboxylate oxygen atom, and its remaining inner-sphere water molecules make hydrogen bonding interactions with the RNA.

Keeping our staff in mind: Dietary results of a lifestyle intervention targeting mental health staff.

ISSUES ADDRESSED: 1) Describe the dietary intake of mental health staff within South Eastern Sydney Local Health District and 2) Evaluate the impact of an individualised staff lifestyle program on the following nutrition parameters; (i) energy, (ii) core food groups and (iii) discretionary foods. METHODS: This was a pragmatic single-arm intervention study, conducted for all staff working in a public mental health service, in Sydney, Australia. A five-session individualised lifestyle intervention delivered over 5 weeks incorporated nutritional counselling delivered by a dietitian. Participants were assessed at baseline, following the intervention, and at follow-up using diet history to assess dietary intake. RESULTS: Eighty-eight staff completed the dietary intervention and follow-up. An intake of core food groups significantly below national recommendations was reported for total vegetables (-1.75 ± 0.14 serves, P < .001), fruit (-0.29 ± 0.11 serves, P = .01), grains (-1.25 ± 0.20 serves, P < .001) and dairy servings (-1.00 ± 1.08 serves, P < .001), and protein-based foods were significantly above national recommendations (0.2 ± 0.09 serves, P = .03). At completion of the program, energy from discretionary foods was reduced by 460 kJ (95% CI -635 to -285, P < .001), and the serves of total vegetables (0.91 serves, 95% CI 0.59-1.22, P < .001) and dairy (0.31 serves, 95% CI 0.11-0.50, P < .001) were increased significantly. CONCLUSIONS: A workplace-based well-being program for staff working in the mental health setting coincided with dietary improvements. SO WHAT: Mental health staff can act as positive role models for clients to promote developing positive physical health behaviours.

Prevalence and correlates of food insecurity in community-based individuals with severe mental illness receiving long-acting injectable antipsychotic treatment.

People with severe mental illness (SMI) have numerous risk factors that may predispose them to food insecurity (FI); however, the prevalence of FI and its effects on health are under-researched in this population. The present study aimed to describe the prevalence of FI and its relationship to lifestyle factors in people with SMI. This cross-sectional study recruited people with SMI receiving long-acting injectable (LAI) antipsychotic medication from community services at three sites in Sydney, Australia. Assessments were completed on physical health and lifestyle factors. χ2 Tests, independent-samples t tests and binary logistic regression analyses were calculated to examine relationships between lifestyle factors and FI. In total, 233 people completed the assessments: 154 were males (66 %), mean age 44·8 (sd 12·7) years, and the majority (70 %) had a diagnosis of schizophrenia. FI was present in 104 participants (45 %). People with FI were less likely to consume fruits (OR 0·42, 95 % CI 0·24, 0·74, P = 0·003), vegetables (OR 0·39, 95 % CI 0·22, 0·69, P = 0·001) and protein-based foods (OR 0·45, 95 % CI 0·25, 0·83, P = 0·011) at least once daily, engaged in less moderate to vigorous physical activity (min) (OR 0·997, 95 % CI 0·993, 1·000, P = 0·044), and were more likely to smoke (OR 1·89, 95 % CI 1·08, 3·32, P = 0·026). FI is highly prevalent among people with SMI receiving LAI antipsychotic medications. Food-insecure people with SMI engage in less healthy lifestyle behaviours, increasing the risk of future non-communicable disease.

Ectogestation ethics: The implications of artificially extending gestation for viability, newborn resuscitation and abortion.

Recent animal research suggests that it may soon be possible to support the human fetus in an artificial uterine environment for part of a pregnancy. A technique of extending gestation in this way ("ectogestation") could be offered to parents of extremely premature infants (EPIs) to improve outcomes for their child. The use of artificial uteruses for ectogestation could generate ethical questions because of the technology's potential impact on the point of "viability"-loosely defined as the stage of pregnancy beyond which the fetus may survive external to the womb. Several medical decisions during the perinatal period are based on the gestation at which infants are considered viable, for example decisions about newborn resuscitation and abortion, and ectogestation has the potential to impact on these. Despite these possible implications, there is little existing evidence or analysis of how this technology would affect medical practice. In this paper, we combine empirical data with ethical analysis. We report a survey of 91 practicing Australian obstetricians and neonatologists; we aimed to assess their conceptual understanding of "viability," and what ethical consequences they envisage arising from improved survival of EPIs. We also assess what the ethical implications of extending gestation should be for newborn and obstetric care. We analyze the concept of viability and argue that while ectogestation might have implications for the permissibility of neonatal life-prolonging treatment at extremely early gestation, it should not necessarily have implications for abortion policy. We compare our ethical findings with the results of the survey.

Upskilling Mental Health Nurses to Address the Burden of Poor Metabolic Health: A Mixed Method Evaluation.

People living with a severe mental illness experience a life expectancy gap compared to the rest of the population that is largely driven by preventable cardiovascular diseases stemming from lifestyle factors, and the side effects of psychotropic medications. Mental health nurses are well positioned to help address the gap using lifestyle interventions. However, many nurses don't prioritise delivering such care, or lack the skills and confidence to implement these strategies. This study used a mixed method approach to determine the effectiveness of 2-day metabolic workshops that aimed to provide nurses with the skills to provide lifestyle interventions. The quantitative component compares pre and post measures of attitudes, confidence, knowledge and perceived barriers of providing metabolic care using a validated tool (M-BACK) and the qualitative component to elicit more details of the needs, expectations and plans of participants. Fifty-six nurse participants demonstrated statistically significant improvements in M-BACK post scores (p < 0.001), with 53 of the 56 participants (95%) achieving improved M-BACK scores. Participants identified three primary barriers to delivering metabolic care, related to individual staff members, consumers, and system issues. Prior to the course participants stated they hoped to improve their knowledge, enhance their practical skills and provide education for others. Following completion of the course participants planned to implement lifestyle education and interventions for consumers, provide education and support to other staff and integrate metabolic health care into clinical reviews and planning. This study demonstrates that education on metabolic health care can be effective in improving the attitudes, confidence, and knowledge of mental health nursing in providing metabolic health care and a decrease in the perceived barriers to delivering that care.

Changing health workforce attitudes to promote improved physical health in mental health service users: Keeping our Staff in Mind (KoSiM).

ISSUE ADDRESSED: People living with mental illness die on average 15 years earlier than the general population, primarily due to preventable and premature cardiovascular disease. Lifestyle interventions can be effective in reducing cardiovascular risk, yet mental health services do not routinely provide targeted lifestyle interventions. Exposing mental health staff to lifestyle interventions prior to targeting patients may be critical to changing culture and improving patient outcomes. This study aimed to improve the physical health of mental health staff through a targeted lifestyle intervention. METHODS: A pragmatic single-arm intervention study was conducted in a public mental health service, including inpatient and community settings, in Sydney, Australia. Participants in this study were n = 212 clinical and non-clinical staff. A five-session individualised lifestyle intervention (delivered over 5 weeks) incorporating physical activity and nutritional counselling was delivered by multidisciplinary teams. Participants were assessed at baseline, following the intervention, and at follow-up (mean = 16.7 weeks). The primary outcome was the barriers, attitudes, knowledge and confidence regarding screening, promoting and intervening to improve physical health outcomes of patients (M-BACK questionnaire). Secondary outcomes included anthropometric measures, cardiorespiratory fitness, sedentary time and nutritional intake. Repeated measures ANCOVAs were performed. RESULTS: A total of 212 staff (79% female) participated in this study. M-BACK total score significantly increased from baseline to follow-up (P < .001). Waist circumference, sedentary time and total energy intake all significantly decreased (all P's < .001) and cardiorespiratory fitness significantly increased (P < .001). CONCLUSION: A brief lifestyle intervention for staff of a public mental health service may increase the capability of the participants to improve their own physical health. SO WHAT?: Improving staff health may be an important strategy in improving the uptake and/or the effectiveness of lifestyle interventions targeting mental health service users.