Comprehensive encoding of conformational and compositional protein structural ensembles through the mmCIF data structure.

In the folded state, biomolecules exchange between multiple conformational states crucial for their function. However, most structural models derived from experiments and computational predictions only encode a single state. To represent biomolecules accurately, we must move towards modeling and predicting structural ensembles. Information about structural ensembles exists within experimental data from X-ray crystallography and cryo-electron microscopy. Although new tools are available to detect conformational and compositional heterogeneity within these ensembles, the legacy PDB data structure does not robustly encapsulate this complexity. We propose modifications to the macromolecular crystallographic information file (mmCIF) to improve the representation and interrelation of conformational and compositional heterogeneity. These modifications will enable the capture of macromolecular ensembles in a human and machine-interpretable way, potentially catalyzing breakthroughs for ensemble-function predictions, analogous to the achievements of AlphaFold with single-structure prediction.

Movement patterns of foraging common terns (Sterna hirundo) breeding in an urban environment in coastal Virginia.

Nesting colonial seabirds are prime examples of central-place foragers, animals that must return to a central location (e.g., a breeding colony) after each bout of foraging. They must balance the costs and benefits of foraging with the need to return to their colonies frequently to form pair bonds during courtship, incubate, provision mates and offspring, and protect and rear young. For some populations, the loss and degradation of suitable breeding habitat due to human activities have necessitated the construction of new breeding sites and/or the restoration of previously occupied sites. South Island, which is part of the Hampton Roads Bridge-Tunnel (HRBT) complex in the Commonwealth of Virginia, U.S.A., is a human-created island that supported Virginia's largest mixed species seabird colony until 2020, when the expansion of the HRBT began and when all nesting seabirds were permanently excluded from the site. We studied the movement patterns of foraging common terns (Sterna hirundo) to determine how travel to and around foraging sites related to their colony location and to inform the siting and construction of a new breeding island. We tracked 18 individual common terns from 07 June to 29 June 2018, and we used a hidden Markov model to assign behavioral states and investigate common tern movements around the HRBT. Common terns spent more than half their time in the colony (58%), followed by time devoted to foraging (22%), and the remainder of their time was spent on outbound (15%) and inbound (5%) transit. Terns traveled as far as 98km from the colony, but on average foraged relatively close to South Island (13.6 ± 0.3km, mean ± 1 SD). Individuals tended to forage in the same locations, but there was variation among individuals. Flying to foraging sites uses energy during the already energetically costly breeding season, thus managers should prioritize placing a new colony site in a location that minimizes the distance traveled to the foraging locations frequented by the South Island birds while accounting for other life-history characteristics. These findings could help in the design and construction of new breeding sites or the restoration of current sites for other, related species, particularly for which these data do not exist.

The impact of Library Size and Scale of Testing on Virtual Screening.

Virtual libraries for ligand discovery have recently increased 10,000-fold, and this is thought to have improved hit rates and potencies from library docking. This idea has not, however, been experimentally tested in direct comparisons of larger-vs-smaller libraries. Meanwhile, though libraries have exploded, the scale of experimental testing has little changed, with often only dozens of high-ranked molecules investigated, making interpretation of hit rates and affinities uncertain. Accordingly, we docked a 1.7 billion molecule virtual library against the model enzyme AmpC ß-lactamase, testing 1,521 new molecules and comparing the results to the same screen with a library of 99 million molecules, where only 44 molecules were tested. Encouragingly, the larger screen outperformed the smaller one: hit rates improved by two-fold, more new scaffolds were discovered, and potency improved. Overall, 50-fold more inhibitors were found, supporting the idea that there are many more compounds to be discovered than are being tested. With so many compounds evaluated, we could ask how the results vary with number tested, sampling smaller sets at random from the 1521. Hit rates and affinities were highly variable when we only sampled dozens of molecules, and it was only when we included several hundred molecules that results converged. As docking scores improved, so too did the likelihood of a molecule binding; hit rates improved steadily with docking score, as did affinities. This also appeared true on re-analysis of large-scale results against the σ2 and dopamine D4 receptors. It may be that as the scale of both the virtual libraries and their testing grows, not only are better ligands found but so too does our ability to rank them.

Automated multiconformer model building for X-ray crystallography and cryo-EM.

In their folded state, biomolecules exchange between multiple conformational states that are crucial for their function. Traditional structural biology methods, such as X-ray crystallography and cryogenic electron microscopy (cryo-EM), produce density maps that are ensemble averages, reflecting molecules in various conformations. Yet, most models derived from these maps explicitly represent only a single conformation, overlooking the complexity of biomolecular structures. To accurately reflect the diversity of biomolecular forms, there is a pressing need to shift toward modeling structural ensembles that mirror the experimental data. However, the challenge of distinguishing signal from noise complicates manual efforts to create these models. In response, we introduce the latest enhancements to qFit, an automated computational strategy designed to incorporate protein conformational heterogeneity into models built into density maps. These algorithmic improvements in qFit are substantiated by superior Rfree and geometry metrics across a wide range of proteins. Importantly, unlike more complex multicopy ensemble models, the multiconformer models produced by qFit can be manually modified in most major model building software (e.g., Coot) and fit can be further improved by refinement using standard pipelines (e.g., Phenix, Refmac, Buster). By reducing the barrier of creating multiconformer models, qFit can foster the development of new hypotheses about the relationship between macromolecular conformational dynamics and function.

Structural characterization of ligand binding and pH-specific enzymatic activity of mouse Acidic Mammalian Chitinase.

Chitin is an abundant biopolymer and pathogen-associated molecular pattern that stimulates a host innate immune response. Mammals express chitin-binding and chitin-degrading proteins to remove chitin from the body. One of these proteins, Acidic Mammalian Chitinase (AMCase), is an enzyme known for its ability to function under acidic conditions in the stomach but is also active in tissues with more neutral pHs, such as the lung. Here, we used a combination of biochemical, structural, and computational modeling approaches to examine how the mouse homolog (mAMCase) can act in both acidic and neutral environments. We measured kinetic properties of mAMCase activity across a broad pH range, quantifying its unusual dual activity optima at pH 2 and 7. We also solved high-resolution crystal structures of mAMCase in complex with oligomeric GlcNAcn, the building block of chitin, where we identified extensive conformational ligand heterogeneity. Leveraging these data, we conducted molecular dynamics simulations that suggest how a key catalytic residue could be protonated via distinct mechanisms in each of the two environmental pH ranges. These results integrate structural, biochemical, and computational approaches to deliver a more complete understanding of the catalytic mechanism governing mAMCase activity at different pH. Engineering proteins with tunable pH optima may provide new opportunities to develop improved enzyme variants, including AMCase, for therapeutic purposes in chitin degradation.

Epidemiology of sarcoptic mange in a geographically constrained insular red fox population.

BACKGROUND: Sarcoptic mange is a skin disease caused by the contagious ectoparasite Sarcoptes scabiei, capable of suppressing and extirpating wild canid populations. Starting in 2015, we observed a multi-year epizootic of sarcoptic mange affecting a red fox (Vulpes vulpes) population on Fire Island, NY, USA. We explored the ecological factors that contributed to the spread of sarcoptic mange and characterized the epizootic in a landscape where red foxes are geographically constrained. METHODS: We tested for the presence of S. scabiei DNA in skin samples collected from deceased red foxes with lesions visibly consistent with sarcoptic mange disease. We deployed 96-100 remote trail camera stations each year to capture red fox occurrences and used generalized linear mixed-effects models to assess the affects of red fox ecology, human and other wildlife activity, and island geography on the frequency of detecting diseased red foxes. We rated the extent of visual lesions in diseased individuals and mapped the severity and variability of the sarcoptic mange disease. RESULTS: Skin samples that we analyzed demonstrated 99.8% similarity to S. scabiei sequences in GenBank. Our top-ranked model (weight = 0.94) showed that diseased red foxes were detected more frequently close to roadways, close to territories of other diseased red foxes, away from human shelters, and in areas with more mammal activity. There was no evidence that detection rates in humans and their dogs or distance to the nearest red fox den explained the detection rates of diseased red foxes. Although detected infrequently, we observed the most severe signs of sarcoptic mange at the periphery of residential villages. The spread of visual signs of the disease was approximately 7.3 ha/week in 2015 and 12.1 ha/week in 2017. CONCLUSIONS: We quantified two separate outbreaks of sarcoptic mange disease that occurred > 40 km apart and were separated by a year. Sarcoptic mange revealed an unfettered spread across the red fox population. The transmission of S. scabiei mites in this system was likely driven by red fox behaviors and contact between individuals, in line with previous studies. Sarcoptic mange is likely an important contributor to red fox population dynamics within barrier island systems.

Rosace: a robust deep mutational scanning analysis framework employing position and mean-variance shrinkage.

Deep mutational scanning (DMS) measures the effects of thousands of genetic variants in a protein simultaneously. The small sample size renders classical statistical methods ineffective. For example, p-values cannot be correctly calibrated when treating variants independently. We propose Rosace, a Bayesian framework for analyzing growth-based DMS data. Rosace leverages amino acid position information to increase power and control the false discovery rate by sharing information across parameters via shrinkage. We also developed Rosette for simulating the distributional properties of DMS. We show that Rosace is robust to the violation of model assumptions and is more powerful than existing tools.

Use of Ultrasound in the Evaluation of Cryptorchidism: A Single-Institution Analysis.

INTRODUCTION: The American Urological Association guidelines recommend against the performance of ultrasound and other imaging modalities in the evaluation of patients with cryptorchidism before expert consultation. We aimed to examine our institutional experience with cryptorchidism and measure adherence to currently available guidelines. METHODS: An institutional review board-approved retrospective review of ultrasound utilization in the evaluation of patients with cryptorchidism was performed from June 1, 2016, to June 30, 2019, at a single tertiary level pediatric hospital. RESULTS: We identified 1796 patients evaluated in surgical clinics for cryptorchidism. Surgical intervention was performed in 75.2% (n = 1351) of the entire cohort. Ultrasound was performed in 42% (n = 754), most of which were ordered by referring physicians (91% n = 686). Of those who received an ultrasound, surgical intervention was performed in 78% (n = 588). Those 166 patients (22%) who did not undergo surgical intervention were referred with ultrasounds suggesting inguinal testes; however, all had normal physical examinations or mildly retractile testes at the time of consultation and were discharged from the outpatient clinic. There were 597 patients referred without an ultrasound, 81% (n = 483) were confirmed to have cryptorchidism at the time of specialist physical examination and underwent definitive surgical intervention, the remainder (19%, n = 114) were discharged from the outpatient clinics. CONCLUSIONS: Ultrasound evaluation of cryptorchidism continues despite high-quality evidence-based guidelines that recommend otherwise, as they should have little to no bearing on the surgeon's decision to operate or the type of operation. Instead, physical examination findings should guide surgical planning.

Artifact suppression and improved signal-to-noise ratio by phase-locked multiplexed coherent imaging.

Laser additive manufacturing (AM) promises direct metal 3D printing, but is held back by defects and process instabilities, giving rise to a need for in situ process monitoring. Inline coherent imaging (ICI) has proven effective for in situ, direct measurements of vapor depression depth and shape in AM and laser welding but struggles to track turbulent interfaces due to poor coupling back into a single-mode fiber and the presence of artifacts. By z-domain multiplexing, we achieve phase-sensitive image consolidation, automatically attenuating autocorrelation artifacts and improving interface tracking rates by 58% in signal-starved applications.

Recommendations for accelerating open preprint peer review to improve the culture of science.

Peer review is an important part of the scientific process, but traditional peer review at journals is coming under increased scrutiny for its inefficiency and lack of transparency. As preprints become more widely used and accepted, they raise the possibility of rethinking the peer-review process. Preprints are enabling new forms of peer review that have the potential to be more thorough, inclusive, and collegial than traditional journal peer review, and to thus fundamentally shift the culture of peer review toward constructive collaboration. In this Consensus View, we make a call to action to stakeholders in the community to accelerate the growing momentum of preprint sharing and provide recommendations to empower researchers to provide open and constructive peer review for preprints.

Structure is beauty, but not always truth.

Structural biology, as powerful as it is, can be misleading. We highlight four fundamental challenges: interpreting raw experimental data; accounting for motion; addressing the misleading nature of in vitro structures; and unraveling interactions between drugs and "anti-targets." Overcoming these challenges will amplify the impact of structural biology on drug discovery.

A national survey of current practice on patients with long term and additional needs in paediatric intensive care units.

BACKGROUND: Patients with long term and additional needs (LEAP) in paediatric intensive care units (PICUs) are a growing and heterogenous cohort that provide unique challenges to clinicians. Currently no standard approach to define and manage this cohort exists. AIM: To analyse bed occupancy, examine current practice, and explore ideas to improve PICU care of patients with long term and additional needs. STUDY DESIGN: Patients with LEAP were defined as meeting two or more of the following criteria: length of stay >14 days; life limiting condition; ≥2 failed extubations; hospital stay >1 month prior to PICU admission; likely to require long-term ventilation. An electronic survey was then sent to all UK PICUs, via the UK Paediatric Critical Care Society, to collect quantitative and qualitative data relating to bed occupancy, length of stay, multidisciplinary and family involvement, and areas of possible improvement. Data collection were occurred between 8 February 2022 and 14 March 2022. Quantitative data were analysed using Microsoft Excel 365 and SPSS Statistics version 28.0. Raw data and descriptive statistics were reported, including percentages and median with interquartile range for non-parametric data. Qualitative raw data were examined using thematic analysis. Analysis was undertaken independently by two authors and results assessed for concordance. RESULTS: 70.1% (17/24) PICUs responded. 25% (67/259) of PICU beds were occupied by patients with long term and additional needs. 29% (5/17) of responding units have tailored management plans to this cohort of patient. A further 11% (2/17) have guidelines for children with generic chronic illness. 12% (2/16) of responding units had a designated area and 81% (13/16) of responding units had designated professionals. The majority (68% and 62%) of responding units engaged families and community professionals in multidisciplinary meetings. When asked how the care of long term and additional needs patients might be improved five themes were identified: consistent, streamlined care pathways; designated transitional care units; designated funding and hospital-to-home commissioning; development of roles to facilitate collaboration between hospital and community teams; proactive discharge planning and parallel planning. CONCLUSIONS: This survey provides a snapshot of UK practice for a cohort of patients that occupies a considerable proportion (29%) of PICU beds. While only a minority of responding PICUs offer specifically tailored management plans, the majority of units have designated professionals. RELEVANCE TO CLINICAL PRACTICE: Opportunities exist to improve PICU care in LEAP patients in areas such as: streamlined care pathways, designated clinical areas, designated funding, and development of defined collaborative roles. Next steps may involve working group convention to develop a consensus definition and share good practice examples.

Uncovering Protein Ensembles: Automated Multiconformer Model Building for X-ray Crystallography and Cryo-EM.

In their folded state, biomolecules exchange between multiple conformational states that are crucial for their function. Traditional structural biology methods, such as X-ray crystallography and cryogenic electron microscopy (cryo-EM), produce density maps that are ensemble averages, reflecting molecules in various conformations. Yet, most models derived from these maps explicitly represent only a single conformation, overlooking the complexity of biomolecular structures. To accurately reflect the diversity of biomolecular forms, there is a pressing need to shift towards modeling structural ensembles that mirror the experimental data. However, the challenge of distinguishing signal from noise complicates manual efforts to create these models. In response, we introduce the latest enhancements to qFit, an automated computational strategy designed to incorporate protein conformational heterogeneity into models built into density maps. These algorithmic improvements in qFit are substantiated by superior Rfree and geometry metrics across a wide range of proteins. Importantly, unlike more complex multicopy ensemble models, the multiconformer models produced by qFit can be manually modified in most major model building software (e.g. Coot) and fit can be further improved by refinement using standard pipelines (e.g. Phenix, Refmac, Buster). By reducing the barrier of creating multiconformer models, qFit can foster the development of new hypotheses about the relationship between macromolecular conformational dynamics and function.

Structural characterization of ligand binding and pH-specific enzymatic activity of mouse Acidic Mammalian Chitinase.

Chitin is an abundant biopolymer and pathogen-associated molecular pattern that stimulates a host innate immune response. Mammals express chitin-binding and chitin-degrading proteins to remove chitin from the body. One of these proteins, Acidic Mammalian Chitinase (AMCase), is an enzyme known for its ability to function under acidic conditions in the stomach but is also active in tissues with more neutral pHs, such as the lung. Here, we used a combination of biochemical, structural, and computational modeling approaches to examine how the mouse homolog (mAMCase) can act in both acidic and neutral environments. We measured kinetic properties of mAMCase activity across a broad pH range, quantifying its unusual dual activity optima at pH 2 and 7. We also solved high resolution crystal structures of mAMCase in complex with oligomeric GlcNAcn, the building block of chitin, where we identified extensive conformational ligand heterogeneity. Leveraging these data, we conducted molecular dynamics simulations that suggest how a key catalytic residue could be protonated via distinct mechanisms in each of the two environmental pH ranges. These results integrate structural, biochemical, and computational approaches to deliver a more complete understanding of the catalytic mechanism governing mAMCase activity at different pH. Engineering proteins with tunable pH optima may provide new opportunities to develop improved enzyme variants, including AMCase, for therapeutic purposes in chitin degradation.

External second opinions: building trust between health professionals and families.

In medicine, external second opinions are frequently sought to inform decisions around a patient's proposed course of treatment. However, they are also sought in more challenging circumstances such as when disagreement arises between the healthcare team and the family, or during complex end-of-life discussions in critically ill children. When done well, external second opinions can help build trust and reduce conflict. However, when done poorly they may antagonise relationships and thwart attempts to bring about consensus. While principles of good medical practice should always be followed, the actual second opinion process itself remains essentially unregulated in all its forms. In this review, we set out what a standardised and transparent second opinion process should look like and recommend key recommendations for healthcare Trusts, Commissioners and professional bodies to support good practice.

Discovery and Clinical Proof-of-Concept of RLY-2608, a First-in-Class Mutant-Selective Allosteric PI3Kα Inhibitor That Decouples Antitumor Activity from Hyperinsulinemia.

PIK3CA (PI3Kα) is a lipid kinase commonly mutated in cancer, including ∼40% of hormone receptor-positive breast cancer. The most frequently observed mutants occur in the kinase and helical domains. Orthosteric PI3Kα inhibitors suffer from poor selectivity leading to undesirable side effects, most prominently hyperglycemia due to inhibition of wild-type (WT) PI3Kα. Here, we used molecular dynamics simulations and cryo-electron microscopy to identify an allosteric network that provides an explanation for how mutations favor PI3Kα activation. A DNA-encoded library screen leveraging electron microscopy-optimized constructs, differential enrichment, and an orthosteric-blocking compound led to the identification of RLY-2608, a first-in-class allosteric mutant-selective inhibitor of PI3Kα. RLY-2608 inhibited tumor growth in PIK3CA-mutant xenograft models with minimal impact on insulin, a marker of dysregulated glucose homeostasis. RLY-2608 elicited objective tumor responses in two patients diagnosed with advanced hormone receptor-positive breast cancer with kinase or helical domain PIK3CA mutations, with no observed WT PI3Kα-related toxicities. SIGNIFICANCE: Treatments for PIK3CA-mutant cancers are limited by toxicities associated with the inhibition of WT PI3Kα. Molecular dynamics, cryo-electron microscopy, and DNA-encoded libraries were used to develop RLY-2608, a first-in-class inhibitor that demonstrates mutant selectivity in patients. This marks the advance of clinical mutant-selective inhibition that overcomes limitations of orthosteric PI3Kα inhibitors. See related commentary by Gong and Vanhaesebroeck, p. 204 . See related article by Varkaris et al., p. 227 . This article is featured in Selected Articles from This Issue, p. 201.

Structure-activity relationships of bensulfuron methyl and its derivatives as novel agents against drug-resistant Candida auris.

With the emergence of the human pathogen Candida auris as a threat to human health, there is a strong demand to identify effective medicines to prevent the harm caused by such drug-tolerant human fungi. Herein, a series of 33 new derivatives of bensulfuron methyl (BSM) were synthesized and characterized by 1 H NMR, 13 C NMR, and HRMS. Among the target compounds, 8a possessed the best Ki value of 1.015 µM against C. auris acetohydroxyacid synthase (CauAHAS) and an MIC value of 6.25 µM against CBS10913, a clinically isolated strain of C. auris. Taken together the structures of BSM and the synthesized compounds, it was found that methoxy groups at both meta-position of pyrimidine ring are likely to provide desirable antifungal activities. Quantum calculations and molecular dockings were performed to understand the structure-activity relationships. The present study has hence provided some interesting clues for the discovery of novel antibiotics with this distinct mode of action.

Determining the Optimal Technique for Bar Fixation in the Repair of Pectus Excavatum.

Introduction: Pectus bar stabilizers are routinely used for bar fixation in the repair of pectus excavatum. We aimed to determine the optimum technique for bar fixation by reviewing our institutional experience with the use of bilateral, unilateral, and no stabilizer placement. Methods: Retrospective single pediatric center review of patients who underwent minimally invasive bar placement for pectus excavatum and subsequent bar removal between December 2001 and July 2019 was performed. Demographic data, details about the surgery, the number of bars and stabilizers used, and follow-up information were collected. Stabilizer-related complications included pain requiring stabilizer removal, surgical site infections (SSIs), and bar displacement. Data are presented as medians with interquartile ranges (IQRs) and frequencies with percentages. Results: A total of 561 patients were included. The cohort was predominantly male (83.1%, n = 466) with a median age at the time of bar placement of 15 years (IQR 12.4, 16.3) and a median Haller index of 3.8 (IQR 3.4, 4.5). Pain attributed to the stabilizer site that required removal was observed only in the bilateral stabilizer group (2.5%, n = 13). SSI related to the stabilizer site occurred in 1.8% (n = 9) of the bilateral stabilizer cases and 2.1% (n = 1) of the unilateral stabilizer cases. Bar displacement was observed in 0.6% (n = 3) of the bilateral stabilizer cases and 2 of those patients also had an SSI. There were no complications in the no stabilizer group. Conclusion: As the trend moves toward unilateral and no stabilizer use, we observe fewer cases of pain requiring stabilizer removal with no increase in bar displacements.

SAGA Complex Subunit Hfi1 Is Important in the Stress Response and Pathogenesis of Cryptococcus neoformans.

The Spt-Ada-Gcn Acetyltransferase (SAGA) complex is a highly conserved co-activator found across eukaryotes. It is composed of a number of modules which can vary between species, but all contain the core module. Hfi1 (known as TADA1 in Homo sapiens) is one of the proteins that forms the core module, and has been shown to play an important role in maintaining complex structural integrity in both brewer's yeast and humans. In this study we successfully identified the gene encoding this protein in the important fungal pathogen, Cryptococcus neoformans, and named it HFI1. The hfi1Δ mutant is highly pleiotropic in vitro, influencing phenotypes, ranging from temperature sensitivity and melanin production to caffeine resistance and titan cell morphogenesis. In the absence of Hfi1, the transcription of several other SAGA genes is impacted, as is the acetylation and deubiquination of several histone residues. Importantly, loss of the gene significantly impacts virulence in a murine inhalation model of cryptococcosis. In summary, we have established that Hfi1 modulates multiple pathways that directly affect virulence and survival in C. neoformans, and provided deeper insight into the importance of the non-enzymatic components of the SAGA complex.