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There has been considerable progress in building models to predict the effect of missense substitutions in protein-coding genes, fueled in large part by progress in applying deep learning methods to sequence data. These models have the potential to enable clinical variant annotation on a large scale and hence increase the impact of patient sequencing in guiding diagnosis and treatment. To realize this potential, it is essential to provide reliable assessments of model performance, scope of applicability, and robustness. As a response to this need, the ClinGen Sequence Variant Interpretation Working Group, Pejaver et al., recently proposed a strategy for validation and calibration of in-silico predictions in the context of guidelines for variant annotation. While this work marks an important step forward, the strategy presented still has important limitations. We propose core principles and recommendations to overcome these limitations that can enable both more reliable and more impactful use of variant effect prediction models in the future., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024. Published by Elsevier Inc.)

When nature evolves a gene over eons at scale, it produces a diversity of homologous sequences with patterns of conservation and change that contain rich structural, functional, and historical information about the gene. However, natural gene diversity accumulates slowly and likely excludes large regions of functional sequence space, limiting the information that is encoded and extractable. We introduce upgraded orthogonal DNA replication (OrthoRep) systems that radically accelerate the evolution of chosen genes under selection in yeast. When applied to a maladapted biosynthetic enzyme, we obtained collections of extensively diverged sequences with patterns that revealed structural and environmental constraints shaping the enzyme's activity. Our upgraded OrthoRep systems should support the discovery of factors influencing gene evolution, uncover previously unknown regions of fitness landscapes, and find broad applications in biomolecular engineering.

Purpose: Children undergoing either posterior spinal fusion (PSF) or index insertion of growing rods for neuromuscular or genetic/syndromic scoliosis may require post-operative care on the paediatric intensive care unit (PICU). Demands on this limited resource result in frequent bed shortage related cancellations. In response, an ad-hoc or 'pop-up' ward-based high-dependency unit (POPUP-HDU) was developed. This converts a ward bed to POP-HDU bed for the required time. This study assesses the safety and efficacy of postoperative management that utilises POPUP-HDU as an alternative to a PICU bed., Methods: Retrospective review of 111 consecutive children undergoing posterior surgery for scoliosis between June 2016 and April 2023. The inclusion criteria included a diagnosis of genetic/syndromic or neuromuscular scoliosis; PSF or primary insertion of distraction-based growth rods and requirement for postoperative care in a PICU. We excluded those children that were mandated to go to PICU post-operatively for any reason by the anaesthetic team., Results: 49 patients (mean age 13.0 years) were managed on PICU, and 62 (mean age 11.4 years) on POPUP-HDU. The groups were matched with respect to body weight, curve magnitude, operative duration, type of fusion procedure performed, the presence of cardiac malformations, the use of home breathing support, the number of operated levels, pelvic instrumentation and intraoperative blood loss. 8 patients in the PICU, and 16 in the POP-HDU groups were readmitted back to PICU following step-down to the hospital ward (p = 0.27). The median PICU length of stay was 1 day in the PICU group and less than a day in POPUP-HDU (for those that needed to be subsequently admitted to PICU). The median total length of hospital stay was 10 days in the PICU group, and 8 days in POPUP-HDU (p < 0.05). 14 patients developed medical complications in the PICU group, compared to 19 in POPUP-HDU. There were no bedshortage cancellations in POPUP-HDU, compared to 23 in PICU., Conclusions: For children with neuromuscular, genetic or syndromic scoliosis undergoing PSF or growth rods that are not deemed suitable for immediate ward-level post-operative care, POPUP-HDU provided a safe alternative to PICU for appropriate patients and was associated with shorter hospital stay and fewer cancellations for lack of PICU beds., Level of Evidence: Therapeutic Level III., (© 2024. The Author(s), under exclusive licence to Scoliosis Research Society.)

Quantitatively mapping enzyme sequence-catalysis landscapes remains a critical challenge in understanding enzyme function, evolution, and design. Here, we expand an emerging microfluidic platform to measure catalytic constants- k cat and K M -for hundreds of diverse naturally occurring sequences and mutants of the model enzyme Adenylate Kinase (ADK). This enables us to dissect the sequence-catalysis landscape's topology, navigability, and mechanistic underpinnings, revealing distinct catalytic peaks organized by structural motifs. These results challenge long-standing hypotheses in enzyme adaptation, demonstrating that thermophilic enzymes are not slower than their mesophilic counterparts. Combining the rich representations of protein sequences provided by deep-learning models with our custom high-throughput kinetic data yields semi-supervised models that significantly outperform existing models at predicting catalytic parameters of naturally occurring ADK sequences. Our work demonstrates a promising strategy for dissecting sequence-catalysis landscapes across enzymatic evolution and building family-specific models capable of accurately predicting catalytic constants, opening new avenues for enzyme engineering and functional prediction.

Introduction: Variants of uncertain significance (VUS) surged with affordable genetic testing, posing challenges for determining pathogenicity. We examine the pathogenicity of a novel VUS P93S in Annexin A11 (ANXA11) - an amyotrophic lateral sclerosis/frontotemporal dementia-associated gene - in a corticobasal syndrome kindred. Established ANXA11 mutations cause ANXA11 aggregation, altered lysosomal-RNA granule co-trafficking, and transactive response DNA binding protein of 43 kDa (TDP-43) mis-localization., Methods: We described the clinical presentation and explored the phenotypic diversity of ANXA11 variants. P93S's effect on ANXA11 function and TDP-43 biology was characterized in induced pluripotent stem cell-derived neurons alongside multiomic neuronal and microglial profiling., Results: ANXA11 mutations were linked to corticobasal syndrome cases. P93S led to decreased lysosome colocalization, neuritic RNA, and nuclear TDP-43 with cryptic exon expression. Multiomic microglial signatures implicated immune dysregulation and interferon signaling pathways., Discussion: This study establishes ANXA11 P93S pathogenicity, broadens the phenotypic spectrum of ANXA11 mutations, underscores neuronal and microglial dysfunction in ANXA11 pathophysiology, and demonstrates the potential of cellular models to determine variant pathogenicity., Highlights: ANXA11 P93S is a pathogenic variant. Corticobasal syndrome is part of the ANXA11 phenotypic spectrum. Hybridization chain reaction fluorescence in situ hybridization (HCR FISH) is a new tool for the detection of cryptic exons due to TDP-43-related loss of splicing regulation. Microglial ANXA11 and related immune pathways are important drivers of disease. Cellular models are powerful tools for adjudicating variants of uncertain significance., (© 2024 The Author(s). Alzheimer's & Dementia published by Wiley Periodicals LLC on behalf of Alzheimer's Association. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

A major challenge in protein design is to augment existing functional proteins with multiple property enhancements. Altering several properties likely necessitates numerous primary sequence changes, and novel methods are needed to accurately predict combinations of mutations that maintain or enhance function. Models of sequence co-variation (e.g., EVcouplings), which leverage extensive information about various protein properties and activities from homologous protein sequences, have proven effective for many applications including structure determination and mutation effect prediction. We apply EVcouplings to computationally design variants of the model protein TEM-1 β-lactamase. Nearly all the 14 experimentally characterized designs were functional, including one with 84 mutations from the nearest natural homolog. The designs also had large increases in thermostability, increased activity on multiple substrates, and nearly identical structure to the wild type enzyme. This study highlights the efficacy of evolutionary models in guiding large sequence alterations to generate functional diversity for protein design applications., (© 2024. The Author(s).)

Hepatitis B virus (HBV) is a small double-stranded DNA virus that chronically infects 296 million people. Over half of its compact genome encodes proteins in two overlapping reading frames, and during evolution, multiple selective pressures can act on shared nucleotides. This study combines an RNA-based HBV cell culture system with deep mutational scanning (DMS) to uncouple cis- and trans-acting sequence requirements in the HBV genome. The results support a leaky ribosome scanning model for polymerase translation, provide a fitness map of the HBV polymerase at single-nucleotide resolution, and identify conserved prolines adjacent to the HBV polymerase termination codon that stall ribosomes. Further experiments indicated that stalled ribosomes tether the nascent polymerase to its template RNA, ensuring cis-preferential RNA packaging and reverse transcription of the HBV genome., Competing Interests: Declaration of interests Y.Y., W.M.S., and C.M.R. filed a patent application, US 62/741,032, with Rockefeller University on September 19, 2019, entitled “RNA-Based Methods to Launch Hepatitis B Virus Infection.” Patent pending. C.M.R. is a shareholder and member of the scientific advisory board at VIR Biotechnology., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The orphan G protein-coupled receptor (GPCR) GPR161 plays a central role in development by suppressing Hedgehog signaling. The fundamental basis of how GPR161 is activated remains unclear. Here, we determined a cryogenic-electron microscopy structure of active human GPR161 bound to heterotrimeric G s . This structure revealed an extracellular loop 2 that occupies the canonical GPCR orthosteric ligand pocket. Furthermore, a sterol that binds adjacent to transmembrane helices 6 and 7 stabilizes a GPR161 conformation required for G s coupling. Mutations that prevent sterol binding to GPR161 suppress G s -mediated signaling. These mutants retain the ability to suppress GLI2 transcription factor accumulation in primary cilia, a key function of ciliary GPR161. By contrast, a protein kinase A-binding site in the GPR161 C terminus is critical in suppressing GLI2 ciliary accumulation. Our work highlights how structural features of GPR161 interface with the Hedgehog pathway and sets a foundation to understand the role of GPR161 function in other signaling pathways., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Analysis across a growing number of single-cell perturbation datasets is hampered by poor data interoperability. To facilitate development and benchmarking of computational methods, we collect a set of 44 publicly available single-cell perturbation-response datasets with molecular readouts, including transcriptomics, proteomics and epigenomics. We apply uniform quality control pipelines and harmonize feature annotations. The resulting information resource, scPerturb, enables development and testing of computational methods, and facilitates comparison and integration across datasets. We describe energy statistics (E-statistics) for quantification of perturbation effects and significance testing, and demonstrate E-distance as a general distance measure between sets of single-cell expression profiles. We illustrate the application of E-statistics for quantifying similarity and efficacy of perturbations. The perturbation-response datasets and E-statistics computation software are publicly available at scperturb.org. This work provides an information resource for researchers working with single-cell perturbation data and recommendations for experimental design, including optimal cell counts and read depth., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Recent developments in protein design rely on large neural networks with up to 100s of millions of parameters, yet it is unclear which residue dependencies are critical for determining protein function. Here, we show that amino acid preferences at individual residues-without accounting for mutation interactions-explain much and sometimes virtually all of the combinatorial mutation effects across 8 datasets (R 2 ~ 78-98%). Hence, few observations (~100 times the number of mutated residues) enable accurate prediction of held-out variant effects (Pearson r > 0.80). We hypothesized that the local structural contexts around a residue could be sufficient to predict mutation preferences, and develop an unsupervised approach termed CoVES (Combinatorial Variant Effects from Structure). Our results suggest that CoVES outperforms not just model-free methods but also similarly to complex models for creating functional and diverse protein variants. CoVES offers an effective alternative to complicated models for identifying functional protein mutations., (© 2024. The Author(s).)