Identifying the minimal sets of distance restraints for FRET-assisted protein structural modeling.

Proteins naturally occur in crowded cellular environments and interact with other proteins, nucleic acids, and organelles. Since most previous experimental protein structure determination techniques require that proteins occur in idealized, non-physiological environments, the effects of realistic cellular environments on protein structure are largely unexplored. Recently, Förster resonance energy transfer (FRET) has been shown to be an effective experimental method for investigating protein structure in vivo. Inter-residue distances measured in vivo can be incorporated as restraints in molecular dynamics (MD) simulations to model protein structural dynamics in vivo. Since most FRET studies only obtain inter-residue separations for a small number of amino acid pairs, it is important to determine the minimum number of restraints in the MD simulations that are required to achieve a given root-mean-square deviation (RMSD) from the experimental structural ensemble. Further, what is the optimal method for selecting these inter-residue restraints? Here, we implement several methods for selecting the most important FRET pairs and determine the number of pairs Nr that are needed to induce conformational changes in proteins between two experimentally determined structures. We find that enforcing only a small fraction of restraints, Nr/N≲0.08, where N is the number of amino acids, can induce the conformational changes. These results establish the efficacy of FRET-assisted MD simulations for atomic scale structural modeling of proteins in vivo. Significance: Determining protein structure in vivo is essential for understanding protein function. Most protein structures have been studied in non-physiological conditions using x-ray crystallography, NMR spectroscopy, and cryo-electron microscopy. Thus, we do not know whether the cellular environment significantly affects protein structure. We emphasize the benefits of FRET-assisted molecular dynamics simulations in characterizing protein structure in vivo at the atomic scale. We identify the minimum number of FRET pairs that can induce conformational changes in several proteins, including one that has been characterized using in-cell NMR.

Inferring B Cell Phylogenies from Paired H and L Chain BCR Sequences with Dowser.

Abs are vital to human immune responses and are composed of genetically variable H and L chains. These structures are initially expressed as BCRs. BCR diversity is shaped through somatic hypermutation and selection during immune responses. This evolutionary process produces B cell clones, cells that descend from a common ancestor but differ by mutations. Phylogenetic trees inferred from BCR sequences can reconstruct the history of mutations within a clone. Until recently, BCR sequencing technologies separated H and L chains, but advancements in single-cell sequencing now pair H and L chains from individual cells. However, it is unclear how these separate genes should be combined to infer B cell phylogenies. In this study, we investigated strategies for using paired H and L chain sequences to build phylogenetic trees. We found that incorporating L chains significantly improved tree accuracy and reproducibility across all methods tested. This improvement was greater than the difference between tree-building methods and persisted even when mixing bulk and single-cell sequencing data. However, we also found that many phylogenetic methods estimated significantly biased branch lengths when some L chains were missing, such as when mixing single-cell and bulk BCR data. This bias was eliminated using maximum likelihood methods with separate branch lengths for H and L chain gene partitions. Thus, we recommend using maximum likelihood methods with separate H and L chain partitions, especially when mixing data types. We implemented these methods in the R package Dowser: https://dowser.readthedocs.io.

Inferring B cell phylogenies from paired heavy and light chain BCR sequences with Dowser.

Antibodies are vital to human immune responses and are composed of genetically variable heavy and light chains. These structures are initially expressed as B cell receptors (BCRs). BCR diversity is shaped through somatic hypermutation and selection during immune responses. This evolutionary process produces B cell clones, cells that descend from a common ancestor but differ by mutations. Phylogenetic trees inferred from BCR sequences can reconstruct the history of mutations within a clone. Until recently, BCR sequencing technologies separated heavy and light chains, but advancements in single cell sequencing now pair heavy and light chains from individual cells. However, it is unclear how these separate genes should be combined to infer B cell phylogenies. In this study, we investigated strategies for using paired heavy and light chain sequences to build phylogenetic trees. We found incorporating light chains significantly improved tree accuracy and reproducibility across all methods tested. This improvement was greater than the difference between tree building methods and persisted even when mixing bulk and single cell sequencing data. However, we also found that many phylogenetic methods estimated significantly biased branch lengths when some light chains were missing, such as when mixing single cell and bulk BCR data. This bias was eliminated using maximum likelihood methods with separate branch lengths for heavy and light chain gene partitions. Thus, we recommend using maximum likelihood methods with separate heavy and light chain partitions, especially when mixing data types. We implemented these methods in the R package Dowser: https://dowser.readthedocs.io.

Spatially confined protein assembly in hierarchical mesoporous metal-organic framework.

Immobilization of biomolecules into porous materials could lead to significantly enhanced performance in terms of stability towards harsh reaction conditions and easier separation for their reuse. Metal-Organic Frameworks (MOFs), offering unique structural features, have emerged as a promising platform for immobilizing large biomolecules. Although many indirect methods have been used to investigate the immobilized biomolecules for diverse applications, understanding their spatial arrangement in the pores of MOFs is still preliminary due to the difficulties in directly monitoring their conformations. To gain insights into the spatial arrangement of biomolecules within the nanopores. We used in situ small-angle neutron scattering (SANS) to probe deuterated green fluorescent protein (d-GFP) entrapped in a mesoporous MOF. Our work revealed that GFP molecules are spatially arranged in adjacent nanosized cavities of MOF-919 to form "assembly" through adsorbate-adsorbate interactions across pore apertures. Our findings, therefore, lay a crucial foundation for the identification of proteins structural basics under confinement environment of MOFs.

DENSS-multiple: A structure reconstruction method using contrast variation of small-angle neutron scattering based on the DENSS algorithm.

The 3D structure of biomacromolecules, such as protein and DNA/RNA, provide keys to understanding their biological functions. Among many structural biology techniques, small-angle scattering techniques with ab initio methods have been widely used to reveal biomolecular structures in relevant solution conditions. Recently, a method called DENsity from Solution Scattering (DENSS) was developed to reconstruct the scattering density directly from biological small-angle X-ray and neutron scattering data instead of using a dummy atom modeling approach. Here, a method named DENSS-Multiple was developed to work simultaneously on multiple datasets from small-angle neutron scattering (SANS) contrast variation data. The easily manipulable neutron contrast has been widely exploited to study the structure and function of biological macromolecules and their complexes in solution. This new method provides a single structural result that includes all the information represented by different contrasts from SANS. The results from DENSS-Multiple generally have better resolution than those from DENSS, and more subtle features are represented by density variations from different phases of a structure. DENSS-Multiple was tested on various examples, including simulated and experimental data. These results, along with DENSS-Multiple's applications and limitations, are discussed herein.

Structure of Diisobutylene Maleic Acid Copolymer (DIBMA) and Its Lipid Particle as a "Stealth" Membrane-Mimetic for Membrane Protein Research.

The study of membrane proteins remains challenging, especially in a native membrane environment. Recently, major progress has been made using maleic acid copolymers, such as styrene maleic acid, to purify membrane proteins and study them directly with native lipids associated with the membrane. Additional maleic acid copolymers, such as diisobutylene maleic acid (DIBMA) membrane-mimetic systems, are being developed and found to have improved spectroscopic properties and pH stability. We studied DIBMA and its lipid particles in solution to better understand its assembly, without and with the lipids, to provide an insight regarding how to use it in solution for better membrane extraction. Using small-angle neutron and X-ray scattering (SANS/SAXS), we show that DIBMA organizes into structures of different size scales at various concentrations and ionic strengths. The polymer performed reasonably well under most solvent conditions except in very low concentrations and high-salt conditions that could result in limited interaction with lipids. To explore DIBMA lipid particles as a suitable membrane-mimetic system for neutron scattering studies of membrane proteins, we measured and determined the contrast-matching point of DIBMA to be ∼12% (v/v) D2O - similar to that of most protiated lipid molecules but distinct from that of regular protiated proteins - providing a natural contrast for separating their neutron scattering signals. Using SANS contrast variation, we demonstrated that the scattering from the whole lipid particle can be annihilated. Further, we determined that a well-defined lipid nanodisc structure with DIBMA was contrast-matched. These results demonstrate that the DIBMA lipid particle is an outstanding "stealth" membrane-mimetic for membrane proteins. The results provide a structural framework for understanding the organization and assembly process of the polymer itself and the lipid molecules. Such an understanding is imperative for structural techniques such as cryo-electron microscopy, nuclear magnetic resonance, small-angle scattering, and other biophysical techniques.

Machine Learning to Predict Lower Extremity Musculoskeletal Injury Risk in Student Athletes.

Injury rates in student athletes are high and often unpredictable. Injury risk factors are not agreed upon and often not validated. Here, we present a random-forest machine learning methodology for identifying the most significant injury risk factors and develop a model of lower extremity musculoskeletal injury risk in student athletes with physical performance metrics spanning joint strength measured with force transducers, postural stability measured using a force plate, and flexibility, measured with a goniometer, combined with previous injury metrics and athlete demographics. We tested our model in a population of 122 student athletes with performance metrics for the lower extremity musculoskeletal system and achieved an injury risk accuracy of 79% and identified significant injury risk factors, that could be used to increase accuracy of injury risk assessments, implement timely interventions, and decrease the number of career-ending or chronic injuries among student athletes.

Dual Antiplatelet Therapy Beyond One Year in Patients After Stent Placement: A Review.

PURPOSE: To review the evidence on the safety and efficacy of the continued use of dual antiplatelet therapy (DAPT) beyond 12 months after stent placement in patients following an acute coronary syndrome (ACS) event. SUMMARY: Recently, the American College of Cardiology (ACC) and the American Heart Association (AHA) released a focused update on the duration of DAPT in patients with coronary artery disease (CAD). The update makes new recommendations about the duration of DAPT in light of recently performed studies investigating this topic. In regard to patients after an ACS event, the update states it is reasonable to continue DAPT beyond 1 year if these patients are not at a high risk of bleeding and had no overt bleeding while on DAPT. Several trials have been released which aim to provide information about the correct duration of DAPT after an ACS event. CONCLUSION: Recent trials have shown a benefit of prolonged (beyond 12 months) DAPT in preventing recurrent cardiovascular (CV) events in patients, mostly in patients who have had a previous myocardial infarction (MI). These benefits must be weighed with the elevated risks of bleeding.