Deep generative models of LDLR protein structure to predict variant pathogenicity.

The complex structure and function of low density lipoprotein receptor (LDLR) makes classification of protein-coding missense variants challenging. Deep generative models, including Evolutionary model of Variant Effect (EVE), Evolutionary Scale Modeling (ESM), and AlphaFold 2 (AF2), have enabled significant progress in the prediction of protein structure and function. ESM and EVE directly estimate the likelihood of a variant sequence but are purely data-driven and challenging to interpret. AF2 predicts LDLR structures, but variant effects are explicitly modeled by estimating changes in stability. We tested the effectiveness of these models for predicting variant pathogenicity compared to established methods. AF2 produced two distinct conformations based on a novel hinge mechanism. Within ESM's hidden space, benign and pathogenic variants had different distributions. In EVE, these distributions were similar. EVE and ESM were comparable to Polyphen-2, SIFT, REVEL, and Primate AI for predicting binary classifications in ClinVar. However, they were more strongly correlated with experimental measures of LDL uptake. AF2 poorly performed in these tasks. Using the UK Biobank to compare association with clinical phenotypes, ESM and EVE were more strongly associated with serum LDL-C than Polyphen-2. ESM was able to identify variants with more extreme LDL-C levels than EVE and had a significantly stronger association with atherosclerotic cardiovascular disease. In conclusion, AF2 predicted LDLR structures do not accurately model variant pathogenicity. ESM and EVE are competitive with prior scoring methods for prediction based on binary classifications in ClinVar but are superior based on correlations with experimental assays and clinical phenotypes.

Sucralose hydrogels: Peering into the reactivity of sucralose versus sucrose under lipase catalyzed trans-esterification.

Sucralose differs from sucrose only by virtue of having three Cl groups instead of OH groups. Its intriguing features include being noncaloric, noncariogenic, ∼600 times sweeter than sucrose, stable at high temperatures/acidic pH's, and void of disagreeable aftertastes. These properties are attractive as food additive, one of which is as hydrogel obtainable via the technique of molecular gelation using a sucralose-derived low-molecular weight gelator (LMWG). Such hydrogels are highly responsive to external stimuli like temperature, because the LMWGs self-assemble via non-covalent interactions and could thus be utilized in applications like control-release. We found that sucralose to be unreactive under lipase biocatalysis, unlike sucrose. Hence, the aim of this work was (i) to use computational simulations to further understand sucralose's lack of enzymatic reactivity and (ii) to synthesize the sucralose-based amphiphiles using conventional chemical synthesis and systematically study their tendency towards hydrogelation. Sucrose and sucralose were docked with a high-resolution atomic structure of lipase B from Candida antarctica, modeling the esterification transition state with an active site serine. In extended molecular dynamics simulations, sucrose remained in the active site due to multiple sugar-protein hydrogen bonds. The oxygen-to-chlorine substitutions in sucralose disrupted this hydrogen bonding network. Consistent with observed lack of enzymatic conversion, in multiple simulations, sucralose would rapidly dissociate from the active site. The sucralose-based LMWGs were subsequently synthesized using base-catalyzed conventional chemical synthesis. Three of the sucralose-based amphiphiles (SL-5, SL-6 and SL-7) proved to be successful hydrogelators. The gelators also showed the ability to gel selected beverages. The LMWGs gelled quantities of water and beverage up to 71 and 55 times their weight, respectively, and remain thermally stable up to 144 °C.

Demographic Trends in Emergency Department Visits for Psychiatric Concerns During the COVID-19 Pandemic.

Objective: To describe changes in emergency department (ED) psychiatric visits during the pandemic in both rural and nonrural regions in the United States. Methods: This cohort study was performed across 22 EDs in the Midwest and Southern United States from January 1, 2019 to April 22, 2021. Prevalence of psychiatric visits before and after the COVID-19 pandemic, defined as starting on March 1, 2020, were compared. Psychiatric and nonpsychiatric visits were defined on the basis of primary clinician-assigned diagnosis. The primary end point was average daily visits normalized to the average daily visit count before the pandemic, labeled as relative mean daily visits (RMDVs). Results: Psychiatric visits decreased by 9% [RMDVs, 0.91; 95% confidence interval (CI), 0.89-0.93] during the pandemic period, whereas nonpsychiatric visits decreased by 17% (RMDVs, 0.83; 95% CI, 0.81-0.84). Black patients were the only demographic group with a significant increase in psychiatric visits during the pandemic (RMDVs, 1.12; 95% CI, 1.04-1.19). Periods of outbreaks of psychiatric emergencies were identified in most demographic groups, including among male and pediatric patients. However, the outbreaks detected among Black patients sustained the longest at 6 months. Unlike older adults who experienced outbreaks in the spring and fall of 2020, outbreaks among pediatric patients were detected later in 2021. Conclusion: In this multisite study, total ED visits declined during the pandemic; however, psychiatric visits declined less than nonpsychiatric visits. Black patients experienced a greater increase in psychiatric emergencies than other demographic groups. There is also a concern for increasing outbreaks of pediatric psychiatric visits as the pandemic progresses.

A Folding Insulator Defines Cryptic Domains in Tropomyosin.

Multidomain proteins are the product of evolutionary selection for diversity of function through concatenation and repurposing of existing modular units of structures. In structures of proteins with multiple domains, components are often globular units stitched together with flexible linkers. Multidomain proteins often fold as multiple distinct order-disorder transitions. However, the relationship between structure and folding is not always straightforward. Tropomyosin binds to actin in muscle and cytoskeletal filaments. The structure is that of a continuous ɑ-helix lacking domain boundaries, but unfolding shows distinct transitions suggesting at least three possible domains do exist. To explore how domains might occur in a continuous structure, we used Lifson-Roig helix-coil models with sequence domains of varying helical nucleation propensities. Of these models, ones with a central folding insulator, separating folding of N- and C-terminal domains, are most consistent with experimental folding studies. The positions of domain boundaries are identified by hydrogen-deuterium exchange mass spectrometry. The presence of structurally cryptic folding domains in tropomyosin could relate to its evolution and explain the uneven distribution of deleterious mutations that lead to various cardiomyopathies.

Characterization of Magnetic Resonance Thermal Imaging Signal Artifact During Magnetic Resonance Guided Laser-Induced Thermal Therapy.

BACKGROUND: Magnetic resonance-guided laser interstitial thermal therapy (MRgLITT) is a minimally invasive procedure that utilizes intraoperative magnetic resonance thermal imaging (MRTI) to generate a thermal damage estimate (TDE) of the ablative area. In select cases, the MRTI contains a signal artifact or defect that distorts the ablative region. No study has attempted to characterize this artifact. OBJECTIVE: To characterize MRTI signal the artifact in select cases to better understand its potential relevance and impact on the ablation procedure. METHODS: All ablations were performed using the Visualase magnetic resonance imaging-guided laser ablation system (Medtronic). Patients were included if the MRTI contained signal artifact that distorted the ablative region during the first thermal dose delivered. Ablation artifact was quantified using MATLAB version R2018a (Mathworks Inc, Natick, Massachusetts). RESULTS: A total of 116 patients undergoing MRgLITT for various surgical indications were examined. MRTI artifact was observed in 37.0% of cases overall. Incidence of artifact was greater at higher powers (P < .001) and with longer ablation times (P = .024), though artifact size did not correlate with laser power or ablation duration. CONCLUSION: MRTI signal artifact is common during LITT. Higher powers and longer ablation times result in greater incidence of ablation artifact, though artifact size is not correlated with power or duration. Future studies should aim to evaluate effects of artifact on postoperative imaging and, most notably, patient outcomes.

Comparative dynamics of tropomyosin in vertebrates and invertebrates.

Tropomyosin (Tpm) is an extended α-helical coiled-coil homodimer that regulates actinomyosin interactions in muscle. Molecular simulations of four Tpms, two from the vertebrate class Mammalia (rat and pig), and two from the invertebrate class Malacostraca (shrimp and lobster), showed that despite extensive sequence and structural homology across metazoans, dynamic behavior-particularly long-range structural fluctuations-were clearly distinct. Vertebrate Tpms were more flexible and sampled complex, multi-state conformational landscapes. Invertebrate Tpms were more rigid, sampling a highly constrained harmonic landscape. Filtering of trajectories by principle component analysis into essential subspaces showed significant overlap within but not between phyla. In vertebrate Tpms, hinge-regions decoupled long-range interhelical motions and suggested distinct domains. In contrast, crustacean Tpms did not exhibit long-range dynamic correlations-behaving more like a single rigid rod on the nanosecond time scale. These observations suggest there may be divergent mechanisms for Tpm binding to actin filaments, where conformational flexibility in mammalian Tpm allows a preorganized shape complementary to the filament surface, and where rigidity in the crustacean Tpm requires concerted bending and binding.

Structural and Dynamic Properties of Allergen and Non-Allergen Forms of Tropomyosin.

To what extent do structural and biophysical features of food allergen proteins distinguish them from other proteins in our diet? Invertebrate tropomyosins (Tpms) as a class are considered "pan-allergens," inducing food allergy to shellfish and respiratory allergy to dust mites. Vertebrate Tpms are not known to elicit allergy or cross-reactivity, despite their high structural similarity and sequence identity to invertebrate homologs. We expect allergens are sufficiently stable against gastrointestinal proteases to survive for immune sensitization in the intestines, and that proteolytic stability will correlate with thermodynamic stability. Thermal denaturation of shrimp Tpm shows that it is more stable than non-allergen vertebrate Tpm. Shrimp Tpm is also more resistant to digestion. Molecular dynamics uncover local dynamics that select epitopes and global differences in flexibility between shrimp and pig Tpm that discriminate allergens from non-allergens. Molecular determinants of allergenicity depend not only on sequence but on contributions of protein structure and dynamics.

A comparison of biophysical characterization techniques in predicting monoclonal antibody stability.

With the rapid growth of biopharmaceutical product development, knowledge of therapeutic protein stability has become increasingly important. We evaluated assays that measure solution-mediated interactions and key molecular characteristics of 9 formulated monoclonal antibody (mAb) therapeutics, to predict their stability behavior. Colloidal interactions, self-association propensity and conformational stability were measured using effective surface charge via zeta potential, diffusion interaction parameter (kD) and differential scanning calorimetry (DSC), respectively. The molecular features of all 9 mAbs were compared to their stability at accelerated (25°C and 40°C) and long-term storage conditions (2-8°C) as measured by size exclusion chromatography. At accelerated storage conditions, the majority of the mAbs in this study degraded via fragmentation rather than aggregation. Our results show that colloidal stability, self-association propensity and conformational characteristics (exposed tryptophan) provide reasonable prediction of accelerated stability, with limited predictive value at 2-8°C stability. While no correlations to stability behavior were observed with onset-of-melting temperatures or domain unfolding temperatures, by DSC, melting of the Fab domain with the CH2 domain suggests lower stability at stressed conditions. The relevance of identifying appropriate biophysical assays based on the primary degradation pathways is discussed.

Dissecting Electrostatic Contributions to Folding and Self-Assembly Using Designed Multicomponent Peptide Systems.

We investigate formation of nano- to microscale peptide fibers and sheets where assembly requires association of two distinct collagen mimetic peptides (CMPs). The multicomponent nature of these designs allows the decoupling of amino acid contributions to peptide folding versus higher-order assembly. While both arginine and lysine containing CMP sequences can favor triple-helix folding, only arginine promotes rapid supramolecular assembly in each of the three two-component systems examined. Unlike lysine, the polyvalent guanidyl group of arginine is capable of both intra- and intermolecular contacts, promoting assembly. This is consistent with the supramolecular diversity of CMP morphologies observed throughout the literature. It also connects CMP self-assembly with a broad range of biomolecular interaction phenomena, providing general principles for modeling and design.

Deletion of muscle GRP94 impairs both muscle and body growth by inhibiting local IGF production.

Insulin-like growth factors (IGFs) are critical for development and growth of skeletal muscles, but because several tissues produce IGFs, it is not clear which source is necessary or sufficient for muscle growth. Because it is critical for production of both IGF-I and IGF-II, we ablated glucose-regulated protein 94 (GRP94) in murine striated muscle to test the necessity of local IGFs for normal muscle growth. These mice exhibited smaller skeletal muscles with diminished IGF contents but with normal contractile function and no apparent endoplasmic reticulum stress response. This result shows that muscles rely on GRP94 primarily to support local production of IGFs, a pool that is necessary for normal muscle growth. In addition, body weights were ∼30% smaller than those of littermate controls, and circulating IGF-I also decreased significantly, yet glucose homeostasis was maintained with little disruption to the growth hormone pathway. The growth defect was complemented on administration of recombinant IGF-I. Thus, unlike liver production of IGF-I, muscle IGF-I is necessary not only locally but also globally for whole-body growth.