Human PLCG2 haploinsufficiency results in a novel natural killer cell immunodeficiency.

BACKGROUND: Although most individuals effectively control herpesvirus infections, some suffer from severe and/or recurrent infections. A subset of these patients possess defects in natural killer (NK) cells, lymphocytes that recognize and lyse herpesvirus-infected cells; however, the genetic etiology is rarely diagnosed. PLCG2 encodes a signaling protein in NK-cell and B-cell signaling. Dominant-negative or gain-of-function variants in PLCG2 cause cold urticaria, antibody deficiency, and autoinflammation. However, loss-of-function variants and haploinsufficiency have not been reported to date. OBJECTIVES: The investigators aimed to identify the genetic cause of NK-cell immunodeficiency in 2 families and herein describe the functional consequences of 2 novel loss-of-function variants in PLCG2. METHODS: The investigators employed whole-exome sequencing in conjunction with mass cytometry, microscopy, functional assays, and a mouse model of PLCG2 haploinsufficiency to investigate 2 families with NK-cell immunodeficiency. RESULTS: The investigators identified novel heterozygous variants in PLCG2 in 2 families with severe and/or recurrent herpesvirus infections. In vitro studies demonstrated that these variants were loss of function due to haploinsufficiency with impaired NK-cell calcium flux and cytotoxicity. In contrast to previous PLCG2 variants, B-cell function remained intact. Plcg2+/- mice also displayed impaired NK-cell function with preserved B-cell function, phenocopying human disease. CONCLUSIONS: PLCG2 haploinsufficiency represents a distinct syndrome from previous variants characterized by NK-cell immunodeficiency with herpesvirus susceptibility, expanding the spectrum of PLCG2-related disease.

Dramatic improvement in the "Bulk" hyperpolarization of 131Xe via spin exchange optical pumping probed using in situ low-field NMR.

We report on hyperpolarization of quadrupolar (I=3/2) 131Xe via spin-exchange optical pumping. Observations of the 131Xe polarization dynamics via in situ low-field NMR show that the estimated alkali-metal/131Xe spin-exchange rates can be large enough to compete with 131Xe spin relaxation. 131Xe polarization up to 7.6±1.5% was achieved in ∼8.5×1020 spins-a ∼100-fold improvement in the total spin angular momentum-potentially enabling various applications, including: measurement of spin-dependent neutron-131Xe s-wave scattering; sensitive searches for time-reversal violation in neutron-131Xe interactions beyond the Standard Model; and surface-sensitive pulmonary MRI.

Does the Doctor-Patient Relationship Affect Enrollment in Clinical Research?

PURPOSE: Recruiting patients for clinical research is challenging, especially for underrepresented populations, and may be influenced by patients' relationships with their physicians, care experiences, and engagement with care. This study sought to understand predictors of enrollment in a research study among socioeconomically diverse participants in studies of care models that promote continuity in the doctor-patient relationship. METHOD: A study of the effects of vitamin D levels and supplementation on COVID-19 risk and outcomes was implemented from 2020 to 2022 within 2 studies of care models at the University of Chicago that promoted continuity of inpatient and outpatient care from the same physician. Hypothesized predictors of vitamin D study enrollment included patient-reported measures of the care experience (quality of relationship with the doctor and their staff, timely receipt of care), engagement in care (scheduling and completing outpatient visits), and engagement with these "parent" studies (follow-up survey completion). The authors used univariate tests and multivariable logistic regression to examine the association of these predictors with enrollment in the vitamin D study among participants in the parent study intervention arms. RESULTS: Among 773 eligible participants, 351/561 (63%) in the parent study intervention arms enrolled in the vitamin D study, versus 35/212 (17%) in the control arms. Among intervention arm participants, vitamin D study enrollment was not associated with reported quality of communication with or trust in the doctor, or helpful/respectful office staff, but was associated with report of receiving timely care, more completed clinic visits, and higher parent study follow-up survey completion. CONCLUSIONS: Study enrollment may be high in care models with high levels of continuity in the doctor-patient relationship. Rates of clinic involvement, parent study engagement, and experience of receiving timely access to care may better predict enrollment than quality of the doctor-patient relationship.

Revisiting the Time Needed to Provide Adult Primary Care.

BACKGROUND: Many patients do not receive guideline-recommended preventive, chronic disease, and acute care. One potential explanation is insufficient time for primary care providers (PCPs) to provide care. OBJECTIVE: To quantify the time needed to provide 2020 preventive care, chronic disease care, and acute care for a nationally representative adult patient panel by a PCP alone, and by a PCP as part of a team-based care model. DESIGN: Simulation study applying preventive and chronic disease care guidelines to hypothetical patient panels. PARTICIPANTS: Hypothetical panels of 2500 patients, representative of the adult US population based on the 2017-2018 National Health and Nutrition Examination Survey. MAIN MEASURES: The mean time required for a PCP to provide guideline-recommended preventive, chronic disease and acute care to the hypothetical patient panels. Estimates were also calculated for visit documentation time and electronic inbox management time. Times were re-estimated in the setting of team-based care. KEY RESULTS: PCPs were estimated to require 26.7 h/day, comprising of 14.1 h/day for preventive care, 7.2 h/day for chronic disease care, 2.2 h/day for acute care, and 3.2 h/day for documentation and inbox management. With team-based care, PCPs were estimated to require 9.3 h per day (2.0 h/day for preventive care and 3.6 h/day for chronic disease care, 1.1 h/day for acute care, and 2.6 h/day for documentation and inbox management). CONCLUSIONS: PCPs do not have enough time to provide the guideline-recommended primary care. With team-based care the time requirements would decrease by over half, but still be excessive.

A cryptic pocket in Ebola VP35 allosterically controls RNA binding.

Protein-protein and protein-nucleic acid interactions are often considered difficult drug targets because the surfaces involved lack obvious druggable pockets. Cryptic pockets could present opportunities for targeting these interactions, but identifying and exploiting these pockets remains challenging. Here, we apply a general pipeline for identifying cryptic pockets to the interferon inhibitory domain (IID) of Ebola virus viral protein 35 (VP35). VP35 plays multiple essential roles in Ebola's replication cycle but lacks pockets that present obvious utility for drug design. Using adaptive sampling simulations and machine learning algorithms, we predict VP35 harbors a cryptic pocket that is allosterically coupled to a key dsRNA-binding interface. Thiol labeling experiments corroborate the predicted pocket and mutating the predicted allosteric network supports our model of allostery. Finally, covalent modifications that mimic drug binding allosterically disrupt dsRNA binding that is essential for immune evasion. Based on these results, we expect this pipeline will be applicable to other proteins.

Naturally Occurring Genetic Variants in the Oxytocin Receptor Alter Receptor Signaling Profiles.

The hormone oxytocin is commonly administered during childbirth to initiate and strengthen uterine contractions and prevent postpartum hemorrhage. However, patients have wide variation in the oxytocin dose required for a clinical response. To begin to uncover the mechanisms underlying this variability, we screened the 11 most prevalent missense genetic variants in the oxytocin receptor (OXTR) gene. We found that five variants, V45L, P108A, L206V, V281M, and E339K, significantly altered oxytocin-induced Ca2+ signaling or ß-arrestin recruitment and proceeded to assess the effects of these variants on OXTR trafficking to the cell membrane, desensitization, and internalization. The variants P108A and L206V increased OXTR localization to the cell membrane, whereas V281M and E339K caused OXTR to be retained inside the cell. We examined how the variants altered the balance between OXTR activation and desensitization, which is critical for appropriate oxytocin dosing. The E339K variant impaired OXTR activation, internalization, and desensitization to roughly equal extents. In contrast, V281M decreased OXTR activation but had no effect on internalization and desensitization. V45L and P108A did not alter OXTR activation but did impair ß-arrestin recruitment, internalization, and desensitization. Molecular dynamics simulations predicted that V45L and P108A prevent extension of the first intracellular loop of OXTR, thus inhibiting ß-arrestin binding. Overall, our data suggest mechanisms by which OXTR genetic variants could alter clinical response to oxytocin.

SARS-CoV-2 simulations go exascale to predict dramatic spike opening and cryptic pockets across the proteome.

SARS-CoV-2 has intricate mechanisms for initiating infection, immune evasion/suppression and replication that depend on the structure and dynamics of its constituent proteins. Many protein structures have been solved, but far less is known about their relevant conformational changes. To address this challenge, over a million citizen scientists banded together through the Folding@home distributed computing project to create the first exascale computer and simulate 0.1 seconds of the viral proteome. Our adaptive sampling simulations predict dramatic opening of the apo spike complex, far beyond that seen experimentally, explaining and predicting the existence of 'cryptic' epitopes. Different spike variants modulate the probabilities of open versus closed structures, balancing receptor binding and immune evasion. We also discover dramatic conformational changes across the proteome, which reveal over 50 'cryptic' pockets that expand targeting options for the design of antivirals. All data and models are freely available online, providing a quantitative structural atlas.

SARS-CoV-2 Simulations Go Exascale to Capture Spike Opening and Reveal Cryptic Pockets Across the Proteome.

SARS-CoV-2 has intricate mechanisms for initiating infection, immune evasion/suppression, and replication, which depend on the structure and dynamics of its constituent proteins. Many protein structures have been solved, but far less is known about their relevant conformational changes. To address this challenge, over a million citizen scientists banded together through the Folding@home distributed computing project to create the first exascale computer and simulate an unprecedented 0.1 seconds of the viral proteome. Our simulations capture dramatic opening of the apo Spike complex, far beyond that seen experimentally, which explains and successfully predicts the existence of 'cryptic' epitopes. Different Spike homologues modulate the probabilities of open versus closed structures, balancing receptor binding and immune evasion. We also observe dramatic conformational changes across the proteome, which reveal over 50 'cryptic' pockets that expand targeting options for the design of antivirals. All data and models are freely available online, providing a quantitative structural atlas.

Conformational distributions of isolated myosin motor domains encode their mechanochemical properties.

Myosin motor domains perform an extraordinary diversity of biological functions despite sharing a common mechanochemical cycle. Motors are adapted to their function, in part, by tuning the thermodynamics and kinetics of steps in this cycle. However, it remains unclear how sequence encodes these differences, since biochemically distinct motors often have nearly indistinguishable crystal structures. We hypothesized that sequences produce distinct biochemical phenotypes by modulating the relative probabilities of an ensemble of conformations primed for different functional roles. To test this hypothesis, we modeled the distribution of conformations for 12 myosin motor domains by building Markov state models (MSMs) from an unprecedented two milliseconds of all-atom, explicit-solvent molecular dynamics simulations. Comparing motors reveals shifts in the balance between nucleotide-favorable and nucleotide-unfavorable P-loop conformations that predict experimentally measured duty ratios and ADP release rates better than sequence or individual structures. This result demonstrates the power of an ensemble perspective for interrogating sequence-function relationships.

Antagonism between substitutions in ß-lactamase explains a path not taken in the evolution of bacterial drug resistance.

CTX-M ß-lactamases are widespread in Gram-negative bacterial pathogens and provide resistance to the cephalosporin cefotaxime but not to the related antibiotic ceftazidime. Nevertheless, variants have emerged that confer resistance to ceftazidime. Two natural mutations, causing P167S and D240G substitutions in the CTX-M enzyme, result in 10-fold increased hydrolysis of ceftazidime. Although the combination of these mutations would be predicted to increase ceftazidime hydrolysis further, the P167S/D240G combination has not been observed in a naturally occurring CTX-M variant. Here, using recombinantly expressed enzymes, minimum inhibitory concentration measurements, steady-state enzyme kinetics, and X-ray crystallography, we show that the P167S/D240G double mutant enzyme exhibits decreased ceftazidime hydrolysis, lower thermostability, and decreased protein expression levels compared with each of the single mutants, indicating negative epistasis. X-ray structures of mutant enzymes with covalently trapped ceftazidime suggested that a change of an active-site Ω-loop to an open conformation accommodates ceftazidime leading to enhanced catalysis. 10-µs molecular dynamics simulations further correlated Ω-loop opening with catalytic activity. We observed that the WT and P167S/D240G variant with acylated ceftazidime both favor a closed conformation not conducive for catalysis. In contrast, the single substitutions dramatically increased the probability of open conformations. We conclude that the antagonism is due to restricting the conformation of the Ω-loop. These results reveal the importance of conformational heterogeneity of active-site loops in controlling catalytic activity and directing evolutionary trajectories.

Cooperative Changes in Solvent Exposure Identify Cryptic Pockets, Switches, and Allosteric Coupling.

Proteins are dynamic molecules that undergo conformational changes to a broad spectrum of different excited states. Unfortunately, the small populations of these states make it difficult to determine their structures or functional implications. Computer simulations are an increasingly powerful means to identify and characterize functionally relevant excited states. However, this advance has uncovered a further challenge: it can be extremely difficult to identify the most salient features of large simulation data sets. We reasoned that many functionally relevant conformational changes are likely to involve large, cooperative changes to the surfaces that are available to interact with potential binding partners. To examine this hypothesis, we introduce a method that returns a prioritized list of potentially functional conformational changes by segmenting protein structures into clusters of residues that undergo cooperative changes in their solvent exposure, along with the hierarchy of interactions between these groups. We term these groups exposons to distinguish them from other types of clusters that arise in this analysis and others. We demonstrate, using three different model systems, that this method identifies experimentally validated and functionally relevant conformational changes, including conformational switches, allosteric coupling, and cryptic pockets. Our results suggest that key functional sites are hubs in the network of exposons. As a further test of the predictive power of this approach, we apply it to discover cryptic allosteric sites in two different ß-lactamase enzymes that are widespread sources of antibiotic resistance. Experimental tests confirm our predictions for both systems. Importantly, we provide the first evidence, to our knowledge, for a cryptic allosteric site in CTX-M-9 ß-lactamase. Experimentally testing this prediction did not require any mutations and revealed that this site exerts the most potent allosteric control over activity of any pockets found in ß-lactamases to date. Discovery of a similar pocket that was previously overlooked in the well-studied TEM-1 ß-lactamase demonstrates the utility of exposons.

Choice of Adaptive Sampling Strategy Impacts State Discovery, Transition Probabilities, and the Apparent Mechanism of Conformational Changes.

Interest in atomically detailed simulations has grown significantly with recent advances in computational hardware and Markov state modeling (MSM) methods, yet outstanding questions remain that hinder their widespread adoption. Namely, how do alternative sampling strategies explore conformational space and how might this influence predictions generated from the data? Here, we seek to answer these questions for four commonly used sampling methods: (1) a single long simulation, (2) many short simulations run in parallel, (3) adaptive sampling, and (4) our recently developed goal-oriented sampling algorithm, FAST. We first develop a theoretical framework for analytically calculating the probability of discovering select states on simple landscapes, where we uncover the drastic effects of varying the number and length of simulations. We then use kinetic Monte Carlo simulations on a variety of physically inspired landscapes to characterize the probability of discovering particular states and transition pathways for each of the four methods. Consistently, we find that FAST simulations discover each target state with the highest probability, while traversing realistic pathways. Furthermore, we uncover the potential pathology that short parallel simulations sometimes predict an incorrect transition pathway by crossing large energy barriers that long simulations would typically circumnavigate. We refer to this pathology as "pathway tunneling". To protect against this phenomenon when using adaptive-sampling and FAST simulations, we introduce the FAST-string method. This method enhances sampling along the highest-flux transition paths to refine an MSMs transition probabilities and discriminate between competing pathways. Additionally, we compare the performance of a variety of MSM estimators in describing accurate thermodynamics and kinetics. For adaptive sampling, we recommend simply normalizing the transition counts out of each state after adding small pseudocounts to avoid creating sources or sinks. Lastly, we evaluate whether our insights from simple landscapes hold for all-atom molecular dynamics simulations of the folding of the λ-repressor protein. Remarkably, we find that FAST-contacts predicts the same folding pathway as a set of long simulations but with orders of magnitude less simulation time.

Selection and Clonal Propagation of High Artemisinin Genotypes of Artemisia annua.

Artemisinin, produced in the glandular trichomes of Artemisia annua L. is a vital antimalarial drug effective against Plasmodium falciparum resistant to quinine-derived medicines. Although work has progressed on the semi-synthetic production of artemisinin, field production of A. annua remains the principal commercial source of the compound. Crop production of artemisia must be increased to meet the growing worldwide demand for artemisinin combination therapies (ACTs) to treat malaria. Grower artemisinin yields rely on plants generated from seeds from open-pollinated parents. Although selection has considerably increased plant artemisinin concentration in the past 15 years, seed-generated plants have highly variable artemisinin content that lowers artemisinin yield per hectare. Breeding efforts to produce improved F1 hybrids have been hampered by the inability to produce inbred lines due to self-incompatibility. An approach combining conventional hybridization and selection with clonal propagation of superior genotypes is proposed as a means to enhance crop yield and artemisinin production. Typical seed-propagated artemisia plants produce less than 1% (dry weight) artemisinin with yields below 25 kg/ha. Genotypes were identified producing high artemisinin levels of over 2% and possessing improved agronomic characteristics such as high leaf area and shoot biomass production. Field studies of clonally-propagated high-artemisinin plants verified enhanced plant uniformity and an estimated gross primary productivity of up to 70 kg/ha artemisinin, with a crop density of one plant m-2. Tissue culture and cutting protocols for the mass clonal propagation of A. annua were developed for shoot regeneration, rooting, acclimatization, and field cultivation. Proof of concept studies showed that both tissue culture-regenerated plants and rooted cutting performed better than plants derived from seed in terms of uniformity, yield, and consistently high artemisinin content. Use of this technology to produce plants with homogeneously-high artemisinin can help farmers markedly increase the artemisinin yield per cultivated area. This would lead to increased profit to farmers and decreased prices of ACT.

Electron Cryo-microscopy Structure of Ebola Virus Nucleoprotein Reveals a Mechanism for Nucleocapsid-like Assembly.

Ebola virus nucleoprotein (eNP) assembles into higher-ordered structures that form the viral nucleocapsid (NC) and serve as the scaffold for viral RNA synthesis. However, molecular insights into the NC assembly process are lacking. Using a hybrid approach, we characterized the NC-like assembly of eNP, identified novel regulatory elements, and described how these elements impact function. We generated a three-dimensional structure of the eNP NC-like assembly at 5.8 Å using electron cryo-microscopy and identified a new regulatory role for eNP helices α22-α23. Biochemical, biophysical, and mutational analyses revealed that inter-eNP contacts within α22-α23 are critical for viral NC assembly and regulate viral RNA synthesis. These observations suggest that the N terminus and α22-α23 of eNP function as context-dependent regulatory modules (CDRMs). Our current study provides a framework for a structural mechanism for NC-like assembly and a new therapeutic target.

Emission of volatile organic compounds from petunia flowers is facilitated by an ABC transporter.

Plants synthesize a diversity of volatile molecules that are important for reproduction and defense, serve as practical products for humans, and influence atmospheric chemistry and climate. Despite progress in deciphering plant volatile biosynthesis, their release from the cell has been poorly understood. The default assumption has been that volatiles passively diffuse out of cells. By characterization of a Petunia hybrida adenosine triphosphate-binding cassette (ABC) transporter, PhABCG1, we demonstrate that passage of volatiles across the plasma membrane relies on active transport. PhABCG1 down-regulation by RNA interference results in decreased emission of volatiles, which accumulate to toxic levels in the plasma membrane. This study provides direct proof of a biologically mediated mechanism of volatile emission.

A Framework to Simplify Combined Sampling Strategies in Rosetta.

A core task in computational structural biology is the search of conformational space for low energy configurations of a biological macromolecule. Because conformational space has a very high dimensionality, the most successful search methods integrate some form of prior knowledge into a general sampling algorithm to reduce the effective dimensionality. However, integrating multiple types of constraints can be challenging. To streamline the incorporation of diverse constraints, we developed the Broker: an extension of the Rosetta macromolecular modeling suite that can express a wide range of protocols using constraints by combining small, independent modules, each of which implements a different set of constraints. We demonstrate expressiveness of the Broker through several code vignettes. The framework enables rapid protocol development in both biomolecular design and structural modeling tasks and thus is an important step towards exposing the rich functionality of Rosetta's core libraries to a growing community of users addressing a diverse set of tasks in computational biology.

Blockade of CD40 ligand for intercellular communication reduces hypertension, placental oxidative stress, and AT1-AA in response to adoptive transfer of CD4+ T lymphocytes from RUPP rats.

Preeclampsia (PE) is associated with altered immune activation during pregnancy. We have previously shown that adoptive transfer of CD4(+) T cells from the reduced uterine perfusion pressure (RUPP) rat model of PE increases blood pressure, oxidative stress (ROS), and inflammation in normal pregnant recipient rats. The objective of this study was to determine if blockade of communication via the CD40-CD40 ligand (CD40L) interaction between placental ischemia-induced CD4(+) T cells with endogenous normal pregnant (NP) cells would improve pathophysiology that was previously observed in NP recipient rats of RUPP CD4(+) T cells. Splenic CD4(+) T lymphocytes were magnetically separated, incubated with 2.5 µg/ml anti-CD40 ligand (αCD40L) overnight, and transferred into NP rats on day 12 of gestation (NP+RUPP CD4(+) T+anti-CD40L). On day 19 of gestation, blood pressure (MAP), blood, and tissues were collected. MAP was 99 ± 2 in NP (n = 13), 116 ± 4 in NP+RUPP CD4(+) T cells (n = 7; P < 0.01); MAP only increased to 104 ± 2 in NP+RUPP CD4(+) T cells+CD40L (n = 24) (P < 0.05 vs. NP+RUPP CD4(+) T cells). Mechanisms of hypertension in response to RUPP CD4(+) T cells include endothelin-1 (ET-1), ROS, and angiotensin II type I receptor (AT1-AA) were analyzed. Inhibition of CD40L binding reduced placental ET-1 to 2.3-fold above NP rats and normalized placental ROS from 318.6 ± 89 in NP+RUPP CD4(+) T cells (P < 0.05) to 118.7 ± 24 in NP+RUPP CD4(+) T+anti-CD40L (P < 0.05). AT1-AA was also normalized with inhibition of CD40L. These data suggest that placental ischemia-induced T-cell communication via the CD40L is one important mechanism leading to much of the pathophysiology of PE.

Simplified technique for injection of Botulinum Toxin to Obturator Internus muscle using ultrasound-guided nerve stimulation for persistent pelvic pain.

Botulinum toxin (BoNT) injections have been used to reduce muscle spasm in the presence of severe pelvic pain. However, while pubococcygeus is easily accessed vaginally, injection to obturator internus is more complex - with variation in operative technique and needle placement confounding the ability to assess outcomes. We describe a simplified technique for BoNT injection to obturator internus using neurostimulation under ultrasound guidance.

Access to Cα backbone dynamics of biological solids by 13C T1 relaxation and molecular dynamics simulation.

We introduce a labeling scheme for magic angle spinning (MAS) solid-state NMR that is based on deuteration in combination with dilution of the carbon spin system. The labeling strategy achieves spectral editing by simplification of the HαCα and aliphatic side chain spectral region. A reduction in both proton and carbon spin density in combination with fast spinning (≥50 kHz) is essential to retrieve artifact-free (13)C-R1 relaxation data for aliphatic carbons. We obtain good agreement between the NMR experimental data and order parameters extracted from a molecular dynamics (MD) trajectory, which indicates that carbon based relaxation parameters can yield complementary information on protein backbone as well as side chain dynamics.

Enodthelin 1 is elevated in plasma and explants from patients having uterine leiomyomas.

OBJECTIVE: To determine a role for endothelin (ET) in progression of uterine fibroids. DESIGN: An in vitro model of fibroid and myometrium cultivation. PATIENTS: A total of 32 women undergoing hysterectomies for uterine fibroids and 11 women undergoing hysterectomies for abnormal uterine bleeding (control population). RESULTS: Women with uterine fibroids were hypertensive and displayed significantly greater circulating ET-1 compared to control patients. Secretion of ET-1 was greater from the fibroids compared to myometrium explants. Endothelin 1 secretion was attenuated with blockade of the angiotensin II type 1 or endothelinA receptors. Hypoxia stimulated ET-1 secretion from both myometrium and fibroid explants. Preproendothelin messenger RNA expression increased with hypoxia from fibroid explants compared to normoxic controls. CONCLUSIONS: These data support the hypothesis that uterine fibroids are associated with hypertension and increased ET-1, which is exacerbated with hypoxia. These data suggest a possible link between mechanisms of blood pressure regulation and development of uterine leiomyoma.