Neutron spin echo shows pHLIP is capable of retarding membrane thickness fluctuations.

Cell membranes are responsible for a range of biological processes that require interactions between lipids and proteins. While the effects of lipids on proteins are becoming better understood, our knowledge of how protein conformational changes influence membrane dynamics remains rudimentary. Here, we performed experiments and computer simulations to study the dynamic response of a lipid membrane to changes in the conformational state of pH-low insertion peptide (pHLIP), which transitions from a surface-associated (SA) state at neutral or basic pH to a transmembrane (TM) α-helix under acidic conditions. Our results show that TM-pHLIP significantly slows down membrane thickness fluctuations due to an increase in effective membrane viscosity. Our findings suggest a possible membrane regulatory mechanism, where the TM helix affects lipid chain conformations, and subsequently alters membrane fluctuations and viscosity.

Biomolecular condensates form spatially inhomogeneous network fluids.

The functions of biomolecular condensates are thought to be influenced by their material properties, and these will be determined by the internal organization of molecules within condensates. However, structural characterizations of condensates are challenging, and rarely reported. Here, we deploy a combination of small angle neutron scattering, fluorescence recovery after photobleaching, and coarse-grained molecular dynamics simulations to provide structural descriptions of model condensates that are formed by macromolecules from nucleolar granular components (GCs). We show that these minimal facsimiles of GCs form condensates that are network fluids featuring spatial inhomogeneities across different length scales that reflect the contributions of distinct protein and peptide domains. The network-like inhomogeneous organization is characterized by a coexistence of liquid- and gas-like macromolecular densities that engenders bimodality of internal molecular dynamics. These insights suggest that condensates formed by multivalent proteins share features with network fluids formed by systems such as patchy or hairy colloids.

An intervention to improve lumbar puncture rates for meningitis surveillance in children at four secondary health facilities in Malawi: A before/after analysis.

OBJECTIVES: A lumbar puncture (LP) procedure plays a key role in meningitis diagnosis. In Malawi and other sub-Saharan African countries, LP completion rates are sometimes poor, making meningitis surveillance challenging. Our objective was to measure LP rates following an intervention to improve these during a sentinel hospital meningitis surveillance exercise in Malawi. METHODS: We conducted a before/after intervention analysis among under-five children admitted to paediatric wards at four secondary health facilities in Malawi. We used local and World Health Organization (WHO) guidelines to determine indications for LP, as these are widely used in low- and middle-income countries (LMIC). The intervention comprised of refresher trainings for facility staff on LP indications and procedure, use of automated reminders to perform LP in real time in the wards, with an electronic data management system, and addition of surveillance-specific clinical officers to support existing health facility staff with performing LPs. Due to the low numbers in the before/after analysis, we also performed a during/after analysis to supplement the findings. RESULTS: A total of 13,375 under-five children were hospitalised over the 21 months window for this analysis. The LP rate was 10.4% (12/115) and 60.4% (32/53) in the before/after analysis, respectively, and 43.8% (441/1006) and 72.5% (424/599) in the supplemental during/after analysis, respectively. In our intervention-specific analysis among the three individual components, there were improvements in the LP rate by 48% (p < 0.001) following the introduction of surveillance-specific clinical officers, 10% (p < 0.001) following the introduction of automated reminders to perform an LP and 13% following refresher training. CONCLUSIONS: This analysis demonstrated a rise in LP rates following our intervention. This intervention package may be considered for planning future facility-based meningitis surveillances in similar low-resource settings.

Biomolecular condensates form spatially inhomogeneous network fluids.

The functions of biomolecular condensates are thought to be influenced by their material properties, and these will be determined by the internal organization of molecules within condensates. However, structural characterizations of condensates are challenging, and rarely reported. Here, we deploy a combination of small angle neutron scattering, fluorescence recovery after photobleaching, and coarse-grained molecular dynamics simulations to provide structural descriptions of model condensates that are formed by macromolecules from nucleolar granular components (GCs). We show that these minimal facsimiles of GCs form condensates that are network fluids featuring spatial inhomogeneities across different length scales that reflect the contributions of distinct protein and peptide domains. The network-like inhomogeneous organization is characterized by a coexistence of liquid- and gas-like macromolecular densities that engenders bimodality of internal molecular dynamics. These insights suggest that condensates formed by multivalent proteins share features with network fluids formed by systems such as patchy or hairy colloids.

Sex, Endothelial Cell Functions, and Peripheral Artery Disease.

Peripheral artery disease (PAD) is caused by blocked arteries due to atherosclerosis and/or thrombosis which reduce blood flow to the lower limbs. It results in major morbidity, including ischemic limb, claudication, and amputation, with patients also suffering a heightened risk of heart attack, stroke, and death. Recent studies suggest women have a higher prevalence of PAD than men, and with worse outcomes after intervention. In addition to a potential unconscious bias faced by women with PAD in the health system, with underdiagnosis, and lower rates of guideline-based therapy, fundamental biological differences between men and women may be important. In this review, we highlight sexual dimorphisms in endothelial cell functions and how they may impact PAD pathophysiology in women. Understanding sex-specific mechanisms in PAD is essential for the development of new therapies and personalized care for patients with PAD.

Biomolecular condensates form spatially inhomogeneous network fluids.

The functions of biomolecular condensates are thought to be influenced by their material properties, and these are in turn determined by the multiscale structural features within condensates. However, structural characterizations of condensates are challenging, and hence rarely reported. Here, we deploy a combination of small angle neutron scattering, fluorescence recovery after photobleaching, and bespoke coarse-grained molecular dynamics simulations to provide structural descriptions of model condensates that mimic nucleolar granular components (GCs). We show that facsimiles of GCs are network fluids featuring spatial inhomogeneities across hierarchies of length scales that reflect the contributions of distinct protein and peptide domains. The network-like inhomogeneous organization is characterized by a coexistence of liquid- and gas-like macromolecular densities that engenders bimodality of internal molecular dynamics. These insights, extracted from a combination of approaches, suggest that condensates formed by multivalent proteins share features with network fluids formed by associative systems such as patchy or hairy colloids.

Children With Bilateral Cerebral Palsy Exhibit Bimanual Asymmetric Motor Deficits and EEG Evidence of Dominant Sensorimotor Hemisphere Overreliance During Reaching.

BACKGROUND: Reaching is a fundamental motor skill often impaired in cerebral palsy (CP). Studies on manual function, intervention, and underlying brain mechanisms largely focus on unilateral CP. This first electroencephalography (EEG) evaluation of reaching exclusively in bilateral CP aims to quantify and relate brain activation patterns to bimanual deficits in this population. METHODS: A total of 15 children with bilateral CP (13.4 ± 2.9 years) and 13 with typical development (TD: 14.3 ± 2.4 years) performed 45 reaches per hand while recording motion capture and EEG data. The Box and Blocks test was administered bilaterally. Cortical sources were identified using independent component analysis and clustered using k-means. Alpha (8-12 Hz) and beta (13-30 Hz) band event-related desynchronization (ERD) values were compared across groups and hands within clusters, between dominant and non-dominant sensorimotor clusters, and related to reach kinematics and the Box and Block test. RESULTS: The group with CP demonstrated bimanual motor deficits with slower reaches, lower Box and Blocks scores, and stronger hand preference than in TD. Beta ERD, representing motor execution, was notably higher in the dominant sensorimotor cluster in CP compared to TD. Both groups demonstrated more contralateral than ipsilateral activity in both hands and clusters, with CP showing a less lateralized (more bilateral) alpha response. Higher brain activation was generally related to better function. CONCLUSION: Bimanual deficits in bilateral CP and related EEG differences warrant more clinical and research attention particularly earlier in life when greater potential for neural and functional recovery exists.

Structural characterization of an intrinsically disordered protein complex using integrated small-angle neutron scattering and computing.

Characterizing structural ensembles of intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) of proteins is essential for studying structure-function relationships. Due to the different neutron scattering lengths of hydrogen and deuterium, selective labeling and contrast matching in small-angle neutron scattering (SANS) becomes an effective tool to study dynamic structures of disordered systems. However, experimental timescales typically capture measurements averaged over multiple conformations, leaving complex SANS data for disentanglement. We hereby demonstrate an integrated method to elucidate the structural ensemble of a complex formed by two IDRs. We use data from both full contrast and contrast matching with residue-specific deuterium labeling SANS experiments, microsecond all-atom molecular dynamics (MD) simulations with four molecular mechanics force fields, and an autoencoder-based deep learning (DL) algorithm. From our combined approach, we show that selective deuteration provides additional information that helps characterize structural ensembles. We find that among the four force fields, a99SB-disp and CHARMM36m show the strongest agreement with SANS and NMR experiments. In addition, our DL algorithm not only complements conventional structural analysis methods but also successfully differentiates NMR and MD structures which are indistinguishable on the free energy surface. Lastly, we present an ensemble that describes experimental SANS and NMR data better than MD ensembles generated by one single force field and reveal three clusters of distinct conformations. Our results demonstrate a new integrated approach for characterizing structural ensembles of IDPs.

Prompt treatment-seeking behaviour varies within communities among guardians of children with malaria-related fever in Malawi.

BACKGROUND: In Malawi, malaria is responsible for 40% of hospital deaths. Prompt diagnosis and effective treatment within 24 h of fever onset is critical to prevent progression from uncomplicated to severe disease and to reduce transmission. METHODS: As part of the large evaluation of the malaria vaccine implementation programme (MVIP), this study analysed survey data to investigate whether prompt treatment-seeking behaviour is clustered at community-level according to socio-economic demographics. RESULTS: From 4563 households included in the survey, 4856 children aged 5-48 months were enrolled. Out of 4732 children with documented gender, 52.2% were female and 47.8% male. Among the 4856 children, 33.8% reported fever in the two weeks prior to the survey. Fever prevalence was high in communities with low socio-economic status (SES) (38.3% [95% CI: 33.7-43.5%]) and low in areas with high SES (29.8% [95% CI: 25.6-34.2%]). Among children with fever, 648 (39.5%) sought treatment promptly i.e., within 24 h from onset of fever symptoms. Children were more likely to be taken for prompt treatment among guardians with secondary education compared to those without formal education (aOR:1.37, 95% CI: 1.11-3.03); in communities with high compared to low SES [aOR: 2.78, 95% CI: 1.27-6.07]. Children were less likely to be taken for prompt treatment if were in communities far beyond 5 km to health facility than within 5 km [aOR: 0.44, 95% CI: 0.21-0.92]. CONCLUSION: The high heterogeneity in prevalence of fever and levels of prompt treatment-seeking behaviour underscore the need to promote community-level malaria control interventions (such as use of long-lasting insecticide-treated nets (LLINs), indoor residual spraying (IRS), intermittent preventive therapy (IPT), presumptive treatment and education). Programmes aimed at improving treatment-seeking behaviour should consider targeting communities with low SES and those far from health facility.

Investigation of brain mechanisms underlying upper limb function in bilateral cerebral palsy using EEG.

OBJECTIVE: Few studies focus on upper limbs in bilateral cerebral palsy (CP) despite potential bimanual deficits. Electroencephalography (EEG) was utilized to investigate brain mechanisms underlying upper limb tasks in bilateral CP and typical development (TD) and relationships to function. METHODS: 26 (14 CP; 12 TD) completed the Box and Blocks Test and transport task with paper, sponge or mixed blocks, while recording EEG and motion data. RESULTS: Group effects for path time, path length and Box and Blocks Test revealed bimanual deficits. Four sensorimotor-related EEG clusters were identified. Group effects were found in premotor and dominant motor clusters with greater beta event-related desynchronization (ERD) in CP. Hand and hand by group effects were found in the dominant motor cluster, showing greater ERD with the more affected hand in CP. Condition effects were prominent in the posterior parietal cluster with higher ERD reflecting greater difficulty in force modulation. CONCLUSIONS: Higher brain activation associated with greater bimanual deficits is similar to our lower limb findings but contrasts studies in TD or unilateral CP linking higher ERD to greater proficiency. SIGNIFICANCE: Bilateral CP shows overreliance on the dominant hemisphere with the less functional hand and higher brain activity presumably related to excessive intracortical connectivity.

Machine Learning-Driven Multiscale Modeling: Bridging the Scales with a Next-Generation Simulation Infrastructure.

Interdependence across time and length scales is common in biology, where atomic interactions can impact larger-scale phenomenon. Such dependence is especially true for a well-known cancer signaling pathway, where the membrane-bound RAS protein binds an effector protein called RAF. To capture the driving forces that bring RAS and RAF (represented as two domains, RBD and CRD) together on the plasma membrane, simulations with the ability to calculate atomic detail while having long time and large length- scales are needed. The Multiscale Machine-Learned Modeling Infrastructure (MuMMI) is able to resolve RAS/RAF protein-membrane interactions that identify specific lipid-protein fingerprints that enhance protein orientations viable for effector binding. MuMMI is a fully automated, ensemble-based multiscale approach connecting three resolution scales: (1) the coarsest scale is a continuum model able to simulate milliseconds of time for a 1 µm2 membrane, (2) the middle scale is a coarse-grained (CG) Martini bead model to explore protein-lipid interactions, and (3) the finest scale is an all-atom (AA) model capturing specific interactions between lipids and proteins. MuMMI dynamically couples adjacent scales in a pairwise manner using machine learning (ML). The dynamic coupling allows for better sampling of the refined scale from the adjacent coarse scale (forward) and on-the-fly feedback to improve the fidelity of the coarser scale from the adjacent refined scale (backward). MuMMI operates efficiently at any scale, from a few compute nodes to the largest supercomputers in the world, and is generalizable to simulate different systems. As computing resources continue to increase and multiscale methods continue to advance, fully automated multiscale simulations (like MuMMI) will be commonly used to address complex science questions.

Destabilization of Atherosclerotic Plaque by Bilirubin Deficiency.

BACKGROUND: The rupture of atherosclerotic plaque contributes significantly to cardiovascular disease. Plasma concentrations of bilirubin-a byproduct of heme catabolism-inversely associate with risk of cardiovascular disease, although the link between bilirubin and atherosclerosis remains unclear. METHODS: To assess the role of bilirubin in atherosclerotic plaque stability, we crossed Bvra-/- with Apoe-/- mice and used the tandem stenosis model of plaque instability. Human coronary arteries were obtained from heart transplant recipients. Analysis of bile pigments, heme metabolism, and proteomics were performed by liquid chromatography tandem mass spectrometry. MPO (myeloperoxidase) activity was determined by in vivo molecular magnetic resonance imaging, liquid chromatography tandem mass spectrometry analysis, and immunohistochemical determination of chlorotyrosine. Systemic oxidative stress was evaluated by plasma concentrations of lipid hydroperoxides and the redox status of circulating Prx2 (peroxiredoxin 2), whereas arterial function was assessed by wire myography. Atherosclerosis and arterial remodeling were quantified by morphometry and plaque stability by fibrous cap thickness, lipid accumulation, infiltration of inflammatory cells, and the presence of intraplaque hemorrhage. RESULTS: Compared with Bvra+/+Apoe-/- tandem stenosis littermates, Bvra-/-Apoe-/- tandem stenosis mice were deficient in bilirubin, showed signs of increased systemic oxidative stress, endothelial dysfunction, as well as hyperlipidemia, and had a higher atherosclerotic plaque burden. Heme metabolism was increased in unstable compared with stable plaque of both Bvra+/+Apoe-/- and Bvra-/-Apoe-/- tandem stenosis mice and in human coronary plaques. In mice, Bvra deletion selectively destabilized unstable plaque, characterized by positive arterial remodeling and increased cap thinning, intraplaque hemorrhage, infiltration of neutrophils, and MPO activity. Proteomic analysis confirmed Bvra deletion enhanced extracellular matrix degradation, recruitment and activation of neutrophils, and associated oxidative stress in unstable plaque. CONCLUSIONS: Bilirubin deficiency, resulting from global Bvra deletion, generates a proatherogenic phenotype and selectively enhances neutrophil-mediated inflammation and destabilization of unstable plaque, thereby providing a link between bilirubin and cardiovascular disease risk.

The Role of Oxidants in Percutaneous Coronary Intervention-Induced Endothelial Dysfunction: Can We Harness Redox Signaling to Improve Clinical Outcomes?

Significance: Coronary artery disease (CAD) is commonly treated using percutaneous coronary interventions (PCI). However, PCI with stent placement damages the endothelium, and failure to restore endothelial function may result in PCI failure with poor patient outcomes. Recent Advances: Oxidative signaling is central to maintaining endothelial function. Potentiation of oxidant production, as observed post-PCI, results in endothelial dysfunction (ED). This review delves into our current understanding of the physiological role that endothelial-derived oxidants play within the vasculature and the effects of altered redox signaling during dysfunction. We then examine the impact of PCI and intracoronary stent placement on oxidant production in the endothelium, which can culminate in stent failure. Finally, we explore how recent advances in PCI and stent technologies aim to mitigate PCI-induced oxidative damage and improve clinical outcomes. Critical Issues: Current PCI technologies exacerbate cellular oxidant levels, driving ED. If left uncontrolled, oxidative signaling leads to increased intravascular inflammation, restenosis, and neoatherosclerosis. Future Directions: Through the development of novel biomaterials and therapeutics, we can limit PCI-induced oxidant production, allowing for the restoration of a healthy endothelium and preventing CAD recurrence.

Training high level balance and stepping responses in atypical progressive supranuclear palsy: a case report.

BACKGROUND: Progressive supranuclear palsy (PSP) is a neurodegenerative condition, typically presenting with, but not limited to, impairments of postural instability, gait, and gaze stability. PURPOSE: This case report describes the multifactorial assessment and rehabilitation of a patient with atypical PSP who has significant gaze deficits, asymmetrical stepping responses, trunk rigidity, and reduced posterior excursion on limits of stability. CASE DESCRIPTION: Evaluation utilized computerized gait and balance assessments, foot clearance analysis, a squat test, and a timed stepping test. The intervention included boxing, stepping tasks, and treadmill training each with eye movement challenges. A total of 15 hours of physical therapy was provided; 1 hour, 2 times a week. OUTCOMES: Post-intervention improvements were noted subjectively, on eye-body coordination, and objectively, on limits of stability, foot clearance, and task performance (squats, timed stepping). Follow-up demonstrated some decline from posttest results; however, patient-reported adherence to the program was less than recommended. CONCLUSION: A multifactorial rehabilitation program can improve balance, eye-body coordination, and strength in a high functioning patient with atypical PSP. Longitudinal randomized controlled studies are suggested to further investigate this interventional approach in high functioning individuals diagnosed with atypical PSP.

Structural analysis and functional evaluation of the disordered ß-hexosyltransferase region from Hamamotoa (Sporobolomyces) singularis.

Hamamotoa (Sporobolomyces) singularis codes for an industrially important membrane bound ß-hexosyltransferase (BHT), (BglA, UniprotKB: Q564N5) that has applications in the production of natural fibers such as galacto-oligosaccharides (GOS) and natural sugars found in human milk. When heterologously expressed by Komagataella phaffii GS115, BHT is found both membrane bound and soluble secreted into the culture medium. In silico structural predictions and crystal structures support a glycosylated homodimeric enzyme and the presence of an intrinsically disordered region (IDR) with membrane binding potential within its novel N-terminal region (1-110 amino acids). Additional in silico analysis showed that the IDR may not be essential for stable homodimerization. Thus, we performed progressive deletion analyses targeting segments within the suspected disordered region, to determine the N-terminal disorder region's impact on the ratio of membrane-bound to secreted soluble enzyme and its contribution to enzyme activity. The ratio of the soluble secreted to membrane-bound enzyme shifted from 40% to 53% after the disordered N-terminal region was completely removed, while the specific activity was unaffected. Furthermore, functional analysis of each glycosylation site found within the C-terminal domain revealed reduced total secreted protein activity by 58%-97% in both the presence and absence of the IDR, indicating that glycosylation at all four locations is required by the host for the secretion of active enzyme and independent of the removed disordered N-terminal region. Overall, the data provides evidence that the disordered region only partially influences the secretion and membrane localization of BHT.

Competing risks modeling of length of hospital stay enhances risk-stratification of patient care: application to under-five children hospitalized in Malawi.

Introduction: Length of hospital stay (LOS), defined as the time from inpatient admission to discharge, death, referral, or abscondment, is one of the key indicators of quality in patient care. Reduced LOS lowers health care expenditure and minimizes the chance of in-hospital acquired infections. Conventional methods for estimating LOS such as the Kaplan-Meier survival curve and the Cox proportional hazards regression for time to discharge cannot account for competing risks such as death, referral, and abscondment. This study applied competing risk methods to investigate factors important for risk-stratifying patients based on LOS in order to enhance patient care. Methods: This study analyzed data from ongoing safety surveillance of the malaria vaccine implementation program in Malawi's four district hospitals of Balaka, Machinga, Mchinji, and Ntchisi. Children aged 1-59 months who were hospitalized (spending at least one night in hospital) with a medical illness were consecutively enrolled between 1 November 2019 and 31 July 2021. Sub-distribution-hazard (SDH) ratios for the cumulative incidence of discharge were estimated using the Fine-Gray competing risk model. Results: Among the 15,463 children hospitalized, 8,607 (55.7%) were male and 6,856 (44.3%) were female. The median age was 22 months [interquartile range (IQR): 12-33 months]. The cumulative incidence of discharge was 40% lower among HIV-positive children compared to HIV-negative (sub-distribution-hazard ratio [SDHR]: 0.60; [95% CI: 0.46-0.76]; P < 0.001); lower among children with severe and cerebral malaria [SDHR: 0.94; (95% CI: 0.86-0.97); P = 0.04], sepsis or septicemia [SDHR: 0.90; (95% CI: 0.82-0.98); P = 0.027], severe anemia related to malaria [SDHR: 0.54; (95% CI: 0.48-0.61); P < 0.001], and meningitis [SDHR: 0.18; (95% CI: 0.09-0.37); P < 0.001] when compared to non-severe malaria; and also 39% lower among malnourished children compared to those that were well-nourished [SDHR: 0.61; (95% CI: 0.55-0.68); P < 0.001]. Conclusions: This study applied the Fine-Gray competing risk approach to more accurately model LOS as the time to discharge when there were significant rates of in-hospital mortality, referrals, and abscondment. Patient care can be enhanced by risk-stratifying by LOS based on children's age, HIV status, diagnosis, and nutritional status.

Endothelial cell dysfunction: Implications for the pathogenesis of peripheral artery disease.

Peripheral artery disease (PAD) is caused by occluded or narrowed arteries that reduce blood flow to the lower limbs. The treatment focuses on lifestyle changes, management of modifiable risk factors and vascular surgery. In this review we focus on how Endothelial Cell (EC) dysfunction contributes to PAD pathophysiology and describe the largely untapped potential of correcting endothelial dysfunction. Moreover, we describe current treatments and clinical trials which improve EC dysfunction and offer insights into where future research efforts could be made. Endothelial dysfunction could represent a target for PAD therapy.

Functional and Structural Brain Connectivity in Children With Bilateral Cerebral Palsy Compared to Age-Related Controls and in Response to Intensive Rapid-Reciprocal Leg Training.

Background: Compared to unilateral cerebral palsy (CP), less is known about brain reorganization and plasticity in bilateral CP especially in relation or response to motor training. The few trials that reported brain imaging results alongside functional outcomes include a handful of studies in unilateral CP, and one pilot trial of three children with bilateral CP. This study is the first locomotor training randomized controlled trial (RCT) in bilateral CP to our knowledge reporting brain imaging outcomes. Methods: Objective was to compare MRI brain volumes, resting state connectivity and white matter integrity using DTI in children with bilateral CP with PVL and preterm birth history (<34 weeks), to age-related controls, and from an RCT of intensive 12 week rapid-reciprocal locomotor training using an elliptical or motor-assisted cycle. We hypothesized that connectivity in CP compared to controls would be greater across sensorimotor-related brain regions and that functional (resting state) and structural (fractional anisotropy) connectivity would improve post intervention. We further anticipated that baseline and post-intervention imaging and functional measures would correlate. Results: Images were acquired with a 3T MRI scanner for 16/27 children with CP in the trial, and 18 controls. No conclusive evidence of training-induced neuroplastic effects were seen. However, analysis of shared variance revealed that greater increases in precentral gyrus connectivity with the thalamus and pons may be associated with larger improvements in the trained device speed. Exploratory analyses also revealed interesting potential relationships between brain integrity and multiple functional outcomes in CP, with functional connectivity between the motor cortex and midbrain showing the strongest potential relationship with mobility. Decreased posterior white matter, corpus callosum and thalamic volumes, and FA in the posterior thalamic radiation were the most prominent group differences with corticospinal tract differences notably not found. Conclusions: Results reinforce the involvement of sensory-related brain areas in bilateral CP. Given the wide individual variability in imaging results and clinical responses to training, a greater focus on neural and other mechanisms related to better or worse outcomes is recommended to enhance rehabilitation results on a patient vs. group level.

Asynchronous Reciprocal Coupling of Martini 2.2 Coarse-Grained and CHARMM36 All-Atom Simulations in an Automated Multiscale Framework.

The appeal of multiscale modeling approaches is predicated on the promise of combinatorial synergy. However, this promise can only be realized when distinct scales are combined with reciprocal consistency. Here, we consider multiscale molecular dynamics (MD) simulations that combine the accuracy and macromolecular flexibility accessible to fixed-charge all-atom (AA) representations with the sampling speed accessible to reductive, coarse-grained (CG) representations. AA-to-CG conversions are relatively straightforward because deterministic routines with unique outcomes are achievable. Conversely, CG-to-AA conversions have many solutions due to a surge in the number of degrees of freedom. While automated tools for biomolecular CG-to-AA transformation exist, we find that one popular option, called Backward, is prone to stochastic failure and the AA models that it does generate frequently have compromised protein structure and incorrect stereochemistry. Although these shortcomings can likely be circumvented by human intervention in isolated instances, automated multiscale coupling requires reliable and robust scale conversion. Here, we detail an extension to Multiscale Machine-learned Modeling Infrastructure (MuMMI), including an improved CG-to-AA conversion tool called sinceCG. This tool is reliable (∼98% weakly correlated repeat success rate), automatable (no unrecoverable hangs), and yields AA models that generally preserve protein secondary structure and maintain correct stereochemistry. We describe how the MuMMI framework identifies CG system configurations of interest, converts them to AA representations, and simulates them at the AA scale while on-the-fly analyses provide feedback to update CG parameters. Application to systems containing the peripheral membrane protein RAS and proximal components of RAF kinase on complex eight-component lipid bilayers with ∼1.5 million atoms is discussed in the context of MuMMI.

Neutron scattering maps the higher-order assembly of NADPH-dependent assimilatory sulfite reductase.

Precursor molecules for biomass incorporation must be imported into cells and made available to the molecular machines that build the cell. Sulfur-containing macromolecules require that sulfur be in its S2- oxidation state before assimilation into amino acids, cofactors, and vitamins that are essential to organisms throughout the biosphere. In α-proteobacteria, NADPH-dependent assimilatory sulfite reductase (SiR) performs the final six-electron reduction of sulfur. SiR is a dodecameric oxidoreductase composed of an octameric flavoprotein reductase (SiRFP) and four hemoprotein metalloenzyme oxidases (SiRHPs). SiR performs the electron transfer reduction reaction to produce sulfide from sulfite through coordinated domain movements and subunit interactions without release of partially reduced intermediates. Efforts to understand the electron transfer mechanism responsible for SiR's efficiency are confounded by structural heterogeneity arising from intrinsically disordered regions throughout its complex, including the flexible linker joining SiRFP's flavin-binding domains. As a result, high-resolution structures of SiR dodecamer and its subcomplexes are unknown, leaving a gap in the fundamental understanding of how SiR performs this uniquely large-volume electron transfer reaction. Here, we use deuterium labeling, in vitro reconstitution, analytical ultracentrifugation (AUC), small-angle neutron scattering (SANS), and neutron contrast variation (NCV) to observe the relative subunit positions within SiR's higher-order assembly. AUC and SANS reveal SiR to be a flexible dodecamer and confirm the mismatched SiRFP and SiRHP subunit stoichiometry. NCV shows that the complex is asymmetric, with SiRHP on the periphery of the complex and the centers of mass between SiRFP and SiRHP components over 100 Å apart. SiRFP undergoes compaction upon assembly into SiR's dodecamer and SiRHP adopts multiple positions in the complex. The resulting map of SiR's higher-order structure supports a cis/trans mechanism for electron transfer between domains of reductase subunits as well as between tightly bound or transiently interacting reductase and oxidase subunits.