The tilting motion of the central core reveals the transport mechanism of the sarco/endoplasmic reticulum Ca2+-ATPase.

The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) transports two Ca2+ ions per ATP hydrolyzed from the cytoplasm to the lumen. However, how the ATP hydrolysis remotely drives the Ca2+ transport is unclear. In the SERCA1a crystal structures, the ATP hydrolysis is accompanied by the notably increasing tilting angle of the central core (CC) and the Ca2+ transport, and the CC tilting angle dramatically decreases in the E2 to E1 transition. We demonstrated that the significantly increasing tilting motion of the CC drove the Ca2+ release in the molecular dynamics simulation of the R836A variant, and the dramatic spontaneous decrease in the CC tilting angle of the E2 state triggers the restart of the SERCA1a's transport cycle. The repulsion between the phosphorylated D351 and the phosphate groups in ADP triggers the release of ADP from the SERCA1a headpiece. We proposed a novel SERCA transport mechanism in which ATP hydrolysis drives a significant tilting motion of the CC, which drives Ca2+ transport and the A domain rotational motion in the E1P-ADP-2Ca2+ to E2P transition. The dramatic spontaneous decrease in the CC tilting angle of the E2 state drives the restart of the transport cycle.

Retrograde amnesia for the stress-induced impairment of extinction: time-dependent and not so forgotten.

We investigated whether retrograde amnesia for the stress-induced impairment of extinction retrieval shares similar characteristics with original acquisition memories. The first experiment demonstrated that cycloheximide administered shortly after a single restraint stress session alleviated the impairment of extinction retrieval but not when administered following a longer delay (i.e., the amnesia for stress is time-dependent). A second experiment showed that the retrograde amnesia for stress could be alleviated by a second brief exposure to the stressor. These results demonstrating that amnesia for stress shares characteristics similar to original memories are explained using a retrieval-based memory integration model of retrograde amnesia.

Impact of e-ASPECTS software on the performance of physicians compared to a consensus ground truth: a multi-reader, multi-case study.

Background: The Alberta Stroke Program Early CT Score (ASPECTS) is used to quantify the extent of injury to the brain following acute ischemic stroke (AIS) and to inform treatment decisions. The e-ASPECTS software uses artificial intelligence methods to automatically process non-contrast CT (NCCT) brain scans from patients with AIS affecting the middle cerebral artery (MCA) territory and generate an ASPECTS. This study aimed to evaluate the impact of e-ASPECTS (Brainomix, Oxford, UK) on the performance of US physicians compared to a consensus ground truth. Methods: The study used a multi-reader, multi-case design. A total of 10 US board-certified physicians (neurologists and neuroradiologists) scored 54 NCCT brain scans of patients with AIS affecting the MCA territory. Each reader scored each scan on two occasions: once with and once without reference to the e-ASPECTS software, in random order. Agreement with a reference standard (expert consensus read with reference to follow-up imaging) was evaluated with and without software support. Results: A comparison of the area under the curve (AUC) for each reader showed a significant improvement from 0.81 to 0.83 (p = 0.028) with the support of the e-ASPECTS tool. The agreement of reader ASPECTS scoring with the reference standard was improved with e-ASPECTS compared to unassisted reading of scans: Cohen's kappa improved from 0.60 to 0.65, and the case-based weighted Kappa improved from 0.70 to 0.81. Conclusion: Decision support with the e-ASPECTS software significantly improves the accuracy of ASPECTS scoring, even by expert US neurologists and neuroradiologists.

Genicular Artery Embolisation in Patients with Osteoarthritis of the Knee (GENESIS 2): Protocol for a Double-Blind Randomised Sham-Controlled Trial.

Knee osteoarthritis is a leading cause of chronic disability and economic burden. In many patients who are not surgical candidates, existing treatment options are insufficient. Clinical evidence for a new treatment approach, genicular artery embolisation (GAE), is currently limited to single arm cohort, or small population randomised studies. This trial will investigate the use of a permanent embolic agent for embolisation of abnormal genicular arterial vasculature to reduce pain in patients with mild to moderate knee osteoarthritis. Up to 110 participants, 45 years or older, with knee pain for ≥ 3 months resistant to conservative treatment will be randomised (1:1) to GAE or a sham procedure. The treatment group will receive embolisation using 100-micron Embozene™ microspheres (Varian, a Siemens Healthineers Company) (investigational use for this indication in the UK), and the sham group will receive 0.9% saline in an otherwise identical procedure. Patients will be followed for 24 months. At 6 months, sham participants will be offered crossover to GAE. The primary endpoint is change of 4 Knee Injury and OA Outcome Score subscales (KOOS4) at 6 months post-randomisation. The study will also evaluate quality of life, health economics, imaging findings, and psychosocial pain outcomes. The primary manuscript will be submitted for publication after all participants complete 6 months of follow-up. The trial is expected to run for 3.5 years. Trial Registration: ClinicalTrials.gov, Identifier: NCT05423587.

Transcatheter embolisation in chronic musculoskeletal disorders.

Chronic musculoskeletal conditions affect millions of patients worldwide resulting in disability, reduced quality of life, and have a profound economic impact on the individual and society. Current treatment strategies fail patients who have not responded to conservative management but are not surgical candidates. Over the last decade, transcatheter embolisation has emerged as a potential treatment for these difficult to treat patients. By exploiting pathological neovascularisation within conditions such as knee osteoarthritis, adhesive capsulitis, and tendinopathy, embolisation has been used to improve patients' pain and function. This review explores the rationale for musculoskeletal transcatheter embolisation, illustrating the technique, and latest evidence for the most common procedures.

Pharmacophore Oriented MP2 Characterization of Charge Distribution for Anti-SARS-CoV-2 Inhibitor Nirmatrelvir.

Quantum mechanical second order Møller-Plesset (MP2) perturbation theory and density functional theory (DFT) Becke, 3-parameter, Lee-Yang-Parr (B3LYP) and Minnesota 2006 local functional (M06L) calculations were performed to optimize structure of nirmatrelvir and compute the Merz-Kollman electrostatic potential (MK ESP), natural population analysis (NPA), Hirshfeld, charge model 5 (CM5), and mulliken partial charges. The mulliken partial charge distribution of nirmatrelvir exhibits a poor correlation with the MK ESP charges in MP2, B3LYP, and M06L calculations respectively. The NPA, Hirshfeld, and CM5 partial charge scheme of nirmatrelvir indicate a reasonable correlation with MK ESP charge assignments in B3LYP and M06L calculations. The above correlations were not improved by the inclusion of implicit solvation model. The MK ESP and CM5 partial charges show a strong correlation between the results of MP2 and two DFT methods. The three optimized structures present a certain degree of differences from the crystal bioactive conformation of nirmatrelvir, suggesting the nirmatrelvir-enzyme complex is formed in the induced-fit model. The Reactivity of warhead electrophilic nitrile is justified by the relatively weaker strength of π bonds in the MP2 calculations. The nirmatrelvir hydrogen bond acceptors consistently show strong delocalization of lone pair electrons in three calculations, whereas hydrogen bond donors are found to have high polarization on the heavy nitrogen atoms in MP2 computations. This work helps to parametrize the force field of nirmatrelvir and improve accuracy of molecular docking and rational inhibitor design.

QM/MM study of N501 involved intermolecular interaction between SARS-CoV-2 receptor binding domain and antibody of human origin.

Intermolecular interaction between key residue N501 of the epitope on SARS-CoV-2 RBD and screening antibody B38 was studied using the QM/MM and QM approach. The QM/MM optimized geometry shows that angle X-H---Y is 165° for O-H---O between mAb light chain S30 and RBD N501. High level MP2 calculations indicated the interaction between RBD N501 and S30 of B38 Fab light chain provide a relatively strong attractive force of - 3.32 kcal/mol, whereas the hydrogen bond between RBD Q498 and S30 was quantified as 0.10 kcal/mol. The decrease in ESP partial charge on hydrogen atom of hydroxyl group on S30 drops from 0.38 a.u. to 0.31 a.u., exhibiting the sharing of 0.07 a.u. from the lone pair electron oxygen of N501 due to hydrogen bond formation. The NBO occupancy of hydrogen atom also decreases from 25.79 % to 22.93 % in the hydroxyl H-O NBO bond of S30. However, the minor change of NBO hybridization of hydroxyl oxygen of S30 from sp3.00 to sp3.05 implies the rigidity of hydrogen bond tetrahedral geometry in the relative dynamic protein complex. The O-H---O angle is 165° which is close but not exactly linear. The structural requirement for sp3 hybridization of oxygen for hydroxyl group on S30 and dimension of protein likely prevent O-H---O from adopting linear geometry. The hydrogen bond strengths were also calculated using a variety of DFT methods, and the result of - 3.33 kcal/mol from the M06L method is the closest to that of the MP2 calculation. Results of this work may aid in the COVID-19 vaccine and drug screening.

Artificial intelligence-based decision support software to improve the efficacy of acute stroke pathway in the NHS: an observational study.

Introduction: In a drip-and-ship model for endovascular thrombectomy (EVT), early identification of large vessel occlusion (LVO) and timely referral to a comprehensive center (CSC) are crucial when patients are admitted to an acute stroke center (ASC). Several artificial intelligence (AI) decision-aid tools are increasingly being used to facilitate the rapid identification of LVO. This retrospective cohort study aimed to evaluate the impact of deploying e-Stroke AI decision support software in the hyperacute stroke pathway on process metrics and patient outcomes at an ASC in the United Kingdom. Methods: Except for the deployment of e-Stroke on 01 March 2020, there were no significant changes made to the stroke pathway at the ASC. The data were obtained from a prospective stroke registry between 01 January 2019 and 31 March 2021. The outcomes were compared between the 14 months before and 12 months after the deployment of AI (pre-e-Stroke cohort vs. post-e-Stroke cohort) on 01 March 2020. Time window analyses were performed using Welch's t-test. Cochran-Mantel-Haenszel test was used to compare changes in disability at 3 months assessed by modified Rankin Score (mRS) ordinal shift analysis, and Fisher's exact test was used for dichotomised mRS analysis. Results: In the pre-e-Stroke cohort, 19 of 22 patients referred received EVT. In the post-e-Stroke cohort, 21 of the 25 patients referred were treated. The mean door-in-door-out (DIDO) and door-to-referral times in pre-e-Stroke vs. post-e-Stroke cohorts were 141 vs. 79 min (difference 62 min, 95% CI 96.9-26.8 min, p < 0.001) and 71 vs. 44 min (difference 27 min, 95% CI 47.4-5.4 min, p = 0.01), respectively. The adjusted odds ratio (age and NIHSS) for mRS ordinal shift analysis at 3 months was 3.14 (95% CI 0.99-10.51, p = 0.06) and the dichotomized mRS 0-2 at 3 months was 16% vs. 48% (p = 0.04) in the pre- vs. post-e-Stroke cohorts, respectively. Conclusion: In this single-center study in the United Kingdom, the DIDO time significantly decreased since the introduction of e-Stroke decision support software into an ASC hyperacute stroke pathway. The reduction in door-in to referral time indicates faster image interpretation and referral for EVT. There was an indication of an increased proportion of patients regaining independent function after EVT. However, this should be interpreted with caution given the small sample size. Larger, prospective studies and further systematic real-world evaluation are needed to demonstrate the widespread generalisability of these findings.

Heterotropic roles of divalent cations in the establishment of allostery and affinity maturation of integrin αXß2.

Allosteric activation and silencing of leukocyte ß2-integrins transpire through cation-dependent structural changes, which mediate integrin biosynthesis and recycling, and are essential to designing leukocyte-specific drugs. Stepwise addition of Mg2+ reveals two mutually coupled events for the αXß2 ligand-binding domain-the αX I-domain-corresponding to allostery establishment and affinity maturation. Electrostatic alterations in the Mg2+-binding site establish long-range couplings, leading to both pH- and Mg2+-occupancy-dependent biphasic stability change in the αX I-domain fold. The ligand-binding sensorgrams show composite affinity events for the αX I-domain accounting for the multiplicity of the αX I-domain conformational states existing in the solution. On cell surfaces, increasing Mg2+ concentration enhanced adhesiveness of αXß2. This work highlights how intrinsically flexible pH- and cation-sensitive architecture endows a unique dynamic continuum to the αI-domain structure on the intact integrin, thereby revealing the importance of allostery establishment and affinity maturation in both extracellular and intracellular integrin events.

Cadaveric and Angiographic Anatomical Considerations in the Genicular Arterial System: Implications for Genicular Artery Embolisation in Patients with Knee Osteoarthritis.

PURPOSE: Genicular artery embolisation (GAE) is a novel treatment for patients with knee osteoarthritis (OA). Cadaveric dissection was undertaken to provide a complete description of the relevant arterial anatomy in order to perform safe and effective GAE. MATERIALS: Twenty human lower limb specimens were dissected. The morphology of the genicular arteries and presence of anastomotic connections was recorded and compared with angiographic images from patients having undergone GAE. Vessels were measured to investigate the risk of non-target embolisation (NTE), taking a diameter of 300 microns as the threshold for significance. RESULTS: The descending genicular artery (DGA) is the dominant vessel in medial OA, with 95% of cases revealing vessel division into muscular, saphenous and osteoarticular branches from a single pedicle. The superior medial genicular artery (SMGA) had a shared origin with the middle genicular artery (MGA) in 25% of cases. NTE to the MGA may damage the cruciate ligaments. In 85% of cases, there was an anastomosis between the DGA and SMGA, often encountered at angiography. The mean diameter of the anastomoses was 850 micron, presenting a risk for NTE. An anastomosis between the Inferior Medial Genicular Artery (IMGA) and medial sural artery was found in 5% of cases; the medial sural artery supplies blood to the tibial nerve and should be avoided. The IMGA and inferior lateral genicular artery provided supply to the patellofemoral joint in 69% and 88% of cases, respectively. CONCLUSION: An in-depth knowledge of genicular artery anatomy is required for interventional radiologists to perform safe and effective GAE in patients with knee osteoarthritis.

Antibody mix-and-read assays based on fluorescence intensity probes.

Antibodies and Fc fusion proteins are a rapidly growing class of pharmaceuticals. Cell culture and purification process development and operation require frequent measurement of product concentrations, commonly by complex enzyme-linked immunosorbent assay and high-performance liquid chromatography methods. Here we report a fast (<30 s), and simple antibody Fc assay based on mix-and-read reporting by fluorescence emission. A soluble fluorescein-labeled Fc-affinity reporter produced by standard peptide synthesis is mixed with an Fc-containing sample to produce an immediate shift in both fluorescence polarization and intensity, compatible with on- and at-line measurements and microbioreactor monitoring. We observed significant shifts in fluorescence intensity in Chinese hamster ovary cell culture fluid spiked with IgG and detected an adalimumab biosimilar down to 100 ng/mL (10-4 g/L), despite the interferents in the complex sample matrix. Neither the fluorescence polarization nor the fluorescence intensity assay is significantly affected by the addition of clarified lysate of 2 million CHO-k1 cells/mL, suggesting applicability even to cultures of low viability. Biochemical and molecular docking approaches suggest that the fluorescence intensity enhancement is caused by changes in the fluorophore's local microenvironment upon binding to IgG Fc, especially by interactions with Fc His433.Abbreviations: CCF: Cell Culture Fluid; CHO: Chinese Hamster Ovary cells; ELISA: Enzyme Linked Immunosorbent Assay; Fc: Fragment Crystallizable of antibody; HPLC: High-Performance Liquid Chromatography; HPßCD: hydroxypropyl-ß-cyclodextrin; IgG: ImmunoglobulinG; mAb: Monoclonal Antibody; PBS: Phosphate-Buffered Saline; PDB: Protein Data Bank; SpA: Staphylococcal protein A; SpG: Staphylococcal protein G.

Screening of Focused Compound Library Targeting Liver X Receptors in Pancreatic Cancer Identified Ligands with Inverse Agonist and Degrader Activity.

Pancreatic ductal adenocarcinoma (PDAC) is the predominant form of pancreatic cancer. PDACs harbor oncogenic mutations in the KRAS gene, and ongoing efforts to directly target its mutant protein product to inhibit tumor growth are a priority not only in pancreatic cancer but in other malignancies such as lung and colorectal cancers where KRAS is also commonly mutated. An alternative strategy to directly targeting KRAS is to identify and target druggable receptors involved in dysregulated cancer hallmarks downstream of KRAS dysregulation. Liver X receptors (LXRs) are members of the nuclear receptor family of ligand-modulated transcription factors and are involved in the regulation of genes which function in key cancer-related processes, including cholesterol transport, lipid and glucose metabolism, and inflammatory and immune responses. Modulation of LXRs via small molecule ligands has emerged as a promising approach for directly targeting tumor cells or the stromal and immune cells within the tumor microenvironment. We have previously shown that only one of the two LXR subtypes (LXRß) is expressed in pancreatic cancer cells, and targeting LXR with available synthetic ligands blocked the proliferation of PDAC cells and tumor formation. In a screen of a focused library of drug-like small molecules predicted to dock in the ligand-binding pocket of LXRß, we identified two novel LXR ligands with more potent antitumor activity than current LXR agonists used in our published studies. Characterization of the two lead compounds (GAC0001E5 and GAC0003A4) indicates that they function as LXR inverse agonists which inhibit their transcriptional activity. Prolonged treatments with novel ligands further revealed their function as LXR "degraders" which significantly reduced LXR protein levels in all three PDAC cell lines tested. These findings support the utility of these novel inhibitors in basic research on ligand design, allosteric mechanisms, and LXR functions and their potential application as treatments for advanced pancreatic cancer and other recalcitrant malignancies.

Population-scale longitudinal mapping of COVID-19 symptoms, behaviour and testing.

Despite the widespread implementation of public health measures, coronavirus disease 2019 (COVID-19) continues to spread in the United States. To facilitate an agile response to the pandemic, we developed How We Feel, a web and mobile application that collects longitudinal self-reported survey responses on health, behaviour and demographics. Here, we report results from over 500,000 users in the United States from 2 April 2020 to 12 May 2020. We show that self-reported surveys can be used to build predictive models to identify likely COVID-19-positive individuals. We find evidence among our users for asymptomatic or presymptomatic presentation; show a variety of exposure, occupational and demographic risk factors for COVID-19 beyond symptoms; reveal factors for which users have been SARS-CoV-2 PCR tested; and highlight the temporal dynamics of symptoms and self-isolation behaviour. These results highlight the utility of collecting a diverse set of symptomatic, demographic, exposure and behavioural self-reported data to fight the COVID-19 pandemic.

Implementing a system for the real-time risk assessment of patients considered for intensive care.

BACKGROUND: Delay in identifying deterioration in hospitalised patients is associated with delayed admission to an intensive care unit (ICU) and poor outcomes. For the HAVEN project (HICF ref.: HICF-R9-524), we have developed a mathematical model that identifies deterioration in hospitalised patients in real time and facilitates the intervention of an ICU outreach team. This paper describes the system that has been designed to implement the model. We have used innovative technologies such as Portable Format for Analytics (PFA) and Open Services Gateway initiative (OSGi) to define the predictive statistical model and implement the system respectively for greater configurability, reliability, and availability. RESULTS: The HAVEN system has been deployed as part of a research project in the Oxford University Hospitals NHS Foundation Trust. The system has so far processed > 164,000 vital signs observations and > 68,000 laboratory results for > 12,500 patients and the algorithm generated score is being evaluated to review patients who are under consideration for transfer to ICU. No clinical decisions are being made based on output from the system. The HAVEN score has been computed using a PFA model for all these patients. The intent is that this score will be displayed on a graphical user interface for clinician review and response. CONCLUSIONS: The system uses a configurable PFA model to compute the HAVEN score which makes the system easily upgradable in terms of enhancing systems' predictive capability. Further system enhancements are planned to handle new data sources and additional management screens.

Population-scale Longitudinal Mapping of COVID-19 Symptoms, Behavior, and Testing Identifies Contributors to Continued Disease Spread in the United States.

Despite social distancing and shelter-in-place policies, COVID-19 continues to spread in the United States. A lack of timely information about factors influencing COVID-19 spread and testing has hampered agile responses to the pandemic. We developed How We Feel, an extensible web and mobile application that aggregates self-reported survey responses, to fill gaps in the collection of COVID-19-related data. How We Feel collects longitudinal and geographically localized information on users' health, behavior, and demographics. Here we report results from over 500,000 users in the United States from April 2, 2020 to May 12, 2020. We show that self- reported surveys can be used to build predictive models of COVID-19 test results, which may aid in identification of likely COVID-19 positive individuals. We find evidence among our users for asymptomatic or presymptomatic presentation, as well as for household and community exposure, occupation, and demographics being strong risk factors for COVID-19. We further reveal factors for which users have been SARS-CoV-2 PCR tested, as well as the temporal dynamics of self- reported symptoms and self-isolation behavior in positive and negative users. These results highlight the utility of collecting a diverse set of symptomatic, demographic, and behavioral self- reported data to fight the COVID-19 pandemic.

Forget the stress: retrograde amnesia for the stress-induced impairment of extinction retrieval.

We investigated whether cycloheximide (CHX) would induce amnesia for the stress-induced impairment of extinction retrieval. First, a single restraint stress session was demonstrated to impair extinction retrieval, but not fear conditioning. A second experiment showed that when CHX was administered immediately after restraint, rats exhibited significant extinction retrieval at test (i.e., retrograde amnesia for the stress). In a third experiment, the stress session impaired various amounts of extinction durations, suggesting that the stress inhibited extinction retrieval rather than enhancing the original fear learning. These results suggest memories for acute stress are susceptible to disruption, which could have clinical implications.

Impact of lymphoma-linked Asn11Tyr point mutation on the interaction between Bcl-2 and a BH3 mimetic: Insights from molecular dynamics simulation.

Frequent mutations in the Bcl-2 anti-apoptotic protein are often implicated in diffuse large B-cell lymphoma (DLBCL), a disease profoundly resistant to drugs. Bcl-2-competitive inhibitors, "BH3 mimetics," activate apoptosis by interfering with the interactions between pro-apoptotic BH3 domains and the hydrophobic groove of Bcl-2. The aim of our research is to determine the potential of DLBCL-linked N11Y mutation to facilitate resistance against a "BH3 mimetic" using molecular dynamics simulation. Binding free energy calculations suggest a significant decrease in the binding affinity in the mutant model. In-depth analysis of the models using residue interaction network, dynamic cross-correlation, and free energy landscape approaches reveal that the mutation modifies the conformations of key residues, thereby altering the shape of the hydrophobic groove. This subsequently changes the ligand orientation and counteracts the phenomenon of LB region unwinding, a crucial event observed in the wild-type model. Lowest frequency motions captured by principal component analysis reflect the stretching of the groove for efficient ligand accommodation in the wild-type complex but not in the mutant model. This is the first in silico study that unravels the mechanism of drug resistance induced by a Bcl-2 mutation, which could be of great relevance while designing and tailoring therapeutics.

RNAdemocracy: an ensemble method for RNA secondary structure prediction using consensus scoring.

The identification of RNA secondary structure has been an important tool for the characterization of nucleic acids. Computational structure prediction has been an effective approach toward this end, but improvement of established methods is often slow and reliant on redundant methodology. Here we present a novel consensus scoring approach, created to incorporate inputs from an array of established methods with the goal of producing outputs that contain mutual structures from these programs. This method is implemented in RNAdemocracy, a python program capable of competing with existing methods. This ensemble approach was limited by commonalities in established methods like parameter sourcing, which may lead to agreement error, an unavoidable outcome due to the limit of available RNA structure datasets. The modular construction of RNAdemocracy allows for its easy upgrading and customization to suit user's needs. RNAdemocracy, while capable of accurate predictions, is best suited to guide users to regions of the sequence space that exhibit agreement instead of a totally reliant predictor of structure. It is also capable of grading predictions for potential accuracy by providing a percentage of consensus between contributing methods in the final structure.