Binding kinetics drive G protein subtype selectivity at the ß1-adrenergic receptor.

G protein-coupled receptors (GPCRs) bind to different G protein α-subtypes with varying degrees of selectivity. The mechanism by which GPCRs achieve this selectivity is still unclear. Using 13C methyl methionine and 19F NMR, we investigate the agonist-bound active state of ß1AR and its ternary complexes with different G proteins in solution. We find the receptor in the ternary complexes adopts very similar conformations. In contrast, the full agonist-bound receptor active state assumes a conformation differing from previously characterised activation intermediates or from ß1AR in ternary complexes. Assessing the kinetics of binding for the agonist-bound receptor with different G proteins, we find the increased affinity of ß1AR for Gs results from its much faster association with the receptor. Consequently, we suggest a kinetic-driven selectivity gate between canonical and secondary coupling which arises from differential favourability of G protein binding to the agonist-bound receptor active state.

How Reproducible is the Synthesis of Zr-Porphyrin Metal-Organic Frameworks? An Interlaboratory Study.

Metal-organic frameworks (MOFs) are a rapidly growing class of materials that offer great promise in various applications. However, the synthesis remains challenging: for example, a range of crystal structures can often be accessed from the same building blocks, which complicates the phase selectivity. Likewise, the high sensitivity to slight changes in synthesis conditions may cause reproducibility issues. This is crucial, as it hampers the research and commercialization of affected MOFs. Here, it presents the first-ever interlaboratory study of the synthetic reproducibility of two Zr-porphyrin MOFs, PCN-222 and PCN-224, to investigate the scope of this problem. For PCN-222, only one sample out of ten was phase pure and of the correct symmetry, while for PCN-224, three are phase pure, although none of these show the spatial linker order characteristic of PCN-224. Instead, these samples resemble dPCN-224 (disordered PCN-224), which has recently been reported. The variability in thermal behavior, defect content, and surface area of the synthesised samples are also studied. The results have important ramifications for field of metal-organic frameworks and their crystallization, by highlighting the synthetic challenges associated with a multi-variable synthesis space and flat energy landscapes characteristic of MOFs.

Antigen-specific depletion of CD4+ T cells by CAR T cells reveals distinct roles of higher- and lower-affinity TCRs during autoimmunity.

Both higher- and lower-affinity self-reactive CD4+ T cells are expanded in autoimmunity; however, their individual contribution to disease remains unclear. We addressed this question using peptide-MHCII chimeric antigen receptor (pMHCII-CAR) T cells to specifically deplete peptide-reactive T cells in mice. Integration of improvements in CAR engineering with TCR repertoire analysis was critical for interrogating in vivo the role of TCR affinity in autoimmunity. Our original MOG35-55 pMHCII-CAR, which targeted only higher-affinity TCRs, could prevent the induction of experimental autoimmune encephalomyelitis (EAE). However, pMHCII-CAR enhancements to pMHCII stability, as well as increased survivability via overexpression of a dominant-negative Fas, were required to target lower-affinity MOG-specific T cells and reverse ongoing clinical EAE. Thus, these data suggest a model in which higher-affinity autoreactive T cells are required to provide the "activation energy" for initiating neuroinflammatory injury, but lower-affinity cells are sufficient to maintain ongoing disease.

Adherence to the 2015 and 2020 British Society of Paediatric Endocrinology and Diabetes guidelines and outcomes in critically ill children with diabetic ketoacidosis: a retrospective cohort study.

OBJECTIVES: To compare clinical management and key outcomes of critically ill children with diabetic ketoacidosis (DKA) in two cohorts (2015 cohort: managed according to the 2015 British Society of Paediatric Endocrinology and Diabetes (BSPED) guidelines; 2020 cohort: managed according to the 2020 BSPED guidelines). DESIGN: Retrospective cohort study using prospectively collected data. SETTING: A critical care advice and transport service based in London, and referring hospitals within the critical care network. PATIENTS: All children 0-17 years referred for advice and/or critical care transport with a clinical diagnosis of DKA over a 30-month period (from September 2018 to March 2021). INTERVENTIONS: None. MAIN OUTCOME MEASURES: Admission to intensive care unit (ICU), clinically diagnosed cerebral oedema and death. RESULTS: There were significant differences in fluid and insulin administration practices between the 2015 and 2020 cohorts (fluid bolus >20 mL/kg: 3% vs 30%, p<0.001; median total fluid given in the first 24 hours: 84 mL/kg vs 117 mL/kg, p<0.01; starting insulin infusion rate 0.1 U/kg/hour: 54% vs 31%, p<0.01). However, these differences were consistent with guideline recommendations (initial fluid infusion rate within 5% of guideline-recommended rate: 80% in the 2015 group vs 84% in the 2020 group). There were no significant differences in outcomes (ICU admission: 26% vs 35%, p=0.2; cerebral oedema: 21% vs 23%, p=0.8). CONCLUSIONS: Our study findings indicate that changes to fluid and insulin administration occurred after the 2020 BSPED guideline publication, with strong adherence to the guideline, but these changes were not associated with changes in key outcomes.

Peripheral and Central/Intraosseous Vasoactive Infusions During and After Pediatric Critical Care Transport: Retrospective Cohort Study of Extravasation Injury.

OBJECTIVES: To compare the prevalence of adverse events related to vasoactive drug infusions administered via a peripheral venous catheter versus a central venous or intraosseous catheter. DESIGN: Retrospective observational study. SETTING: A pediatric critical care transport team, and the PICUs and regional hospitals within the North Thames and East Anglia regions of the United Kingdom. PATIENTS: Children (up to 18 yr old) transported by the Children's Acute Transport Service receiving an infusion of a vasoactive drug (epinephrine, dobutamine, dopamine, norepinephrine, and vasopressin). INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The medical records of all children transported between April 2017 and May 2020 receiving a vasoactive drug infusion were reviewed and cross-referenced with the service critical incident database. The outcome measure was anatomic catheter-related adverse events (including extravasation) reported during transport or in the first 24 hours on the PICU. During the study period, the service undertook 3,836 transports. Vasoactive drugs were administered during 558 patient transports (14.5%). During 198 of 558 transports (35.5%), vasoactive drugs were administered via a peripheral venous catheter, with seven of 198 (3.5%) adverse events. One extravasation event resulted in tissue necrosis. The median time to injury after the infusion was commenced was 60 minutes (interquartile range, 30-60 min). During 360 of 558 transports (64.5%), vasoactive infusions were administered by central venous or intraosseous catheter, with nine of 360 (2.5%) adverse events. CONCLUSIONS: During pediatric critical care transport, we did not find a difference in prevalence of adverse events following the administration of vasoactive drugs via peripheral venous catheters or via central venous and intraosseous catheters.

Beyond online participant crowdsourcing: The benefits and opportunities of big team addiction science.

Participant crowdsourcing platforms (e.g., MTurk, Prolific) offer numerous advantages to addiction science, permitting access to hard-to-reach populations and enhancing the feasibility of complex experimental, longitudinal, and intervention studies. Yet these are met with equal concerns about participant nonnaivety, motivation, and careless responding, which if not considered can greatly compromise data quality. In this article, we discuss an alternative crowdsourcing avenue that overcomes these issues whilst presenting its own unique advantages-crowdsourcing researchers through big team science. First, we review several contemporary efforts within psychology (e.g., ManyLabs, Psychological Science Accelerator) and the benefits these would yield if they were more widely implemented in addiction science. We then outline our own consortium-based approach to empirical dissertations: a grassroots initiative that trains students in reproducible big team addiction science. In doing so, we discuss potential challenges and their remedies, as well as providing resources to help addiction researchers develop these initiatives. Through researcher crowdsourcing, together we can answer fundamental scientific questions about substance use and addiction, build a literature that is representative of a diverse population of researchers and participants, and ultimately achieve our goal of promoting better global health. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Increasing availability of lower energy meals vs. energy labelling in virtual full-service restaurants: two randomized controlled trials in participants of higher and lower socioeconomic position.

BACKGROUND: There are a range of interventions designed to promote healthier food choices in full-service restaurants. However, it is unclear how these interventions affect dietary choices in people of lower and higher socioeconomic position (SEP). METHODS: A total of 2091 US participants recruited online completed Study 1 (n = 1001) and Study 2 (n = 1090). Recruitment was stratified by participant highest education level, resulting in higher SEP and lower SEP groups. In a between-subjects design, participants made hypothetical food choices (main dish, plus optional sides and desserts) from six restaurants menus in the absence vs. presence of menu energy labelling and from menus with baseline (10%) vs. increased availability (50%) of lower energy main dishes. Data were collected and analysed in 2019. Two studies were conducted in order to examine replicability and generalisability of findings across different restaurant menu types. RESULTS: Across both studies, increasing the availability of lower energy main menu options decreased the average energy content of the ordered main dish (- 129 kcal, 95% CI [- 139; - 119]) and total energy ordered (- 117 kcal, 95% CI [- 138; - 95]) in both higher and lower SEP participants. Energy labelling significantly reduced the energy content of ordered main dishes in higher SEP participants (- 41 kcal, 95% CI [- 54; - 29]), but not lower SEP participants (- 5 kcal, 95% CI [- 22; 11]). However, energy labelling reduced total energy ordered (- 83 kcal, 95% CI [- 105; - 60]) irrespective of SEP. CONCLUSIONS: In two virtual experiments, increasing the availability of lower energy restaurant main menu options impacted on main menu dish choice and decreased total energy ordered irrespective of SEP. Energy labelling had a less pronounced effect on total energy ordered and had a larger impact on the energy content of main menu dish choice in higher as opposed to lower SEP participants. TRIAL REGISTRATION: Clinicaltrials.gov NCT04336540 retrospectively registered (7 April, 2020).

A conserved arginine residue is critical for stabilizing the N2 FeS cluster in mitochondrial complex I.

Respiratory complex I (NADH:ubiquinone oxidoreductase), the first enzyme of the electron-transport chain, captures the free energy released by NADH oxidation and ubiquinone reduction to translocate protons across an energy-transducing membrane and drive ATP synthesis during oxidative phosphorylation. The cofactor that transfers the electrons directly to ubiquinone is an iron-sulfur cluster (N2) located in the NDUFS2/NUCM subunit. A nearby arginine residue (R121), which forms part of the second coordination sphere of the N2 cluster, is known to be posttranslationally dimethylated but its functional and structural significance are not known. Here, we show that mutations of this arginine residue (R121M/K) abolish the quinone-reductase activity, concomitant with disappearance of the N2 signature from the electron paramagnetic resonance (EPR) spectrum. Analysis of the cryo-EM structure of NDUFS2-R121M complex I at 3.7 Å resolution identified the absence of the cubane N2 cluster as the cause of the dysfunction, within an otherwise intact enzyme. The mutation further induced localized disorder in nearby elements of the quinone-binding site, consistent with the close connections between the cluster and substrate-binding regions. Our results demonstrate that R121 is required for the formation and/or stability of the N2 cluster and highlight the importance of structural analyses for mechanistic interpretation of biochemical and spectroscopic data on complex I variants.

Structure and Dynamics of GPCRs in Lipid Membranes: Physical Principles and Experimental Approaches.

Over the past decade, the vast amount of information generated through structural and biophysical studies of GPCRs has provided unprecedented mechanistic insight into the complex signalling behaviour of these receptors. With this recent information surge, it has also become increasingly apparent that in order to reproduce the various effects that lipids and membranes exert on the biological function for these allosteric receptors, in vitro studies of GPCRs need to be conducted under conditions that adequately approximate the native lipid bilayer environment. In the first part of this review, we assess some of the more general effects that a membrane environment exerts on lipid bilayer-embedded proteins such as GPCRs. This is then followed by the consideration of more specific effects, including stoichiometric interactions with specific lipid subtypes. In the final section, we survey a range of different membrane mimetics that are currently used for in vitro studies, with a focus on NMR applications.

Conformational plasticity of ligand-bound and ternary GPCR complexes studied by 19F NMR of the ß1-adrenergic receptor.

G-protein-coupled receptors (GPCRs) are allosteric signaling proteins that transmit an extracellular stimulus across the cell membrane. Using 19F NMR and site-specific labelling, we investigate the response of the cytoplasmic region of transmembrane helices 6 and 7 of the ß1-adrenergic receptor to agonist stimulation and coupling to a Gs-protein-mimetic nanobody. Agonist binding shows the receptor in equilibrium between two inactive states and a pre-active form, increasingly populated with higher ligand efficacy. Nanobody coupling leads to a fully active ternary receptor complex present in amounts correlating directly with agonist efficacy, consistent with partial agonism. While for different agonists the helix 6 environment in the active-state ternary complexes resides in a well-defined conformation, showing little conformational mobility, the environment of the highly conserved NPxxY motif on helix 7 remains dynamic adopting diverse, agonist-specific conformations, implying a further role of this region in receptor function. An inactive nanobody-coupled ternary receptor form is also observed.

Clinical and epidemiological factors associated with suicide in colorectal cancer.

PURPOSE: While increased suicidal tendencies among cancer patients have been well documented, this study aims to examine suicide rates and factors associated with suicide specifically in patients with colorectal cancer (CRC). METHODS: Patients diagnosed with CRC between the years of 1988-2010 were selected from the Surveillance, Epidemiology, and End Result (SEER) database. Comparisons with the general population were done using the National Center for Disease Control registry. RESULTS: One thousand three hundred eighty-one suicides among 884,529 patients were identified, with a standardized mortality ratio (SMR) of 1.53 (95% CI, 1.13-1.33) compared to the general population. No statistically significant difference in suicide rate was found with respect to age, marital status, socio-economic status, surgical intervention, histologic subtype, or stage at diagnosis. Within the CRC population, Whites were significantly more likely to commit suicide than non-Whites (OR, 2.28; 95% CI, 1.89-2.75; P < 0.001), and males were significantly more likely than females (OR, 5.635; 95% CI, 4.85-6.54; P < 0.001). Most suicides occurred in patients with distal lesions in the sigmoid/rectosigmoid junction (P < 0.001). SMRs for CRC patients were 4.24 for females (95% CI, 3.69-4.86), 1.35 for males (95% CI, 1.28-1.43), 0.38 for African-Americans (95% CI, 0.28-0.52), 1.77 for Whites (95% CI, 1.68-1.87), and 0.90 for other races (95% CI, 0.72-1.12). CONCLUSION: Identification of risk factors associated with suicide among patients with CRC is an important step in developing screening strategies and management of psychosocial stressors. These results could be helpful in formulating a comprehensive suicide risk scoring system for screening all cancer patients.

Deleting the IF1-like ζ subunit from Paracoccus denitrificans ATP synthase is not sufficient to activate ATP hydrolysis.

In oxidative phosphorylation, ATP synthases interconvert two forms of free energy: they are driven by the proton-motive force across an energy-transducing membrane to synthesize ATP and displace the ADP/ATP ratio from equilibrium. For thermodynamically efficient energy conversion they must be reversible catalysts. However, in many species ATP synthases are unidirectional catalysts (their rates of ATP hydrolysis are negligible), and in others mechanisms have evolved to regulate or minimize hydrolysis. Unidirectional catalysis by Paracoccus denitrificans ATP synthase has been attributed to its unique ζ subunit, which is structurally analogous to the mammalian inhibitor protein IF1 Here, we used homologous recombination to delete the ζ subunit from the P. denitrificans genome, and compared ATP synthesis and hydrolysis by the wild-type and knockout enzymes in inverted membrane vesicles and the F1-ATPase subcomplex. ATP synthesis was not affected by loss of the ζ subunit, and the rate of ATP hydrolysis increased by less than twofold, remaining negligible in comparison with the rates of the Escherichia coli and mammalian enzymes. Therefore, deleting the P. denitrificans ζ subunit is not sufficient to activate ATP hydrolysis. We close by considering our conclusions in the light of reversible catalysis and regulation in ATP synthase enzymes.

Demographic and clinical factors associated with suicide in gastric cancer in the United States.

While increased suicidal tendencies among cancer patients have been well documented, there has been no specific examination of suicide and gastric cancer. The purpose of this study is to characterize suicide incidence among patients diagnosed with gastric cancer from 1973 to 2013 and identify variables associated with higher suicide rates. Patients with gastric cancer were identified in the Surveillance, Epidemiology, and End Results (SEER) database of the National Cancer Institute. The study included clinical and demographic data from 1973 to 2013. Standardized mortality ratios (SMRs) and 95% confidence intervals (95% CIs) were calculated. Comparisons with the general US population were based on mortality data collected by the Centers for Disease Control and Prevention's National Center for Injury Prevention and Control using the Web-based Injury Statistics Query and Reporting System. Multivariable logistic regression models generated odds ratios (ORs) to assess factors associated with increased suicide in gastric malignancy. There were 210 suicides for patients with gastric cancer (SMR, 3.21; 95% CI: 2.80-3.67). Female gender (SMR 8.54), White race (SMR 4.08), age ≤39 years (SMR 3.06), and age 70-79 years (SMR 2.90), were found to be significant for an increased incidence of suicide compared with the general population. There was not a statistically significant relationship between suicide and marital status, income, mode of radiation therapy, and the role of surgical intervention. Approximately 77% of deaths by suicide occurred within the first year following diagnosis. Female gender, White race, age ≤39 years, and age 70-79 years are factors associated with increased risk of suicide in patients with gastric cancer. These results, coupled with further studies and analyses, will be used to formulate a comprehensive suicide risk factor scoring system for screening all cancer patients.

Correlating kinetic and structural data on ubiquinone binding and reduction by respiratory complex I.

Respiratory complex I (NADH:ubiquinone oxidoreductase), one of the largest membrane-bound enzymes in mammalian cells, powers ATP synthesis by using the energy from electron transfer from NADH to ubiquinone-10 to drive protons across the energy-transducing mitochondrial inner membrane. Ubiquinone-10 is extremely hydrophobic, but in complex I the binding site for its redox-active quinone headgroup is ∼20 Šabove the membrane surface. Structural data suggest it accesses the site by a narrow channel, long enough to accommodate almost all of its ∼50-Šisoprenoid chain. However, how ubiquinone/ubiquinol exchange occurs on catalytically relevant timescales, and whether binding/dissociation events are involved in coupling electron transfer to proton translocation, are unknown. Here, we use proteoliposomes containing complex I, together with a quinol oxidase, to determine the kinetics of complex I catalysis with ubiquinones of varying isoprenoid chain length, from 1 to 10 units. We interpret our results using structural data, which show the hydrophobic channel is interrupted by a highly charged region at isoprenoids 4-7. We demonstrate that ubiquinol-10 dissociation is not rate determining and deduce that ubiquinone-10 has both the highest binding affinity and the fastest binding rate. We propose that the charged region and chain directionality assist product dissociation, and that isoprenoid stepping ensures short transit times. These properties of the channel do not benefit the exhange of short-chain quinones, for which product dissociation may become rate limiting. Thus, we discuss how the long channel does not hinder catalysis under physiological conditions and the possible roles of ubiquinone/ubiquinol binding/dissociation in energy conversion.

Using Hyperfine Electron Paramagnetic Resonance Spectroscopy to Define the Proton-Coupled Electron Transfer Reaction at Fe-S Cluster N2 in Respiratory Complex I.

Energy-transducing respiratory complex I (NADH:ubiquinone oxidoreductase) is one of the largest and most complicated enzymes in mammalian cells. Here, we used hyperfine electron paramagnetic resonance (EPR) spectroscopic methods, combined with site-directed mutagenesis, to determine the mechanism of a single proton-coupled electron transfer reaction at one of eight iron-sulfur clusters in complex I, [4Fe-4S] cluster N2. N2 is the terminal cluster of the enzyme's intramolecular electron-transfer chain and the electron donor to ubiquinone. Because of its position and pH-dependent reduction potential, N2 has long been considered a candidate for the elusive "energy-coupling" site in complex I at which energy generated by the redox reaction is used to initiate proton translocation. Here, we used hyperfine sublevel correlation (HYSCORE) spectroscopy, including relaxation-filtered hyperfine and single-matched resonance transfer (SMART) HYSCORE, to detect two weakly coupled exchangeable protons near N2. We assign the larger coupling with A(1H) = [-3.0, -3.0, 8.7] MHz to the exchangeable proton of a conserved histidine and conclude that the histidine is hydrogen-bonded to N2, tuning its reduction potential. The histidine protonation state responds to the cluster oxidation state, but the two are not coupled sufficiently strongly to catalyze a stoichiometric and efficient energy transduction reaction. We thus exclude cluster N2, despite its proton-coupled electron transfer chemistry, as the energy-coupling site in complex I. Our work demonstrates the capability of pulse EPR methods for providing detailed information on the properties of individual protons in even the most challenging of energy-converting enzymes.

Respiratory Complex I in Bos taurus and Paracoccus denitrificans Pumps Four Protons across the Membrane for Every NADH Oxidized.

Respiratory complex I couples electron transfer between NADH and ubiquinone to proton translocation across an energy-transducing membrane to support the proton-motive force that drives ATP synthesis. The proton-pumping stoichiometry of complex I (i.e. the number of protons pumped for each two electrons transferred) underpins all mechanistic proposals. However, it remains controversial and has not been determined for any of the bacterial enzymes that are exploited as model systems for the mammalian enzyme. Here, we describe a simple method for determining the proton-pumping stoichiometry of complex I in inverted membrane vesicles under steady-state ADP-phosphorylating conditions. Our method exploits the rate of ATP synthesis, driven by oxidation of NADH or succinate with different sections of the respiratory chain engaged in catalysis as a proxy for the rate of proton translocation and determines the stoichiometry of complex I by reference to the known stoichiometries of complexes III and IV. Using vesicles prepared from mammalian mitochondria (from Bos taurus) and from the bacterium Paracoccus denitrificans, we show that four protons are pumped for every two electrons transferred in both cases. By confirming the four-proton stoichiometry for mammalian complex I and, for the first time, demonstrating the same value for a bacterial complex, we establish the utility of P. denitrificans complex I as a model system for the mammalian enzyme. P. denitrificans is the first system described in which mutagenesis in any complex I core subunit may be combined with quantitative proton-pumping measurements for mechanistic studies.