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The CC chemokine receptor 6 (CCR6) is a potential target for chronic inflammatory diseases. Previously, we reported an active CCR6 structure in complex with its cognate chemokine CCL20, revealing the molecular basis of CCR6 activation. Here, we present two inactive CCR6 structures in ternary complexes with different allosteric antagonists, CCR6/SQA1/OXM1 and CCR6/SQA1/OXM2. The oxomorpholine analogues, OXM1 and OXM2 are highly selective CCR6 antagonists which bind to an extracellular pocket and disrupt the receptor activation network. An energetically favoured U-shaped conformation in solution that resembles the bound form is observed for the active analogues. SQA1 is a squaramide derivative with close-in analogues reported as antagonists of chemokine receptors including CCR6. SQA1 binds to an intracellular pocket which overlaps with the G protein site, stabilizing a closed pocket that is a hallmark of inactive GPCRs. Minimal communication between the two allosteric pockets is observed, in contrast to the prevalent allosteric cooperativity model of GPCRs. This work highlights the versatility of GPCR antagonism by small molecules, complementing previous knowledge of CCR6 activation, and sheds light on drug discovery targeting CCR6., (© 2024. The Author(s).)

Coronavirus disease 2019 (COVID-19) forecasts from over 100 models are readily available. However, little published information exists regarding the performance of their uncertainty estimates (i.e. probabilistic performance). To evaluate their probabilistic performance, we employ the classical model (CM), an established method typically used to validate expert opinion. In this analysis, we assess both the predictive and probabilistic performance of COVID-19 forecasting models during 2021. We also compare the performance of aggregated forecasts (i.e. ensembles) based on equal and CM performance-based weights to an established ensemble from the Centers for Disease Control and Prevention (CDC). Our analysis of forecasts of COVID-19 mortality from 22 individual models and three ensembles across 49 states indicates that-(i) good predictive performance does not imply good probabilistic performance, and vice versa; (ii) models often provide tight but inaccurate uncertainty estimates; (iii) most models perform worse than a naive baseline model; (iv) both the CDC and CM performance-weighted ensembles perform well; but (v) while the CDC ensemble was more informative, the CM ensemble was more statistically accurate across states. This study presents a worthwhile method for appropriately assessing the performance of probabilistic forecasts and can potentially improve both public health decision-making and COVID-19 modelling., (© 2022 The Authors.)

The ice sheets covering Antarctica and Greenland present the greatest uncertainty in, and largest potential contribution to, future sea level rise. The uncertainty arises from a paucity of suitable observations covering the full range of ice sheet behaviors, incomplete understanding of the influences of diverse processes, and limitations in defining key boundary conditions for the numerical models. To investigate the impact of these uncertainties on ice sheet projections we undertook a structured expert judgement study. Here, we interrogate the findings of that study to identify the dominant drivers of uncertainty in projections and their relative importance as a function of ice sheet and time. We find that for the 21st century, Greenland surface melting, in particular the role of surface albedo effects, and West Antarctic ice dynamics, specifically the role of ice shelf buttressing, dominate the uncertainty. The importance of these effects holds under both a high-end 5°C global warming scenario and another that limits global warming to 2°C. During the 22nd century the dominant drivers of uncertainty shift. Under the 5°C scenario, East Antarctic ice dynamics dominate the uncertainty in projections, driven by the possible role of ice flow instabilities. These dynamic effects only become dominant, however, for a temperature scenario above the Paris Agreement 2°C target and beyond 2100. Our findings identify key processes and factors that need to be addressed in future modeling and observational studies in order to reduce uncertainties in ice sheet projections., (© 2022 The Authors.)

Background: The majority of cases of idiopathic acute pancreatitis (IAP) are thought to result from occult biliary disease. A growing body of evidence suggests that cholecystectomy for IAP reduces the risk of recurrence by up to two thirds. This study examined nationwide uptake and disparities in adoption of cholecystectomy for IAP., Methods: The National Inpatient Sample was queried to identify admissions for IAP between October 2015 and December 2018. Patients who underwent cholecystectomy before discharge and those that did not were compared using Wald χ 2 tests for categorical variables and Student's t test for continuous variables. Patient- and hospital-level predictors of cholecystectomy were identified using weighted multivariable logistic regression., Results: Of 62,305 estimated admissions for IAP, only 665 (1.1%) underwent cholecystectomy before discharge. Female sex, initiation of total parenteral nutrition (TPN), insurance status, and hospital type were associated with cholecystectomy on univariable analysis. On multivariable analysis, Hispanic patients (odds ration [OR] 1.60, 95% confidence interval [CI] 1.01-2.56), patients on TPN (OR 2.70, 95% CI 1.17-6.24), and those with private insurance (OR 2.18, 95% CI 1.48-3.21 versus Medicare/Medicaid) were more likely to receive operations. Small hospitals and hospitals in rural areas were least likely to perform empiric cholecystectomies., Conclusion: Despite increasing evidence supporting cholecystectomy after IAP, the practice remains rare in the United States. Educational efforts and active implementation efforts are needed to promote adoption. Particular attention should be focused on small, rural centers and those that disproportionately care for uninsured patients and patients with public insurance., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Epidemiologic cohort studies have consistently demonstrated that long-term exposure to ambient fine particles (PM 2.5 ) is associated with mortality. Nevertheless, extrapolating results to understudied locations may involve considerable uncertainty. To explore this issue, this review discusses the evidence for (i) the associated risk of mortality, (ii) the shape of the concentration-response function, (iii) a causal interpretation, and (iv) how the source mix/composition of PM 2.5 and population characteristics may alter the effect. The accumulated evidence suggests the following: (i) In the United States, the change in all-cause mortality risk per μg/m 3 is about 0.8%. (ii) The concentration-response function appears nonlinear. (iii) Causation is overwhelmingly supported. (iv) Fossil fuel combustion-related sources are likely more toxic than others, and age, race, and income may modify the effect. To illustrate the use of our findings in support of a risk assessment in an understudied setting, we consider Kuwait. However, given the complexity of this relationship and the heterogeneity in reported effects, it is unreasonable to think that, in such circumstances, point estimates can be meaningful. Consequently, quantitative probabilistic estimates, which cannot be derived objectively, become essential. Formally elicited expert judgment can provide such estimates, and this review provides the evidence to support an elicitation.

Regular vines (R-vines) copulas build high dimensional joint densities from arbitrary one-dimensional margins and (conditional) bivariate copula densities. Vine densities enable the computation of all conditional distributions, though the calculations can be numerically intensive. Saturated continuous nonparametric Bayes nets (CNPBN) are regular vines. Computing regression functions from the vine copula density is termed vine regression. The epicycles of regression-including/excluding covariates, interactions, higher order terms, multicollinearity, model fit, transformations, heteroscedasticity, bias-are dispelled. One simply computes the regressions from the vine copula density. Only the question of finding an adequate vine copula remains. Vine regression is applied to a data set from the National Longitudinal Study of Youth relating breastfeeding to IQ. The expected effects of breastfeeding on IQ depend on IQ, on the baseline level of breastfeeding, on the duration of additional breastfeeding and on the values of other covariates. A child given two weeks breastfeeding can expect to increase his/her IQ by 1.5-2 IQ points by adding 10 weeks of breastfeeding, depending on values of other covariates. A child given two years breastfeeding can expect to gain from 0.48-0.65 IQ points from 10 additional weeks. Adding 10 weeks breastfeeding to each of the 3,179 children in this data set has a net present value $50,700,000 according to the Bayes net, compared to $29,000,000 according to the linear regression., (© 2021 The Authors. Risk Analysis published by Wiley Periodicals LLC on behalf of Society for Risk Analysis.)

Current therapies for Alzheimer's disease seek to correct for defective cholinergic transmission by preventing the breakdown of acetylcholine through inhibition of acetylcholinesterase, these however have limited clinical efficacy. An alternative approach is to directly activate cholinergic receptors responsible for learning and memory. The M1-muscarinic acetylcholine (M1) receptor is the target of choice but has been hampered by adverse effects. Here we aimed to design the drug properties needed for a well-tolerated M1-agonist with the potential to alleviate cognitive loss by taking a stepwise translational approach from atomic structure, cell/tissue-based assays, evaluation in preclinical species, clinical safety testing, and finally establishing activity in memory centers in humans. Through this approach, we rationally designed the optimal properties, including selectivity and partial agonism, into HTL9936-a potential candidate for the treatment of memory loss in Alzheimer's disease. More broadly, this demonstrates a strategy for targeting difficult GPCR targets from structure to clinic., Competing Interests: Declaration of interests T.T. and M.W. are shareholders and board members of Sosei Heptares. The authors A.J.H.B., G.A.B., K.A.B., J.B., J.E.C., M.S.C., R.M.C., J.C.E., E.H., A.J., C.J.L., J.L., F.H.M., P.J.N., K.O., G.O., J.C.P., M.P., N.R., P.R., B.G.T., R.T.S., C.d.G., G.M., and B.T. are or have been employees of Heptares Therapeutics and are shareholders of Sosei Heptares., (Copyright © 2021. Published by Elsevier Inc.)

Drawing on risk methods from volcano crises, we developed a rapid COVID-19 infection model for the partial return of pupils to primary schools in England in June and July 2020, and a full return in September 2020. The model handles uncertainties in key parameters, using a stochastic re-sampling technique, allowing us to evaluate infection levels as a function of COVID-19 prevalence and projected pupil and staff headcounts. Assuming average national adult prevalence, for the first scenario (as at 1 June 2020) we found that between 178 and 924 [90% CI] schools would have at least one infected individual, out of 16 769 primary schools in total. For the second return (July), our estimate ranged between 336 (2%) and 1873 (11%) infected schools. For a full return in September 2020, our projected range was 661 (4%) to 3310 (20%) infected schools, assuming the same prevalence as for 5 June. If national prevalence fell to one-quarter of that, the projected September range would decrease to between 381 (2%) and 900 (5%) schools but would increase to between 2131 (13%) and 9743 (58%) schools if prevalence increased to 4× June level. When regional variations in prevalence and school size distribution were included in the model, a slight decrease in the projected number of infected schools was indicated, but uncertainty on estimates increased markedly. The latter model variant indicated that 82% of infected schools would be in areas where prevalence exceeded the national average and the probability of multiple infected persons in a school would be higher in such areas. Post hoc , our model projections for 1 September 2020 were seen to have been realistic and reasonable (in terms of related uncertainties) when data on schools' infections were released by official agencies following the start of the 2020/2021 academic year., (© 2021 The Authors.)

In this study, we determined the crystal structure of an engineered human adenosine A 2A receptor bound to a partial agonist and compared it to structures cocrystallized with either a full agonist or an antagonist/inverse agonist. The interaction between the partial agonist, belonging to a class of dicyanopyridines, and amino acids in the ligand binding pocket inspired us to develop a small library of derivatives and assess their affinity in radioligand binding studies and potency and intrinsic activity in a functional, label-free, intact cell assay. It appeared that some of the derivatives retained the partial agonist profile, whereas other ligands turned into inverse agonists. We rationalized this remarkable behavior with additional computational docking studies.

Personal contacts drive COVID-19 infections. After being closed (23 March 2020) UK primary schools partially re-opened on 1 June 2020 with social distancing and new risk mitigation strategies. We conducted a structured expert elicitation of teachers to quantify primary school contact patterns and how contact rates changed upon re-opening with risk mitigation measures in place. These rates, with uncertainties, were determined using a performance-based algorithm. We report mean number of contacts per day for four cohorts within schools, with associated 90% confidence ranges. Prior to lockdown, younger children (Reception and Year 1) made 15 contacts per day [range 8.35] within school, older children (Year 6) 18 contacts [range 5.55], teaching staff 25 contacts [range 4.55] and non-classroom staff 11 contacts [range 2.27]. After re-opening, the mean number of contacts was reduced by 53% for young children, 62% for older children, 60% for classroom staff and 64% for other staff. Contacts between teaching and non-teaching staff reduced by 80%. The distributions of contacts per person are asymmetric with heavy tail reflecting a few individuals with high contact numbers. Questions on risk mitigation and supplementary structured interviews elucidated how new measures reduced daily contacts in-school and contribute to infection risk reduction., Competing Interests: We declare we have no competing interests., (© 2021 The Authors.)

Illnesses transmitted by food and water cause a major disease burden in the United States despite advancements in food safety, water treatment, and sanitation. We report estimates from a structured expert judgment study using 48 experts who applied Cooke's classical model of the proportion of disease attributable to 5 major transmission pathways (foodborne, waterborne, person-to-person, animal contact, and environmental) and 6 subpathways (food handler-related, under foodborne; recreational, drinking, and nonrecreational/nondrinking, under waterborne; and presumed person-to-person-associated and presumed animal contact-associated, under environmental). Estimates for 33 pathogens were elicited, including bacteria such as Salmonella enterica, Campylobacter spp., Legionella spp., and Pseudomonas spp.; protozoa such as Acanthamoeba spp., Cyclospora cayetanensis, and Naegleria fowleri; and viruses such as norovirus, rotavirus, and hepatitis A virus. The results highlight the importance of multiple pathways in the transmission of the included pathogens and can be used to guide prioritization of public health interventions.

We present a robust protocol based on iterations of free energy perturbation (FEP) calculations, chemical synthesis, biophysical mapping and X-ray crystallography to reveal the binding mode of an antagonist series to the A 2A adenosine receptor (AR). Eight A 2A AR binding site mutations from biophysical mapping experiments were initially analyzed with sidechain FEP simulations, performed on alternate binding modes. The results distinctively supported one binding mode, which was subsequently used to design new chromone derivatives. Their affinities for the A 2A AR were experimentally determined and investigated through a cycle of ligand-FEP calculations, validating the binding orientation of the different chemical substituents proposed. Subsequent X-ray crystallography of the A 2A AR with a low and a high affinity chromone derivative confirmed the predicted binding orientation. The new molecules and structures here reported were driven by free energy calculations, and provide new insights on antagonist binding to the A 2A AR, an emerging target in immuno-oncology., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

The orexin system, which consists of the two G protein-coupled receptors OX 1 and OX 2 , activated by the neuropeptides OX-A and OX-B, is firmly established as a key regulator of behavioral arousal, sleep, and wakefulness and has been an area of intense research effort over the past two decades. X-ray structures of the receptors in complex with 10 new antagonist ligands from diverse chemotypes are presented, which complement the existing structural information for the system and highlight the critical importance of lipophilic hotspots and water molecules for these peptidergic GPCR targets. Learnings from the structural information regarding the utility of pharmacophore models and how selectivity between OX 1 and OX 2 can be achieved are discussed.

A series of novel allosteric antagonists of the GLP-1 receptor (GLP-1R), exemplified by HTL26119, are described. SBDD approaches were employed to identify HTL26119, exploiting structural understanding of the allosteric binding site of the closely related Glucagon receptor (GCGR) (Jazayeri et al., 2016) and the homology relationships between GCGR and GLP-1R. The region around residue C347 6.36b of the GLP-1R receptor represents a key difference from GCGR and was targeted for selectivity for GLP-1R., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The increase of multidrug resistance and resistance to last-line antibiotics is a major global public health threat. Although surveillance programs provide useful current and historical information on the scale of the problem, the future emergence and spread of antibiotic resistance is uncertain, and quantifying this uncertainty is crucial for guiding decisions about investment in antibiotics and resistance control strategies. Mathematical and statistical models capable of projecting future rates are challenged by the paucity of data and the complexity of the emergence and spread of resistance, but experts have relevant knowledge. We use the Classical Model of structured expert judgment to elicit projections with uncertainty bounds of resistance rates through 2026 for nine pathogen-antibiotic pairs in four European countries and empirically validate the assessments against data on a set of calibration questions. The performance-weighted combination of experts in France, Spain, and the United Kingdom projected that resistance for five pairs on the World Health Organization's priority pathogens list (E. coli and K. pneumoniae resistant to third-generation cephalosporins and carbapenems and MRSA) would remain below 50% in 2026. In Italy, although upper bounds of 90% credible ranges exceed 50% resistance for some pairs, the medians suggest Italy will sustain or improve its current rates. We compare these expert projections to statistical forecasts based on historical data from the European Antimicrobial Resistance Surveillance Network (EARS-Net). Results from the statistical models differ from each other and from the judgmental forecasts in many cases. The judgmental forecasts include information from the experts about the impact of current and future shifts in infection control, antibiotic usage, and other factors that cannot be easily captured in statistical forecasts, demonstrating the potential of structured expert judgment as a tool for better understanding the uncertainty about future antibiotic resistance., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: Alec Morton has received a speakers’ fee from the Office of Health Economics for participation in a workshop on health technology assessment for new antibiotics, sponsored by pharmaceutical companies. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Despite considerable advances in process understanding, numerical modeling, and the observational record of ice sheet contributions to global mean sea-level rise (SLR) since the Fifth Assessment Report (AR5) of the Intergovernmental Panel on Climate Change, severe limitations remain in the predictive capability of ice sheet models. As a consequence, the potential contributions of ice sheets remain the largest source of uncertainty in projecting future SLR. Here, we report the findings of a structured expert judgement study, using unique techniques for modeling correlations between inter- and intra-ice sheet processes and their tail dependences. We find that since the AR5, expert uncertainty has grown, in particular because of uncertain ice dynamic effects. For a +2 °C temperature scenario consistent with the Paris Agreement, we obtain a median estimate of a 26 cm SLR contribution by 2100, with a 95th percentile value of 81 cm. For a +5 °C temperature scenario more consistent with unchecked emissions growth, the corresponding values are 51 and 178 cm, respectively. Inclusion of thermal expansion and glacier contributions results in a global total SLR estimate that exceeds 2 m at the 95th percentile. Our findings support the use of scenarios of 21st century global total SLR exceeding 2 m for planning purposes. Beyond 2100, uncertainty and projected SLR increase rapidly. The 95th percentile ice sheet contribution by 2200, for the +5 °C scenario, is 7.5 m as a result of instabilities coming into play in both West and East Antarctica. Introducing process correlations and tail dependences increases estimates by roughly 15%., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

The discovery of ligands via affinity-mediated selection of DNA-encoded chemical libraries is driven by the quality and concentration of the protein target. G-protein-coupled receptors (GPCRs) and other membrane-bound targets can be difficult to isolate in their functional state and at high concentrations, and therefore have been challenging for affinity-mediated selection. Here, we report a successful selection campaign against protease-activated receptor 2 (PAR2). Using a thermo-stabilized mutant of PAR2, we conducted affinity selection using our >100-billion-compound DNA-encoded library. We observed a number of putative ligands enriched upon selection, and subsequent cellular profiling revealed these ligands to comprise both agonists and antagonists. The agonist series shared structural similarity with known agonists. The antagonists were shown to bind in a novel allosteric binding site on the PAR2 protein. This report serves to demonstrate that cell-free affinity selection against GPCRs can be achieved with mutant stabilized protein targets.

Here we report an efficient method to generate multiple co-structures of the A 2A G protein-coupled receptor (GPCR) with small-molecules from a single preparation of a thermostabilised receptor crystallised in Lipidic Cubic Phase (LCP). Receptor crystallisation is achieved following purification using a low affinity "carrier" ligand (theophylline) and crystals are then soaked in solutions containing the desired (higher affinity) compounds. Complete datasets to high resolution can then be collected from single crystals and seven structures are reported here of which three are novel. The method significantly improves structural throughput for ligand screening using stabilised GPCRs, thereby actively driving Structure-Based Drug Discovery (SBDD).

The complement system is a crucial component of the host response to infection and tissue damage. Activation of the complement cascade generates anaphylatoxins including C5a and C3a. C5a exerts a pro-inflammatory effect via the G-protein-coupled receptor C5a anaphylatoxin chemotactic receptor 1 (C5aR1, also known as CD88) that is expressed on cells of myeloid origin. Inhibitors of the complement system have long been of interest as potential drugs for the treatment of diseases such as sepsis, rheumatoid arthritis, Crohn's disease and ischaemia-reperfusion injuries. More recently, a role of C5a in neurodegenerative conditions such as Alzheimer's disease has been identified. Peptide antagonists based on the C5a ligand have progressed to phase 2 trials in psoriasis and rheumatoid arthritis; however, these compounds exhibited problems with off-target activity, production costs, potential immunogenicity and poor oral bioavailability. Several small-molecule competitive antagonists for C5aR1, such as W-54011 and NDT9513727, have been identified by C5a radioligand-binding assays. NDT9513727 is a non-peptide inverse agonist of C5aR1, and is highly selective for the primate and gerbil receptors over those of other species. Here, to study the mechanism of action of C5a antagonists, we determine the structure of a thermostabilized C5aR1 (known as C5aR1 StaR) in complex with NDT9513727. We found that the small molecule bound between transmembrane helices 3, 4 and 5, outside the helical bundle. One key interaction between the small molecule and residue Trp213 5.49 seems to determine the species selectivity of the compound. The structure demonstrates that NDT9513727 exerts its inverse-agonist activity through an extra-helical mode of action.

Where policy and science intersect, there are always issues of ambiguous and conflicting lines of evidence. Combining disparate information sources is mathematically complex; common heuristics based on simple statistical models easily lead us astray. Here, we use Bayesian Nets (BNs) to illustrate the complexity in reasoning under uncertainty. Data from joint research at Resources for the Future and NASA Langley are used to populate a BN for predicting equilibrium climate sensitivity (ECS). The information sources consist of measuring the rate of decadal temperature rise (DTR) and measuring the rate of percentage change in cloud radiative forcing (CRF), with both the existing configuration of satellites and with a proposed enhanced measuring system. The goal of all measurements is to reduce uncertainty in equilibrium climate sensitivity. Subtle aspects of probabilistic reasoning with concordant and discordant measurements are illustrated. Relative to the current prior distribution on ECS, we show that after 30 years of observing with the current systems, the 2 σ uncertainty band for ECS would be shrunk on average to 73% of its current value. With the enhanced systems over the same time, it would be shrunk to 32% of its current value. The actual shrinkage depends on the values actually observed. These results are based on models recommended by the Social Cost of Carbon methodology and assume a Business as Usual emissions path.

The four adenosine receptors (ARs), A 1 , A 2A , A 2B , and A 3 , constitute a subfamily of G protein-coupled receptors (GPCRs) with exceptional foundations for structure-based ligand design. The vast amount of mutagenesis data, accumulated in the literature since the 1990s, has been recently supplemented with structural information, currently consisting of several inactive and active structures of the A 2A and inactive conformations of the A 1 ARs. We provide the first integrated view of the pharmacological, biochemical, and structural data available for this receptor family, by mapping onto the relevant crystal structures all site-directed mutagenesis data, curated and deposited at the GPCR database (available through http://www.gpcrdb.org). This analysis provides novel insights into ligand binding, allosteric modulation, and signaling of the AR family., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Ligand-protein binding kinetic rates are growing in importance as parameters to consider in drug discovery and lead optimization. In this study we analysed using surface plasmon resonance (SPR) the transition state (TS) properties of a set of six adenosine A 2A receptor inhibitors, belonging to both the xanthine and the triazolo-triazine scaffolds. SPR highlighted interesting differences among the ligands in the enthalpic and entropic components of the TS energy barriers for the binding and unbinding events. To better understand at a molecular level these differences, we developed suMetaD, a novel molecular dynamics (MD)-based approach combining supervised MD and metadynamics. This method allows simulation of the ligand unbinding and binding events. It also provides the system conformation corresponding to the highest energy barrier the ligand is required to overcome to reach the final state. For the six ligands evaluated in this study their TS thermodynamic properties were linked in particular to the role of water molecules in solvating/desolvating the pocket and the small molecules. suMetaD identified kinetic bottleneck conformations near the bound state position or in the vestibule area. In the first case the barrier is mainly enthalpic, requiring the breaking of strong interactions with the protein. In the vestibule TS location the kinetic bottleneck is instead mainly of entropic nature, linked to the solvent behaviour.

Historically, room-temperature structure determination was succeeded by cryo-crystallography to mitigate radiation damage. Here, we demonstrate that serial millisecond crystallography at a synchrotron beamline equipped with high-viscosity injector and high frame-rate detector allows typical crystallographic experiments to be performed at room-temperature. Using a crystal scanning approach, we determine the high-resolution structure of the radiation sensitive molybdenum storage protein, demonstrate soaking of the drug colchicine into tubulin and native sulfur phasing of the human G protein-coupled adenosine receptor. Serial crystallographic data for molecular replacement already converges in 1,000-10,000 diffraction patterns, which we collected in 3 to maximally 82 minutes. Compared with serial data we collected at a free-electron laser, the synchrotron data are of slightly lower resolution, however fewer diffraction patterns are needed for de novo phasing. Overall, the data we collected by room-temperature serial crystallography are of comparable quality to cryo-crystallographic data and can be routinely collected at synchrotrons.Serial crystallography was developed for protein crystal data collection with X-ray free-electron lasers. Here the authors present several examples which show that serial crystallography using high-viscosity injectors can also be routinely employed for room-temperature data collection at synchrotrons.

This corrects the article DOI: 10.1038/nature22800.

The adenosine A 1 and A 2A receptors belong to the purinergic family of G protein-coupled receptors, and regulate diverse functions of the cardiovascular, respiratory, renal, inflammation, and CNS. Xanthines such as caffeine and theophylline are weak, non-selective antagonists of adenosine receptors. Here we report the structure of a thermostabilized human A 1 receptor at 3.3 Å resolution with PSB36, an A 1 -selective xanthine-based antagonist. This is compared with structures of the A 2A receptor with PSB36 (2.8 Å resolution), caffeine (2.1 Å), and theophylline (2.0 Å) to highlight features of ligand recognition which are common across xanthines. The structures of A 1 R and A 2A R were analyzed to identify the differences that are important selectivity determinants for xanthine ligands, and the role of T270 7.35 in A 1 R (M270 7.35 in A 2A R) in conferring selectivity was confirmed by mutagenesis. The structural differences confirmed to lead to selectivity can be utilized in the design of new subtype-selective A 1 R or A 2A R antagonists., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Glucagon-like peptide 1 (GLP-1) regulates glucose homeostasis through the control of insulin release from the pancreas. GLP-1 peptide agonists are efficacious drugs for the treatment of diabetes. To gain insight into the molecular mechanism of action of GLP-1 peptides, here we report the crystal structure of the full-length GLP-1 receptor bound to a truncated peptide agonist. The peptide agonist retains an α-helical conformation as it sits deep within the receptor-binding pocket. The arrangement of the transmembrane helices reveals hallmarks of an active conformation similar to that observed in class A receptors. Guided by this structural information, we design peptide agonists with potent in vivo activity in a mouse model of diabetes.

A considerable number of approved drugs show non-equilibrium binding characteristics, emphasizing the potential role of drug residence times for in vivo efficacy. Therefore, a detailed understanding of the kinetics of association and dissociation of a target-ligand complex might provide crucial insight into the molecular mechanism-of-action of a compound. This deeper understanding will help to improve decision making in drug discovery, thus leading to a better selection of interesting compounds to be profiled further. In this review, we highlight the contributions of the Kinetics for Drug Discovery (K4DD) Consortium, which targets major open questions related to binding kinetics in an industry-driven public-private partnership., (Copyright © 2017. Published by Elsevier Ltd.)

Protease-activated receptors (PARs) are a family of G-protein-coupled receptors (GPCRs) that are irreversibly activated by proteolytic cleavage of the N terminus, which unmasks a tethered peptide ligand that binds and activates the transmembrane receptor domain, eliciting a cellular cascade in response to inflammatory signals and other stimuli. PARs are implicated in a wide range of diseases, such as cancer and inflammation. PARs have been the subject of major pharmaceutical research efforts but the discovery of small-molecule antagonists that effectively bind them has proved challenging. The only marketed drug targeting a PAR is vorapaxar, a selective antagonist of PAR1 used to prevent thrombosis. The structure of PAR1 in complex with vorapaxar has been reported previously. Despite sequence homology across the PAR isoforms, discovery of PAR2 antagonists has been less successful, although GB88 has been described as a weak antagonist. Here we report crystal structures of PAR2 in complex with two distinct antagonists and a blocking antibody. The antagonist AZ8838 binds in a fully occluded pocket near the extracellular surface. Functional and binding studies reveal that AZ8838 exhibits slow binding kinetics, which is an attractive feature for a PAR2 antagonist competing against a tethered ligand. Antagonist AZ3451 binds to a remote allosteric site outside the helical bundle. We propose that antagonist binding prevents structural rearrangements required for receptor activation and signalling. We also show that a blocking antibody antigen-binding fragment binds to the extracellular surface of PAR2, preventing access of the tethered ligand to the peptide-binding site. These structures provide a basis for the development of selective PAR2 antagonists for a range of therapeutic uses.

Chemokines and their G-protein-coupled receptors play a diverse role in immune defence by controlling the migration, activation and survival of immune cells. They are also involved in viral entry, tumour growth and metastasis and hence are important drug targets in a wide range of diseases. Despite very significant efforts by the pharmaceutical industry to develop drugs, with over 50 small-molecule drugs directed at the family entering clinical development, only two compounds have reached the market: maraviroc (CCR5) for HIV infection and plerixafor (CXCR4) for stem-cell mobilization. The high failure rate may in part be due to limited understanding of the mechanism of action of chemokine antagonists and an inability to optimize compounds in the absence of structural information. CC chemokine receptor type 9 (CCR9) activation by CCL25 plays a key role in leukocyte recruitment to the gut and represents a therapeutic target in inflammatory bowel disease. The selective CCR9 antagonist vercirnon progressed to phase 3 clinical trials in Crohn's disease but efficacy was limited, with the need for very high doses to block receptor activation. Here we report the crystal structure of the CCR9 receptor in complex with vercirnon at 2.8 Å resolution. Remarkably, vercirnon binds to the intracellular side of the receptor, exerting allosteric antagonism and preventing G-protein coupling. This binding site explains the need for relatively lipophilic ligands and describes another example of an allosteric site on G-protein-coupled receptors that can be targeted for drug design, not only at CCR9, but potentially extending to other chemokine receptors.

The association and dissociation kinetics of ligands binding to proteins vary considerably, but the mechanisms behind this variability are poorly understood, limiting their utilization for drug discovery. This is particularly so for G protein-coupled receptors (GPCRs) where high resolution structural information is only beginning to emerge. Engineering the human A2A adenosine receptor has allowed structures to be solved in complex with the reference compound ZM241385 and four related ligands at high resolution. Differences between the structures are limited, with the most pronounced being the interaction of each ligand with a salt bridge on the extracellular side of the receptor. Mutagenesis experiments confirm the role of this salt bridge in controlling the dissociation kinetics of the ligands from the receptor, while molecular dynamics simulations demonstrate the ability of ligands to modulate salt bridge stability. These results shed light on a structural determinant of ligand dissociation kinetics and identify a means by which this property may be optimized.

Glucagon is a 29-amino-acid peptide released from the α-cells of the islet of Langerhans, which has a key role in glucose homeostasis. Glucagon action is transduced by the class B G-protein-coupled glucagon receptor (GCGR), which is located on liver, kidney, intestinal smooth muscle, brain, adipose tissue, heart and pancreas cells, and this receptor has been considered an important drug target in the treatment of diabetes. Administration of recently identified small-molecule GCGR antagonists in patients with type 2 diabetes results in a substantial reduction of fasting and postprandial glucose concentrations. Although an X-ray structure of the transmembrane domain of the GCGR has previously been solved, the ligand (NNC0640) was not resolved. Here we report the 2.5 Å structure of human GCGR in complex with the antagonist MK-0893 (ref. 4), which is found to bind to an allosteric site outside the seven transmembrane (7TM) helical bundle in a position between TM6 and TM7 extending into the lipid bilayer. Mutagenesis of key residues identified in the X-ray structure confirms their role in the binding of MK-0893 to the receptor. The unexpected position of the binding site for MK-0893, which is structurally similar to other GCGR antagonists, suggests that glucagon activation of the receptor is prevented by restriction of the outward helical movement of TM6 required for G-protein coupling. Structural knowledge of class B receptors is limited, with only one other ligand-binding site defined--for the corticotropin-releasing hormone receptor 1 (CRF1R)--which was located deep within the 7TM bundle. We describe a completely novel allosteric binding site for class B receptors, providing an opportunity for structure-based drug design for this receptor class and furthering our understanding of the mechanisms of activation of these receptors.

For many societally important science-based decisions, data are inadequate, unreliable or non-existent, and expert advice is sought. In such cases, procedures for eliciting structured expert judgments (SEJ) are increasingly used. This raises questions regarding validity and reproducibility. This paper presents new findings from a large-scale international SEJ study intended to estimate the global burden of foodborne disease on behalf of WHO. The study involved 72 experts distributed over 134 expert panels, with panels comprising thirteen experts on average. Elicitations were conducted in five languages. Performance-based weighted solutions for target questions of interest were formed for each panel. These weights were based on individual expert's statistical accuracy and informativeness, determined using between ten and fifteen calibration variables from the experts' field with known values. Equal weights combinations were also calculated. The main conclusions on expert performance are: (1) SEJ does provide a science-based method for attribution of the global burden of foodborne diseases; (2) equal weighting of experts per panel increased statistical accuracy to acceptable levels, but at the cost of informativeness; (3) performance-based weighting increased informativeness, while retaining accuracy; (4) due to study constraints individual experts' accuracies were generally lower than in other SEJ studies, and (5) there was a negative correlation between experts' informativeness and statistical accuracy which attenuated as accuracy improved, revealing that the least accurate experts drive the negative correlation. It is shown, however, that performance-based weighting has the ability to yield statistically accurate and informative combinations of experts' judgments, thereby offsetting this contrary influence. The present findings suggest that application of SEJ on a large scale is feasible, and motivate the development of enhanced training and tools for remote elicitation of multiple, internationally-dispersed panels.

X-ray structures of G protein-coupled receptors (GPCRs) have now been reported for more than 60 ligands and 20 receptors, including examples from GPCR classes A, B, C and F. The new structures show previously unobtainable details of interactions between GPCRs and ligands, including the roles of lipophilic regions and water molecules as key drivers of binding. In addition, the structures have revealed several surprising ligand-binding modes, including sites outside the orthosteric pocket. This new information is dramatically changing the way we approach GPCR drug discovery., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The options for investigating solubilised G protein-coupled receptors (GPCRs) by biophysical techniques have long been hampered by their instability. A thermostabilised adenosine A2A receptor expressed in insect cells, purified in detergent and reconstituted into high-density lipoprotein (HDL) particles was immobilised onto a Surface Plasmon Resonance sensor chip. This allowed measurement of affinities and kinetics for A2A antagonists with affinities ranging from 50 pM to almost 2 μM. Compared with other formats, reproduction of affinities, and dissociation and association rate constants are good, reasonable and poor respectively, indicating stabilised receptors in HDL particles are useful for investigating specific aspects of GPCR-ligand interactions., (Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Ecosystems often experience multiple environmental stressors simultaneously that can differ widely in their pathways and strengths of impact. Differences in the relative impact of environmental stressors can guide restoration and management prioritization, but few studies have empirically assessed a comprehensive suite of stressors acting on a given ecosystem. To fill this gap in the Laurentian Great Lakes, where considerable restoration investments are currently underway, we used expert elicitation via a detailed online survey to develop ratings of the relative impacts of 50 potential stressors. Highlighting the multiplicity of stressors in this system, experts assessed all 50 stressors as having some impact on ecosystem condition, but ratings differed greatly among stressors. Individual stressors related to invasive and nuisance species (e.g., dreissenid mussels and ballast invasion risk) and climate change were assessed as having the greatest potential impacts. These results mark a shift away from the longstanding emphasis on nonpoint phosphorus and persistent bioaccumulative toxic substances in the Great Lakes. Differences in impact ratings among lakes and ecosystem zones were weak, and experts exhibited surprisingly high levels of agreement on the relative impacts of most stressors. Our results provide a basin-wide, quantitative summary of expert opinion on the present-day influence of all major Great Lakes stressors. The resulting ratings can facilitate prioritizing stressors to achieve management objectives in a given location, as well as providing a baseline for future stressor impact assessments in the Great Lakes and elsewhere.

Identifying which nonindigenous species will become invasive and forecasting the damage they will cause is difficult and presents a significant problem for natural resource management. Often, the data or resources necessary for ecological risk assessment are incomplete or absent, leaving environmental decision makers ill equipped to effectively manage valuable natural resources. Structured expert judgment (SEJ) is a mathematical and performance-based method of eliciting, weighting, and aggregating expert judgments. In contrast to other methods of eliciting and aggregating expert judgments (where, for example, equal weights may be assigned to experts), SEJ weights each expert on the basis of his or her statistical accuracy and informativeness through performance measurement on a set of calibration variables. We used SEJ to forecast impacts of nonindigenous Asian carp (Hypophthalmichthys spp.) in Lake Erie, where it is believed not to be established. Experts quantified Asian carp biomass, production, and consumption and their impact on 4 fish species if Asian carp were to become established. According to experts, in Lake Erie Asian carp have the potential to achieve biomass levels that are similar to the sum of biomasses for several fishes that are harvested commercially or recreationally. However, the impact of Asian carp on the biomass of these fishes was estimated by experts to be small, relative to long term average biomasses, with little uncertainty. Impacts of Asian carp in tributaries and on recreational activities, water quality, or other species were not addressed. SEJ can be used to quantify key uncertainties of invasion biology and also provide a decision-support tool when the necessary information for natural resource management and policy is not available., (© 2014 The Authors. Conservation Biology published by Wiley Periodicals, Inc. on behalf of Society for Conservation Biology.)

G protein-coupled receptors (GPCRs) are of particular importance for drug discovery, being the targets of many existing drugs, and being linked to many diseases where new therapies are required. However, as integral membrane proteins, they are generally unstable when removed from their membrane environment, precluding them from the wide range of structural and biophysical techniques which can be applied to soluble proteins such as kinases. Through the use of protein engineering methods, mutations can be identified which both increase the thermostability of GPCRs when purified in detergent, as well as biasing the receptor toward a specific physiologically relevant conformational state. The resultant stabilized receptor (known as a StaR) can be purified in multiple-milligram quantities, whilst retaining correct folding, thus enabling the generation of reagents suitable for a broad range of structural and biophysical studies. Example protocols for the purification of StaR proteins for analysis, ligand screening with the thiol-specific fluorochrome N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide (CPM), surface plasmon resonance (SPR), and crystallization for structural studies are presented.

This responds to an "evaluation" of the classical model for structured expert judgment by Bolger and Rowe in this issue. This response references extensive expert judgment performance data in the public domain which played no role in their evaluation., (© 2015 Society for Risk Analysis.)

Structured expert judgment (SEJ) is used to quantify the uncertainty of nonindigenous fish (bighead carp [Hypophthalmichthys nobilis] and silver carp [H. molitrix]) establishment in Lake Erie. The classical model for structured expert judgment model is applied. Forming a weighted combination (called a decision maker) of experts' distributions, with weights derived from performance on a set of calibration variables from the experts' field, exhibits greater statistical accuracy and greater informativeness than simple averaging with equal weights. New methods of cross validation are applied and suggest that performance characteristics relative to equal weighting could be predicted with a small number (1-2) of calibration variables. The performance-based decision maker is somewhat degraded on out-of-sample prediction, but remained superior to the equal weight decision maker in terms of statistical accuracy and informativeness., (© 2014 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals, Inc. on behalf of SETAC.)

Metabotropic glutamate receptors are class C G-protein-coupled receptors which respond to the neurotransmitter glutamate. Structural studies have been restricted to the amino-terminal extracellular domain, providing little understanding of the membrane-spanning signal transduction domain. Metabotropic glutamate receptor 5 is of considerable interest as a drug target in the treatment of fragile X syndrome, autism, depression, anxiety, addiction and movement disorders. Here we report the crystal structure of the transmembrane domain of the human receptor in complex with the negative allosteric modulator, mavoglurant. The structure provides detailed insight into the architecture of the transmembrane domain of class C receptors including the precise location of the allosteric binding site within the transmembrane domain and key micro-switches which regulate receptor signalling. This structure also provides a model for all class C G-protein-coupled receptors and may aid in the design of new small-molecule drugs for the treatment of brain disorders.

Purpose: Candidate risk factors for idiopathic rhegmatogenous retinal detachment (RRD) include heavy manual handling (requiring Valsalva's maneuver). We assessed incidence rates of surgically treated idiopathic RRD among manual workers, non-manual workers and housewives resident in Tuscany, Italy., Methods: We retrieved all hospital discharge records bearing a principal diagnosis corresponding to RRD coupled with retinal surgery for any resident of Tuscany during 1997-2009. After elimination of repeated admissions and patients with coexistent, associated conditions (including recent trauma), subjects aged 25-59 years were classified as manual workers, non-manual workers or housewives. Population data were extracted from the 2001 census., Results: We identified 1,946 eligible cases (1,142 men). Among men, manual workers experienced a 1.8-fold higher age-standardized rate per 100,000 person-years than non-manual workers [17.4 (95 % confidence interval (CI) 16.1-18.7) vs. 9.8 (95 % CI 8.8-10.8)]. Age-standardized rates among women were 1.9-fold higher for manual workers [11.1 (95 % CI 9.8-12.3)] and 1.7-fold higher for housewives [9.5 (95 % CI 8.3-10.8)] than in non-manual workers [5.7 (95 % CI 4.8-6.6)]., Conclusions: This large population-based study suggests that manual workers are affected by idiopathic RRD requiring surgical treatment more often than non-manual workers. The higher rates of surgically treated RRD experienced by manual workers are in accord with the hypothesis that heavy manual handling may have a causal role.

Recently, authors have theorized that invasive species prevention is more cost-effective than control in protecting ecosystem services. However, quantification of the effectiveness of prevention is rare because experiments at field scales are expensive or infeasible. We therefore used structured expert judgment to quantify the efficacy of 17 proposed strategies to prevent Asian carp invasion of the Laurentian Great Lakes via the hydrologic connection between the Mississippi and Great Lakes watersheds. Performance-weighted expert estimates indicated that hydrologic separation would prevent 99% (95,100; median, 5th and 95th percentiles) of Asian carp access, while electric and acoustic-bubble-strobe barriers would prevent 92% (85,95) and 92% (75,95), respectively. For all other strategies, estimated effectiveness was lower, with greater uncertainty. When potential invasions by other taxa are considered, the effectiveness of hydrologic separation increases relative to strategies that are effective primarily for fishes. These results could help guide invasive species management in many waterways globally.

Structural analysis of class B G-protein-coupled receptors (GPCRs), cell-surface proteins that respond to peptide hormones, has been restricted to the amino-terminal extracellular domain, thus providing little understanding of the membrane-spanning signal transduction domain. The corticotropin-releasing factor receptor type 1 is a class B receptor which mediates the response to stress and has been considered a drug target for depression and anxiety. Here we report the crystal structure of the transmembrane domain of the human corticotropin-releasing factor receptor type 1 in complex with the small-molecule antagonist CP-376395. The structure provides detailed insight into the architecture of class B receptors. Atomic details of the interactions of the receptor with the non-peptide ligand that binds deep within the receptor are described. This structure provides a model for all class B GPCRs and may aid in the design of new small-molecule drugs for diseases of brain and metabolism.

Nitric oxide (NO) is an important signaling molecule with a variety of functions in the CNS, including a potential role in modulating neuronal growth and synapse formation. In the present study, we used tractable, identified neurons in the CNS of the pond snail Lymnaea stagnalis to study the role of endogenous NO signaling in neuronal growth and synaptic remodeling after nerve injury. Axonal damage of L. stagnalis neurons B1 and B2 induces extensive central growth of neurites that is accompanied by changes in existing electrical connections, the transient formation of novel electrical connections, and the formation of a novel excitatory chemical synapse from B2 to B1 neurons. Partial chronic inhibition of endogenous NO synthesis reduces neurite growth in NO-synthase-expressing B2, but has only minor effects on NOS-negative B1 neurons. Chronic application of an NO donor while inhibiting endogenous NO synthesis rescues neurite extension in B2 neurons and boosts growth of B1 neurons. Blocking soluble guanylate cyclase activity completely suppresses neurite extension and synaptic remodeling after nerve crush, demonstrating the importance of cGMP in these processes. Interestingly, inhibition of cGMP-dependent protein kinase only suppresses chemical synapse formation without effects on neuronal growth and electrical synapse remodeling. We conclude that NO signaling via cGMP is an important modulator of both neurite growth and synaptic remodeling after nerve crush. However, differential effects of cGMP-dependent protein kinase inhibition on neurite growth and synaptic remodeling suggest that these effects are mediated by separate signaling pathways.

Since the publication of the first X-ray structure of a GPCR (G-protein couple receptor) in 2000, the rate at which subsequent ones have appeared has steadily increased. This has required the development of new methodology to overcome the challenges presented by instability of isolated GPCRs, combined with a systematic optimization of existing approaches for protein expression, purification and crystallization. In addition, quality control measures that are predictive of successful outcomes have been identified. Repeated attempts at solving the structures of GPCRs have highlighted experimental approaches that are most likely to lead to success, and have allowed definition of a first-pass protocol for new receptors.

The dynamic economic behavior in most integrated assessment models linking economic growth to climate change involves a differential equation solved by Jacob Bernoulli in 1695. Using the dynamic integrated climate economy (DICE) model and freezing exogenous variables at initial values, this dynamic is shown to produce implausible projections on a 60-year time frame. If world capital started at US$1, after 60 years the world economy would be indistinguishable from one starting with 10 times the current capitalization. Such behavior points to uncertainty at the level of the fundamental dynamics, and suggests that discussions of discounting, utility, damage functions, and ethics should be conducted within a more general modeling vocabulary. Lotka Volterra dynamics is proposed as an alternative with greater prime facie plausibility. With near universality, economists assume that economic growth will go on forever. Lotka Volterra dynamics alert us to the possibility of collapse., (Copyright © 2012 SETAC.)

Background: We investigated a possible association between pharyngeal/tonsillar carcinoma and mixed carcinogen exposures in an asphalt roll company in Italy that used asbestos until 1979, when a new factory was built using a different production process., Methods: We evaluated all workers involved in the entire production history of the company, divided into two subcohorts based on exposure status (workers in the original factory, 1964-1979, and those who worked only in the new factory, 1980-1997). We ascertained the vital status of the study population in February 2001., Results: Among the subset of workers in the earlier subcohort, there were five deaths from pharyngeal/tonsillar carcinoma for a standardized mortality ratio of 21 (95% confidence interval = 8.8-51). No cases were recorded among workers hired after 1979., Conclusion: The increased standardized mortality ratio for this relatively rare cancer among workers exposed before 1979 may have been due to carcinogenic exposures at the plant.

Objective: To compare the yield of hand-searching with optimised electronic search strategies in retrieving occupational health (OH) intervention studies published in a language other than English., Methods: The authors systematically hand-searched and screened reports of OH intervention studies published in Italian in peer-reviewed scientific journals between 1990 and 2008. The authors evaluated how many of them met the Cochrane Occupational Safety and Health Review Groups (OSHRG) definition of being an OH intervention study and how many potentially relevant studies retrieved by hand-searching would not be found by PubMed alone using the OSHRG's most specific and most sensitive search strings., Results: Hand-searching retrieved 25 articles (reporting 27 studies), including nine not indexed in MEDLINE. Most studies (81%, 22/27) had a before-after design and only one was a randomised trial. The OSHRG's most sensitive search string retrieved all 16 articles published in the Italian language journals that were indexed in MEDLINE, while the most specific search strategy retrieved nine articles (56%, 9/16). The most specific search string showed a lower 'number needed to read' value than the most sensitive one (60 vs 132)., Conclusions: These findings suggest that a sensitive electronic search strategy may be able to find most of the OH interventions published in languages other than English that are indexed in MEDLINE. Hand-searching of important national journals not indexed in MEDLINE should be considered when conducting particularly in-depth research.

Purpose: Housework is a form of regular manual work that is often performed by women. Little is known about the entity of biomechanical exposure to the upper limbs during typical housework tasks. This study aims to make an initial quantitative estimate of some such exposures., Methods: We conducted objective assessments of biomechanical exposure to the upper limbs during nine frequent housework tasks performed by 12 women without domestic help. For the main evaluations, we implemented five instruments: the OCRA (Occupational Repetitive Actions) checklist; OREGE (Outil de Repérage et d'Evaluation des Gestes); the American Conference of Governmental Industrial Hygienists (ACGIH) assessment of hand activity levels (HAL); RULA (Rapid Upper Limb Assessment); and the full checklist of the Washington State Department of Labor and Industries., Results: The ACGIH evaluation suggested light/moderate biomechanical exposure levels, as did the RULA. For the OCRA checklist and OREGE, time-weighted average scores (not adjusted by a duration multiplier because women may also routinely undergo biomechanical loads in other domestic or professional settings) were: OCRA checklist, 12.0 ("mild risk", light red) and OREGE, 10.2 ("not recommended"). The full checklist of Washington State Department of Labor and Industries showed that repetitive/similar movements (for >2 h/day) accompanied by other risk factors such as ≥30° bending of the wrists, ≥45° stretched wrists, ≥30° ulnar stretches, and manual force., Conclusions: Housework commonly entails light-moderate biomechanical loads that nevertheless could conceivably contribute to the genesis/worsening of musculoskeletal disorders. Biomechanical loads experienced by women during housework deserve greater consideration in epidemiologic studies of musculoskeletal disorders.