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The ClpAP complex is a conserved bacterial protease that unfolds and degrades proteins targeted for destruction. The ClpA double-ring hexamer powers substrate unfolding and translocation into the ClpP proteolytic chamber. Here, we determined high-resolution structures of wild-type Escherichia coli ClpAP undergoing active substrate unfolding and proteolysis. A spiral of pore loop–substrate contacts spans both ClpA AAA+ domains. Protomers at the spiral seam undergo nucleotide-specific rearrangements, supporting substrate translocation. IGL loops extend flexibly to bind the planar, heptameric ClpP surface with the empty, symmetry-mismatched IGL pocket maintained at the seam. Three different structures identify a binding-pocket switch by the IGL loop of the lowest positioned protomer, involving release and re-engagement with the clockwise pocket. This switch is coupled to a ClpA rotation and a network of conformational changes across the seam, suggesting that ClpA can rotate around the ClpP apical surface during processive steps of translocation and proteolysis.Cryo-EM structures of Escherichia coli ClpAP undergoing active substrate unfolding and proteolysis reveal contacts that drive substrate translocation and a dynamic switch mechanism at the ClpA–ClpP interface.

The ClpAP complex functions as a 'bacterial proteasome' that simultaneously unfolds and degrades proteins targeted for destruction. ClpA utilizes two AAA+ domains per protomer to power substrate unfolding and translocation into the ClpP proteolytic chamber. To understand this mechanism, we determined high-resolution structures of wildtype E. coli ClpAP in distinct substrate-bound states. ClpA forms a spiral with substrate contacts across both AAA+ domains, while protomers at the seam undergo nucleotide-specific rearrangements indicating a conserved rotary mechanism. ClpA IGL loops extend flexibly to bind the planar, heptameric ClpP surface and support a large ClpA-P rotation that re-orients the translocation channel. The symmetry mismatch is maintained at the spiral seam through bind and release states of the IGL loops, which appear precisely coupled to substrate translocation. Thus, ClpA rotates around the apical surface of ClpP to processively translocate substrate into the protease.

Protein conformations are shaped by cellular environments, but how environmental changes alter the conformational landscapes of specific proteins in vivo remains largely uncharacterized, in part due to the challenge of probing protein structures in living cells. Here, we use deep mutational scanning to investigate how a toxic conformation of α-synuclein, a dynamic protein linked to Parkinson's disease, responds to perturbations of cellular proteostasis. In the context of a course for graduate students in the UCSF Integrative Program in Quantitative Biology, we screened a comprehensive library of α-synuclein point mutants in yeast cells treated with a variety of small molecules that perturb cellular processes linked to α-synuclein biology and pathobiology. We found that the conformation of α-synuclein previously shown to drive yeast toxicity - an extended, membrane-bound helix - is largely unaffected by these chemical perturbations, underscoring the importance of this conformational state as a driver of cellular toxicity. On the other hand, the chemical perturbations have a significant effect on the ability of mutations to suppress α-synuclein toxicity. Moreover, we find that sequence determinants of α-synuclein toxicity are well described by a simple structural model of the membrane-bound helix. This model predicts that α-synuclein penetrates the membrane to constant depth across its length but that membrane affinity decreases toward the C terminus, which is consistent with orthogonal biophysical measurements. Finally, we discuss how parallelized chemical genetics experiments can provide a robust framework for inquiry-based graduate coursework. Competing Interest Statement The authors have declared no competing interest.

Understanding potential trajectories in health and drivers of health is crucial to guiding long-term investments and policy implementation. Past work on forecasting has provided an incomplete landscape of future health scenarios, highlighting a need for a more robust modelling platform from which policy options and potential health trajectories can be assessed. This study provides a novel approach to modelling life expectancy, all-cause mortality and cause of death forecasts —and alternative future scenarios—for 250 causes of death from 2016 to 2040 in 195 countries and territories. We modelled 250 causes and cause groups organised by the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) hierarchical cause structure, using GBD 2016 estimates from 1990–2016, to generate predictions for 2017–40. Our modelling framework used data from the GBD 2016 study to systematically account for the relationships between risk factors and health outcomes for 79 independent drivers of health. We developed a three-component model of cause-specific mortality: a component due to changes in risk factors and select interventions; the underlying mortality rate for each cause that is a function of income per capita, educational attainment, and total fertility rate under 25 years and time; and an autoregressive integrated moving average model for unexplained changes correlated with time. We assessed the performance by fitting models with data from 1990–2006 and using these to forecast for 2007–16. Our final model used for generating forecasts and alternative scenarios was fitted to data from 1990–2016. We used this model for 195 countries and territories to generate a reference scenario or forecast through 2040 for each measure by location. Additionally, we generated better health and worse health scenarios based on the 85th and 15th percentiles, respectively, of annualised rates of change across location-years for all the GBD risk factors, income per person, educational attainment, select intervention coverage, and total fertility rate under 25 years in the past. We used the model to generate all-cause age-sex specific mortality, life expectancy, and years of life lost (YLLs) for 250 causes. Scenarios for fertility were also generated and used in a cohort component model to generate population scenarios. For each reference forecast, better health, and worse health scenarios, we generated estimates of mortality and YLLs attributable to each risk factor in the future. Globally, most independent drivers of health were forecast to improve by 2040, but 36 were forecast to worsen. As shown by the better health scenarios, greater progress might be possible, yet for some drivers such as high body-mass index (BMI), their toll will rise in the absence of intervention. We forecasted global life expectancy to increase by 4·4 years (95% UI 2·2 to 6·4) for men and 4·4 years (2·1 to 6·4) for women by 2040, but based on better and worse health scenarios, trajectories could range from a gain of 7·8 years (5·9 to 9·8) to a non-significant loss of 0·4 years (–2·8 to 2·2) for men, and an increase of 7·2 years (5·3 to 9·1) to essentially no change (0·1 years [–2·7 to 2·5]) for women. In 2040, Japan, Singapore, Spain, and Switzerland had a forecasted life expectancy exceeding 85 years for both sexes, and 59 countries including China were projected to surpass a life expectancy of 80 years by 2040. At the same time, Central African Republic, Lesotho, Somalia, and Zimbabwe had projected life expectancies below 65 years in 2040, indicating global disparities in survival are likely to persist if current trends hold. Forecasted YLLs showed a rising toll from several non-communicable diseases (NCDs), partly driven by population growth and ageing. Differences between the reference forecast and alternative scenarios were most striking for HIV/AIDS, for which a potential increase of 120·2% (95% UI 67·2–190·3) in YLLs (nearly 118 million) was projected globally from 2016–40 under the worse health scenario. Compared with 2016, NCDs were forecast to account for a greater proportion of YLLs in all GBD regions by 2040 (67·3% of YLLs [95% UI 61·9–72·3] globally); nonetheless, in many lower-income countries, communicable, maternal, neonatal, and nutritional (CMNN) diseases still accounted for a large share of YLLs in 2040 (eg, 53·5% of YLLs [95% UI 48·3–58·5] in Sub-Saharan Africa). There were large gaps for many health risks between the reference forecast and better health scenario for attributable YLLs. In most countries, metabolic risks amenable to health care (eg, high blood pressure and high plasma fasting glucose) and risks best targeted by population-level or intersectoral interventions (eg, tobacco, high BMI, and ambient particulate matter pollution) had some of the largest differences between reference and better health scenarios. The main exception was sub-Saharan Africa, where many risks associated with poverty and lower levels of development (eg, unsafe water and sanitation, household air pollution, and child malnutrition) were projected to still account for substantive disparities between reference and better health scenarios in 2040. With the present study, we provide a robust, flexible forecasting platform from which reference forecasts and alternative health scenarios can be explored in relation to a wide range of independent drivers of health. Our reference forecast points to overall improvements through 2040 in most countries, yet the range found across better and worse health scenarios renders a precarious vision of the future—a world with accelerating progress from technical innovation but with the potential for worsening health outcomes in the absence of deliberate policy action. For some causes of YLLs, large differences between the reference forecast and alternative scenarios reflect the opportunity to accelerate gains if countries move their trajectories toward better health scenarios—or alarming challenges if countries fall behind their reference forecasts. Generally, decision makers should plan for the likely continued shift toward NCDs and target resources toward the modifiable risks that drive substantial premature mortality. If such modifiable risks are prioritised today, there is opportunity to reduce avoidable mortality in the future. However, CMNN causes and related risks will remain the predominant health priority among lower-income countries. Based on our 2040 worse health scenario, there is a real risk of HIV mortality rebounding if countries lose momentum against the HIV epidemic, jeopardising decades of progress against the disease. Continued technical innovation and increased health spending, including development assistance for health targeted to the world's poorest people, are likely to remain vital components to charting a future where all populations can live full, healthy lives. Bill & Melinda Gates Foundation.

Introduction Racial and language disparities in the United States healthcare system have long undermined the quality of care provided to minority patients. With the projected growth of the Hispanic population, there is an urgent need for medical schools to integrate high-quality medical Spanish and cultural competency content. We propose a comprehensive medical Spanish curriculum aligned with the preclinical curriculum as a solution to these issues. The primary goal of this study is to demonstrate the effectiveness of a clinically focused, culturally competent medical Spanish program and advocate for its widespread adoption in medical institutions nationwide. Methods The study utilized the Kirkpatrick Model to evaluate the success of the medical Spanish curriculum. A total of 111 medical students voluntarily enrolled in the medical Spanish course. Out of these students, 47 completed the final evaluation, which included a Spanish Objective Structured Clinical Examination and a 40-question Multiple-Choice Exam assessing the integration of Spanish language skills and cultural competency. Both assessment methods took place in clinical skills facilities. Descriptive statistics summarized exam results, and two-tailed t-tests compared mean exam scores between students of different proficiency levels. Results and discussion Students achieved a mean score of over 80% on all components of the Spanish Objective Structured Clinical Examination and the Multiple-Choice Exam. Survey data suggest that students felt able to communicate in Spanish with patients after completing the course series. The study also provides a model for a medical Spanish curriculum that applies expert-recommended best practices to meet the needs of Hispanic patient populations. Limitations and conclusions Students who sat for the OSCE and MCE were self-selected. Baseline data on student perceptions and Spanish competency are not sufficient for making comparisons.

The stress‐dominance hypothesis (SDH) is a model of community assembly predicting that the relative importance of environmental filtering increases and competition decreases along a gradient of increasing environmental stress. Tests of the SDH at limited spatial scales have thus far demonstrated equivocal support and no prior study has assessed the generality of the SDH at continental scales. We examined over 53 000 tree communities spanning the eastern United States to determine whether functional trait variation and phylogenetic diversity support the SDH for gradients of water and soil nutrient availability. This analysis incorporated two complementary datasets, those of the U.S. Forest Service Forest Inventory and Analysis National program and the Carolina Vegetation Survey, and was based on three ecologically important traits: leaf nitrogen, seed mass, and wood density. We found that mean trait values were weakly correlated with water and soil nutrient availability, but that trait diversity did not vary consistently along either gradient. This did not conform to trait variation expected under the SDH and instead suggested that environmental filters structure tree communities throughout both gradients, without evidence for an increased role of competition in less stressful environments. Phylogenetic diversity of communities was principally driven by the ratio of angiosperms to gymnosperms and therefore did not exhibit the pattern of variation along stress gradients expected under the SDH. We conclude that the SDH is not a general paradigm for all eastern North American tree communities, although it may operate in certain contexts.

In addition to commonly associated environmental factors, genomic factors may cause cerebral palsy. We performed whole-exome sequencing of 250 parent–offspring trios, and observed enrichment of damaging de novo mutations in cerebral palsy cases. Eight genes had multiple damaging de novo mutations; of these, two ( TUBA1A and CTNNB1 ) met genome-wide significance. We identified two novel monogenic etiologies, FBXO31 and RHOB , and showed that the RHOB mutation enhances active-state Rho effector binding while the FBXO31 mutation diminishes cyclin D levels. Candidate cerebral palsy risk genes overlapped with neurodevelopmental disorder genes. Network analyses identified enrichment of Rho GTPase, extracellular matrix, focal adhesion and cytoskeleton pathways. Cerebral palsy risk genes in enriched pathways were shown to regulate neuromotor function in a Drosophila reverse genetics screen. We estimate that 14% of cases could be attributed to an excess of damaging de novo or recessive variants. These findings provide evidence for genetically mediated dysregulation of early neuronal connectivity in cerebral palsy. Whole-exome sequencing of 250 parent–offspring trios identifies an enrichment of rare damaging de novo mutations in individuals with cerebral palsy and implicates genetically mediated dysregulation of early neuronal connectivity in the etiology of this disorder.

Objective:Neuropsychological evaluations are used to examine a person’s current cognitive functioning. Performance validity tests (PVT) are included in neuropsychological test batteries to ensure that examinees are performing to the best of their abilities and identify non-credible performance. There are two types of PVTs: freestanding and embedded. A freestanding PVT is a cognitive test created to evaluate performance validity and do not measure any type of cognition directly. Meanwhile, an embedded PVT is a task design to evaluate some sort of cognition (e.g., memory) by using traditional neuropsychological tests (e.g., Trail Making Test) and performance validity. Research suggests that undergraduate college students are not always performing to the best of their abilities when completing a comprehensive neuropsychological battery. In fact, in one study where an undergraduate college sample was given three PVTs, it was reported that 56% of the participants failed at least one PVT in their first session and 31% in their second session. Research has also shown that speaking multiple languages can influence cognition. The purpose of this study was to identify in three credible language groups of college students what PVTs does bilingualism influence higher failure rates. It was predicted that bilingual college students would significantly demonstrate higher PVTs failure rates compared to monolingual college students.Participants and Methods:The sample consisted of 70 English first language monolinguals (EFLM), 33 English first language bilinguals (EFLB), and 68 English second language bilinguals (ESLB) that were psychologically and neurologically healthy. All participants completed a comprehensive neuropsychological battery in English. The Rey-Osterrith complex figure copy test, Comalli Stroop part A, B, and C, Trail Making Test part A and B, Symbol Digit Modalities Test written and oral parts, Controlled Oral Word Association Test (COWAT) letter fluency, and Finger Tapping Test were the tasks used as embedded PVTs to evaluate failure rates in our sample. Moreover, all participants were credible (i.e., they did not fail two or more PVTs). PVT cutoff scores were selected for each embedded PVT from previous literature. Chi-square analysis were used to evaluate failure rates between language groups on each PVT.Results:We found no significant failure rate differences between language groups on any of the PVTs. However, while no significant group differences were found, on the COWAT letter fluency results revealed higher failure rates between the three language groups (i.e., 13% EFLM, 24% EFLB, and 22% ESLB) compared to other PVTs.Conclusions:Our data suggested no significant failure rate differences between language groups. It has been suggested in previous studies that linguistic factors impact PVT performance and test interpretation. On the COWAT letter fluency task, it is possible that language is driving higher failure rates between bilingual speakers, even though we found no significant failure rates or performance differences between the three language groups. Future studies should examine language groups and other cultural variables (e.g., time perspective) to determine what may be driving high failure rates on the COWAT letter fluency task in credible participants.

Cancer therapy: stop PARP The discovery that BRCA1/2 mutant cells (defective in the homologous recombination pathway of DNA repair) are spectacularly sensitive to inhibition of the enzyme PARP (involved in base excision repair) suggests a new, low toxicity, approach to the treatment of women with breast cancers caused by BRCA mutations. As the PARP inhibitors have no effect on cells with functional homologous recombination, the hope is that the treatment will be specific for breast cancer cells. PARP-inhibiting chemotherapeutics may be able to make use of a ‘synthetic lethal’ effect as an alternative to conventional nonspecific cytotoxic anticancer treatments. Poly(ADP-ribose) polymerase (PARP1) facilitates DNA repair by binding to DNA breaks and attracting DNA repair proteins to the site of damage 1 , 2 , 3 . Nevertheless, PARP1 -/- mice are viable, fertile and do not develop early onset tumours 4 . Here, we show that PARP inhibitors trigger γ-H2AX and RAD51 foci formation. We propose that, in the absence of PARP1, spontaneous single-strand breaks collapse replication forks and trigger homologous recombination for repair. Furthermore, we show that BRCA2-deficient cells, as a result of their deficiency in homologous recombination, are acutely sensitive to PARP inhibitors, presumably because resultant collapsed replication forks are no longer repaired. Thus, PARP1 activity is essential in homologous recombination-deficient BRCA2 mutant cells. We exploit this requirement in order to kill BRCA2-deficient tumours by PARP inhibition alone. Treatment with PARP inhibitors is likely to be highly tumour specific, because only the tumours (which are BRCA2 -/- ) in BRCA2 +/- patients are defective in homologous recombination. The use of an inhibitor of a DNA repair enzyme alone to selectively kill a tumour, in the absence of an exogenous DNA-damaging agent, represents a new concept in cancer treatment.

Somatic mutations in cancer genomes are caused by multiple mutational processes, each of which generates a characteristic mutational signature 1 . Here, as part of the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium 2 of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA), we characterized mutational signatures using 84,729,690 somatic mutations from 4,645 whole-genome and 19,184 exome sequences that encompass most types of cancer. We identified 49 single-base-substitution, 11 doublet-base-substitution, 4 clustered-base-substitution and 17 small insertion-and-deletion signatures. The substantial size of our dataset, compared with previous analyses 3 – 15 , enabled the discovery of new signatures, the separation of overlapping signatures and the decomposition of signatures into components that may represent associated—but distinct—DNA damage, repair and/or replication mechanisms. By estimating the contribution of each signature to the mutational catalogues of individual cancer genomes, we revealed associations of signatures to exogenous or endogenous exposures, as well as to defective DNA-maintenance processes. However, many signatures are of unknown cause. This analysis provides a systematic perspective on the repertoire of mutational processes that contribute to the development of human cancer. The characterization of 4,645 whole-genome and 19,184 exome sequences, covering most types of cancer, identifies 81 single-base substitution, doublet-base substitution and small-insertion-and-deletion mutational signatures, providing a systematic overview of the mutational processes that contribute to cancer development.