An endoglucanase, GsCelA, from Geobacilus sp. undergoes an intriguing self- truncation process for enhancing activity and thermostability

An endoglucanase, GsCelA, was isolated and cloned from a thermophilic Geobacillus sp. 70PC53 grown in a rice straw compost in southern Taiwan. It was observed that highly purified GsCelA was able to self-truncate, removing a segment of 53 amino acid residues from its C-terminus. The purified GsCelA does not possess any protease activity and this self-truncation process is insensitive to standard protease inhibitors except EDTA and EGTA. This unique self-truncation process takes place at a temperature higher than 10C with an optimal pH between 6-7, and can be further enhanced with certain divalent ions such as Ca+2 and Mg+2. Crystal structure of GsCelA has a typical TIM-barrel configuration with 8 alpha-helices and 8 beta-strands, but with the presence of a divalent ion. Mutations of amino acids residues surrounding this metal ion do not affect the self-truncation process, but some of these mutants have enhanced enzymatic activities. Mutation of the cleavage site between K315 and G316 does not affect the self-truncation process. However, a deletion of ten amino acids near the cleavage site, i.e. from amino acid 310 to 320, slows down the truncation process but does not block it, and a truncated form around 315 amino acids in length eventually appears. This intriguing observation indicates that the self-truncation process is not site specific, but capable of measuring 315 amino acids from the N-terminus as the cleavage site. This self-truncation process also occurs in the native host of this enzyme, Geobacillus sp. 70PC53, with almost all secreted form of this enzyme being self-truncated. The 53 amino-acid-long C-terminal segment removed by this self-truncation process has binding affinity toward both crystal and amorphous cellulose as well as the s cell walls, yet its sequence bears no apparent homology to any known carbohydrate binding motifs. Various other mutation analyses and the structure-based recombination process, SCHEMA, have been carried out, and both the activity and thermostabilty of this enzyme are further improved. The truncated and improved GsCelA has almost twice the activity as the un-truncated form, and its thermostability is also further enhanced with T50 reaching 86C and TA50 higher than 100C, making this enzyme extremely useful in industrial processes carried out at high temperatures, such as the pre-treatment of cellulosic animal feeds during the final drying step. This research was supported by grants from Taiwan Ministry of Science and Technology and from Academia Sinica

Enhancement of activity and thermostability of a Geobacillus endoglucanase via a unique self-truncation process

The complete utilization of lignocellulosic biomass requires the hydrolysis of cellulose fibers via the synergistic action of three enzymes: exoglucanase, endoglucanase and beta-glucosidase. GsCelA is a 368-amino-acid endoglucanase secreted from a thermophilic Geobacillus sp. 70PC53 that was isolated form a rice straw compost in south Taiwan. GsCelA belongs to the glycosyl hydrolase family 5 and has a typical TIM barrel structure. This enzyme has excellent lignocellulolytic activity and high thermostability, with optimal temperature at 60℃ and pH at 5.0. The purified GsCelA is capable of carrying out a unique self-truncation process at temperature higher than 10 ℃ with optimal pH at 6-7. This self-truncation process is not due to the action of contaminating proteases and it can be suppressed by EDTA and EGTA, and enhanced by divalent metal ions. This self-truncation process also takes place in vivo in Geobacillus sp. 70PC53. The spontaneous or engineered C-terminal truncation up to 60 amino acids from the C-terminus improves GsCelA specific activity and renders the enzyme more thermostable. To investigate the importance of specific amino acids on the enzymatic activity of GsCelA, site-directed mutagenesis and protein engineering approach were employed to alter amino acid residues unique to this enzyme. It was demonstrated that point mutations Y195T , D55S, G288T and D289L replacements increase the activity of this enzyme by 30%

Aspirin prevents resistin-induced endothelial dysfunction by modulating AMPK, ROS, and Akt/eNOS signaling

BackgroundResistin, an adipocytokine, plays a potential role in cardiovascular disease and may contribute to increased atherosclerotic risk by modulating the activity of endothelial cells. A growing body of evidence suggests that aspirin is a potent antioxidant. We investigated whether aspirin mitigates resistin-induced endothelial dysfunction via modulation of reactive oxygen species (ROS) generation and explored the role that AMP-activated protein kinase (AMPK), a negative regulator of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, plays in the suppressive effects of aspirin on resistin-induced endothelial dysfunction.MethodsHuman umbilical vein endothelial cells (HUVECs) were pretreated with various doses of aspirin (10-500 μg/mL) for 2 hours and then incubated with resistin (100 ng/mL) for an additional 48 hours. Fluorescence produced by the oxidation of dihydroethidium (DHE) was used to quantify the production of superoxide in situ; superoxide dismutase (SOD) and catalase activities were determined by an enzymatic assay; and protein levels of AMPK-mediated downstream signaling were investigated by Western blot.ResultsTreatment of HUVECs with resistin for 48 hours resulted in a 2.9-fold increase in superoxide production; however, pretreatment with aspirin resulted in a dose-dependent decrease in production of superoxide (10-500 μg/mL; n = 3 experiments; all P < .05). Resistin also suppressed the activity of superoxide dismutase and catalase by nearly 50%; that result, however, was not observed in HUVECs that had been pretreated with aspirin at a concentration of 500 μg/mL. The membrane translocation assay showed that the levels of NADPH oxidase subunits p47phoxand Rac-1 in membrane fractions of HUVECs were threefold to fourfold higher in cells that had been treated with resistin for 1 hour than in untreated cells; however, pretreatment with aspirin markedly inhibited resistin-induced membrane assembly of NADPH oxidase via modulating AMPK-suppressed PKC-α activation. Application of AMPKα1-specific siRNA resulted in increased activation of PKC-α and p47phox. In addition, resistin significantly decreased AMPK-mediated downstream Akt/endothelial nitric oxide synthase (eNOS)/nitric oxide (NO) signaling and induced the phosphorylation of p38 mitogen-activated protein kinases, which in turn activated NF-κB-mediated inflammatory responses such as the release of interleukin (IL)-6 and IL-8, the overexpression of adhesion molecules, and stimulation of monocytic THP-1 cell attachment to HUVECs (2.5-fold vs control; n = 3 experiments). Furthermore, resistin downregulated eNOS and upregulated inducible NO synthase (iNOS) expression, thereby augmenting the formation of NO and protein nitrosylation. Pretreatment with aspirin, however, exerted significant cytoprotective effects in a dose-dependent manner (P < .05).ConclusionsOur findings suggest a direct connection between adipocytokines and endothelial dysfunction and provide further insight into the protective effects of aspirin in obese individuals with endothelial dysfunction.Clinical RelevanceResistin has been reported to involve in the inflammatory process, which is a common feature in metabolic syndrome, insulin resistance status, and vascular diseases. This study underscores the potential clinical benefits and application of aspirin in prevention of obese-associated vascular dysfunction

Exploring the Mechanism Responsible for Cellulase Thermostability by Structure-Guided Recombination

Cellulases from Bacillus and Geobacillus bacteria are potentially useful in the biofuel and animal feed industries. One of the unique characteristics of these enzymes is that they are usually quite thermostable. We previously identified a cellulase, GsCelA, from thermophilic Geobacillus sp. 70PC53, which is much more thermostable than its Bacillus homolog, BsCel5A. Thus, these two cellulases provide a pair of structures ideal for investigating the mechanism regarding how these cellulases can retain activity at high temperature. In the present study, we applied the SCHEMA non-contiguous recombination algorithm as a novel tool, which assigns protein sequences into blocks for domain swapping in a way that lessens structural disruption, to generate a set of chimeric proteins derived from the recombination of GsCelA and BsCel5A. Analyzing the activity and thermostability of this designed library set, which requires only a limited number of chimeras by SCHEMA calculations, revealed that one of the blocks may contribute to the higher thermostability of GsCelA. When tested against swollen Avicel, the highly thermostable chimeric cellulase C10 containing this block showed significantly higher activity (22%-43%) and higher thermostability compared to the parental enzymes. With further structural determinations and mutagenesis analyses, a 3_(10) helix was identified as being responsible for the improved thermostability of this block. Furthermore, in the presence of ionic calcium and crown ether (CR), the chimeric C10 was found to retain 40% residual activity even after heat treatment at 90°C. Combining crystal structure determinations and structure-guided SCHEMA recombination, we have determined the mechanism responsible for the high thermostability of GsCelA, and generated a novel recombinant enzyme with significantly higher activity

The Functional Haplotypes of CHRM3 Modulate mRNA Expression and Associate with Bladder Cancer among a Chinese Han Population in Kaohsiung City

Bladder cancer is one of the major cancer types and both environmental factors and genetic background play important roles in its pathology. Kaohsiung is a high industrialized city in Taiwan, and here we focused on this region to evaluate the genetic effects on bladder cancer. Muscarinic acetylcholine receptor M3 (CHRM3) was reported as a key receptor in different cancer types. CHRM3 is located at 1q42-43 which was reported to associate with bladder cancer. Our study attempted to delineate whether genetic variants of CHRM3 contribute to bladder cancer in Chinese Han population in south Taiwan. Five selected SNPs (rs2165870, rs10802789, rs685550, rs7520974, and rs3738435) were genotyped for 30 bladder cancer patients and 60 control individuals and genetic association studies were performed. Five haplotypes (GTTAT, ATTGT, GCTAC, ACTAC, and ACCAC) were found significantly associated with low CHRM3 mRNA level and contributed to increased susceptibility of bladder cancer in Kaohsiung city after rigid 10000 consecutive permutation tests. To our knowledge, this is the first genetic association study that reveals the genetic contribution of CHRM3 gene in bladder cancer etiology

Establishing an online resource to facilitate global collaboration and inclusion of underrepresented populations:Experience from the MJFF Global Genetic Parkinson's Disease Project

Parkinson's disease (PD) is the fastest-growing neurodegenerative disorder, currently affecting ~7 million people worldwide. PD is clinically and genetically heterogeneous, with at least 10% of all cases explained by a monogenic cause or strong genetic risk factor. However, the vast majority of our present data on monogenic PD is based on the investigation of patients of European White ancestry, leaving a large knowledge gap on monogenic PD in underrepresented populations. Gene-targeted therapies are being developed at a fast pace and have started entering clinical trials. In light of these developments, building a global network of centers working on monogenic PD, fostering collaborative research, and establishing a clinical trial-ready cohort is imperative. Based on a systematic review of the English literature on monogenic PD and a successful team science approach, we have built up a network of 59 sites worldwide and have collected information on the availability of data, biomaterials, and facilities. To enable access to this resource and to foster collaboration across centers, as well as between academia and industry, we have developed an interactive map and online tool allowing for a quick overview of available resources, along with an option to filter for specific items of interest. This initiative is currently being merged with the Global Parkinson's Genetics Program (GP2), which will attract additional centers with a focus on underrepresented sites. This growing resource and tool will facilitate collaborative research and impact the development and testing of new therapies for monogenic and potentially for idiopathic PD patients.</p

Measuring universal health coverage based on an index of effective coverage of health services in 204 countries and territories, 1990–2019 : A systematic analysis for the Global Burden of Disease Study 2019

Background Achieving universal health coverage (UHC) involves all people receiving the health services they need, of high quality, without experiencing financial hardship. Making progress towards UHC is a policy priority for both countries and global institutions, as highlighted by the agenda of the UN Sustainable Development Goals (SDGs) and WHO's Thirteenth General Programme of Work (GPW13). Measuring effective coverage at the health-system level is important for understanding whether health services are aligned with countries' health profiles and are of sufficient quality to produce health gains for populations of all ages. Methods Based on the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2019, we assessed UHC effective coverage for 204 countries and territories from 1990 to 2019. Drawing from a measurement framework developed through WHO's GPW13 consultation, we mapped 23 effective coverage indicators to a matrix representing health service types (eg, promotion, prevention, and treatment) and five population-age groups spanning from reproductive and newborn to older adults (≥65 years). Effective coverage indicators were based on intervention coverage or outcome-based measures such as mortality-to-incidence ratios to approximate access to quality care; outcome-based measures were transformed to values on a scale of 0–100 based on the 2·5th and 97·5th percentile of location-year values. We constructed the UHC effective coverage index by weighting each effective coverage indicator relative to its associated potential health gains, as measured by disability-adjusted life-years for each location-year and population-age group. For three tests of validity (content, known-groups, and convergent), UHC effective coverage index performance was generally better than that of other UHC service coverage indices from WHO (ie, the current metric for SDG indicator 3.8.1 on UHC service coverage), the World Bank, and GBD 2017. We quantified frontiers of UHC effective coverage performance on the basis of pooled health spending per capita, representing UHC effective coverage index levels achieved in 2019 relative to country-level government health spending, prepaid private expenditures, and development assistance for health. To assess current trajectories towards the GPW13 UHC billion target—1 billion more people benefiting from UHC by 2023—we estimated additional population equivalents with UHC effective coverage from 2018 to 2023. Findings Globally, performance on the UHC effective coverage index improved from 45·8 (95% uncertainty interval 44·2–47·5) in 1990 to 60·3 (58·7–61·9) in 2019, yet country-level UHC effective coverage in 2019 still spanned from 95 or higher in Japan and Iceland to lower than 25 in Somalia and the Central African Republic. Since 2010, sub-Saharan Africa showed accelerated gains on the UHC effective coverage index (at an average increase of 2·6% [1·9–3·3] per year up to 2019); by contrast, most other GBD super-regions had slowed rates of progress in 2010–2019 relative to 1990–2010. Many countries showed lagging performance on effective coverage indicators for non-communicable diseases relative to those for communicable diseases and maternal and child health, despite non-communicable diseases accounting for a greater proportion of potential health gains in 2019, suggesting that many health systems are not keeping pace with the rising non-communicable disease burden and associated population health needs. In 2019, the UHC effective coverage index was associated with pooled health spending per capita (r=0·79), although countries across the development spectrum had much lower UHC effective coverage than is potentially achievable relative to their health spending. Under maximum efficiency of translating health spending into UHC effective coverage performance, countries would need to reach $1398 pooled health spending per capita (US$ adjusted for purchasing power parity) in order to achieve 80 on the UHC effective coverage index. From 2018 to 2023, an estimated 388·9 million (358·6–421·3) more population equivalents would have UHC effective coverage, falling well short of the GPW13 target of 1 billion more people benefiting from UHC during this time. Current projections point to an estimated 3·1 billion (3·0–3·2) population equivalents still lacking UHC effective coverage in 2023, with nearly a third (968·1 million [903·5–1040·3]) residing in south Asia. Interpretation The present study demonstrates the utility of measuring effective coverage and its role in supporting improved health outcomes for all people—the ultimate goal of UHC and its achievement. Global ambitions to accelerate progress on UHC service coverage are increasingly unlikely unless concerted action on non-communicable diseases occurs and countries can better translate health spending into improved performance. Focusing on effective coverage and accounting for the world's evolving health needs lays the groundwork for better understanding how close—or how far—all populations are in benefiting from UHC

Global burden of 369 diseases and injuries in 204 countries and territories, 1990–2019: a systematic analysis for the Global Burden of Disease Study 2019

Background: In an era of shifting global agendas and expanded emphasis on non-communicable diseases and injuries along with communicable diseases, sound evidence on trends by cause at the national level is essential. The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) provides a systematic scientific assessment of published, publicly available, and contributed data on incidence, prevalence, and mortality for a mutually exclusive and collectively exhaustive list of diseases and injuries. Methods: GBD estimates incidence, prevalence, mortality, years of life lost (YLLs), years lived with disability (YLDs), and disability-adjusted life-years (DALYs) due to 369 diseases and injuries, for two sexes, and for 204 countries and territories. Input data were extracted from censuses, household surveys, civil registration and vital statistics, disease registries, health service use, air pollution monitors, satellite imaging, disease notifications, and other sources. Cause-specific death rates and cause fractions were calculated using the Cause of Death Ensemble model and spatiotemporal Gaussian process regression. Cause-specific deaths were adjusted to match the total all-cause deaths calculated as part of the GBD population, fertility, and mortality estimates. Deaths were multiplied by standard life expectancy at each age to calculate YLLs. A Bayesian meta-regression modelling tool, DisMod-MR 2.1, was used to ensure consistency between incidence, prevalence, remission, excess mortality, and cause-specific mortality for most causes. Prevalence estimates were multiplied by disability weights for mutually exclusive sequelae of diseases and injuries to calculate YLDs. We considered results in the context of the Socio-demographic Index (SDI), a composite indicator of income per capita, years of schooling, and fertility rate in females younger than 25 years. Uncertainty intervals (UIs) were generated for every metric using the 25th and 975th ordered 1000 draw values of the posterior distribution. Findings: Global health has steadily improved over the past 30 years as measured by age-standardised DALY rates. After taking into account population growth and ageing, the absolute number of DALYs has remained stable. Since 2010, the pace of decline in global age-standardised DALY rates has accelerated in age groups younger than 50 years compared with the 1990–2010 time period, with the greatest annualised rate of decline occurring in the 0–9-year age group. Six infectious diseases were among the top ten causes of DALYs in children younger than 10 years in 2019: lower respiratory infections (ranked second), diarrhoeal diseases (third), malaria (fifth), meningitis (sixth), whooping cough (ninth), and sexually transmitted infections (which, in this age group, is fully accounted for by congenital syphilis; ranked tenth). In adolescents aged 10–24 years, three injury causes were among the top causes of DALYs: road injuries (ranked first), self-harm (third), and interpersonal violence (fifth). Five of the causes that were in the top ten for ages 10–24 years were also in the top ten in the 25–49-year age group: road injuries (ranked first), HIV/AIDS (second), low back pain (fourth), headache disorders (fifth), and depressive disorders (sixth). In 2019, ischaemic heart disease and stroke were the top-ranked causes of DALYs in both the 50–74-year and 75-years-and-older age groups. Since 1990, there has been a marked shift towards a greater proportion of burden due to YLDs from non-communicable diseases and injuries. In 2019, there were 11 countries where non-communicable disease and injury YLDs constituted more than half of all disease burden. Decreases in age-standardised DALY rates have accelerated over the past decade in countries at the lower end of the SDI range, while improvements have started to stagnate or even reverse in countries with higher SDI. Interpretation: As disability becomes an increasingly large component of disease burden and a larger component of health expenditure, greater research and developm nt investment is needed to identify new, more effective intervention strategies. With a rapidly ageing global population, the demands on health services to deal with disabling outcomes, which increase with age, will require policy makers to anticipate these changes. The mix of universal and more geographically specific influences on health reinforces the need for regular reporting on population health in detail and by underlying cause to help decision makers to identify success stories of disease control to emulate, as well as opportunities to improve. Funding: Bill & Melinda Gates Foundation. © 2020 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 licens

Measuring universal health coverage based on an index of effective coverage of health services in 204 countries and territories, 1990–2019: a systematic analysis for the Global Burden of Disease Study 2019

Background Achieving universal health coverage (UHC) involves all people receiving the health services they need, of high quality, without experiencing financial hardship. Making progress towards UHC is a policy priority for both countries and global institutions, as highlighted by the agenda of the UN Sustainable Development Goals (SDGs) and WHO's Thirteenth General Programme of Work (GPW13). Measuring effective coverage at the health-system level is important for understanding whether health services are aligned with countries' health profiles and are of sufficient quality to produce health gains for populations of all ages. Methods Based on the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2019, we assessed UHC effective coverage for 204 countries and territories from 1990 to 2019. Drawing from a measurement framework developed through WHO's GPW13 consultation, we mapped 23 effective coverage indicators to a matrix representing health service types (eg, promotion, prevention, and treatment) and five population-age groups spanning from reproductive and newborn to older adults (≥65 years). Effective coverage indicators were based on intervention coverage or outcome-based measures such as mortality-to-incidence ratios to approximate access to quality care; outcome-based measures were transformed to values on a scale of 0–100 based on the 2·5th and 97·5th percentile of location-year values. We constructed the UHC effective coverage index by weighting each effective coverage indicator relative to its associated potential health gains, as measured by disability-adjusted life-years for each location-year and population-age group. For three tests of validity (content, known-groups, and convergent), UHC effective coverage index performance was generally better than that of other UHC service coverage indices from WHO (ie, the current metric for SDG indicator 3.8.1 on UHC service coverage), the World Bank, and GBD 2017. We quantified frontiers of UHC effective coverage performance on the basis of pooled health spending per capita, representing UHC effective coverage index levels achieved in 2019 relative to country-level government health spending, prepaid private expenditures, and development assistance for health. To assess current trajectories towards the GPW13 UHC billion target—1 billion more people benefiting from UHC by 2023—we estimated additional population equivalents with UHC effective coverage from 2018 to 2023. Findings Globally, performance on the UHC effective coverage index improved from 45·8 (95% uncertainty interval 44·2–47·5) in 1990 to 60·3 (58·7–61·9) in 2019, yet country-level UHC effective coverage in 2019 still spanned from 95 or higher in Japan and Iceland to lower than 25 in Somalia and the Central African Republic. Since 2010, sub-Saharan Africa showed accelerated gains on the UHC effective coverage index (at an average increase of 2·6% [1·9–3·3] per year up to 2019); by contrast, most other GBD super-regions had slowed rates of progress in 2010–2019 relative to 1990–2010. Many countries showed lagging performance on effective coverage indicators for non-communicable diseases relative to those for communicable diseases and maternal and child health, despite non-communicable diseases accounting for a greater proportion of potential health gains in 2019, suggesting that many health systems are not keeping pace with the rising non-communicable disease burden and associated population health needs. In 2019, the UHC effective coverage index was associated with pooled health spending per capita (r=0·79), although countries across the development spectrum had much lower UHC effective coverage than is potentially achievable relative to their health spending. Under maximum efficiency of translating health spending into UHC effective coverage performance, countries would need to reach $1398 pooled health spending per capita (US$ adjusted for purchasing power parity) in order to achieve 80 on the UHC effective coverage index. From 2018 to 2023, an estimated 388·9 million (358·6–421·3) more population equivalents would have UHC effective coverage, falling well short of the GPW13 target of 1 billion more people benefiting from UHC during this time. Current projections point to an estimated 3·1 billion (3·0–3·2) population equivalents still lacking UHC effective coverage in 2023, with nearly a third (968·1 million [903·5–1040·3]) residing in south Asia. Interpretation The present study demonstrates the utility of measuring effective coverage and its role in supporting improved health outcomes for all people—the ultimate goal of UHC and its achievement. Global ambitions to accelerate progress on UHC service coverage are increasingly unlikely unless concerted action on non-communicable diseases occurs and countries can better translate health spending into improved performance. Focusing on effective coverage and accounting for the world's evolving health needs lays the groundwork for better understanding how close—or how far—all populations are in benefiting from UHC