The data-driven future of high-energy-density physics.

High-energy-density physics is the field of physics concerned with studying matter at extremely high temperatures and densities. Such conditions produce highly nonlinear plasmas, in which several phenomena that can normally be treated independently of one another become strongly coupled. The study of these plasmas is important for our understanding of astrophysics, nuclear fusion and fundamental physics-however, the nonlinearities and strong couplings present in these extreme physical systems makes them very difficult to understand theoretically or to optimize experimentally. Here we argue that machine learning models and data-driven methods are in the process of reshaping our exploration of these extreme systems that have hitherto proved far too nonlinear for human researchers. From a fundamental perspective, our understanding can be improved by the way in which machine learning models can rapidly discover complex interactions in large datasets. From a practical point of view, the newest generation of extreme physics facilities can perform experiments multiple times a second (as opposed to approximately daily), thus moving away from human-based control towards automatic control based on real-time interpretation of diagnostic data and updates of the physics model. To make the most of these emerging opportunities, we suggest proposals for the community in terms of research design, training, best practice and support for synthetic diagnostics and data analysis.

A measurement of the equation of state of carbon envelopes of white dwarfs.

White dwarfs represent the final state of evolution for most stars1-3. Certain classes of white dwarfs pulsate4,5, leading to observable brightness variations, and analysis of these variations with theoretical stellar models probes their internal structure. Modelling of these pulsating stars provides stringent tests of white dwarf models and a detailed picture of the outcome of the late stages of stellar evolution6. However, the high-energy-density states that exist in white dwarfs are extremely difficult to reach and to measure in the laboratory, so theoretical predictions are largely untested at these conditions. Here we report measurements of the relationship between pressure and density along the principal shock Hugoniot (equations describing the state of the sample material before and after the passage of the shock derived from conservation laws) of hydrocarbon to within five per cent. The observed maximum compressibility is consistent with theoretical models that include detailed electronic structure. This is relevant for the equation of state of matter at pressures ranging from 100 million to 450 million atmospheres, where the understanding of white dwarf physics is sensitive to the equation of state and where models differ considerably. The measurements test these equation-of-state relations that are used in the modelling of white dwarfs and inertial confinement fusion experiments7,8, and we predict an increase in compressibility due to ionization of the inner-core orbitals of carbon. We also find that a detailed treatment of the electronic structure and the electron degeneracy pressure is required to capture the measured shape of the pressure-density evolution for hydrocarbon before peak compression. Our results illuminate the equation of state of the white dwarf envelope (the region surrounding the stellar core that contains partially ionized and partially degenerate non-ideal plasmas), which is a weak link in the constitutive physics informing the structure and evolution of white dwarf stars9.

Harnessing metabolic plasticity in CHO cells for enhanced perfusion cultivation.

Chinese Hamster Ovary (CHO) cells have rapidly become a cornerstone in biopharmaceutical production. Recently, a reinvigoration of perfusion culture mode in CHO cell cultivation has been observed. However, most cell lines currently in use have been engineered and adapted for fed-batch culture methods, and may not perform optimally under perfusion conditions. To improve the cell's resilience and viability during perfusion culture, we cultured a triple knockout CHO cell line, deficient in three apoptosis related genes BAX, BAK, and BOK in a perfusion system. After 20 days of culture, the cells exhibited a halt in cell proliferation. Interestingly, following this phase of growth arrest, the cells entered a second growth phase. During this phase, the cell numbers nearly doubled, but cell specific productivity decreased. We performed a proteomics investigation, elucidating a distinct correlation between growth arrest and cell cycle arrest and showing an upregulation of the central carbon metabolism and oxidative phosphorylation. The upregulation was partially reverted during the second growth phase, likely caused by intragenerational adaptations to stresses encountered. A phase-dependent response to oxidative stress was noted, indicating glutathione has only a secondary role during cell cycle arrest. Our data provides evidence of metabolic regulation under high cell density culturing conditions and demonstrates that cell growth arrest can be overcome. The acquired insights have the potential to not only enhance our understanding of cellular metabolism but also contribute to the development of superior cell lines for perfusion cultivation.

Plant-environment microscopy tracks interactions of Bacillus subtilis with plant roots across the entire rhizosphere.

Our understanding of plant-microbe interactions in soil is limited by the difficulty of observing processes at the microscopic scale throughout plants' large volume of influence. Here, we present the development of three-dimensional live microscopy for resolving plant-microbe interactions across the environment of an entire seedling growing in a transparent soil in tailor-made mesocosms, maintaining physical conditions for the culture of both plants and microorganisms. A tailor-made, dual-illumination light sheet system acquired photons scattered from the plant while fluorescence emissions were simultaneously captured from transparent soil particles and labeled microorganisms, allowing the generation of quantitative data on samples ∼3,600 mm3 in size, with as good as 5 µm resolution at a rate of up to one scan every 30 min. The system tracked the movement of Bacillus subtilis populations in the rhizosphere of lettuce plants in real time, revealing previously unseen patterns of activity. Motile bacteria favored small pore spaces over the surface of soil particles, colonizing the root in a pulsatile manner. Migrations appeared to be directed toward the root cap, the point of "first contact," before the subsequent colonization of mature epidermis cells. Our findings show that microscopes dedicated to live environmental studies present an invaluable tool to understand plant-microbe interactions.

In situ control of root-bacteria interactions using optical trapping in transparent soil.

Bacterial attachment on root surfaces is an important step preceding the colonization or internalization and subsequent infection of plants by pathogens. Unfortunately, bacterial attachment is not well understood because the phenomenon is difficult to observe. Here we assessed whether this limitation could be overcome using optical trapping approaches. We have developed a system based on counter-propagating beams and studied its ability to guide Pectobacterium atrosepticum (Pba) cells to different root cell types within the interstices of transparent soils. Bacterial cells were successfully trapped and guided to root hair cells, epidermal cells, border cells, and tissues damaged by laser ablation. Finally, we used the system to quantify the bacterial cell detachment rate of Pba cells on root surfaces following reversible attachment. Optical trapping techniques could greatly enhance our ability to deterministically characterize mechanisms linked to attachment and formation of biofilms in the rhizosphere.

Applying the COM-B behaviour change model to a pilot study delivering volatile pyrethroid spatial repellents and insecticide-treated clothing to forest-exposed populations in Mondulkiri Province, Cambodia.

BACKGROUND: Southeast Asia is making tremendous progress towards their 2030 malaria elimination goal but needs new interventions to stop forest malaria. This study trials two new vector control tools, a volatile pyrethroid spatial repellent (VPSR) and insecticide-treated clothing (ITC), amongst forest-exposed populations in Mondulkiri Province Cambodia to inform their potential use for eliminating forest malaria. METHODS: 21 forest-exposed individuals were given a questionnaire on their perceptions of malaria and preventive practices used, after which they trialed two products sequentially. Clothes was treated with ITC by the study team. Mixed methods were used to understand their experience, attitudes, and preferences regarding the products trialed. Quantitative data was summarized and qualitative insights were analysed using thematic analysis, applying the Capability, Opportunity, and Motivation Behaviour Change (COM-B) model and Behaviour Change Wheel Framework to identify intervention functions to support tailored product rollout amongst these populations. RESULTS: Study participants reported a need for protection from mosquito bites in outdoor and forest-exposed settings and perceived both products trialed to be effective for this purpose. The VPSR product was preferred when travel was not required, whereas ITC was preferred for ease of use when going to the forest, especially in rainy conditions. COM-B analysis identified that key enablers for use of both products included their perceived efficacy and ease of use, which required no skill or preparation. For barriers to use, the odour of ITC was sometimes perceived as being toxic, as well as its inability to protect uncovered skin from mosquito bites, while the perceived usefulness of the VPSR product trialed was limited by its water sensitivity in rainy forest settings. Intervention components to encourage appropriate and sustained use of these products include education about how to use these products and what to expect, persuasion to use them from community leaders and targeted channels, and enablement to facilitate convenient and affordable access. CONCLUSION: The rollout of VPSRs and ITC amongst forest-exposed populations can be useful for eliminating malaria in Southeast Asia. Study findings can be applied to increase product uptake among forest exposed populations in Cambodia, while manufacturers can aim to develop products that are rainproof, easy to use in forest settings, and have favourable odour profiles to target users.

Perfusion culture of Chinese Hamster Ovary cells for bioprocessing applications.

Much of the biopharmaceutical industry's success over the past 30 years has relied on products derived from Chinese Hamster Ovary (CHO) cell lines. During this time, improvements in mammalian cell cultures have come from cell line development and process optimization suited for large-scale fed-batch processes. Originally developed for high cell densities and sensitive products, perfusion processes have a long history. Driven by high volumetric titers and a small footprint, perfusion-based bioprocess research has regained an interest from academia and industry. The recent pandemic has further highlighted the need for such intensified biomanufacturing options. In this review, we outline the technical history of research in this field as it applies to biologics production in CHO cells. We demonstrate a number of emerging trends in the literature and corroborate these with underlying drivers in the commercial space. From these trends, we speculate that the future of perfusion bioprocesses is bright and that the fields of media optimization, continuous processing, and cell line engineering hold the greatest potential. Aligning in its continuous setup with the demands for Industry 4.0, perfusion biomanufacturing is likely to be a hot topic in the years to come.

Modeling apoptosis resistance in CHO cells with CRISPR-mediated knockouts of Bak1, Bax, and Bok.

Chinese hamster ovary (CHO) cells are the primary platform for the production of biopharmaceuticals. To increase yields, many CHO cell lines have been genetically engineered to resist cell death. However, the kinetics that governs cell fate in bioreactors are confounded by many variables associated with batch processes. Here, we used CRISPR-Cas9 to create combinatorial knockouts of the three known BCL-2 family effector proteins: Bak1, Bax, and Bok. To assess the response to apoptotic stimuli, cell lines were cultured in the presence of four cytotoxic compounds with different mechanisms of action. A population-based model was developed to describe the behavior of the resulting viable cell dynamics as a function of genotype and treatment. Our results validated the synergistic antiapoptotic nature of Bak1 and Bax, while the deletion of Bok had no significant impact. Importantly, the uniform application of apoptotic stresses permitted direct observation and quantification of a delay in the onset of cell death through Bayesian inference of meaningful model parameters. In addition to the classical death rate, a delay function was found to be essential in the accurate modeling of the cell death response. These findings represent an important bridge between cell line engineering strategies and biological modeling in a bioprocess context.

Uncovering patterns of freshwater positive interactions using meta-analysis: Identifying the roles of common participants, invasive species and environmental context.

Positive interactions are sensitive to human activities, necessitating synthetic approaches to elucidate broad patterns and predict future changes if these interactions are altered or lost. General understanding of freshwater positive interactions has been far outpaced by knowledge of these important relationships in terrestrial and marine ecosystems. We conducted a global meta-analysis to evaluate the magnitude of positive interactions across freshwater habitats. In 340 studies, we found substantial positive effects, with facilitators increasing beneficiaries by, on average, 81% across all taxa and response variables. Mollusks in particular were commonly studied as both facilitators and beneficiaries. Amphibians were one group benefiting the most from positive interactions, yet few studies investigated amphibians. Invasive facilitators had stronger positive effects on beneficiaries than non-invasive facilitators. We compared positive effects between high- and low-stress conditions and found no difference in the magnitude of benefit in the subset of studies that manipulated stressors. Future areas of research include understudied facilitators and beneficiaries, the stress gradient hypothesis, patterns across space or time and the influence of declining taxa whose elimination would jeopardise fragile positive interaction networks. Freshwater positive interactions occur among a wide range of taxa, influence populations, communities and ecosystem processes and deserve further exploration.

'Omics driven discoveries of gene targets for apoptosis attenuation in CHO cells.

Chinese hamster ovary (CHO) cells are widely used in biopharmaceutical production. Improvements to cell lines and bioprocesses are constantly being explored. One of the major limitations of CHO cell culture is that the cells undergo apoptosis, leading to rapid cell death, which impedes reaching high recombinant protein titres. While several genetic engineering strategies have been successfully employed to reduce apoptosis, there is still room to further enhance CHO cell lines performance. 'Omics analysis is a powerful tool to better understand different phenotypes and for the identification of gene targets for engineering. Here, we present a comprehensive review of previous CHO 'omics studies that revealed changes in the expression of apoptosis-related genes. We highlight targets for genetic engineering that have reduced, or have the potential to reduce, apoptosis or to increase cell proliferation in CHO cells, with the final aim of increasing productivity.

Radiological Society of North America/Quantitative Imaging Biomarker Alliance Shear Wave Speed Bias Quantification in Elastic and Viscoelastic Phantoms.

OBJECTIVES: To quantify the bias of shear wave speed (SWS) measurements between different commercial ultrasonic shear elasticity systems and a magnetic resonance elastography (MRE) system in elastic and viscoelastic phantoms. METHODS: Two elastic phantoms, representing healthy through fibrotic liver, were measured with 5 different ultrasound platforms, and 3 viscoelastic phantoms, representing healthy through fibrotic liver tissue, were measured with 12 different ultrasound platforms. Measurements were performed with different systems at different sites, at 3 focal depths, and with different appraisers. The SWS bias across the systems was quantified as a function of the system, site, focal depth, and appraiser. A single MRE research system was also used to characterize these phantoms using discrete frequencies from 60 to 500 Hz. RESULTS: The SWS from different systems had mean difference 95% confidence intervals of ±0.145 m/s (±9.6%) across both elastic phantoms and ± 0.340 m/s (±15.3%) across the viscoelastic phantoms. The focal depth and appraiser were less significant sources of SWS variability than the system and site. Magnetic resonance elastography best matched the ultrasonic SWS in the viscoelastic phantoms using a 140 Hz source but had a - 0.27 ± 0.027-m/s (-12.2% ± 1.2%) bias when using the clinically implemented 60-Hz vibration source. CONCLUSIONS: Shear wave speed reconstruction across different manufacturer systems is more consistent in elastic than viscoelastic phantoms, with a mean difference bias of < ±10% in all cases. Magnetic resonance elastographic measurements in the elastic and viscoelastic phantoms best match the ultrasound systems with a 140-Hz excitation but have a significant negative bias operating at 60 Hz. This study establishes a foundation for meaningful comparison of SWS measurements made with different platforms.

The Role of the Private Sector in Supporting Malaria Control in Resource Development Settings.

Industrial operations of the private sector, such as extraction, agriculture, and construction, can bring large numbers of people into new settlement areas and cause environmental change that promotes the transmission of vector-borne diseases. Industry-related workers and communities unduly exposed to infection risk typically lack the knowledge and means to protect themselves. However, there is a strong business rationale for protecting local resident employees through integrated vector control programs, as well as an ethical responsibility to care for these individuals and the affected communities. We discuss the role and challenges of the private sector in developing malaria control programs, which can include extensive collaborations with the public sector that go on to form the basis of national vector control programs or more broadly support local healthcare systems.

Endoscopic Treatment versus Shunting for Infant Hydrocephalus in Uganda.

BACKGROUND: Postinfectious hydrocephalus in infants is a major health problem in sub-Saharan Africa. The conventional treatment is ventriculoperitoneal shunting, but surgeons are usually not immediately available to revise shunts when they fail. Endoscopic third ventriculostomy with choroid plexus cauterization (ETV-CPC) is an alternative treatment that is less subject to late failure but is also less likely than shunting to result in a reduction in ventricular size that might facilitate better brain growth and cognitive outcomes. METHODS: We conducted a randomized trial to evaluate cognitive outcomes after ETV-CPC versus ventriculoperitoneal shunting in Ugandan infants with postinfectious hydrocephalus. The primary outcome was the Bayley Scales of Infant Development, Third Edition (BSID-3), cognitive scaled score 12 months after surgery (scores range from 1 to 19, with higher scores indicating better performance). The secondary outcomes were BSID-3 motor and language scores, treatment failure (defined as treatment-related death or the need for repeat surgery), and brain volume measured on computed tomography. RESULTS: A total of 100 infants were enrolled; 51 were randomly assigned to undergo ETV-CPC, and 49 were assigned to undergo ventriculoperitoneal shunting. The median BSID-3 cognitive scores at 12 months did not differ significantly between the treatment groups (a score of 4 for ETV-CPC and 2 for ventriculoperitoneal shunting; Hodges-Lehmann estimated difference, 0; 95% confidence interval [CI], -2 to 0; P=0.35). There was no significant difference between the ETV-CPC group and the ventriculoperitoneal-shunt group in BSID-3 motor or language scores, rates of treatment failure (35% and 24%, respectively; hazard ratio, 0.7; 95% CI, 0.3 to 1.5; P=0.24), or brain volume (z score, -2.4 and -2.1, respectively; estimated difference, 0.3; 95% CI, -0.3 to 1.0; P=0.12). CONCLUSIONS: This single-center study involving Ugandan infants with postinfectious hydrocephalus showed no significant difference between endoscopic ETV-CPC and ventriculoperitoneal shunting with regard to cognitive outcomes at 12 months. (Funded by the National Institutes of Health; ClinicalTrials.gov number, NCT01936272 .).

Attenuating apoptosis in Chinese hamster ovary cells for improved biopharmaceutical production.

Chinese hamster ovary (CHO) cells are the predominant host cell line for the production of biopharmaceuticals, a growing industry currently worth more than $188 billion USD in global sales. CHO cells undergo programmed cell death (apoptosis) following different stresses encountered in cell culture, such as substrate limitation, accumulation of toxic by-products, and mechanical shear, hindering production. Genetic engineering strategies to reduce apoptosis in CHO cells have been investigated with mixed results. In this review, a contemporary understanding of the real complexity of apoptotic mechanisms and signaling pathways is described; followed by an overview of antiapoptotic cell line engineering strategies tested so far in CHO cells.

Metformin suppresses gluconeogenesis by inhibiting mitochondrial glycerophosphate dehydrogenase.

Metformin is considered to be one of the most effective therapeutics for treating type 2 diabetes because it specifically reduces hepatic gluconeogenesis without increasing insulin secretion, inducing weight gain or posing a risk of hypoglycaemia. For over half a century, this agent has been prescribed to patients with type 2 diabetes worldwide, yet the underlying mechanism by which metformin inhibits hepatic gluconeogenesis remains unknown. Here we show that metformin non-competitively inhibits the redox shuttle enzyme mitochondrial glycerophosphate dehydrogenase, resulting in an altered hepatocellular redox state, reduced conversion of lactate and glycerol to glucose, and decreased hepatic gluconeogenesis. Acute and chronic low-dose metformin treatment effectively reduced endogenous glucose production, while increasing cytosolic redox and decreasing mitochondrial redox states. Antisense oligonucleotide knockdown of hepatic mitochondrial glycerophosphate dehydrogenase in rats resulted in a phenotype akin to chronic metformin treatment, and abrogated metformin-mediated increases in cytosolic redox state, decreases in plasma glucose concentrations, and inhibition of endogenous glucose production. These findings were replicated in whole-body mitochondrial glycerophosphate dehydrogenase knockout mice. These results have significant implications for understanding the mechanism of metformin's blood glucose lowering effects and provide a new therapeutic target for type 2 diabetes.

Heart Failure With Preserved Ejection Fraction in the Young.

BACKGROUND: Heart failure with preserved ejection fraction (HFpEF), traditionally considered a disease of the elderly, may also affect younger patients. However, little is known about HFpEF in the young. METHODS: We prospectively enrolled 1203 patients with HFpEF (left ventricular ejection fraction ≥50%) from 11 Asian regions. We grouped HFpEF patients into very young (<55 years of age; n=157), young (55-64 years of age; n=284), older (65-74 years of age; n=355), and elderly (≥75 years of age; n=407) and compared clinical and echocardiographic characteristics, quality of life, and outcomes across age groups and between very young individuals with HFpEF and age- and sex-matched control subjects without heart failure. RESULTS: Thirty-seven percent of our HFpEF population was <65 years of age. Younger age was associated with male preponderance and a higher prevalence of obesity (body mass index ≥30 kg/m2; 36% in very young HFpEF versus 16% in elderly) together with less renal impairment, atrial fibrillation, and hypertension (all P<0.001). Left ventricular filling pressures and prevalence of left ventricular hypertrophy were similar in very young and elderly HFpEF. Quality of life was better and death and heart failure hospitalization at 1 year occurred less frequently ( P<0.001) in the very young (7%) compared with elderly (21%) HFpEF. Compared with control subjects, very young HFpEF had a 3-fold higher death rate and twice the prevalence of hypertrophy. CONCLUSIONS: Young and very young patients with HFpEF display similar adverse cardiac remodeling compared with their older counterparts and very poor outcomes compared with control subjects without heart failure. Obesity may be a major driver of HFpEF in a high proportion of HFpEF in the young and very young.

Association of obesity with heart failure outcomes in 11 Asian regions: A cohort study.

BACKGROUND: Asians are predisposed to a lean heart failure (HF) phenotype. Data on the 'obesity paradox', reported in Western populations, are scarce in Asia and have only utilised the traditional classification of body mass index (BMI). We aimed to investigate the association between obesity (defined by BMI and abdominal measures) and HF outcomes in Asia. METHODS AND FINDINGS: Utilising the Asian Sudden Cardiac Death in Heart Failure (ASIAN-HF) registry (11 Asian regions including Taiwan, Hong Kong, China, India, Malaysia, Thailand, Singapore, Indonesia, Philippines, Japan, and Korea; 46 centres with enrolment between 1 October 2012 and 6 October 2016), we prospectively examined 5,964 patients with symptomatic HF (mean age 61.3 ± 13.3 years, 26% women, mean BMI 25.3 ± 5.3 kg/m2, 16% with HF with preserved ejection fraction [HFpEF; ejection fraction ≥ 50%]), among whom 2,051 also had waist-to-height ratio (WHtR) measurements (mean age 60.8 ± 12.9 years, 24% women, mean BMI 25.0 ± 5.2 kg/m2, 7% HFpEF). Patients were categorised by BMI quartiles or WHtR quartiles or 4 combined groups of BMI (low, <24.5 kg/m2 [lean], or high, ≥24.5 kg/m2 [obese]) and WHtR (low, <0.55 [thin], or high, ≥0.55 [fat]). Cox proportional hazards models were used to examine a 1-year composite outcome (HF hospitalisation or mortality). Across BMI quartiles, higher BMI was associated with lower risk of the composite outcome (ptrend < 0.001). Contrastingly, higher WHtR was associated with higher risk of the composite outcome. Individuals in the lean-fat group, with low BMI and high WHtR (13.9%), were more likely to be women (35.4%) and to be from low-income countries (47.7%) (predominantly in South/Southeast Asia), and had higher prevalence of diabetes (46%), worse quality of life scores (63.3 ± 24.2), and a higher rate of the composite outcome (51/232; 22%), compared to the other groups (p < 0.05 for all). Following multivariable adjustment, the lean-fat group had higher adjusted risk of the composite outcome (hazard ratio 1.93, 95% CI 1.17-3.18, p = 0.01), compared to the obese-thin group, with high BMI and low WHtR. Results were consistent across both HF subtypes (HFpEF and HF with reduced ejection fraction [HFrEF]; pinteraction = 0.355). Selection bias and residual confounding are potential limitations of such multinational observational registries. CONCLUSIONS: In this cohort of Asian patients with HF, the 'obesity paradox' is observed only when defined using BMI, with WHtR showing the opposite association with the composite outcome. Lean-fat patients, with high WHtR and low BMI, have the worst outcomes. A direct correlation between high WHtR and the composite outcome is apparent in both HFpEF and HFrEF. TRIAL REGISTRATION: Asian Sudden Cardiac Death in HF (ASIAN-HF) Registry ClinicalTrials.gov Identifier: NCT01633398.

Prediction of Survival in Asian Patients Hospitalized With Heart Failure: Validation of the OPTIMIZE-HF Risk Score.

BACKGROUND: Risk scores predicting in-patient mortality in heart failure patients have not been designed specifically for Asian patients. We aimed to validate and recalibrate the OPTIMIZE-HF risk model for in-hospital mortality in a multiethnic Asian population hospitalized for heart failure. METHODS AND RESULTS: Data from the Singapore Cardiac Databank Heart Failure on patients admitted for heart failure from January 1, 2008, to December 31, 2013, were included. The primary outcome studied was in-hospital mortality. Two models were compared: the original OPTIMIZE-HF risk model and a modified OPTIMIZE-HF risk model (similar variables but with coefficients derived from our cohort). A total of 15,219 patients were included. The overall in-hospital mortality was 1.88% (n = 286). The original model had a C-statistic of 0.739 (95% CI 0.708-0.770) with a good match between predicted and observed mortality rates (Hosmer-Lemeshow statistic 13.8; P = .086). The modified model had a C-statistic of 0.741 (95% CI 0.709-0.773) but a significant difference between predicted and observed mortality rates (Hosmer-Lemeshow statistic 17.2; P = .029). The modified model tended to underestimate risk at the extremes (lowest and highest ends) of risk. CONCLUSIONS: We provide the first independent validation of the OPTIMIZE-HF risk score in an Asian population. This risk model has been shown to perform reliably in our Asian cohort and will potentially provide clinicians with a useful tool to identify high-risk heart failure patients for more intensive management.

Differential contribution of pyruvate carboxylation to anaplerosis and cataplerosis during non-gluconeogenic and gluconeogenic conditions in HepG2 cells.

Pyruvate carboxylase (PC) is an anaplerotic enzyme that supplies oxaloacetate to mitochondria enabling the maintenance of other metabolic intermediates consumed by cataplerosis. Using liquid chromatography mass spectrometry (LC-MS) to measure metabolic intermediates derived from uniformly labeled 13C6-glucose or [3-13C]l-lactate, we investigated the contribution of PC to anaplerosis and cataplerosis in the liver cell line HepG2. Suppression of PC expression by short hairpin RNA lowered incorporation of 13C glucose incorporation into tricarboxylic acid cycle intermediates, aspartate, glutamate and sugar derivatives, indicating impaired cataplerosis. The perturbation of these biosynthetic pathways is accompanied by a marked decrease of cell viability and proliferation. In contrast, under gluconeogenic conditions where the HepG2 cells use lactate as a carbon source, pyruvate carboxylation contributed very little to the maintenance of these metabolites. Suppression of PC did not affect the percent incorporation of 13C-labeled carbon from lactate into citrate, α-ketoglutarate, malate, succinate as well as aspartate and glutamate, suggesting that under gluconeogenic condition, PC does not support cataplerosis from lactate.

Correction: Multimorbidity in patients with heart failure from 11 Asian regions: A prospective cohort study using the ASIAN-HF registry.

[This corrects the article DOI: 10.1371/journal.pmed.1002541.].