Regaining the path to malaria elimination: Lessons from the pandemic.

The stagnation in malaria elimination efforts can be attributed to several contributing reasons: large populations displaced by conflict and severe weather, insecticide and drug resistance, competing priorities with COVID-19 and Ebola. Part of the problem may also be us and our pre-pandemic systems. The accelerated response to the COVID-19 emergency carries lessons for global efforts against the 'other emergency', malaria. Michael has worked in vector control since 1977, beginning with Peace Corps in the Sabah (E. Malaysia) MCP. He earned an Sc.D. from Johns Hopkins researching malaria transmission in Pakistan; lived in Burma, Thailand, Cambodia and Zambia with stints in the US and Geneva supporting programmes throughout Africa and Asia, working for Johns Hopkins and Boston Universities, USAID, WFP, UNHCR, WHO, IVCC and NGOs involved in public health entomology and vector control in Africa and Asia.

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.

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.

Applying the COM-B behavior model to inform the delivery of spatial repellents and insecticide-treated clothing among 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 (VSPR) 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. Mixed methods were used to understand their experience, attitudes, and preferences regarding the products trialed. Quantitative data was summarized and qualitative insights were analyzed using thematic analysis, applying the Capability, Opportunity, Motivation Behavior Change (COM-B) model and Behavior 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 odor 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 ads, and enablement to guarantee 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 in Cambodia, while research efforts can aim to develop products that are rainproof, easy to use in forest settings, and have favorable odor profiles to target users.

Liquid NanoBiosensors Enable One-Pot Electrochemical Detection of Bacteria in Complex Matrices.

There is a need for point-of-care bacterial sensing and identification technologies that are rapid and simple to operate. Technologies that do not rely on growth cultures, nucleic acid amplification, step-wise reagent addition, and complex sample processing are the key for meeting this need. Herein, multiple materials technologies are integrated for overcoming the obstacles in creating rapid and one-pot bacterial sensing platforms. Liquid-infused nanoelectrodes are developed for reducing nonspecific binding on the transducer surface; bacterium-specific RNA-cleaving DNAzymes are used for bacterial identification; and redox DNA barcodes embedded into DNAzymes are used for binding-induced electrochemical signal transduction. The resultant single-step and one-pot assay demonstrates a limit-of-detection of 102 CFU mL-1 , with high specificity in identifying Escherichia coli amongst other Gram positive and negative bacteria including Klebsiella pneumoniae, Staphylococcus aureus, and Bacillus subtilis. Additionally, this assay is evaluated for analyzing 31 clinically obtained urine samples, demonstrating a clinical sensitivity of 100% and specify of 100%. When challenging this assay with nine clinical blood cultures, E. coli-positive and E. coli-negative samples can be distinguished with a probability of p < 0.001.

High-dose intravenous iron reduces myocardial infarction in patients on haemodialysis.

AIMS: To investigate the effect of high-dose iron vs. low-dose intravenous (IV) iron on myocardial infarction (MI) in patients on maintenance haemodialysis. METHODS AND RESULTS: This was a pre-specified analysis of secondary endpoints of the Proactive IV Iron Therapy in Hemodialysis Patients trial (PIVOTAL) randomized, controlled clinical trial. Adults who had started haemodialysis within the previous year, who had a ferritin concentration <400 µg per litre and a transferrin saturation <30% were randomized to high-dose or low-dose IV iron. The main outcome measure for this analysis was fatal or non-fatal MI. Over a median of 2.1 years of follow-up, 8.4% experienced a MI. Rates of type 1 MIs (3.2/100 patient-years) were 2.5 times higher than type 2 MIs (1.3/100 patient-years). Non-ST-elevation MIs (3.3/100 patient-years) were 6 times more common than ST-elevation MIs (0.5/100 patient-years). Mortality was high after non-fatal MI (1- and 2-year mortality of 40% and 60%, respectively). In time-to-first event analyses, proactive high-dose IV iron reduced the composite endpoint of non-fatal and fatal MI [hazard ratio (HR) 0.69, 95% confidence interval (CI) 0.52-0.93, P = 0.01] and non-fatal MI (HR 0.69, 95% CI 0.51-0.93; P = 0.01) when compared with reactive low-dose IV iron. There was less effect of high-dose IV iron on recurrent MI events than on the time-to-first event analysis. CONCLUSION: In total, 8.4% of patients on maintenance haemodialysis had an MI over 2 years. High-dose compared to low-dose IV iron reduced MI in patients receiving haemodialysis. EUDRACT REGISTRATION NUMBER: 2013-002267-25.

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.

Automatic Hemorrhage Detection From Color Doppler Ultrasound Using a Generative Adversarial Network (GAN)-Based Anomaly Detection Method.

Hemorrhage control has been identified as a priority focus area both for civilian and military populations in the United States because exsanguination is the most common cause of preventable death in hemorrhagic injury. Non-compressible torso hemorrhage (NCTH) has high mortality rate and there are currently no broadly available therapies for NCTH outside of a surgical room environment. Novel therapies, which include High Intensity Focused Ultrasound (HIFU) have emerged as promising methods for hemorrhage control as they can non-invasively cauterize bleeding tissue deep within the body without injuring uninvolved regions. A major challenge in the application of HIFU with color Doppler US guidance is the interpretation and optimization of the blood flow images in real-time to identify the hemorrhagic focus. Today, this task requires an expert sonographer, limiting the utility of this therapy in non-clinical environments. In this work, we investigated the feasibility of an automated hemorrhage detection method using a Generative Adversarial Network (GAN) for anomaly detection that learns a manifold of normal blood flow variability and subsequently identifies anomalous flow patterns that fall outside the learned manifold. As an initial feasibility study, we collected ultrasound color Doppler images of femoral arteries in an animal model of vascular injury (N = 11 pigs). Velocity information of the blood flow were extracted from the color Doppler images that were used for training and testing the anomaly detection network. Normotensive images from 8 pigs were used for training, and testing was performed on normotensive, immediately after injury, 10 minutes post-injury and 30 minutes post-injury images from 3 other pigs. The residual images or the reconstructed error maps show promise in detecting hemorrhages with an AUC of 0.90, 0.87, 0.62 immediately, 10 minutes post-injury and 30 minutes post-injury respectively with an overall AUC of 0.83.

Engineering death resistance in CHO cells for improved perfusion culture.

The reliable and cost-efficient manufacturing of monoclonal antibodies (mAbs) is essential to fulfil their ever-growing demand. Cell death in bioreactors reduces productivity and product quality, and is largely attributed to apoptosis. In perfusion bioreactors, this leads to the necessity of a bleed stream, which negatively affects the overall process economy. To combat this limitation, death-resistant Chinese hamster ovary cell lines were developed by simultaneously knocking out the apoptosis effector proteins Bak1, Bax, and Bok with CRISPR technology. These cell lines were cultured in fed-batch and perfusion bioreactors and compared to an unmodified control cell line. In fed-batch, the death-resistant cell lines showed higher cell densities and longer culture durations, lasting nearly a month under standard culture conditions. In perfusion, the death-resistant cell lines showed slower drops in viability and displayed an arrest in cell division after which cell size increased instead. Pertinently, the death-resistant cell lines demonstrated the ability to be cultured for several weeks without bleed, and achieved similar volumetric productivities at lower cell densities than that of the control cell line. Perfusion culture reduced fragmentation of the mAb produced, and the death-resistant cell lines showed increased glycosylation in the light chain in both bioreactor modes. These data demonstrate that rationally engineered death-resistant cell lines are ideal for mAb production in perfusion culture, negating the need to bleed the bioreactor whilst maintaining product quantity and quality.

A Novel Intron-Encoded Neuropilin-1 Isoform in Pancreatic Islets Associated With Very Young Age of Onset of Type 1 Diabetes.

Net synthesis of pancreatic ß-cells peaks before 2 years of life. ß-Cell mass is set within the first 5 years of life. In-frame translational readthrough of the NRP1 gene exon 9 into intron 9 generates a truncated neuropilin-1 protein lacking downstream sequence necessary for binding VEGF that stimulates ß-cell replication. VEGF is critical for developing but not adult islet neogenesis. Herein we show that cells in human pancreatic islets containing the full-length neuropilin-1 possess insulin but cells that contain the truncated neuropilin-1 are devoid of insulin. Decreased insulin cells increases susceptibility to onset of type 1 diabetes at a younger age. We also show that the frequency of a genetic marker in NRP1 intron 9 is higher among patients with onset of type 1 diabetes before age 4 years (31.8%), including those with onset at 0.67-2.00 and 2-4 years, compared with that in patients with onset at 4-8 years, at 8-12 years, and after 16 years (16.1%) with frequency equal to that in subjects without diabetes (16.0%). Decreased insulin cells plus the genetic data are consistent with a low effect mechanism that alters the onset of type 1 diabetes to a very young age in some patients, thus supporting the endotype concept that type 1 diabetes is a heterogeneous disease.

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.

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.

In situ laser manipulation of root tissues in transparent soil.

AIMS: Laser micromanipulation such as dissection or optical trapping enables remote physical modification of the activity of tissues, cells and organelles. To date, applications of laser manipulation to plant roots grown in soil have been limited. Here, we show laser manipulation can be applied in situ when plant roots are grown in transparent soil. METHODS: We have developed a Q-switched laser manipulation and imaging instrument to perform controlled dissection of roots and to study light-induced root growth responses. We performed a detailed characterisation of the properties of the cutting beams through the soil, studying dissection and optical ablation. Furthermore, we also studied the use of low light doses to control the root elongation rate of lettuce seedlings (Lactuca sativa) in air, agar, gel and transparent soil. RESULTS: We show that whilst soil inhomogeneities affect the thickness and circularity of the beam, those distortions are not inherently limiting. The ability to induce changes in root elongation or complete dissection of microscopic regions of the root is robust to substrate heterogeneity and microscopy set up and is maintained following the limited distortions induced by the transparent soil environment. CONCLUSIONS: Our findings show that controlled in situ laser dissection of root tissues is possible with a simple and low-cost optical set-up. We also show that, in the absence of dissection, a reduced laser light power density can provide reversible control of root growth, achieving a precise "point and shoot" method for root manipulation.

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.

Imaging in thick samples, a phased Monte Carlo radiation transfer algorithm.

SIGNIFICANCE: Optical microscopy is characterized by the ability to get high resolution, below 1 µm, high contrast, functional and quantitative images. The use of shaped illumination, such as with lightsheet microscopy, has led to greater three-dimensional isotropic resolution with low phototoxicity. However, in most complex samples and tissues, optical imaging is limited by scattering. Many solutions to this issue have been proposed, from using passive approaches such as Bessel beam illumination to active methods incorporating aberration correction, but making fair comparisons between different approaches has proven to be challenging. AIM: We present a phase-encoded Monte Carlo radiation transfer algorithm (φMC) capable of comparing the merits of different illumination strategies or predicting the performance of an individual approach. APPROACH: We show that φMC is capable of modeling interference phenomena such as Gaussian or Bessel beams and compare the model with experiment. RESULTS: Using this verified model, we show that, for a sample with homogeneously distributed scatterers, there is no inherent advantage to illuminating a sample with a conical wave (Bessel beam) instead of a spherical wave (Gaussian beam), except for maintaining a greater depth of focus. CONCLUSION: φMC is adaptable to any illumination geometry, sample property, or beam type (such as fractal or layered scatterer distribution) and as such provides a powerful predictive tool for optical imaging in thick samples.

Heart Failure Hospitalization in Adults Receiving Hemodialysis and the Effect of Intravenous Iron Therapy.

OBJECTIVES: This study sought to examine the effect of intravenous iron on heart failure events in hemodialysis patients. BACKGROUND: Heart failure is a common and deadly complication in patients receiving hemodialysis and is difficult to diagnose and treat. METHODS: The study analyzed heart failure events in the PIVOTAL (Proactive IV Iron Therapy in Hemodialysis Patients) trial, which compared intravenous iron administered proactively in a high-dose regimen with a low-dose regimen administered reactively. Heart failure hospitalization was an adjudicated outcome, a component of the primary composite outcome, and a prespecified secondary endpoint in the trial. RESULTS: Overall, 2,141 participants were followed for a median of 2.1 years. A first fatal or nonfatal heart failure event occurred in 51 (4.7%) of 1,093 patients in the high-dose iron group and in 70 (6.7%) of 1,048 patients in the low-dose group (HR: 0.66; 95% CI: 0.46-0.94; P = 0.023). There was a total of 63 heart failure events (including first and recurrent events) in the high-dose iron group and 98 in the low-dose group, giving a rate ratio of 0.59 (95% CI: 0.40-0.87; P = 0.0084). Most patients presented with pulmonary edema and were mainly treated by mechanical removal of fluid. History of heart failure and diabetes were independent predictors of a heart failure event. CONCLUSIONS: Compared with a lower-dose regimen, high-dose intravenous iron decreased the occurrence of first and recurrent heart failure events in patients undergoing hemodialysis, with large relative and absolute risk reductions. (UK Multicentre Open-label Randomised Controlled Trial Of IV Iron Therapy In Incident Haemodialysis Patients; 2013-002267-25).

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.

Report of the 2018 annual meeting of the Asia Pacific Malaria Elimination Network Vector Control Working Group: harnessing skills and knowledge for malaria elimination across the Asia Pacific.

The 2018 Asia Pacific Malaria Elimination Network's Vector Control Working Group (APMEN VCWG) annual meeting took place 3-5 September 2018 in Bangkok, Thailand. It was designed to be a forum for entomology and public health specialists from APMEN country programmes (over 90 participants from 30 countries) to discuss current progress and challenges related to planning, implementing, and sustaining effective vector control (VC) strategies for malaria elimination across the region, and to suggest practical and applicable solutions to these moving forward. The meeting was organised as a joint collaboration between the VCWG host institution-Faculty of Tropical Medicine, Mahidol University, Thailand-and leading partner institutions within the VCWG: Malaria Consortium and the Malaria Elimination Initiative at the University of California, San Francisco, Global Health Group (UCSF Global Health Group), under the leadership of the APMEN Director and VCWG Co-Chairs from ministries of health in Malaysia and India. This report provides an introduction to the role and nature of the VCWG, highlights key themes and topics presented and discussed at the meeting, and outlines the future objectives and focal areas for the VCWG and APMEN at large.

Metformin's Therapeutic Efficacy in the Treatment of Diabetes Does Not Involve Inhibition of Mitochondrial Glycerol Phosphate Dehydrogenase.

Mitochondrial glycerol phosphate dehydrogenase (mGPD) is the rate-limiting enzyme of the glycerol phosphate redox shuttle. It was recently claimed that metformin, a first-line drug used for the treatment of type 2 diabetes, inhibits liver mGPD 30-50%, suppressing gluconeogenesis through a redox mechanism. Various factors cast doubt on this idea. Total-body knockout of mGPD in mice has adverse effects in several tissues where the mGPD level is high but has little or no effect in liver, where the mGPD level is the lowest of 10 tissues. Metformin has beneficial effects in humans in tissues with high levels of mGPD, such as pancreatic ß-cells, where the mGPD level is much higher than that in liver. Insulin secretion in mGPD knockout mouse ß-cells is normal because, like liver, ß-cells possess the malate aspartate redox shuttle whose redox action is redundant to the glycerol phosphate shuttle. For these and other reasons, we used four different enzyme assays to reassess whether metformin inhibited mGPD. Metformin did not inhibit mGPD in homogenates or mitochondria from insulin cells or liver cells. If metformin actually inhibited mGPD, adverse effects in tissues where the level of mGPD is much higher than that in the liver could prevent the use of metformin as a diabetes medicine.