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The emergence of human pluripotent stem cell (hPSC) technology over the past two decades has provided a source of normal and diseased human cells for a wide variety of in vitro and in vivo applications. Notably, hPSC-derived cardiomyocytes (hPSC-CMs) are widely used to model human heart development and disease and are in clinical trials for treating heart disease. The success of hPSC-CMs in these applications requires robust, scalable approaches to manufacture large numbers of safe and potent cells. Although significant advances have been made over the past decade in improving the purity and yield of hPSC-CMs and scaling the differentiation process from 2D to 3D, efforts to induce maturation phenotypes during manufacturing have been slow. Process monitoring and closed-loop manufacturing strategies are just being developed. We discuss recent advances in hPSC-CM manufacturing, including differentiation process development and scaling and downstream processes as well as separation and stabilization.

The CREATE labs, a hybrid laboratory experience suitable for remote learning was developed at Imperial College in response to the COVID-19 pandemic. To facilitate the transformation from traditional to remote labs, a systematic review of offered projects was carried out to identify where learning objectives could be met using remote-friendly options such as simulations. Essential physical experiments were performed through the use of various technologies including online collaboration software and first-person point of view cameras to enable a high level of student involvement. Student surveys and interviews confirmed a positive experience comparable to previous years with an improvement in feedback provision.

Transmembrane protein 135 (TMEM135) is thought to participate in the cellular response to increased intracellular lipids yet no defined molecular function for TMEM135 in lipid metabolism has been identified. In this study, we performed a lipid analysis of tissues from Tmem135 mutant mice and found striking reductions of docosahexaenoic acid (DHA) across all Tmem135 mutant tissues, indicating a role of TMEM135 in the production of DHA. Since all enzymes required for DHA synthesis remain intact in Tmem135 mutant mice, we hypothesized that TMEM135 is involved in the export of DHA from peroxisomes. The Tmem135 mutation likely leads to the retention of DHA in peroxisomes, causing DHA to be degraded within peroxisomes by their beta-oxidation machinery. This may lead to generation or alteration of ligands required for the activation of peroxisome proliferator-activated receptor a (PPARa) signaling, which in turn could result in increased peroxisomal number and beta-oxidation enzymes observed in Tmem135 mutant mice. We confirmed this effect of PPARa signaling by detecting decreased peroxisomes and their proteins upon genetic ablation of Ppara in Tmem135 mutant mice. Using Tmem135 mutant mice, we also validated the protective effect of increased peroxisomes and peroxisomal beta-oxidation on the metabolic disease phenotypes of leptin mutant mice which has been observed in previous studies. Thus, we conclude that TMEM135 has a role in lipid homeostasis through its function in peroxisomes.

Mitochondria are dynamic organelles that undergo fission and fusion. While they are essential for cellular metabolism, the effect of dysregulated mitochondrial dynamics on cellular metabolism is not fully understood. We previously found that transmembrane protein 135 ( Tmem135) plays a role in the regulation of mitochondrial dynamics in mice. Mice homozygous for a Tmem135 mutation ( Tmem135FUN025/FUN025) display accelerated aging and age-related disease pathologies in the retina including the retinal pigment epithelium (RPE). We also generated a transgenic mouse line globally overexpressing the Tmem135 gene ( Tmem135 TG). In several tissues and cells that we studied such as the retina, heart, and fibroblast cells, we observed that the Tmem135 mutation causes elongated mitochondria, while overexpression of Tmem135 results in fragmented mitochondria. To investigate how abnormal mitochondrial dynamics affect metabolic signatures of tissues and cells, we identified metabolic changes in primary RPE cell cultures as well as heart, cerebellum, and hippocampus isolated from Tmem135FUN025/FUN025 mice (fusion > fission) and Tmem135 TG mice (fusion FUN025/FUN025 and Tmem135 TG RPE cells associated with osmosis and glucose and phospholipid metabolism. We observed depletion of NAD+ in both Tmem135FUN025/FUN025 and Tmem135 TG RPE cells, indicating that imbalance in mitochondrial dynamics to both directions lowers the cellular NAD+ level. Metabolic changes identified in this study might be associated with imbalanced mitochondrial dynamics in heart tissue and RPE cells which can likely lead to functional abnormalities. Impact statement Mitochondria are dynamic organelles undergoing fission and fusion. Proper regulation of this process is important for healthy aging process, as aberrant mitochondrial dynamics are associated with several age-related diseases/pathologies. However, it is not well understood how imbalanced mitochondrial dynamics may lead to those diseases and pathologies. Here, we aimed to determine metabolic alterations in tissues and cells from mouse models with over-fused (fusion > fission) and over-fragmented (fusion

Experiential learning is an integral component of engineering education. The Chemeng Remote Experience Augmented through TEchnology (CREATE) labs concept was implemented in the academic year 2020–21 in response to COVID19 for first-, second-, and third-year chemical engineering undergraduate students studying at Imperial College London. Using a range of technologies including pan-tilt-zoom cameras and Microsoft HoloLens 2 to provide real-time views of the lab environment from anywhere in the world. Students could control the experiments remotely while graduate teaching assistants (GTAs) operated the equipment based on the students’ instructions. This study is aimed at assessing the effectiveness of this implementation with a focus on student communication and confidence. Students and GTAs were surveyed at the end of labs, and a year-dependent response was observed. The majority of students (>70%) reported experiencing effective communication with team members and GTAs and there was a strong positive correlation between communication and confidence in applying engineering concepts in the labs (χ2 = 79.96; p = 1.69 ×10−10). 5–10% of students from all year groups reported that they disliked the lack of in-person activities. The majority (>90%) of GTAs assisting with experiments stated that they associated their role in the CREATE labs with that of a facilitator. The overall delivery of CREATE labs during academic year 2020–21 was positively received by both students and GTAs with recommendations for in-person activities for first- and second-year students. With minor modifications, CREATE labs has the potential to prepare students for effective remote communication and gain experience in using smart technologies which are key components of Industry 4.0.

Aging is a significant factor in the development of age-related diseases but how aging disrupts cellular homeostasis to cause age-related retinal disease is unknown. Here, we further our studies on transmembrane protein 135 (Tmem135), a gene involved in retinal aging, by examining the transcriptomic profiles of wild-type, heterozygous and homozygous Tmem135 mutant posterior eyecup samples through RNA sequencing (RNA-Seq). We found significant gene expression changes in both heterozygous and homozygous Tmem135 mutant mouse eyecups that correlate with visual function deficits. Further analysis revealed that expression of many genes involved in lipid metabolism are changed due to the Tmem135 mutation. Consistent with these changes, we found increased lipid accumulation in mutant Tmem135 eyecup samples. Since mutant Tmem135 mice have similar ocular pathologies as human age-related macular degeneration (AMD) eyes, we compared our homozygous Tmem135 mutant eyecup RNA-Seq dataset with transcriptomic datasets of human AMD donor eyes. We found similar changes in genes involved in lipid metabolism between the homozygous Tmem135 mutant eyecups and AMD donor eyes. Our study suggests that the Tmem135 mutation affects lipid metabolism as similarly observed in human AMD eyes, thus Tmem135 mutant mice can serve as a good model for the role of dysregulated lipid metabolism in AMD.

Teaching laboratories form an essential component of any engineering education. They enable students to participate in various stages of experiential learning including conceptualization and experimentation followed by reflection, analysis and interpretation of data. However, operating teaching laboratories with social distancing measures poses significant logistical and safety challenges, and alternative modes of delivery may be a realistic way forward in adapting engineering curricula to the post COVID-19 world. Best practices from spaces such as distance learning and virtual / remote laboratories can be leveraged to facilitate educators’ responses. This review is aimed at identifying evidence-based approaches for transforming hands-on labs into virtual or remote operation to achieve desired learning outcomes without compromising on soft skills and student self-efficacy. A critical review of the recent literature on delivering STEMM education laboratories in either a virtual or remote setting or a combination of both is presented here. Commonly emerging approaches are identified and strategies to implement remote or mixed-mode (a combination of remote and traditional lab components) delivery are highlighted. The value of these approaches to the educator is assessed based on claimed learning outcomes, availability of resources, technology, scheduling, and cost factors.

Recent releases of open-source research codes and solvers for numerically solving partial differential equations in Python present a great opportunity for educators to integrate these codes into the classroom in a variety of ways. The ease with which a problem can be implemented and solved using these codes reduce the barrier to entry for users. We demonstrate how one of these codes,FiPy, can be introduced to students through a short course using progression as the guiding philosophy. Four exercises of increasing complexity were developed. Basic concepts from more advanced numerical methods courses are also introduced at appropriate points. To further engage students, we demonstrate how an open research problem can be readily implemented and also incorporate the use of ParaView to post-process their results. Student engagement and learning outcomes were evaluated through a pre and post-course survey and a focus group discussion. Students broadly found the course to be engaging and useful with the ability to easily visualise the solution to PDEs being greatly valued. Due to the introductory nature of the course, due care in terms of set-up and the design of learning activities during the course is essential. This course, if integrated with appropriate level of support, can encourage students to use the provided codes and improve their understanding of concepts used in numerical analysis and PDEs., 12 pages, 9 figures. Fixed references

Background Modern immunosuppressive regimens in paediatric kidney transplant recipients have contributed to improved long-term allograft survival, but at the expense of an increased incidence of viral infections. Here, we describe, for the first time, the incidence, risk factors and clinical outcome of CMV, EBV, BKV and JCV viraemia in a cohort of paediatric allograft recipients treated with a corticosteroid-minimisation immunosuppressive regimen (CMR). Methods: We retrospectively analysed 98 children treated with a CMR (basiliximab induction, corticosteroids until day 4, long-term tacrolimus and mycophenolate mofetil), who received a kidney transplant in our centre between 2009 and 2019. Results: Over the first 4 years post-transplant, the incidences of viraemia were as follows: CMV, 25.5%; EBV, 52.0%; JCV, 16.3%; BKV, 26.5%. Younger children at time of transplant were more likely to develop EBV and BKV viraemia. EBV viraemia was also associated with a regimen involving corticosteroids, but lacking MMF. Recipient CMV serology predicted the development of EBV, BKV and CMV viraemia. Fifty-six percent of CMV viraemia episodes in high-risk patients occurred whilst the graft recipients were still receiving anti-viral prophylaxis or within 3 months of cessation. There was no difference in graft function at latest follow-up between those with and without viraemia. Conclusions: Judicious monitoring of viraemia, coupled with timely clinical intervention, can result in similar long-term outcomes for graft recipients compared to controls. The high incidence of CMV viraemia observed within a short period of cessation of anti-viral prophylaxis supports an extension of the length of prophylactic treatment in high-risk allograft recipients.

Summative year end assessments area major component of student assessment at the Department of Chemical Engineering, Imperial College London.More than 600 studentsparticipate in over40 different exams during the summer term. At the end of the spring term, the college moved to fully remote operation due to COVID-19, leaving the academic community with the challenge of delivering examinationsremotely. At the time pandemic hit the UK, teaching for allmodules in the department had been completed, the exam timetable had already been published and all exam paperspassed the mandatory external quality review. To implement time-limited remote examsas stipulated by the university, the department decided to proceed with anexisting VLE platformfor submission of answer-sheets.This study highlights stakeholder reflections from the academic and student communityduring the implementation of this approachculminating in a mock examination to gauge readiness of the infrastructure as well as the student population.Our survey found that the majority of students (>80%) managed to follow the written instructionsand readily engaged with scanning technologies and the uploading process.In the main, students did not have to adapt their learning or writing style. All stakeholdersprovided constructive suggestions at the end of the mock exam resulting in a relatively smooth transition to this new mode of examination. This study highlights challenges and reflections on making the summer year end examsremote in a very short timeframein a large and diverse Chemical Engineering department at very short notice.

Mitochondria are dynamic organelles that undergo fission and fusion. While they are essential for cellular metabolism, the effect of dysregulated mitochondrial dynamics on cellular metabolism is not fully understood. We previously found that transmembrane protein 135 (Tmem135) plays a role in the regulation of mitochondrial dynamics in mice. Mice homozygous for a Tmem135 mutation (Tmem135(FUN025/FUN025)) display accelerated aging and age-related disease pathologies in the retina including the retinal pigment epithelium (RPE). We also generated a transgenic mouse line globally overexpressing the Tmem135 gene (Tmem135 TG). In several tissues and cells that we studied such as the retina, heart, and fibroblast cells, we observed that the Tmem135 mutation causes elongated mitochondria, while overexpression of Tmem135 results in fragmented mitochondria. To investigate how abnormal mitochondrial dynamics affect metabolic signatures of tissues and cells, we identified metabolic changes in primary RPE cell cultures as well as heart, cerebellum, and hippocampus isolated from Tmem135(FUN025/FUN025) mice (fusion > fission) and Tmem135 TG mice (fusion fission) and over-fragmented (fusion

Background Mannose-binding lectin (MBL) is an important component of innate immune defense. MBL undergoes oligomerization to generate high mol weight (HMW) forms which act as pattern recognition molecules to detect and opsonize various microorganisms. Several post-translational modifications including prolyl hydroxylation are known to affect the oligomerization of MBL. Yet, the enzyme(s) which hydroxylate proline in the collagen-like domain residues have not been identified and the significance of prolyl hydroxylation is incompletely understood. Methods To investigate post-translational modifications of MBL, we stably expressed Myc-DDK tagged MBL in HEK293S cells. We used pharmacologic and genetic inhibition of 2-oxoglutarate–dependent dioxygenases (2OGDD) to identify the enzyme required for prolyl hydroxylation of MBL. We performed mass spectrometry to determine the effects of various inhibitors on MBL modifications. Results Secretion of HMW MBL was impaired by inhibitors of the superfamily of 2OGDD, and was dependent on prolyl-4-hydroxylase subunit α1. Roxadustat and vadadustat, but not molidustat, led to significant suppression of hydroxylation and secretion of HMW forms of MBL. Conclusions These data suggest that prolyl hydroxylation in the collagen-like domain of MBL is mediated by collagen prolyl-4-hydroxylase. Reduced MBL activity is likely to be an off-target effect of some, but not all, prolyl hydroxylase domain (PHD) inhibitors. There may be advantages in selective PHD inhibitors that would not interfere with MBL production.

Here, we describe how to efficiently direct human pluripotent stem cells (hPSCs) differentiation into self-renewing epicardial cells in a completely defined, xeno-free system by temporal modulation of regulators of canonical Wnt signaling. Appropriate differentiation-stage-specific application of Gsk3 inhibitor, Wnt inhibitor, and Gsk3 inhibitor (GiWiGi) is sufficient to produce cells expressing epicardial markers and exhibiting epicardial phenotypes with a high yield and purity from multiple hPSC lines in 16 d. Characterization of differentiated cells is performed via flow cytometry and immunostaining to assess quantitative expression and localization of epicardial cell-specific proteins. In vitro differentiation into fibroblasts and smooth muscle cells (SMCs) is also described. In addition, culture in the presence of transforming growth factor (TGF)-β inhibitors allows long-term expansion of hPSC-derived epicardial cells (for at least 25 population doublings). Functional human epicardial cells differentiated via this protocol may constitute a potential cell source for heart disease modeling, drug screening, and cell-based therapeutic applications.

Stem cells undergo extensive metabolic rewiring during reprogramming, proliferation and differentiation, and numerous studies have demonstrated a significant role of metabolism in controlling stem cell fates. Recent applications of metabolomics, the study of concentrations and fluxes of small molecules in cells, have advanced efforts to characterize and maturate stem cell fates, assess drug toxicity in stem cell tissue models, identify biomarkers, and study the effects of environment on metabolic pathways in stem cells and their progeny. Looking to the future, combining metabolomics with other -omics approaches will provide a deeper understanding of the complex regulatory mechanisms of stem cells.

B lymphocyte development and selection are central to adaptive immunity and self-tolerance. These processes require B cell receptor (BCR) signaling and occur in bone marrow, an environment with variable hypoxia, but whether hypoxia-inducible factor (HIF) is involved is unknown. We show that HIF activity is high in human and murine bone marrow pro-B and pre-B cells and decreases at the immature B cell stage. This stage-specific HIF suppression is required for normal B cell development because genetic activation of HIF-1α in murine B cells led to reduced repertoire diversity, decreased BCR editing and developmental arrest of immature B cells, resulting in reduced peripheral B cell numbers. HIF-1α activation lowered surface BCR, CD19 and B cell-activating factor receptor and increased expression of proapoptotic BIM. BIM deletion rescued the developmental block. Administration of a HIF activator in clinical use markedly reduced bone marrow and transitional B cells, which has therapeutic implications. Together, our work demonstrates that dynamic regulation of HIF-1α is essential for normal B cell development.

The kinetic Monte Carlo (KMC) method is a popular modeling approach for reaching large materials length and time scales. The KMC dynamics is erroneous when atomic processes that are relevant to the dynamics are missing from the KMC model. Recently, we had developed for the first time an error measure for KMC in Bhute and Chatterjee [J. Chem. Phys. 138, 084103 (2013)]. The error measure, which is given in terms of the probability that a missing process will be selected in the correct dynamics, requires estimation of the missing rate. In this work, we present an improved procedure for estimating the missing rate. The estimate found using the new procedure is within an order of magnitude of the correct missing rate, unlike our previous approach where the estimate was larger by orders of magnitude. This enables one to find the error in the KMC model more accurately. In addition, we find the time for which the KMC model can be used before a maximum error in the dynamics has been reached. (C) 2013 AIP Publishing LLC.

Metabolic dysfunction impacts stroke incidence and outcome. However, the intricate association between altered metabolic program due to aging, and focal ischemia in brain, circulation, and peripheral organs is not completely elucidated. Here we identified locally and systemically altered metabolites in brain, liver, and plasma as a result of normal aging, ischemic-stroke, and extended time of reperfusion injury. Comprehensive quantitative metabolic profiling was carried out using nuclear magnetic resonance spectroscopy. Aging, but healthy rats showed significant metabolic alterations in the brain, but only a few metabolic changes in the liver and plasma as compared to younger rats. But, ischemic stroke altered metabolites significantly in liver and plasma of older rats during early acute phase. Major metabolic changes were also seen in the brains of younger rats following ischemic stroke during early acute phase of injury. We further report that metabolic changes occur sequentially in a tissue specific manner during extended reperfusion time of late repair phase. First metabolic alterations occurred in brain due to local injury. Next, changes in circulating metabolites in plasma occurred during acute-repair phase transition time. Lastly, the delayed systemic effect was seen in the peripheral organ, liver that exhibited significant and persistent changes in selected metabolites during later reperfusion time. The metabolic pathways involved in energy/glucose, and amino acid metabolism, inflammation, and oxidative stress were mainly altered as a result of aging and ischemia/reperfusion. Biomarker analysis revealed citrate, lysine, and tyrosine as potential age-independent blood metabolic biomarkers of ischemia/reperfusion. Overall, our study elucidates the complex network of metabolic events as a function of normal aging and acute stroke. We further provide evidence for a clear transition from local to systemic metabolic dysfunction due to ischemic injury in a time dependent manner, which may altogether greatly impact the post-stroke outcome.

B-lymphocyte development and selection is central to adaptive immunity and self-tolerance. These processes require B cell receptor (BCR)-signaling and occur in bone marrow, an environment with variable hypoxia, but whether Hypoxia-inducible factor (HIF) is involved is unknown. We show high HIF-activity in human bone marrow pro/preB cells that decreased at the immature B cell stage. Stage-specific HIF-suppression was required for B cell development, since constitutive HIF-1α-activation in murine B cells led to immature B cell developmental-arrest, limited BCR-editing and reduced repertoire-diversity. Disruption of HIF-1α-regulation reduced surface BCR and CD19 and increased expression of pro-apoptotic BIM. Transient administration of a clinical HIF-activator reduced immature and transitional B cells, confirming the on-going importance of this axis for the maintenance of the established B cell-compartment. Our work identifies a critical role for HIF-1α in tuning BCR-signal strength and demonstrates that dynamic HIF-regulation is essential for normal B cell development, with significant clinical implications.

Background— Cardiac metabolism is critical for the functioning of the heart, and disturbance in this homeostasis is likely to influence cardiac disorders or cardiomyopathy. Our laboratory has previously shown that SNRK (sucrose nonfermenting related kinase) enzyme, which belongs to the AMPK (adenosine monophosphate–activated kinase) family, was essential for cardiac metabolism in mammals. Snrk global homozygous knockout (KO) mice die at postnatal day 0, and conditional deletion of Snrk in cardiomyocytes ( Snrk cmcKO) leads to cardiac failure and death by 8 to 10 months. Methods and Results— We performed additional cardiac functional studies using echocardiography and identified further cardiac functional deficits in Snrk cmcKO mice. Nuclear magnetic resonance-based metabolomics analysis identified key metabolic pathway deficits in SNRK knockdown cardiomyocytes in vitro. Specifically, metabolites involved in lipid metabolism and oxidative phosphorylation are altered, and perturbations in these pathways can result in cardiac function deficits and heart failure. A phosphopeptide-based proteomic screen identified ROCK (Rho-associated kinase) as a putative substrate for SNRK, and mass spec-based fragment analysis confirmed key amino acid residues on ROCK that are phosphorylated by SNRK. Western blot analysis on heart lysates from Snrk cmcKO adult mice and SNRK knockdown cardiomyocytes showed increased ROCK activity. In addition, in vivo inhibition of ROCK partially rescued the in vivo Snrk cmcKO cardiac function deficits. Conclusions— Collectively, our data suggest that SNRK in cardiomyocytes is responsible for maintaining cardiac metabolic homeostasis, which is mediated in part by ROCK, and alteration of this homeostasis influences cardiac function in the adult heart.

Genomic instability is one of the hallmarks of cancer. Several chemotherapeutic drugs and radiotherapy induce DNA damage to prevent cancer cell replication. Cells in turn activate different DNA damage response (DDR) pathways to either repair the damage or induce cell death. These DDR pathways also elicit metabolic alterations which can play a significant role in the proper functioning of the cells. The understanding of these metabolic effects resulting from different types of DNA damage and repair mechanisms is currently lacking. In this study, we used NMR metabolomics to identify metabolic pathways which are altered in response to different DNA damaging agents. By comparing the metabolic responses in MCF-7 cells, we identified the activation of poly (ADP-ribose) polymerase (PARP) in methyl methanesulfonate (MMS)-induced DNA damage. PARP activation led to a significant depletion of NAD+. PARP inhibition using veliparib (ABT-888) was able to successfully restore the NAD+ levels in MMS-treated cells. In addition, double strand break induction by MMS and veliparib exhibited similar metabolic responses as zeocin, suggesting an application of metabolomics to classify the types of DNA damage responses. This prediction was validated by studying the metabolic responses elicited by radiation. Our findings indicate that cancer cell metabolic responses depend on the type of DNA damage responses and can also be used to classify the type of DNA damage.

Background: Metabolic dysregulation associated with aging impacts stroke incidence and outcome. Hyperglycemia occurs in 30% of patients with ischemic stroke, and is associated with poor stroke recovery. On the other hand, depletion of glucose within the brain prevents production of ATP, leading to energy reduction and neuronal death. Thus, balanced glucose metabolism is critical for normal brain function. However, our understanding of the regulation of genes involved in glucose metabolism and relationship to age in the post-stroke brain is elusive. Methods: Using transcriptomic and metabolomics approach, we examined the expression pattern of glucose metabolism pathway specific genes in the naïve and post-stroke brains of 3 month, and 12 month old rats subjected to focal cerebral ischemia by middle cerebral artery occlusion and 2 days re-perfusion. Metabolites were analyzed using Nuclear Magnetic Resonance (NMR) spectroscopy. Results: Our data shows substantial alterations in the glucose metabolism pathway in aged, and particularly in post stroke rat brain. Brains from 12 month old rats showed about 15 fold increase in phosphoenolpyruvate carboxykinase (PCK1), and 2 fold increase in phosphorylase kinase (PHKG1) mRNA as compared to 3 month old rats. In the post-stroke brain, mRNA levels of hexokinase 3 (HK3) was upregulated 9 fold in 3 month old rats. However, HK3 mRNA substantially increased to 26 fold in 12 month old post-stroke brain as compared to control brains. The PCK1 gene was down regulated 2-4 fold in post-stroke brain. Metabolomics studies indicated significant alterations in nucleotide metabolism and decreased antioxidants in the aged brain. Metabolic changes in post-stroke brains included depletion of ADP and AMP, and accumulation of fumarate, O-phospho-ethanolamine, glycerol, glycine, leucine, lysine and malate. Conclusion: Upregulation of PCK1, a key gluconeogenic enzyme may contribute to excess glucose production in aged brain, leading to increased risk of obesity and stroke. The inducible expression of HK3 in post-stroke brain suggests its adaptive role in metabolic responses to stress in the ischemic environment. Overall, HK3 by modulating glycolysis pathway, may lead to metabolic reprogramming in aged and post stroke brain.

The epicardium contributes both multi-lineage descendants and paracrine factors to the heart during cardiogenesis and cardiac repair, underscoring its potential for cardiac regenerative medicine. Yet little is known about the cellular and molecular mechanisms that regulate human epicardial development and regeneration. Here, we show that the temporal modulation of canonical Wnt signaling is sufficient for epicardial induction from 6 different human pluripotent stem cell (hPSC) lines, including a WT1-2A-eGFP knock-in reporter line, under chemically-defined, xeno-free conditions. We also show that treatment with transforming growth factor beta (TGF-β)-signalling inhibitors permitted long-term expansion of the hPSC-derived epicardial cells, resulting in a more than 25 population doublings of WT1+ cells in homogenous monolayers. The hPSC-derived epicardial cells were similar to primary epicardial cells both in vitro and in vivo, as determined by morphological and functional assays, including RNA-seq. Our findings have implications for the understanding of self-renewal mechanisms of the epicardium and for epicardial regeneration using cellular or small-molecule therapies.

Cardiovascular disease is a leading cause of death worldwide. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) hold immense clinical potential and recent studies have enabled generation of virtually pure hPSC-CMs with high efficiency in chemically defined and xeno-free conditions. Despite these advances, hPSC-CMs exhibit an immature phenotype and are arrhythmogenic in vivo, necessitating development of strategies to mature these cells. hPSC-CMs undergo significant metabolic alterations during differentiation and maturation. A detailed analysis of the metabolic changes accompanying maturation of hPSC-CMs may prove useful in identifying new strategies to expedite hPSC-CM maturation and also may provide biomarkers for testing or validating hPSC-CM maturation. In this study we identified global metabolic changes which take place during long-term culture and maturation of hPSC-CMs derived from three different hPSC lines. We have identified several metabolic pathways, including phospholipid metabolism and pantothenate and Coenzyme A metabolism, which showed significant enrichment upon maturation in addition to fatty acid oxidation and metabolism. We also identified increases in glycerophosphocholine and the glycerophosphocholine:phosphocholine ratio as potential metabolic biomarkers of maturation. These biomarkers were also affected in a similar manner during murine heart development in vivo. These results support that hPSC-CM maturation is associated with extensive metabolic changes in metabolic network utilization and understanding the roles of these metabolic changes has the potential to develop novel approaches to monitor and expedite hPSC-CM maturation.

Breast tumors are characterized into subtypes based on their surface marker expression, which affects their prognosis and treatment. Poly (ADP-ribose) polymerase (PARP) inhibitors have shown promising results in clinical trials, both as single agents and in combination with other chemotherapeutics, in several subtypes of breast cancer patients. Here, we used NMR-based metabolomics to probe cell line-specific effects of the PARP inhibitor Veliparib and radiation on metabolism in three breast cancer cell lines. Our data reveal several cell line-independent metabolic changes upon PARP inhibition. Pathway enrichment and topology analysis identified that nitrogen metabolism, glycine, serine and threonine metabolism, aminoacyl-tRNA biosynthesis and taurine and hypotaurine metabolism were enriched after PARP inhibition in all three breast cancer cell lines. Many metabolic changes due to radiation and PARP inhibition were cell line-dependent, highlighting the need to understand how these treatments affect cancer cell response via changes in metabolism. Finally, both PARP inhibition and radiation induced a similar metabolic responses in BRCA-mutant HCC1937 cells, but not in MCF7 and MDAMB231 cells, suggesting that radiation and PARP inhibition share similar interactions with metabolic pathways in BRCA mutant cells. Our study emphasizes the importance of differences in metabolic responses to cancer treatments in different subtypes of cancers.

Introduction: Metabolic dysfunction is a common hallmark of aging, and it influences the neurodegenerative diseases including stroke incidence and outcome. Several metabolomics-based studies have revealed metabolite biomarkers in stroke patients and yet metabolic changes as a result of aging and focal ischemia/reperfusion (I/R) time are not completely elucidated. In this study, we examined the dysregulation of metabolites in the plasma as a function of age and cerebral ischemic injury. Methods: We compared the metabolic pattern in young (3 months) and older rats (12 months) before, and 2 days after I/R injury. Stroke was induced by middle cerebral artery occlusion. For time course study, plasma samples from younger animals were collected at day 1, 2, 3, 7, 14 and 21 of I/R (n=3). We used Nuclear Magnetic Resonance spectroscopy and compared the metabolite peaks using spectral binning and targeted profiling. The data was analyzed using one way analysis of variance, principal component analysis (PCA) and hierarchical clustering. Pathway enrichment and topological analysis tools were used to identify the age and stroke responsive metabolic pathways. Results: PCA revealed global differences in plasma of naïve young and old rats, with old rats showing significantly lower glucose levels, and higher formate levels. Also, older animals showed reduced levels of acetate and formate and very high levels of 3-hydroxybutyrate 2 days after I/R. Younger rats showed significant differences starting from day 3 onwards with lower glucose concentration and increased 3-hydroxybutyrate, creatine, formate and glycine concentrations, which remained at a higher level on day 7 of I/R. Interestingly, the metabolic profile of plasma at day 21 of I/R showed similar pattern as that of control naïve animals. Pathway topology analysis revealed enrichment of citric acid cycle and glycine, serine and threonine metabolic pathways in older animals as early as 2 days after I/R. However, younger animals showed a delayed metabolic response after day 3 of I/R. Conclusions: We have demonstrated that the metabolic profile of plasma is altered as a function of age, ischemic stroke and time of reperfusion. These results suggests an important role for imbalance in metabolites in stroke pathogenesis.

Human pluripotent stem cell (hPSC)-derived endothelial cells and their progenitors are important for vascular research and therapeutic revascularization. Here, we report a completely defined endothelial progenitor differentiation platform that uses a minimalistic medium consisting of Dulbecco's modified eagle medium and ascorbic acid, lacking of albumin and growth factors. Following hPSC treatment with a GSK-3β inhibitor and culture in this medium, this protocol generates more than 30% multipotent CD34+ CD31+ endothelial progenitors that can be purified to >95% CD34+ cells via magnetic activated cell sorting (MACS). These CD34+ progenitors are capable of differentiating into endothelial cells in serum-free inductive media. These hPSC-derived endothelial cells express key endothelial markers including CD31, VE-cadherin, and von Willebrand factor (vWF), exhibit endothelial-specific phenotypes and functions including tube formation and acetylated low-density lipoprotein (Ac-LDL) uptake. This fully defined platform should facilitate production of proliferative, xeno-free endothelial progenitor cells for both research and clinical applications.

Metabolism in cancer cells is significantly altered as compared to their normal counterparts. Metabolic rewiring can assist cancer cells to meet their requirements for uncontrolled proliferation and survival. In addition to their role in protein synthesis and energy generation, metabolites also act as signaling molecules, antioxidants and perform osmoregulatory functions. Differences in genetic makeup and phenotype of cells can lead to differences in these metabolic needs. A detailed analysis of metabolic signatures of different cancer cells can therefore prove useful in understanding metabolic interactions with the phenotype in cancer cells. This study aims at identifying these metabolic differences among distinct breast cancer cell lines and gaining mechanistic insights into possible reasons for differential abundance of metabolites. We used nuclear magnetic resonance (NMR) spectroscopy to quantify intracellular metabolites in five breast cancer cell lines having distinct genetic and phenotypic composition. We show that the intracellular abundance of essential amino acids depends on cell type and medium composition. We also show that myo-inositol abundance was significantly higher in ER-/PR- than ER+/PR+ breast cancer cells due to increased expression of inositol-3-phosphate synthase 1 expression. Myo-inositol is an osmolyte and also a precursor of inositol phosphates which participate in PI3K and calcium signaling pathways. Although the reason ER-/PR- cells contain greater concentrations of myo-inositol than ER+/PR+ cells is not yet known, increased myo-inositol abundance could potentially serve as a marker of ER-/PR- breast cancer cells. We also observed significant changes in phospholipid metabolism, specifically, glycerophosohocholine (GPC) concentration correlated with doubling time. We also found that reducing serum concentration in the growth medium reduced the growth rate of MCF7 cells, which led to increased GPC abundance in the cells. GPC is also an intracellular osmolyte and is a degradation product of phosphatidylcholine (PtdCho). Phospholipid metabolism has been shown to be significantly altered in breast cancer cells and has been used as prognostic marker in breast cancer cells. A positive correlation of doubling time with GPC irrespective of ER/PR status may prove useful in deciding therapeutic options. Our results highlight the differences in metabolic requirements in different breast cancer cell lines and suggest potential for gaining mechanistic understanding of these differences using metabolomics. Note: This abstract was not presented at the meeting. Citation Format: Vijesh J. Bhute, Kylie R. Knutson, Yan Ma, Sean P. Palecek. Differential metabolite requirements in breast cancer cells correlate with ER/PR status and doubling time [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2520. doi:10.1158/1538-7445.AM2017-2520

Background Maintaining DNA integrity is essential for cell survival and proper functioning. Therefore, many cancer therapies, including radiotherapy and several chemotherapeutic drugs, induce damage to the DNA leading to genomic instability [1]. There are several DNA damage responses (DDRs) which can be activated depending on the type and the extent of DNA damage [1]. These responses involve detection of the damage by several proteins including, poly (ADP-ribose) polymerase (PARP), which can directly or indirectly affect metabolism [1].

Markov state models (MSMs) are employed extensively in literature with the kinetic Monte Carlo (KMC) method for studying state-to-state dynamics in a wide range of material systems. A MSM contains a list of atomic processes and their rate constants for different states of the system. In many situations, only few of the possible atomic processes are included in the MSM. The use of an incomplete MSM with the KMC method can lead to an error in the dynamics. In this work, we develop an error measure to assess the accuracy of a MSM generated using dynamical basin escape pathway searches. We show that the error associated with an incomplete MSM depends on the rate constants missing from the MSM. A procedure to estimate the missing rate constants is developed. We demonstrate our approach using some examples., A.C. acknowledges support from BRNS Young Scientist Award from Department of Atomic Energy (DAE-BRNS) No. 2011/36/43-BRNS/1975.

We present a method called local environment kinetic Monte Carlo (LE-KMC) method for efficiently performing off-lattice, self-learning kinetic Monte Carlo (KMC) simulations of activated processes in material systems. Like other off-lattice KMC schemes, new atomic processes can be found on-the-fly in LE-KMC. However, a unique feature of LE-KMC is that as long as the assumption that all processes and rates depend only on the local environment is satisfied, LE-KMC provides a general algorithm for (i) unambiguously describing a process in terms of its local atomic environments, (ii) storing new processes and environments in a catalog for later use with standard KMC, and (iii) updating the system based on the local information once a process has been selected for a KMC move. Search, classification, storage and retrieval steps needed while employing local environments and processes in the LE-KMC method are discussed. The advantages and computational cost of LE-KMC are discussed. We assess the performance of the LE-KMC algorithm by considering test systems involving diffusion in a submonolayer Ag and Ag-Cu alloy films on Ag(001) surface., This work was supported by Indian Institute of Technology Kanpur Start-up grant IITK/CHE/20100105