Comparing the Extraction Performance in Mouse Plasma of Different Biphasic Methods for Polar and Nonpolar Compounds.

Many metabolomic studies are interested in both polar and nonpolar analyses. However, the available sample volume often precludes multiple separate extractions. Therefore, there are major advantages in performing a biphasic extraction and retaining both phases for subsequent separate analyses. To be successful, such approaches require the method to be robust and repeatable for both phases. Hence, we determined the performance of three extraction protocols, plus two variant versions, using 25 µL of commercially available mouse plasma. The preferred option for nonpolar lipids was a modified diluted version of a method employing methyl tert-butyl ether (MTBE) suggested by Matyash and colleagues due to its high repeatability for nonpolar compounds. For polar compounds, the Bligh-Dyer method performs best for sensitivity but with consequentially poorer lipid performance. Overall, the scaled-down version of the MTBE method gave the best overall performance, with high sensitivity for both polar and nonpolar compounds and good repeatability for polar compounds in particular.

Current Practices in LC-MS Untargeted Metabolomics: A Scoping Review on the Use of Pooled Quality Control Samples.

Untargeted metabolomics is an analytical approach with numerous applications serving as an effective metabolic phenotyping platform to characterize small molecules within a biological system. Data quality can be challenging to evaluate and demonstrate in metabolomics experiments. This has driven the use of pooled quality control (QC) samples for monitoring and, if necessary, correcting for analytical variance introduced during sample preparation and data acquisition stages. Described herein is a scoping literature review detailing the use of pooled QC samples in published untargeted liquid chromatography-mass spectrometry (LC-MS) based metabolomics studies. A literature query was performed, the list of papers was filtered, and suitable articles were randomly sampled. In total, 109 papers were each reviewed by at least five reviewers, answering predefined questions surrounding the use of pooled quality control samples. The results of the review indicate that use of pooled QC samples has been relatively widely adopted by the metabolomics community and that it is used at a similar frequency across biological taxa and sample types in both small- and large-scale studies. However, while many studies generated and analyzed pooled QC samples, relatively few reported the use of pooled QC samples to improve data quality. This demonstrates a clear opportunity for the field to more frequently utilize pooled QC samples for quality reporting, feature filtering, analytical drift correction, and metabolite annotation. Additionally, our survey approach enabled us to assess the ambiguity in the reporting of the methods used to describe the generation and use of pooled QC samples. This analysis indicates that many details of the QC framework are missing or unclear, limiting the reader's ability to determine which QC steps have been taken. Collectively, these results capture the current state of pooled QC sample usage and highlight existing strengths and deficiencies as they are applied in untargeted LC-MS metabolomics.

High-Throughput Metabolomics Applications in Pathogenesis and Diagnosis of Valvular Heart Disease.

High-throughput metabolomics techniques are a useful tool to understand many disease conditions including cardiovascular disease such as valvular heart disease(s) (VHD). VHD involves damage to heart valves, mostly presenting as stenosis, regurgitation or prolapse and can be classified into degenerative, rheumatic, congenital, or prosthetic valve disease. Gaps remain in our understanding of the pathogenesis of the common VHD. It is now fitting to place into perspective the contribution of metabolomics in the mechanism of development, diagnosis, and prognosis of VHD. A structured search for metabolomics studies centred on human VHD was undertaken. Biomarkers associated with the pathogenesis of bicuspid aortic valve disease, mitral valve disease, rheumatic heart disease, and degenerative aortic valve stenosis are reviewed and discussed. In addition, metabolic biomarkers reported to prognosticate patient outcomes of post-valve repair or replacement are highlighted. Finally, we also review the pitfalls and limitations to consider when designing metabolomics studies, especially from a clinician's viewpoint. In the future, reliable and simple metabolic biomarker(s) may supplement the existing diagnostic tools in the early diagnosis of VHD.

Inflammation in Children with CKD Linked to Gut Dysbiosis and Metabolite Imbalance.

BACKGROUND: CKD is characterized by a sustained proinflammatory response of the immune system, promoting hypertension and cardiovascular disease. The underlying mechanisms are incompletely understood but may be linked to gut dysbiosis. Dysbiosis has been described in adults with CKD; however, comorbidities limit CKD-specific conclusions. METHODS: We analyzed the fecal microbiome, metabolites, and immune phenotypes in 48 children (with normal kidney function, CKD stage G3-G4, G5 treated by hemodialysis [HD], or kidney transplantation) with a mean±SD age of 10.6±3.8 years. RESULTS: Serum TNF-α and sCD14 were stage-dependently elevated, indicating inflammation, gut barrier dysfunction, and endotoxemia. We observed compositional and functional alterations of the microbiome, including diminished production of short-chain fatty acids. Plasma metabolite analysis revealed a stage-dependent increase of tryptophan metabolites of bacterial origin. Serum from patients on HD activated the aryl hydrocarbon receptor and stimulated TNF-α production in monocytes, corresponding to a proinflammatory shift from classic to nonclassic and intermediate monocytes. Unsupervised analysis of T cells revealed a loss of mucosa-associated invariant T (MAIT) cells and regulatory T cell subtypes in patients on HD. CONCLUSIONS: Gut barrier dysfunction and microbial metabolite imbalance apparently mediate the proinflammatory immune phenotype, thereby driving the susceptibility to cardiovascular disease. The data highlight the importance of the microbiota-immune axis in CKD, irrespective of confounding comorbidities.

Serum Starvation Accelerates Intracellular Metabolism in Endothelial Cells.

Periods of low energy supply are challenging conditions for organisms and cells during fasting or famine. Although changes in nutrient levels in the blood are first sensed by endothelial cells, studies on their metabolic adaptations to diminished energy supply are lacking. We analyzed the dynamic metabolic activity of human umbilical vein endothelial cells (HUVECs) in basal conditions and after serum starvation. Metabolites of glycolysis, the tricarboxylic acid (TCA) cycle, and the glycerol pathway showed lower levels after serum starvation, whereas amino acids had increased levels. A metabolic flux analysis with 13C-glucose or 13C-glutamine labeling for different time points reached a plateau phase of incorporation after 30 h for 13C-glucose and after 8 h for 13C-glutamine under both experimental conditions. Notably, we observed a faster label incorporation for both 13C-glucose and 13C-glutamine after serum starvation. In the linear range of label incorporation after 3 h, we found a significantly faster incorporation of central carbon metabolites after serum starvation compared to the basal state. These findings may indicate that endothelial cells develop increased metabolic activity to cope with energy deficiency. Physiologically, it can be a prerequisite for endothelial cells to form new blood vessels under unfavorable conditions during the process of angiogenesis in vivo.

Deep Learning-Assisted Peak Curation for Large-Scale LC-MS Metabolomics.

Available automated methods for peak detection in untargeted metabolomics suffer from poor precision. We present NeatMS, which uses machine learning based on a convoluted neural network to reduce the number and fraction of false peaks. NeatMS comes with a pre-trained model representing expert knowledge in the differentiation of true chemical signal from noise. Furthermore, it provides all necessary functions to easily train new models or improve existing ones by transfer learning. Thus, the tool improves peak curation and contributes to the robust and scalable analysis of large-scale experiments. We show how to integrate it into different liquid chromatography-mass spectrometry (LC-MS) analysis workflows, quantify its performance, and compare it to various other approaches. NeatMS software is available as open source on github under permissive MIT license and is also provided as easy-to-install PyPi and Bioconda packages.

Quality assurance and quality control reporting in untargeted metabolic phenotyping: mQACC recommendations for analytical quality management.

BACKGROUND: Demonstrating that the data produced in metabolic phenotyping investigations (metabolomics/metabonomics) is of good quality is increasingly seen as a key factor in gaining acceptance for the results of such studies. The use of established quality control (QC) protocols, including appropriate QC samples, is an important and evolving aspect of this process. However, inadequate or incorrect reporting of the QA/QC procedures followed in the study may lead to misinterpretation or overemphasis of the findings and prevent future metanalysis of the body of work. OBJECTIVE: The aim of this guidance is to provide researchers with a framework that encourages them to describe quality assessment and quality control procedures and outcomes in mass spectrometry and nuclear magnetic resonance spectroscopy-based methods in untargeted metabolomics, with a focus on reporting on QC samples in sufficient detail for them to be understood, trusted and replicated. There is no intent to be proscriptive with regard to analytical best practices; rather, guidance for reporting QA/QC procedures is suggested. A template that can be completed as studies progress to ensure that relevant data is collected, and further documents, are provided as on-line resources. KEY REPORTING PRACTICES: Multiple topics should be considered when reporting QA/QC protocols and outcomes for metabolic phenotyping data. Coverage should include the role(s), sources, types, preparation and uses of the QC materials and samples generally employed in the generation of metabolomic data. Details such as sample matrices and sample preparation, the use of test mixtures and system suitability tests, blanks and technique-specific factors are considered and methods for reporting are discussed, including the importance of reporting the acceptance criteria for the QCs. To this end, the reporting of the QC samples and results are considered at two levels of detail: "minimal" and "best reporting practice" levels.

Single cell metabolism: current and future trends.

Single cell metabolomics is an emerging and rapidly developing field that complements developments in single cell analysis by genomics and proteomics. Major goals include mapping and quantifying the metabolome in sufficient detail to provide useful information about cellular function in highly heterogeneous systems such as tissue, ultimately with spatial resolution at the individual cell level. The chemical diversity and dynamic range of metabolites poses particular challenges for detection, identification and quantification. In this review we discuss both significant technical issues of measurement and interpretation, and progress toward addressing them, with recent examples from diverse biological systems. We provide a framework for further directions aimed at improving workflow and robustness so that such analyses may become commonly applied, especially in combination with metabolic imaging and single cell transcriptomics and proteomics.

Improvement in the Prediction of Neonatal Hypoxic-Ischemic Encephalopathy with the Integration of Umbilical Cord Metabolites and Current Clinical Makers.

OBJECTIVE: To validate our previously identified candidate metabolites, and to assess the ability of these metabolites to predict hypoxic-ischemic encephalopathy (HIE) both individually and combined with clinical data. STUDY DESIGN: Term neonates with signs of perinatal asphyxia, with and without HIE, and matched controls were recruited prospectively at birth from 2 large maternity units. Umbilical cord blood was collected for later batch metabolomic analysis by mass spectroscopy along with clinical details. The optimum selection of clinical and metabolites features with the ability to predict the development of HIE was determined using logistic regression modelling and machine learning techniques. Outcome of HIE was determined by clinical Sarnat grading and confirmed by electroencephalogram grade at 24 hours. RESULTS: Fifteen of 27 candidate metabolites showed significant alteration in infants with perinatal asphyxia or HIE when compared with matched controls. Metabolomic data predicted the development of HIE with an area under the curve of 0.67 (95% CI, 0.62-0.71). Lactic acid and alanine were the primary metabolite predictors for the development of HIE, and when combined with clinical data, gave an area under the curve of 0.96 (95% CI, 0.92-0.95). CONCLUSIONS: By combining clinical and metabolic data, accurate identification of infants who will develop HIE is possible shortly after birth, allowing early initiation of therapeutic hypothermia.

Concepts and Software Package for Efficient Quality Control in Targeted Metabolomics Studies: MeTaQuaC.

Targeted quantitative mass spectrometry metabolite profiling is the workhorse of metabolomics research. Robust and reproducible data are essential for confidence in analytical results and are particularly important with large-scale studies. Commercial kits are now available which use carefully calibrated and validated internal and external standards to provide such reliability. However, they are still subject to processing and technical errors in their use and should be subject to a laboratory's routine quality assurance and quality control measures to maintain confidence in the results. We discuss important systematic and random measurement errors when using these kits and suggest measures to detect and quantify them. We demonstrate how wider analysis of the entire data set alongside standard analyses of quality control samples can be used to identify outliers and quantify systematic trends to improve downstream analysis. Finally, we present the MeTaQuaC software which implements the above concepts and methods for Biocrates kits and other target data sets and creates a comprehensive quality control report containing rich visualization and informative scores and summary statistics. Preliminary unsupervised multivariate analysis methods are also included to provide rapid insight into study variables and groups. MeTaQuaC is provided as an open source R package under a permissive MIT license and includes detailed user documentation.

Preanalytical Processing and Biobanking Procedures of Biological Samples for Metabolomics Research: A White Paper, Community Perspective (for "Precision Medicine and Pharmacometabolomics Task Group"-The Metabolomics Society Initiative).

BACKGROUND: The metabolome of any given biological system contains a diverse range of low molecular weight molecules (metabolites), whose abundances can be affected by the timing and method of sample collection, storage, and handling. Thus, it is necessary to consider the requirements for preanalytical processes and biobanking in metabolomics research. Poor practice can create bias and have deleterious effects on the robustness and reproducibility of acquired data. CONTENT: This review presents both current practice and latest evidence on preanalytical processes and biobanking of samples intended for metabolomics measurement of common biofluids and tissues. It highlights areas requiring more validation and research and provides some evidence-based guidelines on best practices. SUMMARY: Although many researchers and biobanking personnel are familiar with the necessity of standardizing sample collection procedures at the axiomatic level (e.g., fasting status, time of day, "time to freezer," sample volume), other less obvious factors can also negatively affect the validity of a study, such as vial size, material and batch, centrifuge speeds, storage temperature, time and conditions, and even environmental changes in the collection room. Any biobank or research study should establish and follow a well-defined and validated protocol for the collection of samples for metabolomics research. This protocol should be fully documented in any resulting study and should involve all stakeholders in its design. The use of samples that have been collected using standardized and validated protocols is a prerequisite to enable robust biological interpretation unhindered by unnecessary preanalytical factors that may complicate data analysis and interpretation.

Mechanical heterogeneity in a soft biomaterial niche controls BMP2 signaling.

The extracellular matrix is known to impact cell function during regeneration by modulating growth factor signaling. However, how the mechanical properties and structure of biomaterials can be used to optimize the cellular response to growth factors is widely neglected. Here, we engineered a macroporous biomaterial to study cellular signaling in environments that mimic the mechanical stiffness but also the mechanical heterogeneity of native extracellular matrix. We found that the mechanical interaction of cells with the heterogeneous and non-linear deformation properties of soft matrices (E < 5 kPa) enhances BMP-2 growth factor signaling with high relevance for tissue regeneration. In contrast, this effect is absent in homogeneous hydrogels that are often used to study cell responses to mechanical cues. Live cell imaging and in silico finite element modeling further revealed that a subpopulation of highly active, fast migrating cells is responsible for most of the material deformation, while a second, less active population experiences this deformation as an extrinsic mechanical stimulation. At an overall low cell density, the active cell population dominates the process, suggesting that it plays a particularly important role in early tissue healing scenarios where cells invade tissue defects or implanted biomaterials. Taken together, our findings demonstrate that the mechanical heterogeneity of the natural extracellular matrix environment plays an important role in triggering regeneration by endogenously acting growth factors. This suggests the inclusion of such mechanical complexity as a design parameter in future biomaterials, in addition to established parameters such as mechanical stiffness and stress relaxation.

Shared and Distinct Gut Microbiota in Spondyloarthritis, Acute Anterior Uveitis, and Crohn's Disease.

OBJECTIVE: Spondyloarthritis (SpA) is a group of immune-mediated diseases highly concomitant with nonmusculoskeletal inflammatory disorders, such as acute anterior uveitis (AAU) and Crohn's disease (CD). The gut microbiome represents a promising avenue to elucidate shared and distinct underlying pathophysiology. METHODS: We performed 16S ribosomal RNA sequencing on stool samples of 277 patients (72 CD, 103 AAU, and 102 SpA) included in the German Spondyloarthritis Inception Cohort and 62 back pain controls without any inflammatory disorder. Discriminatory statistical methods were used to disentangle microbial disease signals from one another and a wide range of potential confounders. Patients were naive to or had not received treatment with biological disease-modifying antirheumatic drugs (DMARDs) for >3 months before enrollment, providing a better approximation of a true baseline disease signal. RESULTS: We identified a shared, immune-mediated disease signal represented by low abundances of Lachnospiraceae taxa relative to controls, most notably Fusicatenibacter, which was most abundant in controls receiving nonsteroidal antiinflammatory drug monotherapy and implied to partially mediate higher serum C-reactive protein. Patients with SpA showed an enrichment of Collinsella, whereas human leukocyte antigen (HLA)-B27+ individuals displayed enriched Faecalibacterium. CD patients had higher abundances of a Ruminococcus taxon, and previous conventional/synthetic DMARD therapy was associated with increased Akkermansia. CONCLUSION: Our work supports the existence of a common gut dysbiosis in SpA and related inflammatory pathologies. We reveal shared and disease-specific microbial associations and suggest potential mediators of disease activity. Validation studies are needed to clarify the role of Fusicatenibacter in gut-joint inflammation, and metagenomic resolution is needed to understand the relationship between Faecalibacterium commensals and HLA-B27.

Prdm16 mutation determines sex-specific cardiac metabolism and identifies two novel cardiac metabolic regulators.

AIMS: Mutation of the PRDM16 gene causes human dilated and non-compaction cardiomyopathy. The PRDM16 protein is a transcriptional regulator that affects cardiac development via Tbx5 and Hand1, thus regulating myocardial structure. The biallelic inactivation of Prdm16 induces severe cardiac dysfunction with post-natal lethality and hypertrophy in mice. The early pathological events that occur upon Prdm16 inactivation have not been explored. METHODS AND RESULTS: This study performed in-depth pathophysiological and molecular analyses of male and female Prdm16csp1/wt mice that carry systemic, monoallelic Prdm16 gene inactivation. We systematically assessed early molecular changes through transcriptomics, proteomics, and metabolomics. Kinetic modelling of cardiac metabolism was performed in silico with CARDIOKIN. Prdm16csp1/wt mice are viable up to 8 months, develop hypoplastic hearts, and diminished systolic performance that is more pronounced in female mice. Prdm16csp1/wt cardiac tissue of both sexes showed reductions in metabolites associated with amino acid as well as glycerol metabolism, glycolysis, and the tricarboxylic acid cycle. Prdm16csp1/wt cardiac tissue revealed diminished glutathione (GSH) and increased inosine monophosphate (IMP) levels indicating oxidative stress and a dysregulated energetics, respectively. An accumulation of triacylglycerides exclusively in male Prdm16csp1/wt hearts suggests a sex-specific metabolic adaptation. Metabolic modelling using CARDIOKIN identified a reduction in fatty acid utilization in males as well as lower glucose utilization in female Prdm16csp1/wt cardiac tissue. On the level of transcripts and protein expression, Prdm16csp1/wt hearts demonstrate an up-regulation of pyridine nucleotide-disulphide oxidoreductase domain 2 (Pyroxd2) and the transcriptional regulator pre-B-cell leukaemia transcription factor interacting protein 1 (Pbxip1). The strongest concordant transcriptional up-regulation was detected for Prdm16 itself, probably through an autoregulatory mechanism. CONCLUSIONS: Monoallelic, global Prdm16 mutation diminishes cardiac performance in Prdm16csp1/wt mice. Metabolic alterations and transcriptional dysregulation in Prdm16csp1/wt affect cardiac tissue. Female Prdm16csp1/wt mice develop a more pronounced phenotype, indicating sexual dimorphism at this early pathological window. This study suggests that metabolic dysregulation is an early event in the PRDM16 associated cardiac pathology.

Quality Assessment by Bile Composition in Normothermic Machine Perfusion of Rat Livers.

Background: The persistent challenge of organ scarcity in liver transplantation leads to an escalating dependence on organs obtained from extended criteria donors (ECD). Normothermic machine perfusion (NMP) is used for improved preservation. Due to the mimicked in vivo conditions during normothermic machine perfusion, the liver is metabolically active, which allows quality assessment during perfusion. Bile seems to be of rising interest in clinical studies, as it is easily collectible for analysis. As there are currently no data on biliary bile acids during NMP, the primary objective of this study was to use our experimental rodent NMP model to assess changes in bile composition through organ damage during perfusion to inform clinical evaluation of donor organs during NMP. Methods: Thirty livers from male Sprague-Dawley rats in five groups underwent 6 h of NMP using either erythrocyte-supplemented DMEM or Steen solution, with or without 30 min of warm ischemia time (WIT). We conducted regular measurements of AST, ALT, LDH, and urea levels in the perfusate at 3-hour intervals. Bile samples were analyzed for biliary pH, LDH, and gamma glutamyltransferase, as well as biliary bile acids via mass spectrometry and UHPLC. Results: Compared with regular livers, liver injury parameters were significantly higher in our donation after circulatory death (DCD) model. Bile production was significantly reduced in livers exposed to WIT, and the bile showed a significantly more alkaline pH. This correlated with the concentration of total bile acids, which was significantly higher in livers experiencing WIT. However, regular livers produced a higher total amount of biliary bile acids during perfusion. Taurocholic acid and its metabolites were most prominent. Secondary bile acids were significantly reduced during perfusion due to the missing enterohepatic circulation. Conclusions: WIT-induced liver injury affects bile composition within our small-animal NMP model. We hypothesize this phenomenon to be attributed to the energy-driven nature of bile secretion, potentially explaining why DCD livers produce less, yet more concentrated, bile. Our results may inform clinical studies, in which biliary bile acids might have a potential as a quantifiable viability marker in human NMP liver transplantation studies.

Promising results of a clinical feasibility study: CIRBP as a potential biomarker in pediatric cardiac surgery.

Objective: Cold-inducible RNA binding Protein (CIRBP) has been shown to be a potent inflammatory mediator and could serve as a novel biomarker for inflammation. Systemic inflammatory response syndrome (SIRS) and capillary leak syndrome (CLS) are frequent complications after pediatric cardiac surgery increasing morbidity, therefore early diagnosis and therapy is crucial. As CIRBP serum levels have not been analyzed in a pediatric population, we conducted a clinical feasibility establishing a customized magnetic bead panel analyzing CIRBP in pediatric patients undergoing cardiac surgery. Methods: A prospective hypothesis generating observational clinical study was conducted at the German Heart Center Berlin during a period of 9 months starting in May 2020 (DRKS00020885, https://drks.de/search/de/trial/DRKS00020885). Serum samples were obtained before the cardiac operation, upon arrival at the pediatric intensive care unit, 6 and 24 h after the operation in patients up to 18 years of age with congenital heart disease (CHD). Customized multiplex magnetic bead-based immunoassay panels were developed to analyze CIRBP, Interleukin-1ß (IL-1ß), Interleukin-6 (IL-6), Interleukin-8 (IL-8), Interleukin-10 (IL-10), Monocyte chemotactic protein 1 (MCP-1), Syndecan-1 (SDC-1), Thrombomodulin (TM), Vascular endothelial growth factor (VEGF-A), Angiopoietin-2 (Ang-2), and Fibroblast growth factor 23 (FGF-23) in 25 µl serum using the Luminex MagPix® system. Results: 19 patients representing a broad range of CHD (10 male patients, median age 2 years, 9 female patients, median age 3 years) were included in the feasibility study. CIRBP was detectable in the whole patient cohort. Relative to individual baseline values, CIRBP concentrations increased 6 h after operation and returned to baseline levels over time. IL-6, IL-8, IL-10, and MCP-1 concentrations were significantly increased after operation and except for MCP-1 concentrations stayed upregulated over time. SDC-1, TM, Ang-2, as well as FGF-23 concentrations were also significantly increased, whereas VEGF-A concentration was significantly decreased after surgery. Discussion: Using customized magnetic bead panels, we were able to detect CIRBP in a minimal serum volume (25 µl) in all enrolled patients. To our knowledge this is the first clinical study to assess CIRBP serum concentrations in a pediatric population.

Sex-specific molecular signature of mouse podocytes in homeostasis and in response to pharmacological challenge with rapamycin.

BACKGROUND: Sex differences exist in the prevalence and progression of major glomerular diseases. Podocytes are the essential cell-type in the kidney which maintain the physiological blood-urine barrier, and pathological changes in podocyte homeostasis are critical accelerators of impairment of kidney function. However, sex-specific molecular signatures of podocytes under physiological and stress conditions remain unknown. This work aimed at identifying sexual dimorphic molecular signatures of podocytes under physiological condition and pharmacologically challenged homeostasis with mechanistic target of rapamycin (mTOR) inhibition. mTOR is a crucial regulator involved in a variety of physiological and pathological stress responses in the kidney and inhibition of this pathway may therefore serve as a general stress challenger to get fundamental insights into sex differences in podocytes. METHODS: The genomic ROSAmT/mG-NPHS2 Cre mouse model was used which allows obtaining highly pure podocyte fractions for cell-specific molecular analyses, and vehicle or pharmacologic treatment with the mTOR inhibitor rapamycin was performed for 3 weeks. Subsequently, deep RNA sequencing and proteomics were performed of the isolated podocytes to identify intrinsic sex differences. Studies were supplemented with metabolomics from kidney cortex tissues. RESULTS: Although kidney function and morphology remained normal in all experimental groups, RNA sequencing, proteomics and metabolomics revealed strong intrinsic sex differences in the expression levels of mitochondrial, translation and structural transcripts, protein abundances and regulation of metabolic pathways. Interestingly, rapamycin abolished prominent sex-specific clustering of podocyte gene expression and induced major changes only in male transcriptome. Several sex-biased transcription factors could be identified as possible upstream regulators of these sexually dimorphic responses. Concordant to transcriptomics, metabolomic changes were more prominent in males. Remarkably, high number of previously reported kidney disease genes showed intrinsic sexual dimorphism and/or different response patterns towards mTOR inhibition. CONCLUSIONS: Our results highlight remarkable intrinsic sex-differences and sex-specific response patterns towards pharmacological challenged podocyte homeostasis which might fundamentally contribute to sex differences in kidney disease susceptibilities and progression. This work provides rationale and an in-depth database for novel targets to be tested in specific kidney disease models to advance with sex-specific treatment strategies.

The global burden of chronic kidney diseases is rapidly increasing and is projected to become the fifth most common cause of years of life lost worldwide by 2040. Sexual dimorphism in kidney diseases and transplantation is well known, yet sex-specific therapeutic strategies are still missing. One reason is the lack of knowledge due to the lack of inclusion of sex as a biological variable in study designs. This work aimed at identification of molecular signatures of male and female podocytes, gate-keepers of the glomerular filtration barrier. Like cardiomyocytes, podocytes are terminally differentiated cells which are highly susceptible towards pathological challenges. Podocytes are the decisive cell-type of the kidney to maintain the physiological blood-urine barrier, and disturbances of their homeostasis critically accelerate kidney function impairment. By help of a genomic mouse model, highly purified podocytes were obtained from male and female mice with and without pharmacological challenge of the mechanistic target of rapamycin (mTOR) signaling pathway which is known to be deregulated in major kidney diseases. Deep RNA sequencing, proteomics and metabolomics revealed strong intrinsic sex differences in the expression levels of mitochondrial, translation and structural transcripts, protein abundances and regulation of metabolic pathways which might fundamentally contribute to sex differences in kidney disease susceptibilities and progression. Remarkably, high number of previously reported kidney disease genes showed so far unknown intrinsic sexual dimorphism and/or different response patterns towards mTOR inhibition. Our work provides an in-depth database for novel targets to be tested in kidney disease models to advance with sex-specific treatment strategies.

ISG15 blocks cardiac glycolysis and ensures sufficient mitochondrial energy production during Coxsackievirus B3 infection.

AIMS: Virus infection triggers inflammation and, may impose nutrient shortage to the heart. Supported by type I interferon (IFN) signalling, cardiomyocytes counteract infection by various effector processes, with the IFN-stimulated gene of 15 kDa (ISG15) system being intensively regulated and protein modification with ISG15 protecting mice Coxsackievirus B3 (CVB3) infection. The underlying molecular aspects how the ISG15 system affects the functional properties of respective protein substrates in the heart are unknown. METHODS AND RESULTS: Based on the protective properties due to protein ISGylation, we set out a study investigating CVB3-infected mice in depth and found cardiac atrophy with lower cardiac output in ISG15-/- mice. By mass spectrometry, we identified the protein targets of the ISG15 conjugation machinery in heart tissue and explored how ISGylation affects their function. The cardiac ISGylome showed a strong enrichment of ISGylation substrates within glycolytic metabolic processes. Two control enzymes of the glycolytic pathway, hexokinase 2 (HK2) and phosphofructokinase muscle form (PFK1), were identified as bona fide ISGylation targets during infection. In an integrative approach complemented with enzymatic functional testing and structural modelling, we demonstrate that protein ISGylation obstructs the activity of HK2 and PFK1. Seahorse-based investigation of glycolysis in cardiomyocytes revealed that, by conjugating proteins, the ISG15 system prevents the infection-/IFN-induced up-regulation of glycolysis. We complemented our analysis with proteomics-based advanced computational modelling of cardiac energy metabolism. Our calculations revealed an ISG15-dependent preservation of the metabolic capacity in cardiac tissue during CVB3 infection. Functional profiling of mitochondrial respiration in cardiomyocytes and mouse heart tissue by Seahorse technology showed an enhanced oxidative activity in cells with a competent ISG15 system. CONCLUSION: Our study demonstrates that ISG15 controls critical nodes in cardiac metabolism. ISG15 reduces the glucose demand, supports higher ATP production capacity in the heart, despite nutrient shortage in infection, and counteracts cardiac atrophy and dysfunction.

A study protocol to characterise pathophysiological and molecular markers of rheumatic heart disease and degenerative aortic stenosis using multiparametric cardiovascular imaging and multiomics techniques.

INTRODUCTION: Rheumatic heart disease (RHD), degenerative aortic stenosis (AS), and congenital valve diseases are prevalent in sub-Saharan Africa. Many knowledge gaps remain in understanding disease mechanisms, stratifying phenotypes, and prognostication. Therefore, we aimed to characterise patients through clinical profiling, imaging, histology, and molecular biomarkers to improve our understanding of the pathophysiology, diagnosis, and prognosis of RHD and AS. METHODS: In this cross-sectional, case-controlled study, we plan to recruit RHD and AS patients and compare them to matched controls. Living participants will undergo clinical assessment, echocardiography, CMR and blood sampling for circulatory biomarker analyses. Tissue samples will be obtained from patients undergoing valve replacement, while healthy tissues will be obtained from cadavers. Immunohistology, proteomics, metabolomics, and transcriptome analyses will be used to analyse circulatory- and tissue-specific biomarkers. Univariate and multivariate statistical analyses will be used for hypothesis testing and identification of important biomarkers. In summary, this study aims to delineate the pathophysiology of RHD and degenerative AS using multiparametric CMR imaging. In addition to discover novel biomarkers and explore the pathomechanisms associated with RHD and AS through high-throughput profiling of the tissue and blood proteome and metabolome and provide a proof of concept of the suitability of using cadaveric tissues as controls for cardiovascular disease studies.