Assessing SOFA score trajectories in sepsis using machine learning: A pragmatic approach to improve the accuracy of mortality prediction.

INTRODUCTION: An increasing amount of longitudinal health data is available on critically ill septic patients in the age of digital medicine, including daily sequential organ failure assessment (SOFA) score measurements. Thus, the assessment in sepsis focuses increasingly on the evaluation of the individual disease's trajectory. Machine learning (ML) algorithms may provide a promising approach here to improve the evaluation of daily SOFA score dynamics. We tested whether ML algorithms can outperform the conventional ΔSOFA score regarding the accuracy of 30-day mortality prediction. METHODS: We used the multicentric SepsisDataNet.NRW study cohort that prospectively enrolled 252 sepsis patients between 03/2018 and 09/2019 for training ML algorithms, i.e. support vector machine (SVM) with polynomial kernel and artificial neural network (aNN). We used the Amsterdam UMC database covering 1,790 sepsis patients for external and independent validation. RESULTS: Both SVM (AUC 0.84; 95% CI: 0.71-0.96) and aNN (AUC 0.82; 95% CI: 0.69-0.95) assessing the SOFA scores of the first seven days led to a more accurate prognosis of 30-day mortality compared to the ΔSOFA score between day 1 and 7 (AUC 0.73; 95% CI: 0.65-0.80; p = 0.02 and p = 0.05, respectively). These differences were even more prominent the shorter the time interval considered. Using the SOFA scores of day 1 to 3 SVM (AUC 0.82; 95% CI: 0.68 0.95) and aNN (AUC 0.80; 95% CI: 0.660.93) led to a more accurate prognosis of 30-day mortality compared to the ΔSOFA score (AUC 0.66; 95% CI: 0.58-0.74; p < 0.01 and p < 0.01, respectively). Strikingly, all these findings could be confirmed in the independent external validation cohort. CONCLUSIONS: The ML-based algorithms using daily SOFA scores markedly improved the accuracy of mortality compared to the conventional ΔSOFA score. Therefore, this approach could provide a promising and automated approach to assess the individual disease trajectory in sepsis. These findings reflect the potential of incorporating ML algorithms as robust and generalizable support tools on intensive care units.

Immortalised murine R349P desmin knock-in myotubes exhibit a reduced proton leak and decreased ADP/ATP translocase levels in purified mitochondria.

Desmin gene mutations cause myopathies and cardiomyopathies. Our previously characterised R349P desminopathy mice, which carry the ortholog of the common human desmin mutation R350P, showed marked alterations in mitochondrial morphology and function in muscle tissue. By isolating skeletal muscle myoblasts from offspring of R349P desminopathy and p53 knock-out mice, we established an immortalised cellular disease model. Heterozygous and homozygous R349P desmin knock-in and wild-type myoblasts could be well differentiated into multinucleated spontaneously contracting myotubes. The desminopathy myoblasts showed the characteristic disruption of the desmin cytoskeleton and desmin protein aggregation, and the desminopathy myotubes showed the characteristic myofibrillar irregularities. Long-term electrical pulse stimulation promoted myotube differentiation and markedly increased their spontaneous contraction rate. In both heterozygous and homozygous R349P desminopathy myotubes, this treatment restored a regular myofibrillar cross-striation pattern as seen in wild-type myotubes. High-resolution respirometry of mitochondria purified from myotubes by density gradient ultracentrifugation revealed normal oxidative phosphorylation capacity, but a significantly reduced proton leak in mitochondria from the homozygous R349P desmin knock-in cells. Consistent with a reduced proton flux across the inner mitochondrial membrane, our quantitative proteomic analysis of the purified mitochondria revealed significantly reduced levels of ADP/ATP translocases in the homozygous R349P desmin knock-in genotype. As this alteration was also detected in the soleus muscle of R349P desminopathy mice, which, in contrast to the mitochondria purified from cultured cells, showed a variety of other dysregulated mitochondrial proteins, we consider this finding to be an early step in the pathogenesis of secondary mitochondriopathy in desminopathy.

Use of Multiple Machine Learning Approaches for Selecting Urothelial Cancer-Specific DNA Methylation Biomarkers in Urine.

Diagnosing urothelial cancer (UCa) via invasive cystoscopy is painful, specifically in men, and can cause infection and bleeding. Because the UCa risk is higher for male patients, urinary non-invasive UCa biomarkers are highly desired to stratify men for invasive cystoscopy. We previously identified multiple DNA methylation sites in urine samples that detect UCa with a high sensitivity and specificity in men. Here, we identified the most relevant markers by employing multiple statistical approaches and machine learning (random forest, boosted trees, LASSO) using a dataset of 251 male UCa patients and 111 controls. Three CpG sites located in ALOX5, TRPS1 and an intergenic region on chromosome 16 have been concordantly selected by all approaches, and their combination in a single decision matrix for clinical use was tested based on their respective thresholds of the individual CpGs. The combination of ALOX5 and TRPS1 yielded the best overall sensitivity (61%) at a pre-set specificity of 95%. This combination exceeded both the diagnostic performance of the most sensitive bioinformatic approach and that of the best single CpG. In summary, we showed that overlap analysis of multiple statistical approaches identifies the most reliable biomarkers for UCa in a male collective. The results may assist in stratifying men for cystoscopy.

The good or the bad: an overview of autoantibodies in traumatic spinal cord injury.

Infections remain the most common cause of death after traumatic spinal cord injury, likely due to a developing immune deficiency syndrome. This, together with a somewhat contradictory development of autoimmunity in many patients, are two major components of the maladaptive systemic immune response. Although the local non-resolving inflammation in the lesioned spinal cord may lead to an antibody formation against autoantigens of the injured spinal cord tissue, there are also natural (pre-existing) autoantibodies independent of the injury. The way in which these autoantibodies with different origins affect the neuronal and functional outcome of spinal cord-injured patients is still controversial.

Investigation of Inter- and Intra-Day Variability of Tear Fluid Regarding Flow Rate, Protein Concentration as well as Protein Composition.

Purpose: The purpose of this study was to present the determination of inter- and intra-day variations in tear flow rate, and tear fluid protein concentration, as well as protein composition regarding their impact for future biomarker studies. Methods: Tear fluid was collected noninvasively from 18 healthy subjects by performing Schirmer tests at 4 different time points repetitive in a period of 2 days. The tear flow rate on the Schirmer test strips was measured. Proteins were extracted from strips and quantified using amino acid analysis. Protein composition was analyzed by the strips data-independent (DIA) based mass spectrometry. To exclude any impairments to health, volunteers underwent a detailed neurological as well as an ophthalmological examination. Results: Whether tear fluid was collected from oculus sinister or oculus dexter did not affect the tear flow rate (P ≈ 0.63) or protein concentration (P ≈ 0.97) of individual subjects. Moreover, protein concentration was independent from the tear volume, so that a change in volume may only influence the total protein amount. When the examination days were compared, investigation of tear flow rate (P ≈ 0.001) and protein concentration (P ≈ 0.0003) indicated significant differences. Further, mass spectrometric analysis of tear fluid revealed 11 differentially regulated proteins when comparing both examination days. Conclusions: Our findings provide evidence of inter-day variation in tear flow rate, tear proteome concentration, and composition in healthy subjects, suggesting that inter-day variation needs to be taken into consideration in biomarker research of tear fluid. Identified proteins were assigned to functions in the immune response, oxidative and reducing processes, as well as mannose metabolism.

Changes in the Proteome of Platelets from Patients with Critical Progression of COVID-19.

Platelets, the smallest cells in human blood, known for their role in primary hemostasis, are also able to interact with pathogens and play a crucial role in the immune response. In severe coronavirus disease 2019 (COVID-19) cases, platelets become overactivated, resulting in the release of granules, exacerbating inflammation and contributing to the cytokine storm. This study aims to further elucidate the role of platelets in COVID-19 progression and to identify predictive biomarkers for disease outcomes. A comparative proteome analysis of highly purified platelets from critically diseased COVID-19 patients with different outcomes (survivors and non-survivors) and age- and sex-matched controls was performed. Platelets from critically diseased COVID-19 patients exhibited significant changes in the levels of proteins associated with protein folding. In addition, a number of proteins with isomerase activity were found to be more highly abundant in patient samples, apparently exerting an influence on platelet activity via the non-genomic properties of the glucocorticoid receptor (GR) and the nuclear factor κ-light-chain-enhancer of activated B cells (NFκB). Moreover, carbonic anhydrase 1 (CA-1) was found to be a candidate biomarker in platelets, showing a significant increase in COVID-19 patients.

Fc N-glycosylation of autoreactive Aß antibodies as a blood-based biomarker for Alzheimer's disease.

INTRODUCTION: Naturally occurring autoantibodies (nAbs) against the pathologic isoform of amyloid beta (Aß42 ) were found in body fluids and indicate a systemic B cell response that may prevent Alzheimer's disease (AD) onset. N-glycans attached to immunoglobulin G-Fab/Fc fragments are features that influence their mechanism of action. The aim was to study the role of N-glycans in nAbs-Aß42 . METHODS: nAbs-Aß42 were isolated from AD patients and age-/sex-matched controls (n = 40) and immunoglobulin preparations. Glycosylated/deglycosylated nAbs-Aß42 were analyzed for their effect on Aß42 's aggregation, toxicity, and phagocytosis. Glycan structure was analyzed using matrix assisted laser desorption ionization time of flight mass spectrometry. RESULTS: Deglycosylation of nAbs-Aß42 had a major impact on Aß42 's aggregation/toxicity/phagocytosis. The glycan structure showed considerable differences between AD and controls. We were able to predict disease status with a sensitivity/specificity of 95% (confidence interval [CI]: 76.4-99.7%)/100% (CI: 83.9-100%). DISCUSSION: N-glycosylation has been identified as a critical attribute maintaining the beneficial effects of autoreactive Aß antibodies. These data have consequences for the development of monocloncal Aß antibodies and may open new avenues for diagnostics.

Lesional Antibody Synthesis and Complement Deposition Associate With De Novo Antineuronal Antibody Synthesis After Spinal Cord Injury.

BACKGROUND AND OBJECTIVES: Spinal cord injury (SCI) disrupts the fine-balanced interaction between the CNS and immune system and can cause maladaptive aberrant immune responses. The study examines emerging autoantibody synthesis after SCI with binding to conformational spinal cord epitopes and surface peptides located on the intact neuronal membrane. METHODS: This is a prospective longitudinal cohort study conducted in acute care and inpatient rehabilitation centers in conjunction with a neuropathologic case-control study in archival tissue samples ranging from acute injury (baseline) to several months thereafter (follow-up). In the cohort study, serum autoantibody binding was examined in a blinded manner using tissue-based assays (TBAs) and dorsal root ganglia (DRG) neuronal cultures. Groups with traumatic motor complete SCI vs motor incomplete SCI vs isolated vertebral fracture without SCI (controls) were compared. In the neuropathologic study, B cell infiltration and antibody synthesis at the spinal lesion site were examined by comparing SCI with neuropathologically unaltered cord tissue. In addition, the CSF in an individual patient was explored. RESULTS: Emerging autoantibody binding in both TBA and DRG assessments was restricted to an SCI patient subpopulation only (16%, 9/55 sera) while being absent in vertebral fracture controls (0%, 0/19 sera). Autoantibody binding to the spinal cord characteristically detected the substantia gelatinosa, a less-myelinated region of high synaptic density involved in sensory-motor integration and pain processing. Autoantibody binding was most frequent after motor complete SCI (grade American Spinal Injury Association impairment scale A/B, 22%, 8/37 sera) and was associated with neuropathic pain medication. In conjunction, the neuropathologic study demonstrated lesional spinal infiltration of B cells (CD20, CD79a) in 27% (6/22) of patients with SCI, the presence of plasma cells (CD138) in 9% (2/22). IgG and IgM antibody syntheses colocalized to areas of activated complement (C9neo) deposition. Longitudinal CSF analysis of an additional single patient demonstrated de novo (IgM) intrathecal antibody synthesis emerging with late reopening of the blood-spinal cord barrier. DISCUSSION: This study provides immunologic, neurobiological, and neuropathologic proof-of-principle for an antibody-mediated autoimmunity response emerging approximately 3 weeks after SCI in a patient subpopulation with a high demand of neuropathic pain medication. Emerging autoimmunity directed against specific spinal cord and neuronal epitopes suggests the existence of paratraumatic CNS autoimmune syndromes.

Quality Control-A Stepchild in Quantitative Proteomics: A Case Study for the Human CSF Proteome.

Proteomic studies using mass spectrometry (MS)-based quantification are a main approach to the discovery of new biomarkers. However, a number of analytical conditions in front and during MS data acquisition can affect the accuracy of the obtained outcome. Therefore, comprehensive quality assessment of the acquired data plays a central role in quantitative proteomics, though, due to the immense complexity of MS data, it is often neglected. Here, we address practically the quality assessment of quantitative MS data, describing key steps for the evaluation, including the levels of raw data, identification and quantification. With this, four independent datasets from cerebrospinal fluid, an important biofluid for neurodegenerative disease biomarker studies, were assessed, demonstrating that sample processing-based differences are already reflected at all three levels but with varying impacts on the quality of the quantitative data. Specifically, we provide guidance to critically interpret the quality of MS data for quantitative proteomics. Moreover, we provide the free and open source quality control tool MaCProQC, enabling systematic, rapid and uncomplicated data comparison of raw data, identification and feature detection levels through defined quality metrics and a step-by-step quality control workflow.

Case Report: Muscle Wasting during Severe Sustained Hypoxia in Two Professional Mountaineers.

PURPOSE: Chronic exposure to hypoxia can induce muscle wasting in unaccustomed individuals. Detailed assessment of the effects of hypoxia on muscle tissue adaptation in elite mountaineers has not been performed. This study aims to assess muscle volume after exposure to normobaric hypoxia. METHODS: Two professional mountaineers (A and B) participated in a 35-d intervention of graded normobaric hypoxia with the aim of 14 d exposure to 8% oxygen corresponding to 7112-m altitude. Volume of the shank, thigh, and hip muscles was assessed by magnetic resonance imaging pre- and postintervention. Dietary intake and physical activity were monitored throughout the study from food images and accelerometry analysis, together with blood analysis and anthropometric measurements. RESULTS: Hypoxia reduced total leg muscle volume by 3.3% ± 6.0% in A and by 9.4% ± 7.3% in B. A lost 288 g and B 642 g of muscle mass, whereas dietary intake only declined by ~23% in the last intervention week. Arterial oxygen saturation declined from 95% and 86% to 77% and 72% in A and B, respectively. In hypoxia, participants could not maintain their physical activity levels. Notably, muscle loss varied substantially across muscle groups amounting to 5.4% ± 3.0%, 8.3% ± 5.2%, and 4.1% ± 8.6% for hip, thigh, and shank muscles, respectively. CONCLUSIONS: Our results indicate that hypoxia and resultant reductions in physical activity and caloric intake lead to substantial loss of muscle mass that was accentuated in proximal muscle as opposed to distal muscles. Surprisingly, thigh muscle wasting during this intervention is comparable with that observed during strict 56-d bed rest.

Proteome analysis of monocytes implicates altered mitochondrial biology in adults reporting adverse childhood experiences.

The experience of adversity in childhood has been associated with poor health outcomes in adulthood. In search of the biological mechanisms underlying these effects, research so far focused on alterations of DNA methylation or shifts in transcriptomic profiles. The level of protein, however, has been largely neglected. We utilized mass spectrometry to investigate the proteome of CD14+ monocytes in healthy adults reporting childhood adversity and a control group before and after psychosocial stress exposure. Particular proteins involved in (i) immune processes, such as neutrophil-related proteins, (ii) protein metabolism, or (iii) proteins related to mitochondrial biology, such as those involved in energy production processes, were upregulated in participants reporting exposure to adversity in childhood. This functional triad was further corroborated by protein interaction- and co-expression analyses, was independent of stress exposure, i.e. observed at both pre- and post-stress time points, and became evident especially in females. In line with the mitochondrial allostatic load model, our findings provide evidence for the long-term effects of childhood adversity on mitochondrial biology.

Target formation in muscle fibres indicates reinnervation - A proteomic study in muscle samples from peripheral neuropathies.

AIMS: Target skeletal muscle fibres - defined by different concentric areas in oxidative enzyme staining - can occur in patients with neurogenic muscular atrophy. Here, we used our established hypothesis-free proteomic approach with the aim of deciphering the protein composition of targets. We also searched for potential novel interactions between target proteins. METHODS: Targets and control areas were laser microdissected from skeletal muscle sections of 20 patients with neurogenic muscular atrophy. Samples were analysed by a highly sensitive mass spectrometry approach, enabling relative protein quantification. The results were validated by immunofluorescence studies. Protein interactions were investigated by yeast two-hybrid assays, coimmunoprecipitation experiments and bimolecular fluorescence complementation. RESULTS: More than 1000 proteins were identified. Among these, 55 proteins were significantly over-represented and 40 proteins were significantly under-represented in targets compared to intraindividual control samples. The majority of over-represented proteins were associated with the myofibrillar Z-disc and actin dynamics, followed by myosin and myosin-associated proteins, proteins involved in protein biosynthesis and chaperones. Under-represented proteins were mainly mitochondrial proteins. Functional studies revealed that the LIM domain of the over-represented protein LIMCH1 interacts with isoform A of Xin actin-binding repeat-containing protein 1 (XinA). CONCLUSIONS: In particular, proteins involved in myofibrillogenesis are over-represented in target structures, which indicate an ongoing process of sarcomere assembly and/or remodelling within this specific area of the muscle fibres. We speculate that target structures are the result of reinnervation processes in which filamin C-associated myofibrillogenesis is tightly regulated by the BAG3-associated protein quality system.

LUBAC assembles a ubiquitin signaling platform at mitochondria for signal amplification and transport of NF-κB to the nucleus.

Mitochondria are increasingly recognized as cellular hubs to orchestrate signaling pathways that regulate metabolism, redox homeostasis, and cell fate decisions. Recent research revealed a role of mitochondria also in innate immune signaling; however, the mechanisms of how mitochondria affect signal transduction are poorly understood. Here, we show that the NF-κB pathway activated by TNF employs mitochondria as a platform for signal amplification and shuttling of activated NF-κB to the nucleus. TNF treatment induces the recruitment of HOIP, the catalytic component of the linear ubiquitin chain assembly complex (LUBAC), and its substrate NEMO to the outer mitochondrial membrane, where M1- and K63-linked ubiquitin chains are generated. NF-κB is locally activated and transported to the nucleus by mitochondria, leading to an increase in mitochondria-nucleus contact sites in a HOIP-dependent manner. Notably, TNF-induced stabilization of the mitochondrial kinase PINK1 furthermore contributes to signal amplification by antagonizing the M1-ubiquitin-specific deubiquitinase OTULIN. Overall, our study reveals a role for mitochondria in amplifying TNF-mediated NF-κB activation, both serving as a signaling platform, as well as a transport mode for activated NF-κB to the nuclear.

The Proteome of Neuromelanin Granules in Dementia with Lewy Bodies.

Neuromelanin granules (NMGs) are organelle-like structures present in the human substantia nigra pars compacta. In addition to neuromelanin, NMGs contain proteins, lipids and metals. As NMG-containing dopaminergic neurons are preferentially lost in Parkinson's disease and dementia with Lewy bodies (DLB), it is assumed that NMGs may play a role in neurodegenerative processes. Until now, this role is not completely understood and needs further investigation. We therefore set up an exploratory proteomic study to identify differences in the proteomic profile of NMGs from DLB patients (n = 5) compared to healthy controls (CTRL, n = 5). We applied a laser microdissection and mass-spectrometry-based approach, in which we used targeted mass spectrometric experiments for validation. In NMG-surrounding (SNSurr.) tissue of DLB patients, we found evidence for ongoing oxidative damage and an impairment of protein degradation. As a potentially disease-related mechanism, we found α-synuclein and protein S100A9 to be enriched in NMGs of DLB cases, while the abundance of several ribosomal proteins was significantly decreased. As S100A9 is known to be able to enhance the formation of toxic α-synuclein fibrils, this finding points towards an involvement of NMGs in pathogenesis, however the exact role of NMGs as either neuroprotective or neurotoxic needs to be further investigated. Nevertheless, our study provides evidence for an impairment of protein degradation, ongoing oxidative damage and accumulation of potentially neurotoxic protein aggregates to be central mechanisms of neurodegeneration in DLB.

Heat Shock Protein Upregulation Supplemental to Complex mRNA Alterations in Autoimmune Glaucoma.

Glaucomatous optic neuropathy is a common cause for blindness. An elevated intraocular pressure is the main risk factor, but also a contribution of the immune system seems likely. In the experimental autoimmune glaucoma model used here, systemic immunization with an optic nerve homogenate antigen (ONA) leads to retinal ganglion cell (RGC) and optic nerve degeneration. We processed retinae for quantitative real-time PCR and immunohistology 28 days after immunization. Furthermore, we performed mRNA profiling in this model for the first time. We detected a significant RGC loss in the ONA retinae. This was accompanied by an upregulation of mRNA expression of genes belonging to the heat shock protein family. Furthermore, mRNA expression levels of the genes of the immune system, such as C1qa, C1qb, Il18, and Nfkb1, were upregulated in ONA animals. After laser microdissection, inner retinal layers were used for mRNA microarrays. Nine of these probes were significantly upregulated in ONA animals (p < 0.05), including Hba-a1 and Cxcl10, while fifteen probes were significantly downregulated in ONA animals (p < 0.05), such as Gdf15 and Wwox. Taken together, these findings provide further insights into the pivotal role of the immune response in glaucomatous optic neuropathy and could help to identify novel diagnostic or therapeutic strategies.

Desmin Knock-Out Cardiomyopathy: A Heart on the Verge of Metabolic Crisis.

Desmin mutations cause familial and sporadic cardiomyopathies. In addition to perturbing the contractile apparatus, both desmin deficiency and mutated desmin negatively impact mitochondria. Impaired myocardial metabolism secondary to mitochondrial defects could conceivably exacerbate cardiac contractile dysfunction. We performed metabolic myocardial phenotyping in left ventricular cardiac muscle tissue in desmin knock-out mice. Our analyses revealed decreased mitochondrial number, ultrastructural mitochondrial defects, and impaired mitochondria-related metabolic pathways including fatty acid transport, activation, and catabolism. Glucose transporter 1 and hexokinase-1 expression and hexokinase activity were increased. While mitochondrial creatine kinase expression was reduced, fetal creatine kinase expression was increased. Proteomic analysis revealed reduced expression of proteins involved in electron transport mainly of complexes I and II, oxidative phosphorylation, citrate cycle, beta-oxidation including auxiliary pathways, amino acid catabolism, and redox reactions and oxidative stress. Thus, desmin deficiency elicits a secondary cardiac mitochondriopathy with severely impaired oxidative phosphorylation and fatty and amino acid metabolism. Increased glucose utilization and fetal creatine kinase upregulation likely portray attempts to maintain myocardial energy supply. It may be prudent to avoid medications worsening mitochondrial function and other metabolic stressors. Therapeutic interventions for mitochondriopathies might also improve the metabolic condition in desmin deficient hearts.

Dataset containing physiological amounts of spike-in proteins into murine C2C12 background as a ground truth quantitative LC-MS/MS reference.

In this article, we present a data dependent acquisition (DDA) dataset which was generated as a reference and ground truth quantitative dataset. While initially used to compare samples measured with DDA and data independent acquisition (DIA) (Barkovits et al., 2020), the presented dataset holds potential value as a benchmark reference for any workflows working on DDA data. The entire dataset consists of 15 LC-MS/MS measurements composed of five distinct spike-in-states, each with three replicates. To generate the data set, a C2C12 (immortalized mouse myoblast) cell lysate was used as a complex background for five different states which were simulated by spiking 13 defined proteins at different concentrations. For this purpose, the cell lysate was used in a constant amount of 20 µg for all samples and different amounts of the 13 selected proteins ranging from 0.1  to 10 pmol were added, reflecting physiological amounts of proteins. Afterwards, all samples were tryptically digested using the same method. From each sample 200 ng tryptic peptides were measured in triplicates on a Q Exactive HF (Thermo Fisher Scientific). The mass range for MS1 was set to 350-1400 m/z with a resolution of 60,000 at 200 m/z. HCD fragmentation of the Top10 abundant precursor ions was performed at 27% NCE. The fragment analysis (MS2) was performed with a resolution of 30,000 at 200 m/z. Additionally to the raw files, the dataset contains centroided mzML files and spectrum identification results for peptide identifications performed by Mascot (Perkins et al., 1999), MS-GF+ (Kim et al., 2010) and X!Tandem (Craig and Beavis, 2004) for each separate MS analysis. The corresponding FASTA containing protein sequences as well as a combination of all identification runs performed by PIA (Uszkoreit et al., 2019, 2015) and a peptide and protein quantification performed by OpenMS (Pfeuffer et al., 2017) is included. All data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (Perez-Riverol et al., 2018) with the dataset identifier PXD012986.

Neuromelanin granules of the substantia nigra: proteomic profile provides links to tyrosine hydroxylase, stress granules and lysosomes.

Neuromelanin is a black-brownish pigment, present in so-called neuromelanin granules (NMGs) in the cell bodies of dopaminergic neurons in the substantia nigra (SN) pars compacta. These neurons are lost in neurodegenerative diseases, such as Parkinson's disease and dementia with Lewy bodies. Although it is known that lipids, proteins, and environmental toxins accumulate in NMGs, the function of NMGs has not yet been finally clarified as well as their origin and the synthesis of neuromelanin. We, therefore, isolated NMGs and surrounding SN tissue from control patients by laser microdissection and analyzed the proteomic profile by tandem mass spectrometry. With our improved workflow, we were able to (1) strengthen the regularly reported link between NMGs and lysosomes, (2) detect tyrosine hydroxylase to be highly abundant in NMGs, which may be related to neuromelanin synthesis and (3) indicate a yet undescribed link between stress granules (SGs) and NMGs. Based on our findings, we cautiously hypothesize, that SGs may be the origin of NMGs or form in close proximity to them, potentially due to the oxidative stress caused by neuromelanin-bound metals.

A Current Encyclopedia of Bioinformatics Tools, Data Formats and Resources for Mass Spectrometry Lipidomics.

Mass spectrometry is a widely used technology to identify and quantify biomolecules such as lipids, metabolites and proteins necessary for biomedical research. In this study, we catalogued freely available software tools, libraries, databases, repositories and resources that support lipidomics data analysis and determined the scope of currently used analytical technologies. Because of the tremendous importance of data interoperability, we assessed the support of standardized data formats in mass spectrometric (MS)-based lipidomics workflows. We included tools in our comparison that support targeted as well as untargeted analysis using direct infusion/shotgun (DI-MS), liquid chromatography-mass spectrometry, ion mobility or MS imaging approaches on MS1 and potentially higher MS levels. As a result, we determined that the Human Proteome Organization-Proteomics Standards Initiative standard data formats, mzML and mzTab-M, are already supported by a substantial number of recent software tools. We further discuss how mzTab-M can serve as a bridge between data acquisition and lipid bioinformatics tools for interpretation, capturing their output and transmitting rich annotated data for downstream processing. However, we identified several challenges of currently available tools and standards. Potential areas for improvement were: adaptation of common nomenclature and standardized reporting to enable high throughput lipidomics and improve its data handling. Finally, we suggest specific areas where tools and repositories need to improve to become FAIRer.

Chronic Hyperglycaemia Inhibits Tricarboxylic Acid Cycle in Rat Cardiomyoblasts Overexpressing Glucose Transporter Type 4.

An oversupply of nutrients with a loss of metabolic flexibility and subsequent cardiac dysfunction are hallmarks of diabetic cardiomyopathy. Even if excess substrate is offered, the heart suffers energy depletion as metabolic fluxes are diminished. To study the effects of a high glucose supply, a stably glucose transporter type 4 (GLUT4)-overexpressing cell line presenting an onset of diabetic cardiomyopathy-like phenotype was established. Long-term hyperglycaemia effects were analysed. Rat cardiomyoblasts overexpressing GLUT4 (H9C2KE2) were cultured under normo- and hyperglycaemic conditions for long-term. Expression profiles of several proteins were compared to non-transfected H9C2 cells (H9C2) using RT-qPCR, proteomics-based analysis, or Western blotting. GLUT4 surface analysis, glucose uptake, and cell morphology changes as well as apoptosis/necrosis measurements were performed using flow cytometry. Additionally, brain natriuretic peptide (BNP) levels, reactive oxygen species (ROS) formation, glucose consumption, and lactate production were quantified. Long-term hyperglycaemia in H9C2KE2 cells induced increased GLUT4 presence on the cell surface and was associated with exaggerated glucose influx and lactate production. On the metabolic level, hyperglycaemia affected the tricarboxylic acid (TCA) cycle with accumulation of fumarate. This was associated with increased BNP-levels, oxidative stress, and lower antioxidant response, resulting in pronounced apoptosis and necrosis. Chronic glucose overload in cardiomyoblasts induced by GLUT4 overexpression and hyperglycaemia resulted in metabolically stimulated proteome profile changes and metabolic alterations on the TCA level.