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Sympathetic system abnormalities have been reported in sepsis-related cardiac dysfunction. The present study aimed at evaluating the potential of the norepinephrine radiolabeled analogue [123I]-meta-iodobenzylguanidine (123I-MIBG) for the noninvasive assessment of modifications in cardiac sympathetic activity occurring in lipopolysaccharide (LPS)-induced experimental acute sepsis by single-photon emission computed tomographic imaging (SPECT).Sepsis was induced in male Wistar rats by intraperitoneal injection of 10 mg·kg−1 lipopolysaccharide (n = 16), whereas control animals (n = 7) were injected with vehicle (NaCl 0.9%). Echocardiography in LPS-injected animals (n = 8) demonstrated systolic and diastolic cardiac dysfunction. 123I-MIBG was injected 1 hour after LPS or vehicle administration (n = 8 and …

Recent advances in mass spectrometry (MS)‐based technologies are now set to transform translational cancer proteomics from an idea to a practice. Here, we present a robust proteomic workflow for the analysis of clinically relevant human cancer tissues that allows quantitation of thousands of tumor proteins in several hours of measuring time and a total turnaround of a few days. We applied it to a chemorefractory metastatic case of the extremely rare urachal carcinoma. Quantitative comparison of lung metastases and surrounding tissue revealed several significantly upregulated proteins, among them lysine‐specific histone demethylase 1 (LSD1/KDM1A). LSD1 is an epigenetic regulator and the target of active development efforts in oncology. Thus, clinical cancer proteomics can rapidly and efficiently identify actionable therapeutic options. While currently described for a single case study, we envision that it can …

We developed EASI-tag (easily abstractable sulfoxide-based isobaric-tag), a new type of amine-derivatizing and sulfoxide-containing isobaric labeling reagents for highly accurate quantitative proteomics analysis using mass spectrometry. We observed that EASI-tag labels dissociate at low collision energy and generate peptide-coupled, interference-free reporter ions with high yield. Efficient isolation of 12C precursors and quantification at the MS2 level allowed accurate determination of quantitative differences between up to six multiplexed samples.

Structural and electrophysiological remodeling of the atria are recognized consequences of sustained atrial arrhythmias, such as atrial fibrillation. The identification of underlying key molecules and signaling pathways has been challenging because of the changing cell type composition during structural remodeling of the atria.Thus, the aims of our study were (1) to search for transcription factors and downstream target genes, which are involved in atrial structural remodeling, (2) to characterize the significance of the transcription factor ETV1 (E twenty-six variant 1) in atrial remodeling and arrhythmia, and (3) to identify ETV1-dependent gene regulatory networks in atrial cardiac myocytes.The transcription factor ETV1 was significantly upregulated in atrial tissue from patients with permanent atrial fibrillation. Mice with cardiac myocyte-specific overexpression of ETV1 …

Antibody-Drug Conjugates (ADCs) are a class of cancer therapeutics that combines antigen specificity and potent cytotoxicity in a single molecule as they are comprised of an engineered antibody linked chemically to a cytotoxic drug. Four ADCs have received approval by the Food and Drug Administration (FDA) and the European Medicine Agency (EMA) and can be prescribed for metastatic conditions while around 60 ADCs are currently enrolled in clinical trials. The efficacy of an ADC greatly relies on its intracellular trafficking and processing of its components to trigger tumor cell death. A limited number of studies have addressed these critical processes that both challenge and help foster the design of ADCs. This review highlights those mechanisms and their relevance for future development of ADCs as cancer therapeutics.

Mitochondrial DNA mutations progressively compromise the respiratory chain of skeletal muscle, resulting in a mosaic of metabolically healthy and defective fibers. The single fiber investigation of this important diagnostic feature has been beyond the capability of large-scale technologies so far. We used laser capture microdissection (LCM) to excise thin sections of individual muscle fibers from frozen biopsies of patients suffering from chronic progressive external ophthalmoplegia. We then applied a highly sensitive mass spectrometry (MS)-based proteomics workflow to analyze healthy and defective muscle fibers within the same biopsy. We quantified more than 4000 proteins in each patient, covering 75% of all respiratory chain subunits, and compared their expression in metabolically healthy and defective muscle fibers. Our findings show that mitochondrial disease causes extensive proteomic rearrangements, affecting the OPA1-dependent cristae remodeling pathway and mitochondrial translation. We provide fiber type-specific information showing that increased expression of fatty acid oxidation enzymes occurs in defective slow but not fast muscle fibers. Our findings shed light on compensatory mechanisms in muscle fibers that struggle with energy shortage and metabolic stress.

Cardiovascular diseases are the leading cause of death worldwide. The molecular mechanisms involved in the underlying pathophysiologies of atherosclerosis and heart related disorders are still poorly known. A closer understanding would greatly benefit clinical outcome predictions and treatment options in future. Two recent studies by Matthias Mann and his team, presented in this review, have addressed cardiovascular diseases using high-resolution mass spectrometry-based proteomics.

Despite the recent success of antibody–drug conjugates (ADCs) in cancer therapy, a detailed understanding of their entry, trafficking, and metabolism in cancer cells is limited. To gain further insight into the activation mechanism of ADCs, we incorporated fluorescence resonance energy transfer (FRET) reporter groups into the linker connecting the antibody to the drug and studied various aspects of intracellular ADC processing mechanisms. When comparing the trafficking of the antibody–FRET drug conjugates in various different model cells, we found that the cellular background plays an important role in how the antigen-mediated antibody is processed. Certain tumor cells showed limited cytosolic transport of the payload despite efficient linker cleavage. Our FRET assay provides a facile and robust assessment of intracellular ADC activation that may have significant implications for the future development of ADCs.

Cancer is the second leading cause of death worldwide and many cancer subtypes remain poorly understood. Most conventional chemotherapeutic treatments are still associated with life-threatening toxic side effects that primarily result from a lack of specificity directed towards cancer cells. Recent breakthroughs in genomic and transcriptomic sequencing technologies have allowed the molecular profiling of thousands of tumors in different cancer types. It has become evident that cancer cannot be considered a singular disease and that its manifestations cannot exclusively be explained by the accumulation of genetic mutations. Instead, epigenetic and proteomic changes as well as posttranslational modifications (PTMs) of proteins are crucial drivers of oncogenesis. In this thesis, I investigated systemwide alterations in cancer at several biological and cellular levels using mass spectrometry (MS). Starting from the nucleus of the cell, I explored the epigenetic changes in lymphoma at the biotechnology company Genentech Inc. We found that the methyltransferase EZH2 is the most significantly over-expressed epigenetic regulator in cancer, and is co-regulated with a cell cycle network. Zooming out from the nuclear level, I analyzed phosphorylation-signaling alterations in primary and secondary glioblastoma cell line models at the University of California, San Francisco (UCSF). Here, I focused on the interplay between the MAPK and PI3K signaling cascades. At the Max Planck Institute (MPI) of Biochemistry, I moved on to translational proteomics, working with human cancer tissues. I optimized an MS-based proteomic workflow for the rapid screening …

Large-scale genetic screens play a key role in the systematic discovery of genes underlying cellular phenotypes. Pooling of genetic perturbations greatly increases screening throughput, but has so far been limited to screens of enrichments defined by cell fitness and flow cytometry, or to comparatively low-throughput single cell gene expression profiles. Although microscopy is a rich source of spatial and temporal information about mammalian cells, high-content imaging screens have been restricted to much less efficient arrayed formats. Here, we introduce an optical method to link perturbations and their phenotypic outcomes at the singlecell level in a pooled setting. Barcoded perturbations are read out by targeted in situ sequencing following image-based phenotyping. We apply this technology to screen a focused set of 952 genes across >3 million cells for involvement in NF-κB activation by imaging the translocation of RelA (p65) to the nucleus, recovering 20 known pathway components and 3 novel candidate positive regulators of IL-1β and TNFα-stimulated immune responses.

Objectives: The aim of the conducted study was the generation of a spatial and cell-type resolved proteomic map of the healthy human heart. A global protein expression “footprint” of the healthy heart can be used as a reference library to compare against diseased hearts in the search for biomarkers, therapeutic targets or disease signatures.Methods: The human heart proteome was measured in 16 anatomical regions from 3 different healthy subjects and in three major cardiac cell types (endothelial cells, fibroblasts and smooth muscle cells) by high-resolution mass spectrometry-based proteomics.Results: The MaxLFQ algorithm quantified a total of 11,163 proteins, 10,751 in the 16 heart regions and 10,447 in the non-cardiomyocyte cell types. For overall assessment of proteomics similarities and differences of the 16 heart regions, we employed principal component analysis (PCA). The major groups cavities …

Identification and quantification of disease-associated proteins in human biofluids is much needed for monitoring disease progression in HD gene expression carriers as well as providing means for monitoring therapeutic intervention. We believe studying biofluids with cutting-edge proteomics tools will help these unmet needs and provide novel leads for further validation and use in the clinic.To this end, the cerebrospinal fluid (CSF) is an accessible source enriched for brain derived peptides as well as proteins, which allows us to investigate changes in the CNS physiology. Similarly, plasma is of great importance for evaluating systemic changes in protein secretion and can act as a secondary source of CSF proteins drained into and found in blood circulation.High-throughput quantitative analysis of plasma and CSF is challenging because of the high dynamic range of protein abundances. However, dramatic …