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 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.

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.

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.

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.

N471D WASH complex subunit strumpellin knock-in mice display mild motor and cardiac abnormalities and BPTF and KLHL11 dysregulation in brain tissue.

AIMS: We investigated N471D WASH complex subunit strumpellin (Washc5) knock-in and Washc5 knock-out mice as models for hereditary spastic paraplegia type 8 (SPG8). METHODS: We generated heterozygous and homozygous N471D Washc5 knock-in mice and subjected them to a comprehensive clinical, morphological and laboratory parameter screen, and gait analyses. Brain tissue was used for proteomic analysis. Furthermore, we generated heterozygous Washc5 knock-out mice. WASH complex subunit strumpellin expression was determined by qPCR and immunoblotting. RESULTS: Homozygous N471D Washc5 knock-in mice showed mild dilated cardiomyopathy, decreased acoustic startle reactivity, thinner eye lenses, increased alkaline phosphatase and potassium levels and increased white blood cell counts. Gait analyses revealed multiple aberrations indicative of locomotor instability. Similarly, the clinical chemistry, haematology and gait parameters of heterozygous mice also deviated from the values expected for healthy animals, albeit to a lesser extent. Proteomic analysis of brain tissue depicted consistent upregulation of BPTF and downregulation of KLHL11 in heterozygous and homozygous knock-in mice. WASHC5-related protein interaction partners and complexes showed no change in abundancies. Heterozygous Washc5 knock-out mice showing normal WASHC5 levels could not be bred to homozygosity. CONCLUSIONS: While biallelic ablation of Washc5 was prenatally lethal, expression of N471D mutated WASHC5 led to several mild clinical and laboratory parameter abnormalities, but not to a typical SPG8 phenotype. The consistent upregulation of BPTF and downregulation of KLHL11 suggest mechanistic links between the expression of N471D mutated WASHC5 and the roles of both proteins in neurodegeneration and protein quality control, respectively.

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.

Reproducibility, Specificity and Accuracy of Relative Quantification Using Spectral Library-based Data-independent Acquisition.

Currently data-dependent acquisition (DDA) is the method of choice for mass spectrometry-based proteomics discovery experiments, but data-independent acquisition (DIA) is steadily becoming more important. One of the most important requirements to perform a DIA analysis is the availability of suitable spectral libraries for peptide identification and quantification. Several studies were performed addressing the evaluation of spectral library performance for protein identification in DIA measurements. But so far only few experiments estimate the effect of these libraries on the quantitative level.In this work we created a gold standard spike-in sample set with known contents and ratios of proteins in a complex protein matrix that allowed a detailed comparison of DIA quantification data obtained with different spectral library approaches. We used in-house generated sample-specific spectral libraries created using varying sample preparation approaches and repeated DDA measurement. In addition, two different search engines were tested for protein identification from DDA data and subsequent library generation. In total, eight different spectral libraries were generated, and the quantification results compared with a library free method, as well as a default DDA analysis. Not only the number of identifications on peptide and protein level in the spectral libraries and the corresponding DIA analysis results was inspected, but also the number of expected and identified differentially abundant protein groups and their ratios.We found, that while libraries of prefractionated samples were generally larger, there was no significant increase in DIA identifications compared with repetitive non-fractionated measurements. Furthermore, we show that the accuracy of the quantification is strongly dependent on the applied spectral library and whether the quantification is based on peptide or protein level. Overall, the reproducibility and accuracy of DIA quantification is superior to DDA in all applied approaches.Data has been deposited to the ProteomeXchange repository with identifiers PXD012986, PXD012987, PXD012988 and PXD014956.

The impact of the COVID-19 pandemic on non-COVID induced sepsis survival.

BACKGROUND: The COVID-19 pandemic has taken a toll on health care systems worldwide, which has led to increased mortality of different diseases like myocardial infarction. This is most likely due to three factors. First, an increased workload per nurse ratio, a factor associated with mortality. Second, patients presenting with COVID-19-like symptoms are isolated, which also decreases survival in cases of emergency. And third, patients hesitate to see a doctor or present themselves at a hospital. To assess if this is also true for sepsis patients, we asked whether non-COVID-19 sepsis patients had an increased 30-day mortality during the COVID-19 pandemic. METHODS: This is a post hoc analysis of the SepsisDataNet.NRW study, a multicentric, prospective study that includes septic patients fulfilling the SEPSIS-3 criteria. Within this study, we compared the 30-day mortality and disease severity of patients recruited pre-pandemic (recruited from March 2018 until February 2020) with non-COVID-19 septic patients recruited during the pandemic (recruited from March 2020 till December 2020). RESULTS: Comparing septic patients recruited before the pandemic to those recruited during the pandemic, we found an increased raw 30-day mortality in sepsis-patients recruited during the pandemic (33% vs. 52%, p = 0.004). We also found a significant difference in the severity of disease at recruitment (SOFA score pre-pandemic: 8 (5 - 11) vs. pandemic: 10 (8 - 13); p < 0.001). When adjusted for this, the 30-day mortality rates were not significantly different between the two groups (52% vs. 52% pre-pandemic and pandemic, p = 0.798). CONCLUSIONS: This led us to believe that the higher mortality of non-COVID19 sepsis patients during the pandemic might be attributed to a more severe septic disease at the time of recruitment. We note that patients may experience a delayed admission, as indicated by elevated SOFA scores. This could explain the higher mortality during the pandemic and we found no evidence for a diminished quality of care for critically ill sepsis patients in German intensive care units.

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.

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.

MaCPepDB: A Database to Quickly Access All Tryptic Peptides of the UniProtKB.

Protein sequence databases play a crucial role in the majority of the currently applied mass-spectrometry-based proteomics workflows. Here UniProtKB serves as one of the major sources, as it combines the information of several smaller databases and enriches the entries with additional biological information. For the identification of peptides in a sample by tandem mass spectra, as generated by data-dependent acquisition, protein sequence databases provide the basis for most spectrum identification search engines. In addition, for targeted proteomics approaches like selected reaction monitoring (SRM) and parallel reaction monitoring (PRM), knowledge of the peptide sequences, their masses, and whether they are unique for a protein is essential. Because most bottom-up proteomics approaches use trypsin to cleave the proteins in a sample, the tryptic peptides contained in a protein database are of great interest. We present a database, called MaCPepDB (mass-centric peptide database), that consists of the complete tryptic digest of the Swiss-Prot and TrEMBL parts of UniProtKB. This database is especially designed to not only allow queries of peptide sequences and return the respective information about connected proteins and thus whether a peptide is unique but also allow queries of specific masses of peptides or precursors of MS/MS spectra. Furthermore, posttranslational modifications can be considered in a query as well as different mass deviations for posttranslational modifications. Hence the database can be used by a sequence query not only to, for example, check in which proteins of the UniProt database a tryptic peptide can be found but also to find possibly interfering peptides in PRM/SRM experiments using the mass query. The complete database contains currently 5 939 244 990 peptides from 185 561 610 proteins (UniProt version 2020_03), for which a single query usually takes less than 1 s. For easy exploration of the data, a web interface was developed. A REST application programming interface (API) for programmatic and workflow access is also available at https://macpepdb.mpc.rub.de.

A generally applicable lightweight method for calculating a value structure for tools and services in bioinformatics infrastructure projects.

Sustainable noncommercial bioinformatics infrastructures are a prerequisite to use and take advantage of the potential of big data analysis for research and economy. Consequently, funders, universities and institutes as well as users ask for a transparent value model for the tools and services offered. In this article, a generally applicable lightweight method is described by which bioinformatics infrastructure projects can estimate the value of tools and services offered without determining exactly the total costs of ownership. Five representative scenarios for value estimation from a rough estimation to a detailed breakdown of costs are presented. To account for the diversity in bioinformatics applications and services, the notion of service-specific 'service provision units' is introduced together with the factors influencing them and the main underlying assumptions for these 'value influencing factors'. Special attention is given on how to handle personnel costs and indirect costs such as electricity. Four examples are presented for the calculation of the value of tools and services provided by the German Network for Bioinformatics Infrastructure (de.NBI): one for tool usage, one for (Web-based) database analyses, one for consulting services and one for bioinformatics training events. Finally, from the discussed values, the costs of direct funding and the costs of payment of services by funded projects are calculated and compared.

The process of somatic hypermutation increases polyreactivity for central nervous system antigens in primary central nervous system lymphoma.

The immunoglobulin (Ig) heavy and light chain variable gene mutational pattern of the B cell receptor (BCR) in primary central nervous system (CNS) lymphoma (PCNSL) cells suggests antigenic selection to drive pathogenesis and confinement to the CNS. This hypothesis is supported by the observation that the tumor B cell receptor (tBCR) of PCNSL is polyreactive and may be stimulated by CNS proteins. To obtain further insight into the role of the germinal center (GC) reaction on BCR reactivity, we constructed recombinant antibodies (recAb) with Ig heavy and light chain sequences of the corresponding naive BCR (nBCR) by reverting tBCR somatic mutations in 10 PCNSL. Analysis of nBCR-derived recAb reactivity by a protein microarray and immunoprecipitation demonstrated auto- and polyreactivity in all cases. Self-/polyreactivity was not lost during the GC reaction; surprisingly, tBCR significantly increased self-/polyreactivity. In addition to proteins recognized by both the nBCR and tBCR, tBCR gained self-/polyreactivity particularly for proteins expressed in the CNS including proteins of oligodendrocytes/myelin, the S100 protein family, and splicing factors. Thus, in PCNSL pathogenesis, a faulty GC reaction may increase self-/polyreactivity, hereby facilitating BCR signaling via multiple CNS antigens, and may ultimately foster tumor cell survival in the CNS.

New Approaches to Critical Illness Polyneuromyopathy: High-Resolution Neuromuscular Ultrasound Characteristics and Cytokine Profiling.

BACKGROUND: Diagnosis of intensive care unit acquired weakness (ICUAW) is challenging. Pathogenesis of underlying critical illness polyneuromyopathy (CIPNM) remains incompletely understood. This exploratory study investigated whether longitudinal neuromuscular ultrasound examinations and cytokine analyses in correlation to classical clinical and electrophysiological assessment contribute to the understanding of CIPNM. METHODS: Intensive care unit patients were examined every 7 days until discharge from hospital. Clinical status, nerve conduction studies, electromyography as well as ultrasound of peripheral nerves and tibial anterior muscle were performed. Cytokine levels were analyzed by a bead-based multiplex assay system. RESULTS: Of 248 screened patients, 35 patients were included at median of 6 days (IQR: 8) after admission to intensive care unit. Axonal damage was the main feature of CIPNM. At the peak of CIPNM (7 days after inclusion), nerve ultrasound showed cross-sectional area increase of tibial nerve as a sign of inflammatory edema as well as hypoechoic nerves as a possible sign of inflammation. Cytokine analyses showed signs of monocyte and macrophage activation at this stage. Fourteen days after inclusion, cytokines indicated systemic immune response as well as profiles associated to neovascularization and regeneration. CONCLUSIONS: Exploratory neuromuscular ultrasound and cytokine analyses showed signs of inflammation like macrophage and monocyte activation at the peak of CIPNM followed by a systemic immune response parallel to axonal damage. This underlines the role of both axonal damage and inflammation in pathogenesis of CIPNM.

CalibraCurve: A Tool for Calibration of Targeted MS-Based Measurements.

Targeted proteomics techniques allow accurate quantitative measurements of analytes in complex matrices with dynamic linear ranges that span up to 4-5 orders of magnitude. Hence, targeted methods are promising for the development of robust protein assays in several sensitive areas, for example, in health care. However, exploiting the full method potential requires reliable determination of the dynamic range along with related quantification limits for each analyte. Here, a software named CalibraCurve that enables an automated batch-mode determination of dynamic linear ranges and quantification limits for both targeted proteomics and similar assays is presented. The software uses a variety of measures to assess the accuracy of the calibration, namely precision and trueness. Two different kinds of customizable graphs are created (calibration curves and response factor plots). The accuracy measures and the graphs offer an intuitive, detailed, and reliable opportunity to assess the quality of the model fit. Thus, CalibraCurve is deemed a highly useful and flexible tool to facilitate the development and control of reliable SRM/MRM-MS-based proteomics assays.

Comparative proteomic analysis of osteogenic differentiated human adipose tissue and bone marrow-derived stromal cells.

Mesenchymal stromal cells are promising candidates for regenerative applications upon treatment of bone defects. Bone marrow-derived stromal cells (BMSCs) are limited by yield and donor morbidity but show superior osteogenic capacity compared to adipose-derived stromal cells (ASCs), which are highly abundant and easy to harvest. The underlying reasons for this difference on a proteomic level have not been studied yet. Human ASCs and BMSCs were characterized by FACS analysis and tri-lineage differentiation, followed by an intraindividual comparative proteomic analysis upon osteogenic differentiation. Results of the proteomic analysis were followed by functional pathway analysis. 29 patients were included with a total of 58 specimen analysed. In these, out of 5148 identified proteins 2095 could be quantified in >80% of samples of both cell types, 427 in >80% of ASCs only and 102 in >80% of BMSCs only. 281 proteins were differentially regulated with a fold change of >1.5 of which 204 were higher abundant in BMSCs and 77 in ASCs. Integrin cell surface interactions were the most overrepresented pathway with 5 integrins being among the proteins with highest fold change. Integrin 11a, a known key protein for osteogenesis, could be identified as strongly up-regulated in BMSC confirmed by Western blotting. The integrin expression profile is one of the key distinctive features of osteogenic differentiated BMSCs and ASCs. Thus, they represent a promising target for modifications of ASCs aiming to improve their osteogenic capacity and approximate them to that of BMSCs.

Integrated Fourier Transform Infrared Imaging and Proteomics for Identification of a Candidate Histochemical Biomarker in Bladder Cancer.

Histopathological differentiation between severe urocystitis with reactive urothelial atypia and carcinoma in situ (CIS) can be difficult, particularly after a treatment that deliberately induces an inflammatory reaction, such as intravesical instillation of Bacillus Calmette-Guèrin. However, precise grading in bladder cancer is critical for therapeutic decision making and thus requires reliable immunohistochemical biomarkers. Herein, an exemplary potential biomarker in bladder cancer was identified by the novel approach of Fourier transform infrared imaging for label-free tissue annotation of tissue thin sections. Identified regions of interest are collected by laser microdissection to provide homogeneous samples for liquid chromatography-tandem mass spectrometry-based proteomic analysis. This approach afforded label-free spatial classification with a high accuracy and without interobserver variability, along with the molecular resolution of the proteomic analysis. Cystitis and invasive high-grade urothelial carcinoma samples were analyzed. Three candidate biomarkers were identified and verified by immunohistochemistry in a small cohort, including low-grade urothelial carcinoma samples. The best-performing candidate AHNAK2 was further evaluated in a much larger independent verification cohort that also included CIS samples. Reactive urothelial atypia and CIS were distinguishable on the basis of the expression of this newly identified and verified immunohistochemical biomarker, with a sensitivity of 97% and a specificity of 69%. AHNAK2 can differentiate between reactive urothelial atypia in the setting of an acute or chronic cystitis and nonmuscle invasive-type CIS.

Chaperones in sporadic inclusion body myositis-Validation of proteomic data.

INTRODUCTION: Sporadic inclusion body myositis (sIBM) is characterized by myopathological features including rimmed vacuoles (RVs) and proteins associated with protein aggregation, autophagy, and inflammation. Previous proteomic studies of RV areas revealed an overrepresentation of several chaperones and subunits of the T-complex protein 1 (TCP-1), which is involved in prevention of protein aggregation. METHODS: To validate our proteomic findings, immunofluorescence analyses of selected chaperones and quantitative Western blot analysis of TCP-1 proteins were performed in five sIBM patients and five healthy controls. RESULTS: Immunofluorescence studies confirmed increased immunoreactivity for VCP, UNC45B, GRP-75, αB-crystallin, LAMP-2, Rab-7a, and TCP-1α and TCP-θ in RVs. Quantitative Western blot analysis revealed a significantly higher level of TCP-1 in sIBM muscle tissue when compared with healthy controls. DISCUSSION: Our study findings validate new insights in protein quality control and degradation processes that seem to be relevant in sIBM. These data provide an important basis for future functional and therapeutic studies.

miR-129-5p and miR-130a-3p Regulate VEGFR-2 Expression in Sensory and Motor Neurons during Development.

The wide-ranging influence of vascular endothelial growth factor (VEGF) within the central (CNS) and peripheral nervous system (PNS), for example through effects on axonal growth or neuronal cell survival, is mainly mediated by VEGF receptor 2 (VEGFR-2). However, the regulation of VEGFR-2 expression during development is not yet well understood. As microRNAs are considered to be key players during neuronal maturation and regenerative processes, we identified the two microRNAs (miRNAs)-miR-129-5p and miR-130a-3p-that may have an impact on VEGFR-2 expression in young and mature sensory and lower motor neurons. The expression level of VEGFR-2 was analyzed by using in situ hybridization, RT-qPCR, Western blot, and immunohistochemistry in developing rats. microRNAs were validated within the spinal cord and dorsal root ganglia. To unveil the molecular impact of our candidate microRNAs, dissociated cell cultures of sensory and lower motor neurons were transfected with mimics and inhibitors. We depicted age-dependent VEGFR-2 expression in sensory and lower motor neurons. In detail, in lower motor neurons, VEGFR-2 expression was significantly reduced during maturation, in conjunction with an increased level of miR-129-5p. In sensory dorsal root ganglia, VEGFR-2 expression increased during maturation and was accompanied by an overexpression of miR-130a-3p. In a second step, the functional significance of these microRNAs with respect to VEGFR-2 expression was proven. Whereas miR-129-5p seems to decrease VEGFR-2 expression in a direct manner in the CNS, miR-130a-3p might indirectly control VEGFR-2 expression in the PNS. A detailed understanding of genetic VEGFR-2 expression control might promote new strategies for the treatment of severe neurological diseases like ischemia or peripheral nerve injury.