Standardized peptidome profiling of human urine by magnetic bead separation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

BACKGROUND: Peptidome profiling of human urine is a promising tool to identify novel disease-associated biomarkers; however, a wide range of preanalytical variables influence the results of peptidome analysis. Our aim was to develop a standardized protocol for reproducible urine peptidome profiling by means of magnetic bead (MB) separation followed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). METHODS: MBs with defined surface functionalities (hydrophobic interaction, cation exchange, and metal ion affinity) were used for peptide fractionation of urine. Mass accuracy and imprecision were calculated for 9 characteristic mass signals (M(r), 1000-10,000). Exogenous variables (instrument performance, urine sampling/storage conditions, freezing conditions, and freeze-thaw cycles) and endogenous variables (pH, urine salt and protein concentrations, and blood and bacteria interferences) were investigated with urine samples from 10 male and 10 female volunteers. RESULTS: We detected 427 different mass signals in the urine of healthy donors. Within- and between-day imprecision in relative signal intensities ranged from 1% to 14% and from 4% to 16%, respectively. Weak cation-exchange and metal ion affinity MB preparations required adjustment of the urinary pH to 7. Storage time, storage temperature, the number of freeze-thaw cycles, and bacterial and blood contamination significantly influenced urine peptide patterns. Individual urine peptide patterns differed significantly within and between days. This imprecision was diminished by normalization to a urinary protein content of 3.5 microg. CONCLUSION: This reliable pretreatment protocol allows standardization of preanalytical modalities and facilitates reproducible peptidome profiling of human urine by means of MB separation in combination with MALDI-TOF MS.     

Interim thymus and activation regulated chemokine versus interim 18F-fluorodeoxyglucose positron-emission tomography in classical Hodgkin lymphoma response evaluation

Serum thymus and activation regulated chemokine (TARC) levels reflect classical Hodgkin lymphoma (cHL) disease activity and correspond with treatment response. We compared mid-treatment interim TARC (iTARC) with interim ¹⁸F-fluorodeoxyglucose positron-emission tomography (iPET) imaging to predict modified progression-free survival (mPFS) in a group of 95 patients with cHL. High iTARC levels were found in nine and positive iPET in 17 patients. The positive predictive value (PPV) of iTARC for a 5-year mPFS event was 88% compared to 47% for iPET. The negative predictive value was comparable at 86% for iTARC and 85% for iPET. Serum iTARC levels more accurately reflect treatment response with a higher PPV compared to iPET.     

From the Cover: Trypanosomal TAC40 constitutes a novel subclass of mitochondrial β-barrel proteins specialized in mitochondrial genome inheritance

Mitochondria cannot form de novo but require mechanisms allowing their inheritance to daughter cells. In contrast to most other eukaryotes Trypanosoma brucei has a single mitochondrion whose single-unit genome is physically connected to the flagellum. Here we identify a β-barrel mitochondrial outer membrane protein, termed tripartite attachment complex 40 (TAC40), that localizes to this connection. TAC40 is essential for mitochondrial DNA inheritance and belongs to the mitochondrial porin protein family. However, it is not specifically related to any of the three subclasses of mitochondrial porins represented by the metabolite transporter voltage-dependent anion channel (VDAC), the protein translocator of the outer membrane 40 (TOM40), or the fungi-specific MDM10, a component of the endoplasmic reticulum–mitochondria encounter structure (ERMES). MDM10 and TAC40 mediate cellular architecture and participate in transmembrane complexes that are essential for mitochondrial DNA inheritance. In yeast MDM10, in the context of the ERMES, is postulated to connect the mitochondrial genomes to actin filaments, whereas in trypanosomes TAC40 mediates the linkage of the mitochondrial DNA to the basal body of the flagellum. However, TAC40 does not colocalize with trypanosomal orthologs of ERMES components and, unlike MDM10, it regulates neither mitochondrial morphology nor the assembly of the protein translocase. TAC40 therefore defines a novel subclass of mitochondrial porins that is distinct from VDAC, TOM40, and MDM10. However, whereas the architecture of the TAC40-containing complex in trypanosomes and the MDM10-containing ERMES in yeast is very different, both are organized around a β-barrel protein of the mitochondrial porin family that mediates a DNA–cytoskeleton linkage that is essential for mitochondrial DNA inheritance.Mitochondria are a hallmark of all eukaroytic cells. They derive from an endosymbiontic event between a free-living bacterium and a presumably prokaryotic host cell. More than 1.5 billion years of evolution resulted in a great diversification of mitochondria. As a consequence, the shape and number of organelles per cell as well as size, content, copy number, and organization of their genomes vary greatly between different taxons (1). However, all eukaryotes must be able to faithfully transmit mitochondria to their offspring (2, 3).Unlike most other eukaryotes, the parasitic protozoa Trypanosoma brucei has a single mitochondrion throughout its life and its cell cycle. Due to the single-unit nature of the mitochondrion, its duplication must be coordinated with the duplication of the nucleus (4). The mitochondrial genome of T. brucei, termed kinetoplast DNA (kDNA), is essential for growth of both the procyclic insect stage and the bloodstream form of the parasite (5). It consists of a disk-shaped single-unit kDNA network that localizes to a distinct region within the mitochondrial matrix (6). The kDNA is physically connected with the cytosolic basal body, the organizing center of the eukaryotic flagellum, via a high-order transmembrane structure termed tripartite attachment complex (TAC) (7) of which only few components have been identified (8–10). Replication of the kDNA network occurs at a defined stage of the cell cycle shortly before the onset of the nuclear S phase. After replication, the kDNA networks need to be correctly positioned so that during cell and mitochondrial division each daughter cell receives a single organelle with a single kDNA network. This process requires an intact TAC and is mediated by the movement of the basal body: one kDNA network remains connected to the basal body of the old flagellum whereas the other one segregates with the basal body of the new flagellum (7, 11).Unlike trypanosomes, Saccharomyces cerevisiae propagates by budding and contains highly dynamic mitochondria that constantly divide and fuse (12, 13). Mitochondrial inheritance in budding yeast therefore requires a mechanism to move mitochondria and their genomes from the mother cell into the growing bud. The protein-associated mitochondrial genomes of S. cerevisiae, termed nucleoids, localize to dozens of globular foci that are distributed all over the organelles. Most actively replicating nucleoids are associated with a protein complex that includes the outer membrane (OM) protein MDM10 as a central unit, as well as the proteins MDM12, MDM34, and MMM1 (14–16). The protein complex forms the endoplasmic reticulum (ER)–mitochondria encounter structure (ERMES) tethering the ER to the mitochondrion (17). The ERMES has also been suggested to connect to cytosolic actin fibers that mediate the movement of mitochondria to the bud of dividing yeast cells (14, 18, 19). Besides its role in mitochondrial inheritance, the ERMES has been implicated in maintenance of mitochondrial morphology and in phospholipid and calcium exchange as well as in the assembly of the protein translocase of the mitochondrial OM (TOM) (20, 21). Some of the proposed ERMES functions are controversial and there is evidence that some of them might be due to secondary effects caused by the drastically altered mitochondrial morphology (22).The central ERMES subunit, the β-barrel protein MDM10 belongs to the mitochondrial porin superfamily, which comprises the three members voltage-dependent anion channel (VDAC), Tom40, and MDM10. Whereas VDAC and Tom40 have so far been found in all eukaryotes, including T. brucei (23, 24), MDM10 is specific to the fungal clade.In this study we identify a mitochondrial OM protein of T. brucei as a novel component of the TAC. We show that the protein defines a novel subclass of the mitochondrial porin superfamily that is specialized in mitochondrial DNA inheritance.     

From embouchure problems to embouchure dystonia? A survey of self-reported embouchure disorders in 585 professional orchestra brass players

Objectives

Data concerning embouchure problems in professional brass players are scarce. Embouchure problems can potentially lead to focal dystonia. The aim of this study was to investigate the frequency of distinct embouchure problems in professional brass players. Furthermore, the frequency of “cramping”, a distinct symptom of embouchure dystonia, was evaluated in the context of established embouchure dystonia risk factors.

Methods

Five hundred and eighty-five professional brass players participated in a cross-sectional study concerning embouchure problems. A self-administered questionnaire was developed to evaluate embouchure fatigue, embouchure disorders and their consequences. To study the association between risk factors and cramping (a symptom of embouchure dystonia), a log-binomial regression analysis was conducted, enabling estimation of prevalence ratios (PR) and 95 % confidence intervals (95 % CI).

Results

Thirty percent (95 % CI 25.9–33.3) reported embouchure fatigue. The relative frequency of embouchure disorders was 59 % (95 % CI 54.6–63.6), with 26 % (95 % CI 22.4–29.5) reporting embouchure cramping. Embouchure disorders resulted in sick leave in 16 % (95 % CI 12.7–20.6). Female brass players (PR 2.0, 95 % CI 0.98–3.98) and musicians with a prior change in their embouchure (PR 2.4, 95 % CI 1.38–4.05) or breathing technique (PR 2.2, 95 % CI 1.25–3.72) and musicians with embouchure fatigue (PR 1.9, 95 % CI 1.18–2.93) presented more frequently with embouchure cramping than musicians with other or without risk factors.

Conclusion

This study shows a high relative frequency of embouchure problems in professional brass players. Given that embouchure dystonia is often preceded by embouchure problems, these findings may assist in gaining further insight into the characteristics of embouchure dystonia and the development of preventive strategies.