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- ItemSurface-assisted laser desorption/ionization mass spectrometry using ordered silicon nanopillar arrays(Cambridge : Royal Society of Chemistry, 2014) Alhmoud, Hashim Z.; Guinan, Taryn M.; Elnathan, Roey; Kobus, Hilton; Voelcker, Nicolas H.Surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) is ideally suited for the high-throughput analysis of small molecules in bodily fluids (e.g. saliva, urine, and blood plasma). A key application for this technique is the testing of drug consumption in the context of workplace, roadside, athlete sports and anti-addictive drug compliance. Here, we show that vertically-aligned ordered silicon nanopillar (SiNP) arrays fabricated using nanosphere lithography followed by metal-assisted chemical etching (MACE) are suitable substrates for the SALDI-MS detection of methadone and small peptides. Porosity, length and diameter are fabrication parameters that we have explored here in order to optimize analytical performance. We demonstrate the quantitative analysis of methadone in MilliQ water down to 32 ng mL-1. Finally, the capability of SiNP arrays to facilitate the detection of methadone in clinical samples is also demonstrated.
- ItemProteome-wide analysis reveals an age-associated cellular phenotype of in situ aged human fibroblasts(Orchard Park : Impact Journals, 2014) Waldera-Lupa, Daniel M.; Kalfalah, Faiza; Florea, Ana-Maria; Sass, Steffen; Kruse, Fabian; Rieder, Vera; Tigges, Julia; Fritsche, Ellen; Krutmann, Jean; Busch, Hauke; Boerries, Melanie; Meyer, Helmut E.; Boege, Fritz; Theis, Fabian; Reifenberger, Guido; Stühle, KaiWe analyzed an ex vivo model of in situ aged human dermal fibroblasts, obtained from 15 adult healthy donors from three different age groups using an unbiased quantitative proteome-wide approach applying label-free mass spectrometry. Thereby, we identified 2409 proteins, including 43 proteins with an age-associated abundance change. Most of the differentially abundant proteins have not been described in the context of fibroblasts' aging before, but the deduced biological processes confirmed known hallmarks of aging and led to a consistent picture of eight biological categories involved in fibroblast aging, namely proteostasis, cell cycle and proliferation, development and differentiation, cell death, cell organization and cytoskeleton, response to stress, cell communication and signal transduction, as well as RNA metabolism and translation. The exhaustive analysis of protein and mRNA data revealed that 77 % of the age-associated proteins were not linked to expression changes of the corresponding transcripts. This is in line with an associated miRNA study and led us to the conclusion that most of the age-associated alterations detected at the proteome level are likely caused post-transcriptionally rather than by differential gene expression. In summary, our findings led to the characterization of novel proteins potentially associated with fibroblast aging and revealed that primary cultures of in situ aged fibroblasts are characterized by moderate age-related proteomic changes comprising the multifactorial process of aging.
- ItemRegulation of the tumor-suppressor function of the class III phosphatidylinositol 3-kinase complex by ubiquitin and SUMO(Basel : MDPI, 2014) Reidick, Christina; El Magraoui, Fouzi; Meyer, Helmut E.; Stenmark, Harald; Platta, Harald W.The occurrence of cancer is often associated with a dysfunction in one of the three central membrane-involution processes—autophagy, endocytosis or cytokinesis. Interestingly, all three pathways are controlled by the same central signaling module: the class III phosphatidylinositol 3-kinase (PI3K-III) complex and its catalytic product, the phosphorylated lipid phosphatidylinositol 3-phosphate (PtdIns3P). The activity of the catalytic subunit of the PI3K-III complex, the lipid-kinase VPS34, requires the presence of the membrane-targeting factor VPS15 as well as the adaptor protein Beclin 1. Furthermore, a growing list of regulatory proteins associates with VPS34 via Beclin 1. These accessory factors define distinct subunit compositions and thereby guide the PI3K-III complex to its different cellular and physiological roles. Here we discuss the regulation of the PI3K-III complex components by ubiquitination and SUMOylation. Especially Beclin 1 has emerged as a highly regulated protein, which can be modified with Lys11-, Lys48- or Lys63-linked polyubiquitin chains catalyzed by distinct E3 ligases from the RING-, HECT-, RBR- or Cullin-type. We also point out other cross-links of these ligases with autophagy in order to discuss how these data might be merged into a general concept.
- ItemMultimodal nonlinear imaging of atherosclerotic plaques differentiation of triglyceride and cholesterol deposits(Singapore [u.a.] : World Scientific Publishing, 2014) Matthäus, C.; Cicchi, R.; Meyer, T.; Lattermann, A.; Schmitt, M.; Romeike, B.F.M.; Krafft, C.; Dietzek, B.; Brehm, B.R.; Pavone, F.S.; Popp, J.Cardiovascular diseases in general and atherothrombosis as the most common of its individual disease entities is the leading cause of death in the developed countries. Therefore, visualization and characterization of inner arterial plaque composition is of vital diagnostic interest, especially for the early recognition of vulnerable plaques. Established clinical techniques provide valuable morphological information but cannot deliver information about the chemical composition of individual plaques. Therefore, spectroscopic imaging techniques have recently drawn considerable attention. Based on the spectroscopic properties of the individual plaque components, as for instance different types of lipids, the composition of atherosclerotic plaques can be analyzed qualitatively as well as quantitatively. Here, we compare the feasibility of multimodal nonlinear imaging combining two-photon fluorescence (TPF), coherent anti-Stokes Raman scattering (CARS) and second-harmonic generation (SHG) microscopy to contrast composition and morphology of lipid deposits against the surrounding matrix of connective tissue with diffraction limited spatial resolution. In this contribution, the spatial distribution of major constituents of the arterial wall and atherosclerotic plaques like elastin, collagen, triglycerides and cholesterol can be simultaneously visualized by a combination of nonlinear imaging methods, providing a powerful label-free complement to standard histopathological methods with great potential for in vivo application.
- ItemProteome analyses of hepatocellular carcinoma(Sugar Land, TX : Xia & He Publishing, 2014) Megger, Dominik A.; Naboulsi, Naboulsi; Meyer, Helmut E.; Sitek, BarbaraProteomics has evolved into a powerful and widely used bioanalytical technique in the study of cancer, especially hepatocellular carcinoma (HCC). In this review, we provide an up to date overview of feasible proteome-analytical techniques for clinical questions. In addition, we present a broad summary of proteomic studies of HCC utilizing various technical approaches for the analysis of samples derived from diverse sources like HCC cell lines, animal models, human tissue and body fluids.
- ItemHighly immunoreactive IgG antibodies directed against a set of twenty human proteins in the sera of patients with amyotrophic lateral sclerosis identified by protein array(San Francisco, CA : Public Library of Science, 2014) May, Caroline; Nordhoff, Eckhard; Casjens, Swaantje; Turewicz, Michael; Eisenacher, Martin; Gold, Ralf; Brüning, Thomas; Pesch, Beate; Stephan, Christian; Woitalla, Dirk; Penke, Botond; Janáky, Tamás; Virók, Dezső; Siklós, László; Engelhardt, Jozsef I.; Meyer, Helmut E.Amyotrophic lateral sclerosis (ALS), the most common adult-onset motor neuron disorder, is characterized by the progressive and selective loss of upper and lower motor neurons. Diagnosis of this disorder is based on clinical assessment, and the average survival time is less than 3 years. Injections of IgG from ALS patients into mice are known to specifically mark motor neurons. Moreover, IgG has been found in upper and lower motor neurons in ALS patients. These results led us to perform a case-control study using human protein microarrays to identify the antibody profiles of serum samples from 20 ALS patients and 20 healthy controls. We demonstrated high levels of 20 IgG antibodies that distinguished the patients from the controls. These findings suggest that a panel of antibodies may serve as a potential diagnostic biomarker for ALS.
- ItemCardio-respiratory coordination increases during sleep apnea(San Francisco, CA : Public Library of Science (PLoS), 2014) Riedl, M.; Müller, A.; Kraemer, J.F.; Penzel, T.; Kurths, J.; Wessel, N.Cardiovascular diseases are the main source of morbidity and mortality in the United States with costs of more than $170 billion. Repetitive respiratory disorders during sleep are assumed to be a major cause of these diseases. Therefore, the understanding of the cardio-respiratory regulation during these events is of high public interest. One of the governing mechanisms is the mutual influence of the cardiac and respiratory oscillations on their respective onsets, the cardiorespiratory coordination (CRC). We analyze this mechanism based on nocturnal measurements of 27 males suffering from obstructive sleep apnea syndrome. Here we find, by using an advanced analysis technique, the coordigram, not only that the occurrence of CRC is significantly more frequent during respiratory sleep disturbances than in normal respiration (p-value<10-51) but also more frequent after these events (p-value<10-15). Especially, the latter finding contradicts the common assumption that spontaneous CRC can only be observed in epochs of relaxed conditions, while our newly discovered epochs of CRC after disturbances are characterized by high autonomic stress. Our findings on the connection between CRC and the appearance of sleep-disordered events require a substantial extension of the current understanding of obstructive sleep apneas and hypopneas.
- ItemThe cytosolic domain of Pex22p stimulates the Pex4p-dependent ubiquitination of the PTS1-receptor(San Francisco, CA : Public Library of Science, 2014) El Magraoui, Fouzi; Schrötter, Andreas; Brinkmeier, Rebecca; Kunst, Lena; Mastalski, Thomas; Müller, Thorsten; Marcus, Katrin; Meyer, Helmut E.; Girzalsky, Wolfgang; Erdmann, Ralf; Platta, Harald W.Peroxisomal biogenesis is an ubiquitin-dependent process because the receptors required for the import of peroxisomal matrix proteins are controlled via their ubiquitination status. A key step is the monoubiquitination of the import receptor Pex5p by the ubiquitin-conjugating enzyme (E2) Pex4p. This monoubiquitination is supposed to take place after Pex5p has released the cargo into the peroxisomal matrix and primes Pex5p for the extraction from the membrane by the mechano-enzymes Pex1p/Pex6p. These two AAA-type ATPases export Pex5p back to the cytosol for further rounds of matrix protein import. Recently, it has been reported that the soluble Pex4p requires the interaction to its peroxisomal membrane-anchor Pex22p to display full activity. Here we demonstrate that the soluble C-terminal domain of Pex22p harbours its biological activity and that this activity is independent from its function as membrane-anchor of Pex4p. We show that Pex4p can be functionally fused to the trans-membrane segment of the membrane protein Pex3p, which is not directly involved in Pex5p-ubiquitination and matrix protein import. However, this Pex3(N)-Pex4p chimera can only complement the double-deletion strain pex4Δ/pex22Δ and ensure optimal Pex5p-ubiquitination when the C-terminal part of Pex22p is additionally expressed in the cell. Thus, while the membrane-bound portion Pex22(N)p is not required when Pex4p is fused to Pex3(N)p, the soluble Pex22(C)p is essential for peroxisomal biogenesis and efficient monoubiquitination of the import receptor Pex5p by the E3-ligase Pex12p in vivo and in vitro. The results merge into a picture of an ubiquitin-conjugating complex at the peroxisomal membrane consisting of three domains: the ubiquitin-conjugating domain (Pex4p), a membrane-anchor domain (Pex22(N)p) and an enhancing domain (Pex22(C)p), with the membrane-anchor domain being mutually exchangeable, while the Ubc- and enhancer-domains are essential.
- ItemSleep apnea-hypopnea quantification by cardiovascular data analysis(San Francisco, CA : Public Library of Science (PLoS), 2014) Camargo, S.; Riedl, M.; Anteneodo, C.; Kurths, J.; Penzel, T.; Wessel, N.Sleep disorders are a major risk factor for cardiovascular diseases. Sleep apnea is the most common sleep disturbance and its detection relies on a polysomnography, i.e., a combination of several medical examinations performed during a monitored sleep night. In order to detect occurrences of sleep apnea without the need of combined recordings, we focus our efforts on extracting a quantifier related to the events of sleep apnea from a cardiovascular time series, namely systolic blood pressure (SBP). Physiologic time series are generally highly nonstationary and entrap the application of conventional tools that require a stationary condition. In our study, data nonstationarities are uncovered by a segmentation procedure which splits the signal into stationary patches, providing local quantities such as mean and variance of the SBP signal in each stationary patch, as well as its duration L. We analysed the data of 26 apneic diagnosed individuals, divided into hypertensive and normotensive groups, and compared the results with those of a control group. From the segmentation procedure, we identified that the average duration 〈L〉, as well as the average variance 〈σ2〉, are correlated to the apnea-hypoapnea index (AHI), previously obtained by polysomnographic exams. Moreover, our results unveil an oscillatory pattern in apneic subjects, whose amplitude S∗ is also correlated with AHI. All these quantities allow to separate apneic individuals, with an accuracy of at least 79%. Therefore, they provide alternative criteria to detect sleep apnea based on a single time series, the systolic blood pressure.
- ItemGuidance of mesenchymal stem cells on fibronectin structured hydrogel films(San Francisco, California, US : PLOS, 2014) Kasten, Annika; Naser, Tamara; Brüllhoff, Kristina; Fiedler, Jörg; Müller, Petra; Möller, Martin; Rychly, Joachim; Groll, Jürgen; Brenner, Rolf E.; Engler, Adam J.Designing of implant surfaces using a suitable ligand for cell adhesion to stimulate specific biological responses of stem cells will boost the application of regenerative implants. For example, materials that facilitate rapid and guided migration of stem cells would promote tissue regeneration. When seeded on fibronectin (FN) that was homogeneously immmobilized to NCO-sP(EO-stat-PO), which otherwise prevents protein binding and cell adhesion, human mesenchymal stem cells (MSC) revealed a faster migration, increased spreading and a more rapid organization of different cellular components for cell adhesion on fibronectin than on a glass surface. To further explore, how a structural organization of FN controls the behavior of MSC, adhesive lines of FN with varying width between 10 µm and 80 µm and spacings between 5 µm and 20 µm that did not allow cell adhesion were generated. In dependance on both line width and gaps, cells formed adjacent cell contacts, were individually organized in lines, or bridged the lines. With decreasing sizes of FN lines, speed and directionality of cell migration increased, which correlated with organization of the actin cytoskeleton, size and shape of the nuclei as well as of focal adhesions. Together, defined FN lines and gaps enabled a fine tuning of the structural organization of cellular components and migration. Microstructured adhesive substrates can mimic the extracellular matrix in vivo and stimulate cellular mechanisms which play a role in tissue regeneration.