Proteomic characterization of thymocyte-derived microvesicles and apoptotic bodies in BALB/c mice

Several studies have characterized exosomes derived from different cell sources. In this work we set the goal of proteomic characterization of two less studied populations of membrane vesicles, microvesicles (100-800 nm) and apoptotic bodies (> 800 nm) released by thymus cells of BALB/c mice. The vesicles were isolated by the combination of differential centrifugation and gravity driven multistep filtration of the supernatant of thymus cell cultures. The size distribution of vesicle preparations was determined by transmission electron microscopy. Proteins were released from the vesicles, digested in solution, and analyzed using nano-HPLC/MS(MS). Ingenuity pathway analysis was used to identify functions related to membrane vesicle proteins. In apoptotic bodies and microvesicles we have identified 142 and 195 proteins, respectively. A striking overlap was detected between the proteomic compositions of the two subcellular structures as 108 proteins were detected in both preparations. Identified proteins included autoantigens implicated in human autoimmune diseases, key regulators of T-cell activation, molecules involved in known immune functions or in leukocyte rolling and transendothelial transmigration. The presence and abundance of proteins with high immunological relevance within thymocyte-derived apoptotic bodies and microvesicles raise the possibility that these subcellular structures may substantially modulate T-cell maturation processes within the thymus.     

Improved Flow Cytometric Assessment Reveals Distinct Microvesicle (Cell-Derived Microparticle) Signatures in Joint Diseases

Introduction

Microvesicles (MVs), earlier referred to as microparticles, represent a major type of extracellular vesicles currently considered as novel biomarkers in various clinical settings such as autoimmune disorders. However, the analysis of MVs in body fluids has not been fully standardized yet, and there are numerous pitfalls that hinder the correct assessment of these structures.

Methods

In this study, we analyzed synovial fluid (SF) samples of patients with osteoarthritis (OA), rheumatoid arthritis (RA) and juvenile idiopathic arthritis (JIA). To assess factors that may confound MV detection in joint diseases, we used electron microscopy (EM), Nanoparticle Tracking Analysis (NTA) and mass spectrometry (MS). For flow cytometry, a method commonly used for phenotyping and enumeration of MVs, we combined recent advances in the field, and used a novel approach of differential detergent lysis for the exclusion of MV-mimicking non-vesicular signals.

Results

EM and NTA showed that substantial amounts of particles other than MVs were present in SF samples. Beyond known MV-associated proteins, MS analysis also revealed abundant plasma- and immune complex-related proteins in MV preparations. Applying improved flow cytometric analysis, we demonstrate for the first time that CD3⁺ and CD8⁺ T-cell derived SF MVs are highly elevated in patients with RA compared to OA patients (p = 0.027 and p = 0.009, respectively, after Bonferroni corrections). In JIA, we identified reduced numbers of B cell-derived MVs (p = 0.009, after Bonferroni correction).

Conclusions

Our results suggest that improved flow cytometric assessment of MVs facilitates the detection of previously unrecognized disease-associated vesicular signatures.     

Hierarchy measure for complex networks

Nature, technology and society are full of complexity arising from the intricate web of the interactions among the units of the related systems (e.g., proteins, computers, people). Consequently, one of the most successful recent approaches to capturing the fundamental features of the structure and dynamics of complex systems has been the investigation of the networks associated with the above units (nodes) together with their relations (edges). Most complex systems have an inherently hierarchical organization and, correspondingly, the networks behind them also exhibit hierarchical features. Indeed, several papers have been devoted to describing this essential aspect of networks, however, without resulting in a widely accepted, converging concept concerning the quantitative characterization of the level of their hierarchy. Here we develop an approach and propose a quantity (measure) which is simple enough to be widely applicable, reveals a number of universal features of the organization of real-world networks and, as we demonstrate, is capable of capturing the essential features of the structure and the degree of hierarchy in a complex network. The measure we introduce is based on a generalization of the m-reach centrality, which we first extend to directed/partially directed graphs. Then, we define the global reaching centrality (GRC), which is the difference between the maximum and the average value of the generalized reach centralities over the network. We investigate the behavior of the GRC considering both a synthetic model with an adjustable level of hierarchy and real networks. Results for real networks show that our hierarchy measure is related to the controllability of the given system. We also propose a visualization procedure for large complex networks that can be used to obtain an overall qualitative picture about the nature of their hierarchical structure.     

Evaluation of a partial genome screening of two asthma susceptibility regions using bayesian network based bayesian multilevel analysis of relevance

Genetic studies indicate high number of potential factors related to asthma. Based on earlier linkage analyses we selected the 11q13 and 14q22 asthma susceptibility regions, for which we designed a partial genome screening study using 145 SNPs in 1201 individuals (436 asthmatic children and 765 controls). The results were evaluated with traditional frequentist methods and we applied a new statistical method, called Bayesian network based Bayesian multilevel analysis of relevance (BN-BMLA). This method uses Bayesian network representation to provide detailed characterization of the relevance of factors, such as joint significance, the type of dependency, and multi-target aspects. We estimated posteriors for these relations within the Bayesian statistical framework, in order to estimate the posteriors whether a variable is directly relevant or its association is only mediated.With frequentist methods one SNP (rs3751464 in the FRMD6 gene) provided evidence for an association with asthma (OR = 1.43(1.2–1.8); p = 3×10⁻⁴). The possible role of the FRMD6 gene in asthma was also confirmed in an animal model and human asthmatics.In the BN-BMLA analysis altogether 5 SNPs in 4 genes were found relevant in connection with asthma phenotype: PRPF19 on chromosome 11, and FRMD6, PTGER2 and PTGDR on chromosome 14. In a subsequent step a partial dataset containing rhinitis and further clinical parameters was used, which allowed the analysis of relevance of SNPs for asthma and multiple targets. These analyses suggested that SNPs in the AHNAK and MS4A2 genes were indirectly associated with asthma. This paper indicates that BN-BMLA explores the relevant factors more comprehensively than traditional statistical methods and extends the scope of strong relevance based methods to include partial relevance, global characterization of relevance and multi-target relevance.     

Aldosterone antagonists in monotherapy are protective against streptozotocin-induced diabetic nephropathy in rats

Angiotensin converting enzyme inhibitors (ACEi) and angiotensin II receptor blockers (ARB) are the standard clinical therapy of diabetic nephropathy (DN), while aldosterone antagonists are only used as adjuncts. Previously in experimental DN we showed that Na/K ATPase (NKA) is mislocated and angiotensin II leads to superimposed renal progression. Here we investigated the monotherapeutic effect of aldosterone blockers on the progression of DN and renal NKA alteration in comparison to ACEi and ARBs. Streptozotocin-diabetic rats developing DN were treated with aldosterone antagonists; ACEi and ARB. Renal function, morphology, protein level and tubular localization of NKA were analyzed. To evaluate the effect of high glucose per se; HK-2 proximal tubular cells were cultured in normal or high concentration of glucose and treated with the same agents. Aldosterone antagonists were the most effective in ameliorating functional and structural kidney damage and they normalized diabetes induced bradycardia and weight loss. Aldosterone blockers also prevented hyperglycemia and diabetes induced increase in NKA protein level and enzyme mislocation. A monotherapy with aldosterone antagonists might be as, or more effective than ACEi or ARBs in the prevention of STZ-induced DN. Furthermore the alteration of the NKA could represent a novel pathophysiological feature of DN and might serve as an additional target of aldosterone blockers.     

Quackery versus professionalism? Characters,places and media of medical knowledge in eighteenth-century Hungary

This essay discusses the question of health in the Kingdom of Hungary during the Age of Enlightenment. It explores the relationships and tensions between central theories of medical police and the local expectations of government administrators, as well as those between academic or official knowledge and implicit or alternative knowledge about health. The reigns of Maria Theresia and Joseph II marked the moment at which particular kinds of folk and practical knowledge about healing became visible and above all legible. This is to be seen in the enormous rise in book production, which in itself represented an 'approved knowledge' that found legitimation in new academic and bureaucratic institutions, such as the reformed medical faculty of the University of Vienna, the newly-founded medical faculty at Tyrnau, the establishment of a health department within the Hungarian Statthalterei, as well as in the emission of royal legislation supporting the agendas of the new enlightened science of 'medical police'.     

Comparative large scale characterization of plant versus mammal proteins reveals similar and idiosyncratic N-α-acetylation features

N-terminal modifications play a major role in the fate of proteins in terms of activity, stability, or subcellular compartmentalization. Such modifications remain poorly described and badly characterized in proteomic studies, and only a few comparison studies among organisms have been made available so far. Recent advances in the field now allow the enrichment and selection of N-terminal peptides in the course of proteome-wide mass spectrometry analyses. These targeted approaches unravel as a result the extent and nature of the protein N-terminal modifications. Here, we aimed at studying such modifications in the model plant Arabidopsis thaliana to compare these results with those obtained from a human sample analyzed in parallel. We applied large scale analysis to compile robust conclusions on both data sets. Our data show strong convergence of the characterized modifications especially for protein N-terminal methionine excision, co-translational N-α-acetylation, or N-myristoylation between animal and plant kingdoms. Because of the convergence of both the substrates and the N-α-acetylation machinery, it was possible to identify the N-acetyltransferases involved in such modifications for a small number of model plants. Finally, a high proportion of nuclear-encoded chloroplast proteins feature post-translational N-α-acetylation of the mature protein after removal of the transit peptide. Unlike animals, plants feature in a dedicated pathway for post-translational acetylation of organelle-targeted proteins. The corresponding machinery is yet to be discovered.     

Erythropoietic protein supportive treatment in small cell lung cancer

Anemia is common among patients with malignant tumors, due to the disease and chemotherapy. Anemia decreases patient's quality of life, and worsens the dose intensity of chemotherapy. The aim of this retrospective data-analysis was to determine the rate of transfusions and the maintenance of chemotherapeutic dose intensity in 9 small cell lung cancer patients receiving beta-erythropoietin, due to anemia observed after the first cycle of chemotherapy. The mean pre-treatment hemoglobin concentration of the patients was 116.67+/-8.17 g/L (mean+/-SD). The mean pre-erythropoietin hemoglobin concentration at baseline was 103.11+/-7.52 g/L. Six cycles of platinum compounds and etoposide were used. The post-treatment hemoglobin concentration of patients was 110.11+/-5.37 g/L (p = 0,028 vs. baseline). During these 54 chemotherapeutic cycles, only 2 patients needed transfusion, each of them once. According to our experience, the use of beta-erythropoietin in 9 anemic small cell lung cancer patients resulted in a low rate of transfusions and maintenance of cytotoxic treatment dose intensity. The adequate use of beta-erythropoietin is of great help to the physician in the management of small cell lung cancer patients.     

Robustness of central carbohydrate metabolism in developing maize kernels

The central carbohydrate metabolism provides the precursors for the syntheses of various storage products in seeds. While the underlying biochemical map is well established, little is known about the organization and flexibility of carbohydrate metabolic fluxes in the face of changing biosynthetic demands or other perturbations. This question was addressed in developing kernels of maize (Zea mays L.), a model system for the study of starch and sugar metabolism. ¹³C-labeling experiments were carried out with inbred lines, heterotic hybrids, and starch-deficient mutants that were selected to cover a wide range of performances and kernel phenotypes. In total, 46 labeling experiments were carried out using either [U-¹³C₆]glucose or [U-¹³C₁₂]sucrose and up to three stages of kernel development. Carbohydrate flux distributions were estimated based on glucose isotopologue abundances, which were determined in hydrolysates of starch by using quantitative ¹³C-NMR and GC-MS. Similar labeling patterns in all samples indicated robustness of carbohydrate fluxes in maize endosperm, and fluxes were rather stable in response to glucose or sucrose feeding and during development. A lack of ADP-glucose pyrophosphorylase in the bt2 and sh2 mutants triggered significantly increased hexose cycling. In contrast, other mutations with similar kernel phenotypes had no effect. Thus, the distribution of carbohydrate fluxes is stable and not determined by sink strength in maize kernels.     

Muscular ETB receptors develop postnatally and are differentially distributed in specific segments of the rat vasculature.

The endothelin/endothelin-receptor system is a key player in the regulation of vascular tone in mammals. We raised and characterized an antiserum against rat ETB receptor and investigated the distribution of ETB receptors in different vascular beds during postnatal development (day 0 through day 28) and in the adult rat. We report the tissue-specific and age-dependent presence of vasoconstrictor ETB receptors. At the time of birth, vascular smooth muscle cells from all tissues examined did not exhibit ETB receptor immunoreactivity. The occurrence of ETB receptor immunoreactivity in the postnatal development was time dependent and started in small coronary and meningeal arteries at day 5, followed by small mesenteric arteries as well as brachial artery and vein at day 14. At day 21, ETB receptors were present in the media of muscular segments of pulmonary artery, large coronary arteries, and intracerebral arterioles. At day 28, ETB receptor immunoreactivity was evident in interlobular renal arteries, vas afferens, and efferens. Large renal arteries, mesenteric artery, and elastic segments of pulmonary arteries, as well as coronary and mesenteric veins, did not exhibit ETB receptor immunoreactivity. These data demonstrate the age-dependent and tissue-specific presence of ETB receptors, mainly on arterial smooth muscle cells in the vascular system of the rat.