DIGE proteome analysis reveals suitability of ischemic cardiac in vitro model for studying cellular response to acute ischemia and regeneration

Proteomic analysis of myocardial tissue from patient population is suited to yield insights into cellular and molecular mechanisms taking place in cardiovascular diseases. However, it has been limited by small sized biopsies and complicated by high variances between patients. Therefore, there is a high demand for suitable model systems with the capability to simulate ischemic and cardiotoxic effects in vitro, under defined conditions. In this context, we established an in vitro ischemia/reperfusion cardiac disease model based on the contractile HL-1 cell line. To identify pathways involved in the cellular alterations induced by ischemia and thereby defining disease-specific biomarkers and potential target structures for new drug candidates we used fluorescence 2D-difference gel electrophoresis. By comparing spot density changes in ischemic and reperfusion samples we detected several protein spots that were differentially abundant. Using MALDI-TOF/TOF-MS and ESI-MS the proteins were identified and subsequently grouped by functionality. Most prominent were changes in apoptosis signalling, cell structure and energy-metabolism. Alterations were confirmed by analysis of human biopsies from patients with ischemic cardiomyopathy.With the establishment of our in vitro disease model for ischemia injury target identification via proteomic research becomes independent from rare human material and will create new possibilities in cardiac research.     

Haemoglobin variants and Plasmodium falciparum malaria in children under five years of age living in a high and seasonal malaria transmission area of Burkina Faso

ABSTRACT: BACKGROUND: Genetic factors play a key role in determining resistance/susceptibility to infectious disease. Susceptibility of the human host to malaria infection has been reported to be influenced by genetic factors, which could be confounders if not taken into account in the assessment of the efficacy of interventions against malaria. This study aimed to assess the relationship between haemoglobin genotypes and malaria in children under five years in a site being characterized for future malaria vaccine trials. METHODS: The study population consisted of 452 children living in four rural villages. Hb genotype was determined at enrolment. Clinical malaria incidence was evaluated over a one-year period using combined active and passive surveillance. Prevalence of infection was evaluated via bi-annual cross-sectional surveys. At each follow-up visit, children received a brief clinical examination and thick and thin blood films were prepared for malaria diagnosis. A clinical malaria was defined as Plasmodium falciparum parasitaemia >2,500 parasites/ul and axillary temperature [greater than or equal to]37.5degreesC or reported fever over the previous 24 hours. RESULTS: Frequencies of Hb genotypes were 73.2% AA; 15.0% AC; 8.2% AS; 2.2% CC; 1.1% CS and 0.2% SS. Prevalence of infection at enrolment ranged from 61.9%-54.1% among AA, AC and AS children. After one year follow-up, clinical malaria incidence (95% CI) (episodes per person-year) was 1.9 (1.7-2.0) in AA, 1.6 (1.4-2.1) in AC, and 1.7 (1.4-2.0) in AS children. AC genotype was associated with lower incidence of clinical malaria relative to AA genotype among children aged 1-2 years [rate ratio (95% CI) 0.66 (0.42-1.05)] and 2-3 years [rate ratio (95% CI) 0.37 (0.18-0.75)]; an association of opposite direction was however apparent among children aged 3-4 years. AS genotype was associated with lower incidence of clinical malaria relative to AA genotype among children aged 2-3 years [rate ratio (95% CI) 0.63 (0.40-1.01)]. CONCLUSIONS: In this cohort of children, AC or AS genotype was associated with lower risk of clinical malaria relative to AA genotype only among children aged one to three years. It would be advisable for clinical studies of malaria in endemic regions to consider haemoglobin gene differences as a potentially important confounder, particularly among younger children.     

Protein-protein-interaction Network Organization of the Hypusine Modification System

Hypusine modification of eukaryotic initiation factor 5A (eIF-5A) represents a unique and highly specific post-translational modification with regulatory functions in cancer, diabetes, and infectious diseases. However, the specific cellular pathways that are influenced by the hypusine modification remain largely unknown. To globally characterize eIF-5A and hypusine-dependent pathways, we used an approach that combines large-scale bioreactor cell culture with tandem affinity purification and mass spectrometry: “bioreactor-TAP-MS/MS.” By applying this approach systematically to all four components of the hypusine modification system (eIF-5A1, eIF-5A2, DHS, and DOHH), we identified 248 interacting proteins as components of the cellular hypusine network, with diverse functions including regulation of translation, mRNA processing, DNA replication, and cell cycle regulation. Network analysis of this data set enabled us to provide a comprehensive overview of the protein-protein interaction landscape of the hypusine modification system. In addition, we validated the interaction of eIF-5A with some of the newly identified associated proteins in more detail. Our analysis has revealed numerous novel interactions, and thus provides a valuable resource for understanding how this crucial homeostatic signaling pathway affects different cellular functions.Cellular homeostasis is controlled by signaling networks that communicate through post-translational modifications (PTM)¹ of proteins, including phosphorylation, acetylation and methylation (1–3). These modifications are typically attached to various types of proteins by multiple independent enzymes, and thereby simultaneously regulate a wide range of protein functions. Consequently, most signaling pathways are highly redundant, enabling maintenance of cellular integrity even if the modification of a single signaling molecule is disrupted (4). A striking exception is hypusine. This essential PTM is limited to a single protein: the eukaryotic initiation factor 5A (eIF-5A) (5). Disruption of this PTM leads to growth arrest in proliferating eukaryotic cells and is fatal for the developing mammalian embryo (6, 7). During hypusine biosynthesis, the lysine residue at position 50 (Lys₅₀) in eIF-5A is converted into the unusual amino acid hypusine (N^ε-(4-amino-2-hydroxybutyl)lysine; depicted in Fig. 1A) (5). This process activates eIF-5A and is mediated by two enzymatic reactions: first, deoxyhypusine synthase (DHS) catalyzes the transfer of the 4-aminobutyl moiety of spermidine to the ε-amino group of Lys₅₀ to form an intermediate residue, deoxyhypusine (Dhp₅₀) (8). Subsequently, deoxyhypusine hydroxylase (DOHH) mediates the formation of hypusine (Hyp₅₀) by addition of a hydroxyl group to the deoxyhypusine residue (9). eIF-5A, DHS and DOHH are all essential for proliferation of higher eukaryotic cells (10, 11), and eIF-5A is strictly conserved throughout eukaryotic evolution (12).Open in a separate windowFig. 1.The hypusine modification and TAP fusion proteins employed in this study. A, The hypusine modification pathway and major proposed eIF-5A functions. B, Structure of the plasmid inserts coding for SG-tagged bait proteins. The amino acid positions of eIF-5A mutants are indicated in italic. SBP, streptavidin binding peptide. C, Metabolic incorporation of ³H-labeled spermidine into eIF-5A. Arrowheads indicate bands of SG-tagged and endogenous eIF-5A proteins. D, Anti-Myc-tag Western blot of cell lysates from retrovirally transduced Ba/F3 p210 cell lines for the quantification of constitutively expressed SG-tagged bait proteins. E, Representative TAP outputs for MS/MS analysis, after 1D PAGE separation and Coomassie staining. Separation distance varies from ∼2 to 4 cm.The eIF-5A protein has been proposed to promote various different cellular processes that potentially regulate proliferation, including translation initiation (13) and elongation (14) as well as nucleocytoplasmic transport of RNA or other cargoes (15, 16). Using inhibitors of DHS and DOHH or eIF-5A mutants deficient for hypusine modification, it has also been shown that this modification is a prerequisite of at least a subset of known eIF-5A functions (10, 11, 17, 18). The eIF-5A protein has also been implicated in numerous pathologic conditions including various types of cancer (19–23), β-cell inflammation (and therefore diabetes) (24) and HIV-1 infection (25). Human and rodent cells carry two highly homologous eIF-5A genes coding for distinct isoforms. Although eIF-5A1 is expressed at high levels throughout all tissues, eIF-5A2 is detectable only in a few embryonic tissues as well as adult testis, central nervous system (26), and cancer tissue (21, 22, 27–29).Although there have been ample reports suggesting eIF-5A is involved in translational control, the molecular mechanisms through which it ultimately influences cellular physiology and leads to disease remain unclear. Moreover, it remains equally possible that at least some of eIF-5A''s effects on cellular functions might not involve direct effects on translation. Also, there is no information available on whether the two isoforms of mammalian eIF-5A are functionally congruent.To address these fundamental questions systematically and comprehensively, we employed a bioreactor-based tandem affinity purification (TAP) approach followed by MS identification of purified protein complexes (“bioreactor-TAP-MS/MS”). To obtain a complete interaction map of the proteins involved in hypusine modification, we used this approach to identify interaction partners of both isoforms of eIF-5A, as well as the hypusine modification enzymes DHS and DOHH. In total, we identified 248 proteins that either directly interact with these bait proteins or are components of higher complexes containing the aforementioned proteins. Furthermore, we validated a subset of putative interaction partners of both eIF-5A isoforms, using Western blots of reciprocal TAP experiments, as well as a live-cell protein-fragment complementation assay (PCA). Our analysis provides a molecular framework for a detailed understanding on how this signal transduction pathway affects different crucial cellular functions.     

Identification of a 4-microRNA signature for clear cell renal cell carcinoma metastasis and prognosis

Renal cell carcinoma (RCC) metastasis portends a poor prognosis and cannot be reliably predicted. Early determination of the metastatic potential of RCC may help guide proper treatment. We analyzed microRNA (miRNA) expression in clear cell RCC (ccRCC) for the purpose of developing a miRNA expression signature to determine the risk of metastasis and prognosis. We used the microarray technology to profile miRNA expression of 78 benign kidney and ccRCC samples. Using 28 localized and metastatic ccRCC specimens as the training cohort and the univariate logistic regression and risk score methods, we developed a miRNA signature model in which the expression levels of miR-10b, miR-139-5p, miR-130b and miR-199b-5p were used to determine the status of ccRCC metastasis. We validated the signature in an independent 40-sample testing cohort of different stages of primary ccRCCs using the microarray data. Within the testing cohort patients who had at least 5 years follow-up if no metastasis developed, the signature showed a high sensitivity and specificity. The risk status was proven to be associated with the cancer-specific survival. Using the most stably expressed miRNA among benign and tumorous kidney tissue as the internal reference for normalization, we successfully converted his signature to be a quantitative PCR (qPCR)-based assay, which showed the same high sensitivity and specificity. The 4-miRNA is associated with ccRCC metastasis and prognosis. The signature is ready for and will benefit from further large clinical cohort validation and has the potential for clinical application.     

Adaptation to aridity in the malaria mosquito Anopheles gambiae: chromosomal inversion polymorphism and body size influence resistance to desiccation

Chromosomal inversions are thought to confer a selective advantage in alternative habitats by protecting co-adapted alleles from recombination. The frequencies of two inversions (2La and 2Rb) of the afro-tropical malaria mosquito Anopheles gambiae change gradually along geographical clines, increasing in frequency with degree of aridity. Such clines can result from gene flow and local selection acting upon alternative karyotypes along the cline, suggesting that these inversions may be associated with tolerance to xeric conditions. Since water loss represents a major challenge in xeric habitats, it can be supposed that genes inside these inversions are involved in water homeostasis. To test this hypothesis, we compared the desiccation resistance of alternative karyotypes from a colonised 2Rb/2La polymorphic population of A. gambiae from Cameroon. The strain included only the molecular form S, one of the genetic units marking incipient speciation in this taxon. Day-old mosquitoes of both sexes were assayed individually for time to death in a dry environment and the karyotype of each was determined post-mortem using molecular diagnostic assays for each inversion. In agreement with expectations based on their eco-geographical distribution, we found that 2La homokaryotypes survived significantly longer (1.3 hours) than the other karyotypes. However, there was weak support for the effect of 2Rb on desiccation resistance. Larger mosquitoes survived longer than smaller ones. Median survival of females was greater than males, but the effect of sex on desiccation resistance was weakly supported, indicating that differential survival was correlated to differences between sexes in average size. We found weak evidence for a heterotic effect of 2La karyotype on size in females. These results support the notion that genes located inside the 2La inversion are involved in water balance, contributing towards local adaptation of A. gambiae to xeric habitats, beyond the adaptive value conferred by a larger body size.     

COMP-angiopoietin-1 recovers molecular biomarkers of neuropathy and improves vascularisation in sciatic nerve of ob/ob mice

Background

Leptin-deficient ob/ob mice are a model of type 2 diabetes induced peripheral neuropathy. Ob/ob mice exhibit obesity, insulin resistance, hyperglycaemia, and alterations of peripheral nerve fibres and endoneural microvessels. Here we test the hypothesis that cartilage oligomeric matrix protein (COMP)-Ang-1, a soluble and stabile form of Ang-1 which promotes angiogenesis and nerve growth, improves regeneration of nerve fibres and endoneural microvessels in ob/ob mice.

Methods and Findings

COMP-Ang-1 (100 ng/ml) or NaCl were intraperitoneally (i.p.) injected into male (N = 184), 3-month old, ob/ob or ob/+ mice for 7 and 21 days. We measured expression of Nf68, GAP43, Cx32, Cx26, Cx43, and TNFα in sciatic nerves using Western blot analysis. To investigate the inflammation in sciatic nerves, numbers of macrophages and T-cells were counted after immunofluorescence staining. In ultrathin section, number of myelinated/non-mylinated nerve fibers, g-ratio, the thickness of Schwann cell basal lamina and microvessel endothelium were investigated.Endoneural microvessels were reconstructed with intracardial FITC injection. Treatment with COMP-Ang-1 over 21 days significantly reduced fasting blood glucose and plasma cholesterol concentrations compared to saline treated ob/ob mice. In addition, COMP-Ang-1 treatment: 1) up-regulated expression of Nf68 and GAP43; 2) improved expression of gap junction proteins including connexin 32 and 26; 3) suppressed the expression of TNFα and Cx43 and 4) led to decreased macrophage and T-cell infiltration in sciatic nerve of ob/ob mice. The significant changes of sciatic nerve ultrastructure were not observed after 21-day long COMP-Ang-1 treatment. COMP-Ang-1 treated ob/ob mice displayed regeneration of small-diameter endoneural microvessels. Effects of COMP-Ang-1 corresponded to increased phosphorylation of Akt and p38 MAPK upon Tie-2 receptor.

Conclusions

COMP-Ang-1 recovers molecular biomarkers of neuropathy, promotes angiogenesis and suppresses inflammation in sciatic nerves of ob/ob mice suggesting COMP-Ang-1 as novel treatment option to improve morphologic and protein expression changes associated with diabetic neuropathy.     

Fungemia in hospitals of the City of Buenos Aires, Argentina

BackgroundThe incidence of fungi like pathogens in hospitals varies by regions.ObjectivesOur goal was not only to record the incidence and etiology of fungaemia, but also the change during the 4 years analysed, to determine the time of detection in automated blood culture and by lysis-centrifugation, and finally to assess the gender, age and underlying disease of the patients with fungaemia.MethodsAn observational multicentre study of fungaemia was conducted in hospitals in the Mycology Network of Buenos Aires.ResultsA total of 190,920 blood cultures were processed: 182,050 automated blood culture and 8,870 lysis-centrifugation. Fungi were recovered in 1,020 episodes. The overall incidence of fungaemia was 1.72/1,000 admissions; 683 episodes were due to Candida (68%), and 325 (32%) to other fungi: 214 Cryptococcus, 105 Histoplasma, 7 Rhodotorula, 5 Trichosporon, 2 Pichia, 2 Acremonium, one Saccharomyces and one Fusarium. The incidence of candidaemia was 1.15/1,000 admissions with a wide variation between centres (0.35 to 2.65). Most Candida isolates (97%) were detected in the first 2 days of incubation. Candida albicans was recovered in 43% of the episodes. In fungaemia other than candidaemia, the predominant fungi were Cryptococcus and Histoplasma capsulatum.ConclusionsThe incidence remained stable during the study period. Fungaemia by Candida were predominant. C. albicans was involved in less than a half of the episodes. The recovery of Cryptoccocus and H. capsulatum is strongly associated with HIV patients.     

An N-Myristoylated Globin with a Redox-Sensing Function That Regulates the Defecation Cycle in Caenorhabditis elegans

Globins occur in all kingdoms of life where they fulfill a wide variety of functions. In the past they used to be primarily characterized as oxygen transport/storage proteins, but since the discovery of new members of the globin family like neuroglobin and cytoglobin, more diverse and complex functions have been assigned to this heterogeneous family. Here we propose a function for a membrane-bound globin of C. elegans, GLB-26. This globin was predicted to be myristoylated at its N-terminus, a post-translational modification only recently described in the globin family. In vivo, this globin is found in the membrane of the head mesodermal cell and in the tail stomato-intestinal and anal depressor muscle cells. Since GLB-26 is almost directly oxidized when exposed to oxygen, we postulate a possible function as electron transfer protein. Phenotypical studies show that GLB-26 takes part in regulating the length of the defecation cycle in C. elegans under oxidative stress conditions.