EC Number Research Articles

ABO-incompatible kidney transplant recipients have a higher bleeding risk after antigen-specific immunoadsorption

Pretransplant removal of antiblood group ABO antibodies is the cornerstone of all current ABO-incompatible (ABOi) transplantation programmes. In our protocol, plasmapheresis (PP) is performed with a plasmafilter followed by immunoadsorption (IA) of anti-ABO antibodies. The bleeding complications of this technique are not known. We analysed the data of all 65 consecutive ABOi kidney transplantations between March 2006 and October 2013 and compared these with matched 130 ABO-compatible (ABOc) kidney transplantations. Cases differed from controls in the pre-operative regimen, which included IA-PP and rituximab, tacrolimus, mycophenolate mofetil, prednisone and immunoglobulines. Data on platelet count, blood loss and red blood cell (EC) transfusions during 48h postoperatively were collected. ABOi patients received EC transfusions more frequently than controls (29% vs. 12%, P=0.005). Intra-operative blood loss was higher (544 vs. 355ml, P<0.005) and they experienced more major bleeding (≥3 EC within 24h, 15% vs. 2%, P<0.0005). Platelet count decreased by 28% after the pre-operative IA. In a multivariate model, only the number of pre-operative IAs was associated with the number of ECs given (OR per IA 1.9, P<0.05). ABOi kidney transplant recipients have a high postoperative bleeding risk, correlating with the number of pre-operative IA sessions performed.

Proteomics-based metabolic modeling and characterization of the cellulolytic bacterium Thermobifida fusca.

BackgroundThermobifida fusca is a cellulolytic bacterium with potential to be used as a platform organism for sustainable industrial production of biofuels, pharmaceutical ingredients and other bioprocesses due to its capability of potential to convert plant biomass to value-added chemicals. To best develop T. fusca as a bioprocess organism, it is important to understand its native cellular processes. In the current study, we characterize the metabolic network of T. fusca through reconstruction of a genome-scale metabolic model and proteomics data. The overall goal of this study was to use multiple metabolic models generated by different methods and comparison to experimental data to gain a high-confidence understanding of the T. fusca metabolic network.ResultsWe report the generation of three versions of a metabolic model of Thermobifida fusca sp. XY developed using three different approaches (automated, semi-automated, and proteomics-derived). The model closest to in vivo growth was the proteomics-derived model that consists of 975 reactions involving 1382 metabolites and account for 316 EC numbers (296 genes). The model was optimized for biomass production with the optimal flux of 0.48 doublings per hour when grown on cellobiose with a substrate uptake rate of 0.25 mmole/h. In vivo activity of the DXP pathway for terpenoid biosynthesis was also confirmed using real-time PCR.ConclusionsiTfu296 provides a platform to understand and explore the metabolic capabilities of the actinomycete T. fusca for the potential use in bioprocess industries for the production of biofuel and pharmaceutical ingredients. By comparing different model reconstruction methods, the use of high-throughput proteomics data as a starting point proved to be the most accurate to in vivo growth.

Computational Approaches for Enzyme Functional Class Prediction: A Review

Numerous genome sequence projects of various organisms have resulted in generation of large amount of data on genes and proteins sequence information. Functional annotation of these proteins is important to bridge gap between sequence information and functional characterization. As experimental approaches for characterizing the functional class of an enzyme are expensive and time consuming, computational prediction methods are an effective alternative. Various approaches like homology-based function transfer and machine learning methods have been utilized for in silico enzyme functional classifications in terms of Enzyme Commission number (EC number) for a protein. Different types of features have been used in various machine learning techniques and each has its own advantages and limitations. The critical evaluation of performance measure in terms of predictive ability of these methods is necessary. Here, a systematic review on the various approaches used by different research groups, their utility and inference is presented.

Placenta Growth Factor and Vascular Endothelial Growth Factor A Have Differential, Cell‐Type Specific Patterns of Expression in Vascular Cells

PLGF, a VEGF-A related protein, mediates collateral enlargement via monocytes but plays little role in capillary proliferation. In contrast, VEGF-A mediates both collateral enlargement and capillary proliferation. PLGF has been less thoroughly studied than VEGF-A, and questions remain regarding its regulation and function. Therefore, our goal was to characterize the expression of PLGF by vascular cells. We hypothesized that vascular SMC would express more PLGF than EC, since VEGF-A is primarily expressed by non-EC. We compared PLGF and VEGF-A across eight EC and SMC lines, then knocked down PLGF and evaluated cell function. We also assessed the effect of hypoxia on PLGF expression and promoter activity. PLGF was most highly expressed in EC, whereas VEGF-A was most highly expressed in SMC. PLGF knockdown did not affect EC number, migration, or tube formation, but reduced monocyte migration toward EC. Monocyte migration was rescued by exogenous PLGF. Hypoxia increased PLGF protein without activating PLGF gene transcription. PLGF and VEGF-A have distinct patterns of expression in vascular cells. EC derived PLGF may function primarily in communication between EC and circulating cells. Hypoxia increases EC PLGF expression posttranscriptionally.

Engineered cytidine triphosphate synthetase with reduced product inhibition.

Cytidine triphosphate (CTP) synthetase (CTPS) (EC number 6.3.4.2) is a key enzyme involved in de novo synthesis of CTP. It catalyzes the rate-limiting step of the process due to the product inhibition effects on the enzyme. In this study, a novel CTPS from Corynebacterium glutamicum ATCC 13032 (CgCTPS) was cloned, expressed and characterized. A series of mutagenesis in its N-terminal ammonia ligase (ALase) domain was performed in order to reduce CTP product inhibition. All single mutation variants (D160E, E162A, E168K) lowered product inhibition by lowering the enzyme's binding affinity for CTP. The homology model of CgCTPS showed that D160E mutant caused steric hindrance for the pyrimidine ring of CTP stacking, E162A disrupted the hydrogen bond between CTP ribose and side chain and D168K caused minor localized structure perturbations of CTP binding pocket. The triple mutant of CTPS (D160E-E162A-E168K) with halved Km, doubled Vmax and the 23.5-fold increased IC50 for CTP shows a potential for use in industrial-scale CTP production by its better performance in enzyme kinetics and product inhibition.

On the relation between explosive cyclones affecting Europe and the North Atlantic Oscillation

Intense winter cyclones often lead to hazardous weather over Europe. Previous studies have pointed to a link between the North Atlantic Oscillation (NAO) and strong European windstorms. However, the robustness of this relation for cyclones of varying intensities remains largely unexplored. In this paper, the bi-directional relation between the NAO and cyclones impacting Europe is analyzed for the period 1950–2010 focusing on the sensitivity to storm intensity. Evidence is given that explosive (EC) and non-explosive cyclones (NoEC) predominantly develop under different large-scale circulation conditions over the North Atlantic. Whereas NoEC evolve more frequently under negative and neutral NAO phases, the number of EC is larger under a positive NAO phase, typically characterized by an intensified jet toward Western Europe. Important differences are also found on the dynamics of NAO evolution after peak intensity for both cyclone populations.

Space-based observations of fire NO&amp;lt;sub&amp;gt;x&amp;lt;/sub&amp;gt; emission coefficients: a global biome-scale comparison

Abstract. Biomass burning represents both a significant and highly variable source of NOx to the atmosphere. This variability stems from both the episodic nature of fires, and from fire conditions such as the modified combustion efficiency of the fire, the nitrogen content of the fuel and possibly other factors that have not been identified or evaluated by comparison with observations. Satellite instruments offer an opportunity to observe emissions from wildfires, providing a large suite of measurements which allow us to study mean behavior and variability on the regional scale in a statistically rigorous manner. Here we use space-based measurements of fire radiative power from the Moderate Resolution Imaging Spectroradiometer in combination with NO2 tropospheric column densities from the Ozone Monitoring Instrument to measure mean emission coefficients (ECs in g NO MJ−1) from fires for global biomes, and across a wide range of smaller-scale ecoregions, defined as spatially-distinct clusters of fires with similar fuel type. Mean ECs for all biomes fall between 0.250–0.362 g NO MJ−1, a range that is smaller than found in previous studies of biome-scale emission factors. The majority of ecoregion ECs fall within or near this range, implying that under most conditions, mean fire emissions of NOx per unit energy are similar between different regions regardless of fuel type or spatial variability. In contrast to these similarities, we find that about 24% of individual ecoregion ECs deviate significantly (with 95% confidence) from the mean EC for the associated biome, and a similar number of ecoregion ECs falls outside the range of all mean biome ECs, implying that there are some regions where fuel type-specific global emission parameterizations fail to capture local fire NOx emissions.

Analysis of the Skin Transcriptome in Two Oujiang Color Varieties of Common Carp

BackgroundBody color and coloration patterns are important phenotypic traits to maintain survival and reproduction activities. The Oujiang color varieties of common carp (Cyprinus carpio var. color), with a narrow distribution in Zhejiang Province of China and a history of aquaculture for over 1,200 years, consistently exhibit a variety of body color patterns. The molecular mechanism underlying diverse color patterns in these variants is unknown. To the practical end, it is essential to develop molecular markers that can distinguish different phenotypes and assist selective breeding.Methodology/Principal FindingsIn this exploratory study, we conducted Roche 454 transcriptome sequencing of two pooled skin tissue samples of Oujiang common carp, which correspond to distinct color patterns, red with big black spots (RB) and whole white (WW), and a total of 737,525 sequence reads were generated. The reads obtained in this study were co-assembled jointly with common carp Roche 454 sequencing reads downloaded from NCBI SRA database, resulting in 43,923 isotigs and 546,676 singletons. Over 31 thousand (31,445; 71.6%) isotigs were found with significant BLAST matches (E<1e-10) to the nr protein database, which corresponds to 12,597 annotated zebrafish genes. A total of 70,947 isotigs and singletons (transcripts) were annotated with Gene Ontology, and 60,221 transcripts were found with corresponding EC numbers. Out of 145 zebrafish pigmentation genes, orthologs for 117 were recovered in Oujiang color carp transcriptome, including 18 found only among singletons. Our transcriptome analysis revealed over 52,902 SNPs in Oujiang common carp, and identified 63 SNP markers that are putatively unique either for RB or WW.ConclusionsThe transcriptome of Oujiang color varieties of common carp obtained through this study, along with the pigmentation genes recovered and the color pattern-specific molecular markers developed, will facilitate future research on the molecular mechanism of color patterns and promote aquaculture of Oujiang color varieties of common carp through molecular marker assisted-selective breeding.

ENZYMAP: Exploiting Protein Annotation for Modeling and Predicting EC Number Changes in UniProt/Swiss-Prot

The volume and diversity of biological data are increasing at very high rates. Vast amounts of protein sequences and structures, protein and genetic interactions and phenotype studies have been produced. The majority of data generated by high-throughput devices is automatically annotated because manually annotating them is not possible. Thus, efficient and precise automatic annotation methods are required to ensure the quality and reliability of both the biological data and associated annotations. We proposed ENZYMatic Annotation Predictor (ENZYMAP), a technique to characterize and predict EC number changes based on annotations from UniProt/Swiss-Prot using a supervised learning approach. We evaluated ENZYMAP experimentally, using test data sets from both UniProt/Swiss-Prot and UniProt/TrEMBL, and showed that predicting EC changes using selected types of annotation is possible. Finally, we compared ENZYMAP and DETECT with respect to their predictions and checked both against the UniProt/Swiss-Prot annotations. ENZYMAP was shown to be more accurate than DETECT, coming closer to the actual changes in UniProt/Swiss-Prot. Our proposal is intended to be an automatic complementary method (that can be used together with other techniques like the ones based on protein sequence and structure) that helps to improve the quality and reliability of enzyme annotations over time, suggesting possible corrections, anticipating annotation changes and propagating the implicit knowledge for the whole dataset.

Characterization of hydantoin racemase predicted from the genome sequence ofLactobacillus pentosusKCA1

Background: Hydantoin racemase from Lactobacillus species of human origin has not been reported and characterized. The genome of Lactobacillus pentosus KCA1 has been sequenced and found to possess gene cassettes and open reading frames encoding the hydantoinase machinery, including a putative hydantoin racemase. Aims: To use bioinformatic tools to characterize the new hydantoin racemase predicted in the genome sequence of L. pentosus KCA1. Materials and Methods: Bioinformatic tools such as ClustalW algorithm was used to align hydantoin racemase from L. pentosus KCA1 with other hydantoin racemases extracted from the uniprot΢ database. I-TASSER was used for the prediction of secondary structure, 3-D model, similarity structure in PDB, and functional active binding site residues. Results: L. pentosus KCA1 hydantoin racemase showed significant amino acid sequence identity with hydantoin racemase from the selected bacterial organisms in the protein databank (PDB). The predicted secondary structure revealed 9 alpha-helices and 8 beta-strands. Functional prediction using enzyme partners predicted EC number 5.1.1.13 as the corresponding enzyme homolog (3eq5A) showing Cys83 and Cys187 as the potential active residues in KCA1 hydantoin racemase. The 3-D structure of KCA1 hydantoin racemase has a confidence score (C-score) of 1.2 that reflects a model of better quality, based on 3qvjA from PDB. Conclusion: The in silico data presented provides new insights into the potential activity and substrate specificity of hydantoin racemase from L. pentosus KCA1 and has proposed a mechanism for racemization of hydantoin derivatives that is consistent with the two-base process observed in other members of the Aspartate/Glutamate superfamily.

Re-annotation of the genome sequence of Helicobacter pylori 26695

Summary Helicobacter pylori is a pathogenic bacterium that colonizes the human epithelia, causing duodenal and gastric ulcers, and gastric cancer. The genome of H. pylori 26695 has been previously sequenced and annotated. In addition, two genome-scale metabolic models have been developed. In order to maintain accurate and relevant information on coding sequences (CDS) and to retrieve new information, the assignment of new functions to Helicobacter pylori 26695s genes was performed in this work. The use of software tools, on-line databases and an annotation pipeline for inspecting each gene allowed the attribution of validated EC numbers and TC numbers to metabolic genes encoding enzymes and transport proteins, respectively. 1212 genes encoding proteins were identified in this annotation, being 712 metabolic genes and 500 non-metabolic, while 191 new functions were assignment to the CDS of this bacterium. This information provides relevant biological information for the scientific community dealing with this organism and can be used as the basis for a new metabolic model reconstruction.

Re-annotation of the genome sequence of Helicobacter pylori 26695.

Helicobacter pylori is a pathogenic bacterium that colonizes the human epithelia, causing duodenal and gastric ulcers, and gastric cancer. The genome of H. pylori 26695 has been previously sequenced and annotated. In addition, two genome-scale metabolic models have been developed. In order to maintain accurate and relevant information on coding sequences (CDS) and to retrieve new information, the assignment of new functions to Helicobacter pylori 26695s genes was performed in this work. The use of software tools, on-line databases and an annotation pipeline for inspecting each gene allowed the attribution of validated EC numbers and TC numbers to metabolic genes encoding enzymes and transport proteins, respectively. 1212 genes encoding proteins were identified in this annotation, being 712 metabolic genes and 500 non-metabolic, while 191 new functions were assignment to the CDS of this bacterium. This information provides relevant biological information for the scientific community dealing with this organism and can be used as the basis for a new metabolic model reconstruction.

Anti-atherogenic effects of statins: Impact on angiopoietin-2 release from endothelial cells

Beyond lipid lowering, statins are supposed to exert pleiotropic effects positively influencing the progression of atherosclerotic lesions. The development of such lesions is associated with increased release of angiopoietin-2 (Ang-2), an endothelial cell-specific protein growth factor stored in Weibel-Palade bodies (WPBs). The aim of our study was to examine whether statin pretreatment influences the release of Ang-2 from endothelial cells. Stimulation of HUVECs and HMVECs with PMA, thrombin or histamine resulted in significant release of Ang-2, as evidenced by ELISA. Pretreatment with simvastatin and mevastatin suppressed this release to basal level, while pravastatin had no effect. Simvastatin itself increased nitric oxide (NO, EC number 1.14.13.39) synthesis, measured by Griess reaction. Combining the statin pretreatment with the eNOS inhibitor L-NNA as well as bypassing the HMG-CoA reductase (EC number: 1.1.1.34) by adding mevalonic acid or geranyl pyrophosphate restored the exocytotic effect of PMA. Immunofluorescence microscopy showed that depletion of WPBs upon PMA stimulation ceased after pretreatment with simvastatin. This study demonstrates a potent suppressive effect of statins on the release of Ang-2 from endothelial cells. Regarding its harmful effects in the development of atherosclerotic lesions, our data provide further insight into the mechanisms of the anti-atherogenic potential of statins.

Gonadotropin surge stimulate ovarian osteopontin expression via epidermal growth factor receptor signaling to enhance corpus luteum function

cific angiocrine factors, creating an instructive vascular niche which promotes organ development, repair and regeneration. Studying the role of primary ovarian ECs in the regulation of physiological processes in the ovary has been hampered by the need for growth factor deprivation during culture, which leads to apoptosis. Here, we aim to isolate, purify, characterize and culture ovarian ECs in serum-free conditions, creating a platform for the exploration of endothelial-follicular communication. DESIGN: Laboratory study. MATERIALS AND METHODS: Mouse ovarian ECs were isolated via magnetic cell sorting using CD-31 antibody. After a culture period of 5-7 days, EC activation was initiated via lentivirus transduction, allowing prolonged culture in serum-free conditions. Purity of cells was confirmed with flow cytometry using CD31, CD45 and VE-Cadherin antibodies (VECAD). Gene expression was assessed with qPCR. In order to obtain adequate numbers of fresh ECs for comparison, a minimal stimulation protocol was applied. Intravital staining using VECAD and Isolectin was performed, allowing ovarian EC isolation using florescence activated cell sorting (FACS). Gene expression of fresh ECs was assessed with qPCR and compared to cultured counterparts. RESULTS: Flow cytometry showed that 98% of cultured activated ovarian ECs were CD31+, VECAD+ and CD45-. Relative expression of dll1, mmp10 and fgf1 was higher in cultured ECs, while expression of Jagged-1, VECAD and PDGF was comparable in both groups. Expression of Jagged-2, Notch14, dll4, VEGFR1, VEGFR2, mmp4, il6, and il8 was lower in activated, cultured ECs in comparison to fresh ECs but fairly high compared to its own housekeeping gene. CONCLUSION: Here, we report successful isolation and durable culture of ovarian ECs with an angiocrine profile matching that of fresh ovarian ECs, thus providing a novel tool with which to study cell-cell communication and vascularization within the ovary. Supported by: Institutional.

Research of smooth muscle cells response to fluid flow shear stress by hyaluronic acid micro-pattern on a titanium surface

The morphology of vascular smooth muscle cells (SMCs) in the normal physiological state depends on cytoskeletal distribution and topology beneath, and presents vertical to the direction of blood flow shear stress (FFSS) although SMCs physiologically are not directly exposed to the shear conditions of blood flow. However, this condition is relevant for arteriosclerotic plaques and the sites of a vascular stent, and little of this condition in vitro has been studied and reported till now. It is unclear what will happen to SMC morphology, phenotype and function when the direction of the blood flow changed. In this paper, the distribution of SMCs in a specific area on Ti surface was regulated by micro-strips of hyaluronic acid (HA). Cell morphology depended on the distribution of the cytoskeleton extending along the micrographic direction. Simulated vascular FFSS was perpendicular or parallel to the direction of the cytoskeleton distribution. Based on investigating the morphology, apoptotic number, phenotypes and functional factors of SMCs, it was obtained that SMCs of vertical groups showed more apoptosis, expressed more contractile types and secreted less TGF-β1 factor compared with SMCs of parallel groups, the number of ECs cultured by medium from SMCs of parallel groups was larger than vertical groups. This study could help to understand the effect of direction change of FFSS on patterned SMC morphology, phenotype and function.

Consensus and conflict cards for metabolic pathway databases

BackgroundThe metabolic network of H. sapiens and many other organisms is described in multiple pathway databases. The level of agreement between these descriptions, however, has proven to be low. We can use these different descriptions to our advantage by identifying conflicting information and combining their knowledge into a single, more accurate, and more complete description. This task is, however, far from trivial.ResultsWe introduce the concept of Consensus and Conflict Cards (C2Cards) to provide concise overviews of what the databases do or do not agree on. Each card is centered at a single gene, EC number or reaction. These three complementary perspectives make it possible to distinguish disagreements on the underlying biology of a metabolic process from differences that can be explained by different decisions on how and in what detail to represent knowledge. As a proof-of-concept, we implemented C2CardsHuman, as a web application http://www.molgenis.org/c2cards, covering five human pathway databases.ConclusionsC2Cards can contribute to ongoing reconciliation efforts by simplifying the identification of consensus and conflicts between pathway databases and lowering the threshold for experts to contribute. Several case studies illustrate the potential of the C2Cards in identifying disagreements on the underlying biology of a metabolic process. The overviews may also point out controversial biological knowledge that should be subject of further research. Finally, the examples provided emphasize the importance of manual curation and the need for a broad community involvement.

Co-operation between Europe and some Southern and Eastern Mediterranean Countries on the use of ICT in Education: constrains and opportunities learnt from several European projects

Based on previous experience in Southern Europe and Middle-East, potentiality and constraints in collaborative projects are presented. More specifically, a number of EC funded ICT projects, undertaking by countries of these regions and implemented the last seven years, are presented in brief. By the introduction of quality issues and carefully examining a number of constraints and difficulties appeared during the development of these projects, we propose specific recommendations for successful co-operation, especially at a moment of deep and strong changes in these regions.

P53: Nitrite dismutase – A “nitrite-only” source for NO

Background Nitrophorins (NPs) are NO transporting proteins from the saliva of the blood-sucking insect Rhodnius prolixus . The delivery of NO is accomplished by storage on the protein’s ferriheme cofactor and the gas molecule is released when the protein is injected into a victim’s tissue. However, we demonstrated that nitrophorins can also produce NO from nitrite as the only substrate, i.e., in this reaction nitrite serves as both electron donor and electron acceptor. The enzymatic activity was termed nitrite dismutase activity and is now classified with the EC number 1.7.6.1. This study provides insight into the structural features required for the catalytic activity. Methods Proteins were recombinantly expressed in Escherichia coli cells. They were characterized by X-ray crystallography, EPR spectroscopy, UV/vis spectroscopy, resonance Raman spectroscopy, and NMR spectroscopy. The enzymatic reaction was studied by absorbance detected stopped-flow kinetics. Results The binding mode of nitrite to the NP Fe (III) is clearly different compared to that of the globins, i.e., N-bound. Contrary to the globins, the insertion of an H-donating residue (NP4(L130R)) into the protein pocket does not affect the nitrite binding mode; however, it cancels the enzymatic activity. On the other hand, mutation of the proximal Asp70 residue, which is H-bonded via a water to the iron coordinating His59, also affects the enzymatic activity, indicating that the charge distribution on the heme cofactor has a major influence on the iron reactivity. Conclusion The nitrophorins are good model systems for the characterization of the nitrite dismutase catalysis. The absence of H-donating residues and the charge density on the proximal His were identified as important structural features that are required for the nitrite dismutase reaction. Among the many heme proteins expressed in the human organism some might bear a similar catalytic activity. We suggest the group of heme peroxidases as potential candidates for enzymatic “nitrite-only” NO production. Disclosure Supported by the Deutsche Forschungsgemeinschaft (DFG), Grants KN 951-1/1 and 2.

P22: Sodium nitrite promotes the viability and proliferation of endothelial cells but inhibits the growth of smooth muscle cells under hypoxia

Percutaneous coronary intervention (PCI) is essential following myocardial infarction to restore cardiac perfusion. However, in the long term, the beneficial effects of PCI with stent insertion are limited by restenosis (associated with vascular injury and smooth muscle cell proliferation) and stent thrombosis (associated with inhibition of/delayed re-endothelialisation). Pre-clinical studies suggest that vascular injury and intimal hyperplasia are associated with lack of endogenous nitric oxide (NO) and that nitrite, via its conversion to NO decreases intimal hyperplasia following vascular injury (Alef et al., J. Clin. Invest. 121 (2011) 1646–1656). The aim of this study was to determine the effect of nitrite on the proliferation/viability of EC and VSMC in normoxia, hypoxia and ischaemia/reperfusion (I/R). Human umbilical vein (HUVEC) and coronary artery (HCAEC) endothelial cells and aortic VSMC (HAVSMC) were incubated for 24 h in the absence or presence of nitrite (0.1–10 μM) under normoxic (21% O 2 ) or hypoxic (1–5% O 2 ) conditions. In a separate series of experiments, ECs were subjected to 24 h of 0.1% O 2 followed by 48 h of normoxia to simulate I/R injury. Cell proliferation (cell count) and cell viability (flow cytometry with apoptosis assay-staining for annexin V/propidium iodide) were determined in control cells or cells treated with inhibitors of two potential nitrite reductases: xanthine oxidoreductase and endothelial nitric oxide synthase (eNOS) using allopurinol (100 μM) or l -NMMA (300 μM), respectively. Hypoxia attenuated proliferation of HUVECs with 5% O 2 causing a 21.2 ± 2.8% and 1% O 2 a 24.1 ± 3.1% reduction ( P < 0.001, n = 10) compared to normoxia. Similarly, with HCAECs 5% O 2 caused 30.1 ± 4.1% and 1% a 31.4 ± 3.6% reduction ( P < 0.001, n = 10). This effect on cell number was associated with an increase in the numbers of apoptotic cells by 7.4 ± 1.4% in 5% O 2 and 9.0 ± 1.8% at 1% in HUVECs and 9.5 ± 1.9% and 9.6 ± 1.3% in HCAECs ( P < 0.001, n = 10). Treatment of cells with nitrite caused a concentration-dependent increase in EC number and viability in hypoxia with a peak effect (near complete reversal) occurring at 3 μM in both HUVECs and HCAECs ( P < 0.001, n = 10). This effect of nitrite was attenuated by l -NMMA by ∼85% ( P < 0.001, n = 10) or allopurinol (∼80.0%, P < 0.001, n = 10). Reperfusion with nitrite (3 μM) following 0.1% O 2 for 24 h resulted in a significant concentration dependent improvement in cell growth compared to reperfusion alone at all timepoints ( P < 0.01). In contrast, nitrite had no effect on the proliferation or viability of either cell type under normoxic conditions ( n = 12). Hypoxia also caused a decrease in HAVSMC numbers although this was not associated with an increase in apoptosis ( n = 7). The addition of nitrite caused a concentration-dependent decrease in cell growth with a peak effect occurring at 1 μM of 29 ± 13% ( n = 7) vs normoxia. These studies demonstrate that nitrite protects ECs from the damaging effects of ischaemia or I/R injury but exerts an opposing repressive influence over smooth muscle cell growth. These results suggest that in an environment where improved EC growth but repressed SMC growth is desired, such as in restenosis following stent implantation, raising circulating nitrite levels may be of therapeutic utility. D.A.J. and this work are funded by an NIHR Doctoral Fellowship. Supported by NIHR Doctoral Research fellowship.

Discovery-2: an interactive resource for the rational selection and comparison of putative drug target proteins in malaria

BackgroundDrug resistance to anti-malarial compounds remains a serious problem, with resistance to newer pharmaceuticals developing at an alarming rate. The development of new anti-malarials remains a priority, and the rational selection of putative targets is a key element of this process. Discovery-2 is an update of the original Discovery in silico resource for the rational selection of putative drug target proteins, enabling researchers to obtain information for a protein which may be useful for the selection of putative drug targets, and to perform advanced filtering of proteins encoded by the malaria genome based on a series of molecular properties.MethodsAn updated in silico resource has been developed where researchers are able to mine information on malaria proteins and predicted ligands, as well as perform comparisons to the human and mosquito host characteristics. Protein properties used include: domains, motifs, EC numbers, GO terms, orthologs, protein-protein interactions, protein-ligand interactions. Newly added features include drugability measures from ChEMBL, automated literature relations and links to clinical trial information. Searching by chemical structure is also available.ResultsThe updated functionality of the Discovery-2 resource is presented, together with a detailed case study of the Plasmodium falciparum S-adenosyl-L-homocysteine hydrolase (PfSAHH) protein. A short example of a chemical search with pyrimethamine is also illustrated.ConclusionThe updated Discovery-2 resource allows researchers to obtain detailed properties of proteins from the malaria genome, which may be of interest in the target selection process, and to perform advanced filtering and selection of proteins based on a relevant range of molecular characteristics.