Dissecting the Active Site of the Collagenolytic Cathepsin L3 Protease of the Invasive Stage of Fasciola hepatica

Background: A family of secreted cathepsin L proteases with differential activities is essential for host colonization and survival in the parasitic flatworm Fasciola hepatica. While the blood feeding adult secretes predominantly FheCL1, an enzyme with a strong preference for Leu at the S2 pocket of the active site, the infective stage produces FheCL3, a unique enzyme with collagenolytic activity that favours Pro at P2. Methodology/Principal Findings: Using a novel unbiased multiplex substrate profiling and mass spectrometry methodology (MSP-MS), we compared the preferences of FheCL1 and FheCL3 along the complete active site cleft and confirm that while the S2 imposes the greatest influence on substrate selectivity, preferences can be indicated on other active site subsites. Notably, we discovered that the activity of FheCL1 and FheCL3 enzymes is very different, sharing only 50% of the cleavage sites, supporting the idea of functional specialization. We generated variants of FheCL1 and FheCL3 with S2 and S3 residues by mutagenesis and evaluated their substrate specificity using positional scanning synthetic combinatorial libraries (PS-SCL). Besides the rare P2 Pro preference, FheCL3 showed a distinctive specificity at the S3 pocket, accommodating preferentially the small Gly residue. Both P2 Pro and P3 Gly preferences were strongly reduced when Trp67 of FheCL3 was replaced by Leu, rendering the enzyme incapable of digesting collagen. In contrast, the inverse Leu67Trp substitution in FheCL1 only slightly reduced its Leu preference and improved Pro acceptance in P2, but greatly increased accommodation of Gly at S3. Conclusions/Significance: These data reveal the significance of S2 and S3 interactions in substrate binding emphasizing the role for residue 67 in modulating both sites, providing a plausible explanation for the FheCL3 collagenolytic activity essential to host invasion. The unique specificity of FheCL3 could be exploited in the design of specific inhibitors selectively directed to specific infective stage parasite proteinases. © 2013 Corvo et al

Development of an international standard set of outcome measures for patients with atrial fibrillation: a report of the International Consortium for Health Outcomes Measurement (ICHOM) atrial fibrillation working group.

AIMS: As health systems around the world increasingly look to measure and improve the value of care that they provide to patients, being able to measure the outcomes that matter most to patients is vital. To support the shift towards value-based health care in atrial fibrillation (AF), the International Consortium for Health Outcomes Measurement (ICHOM) assembled an international Working Group (WG) of 30 volunteers, including health professionals and patient representatives to develop a standardized minimum set of outcomes for benchmarking care delivery in clinical settings. METHODS AND RESULTS: Using an online-modified Delphi process, outcomes important to patients and health professionals were selected and categorized into (i) long-term consequences of disease outcomes, (ii) complications of treatment outcomes, and (iii) patient-reported outcomes. The WG identified demographic and clinical variables for use as case-mix risk adjusters. These included baseline demographics, comorbidities, cognitive function, date of diagnosis, disease duration, medications prescribed and AF procedures, as well as smoking, body mass index (BMI), alcohol intake, and physical activity. Where appropriate, and for ease of implementation, standardization of outcomes and case-mix variables was achieved using ICD codes. The standard set underwent an open review process in which over 80% of patients surveyed agreed with the outcomes captured by the standard set. CONCLUSION: Implementation of these consensus recommendations could help institutions to monitor, compare and improve the quality and delivery of chronic AF care. Their consistent definition and collection, using ICD codes where applicable, could also broaden the implementation of more patient-centric clinical outcomes research in AF

Study of 2 beta-decay of Mo-100 and Se-82 using the NEMO3 detector

After analysis of 5797 h of data from the detector NEMO3, new limits on neutrinoless double beta decay of Mo-100 (T-1/2 > 3.1 x 10(23) y, 90% CL) and Se-82 (T-1/2 > 1.4 x 10(23) y, 90% CL) have been obtained. The corresponding limits on the effective majorana neutrino mass are: 1.4 x 10(22) y (90% CL) for Mo-100 and T-1/2 > 1.2 x 10(22) y (90% CL) for Se-82. Corresponding bounds on the Majoron-neutrino coupling constant are < (0.5-0.9) x 10(- 4) and <(0.7-1.6) x 10(- 4). Two-neutrino 2beta-decay half-lives have been measured with a high accuracy, (T1/2Mo)-Mo-100 = [7.68 +/- 0.02(stat) +/- 0.54(syst)] x 10(18) y and (T1/2Se)-Se-82 = [10.3 +/- 0.3(stat) +/- 0.7(syst)] x 10(19) y. (C) 2004 MAIK "Nauka/Interperiodica"

Measurement of double beta decay of ¹⁰⁰Mo to excited states in the NEMO 3 experiment

The double beta decay of ¹⁰⁰Mo to the 0_{1}^{+} and 2_{1}^{+} excited states of ¹⁰⁰Ru is studied using the NEMO 3 data. After the analysis of 8024 h of data the half-life for the two-neutrino double beta decay of ¹⁰⁰Mo to the excited 0_{1}^{+} state is measured to be T_{1/2}^{2v} = [5.7_{-0.9}^{+1.3} (stat.) ± 0.8 (syst.)] x 10²⁰ y. The signal-to-background ratio is equal to 3. Information about energy and angular distributions of emitted electrons is also obtained. No evidence for neutrinoless double beta decay to the excited 0_{1}^{+} state has been found. The corresponding half-life limit is T_{1/2}^{0v} (0⁺→0_{1}^{+}) > 8.9 x 10²² y (at 90% C.L.). The search for the double beta decay to the 2_{1}^{+} excited state has allowed the determination of limits on the half-life for the two neutrino mode T_{1/2}^{0v} (0⁺→2_{1}^{+}) > 1.1 x 10²¹ y (at 90% C.L.) and for the neutrinoless mode T_{1/2}^{0v} (0⁺→2_{1}^{+}) > 1.6 x 10²³ y (at 90% C.L.)

Natural enemies from South Africa for biological control of Lagarosiphon major (Ridl.) Moss ex Wager (Hydrocharitaceae) in Europe

The non-native invasive plant, Lagarosiphon major (Hydrocharitaceae) is a submersed aquatic macrophyte that poses a significant threat to water bodies in Europe. Dense infestations prove difficult to manage using traditional methods. In order to initiate a biocontrol programme, a survey for natural enemies of Lagarosiphon was conducted in South Africa. Several phytophagous species were recorded for the first time, with at least three showing notable promise as candidate agents. Amongst these, a leaf-mining fly, Hydrellia sp. (Ephydridae) that occurred over a wide distribution causes significant leaf damage despite high levels of parasitism by braconid wasps. Another yet unidentified fly was recorded mining the stem of L. major. Two leaf-feeding and shoot boring weevils, cf. Bagous sp. (Curculionidae) were recorded damaging the shoot tips and stunting the growth of the stem. Several leaf-feeding lepidopteran species (Nymphulinae) were frequently recorded, but are expected to feed on a wide range of plant species and are not considered for importation before other candidates are assessed. The discovery of several natural enemies in the country of origin improves the biological control prospects of L. major in Europe

SmCL3, a Gastrodermal Cysteine Protease of the Human Blood Fluke Schistosoma mansoni

Parasitic infection caused by blood flukes of the genus Schistosoma is a major global health problem. More than 200 million people are infected. Identifying and characterizing the constituent enzymes of the parasite's biochemical pathways should reveal opportunities for developing new therapies (i.e., vaccines, drugs). Schistosomes feed on host blood, and a number of proteolytic enzymes (proteases) contribute to this process. We have identified and characterized a new protease, SmCL3 (for Schistosoma mansoni cathepsin L3), that is found within the gut tissue of the parasite. We have employed various biochemical and molecular biological methods and sequence similarity analyses to characterize SmCL3 and obtain insights into its possible functions in the parasite, as well as its evolutionary position among cathepsin L proteases in general. SmCL3 hydrolyzes major host blood proteins (serum albumin and hemoglobin) and is expressed in parasite life stages infecting the mammalian host. Enzyme substrate specificity detected by positional scanning-synthetic combinatorial library was confirmed by molecular modeling. A sequence analysis placed SmCL3 to the cluster of other cathepsins L in accordance with previous phylogenetic analyses

Using structural motif descriptors for sequence-based binding site prediction

All authors are with the Biotechnological Center, TU Dresden, Tatzberg 47-51, 01307 Dresden, Germany and -- Wan Kyu Kim is with the Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USABackground: Many protein sequences are still poorly annotated. Functional characterization of a protein is often improved by the identification of its interaction partners. Here, we aim to predict protein-protein interactions (PPI) and protein-ligand interactions (PLI) on sequence level using 3D information. To this end, we use machine learning to compile sequential segments that constitute structural features of an interaction site into one profile Hidden Markov Model descriptor. The resulting collection of descriptors can be used to screen sequence databases in order to predict functional sites. -- Results: We generate descriptors for 740 classified types of protein-protein binding sites and for more than 3,000 protein-ligand binding sites. Cross validation reveals that two thirds of the PPI descriptors are sufficiently conserved and significant enough to be used for binding site recognition. We further validate 230 PPIs that were extracted from the literature, where we additionally identify the interface residues. Finally we test ligand-binding descriptors for the case of ATP. From sequences with Swiss-Prot annotation "ATP-binding", we achieve a recall of 25% with a precision of 89%, whereas Prosite's P-loop motif recognizes an equal amount of hits at the expense of a much higher number of false positives (precision: 57%). Our method yields 771 hits with a precision of 96% that were not previously picked up by any Prosite-pattern. -- Conclusion: The automatically generated descriptors are a useful complement to known Prosite/InterPro motifs. They serve to predict protein-protein as well as protein-ligand interactions along with their binding site residues for proteins where merely sequence information is available.Institute for Cellular and Molecular [email protected]

Identification of Attractive Drug Targets in Neglected-Disease Pathogens Using an In Silico Approach

In cell-based drug development, researchers attempt to create drugs that kill a pathogen without necessarily understanding the details of how the drugs work. In contrast, target-based drug development entails the search for compounds that act on a specific intracellular target—often a protein known or suspected to be required for survival of the pathogen. The latter approach to drug development has been facilitated greatly by the sequencing of many pathogen genomes and the incorporation of genome data into user-friendly databases. The present paper shows how the database TDRtargets.org can identify proteins that might be considered good drug targets for diseases such as African sleeping sickness, Chagas disease, parasitic worm infections, tuberculosis, and malaria. These proteins may score highly in searches of the database because they are dissimilar to human proteins, are structurally similar to other “druggable” proteins, have functions that are easy to measure, and/or fulfill other criteria. Researchers can use the lists of high-scoring proteins as a basis for deciding which potential drug targets to pursue experimentally

Evolutionary History of the Vertebrate Mitogen Activated Protein Kinases Family

Background: The mitogen activated protein kinases (MAPK) family pathway is implicated in diverse cellular processes and pathways essential to most organisms. Its evolution is conserved throughout the eukaryotic kingdoms. However, the detailed evolutionary history of the vertebrate MAPK family is largely unclear. Methodology/Principal Findings: The MAPK family members were collected from literatures or by searching the genomes of several vertebrates and invertebrates with the known MAPK sequences as queries. We found that vertebrates had significantly more MAPK family members than invertebrates, and the vertebrate MAPK family originated from 3 progenitors, suggesting that a burst of gene duplication events had occurred after the divergence of vertebrates from invertebrates. Conservation of evolutionary synteny was observed in the vertebrate MAPK subfamilies 4, 6, 7, and 11 to 14. Based on synteny and phylogenetic relationships, MAPK12 appeared to have arisen from a tandem duplication of MAPK11 and the MAPK13-MAPK14 gene unit was from a segmental duplication of the MAPK11-MAPK12 gene unit. Adaptive evolution analyses reveal that purifying selection drove the evolution of MAPK family, implying strong functional constraints of MAPK genes. Intriguingly, however, intron losses were specifically observed in the MAPK4 and MAPK7 genes, but not in their flanking genes, during the evolution from teleosts to amphibians and mammals. The specific occurrence of intron losses in the MAPK4 and MAPK7 subfamilies might be associated with adaptive evolution of the vertebrates by enhancing the gen