Monoclonal antibody to dengue capsid protein: Its application in dengue studies

Dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) are considered the most important arthropod-borne viral diseases in terms of morbidity and mortality. The emergency and severity of dengue (Den) infections increase the necessity of an early, quick and effective dengue laboratory diagnostic. Viral isolation is considered a gold standard for diagnosis of dengue infection using monoclonal antibodies (mAbs) as a tool for determining serotype specificity. Alternatives have been used to improve sensitivity and time to dengue diagnosis. Based on the early expression of dengue C protein in the life cycle, we focused our study on the application of an anti-dengue 2 virus capsid protein mAb in dengue diagnosis. The kinetic expression of dengue-2 capsid in mosquito cells and its immuno-localization in experimentally infected suckling albin Swiss (OF-1) mice brain tissues was established. The results demonstrate the possible utility of this mAb in early dengue diagnosis versus traditional isolation. In addition, a preliminary study of an enzyme immunoassay method using 8H8 mAb for specific detection of dengue C protein antigen was performed, making possible recombinant C protein quantification. The results suggest that detection of dengue capsid protein could be useful in the diagnosis of early dengue infection.Key words: monoclonal antibodies, capsid protein, dengue virus, diagnosis, immunoassays     

KINALYZER, a computer program for reconstructing sibling groups

A software suite KINALYZER reconstructs full-sibling groups without parental information using data from codominant marker loci such as microsatellites. KINALYZER utilizes a new algorithm for sibling reconstruction in diploid organisms based on combinatorial optimization. KINALYZER makes use of a Minimum 2-Allele Set Cover approach based on Mendelian inheritance rules and finds the smallest number of sibling groups that contain all the individuals in the sample. Also available is a 'Greedy Consensus' approach that reconstructs sibgroups using subsets of loci and finds the consensus of the partial solutions. Unlike likelihood methods for sibling reconstruction, KINALYZER does not require information about population allele frequencies and it makes no assumptions regarding the mating system of the species. KINALYZER is freely available as a web-based service.     

Dynamics of deletion genotypes in an experimental insect virus population

Defective viruses, that are deficient in certain essential genes, are maintained in the population by trans-complementation, exploiting the gene products of complete genotypes in co-infected cells. This process becomes prevalent only when cells are frequently infected by several virus particles, and only then will the fitness of defective viruses be subjected to frequency-dependent selection. Deletion variants that are not infectious per os are present in a multicapsid nucleopolyhedrovirus (SfMNPV, Baculoviridae) that infects the fall army worm, Spodoptera frugiperda. These variants enhance the pathogenicity and, therefore, the likelihood of transmission of the virus when co-infecting cells with complete genotypes, resulting in occlusion bodies (OBs) that may contain both genotypes co-occluded. Mixtures of complete (B) and defective (C) variants in ratios of 90% B+10% C, 50% B+50% C and 10% B+90% C were used to inoculate by injection S. frugiperda larvae. Viral OBs extracted from diseased insects were subjected to four or five successive rounds of per os infection. Following successive passages, genotype frequencies in all three experimental populations converged to a single equilibrium frequency comprising approximately 20% of deletion genotype C and approximately 80% of complete genotype B. This mirrors the relative proportions of deletion (22%) and complete (78%) genotypes observed in the wild-type SfMNPV population. The pathogenicity of experimental populations at the final passage was not significantly different from that of the wild-type isolate. In contrast, OBs of all genotype mixtures were significantly more pathogenic than OBs of genotype B alone. A population genetics model, in which virus populations were assigned linear frequency-dependent transmissibility values, was in remarkably close agreement to empirical data. Clearly, non-infectious deletion variants can profoundly affect the likelihood of transmission and the genetic structure and stability of virus populations.     

2D Autocorrelation modeling of the negative inotropic activity of calcium entry blockers using Bayesian-regularized genetic neural networks

Negative inotropic potency of 60 benzothiazepine-like calcium entry blockers (CEBs), Diltiazem analogs, was successfully modeled using Bayesian-regularized genetic neural networks (BRGNNs) and 2D autocorrelation vectors. This approach yielded reliable and robust models whilst by means of a linear genetic algorithm (GA) search routine no multilinear regression model was found describing more than 50% of the training set. On the contrary, the optimum neural network predictor with five inputs described about 84% and 65% variances of 50 randomly selected training and test sets. Autocorrelation vectors in the nonlinear model contained information regarding 2D spatial distributions on the CEB structure of van der Waals volumes, electronegativities, and polarizabilities. However, a sensitivity analysis of the network inputs pointed out to the electronegativity and polarizability 2D topological distributions at substructural fragments of sizes 3 and 4 as the most relevant features governing the nonlinear modeling of the negative inotropic potency.     

Epithelial cadherin is present in bovine oviduct epithelial cells and gametes,and is involved in fertilization-related events

Fertilization is a calcium-dependent process that involves sequential cell–cell adhesion events of spermatozoa with oviduct epithelial cells (OECs) and with cumulus-oocyte complexes (COCs). Epithelial cadherin (E-cadherin) participates in calcium-dependent somatic cell adhesion; the adaptor protein β-catenin binds to the E-cadherin cytoplasmic domain and links the adhesion protein to the cytoskeleton. The study was conducted to immunodetect E-cadherin and β-catenin in bovine gametes and oviduct (tissue sections and OEC monolayers), and to assess E-cadherin participation in fertilization-related events. Epithelial cadherin was found in spermatozoa, oocytes, cumulus cells, and OEC. In acrosome-intact noncapacitated spermatozoa, E-cadherin was mainly localized in the apical ridge and acrosomal cap (E1-pattern; 84 ± 9%; mean ± standard deviation of the mean). After sperm treatment with heparin to promote capacitation, the percentage of cells with E1-pattern (56 ± 12%) significantly decreased; concomitantly, the percentage of spermatozoa depicting an E-cadherin staining pattern similar to E1-pattern but showing a signal loss in the acrosomal cap (E2-pattern: 40 ± 11%) increased. After l-α-lysophosphatidylcholine–induced acrosome reaction, E-cadherin signal was mainly localized in the inner acrosomal membrane (E3-pattern: 67 ± 22%). In IVM COC, E-cadherin was immunodetected in the plasma membrane of cumulus cells and oocytes, but was absent in the polar body. The 120 KDa mature protein form was found in protein extracts from spermatozoa, oocytes, cumulus cells, and OEC. β-Catenin distribution followed E-cadherin's in all cells evaluated. Epithelial cadherin participation in cell–cell interaction was evaluated using specific blocking monoclonal antibody DECMA-1. Sperm incubation with DECMA-1 impaired sperm–OEC binding (the number of sperm bound to OEC: DECMA-1 = 6.7 ± 6.1 vs. control = 29.6 ± 20.1; P < 0.001), fertilization with COC (% fertilized COC: DECMA-1 = 68.8 ± 10.4 vs. control = 90.7 ± 3.1; P < 0.05) or denuded oocytes (% fertilized oocytes: DECMA-1 = 57.0 ± 15.2 vs. control = 89.2 ± 9.8; P < 0.05) and binding to the oolemma (the number of sperm bound to oolemma: DECMA-1 = 2.2 ± 1.1 vs. control = 11.1 ± 4.8; P < 0.05). This study describes, for the first time, the presence of E-cadherin in bovine spermatozoa, COC, and OEC, and shows evidence of its participation in sperm interaction with the oviduct and the oocyte during fertilization.     

Solvent-dependent 2D-coordination polymers of Cu(I) containing a bridging triazolopyrimidine ligand

Two 2D-polymers of Cu(I), [Cu₂(μ-tp)(CH₃CN)(μ-I)(μ₃-I)]_n (1) and [Cu₂(μ-tp)(μ₃-I)₂]_n (2), both obtained from the reaction between copper iodide and 1,2,4-triazolo[1,5-a]pyrimidine (tp) in the presence of KI are described. While in compound 1, tp acts as bridging ligand via N1 and N3 between chair-like [Cu₄I₄] units, in compound 2, tp moieties are placed at both sides of a [Cu_nI_n] kinked layer, displaying also a bridging mode but, in this case, through its atoms N3 and N4. These structural differences between both compounds seem to be due to the solvent of crystallization, being acetonitrile for 1 and water for 2. These polymers are the first examples of Cu(I) compounds based on the nucleobase-analog ligand tp.     

The strawberry plant defense mechanism: a molecular review

Strawberry, a small fruit crop of great importance throughout the world, has been considered a model plant system for Rosaceae, and is susceptible to a large variety of phytopathogenic organisms. Most components and mechanisms of the strawberry defense network remain poorly understood. However, from current knowledge, it seems clear that the ability of a strawberry plant to respond efficiently to pathogens relies first on the physiological status of injured tissue (pre-formed mechanisms of defense) and secondly on the general ability to recognize and identify the invaders by surface plant receptors, followed by a broad range of induced mechanisms, which include cell wall reinforcement, production of reactive oxygen species, phytoalexin generation and pathogenesis-related protein accumulation. Dissection of these physiological responses at a molecular level will provide valuable information to improve future breeding strategies for new strawberry varieties and to engineer strawberry plants for durable and broad-spectrum disease resistance. In turn, this will lead to a reduction in use of chemicals and in environmental risks. Advances in the understanding of the molecular interplay between plant (mainly those considered model systems) and various classes of microbial pathogens have been made in the last two decades. However, major progress in the genetics and molecular biology of strawberry is still needed to uncover fully the way in which this elaborate plant innate immune system works. These fundamental insights will provide a conceptual framework for rational human intervention through new strawberry research approaches. In this review, we will provide a comprehensive overview and discuss recent advances in molecular research on strawberry defense mechanisms against pathogens.     

Taxonomy of Pluteus eugraptus and morphologically similar taxa

The status of the taxa morphologically similar to Pluteus eugraptus (Basidiomycota, Agaricales) was investigated with morphological and molecular (ITS region) characters. This group of species belongs in Pluteus sect. Celluloderma based on morphological and molecular characters. Two species, Pluteus multiformis, from Spain and Pluteus eludens from Madeira, Portugal, Russia and USA, are described as new. Both species share pigmented cheilocystidia and a pileipellis composed of both clavate-spheropedunculate and elongated elements with P. eugraptus, but they can be separated based on the characteristics of the cystidia and pileipellis. Pluteus multiformis is characterized by the scarce pleurocystidia, clavate cheilocystidia and caulocystidia and highly polymorphic elements of the pileipellis. Pluteus eludens is characterized mainly by utriform pleurocystidia. Pluteus eugraptus is known only with certainty from the type collection (Sri Lanka), which has been re-examined here, and it is characterized by narrowly lageniform pleurocystidia. Phylogenetic analyses based on ITS region sequence data supported the separation of P. multiformis, P. eludens and an additional collection from Japan that likely represents the true P. eugraptus.     

In Candida albicans hyphae,Sec2p is physically associated with SEC2 mRNA on secretory vesicles

Candida albicans hyphae grow in a highly polarized fashion from their tips. This polarized growth requires the continuous delivery of secretory vesicles to the tip region. Vesicle delivery depends on Sec2p, the Guanine Exchange Factor (GEF) for the Rab GTPase Sec4p. GTP bound Sec4p is required for the transit of secretory vesicles from the trans‐Golgi to sites of polarized growth. We previously showed that phosphorylation of Sec2p at residue S584 was necessary for Sec2p to support hyphal, but not yeast growth. Here we show that on secretory vesicles SEC2 mRNA is physically associated with Sec2p. Moreover, we show that the phosphorylation of S584 allows SEC2 mRNA to dissociate from Sec2p and we speculate that this is necessary for Sec2p function and/or translation. During hyphal extension, the growing tip may be separated from the nucleus by up to 15 μm. Transport of SEC2 mRNA on secretory vesicles to the tip localizes SEC2 translation to tip allowing a sufficient accumulation of this key protein at the site of polarized growth.