Metabolomics Study of Immune Responses of New Zealand Greenshell™ Mussels (<Emphasis Type="Italic">Perna canaliculus</Emphasis>) Infected with Pathogenic <Emphasis Type="Italic">Vibrio</Emphasis> sp.

Vibrio coralliilyticus is a bacterial pathogen which can affect a range of marine organisms, such as corals, fish and shellfish, with sometimes devastating consequences. However, little is known about the mechanisms involved in the host-pathogen interaction, especially within molluscan models. We applied gas chromatography-mass spectrometry (GC-MS)-based metabolomics to characterize the physiological responses in haemolymph of New Zealand Greenshell? mussels (Perna canaliculus) injected with Vibrio sp. DO1 (V. coralliilyticus/neptunius-like isolate). Univariate data analyses of metabolite profiles in Vibrio-exposed mussels revealed significant changes in 22 metabolites at 6 h post-infection, compared to non-exposed mussels. Among them, 10 metabolites were up-regulated, while 12 metabolites were down-regulated in infected mussels. Multivariate analyses showed a clear distinction between infected and non-infected mussels. In addition, secondary pathway analyses indicated perturbations of the host innate immune system following infection, including oxidative stress, inflammation and disruption of the TCA cycle, change in amino acid metabolism and protein synthesis. These findings provide new insights into the pathogenic mechanisms of Vibrio infection of mussels and demonstrate our ability to detect detailed and rapid host responses from haemolymph samples using a metabolomics approach.     

Phylogeny of Cerberus (Serpentes: Homalopsinae) and phylogeography of Cerberus rynchops: diversification of a coastal marine snake in Southeast Asia

Aim The biogeography of Southeast Asia has been greatly affected by plate tectonic events over the last 10 Myr and changing sea levels during the Quaternary. We investigated how these events may have influenced the evolution of Cerberus Cuvier, a marine coastal snake belonging to the Homalopsinae (Oriental‐Australian Rear‐fanged Water Snakes). This study is an expansion of a previous study on the biogeography and systematics of Cerberus. Location We obtained species from localities across the range of the widely distributed Cerberus: India, Sri Lanka, the Andaman islands, Myanmar, the Philippines, Borneo, Suluwesi, Sumatra, Vietnam, Thailand, Singapore and Australia. Methods We analysed mtDNA sequences (12S, ND3, ATPase, 2338 nucleotide characters) from 21 localities. The sample consisted of 65 Cerberus rynchops (Schneider), three Cerberus australis (Gray) and four Cerberus microlepis Boulenger. One Homalopsis buccata (Linnaeus), one Bitia hydroides Gray, one Enhydris enhydris (Schneider), and two Enhydris plumbea (Boie) were used as outgroups. Results We produced phylogenetic trees based on parsimony, maximum likelihood and Bayesian analysis. We did not find unambiguous support for the monophly of Cerberus. Cerberus austalis, H. buccata and all other Cerberus populations formed a three‐way basal polytomy under parsimony and C. australis formed the sister group to a clade consisting of H. buccata and all other Cerberus in likelihood and Bayesian analysis. The non‐Australian Cerberus were monophyletic and consisted of four primary biogeographical clades: Indian and Mayanmar, Philippines, Greater Sunda Islands and Suluwesi, and the Thai‐Malay peninsula and Gulf of Thailand. The range of genetic divergence between these clades and Australian Cerberus was 0.06–0.12. Genetic divergence among clades to the west of Australia was less pronounced (Thai‐Malay peninsula and Gulf of Thailand = 0.02–0.05; Sunda Islands and Suluwesi = 0.02–0.05; Philippines = 0.02–0.06; India and Myanmar = 0.04–0.06, Philippines = 0.02–0.5). Main conclusions Gyi [University of Kansas Publications, Museum of Natural History 20 (1970), 47] recognized three species of Cerberus: C. australis (from Australia), C. microlepis (known only from Lake Buhi in the Philippines), and the widely distributed C. rynchops (India to Wallacea). We did not find strong support for the monophyly of the genus. Cerberus australis is highly divergent from all other Cerberus lineages sampled from this region. The geographically widespread C. rynchops is resolved into four biogeographical clades (Indian and Myanmar, Philippines, Greater Sunda Islands and Suluwesi, and the Thai‐Malay Peninsula and Gulf of Thailand). We discuss how the dispersal biology of a salt‐water tolerant, coastal marine taxon and the complex geological history of the region (Tertiary plate tectonic movements and Quaternary sea‐level changes) could produce the observed patterns of diversification.     

Antimetastatic effects of liposome entrapped indomethacin

The "in vivo"administration of sized liposomes encapsulating indomethacin to mice bearing 3LL tumor, significantly reduced the incidence and/or number of superficial lung metastases. Also liposomes encapsulating indomethacin had significant inhibitory effects on the experimentally induced lung metastases. We conclude: i) indomethacin encapsulated in liposomes is more efficient than the free drug in mediating the antimetastatic effects and ii) liposomes are an valuable vehicle in evading the side metastatic effects of this drug during indomethacin treatment of tumor bearing mice.     

Effect of C-fiber-mediated, ozone-induced rapid shallow breathing on airway epithelial injury in rats.

We examined the relationship between C-fiber-mediated, ozone-induced rapid shallow breathing and airway epithelial cell injury at different airway sites within the lower respiratory tract of conscious Wistar rats (n = 24). We combined an acute 8-h ozone inhalation with vagal perineural capsaicin treatment, a selective C-fiber conduction block, and 5-bromo-2'-deoxyuridine (BrdU) labeling as an index of epithelial injury. Vehicle-treated rats that inhaled ozone developed a rapid shallow breathing pattern during ozone inhalation, whereas the capsaicin-treated rats that inhaled ozone showed no changes in respiratory frequency. In vehicle-treated, ozone-exposed rats that developed rapid shallow breathing, a progressive increase in BrdU-labeling density (no. of BrdU-labeled cells/mm(2) airway) was observed starting at the bifurcation of the left main stem bronchi (central airway) and going down either a short or long airway path. In vehicle-treated, ozone-exposed rats, terminal bronchioles supplied by short and long airway paths had a similar degree of BrdU-labeling density that was significantly (P < 0.05) greater than the BrdU-labeling density of the proximal airways that supply them. In contrast, the attenuation of rapid shallow breathing produced by capsaicin treatment resulted in a significantly reduced BrdU-labeling density in the terminal bronchioles supplied by short airway paths compared with the terminal bronchioles supplied by long airway paths. Our data indicate that ozone-induced rapid shallow breathing protects large conducting airways while producing a more even distribution of injury to terminal bronchioles.     

Benzimidazole derivatives. Part 1: Synthesis and structure-activity relationships of new benzimidazole-4-carboxamides and carboxylates as potent and selective 5-HT4 receptor antagonists

New benzimidazole-4-carboxamides 1-16 and -carboxylates 17-26 were synthesized and evaluated for binding affinity at serotonergic 5-HT4 and 5-HT3 receptors in the CNS. Most of the synthesized compounds exhibited moderate-to-very high affinity (in many cases subnanomolar) for the 5-HT4 binding site and no significant affinity for the 5-HT3 receptor. SAR observations and structural analyses (molecular modeling, INSIGHT II) indicated that the presence of a voluminous substituent in the basic nitrogen atom of the amino moiety and a distance of ca. 8.0 A from this nitrogen to the aromatic ring are of great importance for high affinity and selectivity for 5-HT4 receptors. These results confirm our recently proposed model for recognition by the 5-HT4 binding site. Amides 12-15 and esters 24 and 25 bound at central 5-HT4 sites with very high affinity (Ki = 0.11-2.9 nM) and excellent selectivity over serotonin 5-HT3, 5-HT2A, and 5-HT1A receptors (Ki > 1000-10,000 nM). Analogues 12 (Ki(5-HT4) = 0.32 nM), 13 (Ki(5-HT4) = 0.11 nM), 14 (Ki(5-HT4) = 0.29 nM) and 15 (Ki(5-HT4) = 0.54 nM) were pharmacologically characterized as selective 5-HT4 antagonists in the isolated guinea pig ileum (pA2 = 7.6, 7.9, 8.2 and 7.9, respectively), with a potency comparable to the 5-HT4 receptor antagonist RS 39604 (pA2 = 8.2). The benzimidazole-4-carboxylic acid derivatives described in this paper represent a novel class of potent and selective 5-HT4 receptor antagonists. In particular, compounds 12-15 could be interesting pharmacological tools for the understanding of the role of 5-HT4 receptors.     

Treatment of schizophrenic disorder,paranoid type,with intramuscular recombinant alpha-2b interferon

Twenty patients meeting DSM-III criteria for Schizophrenic Disorder, Paranoid Type were studied. After a 30 day drug-free period the patients were randomized in two groups. During a year, patients in Group 1 received intramuscular injections of a placebo while Group 2 received recombinant IFN alpha-2b. Both groups took anti-psychotic medication (APM) as needed on an individual basis, depending on their psychiatric symptomatology. Double blind evaluations, were performed at the beginning and at the end of the trial, using the Brief Psychiatric Rating Scale (BPRS) and the Reyes Scale for Social Evolution (RSSE). Information about relapses was gathered such as months without relapse, number and duration of the relapses and maximum dosages of APM given for relapse control. The statistical analysis of the results was performed with a matched pairs sign or Student's t tests for comparisons of each group before and after treatment. Groups were compared between them using the Fisher's exact test for frequencies and Student's t test for continuous variables. In Group 1: only one patient improved on the BPRS score; two had improved ratings on the RSSE; 2 patients got worse; and there were no changes in the rest of the group; three patients had no relapses and one increased in relapse frequency. These changes were not significant. The rate of relapses per year and their duration were not significantly modified in Group 1. The maximum dose of APM required for their relapse control was larger than before treatment although not significantly. All these patients required continuous APM. In Group 2 (IFN treated): 6 patients had improved BPRS scores (N=6, K=6, p<0.01) and 5 improved their RSSE scores (n=5, K=5, p<0.05). In 5 patients there were no relapses on their frequency decreased. There was significant reduction in the duration of the relapses (37.8±14.6 to 20.7±12.5 days; t=4.83; d.f.=9; p=0.0009) after treatment. Only 3 patients in Group 2 needed continuous APM after a relapse and the maximum dose required for control was significantly less (1281±527 to 687±552 chlorpromazine-equivalent mg. per day; t=5.56, p < 0.001). Comparisons between groups showed advantage for the IFN treated group in the BPRS change, proportion of patients needing continuous APM and integral evaluation. These results indicate that alpha IFN may be useful in the treatment of Schizophrenic Disorder, Paranoid Type.Abbreviations IFN Interferon - CSF cerebrospinal fluid - RSSE Reyes Scale for Social Evolution - APM antipsychotic medication - BBB blood-brain barrier - mRNA ribonucleic acid messenger - DSM-III Diagnostic and Statistical Manual of Mental Disorders - JCG Juan Cordero Gutierrez - OFL Odalys Fernandez Lopez - BPRS Brief Psychiatric Rating Scale - ECT Electroconvulsive treatment - IgG immunoglobulin G - EEG Electroencephalogram - SD standard deviation - CNS Central Nervous System     

Morphological convergence as a consequence of extreme functional demands: examples from the feeding system of natricine snakes

Despite repeated acquisitions of aquatic or semi-aquatic lifestyles revolving around piscivory, snakes have not evolved suction feeding. Instead, snakes use frontally or laterally directed strikes to capture prey under water. If the aquatic medium constrains strike performance because of its physical properties, we predict morphological and functional convergence in snakes that use similar strike behaviours. Here we use natricine snakes to test for such patterns of convergence in morphology and function. Our data show that frontal strikers have converged on a similar morphology characterized by narrow elongate heads with a reduced projected frontal surface area. Moreover, simple computational fluid dynamics models show that the observed morphological differences are likely biologically relevant as they affect the flow of water around the head. In general, our data suggest that the direction of evolution may be predictable if constraints are strong and evolutionary solutions limited.     

Inoculation of cucumber,melon and zucchini varieties with Tomato leaf curl New Delhi virus and evaluation of infection using different detection methods

The disease caused by Tomato leaf curl New Delhi virus (ToLCNDV), which is naturally transmitted by the whitefly Bemisia tabaci, causes important economic losses in cucurbit crops. The availability of simple and efficient inoculation protocols and detection methods is necessary for screening varieties and germplasm collections as well as for breeding populations. We evaluated the infectivity of ToLCNDV inocula prepared using three different buffers for mechanical sap inoculation in a susceptible variety of zucchini. We found that inoculum prepared with buffer III, which contains polyvinylpyrrolidone, is highly efficient for mechanical inoculation, with 100% of plants displaying severe symptoms 21 days post‐inoculation. Using this buffer, we mechanically inoculated 19 commercial varieties of cucurbit crops (six of cucumber, six of melon and seven of zucchini), evaluated the evolution of symptoms and diagnosed infection using nine different ToLCNDV detection methods (four based on serology, four based on molecular hybridization and one based on PCR detection). The results revealed that all varieties are susceptible, although cucumber varieties display less severe symptoms than those of melon or zucchini. All detection methods were highly efficient (more than 85% of plants testing positive) in melon and zucchini, but in cucumber, the percentage of positive plants detected with serology and molecular hybridization methods ranged from 20.4% with Squash leaf curl virus (SLCV) antiserum, to 78.5% with DNA extract hybridization. Overall, the best detection results were obtained with PCR, with 92.6%, 92.4% and 98.4% cucumber, melon and zucchini plants, respectively, testing positive. When considering the overall results in the three crops, the best serology and molecular hybridization methods were those using Watermelon chlorotic stunt virus (WmCSV) antiserum and DNA extract, respectively. The inoculation methodology developed and the information on detection methods are of great relevance for the selection and breeding of varieties of cucurbit crops that are tolerant or resistant to ToLCNDV.     

A possible role for flowering locus T‐encoding genes in interpreting environmental and internal cues affecting olive (Olea europaea L.) flower induction

Olive (Olea europaea L.) inflorescences, formed in lateral buds, flower in spring. However, there is some debate regarding time of flower induction and inflorescence initiation. Olive juvenility and seasonality of flowering were altered by overexpressing genes encoding flowering locus T (FT). OeFT1 and OeFT2 caused early flowering under short days when expressed in Arabidopsis. Expression of OeFT1/2 in olive leaves and OeFT2 in buds increased in winter, while initiation of inflorescences occurred i n late winter. Trees exposed to an artificial warm winter expressed low levels of OeFT1/2 in leaves and did not flower. Olive flower induction thus seems to be mediated by an increase in FT levels in response to cold winters. Olive flowering is dependent on additional internal factors. It was severely reduced in trees that carried a heavy fruit load the previous season (harvested in November) and in trees without fruit to which cold temperatures were artificially applied in summer. Expression analysis suggested that these internal factors work either by reducing the increase in OeFT1/2 expression or through putative flowering repressors such as TFL1. With expected warmer winters, future consumption of olive oil, as part of a healthy Mediterranean diet, should benefit from better understanding these factors.