The role of WNT singling activation and its blockage in the focal retinal lesion of Ccl2-/-/Cx3cr1-/- mice with and without rd8 background

Purpose: The canonical Wnt signaling is activated by retinal injury. Under disease conditions, the Wnt mediates inflammatory responses. Inflammation has been detected in age-related macular degeneration (AMD) retinas and Ccl2-/-/Cx3cr1-/- (DKO) mice with or without rd8 background, a model with progressive AMD-like lesions including focal photoreceptor/RPE degeneration and A2E accumulation. We evaluated the effects of Wnt-β-catenin activation and an antibody against LRP6, the co-receptor of Wnt on these two models.

Methods: anti-LRP6 antibody (2F1, 1 μl of 5 μg/μL) was intravitreally injected into the right eyes in 3 separate experiments (DKOrd8, N=35; DKO, N=10). The left eyes were injected with mouse IgG as controls. Fundoscopy was taken before injection and sequentially monthly after injection. Two months after injection, light-adapted ERG responses were recorded; then the eyes were harvested for histopathology, the determination of retinal A2E, and molecular analysis. The microarray of ocular mRNA of 92 Wnt genes was compared between the treated and the control eyes. The phosphorylated types of LRP6 and β-catenin and endogenous forms of the proteins were assayed by Western blotting.

Results: For DKOrd8 mice, the fundus showed a slower progression or alleviation of retinal lesions in the right eyes as compared to the left eyes. Among 35 pairs of eyes, 26 (74.3%) were improved, 7 (20%) stayed the same and 2 (5.7%) remained progressing. Histology confirmed the clinical observation. Light-adapted ERG of the treated eyes exhibited larger amplitudes compared to control eyes (n=6), with greater improvements under UV light stimulus. There was a significantly lower A2E in the treated eyes compared to controls. Microarray of 92 Wnt genes expression pattern was similar in both eyes. Western blotting indicated local administration of 2F1 antibody to suppress the activation of Wnt pathway in the retina. For DKO mice, the treatment improved ERG but less effect on RPE degeneration.

Conclusions: The canonical Wnt signaling plays a role in the focal retina lesion of both DKOrd8 and DKO mice; and intravitreal anti-LRP6 antibody might be neuroprotective via deactivation of canonical Wnt pathway.

XBP 1-deficiency abrogates neointimal lesion of injured vessels via cross talk with the PDGF signaling

Objective: Smooth muscle cell (SMC) migration and proliferation play an essential role in neointimal formation after vascular injury. In this study, we intended to investigate whether the X-box-binding protein 1 (XBP1) was involved in these processes.

Approach and Results: In vivo studies on femoral artery injury models revealed that vascular injury triggered an immediate upregulation of XBP1 expression and splicing in vascular SMCs and that XBP1 deficiency in SMCs significantly abrogated neointimal formation in the injured vessels. In vitro studies indicated that platelet-derived growth factor-BB triggered XBP1 splicing in SMCs via the interaction between platelet-derived growth factor receptor β and the inositol-requiring enzyme 1α. The spliced XBP1 (XBP1s) increased SMC migration via PI3K/Akt activation and proliferation via downregulating calponin h1 (CNN1). XBP1s directed the transcription of mir-1274B that targeted CNN1 mRNA degradation. Proteomic analysis of culture media revealed that XBP1s decreased transforming growth factor (TGF)-β family proteins secretion via transcriptional suppression. TGF-β3 but not TGF-β1 or TGF-β2 attenuated XBP1s-induced CNN1 decrease and SMC proliferation.

Conclusions: This study demonstrates for the first time that XBP1 is crucial for SMC proliferation via modulating the platelet-derived growth factor/TGF-β pathways, leading to neointimal formation.

New insights into the FLPergic complements of parasitic nematodes: Informing deorphanisation approaches

FMRFamide-like peptide (FLP) receptors are appealing as putative anthelmintic targets. To date, 31 flp-encoding genes have been identified in Caenorhabditis elegans and thirteen FLP-activated G-protein coupled receptors (FLP-GPCRs) have been reported. The lack of knowledge on FLPs and FLP-GPCRs in parasites impedes their functional characterisation and chemotherapeutic exploitation. Using homology-based BLAST searches and phylogenetic analyses this study describes the identification of putative flp and flp-GPCR gene homologues in 17 nematode parasites providing the first pan-phylum genome-based overview of the FLPergic complement. These data will facilitate FLP-receptor deorphanisation efforts in the quest for novel control targets for nematode parasites.

POPULATION DYNAMICS OF PLANT PARASITIC NEMATODES AND THEIR RELATIONSHIPS WITH SOIL PHYSICO-CHEMICAL PARAMETERS DURING THE FALLOW PERIOD AFTER SUGARBEET AND LEADING INTO TOMATO CULTIVATION

The main objective of this research was the study of the soil nematode community, and in particular plant parasitic nematodes (PPN), from a field located in Portugal’s southern region, used for sugarbeet production. The study was performed from February to July 2003, covering part of the fallow period previous to tomato cultivation, the alternative crop in the rotation. The end of the fallow period in March and the soil preparation period in May were marked by a significant reduction in the numbers of PPN, whereas their numbers increased on the following tomato crop. The genus Helicotylenchus stood out as the most representative group, forming 90% of all PPN counted each month. The genus Heterodera was relatively abundant in the months following the previous sugarbeet crop, and numbers of the genus Meloidogyne increased during the tomato crop. The correlations between these group and environmental parameters show that, apart from the direct influence of the host, pH, organic matter, temperature and soil moisture significantly influenced nematode abundance and community composition.

Taxonomic databases for Bursaphelenchus and other aphelenchoid nematodes

Nematodes are the most abundant metazoans, comprising more than 80% of all animals alive today. Since 1743, when Needham (Needham, 1743) described the first nematode, approximately 20,000 - 30,000 species have been named, with estimates of species remaining to be described ranging from 100,000 to 1 million (Blaxter, 2004; De Ley, 2000). Unfortunately, the taxonomic community is woefully inadequate for this task. The number of taxonomists currently describing new species of nematodes around the world is less than 100, and significant increases are not expected. If each of these taxonomists were able to describe 10 new species every year, it would take between 100 to 1,000 years to name these yet to be described species.

List of the terrestriaL nematodes (nematoda) from Azores

Os nemátodes são um grupo de invertebrados, não segmentados que formam um Filo (Nematoda) bem definido e claramente distinto dos outros grupos de organismos. Este Filo constitui um dos grupos animais mais disseminados no planeta, e em termos de número de indivíduos os nemátodes são o grupo animal mais abundante na Terra: quatro em cada cinco animais da Biosfera são nemátodes. Apesar de microscópicos, os animais multicelulares que constituem este grupo são capazes de explorar uma enorme variedade de habitats, nos mares, nas águas doces, nos solos, como parasitas de animais ou de plantas, ou mesmo em condições extremas, como nos solos secos da Antárctida ou em fontes termais (Baldwin et al. 1999). A actual informação sobre a nematofauna do arquipélago dos Açores encontra-se fraccionada e espalhada em diversas publicações científicas, mas igualmente em dados ainda não publicados (Sturhan, comunicação pessoal). Entre as diversas publicações de nemátodes para os Açores, há que salientar os trabalhos realizados por Sturhan (1973, 1975, 1983) e Macara (1994), que muito contribuíram para o conhecimento e distribuição actual das espécies de vida livre e parasitas de plantas; bem como os trabalhos de Afonso-Roque (1995) e Casanova et al. (1996) relativamente a espécies parasitas de animais, reportadas para as diferentes ilhas do arquipélago. A lista de nemátodes apresentada neste capítulo, tem como base as publicações conhecidas para o arquipélago (ver listagem de publicações até 2010 em www. naturdata.com), bem como o registo de espécies assinaladas pela primeira vez para o arquipélago (Sturhan, comunicação pessoal). A classificação utilizada baseia-se na recente revisão da sistemática e filogenia proposta por De Ley & Blaxter (2002) para todo o Filo, até ao nível da Família. A classificação dos restantes taxa segue o critério utilizado para a fauna terrestre da Fauna Europaea (http://www. faunaeur.org). É apresentada a distribuição das espécies nas nove ilhas dos Açores, usando-se a seguinte simbologia: COR – Corvo; FLO – Flores; FAI – Faial; PIC – Pico; GRA – Graciosa; SJG – São Jorge; TER – Terceira; SMG – São Miguel e SMR – Santa Maria.

Genetic diversity of entomopathogenic nematodes (Nematoda: Steinernematidae and Heterorhabditidae) and the nematode Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae) from continental Portugal

“Diversidade genética dos nemátodes entomopatogénicos (Nematoda: Steinernematidae e Heterorhabditidae) e do nemátode Bursaphelenchus xylophilus (Nematoda: Aphelenchoididade) em Portugal continental” Os nematodes entomopatogénicos são utilizados como agentes de controlo biológico. Para compreender a sua diversidade, foi realizada uma prospecção em Portugal. Cinco espécies, nomeadamente Steinernema feltiae, S. intermedium, S. kraussei, Steinernema sp. e Heterorhabditis bacteriophora foram identificadas. As sequências de ITS, região D2D3 do 28S rRNA, COXI e cytb foram utilizadas para estudar a diversidade genética das duas espécies mais abundantes, S. feltiae and H. bacteriophora, não tendo sido encontradas diferenças significativas entre isolados. O nemátode da madeira do pinheiro, Bursaphelenchus xylophilus, provoca doença nos pinheiros tendo sido detectada pela primeira vez na Europa e em Portugal em 1999. Para avaliar a diversidade genética dos isolados Portugueses e identificar o padrão de propagação da doença, foram utilizadas a sequência da região IGS do 5.8S rRNA, e os genes cytb e cellulase, combinados com os padrões ISSR. Os padrões de ISSR mostraram elevada diversidade genética entre os recentes isolados Portugueses, sugerindo a possibilidade de uma nova introdução. As árvores filogenéticas dos genes da celulase e cytb sugeriram uma origem Asiática para os isolados Portugueses; ABSTRACT: Entomopathogenic nematodes are used as biocontrol agents. To understand their diversity, a survey was undertaken in Portugal. Five species, namely Steinernema feltiae, S. intermedium, S. kraussei, Steinernema sp. and Heterorhabditis bacteriophora were identified. The ITS, 28S rRNA D2D3 region, COXI and cytb sequences, used to study the genetic diversity of the two most abundant species, S. feltiae and H. bacteriophora, showed no significant differences among the isolates. Bursaphelenchus xylophilus causes severe disease in pine trees and was detected for the first time in Europe and in Portugal in 1999. To evaluate the genetic diversity of Portuguese isolates and identify disease spread pathways, the sequence of 5.8S rRNA IGS region, cytb and cellulase genes, combined with ISSR fingerprints were used. ISSR fingerprints show a high genetic variability among recent Portuguese isolates, suggesting the possibility of a new introduction. Phylogenetic trees based on cellulase and cytb genes suggests an Asian origin for Portuguese isolates.

Cortical origin of functional recovery in the somatosensory cortex of the adult mouse after thalamic lesion

Résumé L'accident vasculaire cérébral sensoriel pur est un des syndromes lacunaires, dû à l'occlusion de petits vaisseaux cérébraux, souvent dans le cadre d'une lésion intéressant le noyau ventro-caudal du thalamus. Il produit un hémisyndrome sensitif pur, et parfois un syndrome douloureux se développe à distance de l'événement aigu. Afin d'étudier la récupération fonctionnelle dans le cortex somatosensoriel (SI) après une telle lésion dans le thalamus, un modèle de lésion excitotoxique a été développé dans le système somatosensoriel de la souris adulte, caractérisé par la présence de formations cytoarchitectoniques dans SI appelées "tonneaux". Chacun de ces tonneaux correspond à la représentation corticale d'une vibrisse du museau. L'activité métabolique a été mesurée dans SI à différents intervalles après la lésion, à l'aide de déoxyglucose marqué radioactivement. Dans les deux premiers jours suivant celle-ci, l'activité métabolique diminue de manière importante dans toutes les couches corticales, avec une atteinte plus marquée dans la couche IV, principale projection des axones thalamo-corticaux. Une récupération de l'activité métabolique se produit ensuite, d'autant plus marquée que le délai après la lésion est grand. Cette récupération s'observe dans toutes les couches coticales, les couches I et Vb récupérant plus rapidement que les couches II, III, IV, Va et VI. Cinq semaines après la lésion, l'absence des vibrisses correspondant à la partie déafférentée de SI diminue l'activité métabolique corticale de 32% et démontre l'activation par la périphérie de cette partie de l'écorce, malgré la perte des axones thalamo-corticaux provenant du noyau ventro-caudal. Des expériences de traçage rétrograde ont montré une augmentation des projections intracorticales sur la partie déafférentée de l'écorce, en particulier de longue distance, ainsi que des projections interhémisphériques, mais n'ont pas permis de mettre en évidence de nouvelle projection thalamique, indiquant une origine corticale à la récupération fonctionnelle observée. Abstract To study the degree and time course of the functional recovery in the somatosensory cortex (SI) after an excitotoxic lesion in the adult mouse thalamus, metabolic activity was determined in SI at various times points post lesion. Immediately after the lesion, metabolic activity in the thalamically deafferented part of SI was at its lowest value but increased progressively at subsequent time points. This was seen in all cortical layers, however, layers I and Vb recover more rapidly than layers II, III, IV, Va and VI. Removal of the mystacial whiskers corresponding to the deafferented area, 5 weeks after cortical recovery, produced a subsequent 32% drop in metabolic activity, demonstrating peripheral sensory activation of this part of the cortex. Tracing experiments revealed that the deafferented cortex did not receive a novel thalamic input, but cortico-cortical and contralateral barrel cortex projections to this area were reinforced. We conclude that the cortical functional recovery after a thalamic lesion is, at least partially, due to modified cortico-cortical and callosal projections to the deafferented cortical area.

Alveolar echinococcosis of the liver: Diffusion-weighted MRI findings and potential role in lesion characterisation

PURPOSE: To report the diffusion-weighted MRI findings in alveolar echinococcosis (AE) of the liver and evaluate the potential role of apparent diffusion coefficients (ADCs) in the characterisation of lesions. MATERIALS AND METHODS: We retrospectively included 22 patients with 63 AE liver lesions (≥1cm), examined with 3-T liver MRI, including a free-breathing diffusion-weighted single-shot echo-planar imaging sequence (b-values=50, 300 and 600s/mm(2)). Two radiologists jointly assessed the following lesion features: size, location, presence of cystic and/or solid components (according to Kodama's classification system), relative contrast enhancement, and calcifications (on CT). The ADCtotal, ADCmin and ADCmax were measured in each lesion and the surrounding liver parenchyma. RESULTS: Three type 1, 19 type 2, 17 type 3, three type 4 and 21 type 5 lesions were identified. The mean (±SD) ADCtotal, ADCmin and ADCmax for all lesions were 1.73±0.50, 0.76±0.38 and 2.63±0.76×10(-3)mm(2)/s, respectively. The mean ADCtotal for type 1, type 2, type 3, type 4 and type 5 lesions were 1.97±1.01, 1.76±0.53, 1.73±0.41, 1.15±0.42 and 1.76±0.44×10(-3)mm(2)/s, respectively. No significant differences were found between the five lesion types, except for type 4 (p=0.0363). There was a significant correlation between the presence of a solid component and low ADCmin (r=0.39, p=0.0016), whereas an inverse correlation was found between the relative contrast enhancement and ADCtotal (r=-0.34, p=0.0072). CONCLUSION: The ADCs of AE lesions are relatively low compared to other cystic liver lesions, which may help in the differential diagnosis. Although ADCs are of little use to distinguish between the five lesion types, their low value reflects the underlying solid component.

The determination of sinigrin in Brassica, and investigations into the use of allyl-isothiocyanate as a nematicide

The goal of this thesis was to study factors related to the development of Brassica juncea as a sustainable nematicide. Brassica juncea is characterized by the glycoside (glucosinolate) sinigrin. Various methods were developed for the determination of sinigrin in Brassica juncea tissue extracts. Sinigrin concentrations in plant tissues at various stages of growth were monitored. Sinigrin enzymatically breaks down into allylisothiocyanate (AITC). AITC is unstable in aqueous solution and degradation was studied in water and in soil. Finally, the toxicity of AITC against the root-lesion nematode (Pratylenchus penetrans) was determined. A method was developed to extract sinigrin from whole Brassica j uncea tissues. The optimal time of extraction wi th boiling phosphate buffer (0.7mM, pH=6.38) and methanol/water (70:30 v/v) solutions were both 25 minutes. Methanol/water extracted 13% greater amount of sinigrin than phosphate buffer solution. Degradation of sinigrin in boiling phosphate buffer solution (0.13%/minute) was similar to the loss of sinigrin during the extraction procedure. The loss of sinigrin from boiling methanol/water was estimated to be O.Ol%/minute. Brassica juncea extract clean up was accomplished by an ion-pair solid phase extraction (SPE) method. The recovery of sinigrin was 92.6% and coextractive impurities were not detected in the cleaned up extract. Several high performance liquid chromatography (HPLC) methods were developed for the determination of sinigrin. All the developed methods employed an isocratic mobile phase system wi th a low concentration of phosphate buffer solution, ammonium acetate solution or an ion-pair reagent solution. A step gradient system was also developed. The method involved preconditioning the analytical column with phosphate buffer solution and then switching the mobile phase to 100% water after sample injection.Sinigrin and benzyl-glucosinolate were both studied by HPLC particle beam negative chemical ionization mass spectrometry (HPLCPB- NCI-MS). Comparison of the mass spectra revealed the presence of fragments arising from the ~hioglucose moiety and glucosinolate side-chain. Variation in the slnlgrin concentration within Brassica juncea plants was studied (Domo and Cutlass cuItivars). The sinigrin concentration in the top three leaves was studied during growth of each cultivar. For Cutlass, the minimum (200~100~g/g) and maximum (1300~200~g/g) concentrations were observed at the third and seventh week after planting, respectively. For Domo, the minimum (190~70~g/g) and maximum (1100~400~g/g) concentrations were observed at the fourth and eighth week after planting, respectively. The highest sinigrin concentration was observed in flower tissues 2050±90~g/g and 2300±100~g/g for Cutlass and Domo cultivars, respectively. Physical properties of AITC were studied. The solubility of AITC in water was determined to be approximately 1290~g/ml at 24°C. An HPLC method was developed for the separation of degradation compounds from aqueous AITC sample solutions. Some of the degradation compounds identified have not been reported in the literature: allyl-thiourea, allyl-thiocyanate and diallyl-sulfide. In water, AITC degradation to' diallyl-thiourea was favored at basic pH (9.07) and degradation to diallyl-sulfide was favored at acidic pH (4 . 97). It wap necessary to amend the aqueous AITC sample solution with acetonitrile ?efore injection into the HPLC system. The acetonitrile amendment considerably improved AITC recovery and the reproducibility of the results. The half-life of aqueous AITC degradation at room temperature did not follow first-order kinetics. Beginning with a 1084~g/ml solution, the half-life was 633 hours. Wi th an ini tial AITC concentration of 335~g/ml the half-life was 865 hours. At 35°C the half-life AITC was 76+4 hours essentially independent of the iiisolution pH over the range of pH=4.97 to 9.07 (1000~g/ml). AITC degradation was also studied in soil at 35°C; after 24 hours approximately 75% of the initial AITC addition was unrecoverable by water extraction. The ECso of aqueous AITC against the root-lesion nematode (Pratylenchus penetrans) was determined to be approximately 20~g/ml at one hour exposure of the nematode to the test solution. The toxicological study was also performed with a myrosinase treated Brassica juncea extract. Myrosinase treatment of the Brassica juncea extract gave nearly quantitative conversion of sinigrin into AITC. The myrosinase treated extract was of the same efficacy as an aqueous AITC solution of equivalent concentration. The work of this thesis was focused upon understanding parameters relevant to the development of Brassica juncea as a sustainable nematicide. The broad range of experiments were undertaken in support of a research priority at Agriculture and Agri-Food Canada.

The effect of lesion size on cortical reorganization in the ipsi and contralesional hemispheres.

Bien que la plasticité ipsilesionnelle suite à un accident vasculo-cérébral (AVC) soit bien établie, la réorganisation du cortex contralésionnel et son effet sur la récupération fonctionnelle restent toujours non élucidés. Les études publiées présentent des points de vue contradictoires sur le rôle du cortex contralésionnel dans la récupération fonctionnelle. La taille de lésion pourrait être le facteur déterminant la réorganisation de ce dernier. Le but principal de cette étude fut donc d’évaluer l’effet des AVC de tailles différentes dans la région caudal forelimb area (CFA) du rat sur la réorganisation physiologique et la récupération comportementale de la main. Suite à une période de récupération spontanée pendant laquelle la performance motrice des deux membres antérieurs fut observée, les cartes motrices bilatérales du CFA et du rostral forelimb area (RFA) furent obtenues. Nous avons trouvé que le volume de lésion était en corrélation avec le niveau de récupération comportementale et l’étendue de la réorganisation des RFA bilatéraux. Aussi, les rats ayant de grandes lésions avaient des plus grandes représentations de la main dans le RFA de l’hémisphère ipsilésionnel et un déficit de fonctionnement plus persistant de la main parétique. Dans l’hémisphère contralésionnel nous avons trouvé que les rats avec des plus grandes représentations de la main dans le RFA avaient des lésions plus grandes et une récupération incomplète de la main parétique. Nos résultats confirment l’effet du volume de lésion sur la réorganisation du cortex contralésionnel et soulignent que le RFA est l’aire motrice la plus influencée dans le cortex contralésionnel.

Identification of a novel herpesvirus associated with a penile proliferative lesion in a beluga (Delphinapterus leucas).

The carcass of an adult male beluga (Delphinapterus leucas) was found beach cast in 2008 on the shore of the St. Lawrence Estuary at Rivière-Ouelle, Quebec, Canada. The carcass was transported to the Faculté de médecine vétérinaire of the Université de Montréal for postmortem examination. Aspiration pneumonia was the probable cause of death. Necropsy revealed a focal papilloma-like penile lesion, characterized by focal mucosal thickening with disorganization of the epithelial layers and lymphoplasmacytic infiltration. A pan-herpesvirus nested PCR assay on frozen tissue from the penile lesion was positive. The PCR product sequencing revealed a partial herpesvirus DNA polymerase (DPOL) gene sequence of 600 nucleotides. Its nearest nucleotide identity was with the partial DPOL gene of an alphaherpesvirus, bovine herpesvirus 5 (79.5% identity). It also shared high identity with several other marine mammal herpesviruses (50.2 to 77.3% identity). This new herpesvirus was tentatively named beluga whale herpesvirus (BWHV). Virus isolation was unsuccessful. The pathogenic potential of BWHV is unknown, but the evaluation of archived tissues suggests that the virus is endemic in the St. Lawrence Estuary beluga population.

Meiobenthos of the Shelf Waters of West Coast of India With Special Reference to Free-Living Marine Nematodes

Department of Marine Biology, Microbiology and Biotechnology, Cochin University of Science and Technology