Distribution, occurrence and biotoxin composition of the main shellfish toxin producing microalgae within European waters: A comparison of methods of analysis.

Harmful algal blooms (HABs) are a natural global phenomena emerging in severity and extent. Incidents have many economic, ecological and human health impacts. Monitoring and providing early warning of toxic HABs are critical for protecting public health. Current monitoring programmes include measuring the number of toxic phytoplankton cells in the water and biotoxin levels in shellfish tissue. As these efforts are demanding and labour intensive, methods which improve the efficiency are essential. This study compares the utilisation of a multitoxin surface plasmon resonance (multitoxin SPR) biosensor with enzyme-linked immunosorbent assay (ELISA) and analytical methods such as high performance liquid chromatography with fluorescence detection (HPLC-FLD) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) for toxic HAB monitoring efforts in Europe. Seawater samples (n = 256) from European waters, collected 2009–2011, were analysed for biotoxins: saxitoxin and analogues, okadaic acid and dinophysistoxins 1/2 (DTX1/DTX2) and domoic acid responsible for paralytic shellfish poisoning (PSP), diarrheic shellfish poisoning (DSP) and amnesic shellfish poisoning (ASP), respectively. Biotoxins were detected mainly in samples from Spain and Ireland. France and Norway appeared to have the lowest number of toxic samples. Both the multitoxin SPR biosensor and the RNA microarray were more sensitive at detecting toxic HABs than standard light microscopy phytoplankton monitoring. Correlations between each of the detection methods were performed with the overall agreement, based on statistical 2 × 2 comparison tables, between each testing platform ranging between 32% and 74% for all three toxin families illustrating that one individual testing method may not be an ideal solution. An efficient early warning monitoring system for the detection of toxic HABs could therefore be achieved by combining both the multitoxin SPR biosensor and RNA microarray.

The enteropathogenic Escherichia coli effector NleH inhibits apoptosis induced by Clostridium difficile toxin B

Clostridium difficile is a leading cause of nosocomial infections, causing a spectrum of diseases ranging from diarrhoea to pseudomembranous colitis triggered by a range of virulence factors including C. difficile toxins A (TcdA) and B (TcdB). TcdA and TcdB are monoglucosyltransferases that irreversibly glycosylate small Rho GTPases, inhibiting their ability to interact with their effectors, guanine nucleotide exchange factors, and membrane partners, leading to disruption of downstream signalling pathways and cell death. In addition, TcdB targets the mitochondria, inducing the intrinsic apoptotic pathway resulting in TcdB-mediated apoptosis. Modulation of apoptosis is a common strategy used by infectious agents. Recently, we have shown that the enteropathogenic Escherichia coli (EPEC) type III secretion system effector NleH has a broad-range anti-apoptotic activity. In this study we examined the effects of NleH on cells challenged with TcdB. During infection with wild-type EPEC, NleH inhibited TcdB-induced apoptosis at both low and high toxin concentrations. Transfected nleH1 alone was sufficient to block TcdB-induced cell rounding, nuclear condensation, mitochondrial swelling and lysis, and activation of caspase-3. These results show that NleH acts via a global anti-apoptotic pathway.

Erection induced by Tx2-6 toxin of Phoneutria nigriventer spider: expression profile of genes in the nitric oxide pathway of penile tissue of mice

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Coma caused by Shigella toxin-producing Escherichia coli in the absence of haemorrhagic uraemic syndrome (HUS)

Shigella toxin-producing Escherichia coli (STEC) is well known for its complications such as haemolytic uraemic syndrome (HUS), but neurological symptoms have also been reported. While most cases of infection with STEC occur with concurrent HUS, we describe a patient with severe neurological symptoms in the absence of HUS.

Toxin and Growth Responses of the Neurotoxic Dinoflagellate Vulcanodinium rugosum to Varying Temperature and Salinity

Vulcanodinium rugosum, a recently described species, produces pinnatoxins. The IFR-VRU-01 strain, isolated from a French Mediterranean lagoon in 2010 and identified as the causative dinoflagellate contaminating mussels in the Ingril Lagoon (French Mediterranean) with pinnatoxin-G, was grown in an enriched natural seawater medium. We tested the effect of temperature and salinity on growth, pinnatoxin-G production and chlorophyll a levels of this dinoflagellate. These factors were tested in combinations of five temperatures (15, 20, 25, 30 and 35 °C) and five salinities (20, 25, 30, 35 and 40) at an irradiance of 100 µmol photon m−2 s−1. V. rugosum can grow at temperatures and salinities ranging from 20 °C to 30 °C and 20 to 40, respectively. The optimal combination for growth (0.39 ± 0.11 d−1) was a temperature of 25 °C and a salinity of 40. Results suggest that V. rugosum is euryhaline and thermophile which could explain why this dinoflagellate develops in situ only from June to September. V. rugosum growth rate and pinnatoxin-G production were highest at temperatures ranging between 25 and 30 °C. This suggests that the dinoflagellate may give rise to extensive blooms in the coming decades caused by the climate change-related increases in temperature expected in the Mediterranean coasts.

Molecular Profiling of Shiga Toxin-Producing Escherichia coli and Enteropathogenic E. coli Strains Isolated from French Coastal Environments

Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) strains may be responsible for food-borne infections in humans. Twenty-eight STEC and 75 EPEC strains previously isolated from French shellfish-harvesting areas and their watersheds and belonging to 68 distinguishable serotypes were characterized in this study. High-throughput real-time PCR was used to search for the presence of 75 E. coli virulence-associated gene targets, and genes encoding Shiga toxin (stx) and intimin (eae) were subtyped using PCR tests and DNA sequencing, respectively. The results showed a high level of diversity between strains, with 17 unique virulence gene profiles for STEC and 56 for EPEC. Seven STEC and 15 EPEC strains were found to display a large number or a particular combination of genetic markers of virulence and the presence of stx and/or eae variants, suggesting their potential pathogenicity for humans. Among these, an O26:H11 stx1a eae-β1 strain was associated with a large number of virulence-associated genes (n = 47), including genes carried on the locus of enterocyte effacement (LEE) or other pathogenicity islands, such as OI-122, OI-71, OI-43/48, OI-50, OI-57, and the high-pathogenicity island (HPI). One O91:H21 STEC strain containing 4 stx variants (stx1a, stx2a, stx2c, and stx2d) was found to possess genes associated with pathogenicity islands OI-122, OI-43/48, and OI-15. Among EPEC strains harboring a large number of virulence genes (n, 34 to 50), eight belonged to serotype O26:H11, O103:H2, O103:H25, O145:H28, O157:H7, or O153:H2.

Role of phospholipase A(2) and tyrosine kinase in Clostridium difficile toxin A-induced disruption of epithelial integrity, histologic inflammatory damage and intestinal secretion

Clostridium difficile-associated disease causes diarrhea to fulminant colitis and death. We investigated the role of phospholipase A(2) (PLA(2)) inhibitors, aristolochic acid (AA), bromophenacyl bromide BPB and quinacrine (QUIN) on the C. difficile toxin A-induced disruption of epithelial integrity, histologic inflammatory damage and intestinal secretion. Toxin A caused severe hemorrhagic and inflammatory fluid secretion at 6-8 h in rabbit ileal segments, an effect that was significantly inhibited by QUIN (71%, P < 0.01), AA (87%, P < 0.0001) or by BPB (51%, P < 0.01). The secretory effect of toxin A was also inhibited in segments adjacent to those with AA (89%, P < 0.01). Furthermore, QUIN or AA substantially reduced the histologic damage seen after 6-8 h in rabbit ileal segments. The cyclooxygenase inhibitor, indomethacin, also significantly inhibited (96%; n = 6) the secretory effects of toxin A in ligated rabbit intestinal segments. The destruction by toxin A of F-actin at the light junctions of T-84 cell monolayers was not inhibited by AA or BPB. AA or QUIN had no effect on the T-84 cell tissue resistance reduction over 8-24 h after toxin A exposure. All the inhibitors were shown to be effective in the doses administered direct in ileal loops to inhibit PLA(2) activity. The data suggest that PLA(2) is involved in the major pathway of toxin A-induced histologic inflammatory damage and hemorrhagic fluid secretion. Cop. right (C) 2008 John Wiley & Sons, Ltd.

Crystallization and preliminary X-ray diffraction analysis of a eumenine mastoparan toxin: a new class of mast-cell degranulating peptide in the wasp venom

Mastoparans are tetradecapeptides found to be the major component of vespid venoms. A mastoparan toxin isolated from the venom of Anterhynchium flavomarginatum micado has been crystallized and X-ray diffraction data collected to 2.7 Angstrom resolution using a synchrotron-radiation source. Crystals were determined to belong to the space group P6(2)22 (P6(4)22). This is the first mastoparan to be crystallized and will provide further insights into the conformational significance of mastoparan toxins with respect to their potency and activity in G-protein regulation.

Preliminary cryocrystallography analysis of an eumenine mastoparan toxin isolated from the venom of the wasp Anterhynchium flavomarginatum micado

Mastoparans are tetradecapeptides found to be the major component of vespid venoms. These peptides present a wide spectrum of biological activities, such as mast cell degranulation, hemolytic activity and also reveals antimicrobial activity. A mastoparan toxin isolated from the venom of Anterhynchium flavomarginatum micado has been crystallized. At room temperature these crystals diffracted to 2.8 Angstrom resolution. However, upon cooling to cryogenic temperature around 85 K, the original resolution limit could be improved to 2.0 Angstrom. Crystals were determined to belong to the space group P3(1) (P3(2)). This is the first mastoparan to be crystallized and it will provide further insights in the conformational significance of mastoparan toxins, with respect to their potency and activity in G protein regulation. (C) 3001 Elsevier B.V. B.V. All rights reserved.

Characterization of a new toxin from the entomopathogenic fungus Metarhizium anisopliae: the ribotoxin anisoplin

Metarhizium anisopliae is an entomopathogenic fungus relevant in biotechnology with applications like malaria vector control. Studies of its virulence factors are therefore of great interest. Fungal ribotoxins are toxic ribonucleases with extraordinary efficiency against target ribosomes and suggested as potential insecticides. Here, we describe this ribotoxin characteristic activity in M. anisopliae cultures. Anisoplin has been obtained as a recombinant protein and further characterized. It is structurally similar to hirsutellin A, the ribotoxin from the entomopathogen Hirsutella thompsonii. Moreover, anisoplin shows the ribonucleolytic activity typical of ribotoxins and cytotoxicity against insect cells. How Metarhizium uses this toxin and possible applications are on perspective.

Establishment of a mouse model of colitis and its use to evaluate the anti-inflammatory effects of two ghrelin peptides

Ghrelin is a gut-brain peptide hormone that induces appetite, stimulates the release of growth hormone, and has recently been shown to ameliorate inflammation. Recent studies have suggested that ghrelin may play a potential role in inflammation-related diseases such as inflammatory bowel diseases (IBD). A previous study with ghrelin in the TNBS mouse model of colitis demonstrated that ghrelin treatment decreased the clinical severity of colitis and inflammation and prevented the recurrence of disease. Ghrelin may be acting at the immunological and epithelial level as the ghrelin receptor (GHSR) is expressed by immune cells and intestinal epithelial cells. The current project investigated the effect of ghrelin in a different mouse model of colitis using dextran sodium sulphate (DSS) – a luminal toxin. Two molecular weight forms of DSS were used as they give differing effects (5kDa and 40kDa). Ghrelin treatment significantly improved clinical colitis scores (p=0.012) in the C57BL/6 mouse strain with colitis induced by 2% DSS (5kDa). Treatment with ghrelin suppressed colitis in the proximal colon as indicated by reduced accumulative histopathology scores (p=0.03). Whilst there was a trend toward reduced scores in the mid and distal colon in these mice this did not reach significance. Ghrelin did not affect histopathology scores in the 40kDa model. There was no significant effect on the number of regulatory T cells or TNF-α secretion from cultured lymph node cells from these mice. The discovery of C-terminal ghrelin peptides, for example, obestatin and the peptide derived from exon 4 deleted proghrelin (Δ4 preproghrelin peptide) have raised questions regarding their potential role in biological functions. The current project investigated the effect of Δ4 peptide in the DSS model of colitis however no significant suppression of colitis was observed. In vitro epithelial wound healing assays were also undertaken to determine the effect of ghrelin on intestinal epithelial cell migration. Ghrelin did not significantly improve wound healing in these assays. In conclusion, ghrelin treatment displays a mild anti-inflammatory effect in the 5kDa DSS model. The potential mechanisms behind this effect and the disparity between these results and those published previously will be discussed.

Investigation of molecular mechanisms regulating biomineralization of pearl oyster Pinctada maxima

Biomineralization is a process encompassing all mineral containing tissues produced within an organism. The most dynamic example of this process is the formation of the mollusk shell, comprising a variety of crystal phases and microstructures. The organic component incorporated within the shell is said to dictate this remarkable architecture. Subsequently, for the past decade considerable research have been undertaken to identify and characterize the protein components involved in biomineralization. Despite these efforts the general understanding of the process remains ambiguous. This study employs a novel molecular approach to further the elucidation of the shell biomineralization. A microarray platform has been custom generated (PmaxArray 1.0) from the pearl oyster Pinctada maxima. PmaxArray 1.0 consists of 4992 expressed sequence tags (ESTs) originating from the mantle, an organ involved in shell formation. This microarray has been used as the primary tool for three separate investigations in an effort to associate transcriptional gene expression from P. maxima to the process of shell biomineralization. The first investigation analyzes the spatial expression of ESTs throughout the mantle organ. The mantle was dissected into five discrete regions and each analyzed for gene expression with PmaxArray 1.0. Over 2000 ESTs were differentially expressed among the tissue sections, identifying five major expression regions. Three of these regions have been proposed to have shell formation functions belonging to nacre, prismatic calcite and periostracum. The spatial gene expression map was confirmed by in situ hybridization, localizing a subset of ESTs from each expression region to the same mantle area. Comparative sequence analysis of ESTs expressed in the proposed shell formation regions with the BLAST tool, revealed a number of the transcripts were novel while others showed significant sequence similarities to previously characterized shell formation genes. The second investigation correlates temporal EST expression during P. maxima larval ontogeny with transitions in shell mineralization during the same period. A timeline documenting the morphologicat microstructural and mineralogical shell characteristics of P. maxima throughout larval ontogeny has been established. Three different shell types were noted based on the physical characters and termed, prodissoconch I, prodissoconch 11 and dissoconch. PmaxArray 1.0 analyzed ESTs expression of animals throughout the larval development of P. maxima, noting up-regulation of 359 ESTs in association with the shell transitions from prodissoconch 1 to prodissoconch 11 to dissoconch. Comparative sequence analysis of these ESTs indicates a number of the transcripts are novel as well as showing significant sequence similarities between ESTs and known shell matrix associated genes and proteins. These ESTs are discussed in relation to the shell characters associated with their temporal expression. The third investigation uses PmaxArray 1.0 to analyze gene expression in the mantle tissue of P. maxima specimens exposed to sub-lethal concentrations of a shell-deforming toxin, tributyltin (TBT). The shell specific effects of TBT are used in this investigation to interpret differential expression of ESTs with respect to shell formation functions. A lethal and sublethal TBT concentration range was established for P. maxima, noting a concentration of 50 ng L- 1 TBT as sub-lethal over a 21 day period. Mantle tissue from P. maxima animals treated with 50 ng L- 1 TBT was assessed for differential EST expression with untreated control animals. A total of 102 ESTs were identified as differentially expressed in association with TBT exposure, comparative sequence identities included an up-regulation of immunity and detoxification related genes and down-regulation of several shell matrix genes. A number of transcripts encoding novel peptides were additionally identified. The potential actions of these genes are discussed with reference to TBT toxicity and shell biomineralization. This thesis has used a microarray platform to analyze gene expression in spatial, temporal and toxicity investigations, revealing the involvement of numerous gene transcripts in specific shell formation functions. Investigation of thousands of transcripts simultaneously has provided a holistic interpretation of the organic components regulating shell biomineralization.

CTA1-DD is an effective adjuvant for targeting anti-chlamydial immunity to the murine genital mucosa

Chlamydia trachomatis is a significant human pathogen with potentially severe disease sequelae in the genital tract, including infertility. A successful vaccine will need to effectively target immunity to the genital mucosa. Intranasal immunisation with cholera toxin (CT) can target immunity to the genital tract, but has the potential to cause neurological side effects. CTA1-DD is a non-toxic potent mucosal adjuvant which combines the enzymatic properties of CT, with a B cell targeting moiety. Here, we demonstrate that intranasal immunisation with CTA1-DD and chlamydial Major Outer Membrane Protein (MOMP) results in the induction of neutralising systemic and mucosal antibodies, and reduces the level of chlamydial shedding following intravaginal challenge with Chlamydia muridarum. Thus, CTA1-DD is an effective adjuvant for vaccine development against Chlamydia trachomatis, and possibly also a range of other genital pathogens.

An egg apparatus-specific enhancer of Arabidopsis, identified by enhancer detection

Despite a central role in angiosperm reproduction, few gametophyte-specific genes and promoters have been isolated, particularly for the inaccessible female gametophyte (embryo sac). Using the Ds-based enhancer-detector line ET253, we have cloned an egg apparatus-specific enhancer (EASE) from Arabidopsis (Arabidopsis thaliana). The genomic region flanking the Ds insertion site was further analyzed by examining its capability to control gusA and GFP reporter gene expression in the embryo sac in a transgenic context. Through analysis of a 5' and 3' deletion series in transgenic Arabidopsis, the sequence responsible for egg apparatus-specific expression was delineated to 77 bp. Our data showed that this enhancer is unique in the Arabidopsis genome, is conserved among different accessions, and shows an unusual pattern of sequence variation. This EASE works independently of position and orientation in Arabidopsis but is probably not associated with any nearby gene, suggesting either that it acts over a large distance or that a cryptic element was detected. Embryo-specific ablation in Arabidopsis was achieved by transactivation of a diphtheria toxin gene under the control of the EASE. The potential application of the EASE element and similar control elements as part of an open-source biotechnology toolkit for apomixis is discussed.