J-filter: An experiment to simplify and isolate specific signals in 1 H NMR spectra of complex mixtures based on scalar coupling constants.

One-dimensional selective NMR experiments relying on a J-filter element are proposed to isolate specific signals in crowded 1 H spectral regions. The J-filter allows the edition or filtering of signals in a region of interest of the spectrum by exploiting the specific values of their 1 H-1 H coupling constants and certain parameters of protons coupled to them that appear in less congested parts of the spectrum (chemical shifts and coupling constants). The new experiments permitted the isolation of specific peaks of phytosterol components in a sample obtained from a liquid nutraceutical recommended for lowering blood cholesterol levels in regions with complete overlap in the 1 H spectrum.

Concurrent Akt, ERK1/2 and AMPK Activation by Obestatin Inhibits Apoptotic Signaling Cascades on Nutrient-Deprived PC12 Cells.

Targeting apoptosis in the ischemic penumbra is a rational therapeutic approach for restricting cerebral infarct volume after clinical stroke. The present work explored the capability of the obestatin peptide, as a novel approach to inhibit apoptotic signaling cascades on PC12 cells. According to the results, obestatin treatment significantly reduced nutrient deprivation-induced apoptotic cell death. The protective effects were related to the regulation of the anti-apoptotic protein, BCL-2, and the apoptotic protein caspase-3. This encompasses the control of apoptosis by the interplay between Akt, ERK1/2 and AMPK signaling pathways. The activation of Akt and AMPK was concomitant with the phosphorylation of their downstream targets, GSK3 and ACC, respectively. Besides, obestatin also causes FoxO1 nuclear export supporting the prevention of the apoptosome formation. The concurrent activation of Akt and AMPK by obestatin via the GPR39 receptor, supports a role for this system in the balance concerning the catabolic and the anabolic signaling to sustain cellular function and viability. Furthermore, these results provide both an insight into how the obestatin/GPR39 system regulates anti-apoptotic pathways, and a framework for ascertaining how this system can be optimally targeted in treatment of brain cell death after stroke.

Obestatin Increases the Regenerative Capacity of Human Myoblasts Transplanted Intramuscularly in an Immunodeficient Mouse Model.

Although cell-based therapy is considered a promising method aiming at treating different muscular disorders, little clinical benefit has been reported. One of major hurdles limiting the efficiency of myoblast transfer therapy is the poor survival of the transplanted cells. Any intervention upon the donor cells focused on enhancing in vivo survival, proliferation, and expansion is essential to improve the effectiveness of such therapies in regenerative medicine. In the present work, we investigated the potential role of obestatin, an autocrine peptide factor regulating skeletal muscle growth and repair, to improve the outcome of myoblast-based therapy by xenotransplanting primary human myoblasts into immunodeficient mice. The data proved that short in vivo obestatin treatment of primary human myoblasts not only enhances the efficiency of engraftment, but also facilitates an even distribution of myoblasts in the host muscle. Moreover, this treatment leads to a hypertrophic response of the human-derived regenerating myofibers. Taken together, the activation of the obestatin/GPR39 pathway resulted in an overall improvement of the efficacy of cell engraftment within the host's skeletal muscle. These data suggest considerable potential for future therapeutic applications and highlight the importance of combinatorial therapies.

ß-Arrestin scaffolds and signaling elements essential for the obestatin/GPR39 system that determine the myogenic program in human myoblast cells.

Obestatin/GPR39 signaling stimulates skeletal muscle repair by inducing the expansion of satellite stem cells as well as myofiber hypertrophy. Here, we describe that the obestatin/GPR39 system acts as autocrine/paracrine factor on human myogenesis. Obestatin regulated multiple steps of myogenesis: myoblast proliferation, cell cycle exit, differentiation and recruitment to fuse and form multinucleated hypertrophic myotubes. Obestatin-induced mitogenic action was mediated by ERK1/2 and JunD activity, being orchestrated by a G-dependent mechanism. At a later stage of myogenesis, scaffolding proteins ß-arrestin 1 and 2 were essential for the activation of cell cycle exit and differentiation through the transactivation of the epidermal growth factor receptor (EGFR). Upon obestatin stimulus, ß-arrestins are recruited to the membrane, where they functionally interact with GPR39 leading to Src activation and signalplex formation to EGFR transactivation by matrix metalloproteinases. This signalplex regulated the mitotic arrest by p21 and p57 expression and the mid- to late stages of differentiation through JNK/c-Jun, CAMKII, Akt and p38 pathways. This finding not only provides the first functional activity for ß-arrestins in myogenesis but also identify potential targets for therapeutic approaches by triggering specific signaling arms of the GPR39 signaling involved in myogenesis.

Action of obestatin in skeletal muscle repair: stem cell expansion, muscle growth, and microenvironment remodeling.

The development of therapeutic strategies for skeletal muscle diseases, such as physical injuries and myopathies, depends on the knowledge of regulatory signals that control the myogenic process. The obestatin/GPR39 system operates as an autocrine signal in the regulation of skeletal myogenesis. Using a mouse model of skeletal muscle regeneration after injury and several cellular strategies, we explored the potential use of obestatin as a therapeutic agent for the treatment of trauma-induced muscle injuries. Our results evidenced that the overexpression of the preproghrelin, and thus obestatin, and GPR39 in skeletal muscle increased regeneration after muscle injury. More importantly, the intramuscular injection of obestatin significantly enhanced muscle regeneration by simulating satellite stem cell expansion as well as myofiber hypertrophy through a kinase hierarchy. Added to the myogenic action, the obestatin administration resulted in an increased expression of vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor 2 (VEGFR2) and the consequent microvascularization, with no effect on collagen deposition in skeletal muscle. Furthermore, the potential inhibition of myostatin during obestatin treatment might contribute to its myogenic action improving muscle growth and regeneration. Overall, our data demonstrate successful improvement of muscle regeneration, indicating obestatin is a potential therapeutic agent for skeletal muscle injury and would benefit other myopathies related to muscle regeneration.

The obestatin/GPR39 system is up-regulated by muscle injury and functions as an autocrine regenerative system.

The maintenance and repair of skeletal muscle are attributable to an elaborate interaction between extrinsic and intrinsic regulatory signals that regulate the myogenic process. In the present work, we showed that obestatin, a 23-amino acid peptide encoded by the ghrelin gene, and the GPR39 receptor are expressed in rat skeletal muscle and are up-regulated upon experimental injury. To define their roles in muscle regeneration, L6E9 cells were used to perform in vitro assays. For the in vivo assays, skeletal muscle tissue was obtained from male rats and maintained under continuous subcutaneous infusion of obestatin. In differentiating L6E9 cells, preproghrelin expression and correspondingly obestatin increased during myogenesis being sustained throughout terminal differentiation. Autocrine action was demonstrated by neutralization of the endogenous obestatin secreted by differentiating L6E9 cells using a specific anti-obestatin antibody. Knockdown experiments by preproghrelin siRNA confirmed the contribution of obestatin to the myogenic program. Furthermore, GPR39 siRNA reduced obestatin action and myogenic differentiation. Exogenous obestatin stimulation was also shown to regulate myoblast migration and proliferation. Furthermore, the addition of obestatin to the differentiation medium increased myogenic differentiation of L6E9 cells. The relevance of the actions of obestatin was confirmed in vivo by the up-regulation of Pax-7, MyoD, Myf5, Myf6, myogenin, and myosin heavy chain (MHC) in obestatin-infused rats when compared with saline-infused rats. These data elucidate a novel mechanism whereby the obestatin/GPR39 system is coordinately regulated as part of the myogenic program and operates as an autocrine signal regulating skeletal myogenesis.

Evaluation of passive samplers as a monitoring tool for early warning of Dinophysis toxins in shellfish.

From June 2006 to January 2007 passive samplers (solid phase adsorbing toxin tracking, SPATT) were tested as a monitoring tool with weekly monitoring of phytoplankton and toxin content (liquid chromatography-mass spectrometry, LC-MS) in picked cells of Dinophysis and plankton concentrates. Successive blooms of Dinophysis acuminata, D. acuta and D. caudata in 2006 caused a long mussel harvesting closure (4.5 months) in the Galician Rías (NW Spain) and a record (up to 9246 ng·g resin-week-1) accumulation of toxins in SPATT discs. Best fit of a toxin accumulation model was between toxin accumulation in SPATT and the product of cell densities by a constant value, for each species of Dinophysis, of toxin content (average) in picked cells. Detection of Dinophysis populations provided earlier warning of oncoming diarrhetic shellfish poisoning (DSP) outbreaks than the SPATT, which at times overestimated the expected toxin levels in shellfish because: (i) SPATT accumulated toxins did not include biotransformation and depuration loss terms and (ii) accumulation of toxins not available to mussels continued for weeks after Dinophysis cells were undetectable and mussels were toxin-free. SPATT may be a valuable environmental monitoring and research tool for toxin dynamics, in particular in areas with no aquaculture, but does not provide a practical gain for early warning of DSP outbreaks.

Climate variability and oceanographic settings associated with interannual variability in the initiation of Dinophysis acuminata blooms.

In 2012, there were exceptional blooms of D. acuminata in early spring in what appeared to be a mesoscale event affecting Western Iberia and the Bay of Biscay. The objective of this work was to identify common climatic patterns to explain the observed anomalies in two important aquaculture sites, the Galician Rías Baixas (NW Spain) and Arcachon Bay (SW France). Here, we examine climate variability through physical-biological couplings, Sea Surface Temperature (SST) anomalies and time of initiation of the upwelling season and its intensity over several decades. In 2012, the mesoscale features common to the two sites were positive anomalies in SST and unusual wind patterns. These led to an atypical predominance of upwelling in winter in the Galician Rías, and increased haline stratification associated with a southward advection of the Gironde plume in Arcachon Bay. Both scenarios promoted an early phytoplankton growth season and increased stability that enhanced D. acuminata growth. Therefore, a common climate anomaly caused exceptional blooms of D. acuminata in two distant regions through different triggering mechanisms. These results increase our capability to predict intense diarrhetic shellfish poisoning outbreaks in the early spring from observations in the preceding winter.

Oceanographical Context of the First Bloom of the Silicoflagellate Octactis speculum (Ehrenberg) Recorded to Cause Salmon Mortality in a Galician Ria: Was This Bloom a Rare Event in the Iberian Coast?

Harmful algal blooms are one of the leading causes of mortality in salmon aquaculture, with significant economic consequences. From 15 to 31 October 1996, a bloom of the skeletonized form of Octactis speculum (Ehrenberg) F.H. Chang, J.M. Grieve & J.E. Sutherland was detected in the small Merexo inlet (1.7 km2 area), located on the southern shore of the Ria of Muxía (Galicia, NW Spain). The O. speculum population inside the inlet (data period: 1992-1996) seldom exceeded 4·103 cell·L-1. However, its concentration reached 2·105 cell·L-1 during the bloom, coinciding with a decrease in light penetration from 5 to 2 m deep, as measured using a Secchi disk. Although similar concentrations were reported during late October 1992, this was the first time that a bloom was associated with caged salmon (Salmo salar, Linnaeus 1758) mortality in the Galician coastal waters. This mortality was not associated with anoxia in the water column, but with fish gill irritations and mucus segregation due to gill clogging. Excess nitrate and silicate, the latter being essential for skeleton formation, were measured in the inlet during the bloom, with phosphate acting as the limiting nutrient (high negative correlation). Blooms of O. speculum occurred in autumn-winter, when water was retained within the inlet under meteorological conditions of southwest winds (which prompted downwelling conditions) and clear skies. A review of the oceanographic database of the Galician rias showed that massive O. speculum proliferations are also commonplace in other rias with similar environmental conditions, such as the Ria of Ares-Betanzos, and can therefore constitute a threat for the development of salmon aquaculture on this coast.

Twenty-Five Years of PSP Toxicity in Galician (NW Spain) Bivalves: Spatial, Temporal, and Interspecific Variations.

Twenty-five years of paralytic shellfish poisoning (PSP) toxicity in Galician bivalves have been studied. PSP was detected in 4785 out of 73,740 samples of the commercially important bivalve species analyzed from 1995 to 2020. Its general prevalence in the area was 6.5%. Only 1.6% of all samples tested were over the regulatory limit (incidence). The maximum level of PSP in the area, 40,800 µg STX 2HCl-eq kg-1, was recorded in raft mussels from Bueu (PON-II, Pontevedra) in December 2005. The highest maximum PSP values were found in mussels, which were mostly affected by Gymnodinium catenatum, but not those of prevalence and incidence which were recorded in clams, mostly affected by Alexandrium. Average levels in mussels were higher than in any other studied species. Spatially, in general, the prevalence, incidence, maximum, and average PSP toxicity during episodes tend to decrease from south to northeast, but some hot points with high levels can be identified. PCA analysis separates the southern rías, associated to G. catenatum blooms, from the middle and northern ones, associated to Alexandrium blooms. Along the year, two main peaks of the four variables are observed, the first one in late autumn-winter and the other in summer, the summer peak being much more important for the infaunal species than for raft mussels. In the seasonal pattern obtained by time series analysis of the average PSP toxicity, the autumn-winter peak was only maintained (and very reduced) in the southern rías, indicating that this peak is seasonally much less important than the summer peak. The observed seasonality is expected based on the timing of the blooms of the two PSP-producing phytoplankton groups present in the area. Over the 25 years of monitoring, large differences in PSP toxicity have been observed. Apart from some special years, an ascending trend in prevalence and incidence seems to be present from 2011 to 2020. No trend seems to exist during the same period for average or maximum toxicity.

Obestatin as a regulator of adipocyte metabolism and adipogenesis.

The role of obestatin, a 23-amino-acid peptide encoded by the ghrelin gene, on the control of the metabolism of pre-adipocyte and adipocytes as well as on adipogenesis was determined. For in vitro assays, pre-adipocyte and adipocyte 3T3-L1 cells were used to assess the obestatin effect on cell metabolism and adipogenesis based on the regulation of the key enzymatic nodes, Akt and AMPK and their downstream targets. For in vivo assays, white adipose tissue (WAT) was obtained from male rats under continuous subcutaneous infusion of obestatin. Obestatin activated Akt and its downstream targets, GSK3α/ß, mTOR and S6K1, in 3T3-L1 adipocyte cells. Simultaneously, obestatin inactivated AMPK in this cell model. In keeping with this, ACC phosphorylation was also decreased. This fact was confirmed in vivo in white adipose tissue (omental, subcutaneous and gonadal) obtained from male rats under continuous sc infusion of obestatin (24 and 72 hrs). The relevance of obestatin as regulator of adipocyte metabolism was supported by AS160 phosphorylation, GLUT4 translocation and augment of glucose uptake in 3T3-L1 adipocyte cells. In contrast, obestatin failed to modify translocation of fatty acid transporters, FATP1, FATP4 and FAT/CD36, to plasma membrane. Obestatin treatment in combination with IBMX and DEX showed to regulate the expression of C/EBPα, C/EBPß, C/EBPδ and PPARγ promoting adipogenesis. Remarkable, preproghrelin expression, and thus obestatin expression, increased during adipogenesis being sustained throughout terminal differentiation. Neutralization of endogenous obestatin secreted by 3T3-L1 cells by anti-obestatin antibody decreased adipocyte differentiation. Furthermore, knockdown experiments by preproghrelin siRNA supported that obestatin contributes to adipogenesis. In summary, obestatin promotes adipogenesis in an autocrine/paracrine manner, being a regulator of adipocyte metabolism. These data point to a putative role in the pathogenesis of metabolic syndrome.

Growth hormone-releasing hormone as an agonist of the ghrelin receptor GHS-R1a.

Ghrelin synergizes with growth hormone-releasing hormone (GHRH) to potentiate growth hormone (GH) response through a mechanism not yet fully characterized. This study was conducted to analyze the role of GHRH as a potential ligand of the ghrelin receptor, GHS-R1a. The results show that hGHRH(1-29)NH(2) (GHRH) induces a dose-dependent calcium mobilization in HEK 293 cells stably transfected with GHS-R1a an effect not observed in wild-type HEK 293 cells. This calcium rise is also observed using the GHRH receptor agonists JI-34 and JI-36. Radioligand binding and cross-linking studies revealed that calcium response to GHRH is mediated by the ghrelin receptor GHS-R1a. GHRH activates the signaling route of inositol phosphate and potentiates the maximal response to ghrelin measured in inositol phosphate turnover. The presence of GHRH increases the binding capacity of (125)I-ghrelin in a dose dependent-fashion showing a positive binding cooperativity. In addition, confocal microscopy in CHO cells transfected with GHS-R1a tagged with enhanced green fluorescent protein shows that GHRH activates the GHS-R1a endocytosis. Furthermore, the selective GHRH-R antagonists, JV-1-42 and JMR-132, act also as antagonists of the ghrelin receptor GHS-R1a. Our findings suggest that GHRH interacts with ghrelin receptor GHS-R1a, and, in consequence, modifies the ghrelin-associated intracellular signaling pathway. This interaction may represent a form of regulation, which could play a putative role in the physiology of GH regulation and appetite control.

Obestatin signalling counteracts glucocorticoid-induced skeletal muscle atrophy via NEDD4/KLF15 axis.

BACKGROUND: A therapeutic approach for the treatment of glucocorticoid-induced skeletal muscle atrophy should be based on the knowledge of the molecular mechanisms determining the unbalance between anabolic and catabolic processes and how to re-establish this balance. Here, we investigated whether the obestatin/GPR39 system, an autocrine signalling system acting on myogenesis and with anabolic effects on the skeletal muscle, could protect against chronic glucocorticoid-induced muscle atrophy. METHODS: In this study, we used an in vivo model of muscle atrophy induced by the synthetic glucocorticoid dexamethasone to examine the liaison molecules that define the interaction between the glucocorticoid receptor and the obestatin/GPR39 systems. The findings were extended to in vitro effects on human atrophy using human KM155C25 myotubes. RESULTS: KLF15 and FoxO transcription factors were identified as direct targets of obestatin signalling in the control of proteostasis in skeletal muscle. The KLF15-triggered gene expression program, including atrogenes and FoxOs, was regulated via KLF15 ubiquitination by the E3 ubiquitin ligase NEDD4. Additionally, a specific pattern of FoxO post-translational modification, including FoxO4 phosphorylation by Akt pathway, was critical in the regulation of the ubiquitin-proteasome system. The functional cooperativity between Akt and NEDD4 in the regulation of FoxO and KLF15 provides integrated cues to counteract muscle proteostasis and re-establish protein synthesis. CONCLUSIONS: The effective control of FoxO activity in response to glucocorticoid is critical to counteract muscle-related pathologies. These results highlight the potential of the obestatin/GPR39 system to fine-tune the effects of glucocorticoids on skeletal muscle wasting.

Diversity and regional distribution of harmful algal events along the Atlantic margin of Europe.

The IOC-ICES-PICES Harmful Algal Event Database (HAEDAT) was used to describe the diversity and spatiotemporal distribution of harmful algal events along the Atlantic margin of Europe from 1987 - 2018. The majority of events recorded are caused by Diarrhetic Shellfish Toxins (DSTs). These events are recorded annually over a wide geographic area from southern Spain to northern Scotland and Iceland, and are responsible for annual closures of many shellfish harvesting areas. The dominant causative dinoflagellates, members of the morphospecies 'Dinophysis acuminata complex' and D. acuta, are common in the waters of the majority of countries affected. There are regional differences in the causative species associated with PST events; the coasts of Spain and Portugal with the dinoflagellates Alexandrium minutum and Gymnodinium catenatum, north west France/south west England/south Ireland with A. minutum, and Scotland/Faroe Islands/Iceland with A. catenella. This can influence the duration and spatial scale of PST events as well as the toxicity of shellfish. The diatom Pseudo-nitzschia australis is the most widespread Domoic Acid (DA) producer, with records coming from Spain, Portugal, France, Ireland and the UK. Amnesic Shellfish Toxins (ASTs) have caused prolonged closures for the scallop fishing industry due to the slow depuration rate of DA. Amendments to EU shellfish hygiene regulations introduced between 2002 and 2005 facilitated end-product testing and sale of adductor muscle. This reduced the impact of ASTs on the scallop fishing industry and thus the number of recorded HAEDAT events. Azaspiracids (AZAs) are the most recent toxin group responsible for events to be characterised in the ICES area. Events associated with AZAs have a discrete distribution with the majority recorded along the west coast of Ireland. Ciguatera Poisoning (CP) has been an emerging issue in the Canary Islands and Madeira since 2004. The majority of aquaculture and wild fish mortality events are associated with blooms of the dinoflagellate Karenia mikimotoi and raphidophyte Heterosigma akashiwo. Such fish killing events occur infrequently yet can cause significant mortalities. Interannual variability was observed in the annual number of HAEDAT areas with events associated with individual shellfish toxin groups. HABs represent a continued risk for the aquaculture industry along the Atlantic margin of Europe and should be accounted for when considering expansion of the industry or operational shifts to offshore areas.

Interaction between ghrelin and the ghrelin receptor (GHS-R1a), a NMR study using living cells.

The study of the interaction of ghrelin (1), the endogenous ligand for the GH secretagogues receptor (GHS-R1a), and des-acyl ghrelin (2) with the GHS-R1a by NMR using living cells is presented, using GHS-R1a stably transfected cell lines (CHO and HEK 293) and wild type cells. Therefore, the interaction of 1 and 2 with the GHS-R1a receptor has been performed using quasi-physiological conditions. Ghrelin (1), showed a higher number of residues affected by chemical shift perturbation (CSP) or chemical shift exchange (CSE) effects: Ser3, Phe4, Leu5, Val12, Gln13/Gln14, Lys16/Lys19, Glu17 and Lys24 were much more affected in 1 than in des-acyl ghrelin (2). The chemical shift index CSI values indicated the presence of a possible alpha-helical region between Glu8 and Lys20 for ghrelin (1). After analysing the NMR data, two possible structures have arisen, which present different proline rotamers: the EEZE and the EZEZ conformers, at positions Pro7, Pro21, Pro22 and Pro27, respectively, keeping a left-handed alpha-helix from Glu8 to Lys20. These experimental evidences might imply that the GHS-R1a receptor is acting as a prolyl-cis/trans isomerase.

Obestatin stimulates Akt signalling in gastric cancer cells through beta-arrestin-mediated epidermal growth factor receptor transactivation.

Obestatin was identified as a gut peptide encoded by the ghrelin gene that interacts with the G protein-coupled receptor, GPR39. In this work, a sequential analysis of its transmembrane signalling pathway has been undertaken to characterize the intracellular mechanisms responsible for Akt activation. The results show that Akt activation requires the phosphorylation of T308 in the A-loop by the phosphoinositide-dependent kinase 1 (PDK1) and S473 within the HM by the mammalian target of rapamycin (mTOR) kinase complex 2 (mTORC2: Rictor, mLST8, mSin1, mTOR kinase) with participation neither of G(i)(/o)-protein nor Gbetagamma dimers. Obestatin induces the association of GPR39/beta-arrestin 1/Src signalling complex resulting in the transactivation of the epidermal growth factor receptor (EGFR) and downstream Akt signalling. Upon administration of obestatin, phosphorylation of mTOR (S2448) and p70S6K1 (T389) rise with a time course that parallels that of Akt activation. Based on the experimental data obtained, a signalling pathway involving a beta-arrestin 1 scaffolding complex and EGFR to activate Akt signalling is proposed.

Role of obestatin on growth hormone secretion: An in vitro approach.

Obestatin, the ghrelin-associated peptide, showed to activate MAPK signaling with no effect on Akt nor cell proliferating activity in rat tumor somatotroph cells (growth cells, GC). A sequential analysis of the obestatin transmembrane signaling pathway indicated a route involving the consecutive activation of G(i), PI3k, novel PKCepsilon, and Src for ERK1/2 activation. Furthermore, obestatin treatment triggers growth hormone (GH) release in the first 30min, being more acute at 15min. At 1h, obestatin treated cells showed the same levels in GH secretion than controls. Added to this functionality, obestatin was secreted by GC cells. Based on the capacity to stimulate GH release from somatotroph cells, obestatin may act directly in the pituitary through an autocrine/paracrine mechanism.

Environmental determinants of the occurrence and distribution of Vibrio parahaemolyticus in the rias of Galicia, Spain.

Infections associated with Vibrio parahaemolyticus on the coast of Galicia (in northwestern Spain) were reported to be linked to large outbreaks of illness during 1999 and 2000. Little information is available about the ecological factors that influence the emergence of V. parahaemolyticus infections in this temperate region. We carried out a 3-year study to investigate the occurrence and distribution of V. parahaemolyticus at 26 sites located in the four main rias of Galicia in association with environmental and oceanographic variables. V. parahaemolyticus was detected in all the areas investigated and throughout the complete period of study with an overall incidence of 12.5%. Salinity was the primary factor governing the temporal and spatial distribution of V. parahaemolyticus, whereas seawater temperature had a secondary effect and only modulated the abundance in periods and areas of reduced salinities. Higher occurrence of V. parahaemolyticus was observed during periods of lower salinity in autumn, with a total of 61 positive samples (18%) and a mean density of 1,234 most probable number/100 g. V. parahaemolyticus was primarily detected in areas of reduced salinity close to freshwater discharge points, where it was found in up to 45% of the samples. Characterization of the isolates obtained from the study resulted in the first identification of two pathogenic tdh-positive strains of V. parahaemolyticus recovered from the marine environment in Galicia. These isolates showed serotypes identical to and DNA profiles indistinguishable from those of the clinical clone of V. parahaemolyticus dominant in infections in Spain in the last 10 years.

Self-assembled monolayer-based immunoassays for okadaic acid detection in seawater as monitoring tools.

Rapid and cost-effective methods to monitor the presence of diarrhetic shellfish poisoning (DSP) toxins in seawater samples in an easy and reliable manner are required to protect human health and avoid economic losses to shellfish industry. Immunoassays for the detection of okadaic acid (OA) and dinophysistoxin-1 and dinophysistoxin-2 are developed by immobilising OA on self-assembled monothiols or dithiols in an ordered and oriented way, providing an effective limit of detection of ∼1 ng OA equiv./mL seawater. The immunoassays are applied to the analysis of the particulate fraction of seawater samples from two Catalan harbours (NW Mediterranean) and samples collected periodically from the Galician Rias (E Atlantic), as well as a reference mussel sample. Results are in agreement with LC-MS/MS and the certified values. OA concentration in seawater correlates with Dinophysis cell abundance, with a 1-2 weeks lag. The immunoassays provide powerful high-throughput analytical methods potentially applicable as alternative monitoring tools.

Improvement of Duchenne muscular dystrophy phenotype following obestatin treatment.

BACKGROUND: This study was performed to test the therapeutic potential of obestatin, an autocrine anabolic factor regulating skeletal muscle repair, to ameliorate the Duchenne muscular dystrophy (DMD) phenotype. METHODS AND RESULTS: Using a multidisciplinary approach, we characterized the ageing-related preproghrelin/GPR39 expression patterns in tibialis anterior (TA) muscles of 4-, 8-, and 18-week-old mdx mice (n = 3/group) and established the effects of obestatin administration at this level in 8-week-old mdx mice (n = 5/group). The findings were extended to in vitro effects on human immortalized DMD myotubes. An analysis of TAs revealed an age-related loss of preproghrelin expression, as precursor of obestatin, in mdx mice. Administration of obestatin resulted in a significant increase in tetanic specific force (33.0% ± 1.5%, P < 0.05), compared with control mdx mice. Obestatin-treated TAs were characterized by reduction of fibres with centrally located nuclei (10.0% ± 1.2%, P < 0.05) together with an increase in the number of type I fibres (25.2% ± 1.7%, P < 0.05) associated to histone deacetylases/myocyte enhancer factor-2 and peroxisome proliferator-activated receptor-gamma coactivator 1α axis, and down-regulation of ubiquitin E3-ligases by inactivation of FoxO1/4, indexes of muscle atrophy. Obestatin reduced the level of contractile damage and tissue fibrosis. These observations correlated with decline in serum creatine kinase (58.8 ± 15.2, P < 0.05). Obestatin led to stabilization of the sarcolemma by up-regulation of utrophin, α-syntrophin, ß-dystroglycan, and α7ß1-integrin proteins. These pathways were also operative in human DMD myotubes. CONCLUSIONS: These results highlight the potential of obestatin as a peptide therapeutic for preserving muscle integrity in DMD, thus allowing a better efficiency of gene or cell therapy in a combined therapeutic approach.