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.

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.

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.

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.

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.

Publisher Correction: Obestatin controls skeletal muscle fiber-type determination.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

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.

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.

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.

Obestatin controls the ubiquitin-proteasome and autophagy-lysosome systems in glucocorticoid-induced muscle cell atrophy.

BACKGROUND: Many pathological states characterized by muscle atrophy are associated with an increase in circulating glucocorticoids and poor patient prognosis, making it an important target for treatment. The development of treatments for glucocorticoid-induced and wasting disorder-related skeletal muscle atrophy should be designed based on how the particular transcriptional program is orchestrated and how the balance of muscle protein synthesis and degradation is deregulated. Here, we investigated whether the obestatin/GPR39 system, an autocrine/paracrine signaling system acting on myogenesis and with anabolic effects on the skeletal muscle, could protect against glucocorticoid-induced muscle cell atrophy. METHODS: In the present study, we have utilized mouse C2C12 myotube cultures to examine whether the obestatin/GPR39 signaling pathways can affect the atrophy induced by the synthetic glucocorticoid dexamethasone. We have extended these findings to in vitro effects on human atrophy using human KM155C25 myotubes. RESULTS: The activation of the obestatin/GPR39 system protects from glucocorticoid-induced atrophy by regulation of Akt, PKD/PKCµ, CAMKII and AMPK signaling and its downstream targets in the control of protein synthesis, ubiquitin-proteasome system and autophagy-lysosome system in mouse cells. We compared mouse and human myotube cells in their response to glucocorticoid and identified differences in both the triggering of the atrophic program and the response to obestatin stimulation. Notably, we demonstrate that specific patterns of post-translational modifications of FoxO4 and FoxO1 play a key role in directing FoxO activity in response to obestatin in human myotubes. CONCLUSIONS: Our findings emphasize the function of the obestatin/GPR39 system in coordinating a variety of pathways involved in the regulation of protein degradation during catabolic conditions.

Obestatin controls skeletal muscle fiber-type determination.

Obestatin/GPR39 signaling stimulates skeletal muscle growth and repair by inducing both G-protein-dependent and -independent mechanisms linking the activated GPR39 receptor with distinct sets of accessory and effector proteins. In this work, we describe a new level of activity where obestatin signaling plays a role in the formation, contractile properties and metabolic profile of skeletal muscle through determination of oxidative fiber type. Our data indicate that obestatin regulates Mef2 activity and PGC-1α expression. Both mechanisms result in a shift in muscle metabolism and function. The increase in Mef2 and PGC-1α signaling activates oxidative capacity, whereas Akt/mTOR signaling positively regulates myofiber growth. Taken together, these data indicate that the obestatin signaling acts on muscle fiber-type program in skeletal muscle.

Distinct phosphorylation sites on the ghrelin receptor, GHSR1a, establish a code that determines the functions of ß-arrestins.

The growth hormone secretagogue receptor, GHSR1a, mediates the biological activities of ghrelin, which includes the secretion of growth hormone, as well as the stimulation of appetite, food intake and maintenance of energy homeostasis. Mapping phosphorylation sites on GHSR1a and knowledge of how these sites control specific functional consequences unlocks new strategies for the development of therapeutic agents targeting individual functions. Herein, we have identified the phosphorylation of different sets of sites within GHSR1a which engender distinct functionality of ß-arrestins. More specifically, the Ser(362), Ser(363) and Thr(366) residues at the carboxyl-terminal tail were primarily responsible for ß-arrestin 1 and 2 binding, internalization and ß-arrestin-mediated proliferation and adipogenesis. The Thr(350) and Ser(349) are not necessary for ß-arrestin recruitment, but are involved in the stabilization of the GHSR1a-ß-arrestin complex in a manner that determines the ultimate cellular consequences of ß-arrestin signaling. We further demonstrated that the mitogenic and adipogenic effect of ghrelin were mainly dependent on the ß-arrestin bound to the phosphorylated GHSR1a. In contrast, the ghrelin function on GH secretion was entirely mediated by G protein signaling. Our data is consistent with the hypothesis that the phosphorylation pattern on the C terminus of GHSR1a determines the signaling and physiological output.

The role of the obestatin/GPR39 system in human gastric adenocarcinomas.

Obestatin, a 23-amino acid peptide encoded by the ghrelin gene, and the GPR39 receptor were reported to be involved in the control of mitogenesis of gastric cancer cell lines; however, the relationship between the obestatin/GPR39 system and gastric cancer progression remains unknown. In the present study, we determined the expression levels of the obestatin/GPR39 system in human gastric adenocarcinomas and explored their potential functional roles. Twenty-eight patients with gastric adenocarcinomas were retrospectively studied, and clinical data were obtained. The role of obestatin/GPR39 in gastric cancer progression was studied in vitro using the human gastric adenocarcinoma AGS cell line. Obestatin exogenous administration in these GPR39-bearing cells deregulated the expression of several hallmarks of the epithelial-mesenchymal transition (EMT) and angiogenesis. Moreover, obestatin signaling promoted phenotypic changes via GPR39, increasingly impacting on the cell morphology, proliferation, migration and invasion of these cells. In healthy human stomachs, obestatin expression was observed in the neuroendocrine cells and GPR39 expression was localized mainly in the chief cells of the oxyntic glands. In human gastric adenocarcinomas, no obestatin expression was found; however, an aberrant pattern of GPR39 expression was discovered, correlating to the dedifferentiation of the tumor. Altogether, our data strongly suggest the involvement of the obestatin/GPR39 system in the pathogenesis and/or clinical outcome of human gastric adenocarcinomas and highlight the potential usefulness of GPR39 as a prognostic marker in gastric cancer.

Climate variability and Dinophysis acuta blooms in an upwelling system.

Dinophysis acuta is a frequent seasonal lipophilic toxin producer in European Atlantic coastal waters associated with thermal stratification. In the Galician Rías, populations of D. acuta with their epicentre located off Aveiro (northern Portugal), typically co-occur with and follow those of Dinophysis acuminata during the upwelling transition (early autumn) as a result of longshore transport. During hotter than average summers, D. acuta blooms also occur in August in the Rías, when they replace D. acuminata. Here we examined a 30-year (1985-2014) time series of D. acuta from samples collected by the same method in the Galician Rías. Our main objective was to identify patterns of distribution and their relation with climate variability, and to explain the exceptional summer blooms of D. acuta in 1989-1990. A dome-shaped relationship was found between summer upwelling intensity and D. acuta blooms; cell maxima were associated with conditions where the balance between upwelling intensity and heating, leading to deepened thermoclines, combined with tidal phase (3 days after neap tides) created windows of opportunity for this species. The application of a generalized additive model based on biological (D. acuta inoculum) and environmental predictors (Cumulative June-August upwelling CUIJJA, average June-August SSTJJA and tidal range) explained more than 70% of the deviance for the exceptional summer blooms of D. acuta, through a combination of moderate (35,000-50,000m3s-1km-1) summer upwelling (CUIJJA), thermal stratification (SSTJJA>17°C) and moderate tidal range (∼2.5m), provided D. acuta cells (inoculum) were present in July. There was no evidence of increasing trends in D. acuta bloom frequency/intensity nor a clear relationship with NAO or other long-term climatic cycles. Instead, the exceptional summer blooms of 1989-1990 appeared linked to extreme hydroclimatic anomalies (high positive anomalies in SST and NAO index), which affected most of the European Atlantic coast.

Toward predicting Dinophysis blooms off NW Iberia: A decade of events.

Dinophysis acuminata and Dinophysis acuta are recurrent species off NW Iberia but their outbreaks occur under different conditions. A decade (2004-2013) of weekly data for each species at two sentinel stations located at the entrance of Rias de Aveiro-AV (NW Portugal, 40°38.6'N) and Pontevedra-PO (Galicia, Spain, 42°21.5'N), were used to investigate the regional synchronism and mesoscale differences related to species detection, bloom (>200cellsL-1) initiation and development. Results highlight the high interannual variability of bloom events and summarize the associated meteorological/oceanographic conditions. D. acuta blooms were observed in 2004-2008 and 2013, and the species highest maxima at AV occurred after the highest maxima of its prey Mesodinium, with a time-lag of 2-3 weeks. D. acuminata blooms were observed every year at both stations. The cell concentration time series shows that the blooms generally present a sequence starting in March with D. acuminata in PO and three weeks later in AV, followed by D. acuta that starts at AV and three months later in PO. Exceptionally, D. acuminata blooms occurred earlier at AV than PO, namely in high spring upwelling (2007) or river runoff (2010) years. A four-year gap (2009-2012) of D. acuta blooms occurred after an anomalous 2008 autumn with intense upwelling which is interpreted as the result of an equatorward displacement of the population core. Numerical model solutions are used to analyze monthly alongshore current anomalies and test transport hypotheses for selected events. The results show a strong interannual variability in the poleward/equatorward currents associated with changes in upwelling forcing winds, the advection of D. acuta blooms from AV to PO and the possibility that D. acuminata blooms at AV might result from inocula advected southward from PO. However, the sensitivity of the results to vertical position of the lagrangian tracers call for more studies on species distribution at the various bloom stages.

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.

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.

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.