Interference with the retinoic acid signalling pathway inhibits the initiation of teeth and caudal primary scales in the small-spotted catshark Scyliorhinus canicula.

The retinoic acid (RA) pathway was shown to be important for tooth development in mammals, and suspected to play a key role in tooth evolution in teleosts. The general modalities of development of tooth and "tooth-like" structures (collectively named odontodes) seem to be conserved among all jawed vertebrates, both with regard to histogenesis and genetic regulation. We investigated the putative function of RA signalling in tooth and scale initiation in a cartilaginous fish, the small-spotted catshark Scyliorhinus canicula. To address this issue, we identified the expression pattern of genes from the RA pathway during both tooth and scale development and performed functional experiments by exposing small-spotted catshark embryos to exogenous RA or an inhibitor of RA synthesis. Our results showed that inhibiting RA synthesis affects tooth but not caudal primary scale development while exposure to exogenous RA inhibited both. We also showed that the reduced number of teeth observed with RA exposure is probably due to a specific inhibition of tooth bud initiation while the observed effects of the RA synthesis inhibitor is related to a general delay in embryonic development that interacts with tooth development. This study provides data complementary to previous studies of bony vertebrates and support an involvement of the RA signalling pathway toolkit in odontode initiation in all jawed vertebrates. However, the modalities of RA signalling may vary depending on the target location along the body, and depending on the species lineage.

Determination of Mercury, Methylmercury and Selenium Concentrations in Elasmobranch Meat: Fish Consumption Safety.

This study measures total mercury (THg), methylmercury (MeHg) and selenium (Se) concentrations in elasmobranch fish from an Italian market with the aim of evaluating the risk-benefit associated with their consumption, using estimated weekly intake (EWI), permissible safety level (MeHgPSL), selenium health benefit value (HBVSe) and monthly consumption rate limit (CRmm) for each species. THg and Se were analysed by atomic absorption spectrometry, while MeHg was determined by HrGc/Ms. THg and MeHg concentrations ranged from 0.61 to 1.25 µg g-1 w.w. and from 0.57 to 0.97 µg g-1 w.w., respectively, whereas Se levels were 0.49-0.65 µg g-1 w.w. In most samples European Community limits for THg were surpassed, while for MeHg none of the fish had levels above the limit adopted by FAO/WHO. EWIs for THg and MeHg in many cases were above the provisional tolerable weekly intakes (PTWIs). MeHgPSL estimate showed that fish should contain approximately 50% of the concentration measured to avoid exceeding the PTWI. Nevertheless, the HBVSe index indicated that solely skates were safe for human consumption (HBVSe = 3.57-6.22). Our results highlight the importance of a constant monitoring of THg and MeHg level in fish, especially in apex predators, to avoid the risk of overexposure for consumers.

Dissecting the Interaction Deficiency of a Cartilaginous Fish Digestive Lipase with Pancreatic Colipase: Biochemical and Structural Insights.

A full-length cDNA encoding digestive lipase (SmDL) was cloned from the pancreas of the smooth-hound (Mustelus mustelus). The obtained cDNA was 1350 bp long encoding 451 amino acids. The deduced amino acid sequence has high similarity with known pancreatic lipases. Catalytic triad and disulphide bond positions are also conserved. According to the established phylogeny, the SmDL was grouped with those of tuna and Sparidae lipases into one fish digestive lipase cluster. The recently purified enzyme shows no dependence for bile salts and colipase. For this, the residue-level interactions between lipase-colipase are yet to be clearly understood. The structural model of the SmDL was built, and several dissimilarities were noticed when analyzing the SmDL amino acids corresponding to those involved in HPL binding to colipase. Interestingly, the C-terminal domain of SmDL which holds the colipase shows a significant role for colipase interaction. This is apt to prevent the interaction between fish lipase and the pancreatic colipase which and can provide more explanation on the fact that the classical colipase is unable to activate the SmDL.

A new fluorescent probe for sensing of biothiols and screening of acetylcholinesterase inhibitors.

A new N2O-type BODIPY probe (LF-Bop) has been proposed for the selective and sensitive detection of biologically relevant small molecular thiols. This detection is based on the Michael addition reaction between the thiol and nitrostyrene groups in the probe, which decreases the quenching effect from the nitro group, thus resulting in the recovery of the deep-red fluorescence from the BODIPY structure. The results show that LF-Bop is able to detect all tested free thiols through a fluorescence turn-on assay. The lowest limit of detection (LOD) for glutathione was found to be down to nanomolar levels (220 nM). Based on this probe, we have developed a new fluorescence assay for the screening of acetylcholinesterase inhibitors. In total, 11 natural and synthetic alkaloids have been evaluated. Both experimental measurements and theoretical molecular docking results reveal that both natural berberine and its synthetic derivative dihydroberberine are potential inhibitors of acetylcholinesterase.

Trauma and envenoming caused by stingrays and other fish in a fishing community in Pontal do Paranapanema, state of São Paulo, Brazil: epidemiology, clinical aspects, and therapeutic and preventive measures.

INTRODUCTION: Accidents caused by fish are common in inland fishing communities in Brazil, being work-related injuries in the majority of cases. These populations have no information on the mechanisms of trauma or envenoming. METHODS: Through a questionnaire administered to fishermen, we obtained clinical and epidemiological data on accidents in Rosana, Pontal do Paranapanema, State of São Paulo, Brazil. These data were analyzed and converted into an easily understood prevention and treatment program for the colony. RESULTS: Thirty-nine fishermen replied to the survey. All of the patients had been hurt by fish. Of those mentioned, the yellow catfish (Pimelodus maculatus) was the main fish species associated with injuries, but others also caused trauma to the fishermen. Six fishermen had been envenomed by stingrays. Pain and ulcers were the main symptoms and were described as intolerable. Approximately half of those injured were treated using traditional folk remedies. CONCLUSIONS: The fishermen suffered multiple accidents with catfish, which are venomous and cause intense pain, as well as trauma due to other fish, such as surubins, traíras, freshwater croakers, and piranhas. Approximately 16% of those interviewed presented with envenomation from stingrays. Our data and previous experience in the area led to the creation of a pamphlet with clear language that can effectively help fishermen in the region, an area in need of health services and disease prevention. This initiative also applies to the whole La Plata River basin, which has similar fauna.

Trauma and envenoming caused by stingrays and other fish in a fishing community in Pontal do Paranapanema, state of São Paulo, Brazil: epidemiology, clinical aspects, and therapeutic and preventive measures / Traumas e envenenamentos por arraias e outros peixes em uma colônia de pescadores no pontal do Paranapanema, estado de São Paulo, Brasil: epidemiologia, aspectos clínicos e propostas terapêuticas e preventivas

INTRODUCTION: Accidents caused by fish are common in inland fishing communities in Brazil, being work-related injuries in the majority of cases. These populations have no information on the mechanisms of trauma or envenoming. METHODS: Through a questionnaire administered to fishermen, we obtained clinical and epidemiological data on accidents in Rosana, Pontal do Paranapanema, State of São Paulo, Brazil. These data were analyzed and converted into an easily understood prevention and treatment program for the colony. RESULTS: Thirty-nine fishermen replied to the survey. All of the patients had been hurt by fish. Of those mentioned, the yellow catfish (Pimelodus maculatus) was the main fish species associated with injuries, but others also caused trauma to the fishermen. Six fishermen had been envenomed by stingrays. Pain and ulcers were the main symptoms and were described as intolerable. Approximately half of those injured were treated using traditional folk remedies. CONCLUSIONS: The fishermen suffered multiple accidents with catfish, which are venomous and cause intense pain, as well as trauma due to other fish, such as surubins, traíras, freshwater croakers, and piranhas. Approximately 16% of those interviewed presented with envenomation from stingrays. Our data and previous experience in the area led to the creation of a pamphlet with clear language that can effectively help fishermen in the region, an area in need of health services and disease prevention. This initiative also applies to the whole La Plata River basin, which has similar fauna.

INTRODUÇÃO: Os acidentes causados por peixes são comuns em comunidades de pescadores fluviais no Brasil, sendo acidentes ocupacionais na maioria das vezes. Estas populações não têm informações dos mecanismos de trauma e envenenamento. MÉTODOS: Através de um questionário aplicado aos pescadores, foram obtidos dados clínicos e epidemiológicos sobre acidentes em Rosana, Pontal do Paranapanema, Estado de São Paulo, Brasil. Estes dados foram analisados e convertidos em um programa de prevenção e tratamento de acidentes através de um folheto de fácil assimilação RESULTADOS: Trinta e nove pescadores responderam o questionário. Todos os pacientes apresentaram ferimentos causados por algum peixe. Dos peixes mencionados, mandijubas (Pimelodus maculatus) foram os mais associados aos ferimentos, mas outros também causaram traumas. Em relação às arraias, seis pescadores tinham sido envenenados. Dor intensa e úlceras foram os principais sintomas. Aproximadamente metade dos acidentados usou apenas medidas de tratamentos populares. CONCLUSÕES: Os pescadores apresentaram acidentes múltiplos por mandis, que são peçonhentos e causam dor intensa, assim como traumas por outros peixes, como surubins, traíras, corvinas de água doce e piranhas. Cerca de 16% dos entrevistados apresentaram envenenamento por arraias. Nossos dados e experiência prévia permitiram a criação de um folheto de fácil assimilação pelas populações locais que pode ajudar os pescadores de forma efetiva, em uma área extremamente carente de serviços de saúde e prevenção de doenças. Esta iniciativa é aplicável a toda a bacia do Rio da Prata, área extensa e de fauna similar.

Evolutionary changes of astroglia in Elasmobranchii comparing to amniotes: a study based on three immunohistochemical markers (GFAP, S-100, and glutamine synthetase).

This paper supplements former studies on elasmobranch species with an immunohistochemical investigation into glutamine synthetase and S-100 protein, in addition to GFAP, and extends its scope to the representatives of almost every group of Elasmobranchii: squalomorph sharks, galeomorph sharks, skates (Rajiformes) and rays (Torpediniformes and Myliobatifomes). More glial elements were labeled by S-100 protein, and even more so by using glutamine synthetase immunostaining than by GFAP: more astrocytes (mainly non-perivascular ones) were detected in the telencephalon of sharks, skates and rays. Only the markers S-100 and glutamine synthetase, but not GFAP, characterized the Bergmann-glia of skates and rays and astrocyte-like non-ependymal cells in Squalus acanthias. Another squalomorph shark species, Pristiophorus cirratus, however, had GFAP immunopositive astrocytes. Of all the species studied, the greatest number of GFAP positive astrocytes could be observed in Mobula japanica (order Myliobatiformes), in each major brain part. According to anatomical location, perivascular glia comprised varied types, including even a location in Mobula, which can also be found in mammals. Remnants of radial glia were found in confined areas of skates, less so in rays. In the rhombencephalon and in the spinal cord modified ependymoglia predominated in every group. In conclusion, there was no meaningful difference between the astroglial architectures of squalomorph and galeomorph sharks. The difference in the astroglial structure between sharks and batoids, however, was confined to the telencephalon and mesencephalon, and did not take place in the rhombencephalon, the latter structure being quite similar in all the species studied. The appearance of astrocytes in the relatively thin-walled shark telencephalon, however, indicates that the brain thickening promoted the preponderance of astrocytes rather than their appearance itself. Although the evolutionary changes of astroglia had some similarities in Elasmobranchii and Amniota, there was one meaningful difference: in Elasmobranchii astrocytes did not prevail in conservative brain regions as they did in the progressive brain regions.

New insights on Saccus vasculosus evolution: a developmental and immunohistochemical study in elasmobranchs.

The saccus vasculosus (SV) is a circumventricular organ of the hypothalamus of many jawed fishes whose functions have not yet been clarified. It is a vascularized neuroepithelium that consists of coronet cells, cerebrospinal fluid-contacting (CSF-c) neurons and supporting cells. To assess the organization, development and evolution of the SV, the expression of glial fibrillary acidic protein (GFAP) and the neuronal markers gamma-aminobutyric acid (GABA), glutamic acid decarboxylase (GAD; the GABA synthesizing enzyme), neuropeptide Y (NPY), neurophysin II (NPH), tyrosine hydroxylase (TH; the rate-limiting catecholamine-synthesizing enzyme) and serotonin (5-HT), were investigated by immunohistochemistry in developing and adult sharks. Coronet cells showed GFAP immunoreactivity from embryos at stage 31 to adults, indicating a glial nature. GABAergic CSF-c neurons were evidenced just when the primordium of the SV becomes detectable (at stage 29). Double immunolabeling revealed colocalization of NPY and GAD in these cells. Some CSF-c cells showed TH immunoreactivity in postembryonic stages. Saccofugal GABAergic fibers formed a defined SV tract from the stage 30 and scattered neurosecretory (NPH-immunoreactive) and monoaminergic (5-HT- and TH-immunoreactive) saccopetal fibers were first detected at stages 31 and 32, respectively. The early differentiation of GABAergic neurons and the presence of a conspicuous GABAergic saccofugal system are shared by elasmobranch and teleosts (trout), suggesting that GABA plays a key function in the SV circuitry. Monoaminergic structures have not been reported in the SV of bony fishes, and were probably acquired secondarily in sharks. The existence of saccopetal monoaminergic and neurosecretory fibers reveals reciprocal connections between the SV and hypothalamic structures which have not been previously detected in teleosts.

Hyperbaric oxygen therapy in the treatment of soft tissue necrosis resulting from a stingray puncture.

Necrotizing soft tissue processes of the foot secondary to an acute stingray envenomation can be a challenge to manage. Very little is reported in the orthopaedic literature to aid the practicing surgeon faced with this problem. In this case report, we describe the wound management and team approach employed in this patient's care, including the indications for hyperbaric oxygen therapy, which, in this case, was ultimately successful.

Hypophysial and extrahypophysial projections of the neurosecretory system of cartilaginous fishes: an immunocytochemical study using a polyclonal antibody against dogfish neurophysin.

Immunocytochemistry using antibodies against the neurohypophysial nonapeptides has given equivocal results regarding relevant aspects of the classical neurosecretory system of elasmobranchs. The lack of antibodies reacting with the elasmobranch neurophysins (Nps) has prevented the study of this neurosecretory system by Nps immunocytochemistry. This led us to purify Nps from Scyliorhinus canicula, and to use them to raise a polyclonal antibody. This antibody reacted strongly with the elasmobranch neurophysin neurons, revealing their most delicate and distant hypophysial and extrahypophysial projections. A detailed mapping of the neurosecretory system of five elasmobranch species (Etmopterus spinax, Squalus acanthias, Scyliorhinus canicula, Galeus melanostomus, Raja radiata) and one holocephalian species (Hydrolagus colliei) was performed. In elasmobranchs, the magnocellular neurophysin cells formed a distinct preoptic nucleus, whereas in Hydrolagus the immunoreactive cells were scattered. Distinct parvicellular neurophysin cells were present in the preoptic nucleus. In Raja the nucleus "O" contained parvicellular Nps-immunoreactive neurons. The findings at the pituitary level point to the possibility that neurophysin neurons, in addition to releasing nonapeptides into the systemic capillaries of the neural lobe, also participate in the regulation of the function of the rostral, medial and intermediate lobes of the adenohypophysis by a dual mechanism, i.e., a neurovascular pathway and a direct neural input. The extrahypophysial projections of the neurophysin neurons were highly developed to a degree not comparable to any other vertebrate group. The targets of these projections were located in the telencephalon, diencephalon and hindbrain. The evolutionary and functional implications of this phenomenon are discussed.

Macrophage-lymphocyte cell clusters in the hypothalamic ventricle of some elasmobranch fish: ultrastructural analysis and possible functional significance.

BACKGROUND: Previous studies have demonstrated the existence of lympho-haemopoietic tissue in the meninges and choroid plexuses of various primitive vertebrates, including the stingray Dasyatis akajei and in early human embryos. In the present study, we extend these results analyzing macrophage-lymphocyte cell clusters found in the floor of the hypothalamic ventricle of several specimens of elasmobranchs. METHODS: After aseptical isolation of the brain from several specimens of smooth dogfish Triakis scyllia, cloudy dogfish Scyliorhinus torazame, gummy shark Mustelus manazo, and stingray Dasyatis akajei their hypothalamic regions were processed routinely by light, scanning, and transmission electron microscopy. RESULTS: The study of serial histological sections demonstrated that the macrophage-lymphocyte cell clusters proceeded from the meningeal lymphohaemopoietic tissue, reaching the ventricular lumen along large blood vessels. In this tissue, macrophages, different sized lymphocytes, lymphoblasts, granulocytes, monocytes, and developing and mature plasma cells were closely packed among a meshwork of fibroblastic reticular cell processes. It never invaded the brain parenchyma. A cell layer of glial elements and a continuous basement membrane interposed between the lymphoid tissue and the neural elements although some macrophages had migrated across the ependymal cell layer. In the ventricular lumen very irregular macrophages with long cell processes and containing abundant engulfed material of unknown origin formed big cell clusters with neighboring lymphocytes, lymphoblasts, and plasma cells, similar to those described during the immune response. Moreover, electron lucent cells which resembled the antigen-presenting cells of higher vertebrates established intimate surface cell contacts with the surrounding lymphocytes. In the third ventricle of several specimens of gummy shark, Mustelus manazo, morphologically similar cell clusters appeared but these were not connected to the meningeal lympho-haemopoietic tissue. No intraventricular cell aggregates were found in the stingray brain. CONCLUSIONS: Although we cannot rule out that these macrophage-lymphocyte cell clusters represent a permanent structure in the elasmobranch brain they rather seem to be only established after specific stimulation for preventing the entrance of noxious, foreign materials into the elasmobranch brain parenchyma.