Exploring the molecular pathways linking sleep phenotypes and POGZ-associated neurodevelopmental disorder.

Pogo transposable element-derived protein with ZNF domain (POGZ) gene encodes a chromatin regulator and rare variants on this gene have been associated with a broad spectrum of neurodevelopmental disorders, such as White-Sutton syndrome. Patient clinical manifestations frequently include developmental delay, autism spectrum disorder and obesity. Sleep disturbances are also commonly observed in these patients, yet the biological pathways which link sleep traits to the POGZ-associated syndrome remain unclear. We screened for sleep implications among individuals with causative POGZ variants previously described. Sleep disturbances were observed in 52% of patients, and being obese was not observed as a risk factor for sleep problems. Next, we identified genes associated with sleep-associated traits among the POGZ regulatory targets, aiming to uncover the molecular pathways that, when disrupted by POGZ loss of function, contribute to the aetiology of sleep phenotypes in these patients. The intersect between POGZ targets and sleep-related genes was used in a pathway enrichment analysis. Relevant pathways among these overlapping genes are involved in the regulation of circadian rhythm, tau protein binding, ATPase activator activity. This study may represent the beginning for novel functional investigations on shared molecular mechanisms between sleep disturbances and rare developmental syndromes related to POGZ and its regulatory targets.

Environmental salinity modulates olfactory sensitivity in the euryhaline European seabass, Dicentrarchus labrax, acclimated to seawater and brackish water.

The olfactory epithelium of fish is - of necessity - in intimate contact with the surrounding water. In euryhaline fish, movement from seawater to freshwater (and vice versa) exposes the epithelium to massive changes in salinity and ionic concentrations. How does the olfactory system function in the face of such changes? The current study compared olfactory sensitivity in seawater- (35‰) and brackish water-adapted seabass (5‰) using extracellular multi-unit recording from the olfactory nerve. Seawater-adapted bass had higher olfactory sensitivity to amino acid odorants when delivered in seawater than in freshwater. Conversely, brackish water-adapted bass had largely similar sensitivities to the same odorants when delivered in seawater or freshwater, although sensitivity was still slightly higher in seawater. The olfactory system of seawater-adapted bass was sensitive to decreases in external [Ca2+], whereas brackish water-adapted bass responded to increases in [Ca2+]; both seawater- and brackish water-adapted bass responded to increases in external [Na+] but the sensitivity was markedly higher in brackish water-adapted bass. In seawater-adapted bass, olfactory sensitivity to l-alanine depended on external Ca2+ ions, but not Na+; brackish water-adapted bass did respond to l-alanine in the absence of Ca2+, albeit with lower sensitivity, whereas sensitivity was unaffected by removal of Na+ ions. A possible adaptation of the olfactory epithelium was the higher number of mucous cells in brackish water-adapted bass. The olfactory system of seabass is able to adapt to low salinities, but this is not immediate; further studies are needed to identify the processes involved.

Differential tissue immune stimulation through immersion in bacterial and viral agonists in the Antarctic Notothenia rossii.

The genome evolution of Antarctic notothenioids has been modulated by their extreme environment over millennia and more recently by human-caused constraints such as overfishing and climate change. Here we investigated the characteristics of the immune system in Notothenia rossii and how it responds to 8 h immersion in viral (Poly I:C, polyinosinic: polycytidylic acid) and bacterial (LPS, lipopolysaccharide) proxies. Blood plasma antiprotease activity and haematocrit were reduced in Poly I:C-treated fish only, while plasma protein, lysozyme activity and cortisol were unchanged with both treatments. The skin and duodenum transcriptomes responded strongly to the treatments, unlike the liver and spleen which had a mild response. Furthermore, the skin transcriptome responded most to the bacterial proxy (cell adhesion, metabolism and immune response processes) and the duodenum (metabolism, response to stress, regulation of intracellular signal transduction, and immune system responses) to the viral proxy. The differential tissue response to the two proxy challenges is indicative of immune specialisation of the duodenum and the skin towards pathogens. NOD-like and C-type lectin receptors may be central in recognising LPS and Poly I:C. Other antimicrobial compounds such as iron and selenium-related genes are essential defence mechanisms to protect the host from sepsis. In conclusion, our study revealed a specific response of two immune barrier tissue, the skin and duodenum, in Notothenia rossii when exposed to pathogen proxies by immersion, and this may represent an adaptation to pathogen infective strategies.

Anatomy of the olfactory system and potential role for chemical communication in the sound-producing Lusitanian toadfish, Halobatrachus didactylus.

The current study investigated the structure and function of the olfactory system of the Lusitanian toadfish, Halobatrachus didactylus, using histology and electrophysiology (electro-olfactogram [EOG]), respectively. The olfactory system consists of a digitated anterior peduncle, of unknown function, containing the inhalant nostril. This then leads to a U-shaped olfactory chamber with the olfactory epithelium-identified by Gαolf-immunoreactivity-on the ventral surface. A large lacrimal sac is connected to this tube and is likely involved in generating water movement through the olfactory chamber (this species is largely sedentary). The exhalent nostril lies by the eye and is preceded by a bicuspid valve to ensure one-way flow of water. As do other teleosts, H. didactylus had olfactory sensitivity to amino acids and bile acids. Large-amplitude EOG responses were evoked by fluid from the anterior and posterior testicular accessory glands, and bile and intestinal fluids. Anterior gland and intestinal fluids from reproductive males were significantly more potent than those from non-reproductive males. Male urine and skin mucus proved to be the least potent body fluids tested. These results suggest that chemical communication-as well as acoustic communication-may be important in the reproduction of this species and that this may be mediated by the accessory glands and intestinal fluid.

Genetic basis of sleep phenotypes and rare neurodevelopmental syndromes reveal shared molecular pathways.

Sleep-related phenotypes have been frequently reported in early on-set epileptic encephalopathies and in developmental delay syndromes, in particular in syndromes related to autism spectrum disorder. Yet the convergent pathogenetic mechanisms between these comorbidities are largely unknown. We first performed a gene enrichment study that identified shared risk genes among rare epileptic encephalopathies/neurodevelopmental disorders, rare developmental delay genetic syndromes and sleep disturbances. We then determined cellular and molecular pathways enriched among genes shared between sleep phenotypes and those two early onset mental illnesses, aiming to identify genetic disparities and commonalities among these phenotypic groups. The sleep gene set was observed as significantly overlapped with the two gene lists associated to rare genetic syndromes (i.e., epileptic encephalopathies/neurodevelopmental disorders and developmental delay gene sets), suggesting shared genetic contribution. Similarities across significantly enriched pathways between the two intersect lists comprehended mostly synapse-related pathways, such as retrograde endocannabinoid signaling, serotonergic, and GABAergic synapse. Network analysis indicates epileptic encephalopathies/neurodevelopmental disorders versus sleep-specific clusters and developmental delay versus sleep-specific clusters related to synaptic and transcriptional regulation, respectively. Longstanding functional patterns previously described in epileptic encephalopathies and neurodevelopmental disorders genetic architecture were recaptured after dissecting the overlap between the genes associated to those developmental phenotypes and sleep disturbances, suggesting that during neurodevelopment different molecular and functional mechanisms are related to alterations on circadian rhythm. The overlapping gene set and biological pathways highlighted by this study may serve as a primer for new functional investigations of shared molecular mechanisms between sleep disturbances and rare developmental syndromes.

Assessment of male reproductive traits in endangered leuciscids from the Iberian Peninsula: first attempts to store gametes both at short- and long-term.

During the spring of 2022, several endangered leuciscid species (Anaecypris hispanica, Squalius aradensis, Anachondrostoma Occidentale, and Iberochondrostoma lusitanicum) were sampled both at the Vasco da Gama aquarium facilities and in some rivers of the Algarve region, Portugal. Sperm samples were extracted by gentle abdominal pressure and sperm motion parameters were assessed for the first time in four species, using a computerized analysis system. The results obtained showed that spermatozoa kinetic patterns were similar for all 4 species, with high motility and velocity values after the sperm activation time and with a marked decrease after 20. On the other hand, sperm longevity was highly variable between species, with short longevities (around 40 s) for A. hispanica and S. aradensis, and longer longevities (100-120 s) for A. occidentale and I. lusitanicum, which could indicate a latitudinal pattern in terms of sperm longevity. At the same time, morphometric analysis was carried out for the four target species, revealing that spermatozoa showed similar sizes and shapes to other external fertilizers belonging to Leuscididae, with small spherical heads, uniflagellate, and without acrosomes. In addition, a short-term gamete storage trail was performed by diluting sperm in 1:9 (sperm:extender) and storing them at 4ºC. Although the results obtained were uneven among the species studied, the dilution and extender used generated motilities above 40% up to day 4 of storage in S. aradensis and I. lusitanicum, and up to days 1-2 in A. hispanica and A. occidentale, respectively. Finally, gamete cryopreservation trials were also carried out on these threatened species. Although cryopreserved samples showed significantly lower motility than fresh samples, some protocols generate acceptable percentages of viability, DNA integrity, and sperm motility in some species such as I. lusitanicum and A. occidentale. The data revealed that the protocol based on 10% DMSO plus 7.5% egg yolk generated the best results.This study is the first to assess the reproductive traits of wild and captive populations of endangered leuciscids endemic from the Iberian Peninsula, describing the spermatozoa kinetics and developing protocols for managing male gametes both in short- and long-term storage. Outcomes will provide new and useful tools to complement the management and conservation of ex situ breeding programs that are being developed for these four endangered species.

Freshening effect on the osmotic response of the Antarctic spiny plunderfish Harpagifer antarcticus.

Global warming is having a significant impact around the world, modifying environmental conditions in many areas, including in zones that have been thermally stable for thousands of years, such as Antarctica. Stenothermal sedentary intertidal fish species may suffer due to warming, notably if this causes water freshening from increased freshwater inputs. Acute decreases in salinity, from 33 down to 5, were used to assess osmotic responses to environmental salinity fluctuations in Antarctic spiny plunderfish Harpagifer antarcticus, in particular to evaluate if H. antarcticus is able to cope with freshening and to describe osmoregulatory responses at different levels (haematological variables, muscle water content, gene expression, NKA activity). H. antarcticus were acclimated to a range of salinities (33 as control, 20, 15, 10 and 5) for 1 week. At 5, plasma osmolality and calcium concentration were both at their lowest, while plasma cortisol and percentage muscle water content were at their highest. At the same salinity, gill and intestine Na+ -K+ -ATPase (NKA) activities were at their lowest and highest, respectively. In kidney, NKA activity was highest at intermediate salinities (15 and 10). The salinity-dependent NKA mRNA expression patterns differed depending on the tissue. Marked changes were also observed in the expression of genes coding membrane proteins associated with ion and water transport, such as NKCC2, CFTR and AQP8, and in the expression of mRNA for the regulatory hormone prolactin (PRL) and its receptor (PRLr). Our results demonstrate that freshening causes osmotic imbalances in H. antarcticus, apparently due to reduced capacity of both transport and regulatory mechanisms of key organs to maintain homeostasis. This has implications for fish species that have evolved in stable environmental conditions in the Antarctic, now threatened by climate change.

Pth4, an ancient parathyroid hormone lost in eutherian mammals, reveals a new brain-to-bone signaling pathway.

Regulation of bone development, growth, and remodeling traditionally has been thought to depend on endocrine and autocrine/paracrine modulators. Recently, however, brain-derived signals have emerged as key regulators of bone metabolism, although their mechanisms of action have been poorly understood. We reveal the existence of an ancient parathyroid hormone (Pth)4 in zebrafish that was secondarily lost in the eutherian mammals' lineage, including humans, and that is specifically expressed in neurons of the hypothalamus and appears to be a central neural regulator of bone development and mineral homeostasis. Transgenic fish lines enabled mapping of axonal projections leading from the hypothalamus to the brainstem and spinal cord. Targeted laser ablation demonstrated an essential role for of pth4-expressing neurons in larval bone mineralization. Moreover, we show that Runx2 is a direct regulator of pth4 expression and that Pth4 can activate cAMP signaling mediated by Pth receptors. Finally, gain-of-function experiments show that Pth4 can alter calcium/phosphorus levels and affect expression of genes involved in phosphate homeostasis. Based on our discovery and characterization of Pth4, we propose a model for evolution of bone homeostasis in the context of the vertebrate transition from an aquatic to a terrestrial lifestyle.-Suarez-Bregua, P., Torres-Nuñez, E., Saxena, A., Guerreiro, P., Braasch, I., Prober, D. A., Moran, P., Cerda-Reverter, J. M., Du, S. J., Adrio, F., Power, D. M., Canario, A. V. M., Postlethwait, J. H., Bronner, M E., Cañestro, C., Rotllant, J. Pth4, an ancient parathyroid hormone lost in eutherian mammals, reveals a new brain-to-bone signaling pathway.

Active removal of inorganic phosphate from cerebrospinal fluid by the choroid plexus.

The P(i) concentration of mammalian cerebrospinal fluid (CSF) is about one-half that of plasma, a phenomenon also shown here in the spiny dogfish, Squalus acanthias. The objective of the present study was to characterize the possible role of the choroid plexus (CP) in determining CSF P(i) concentration. The large sheet-like fourth CP of the shark was mounted in Ussing chambers where unidirectional (33)P(i) fluxes revealed potent active transport from CSF to the blood side under short-circuited conditions. The flux ratio was 8:1 with an average transepithelial resistance of 87 ± 17.9 Ω·cm(2) and electrical potential difference of +0.9 ± 0.17 mV (CSF side positive). Active P(i) absorption from CSF was inhibited by 10 mM arsenate, 0.2 mM ouabain, Na(+)-free medium, and increasing the K(+) concentration from 5 to 100 mM. Li(+) stimulated transport twofold compared with Na(+)-free medium. Phosphonoformic acid (1 mM) had no effect on active P(i) transport. RT-PCR revealed both P(i) transporter (PiT)1 and PiT2 (SLC20 family) gene expression, but no Na(+)-P(i) cotransporter II (SLC34 family) expression, in the shark CP. PiT2 immunoreactivity was shown by immunoblot analysis and localized by immunohistochemistry in (or near) the CP apical microvillar membranes of both the shark and rat. PiT1 appeared to be localized primarily to vascular endothelial cells. Taken together, these data indicate that the CP actively removes P(i) from CSF. This process has transport properties consistent with a PiT2, Na(+)-dependent transporter that is located in the apical region of the CP epithelium.

Novel galanin receptors in teleost fish: identification, expression and regulation by sex steroids.

In fish, the onset of puberty, the transition from juvenile to sexually reproductive adult animals, is triggered by the activation of pituitary gonadotropin secretion and its timing is influenced by external and internal factors that include the growth/adiposity status of the animal. Kisspeptins have been implicated in the activation of puberty but peripheral signals coming from the immature gonad or associated to the metabolic/nutritional status are also thought to be involved. Therefore we hypothesize the importance of the galinergic system in the brain and testis of pre-pubertal male sea bass as a candidate to translate the signals leading to activation of testicular maturation. Here, the transcripts for four galanin receptors (GALR), named GALR1a, 1b, 2a and 2b, were isolated from European sea bass, Dicentrarchus labrax. Phylogenetic analysis confirmed the previously reported duplication of GALR1 in teleost fish, and unravelled the duplication of GALR2 in teleost fish and in some tetrapod species. Comparison with human showed that the key amino acids involved in ligand binding are present in the corresponding GALR1 and GALR2 orthologs. Transcripts for all four receptors are expressed in brain and testes of adult fish with GALR1a and GALR1b abundant in testes and hardly detected in ovaries. In order to investigate whether GALR1 dimorphic expression was dependent on steroid context we evaluated the effect of 11-ketotestosterone and 17ß-estradiol treatments on the receptor expression in brain and testes of pre-pubertal males. Interestingly, steroid treatments had no effect on the expression of GALRs in the brain while in the testes, GALR1a and GALR1b were significantly up regulated by 11KT. Altogether, these results support a role for the galaninergic system, in particular the GALR1 paralog, in fish reproductive function.

Dynamics of scale regeneration in seawater- and brackish water-acclimated sea bass, Dicentrarchus labrax.

Scale loss is a common occurrence in both wild fish and those in aquaculture production systems, and regeneration of scales has been described in several freshwater species. Relatively little information exists about this process in marine fish, and in the present study, the chronology of scale regeneration was characterized in juvenile sea bass Dicentrarchus labrax, maintained in full seawater (SW; 36‰, 11.2 mM Ca(2+)) or brackish water (BW; 3.5‰, 1.1 mM Ca(2+)). Despite the significant differences in plasma osmolality, plasma calcium (Ca) and phosphorus (P) were similar between SW and BW. Removal of scales (2.25 cm(2)) did not modify plasma osmolality but a significant reduction in plasma Ca occurred in sea bass in BW during early phases of scale regeneration. Scale Ca and P contents changed during regeneration in SW and BW fish, and in fully regenerated scales, their concentration was comparable to ontogenic scales which had a Ca/P ratio around 1.0. Regenerating scales were first visible at 3 days after removal, and by 6 days, the descaled area was covered with new scales in both SW and BW fish. From this stage onwards, while scale area and mass rose gradually, a rapid accretion of calcium crystals was observed over the basal plate, especially in SW, and Ca and P content per scale increased at a very high rate, decreasing only at the later stages, and between 21 and 30 days the newly formed scales had already ontogenic-like morphometric features. Scale tartrate-resistant acid phosphatase (TRAP) or alkaline phosphatase (ALP) activity was initially similar in SW and BW. However, during regeneration, TRAP was significantly higher in scales of sea bass in SW compared to those in BW where ALP predominated; these differences persisted at 30 days.

Metabolic Responses and Resilience to Environmental Challenges in the Sedentary Batrachoid Halobatrachus didactylus (Bloch & Schneider, 1801).

In the context of climate change, warming of the seas and expansion of hypoxic zones are challenges that most species of fish are, or will be subjected to. Understanding how different species cope with these changes in their environment at the individual level can shed light on how populations and ecosystems will be affected. We provide first-time estimates on the metabolic rates, thermal, and oxygen-related limits for Halobatrachus didactylus, a coastal sedentary fish that lives in intertidal environments of the Northeast Atlantic. Using respirometry in different experimental designs, we found that this species is highly resistant to acute thermal stress (CTmax: 34.82 ± 0.66 °C) and acute hypoxia (Pcrit: 0.59-1.97 mg O2 L-1). We found size-specific differences in this stress response, with smaller individuals being more sensitive. We also quantified its aerobic scope and daily activity patterns, finding this fish to be extremely sedentary, with one of the lowest standard metabolic rates found in temperate fish (SMR: 14.96 mg O2 kg-1h-1). H. didactylus activity increases at night, when its metabolic rate increases drastically (RMR: 36.01 mg O2 kg-1h-1). The maximum metabolic rate of H. didactylus was estimated to be 67.31 mg O2 kg-1h-1, producing an aerobic scope of 52.35 mg O2 kg-1h-1 (77.8% increase). The metrics obtained in this study prove that H. didactylus is remarkably resilient to acute environmental variations in temperature and oxygen content, which might enable it to adapt to the extreme abiotic conditions forecasted for the world's oceans in the near future.

The relationship between neurodevelopmental transcriptional programs and insomnia: From Rubinstein-Taybi syndrome into energy metabolism.

Neurodevelopmental disorders (NDD) are characterized by cognitive, emotional, and/or motor skills impairment since childhood, and sleep disturbances are a common comorbidity. Rubinstein-Taybi syndrome (RSTS), a rare genetic syndrome associated with NDD, is caused by CREBBP haploinsufficiency. This gene encodes an acetyltransferase with crucial role on the establishment of transcriptional programs during neurodevelopment. Although insomnia has been reported in RSTS patients, the convergent mechanisms between this sleep disturbance and CREBBP loss-of-function are not fully understood. We tested weather the genetic architecture underlying CREBBP regulatory targets and insomnia-associated genes is significantly shared. We then identified the biological pathways enriched among these shared genes. The intersection between CREBBP regulatory targets and genes associated with insomnia included 7 overlapping genes, indicating significantly more overlap than expected by chance. An over-representation analysis on these intersect genes identified pathways related to mitochondrial activity. This finding indicates that the transcriptional programs established by CREBBP might impact insomnia-related biological pathways through the modulation of energy metabolism. The overlapping gene set and biological pathways highlighted by this study may serve as a primer for new functional investigations of shared molecular mechanisms between insomnia and CREBBP regulatory targets.

Endocrine and Epigenetic Regulation as Common Pathways Underlying the Genetic Basis of Sleep Traits and Longevity.

The amount of sleep needed over one's lifespan is age dependent and not sleeping enough or sleeping in excess is associated with increased morbidity and mortality. Yet, the convergent molecular mechanisms that link longevity and sleep are largely unknown. We performed a gene enrichment study that (1) identified genes associated with both longevity and sleep traits and (2) determined molecular pathways enriched among these shared genes. We manually curated two sets of genes, one associated with longevity and aging and the other with sleep traits (e.g., insomnia, narcolepsy, sleep duration, chronotype, among others), with both gene lists heavily driven by hits from recent large-scale Genome-Wide Association Studies. There were 47 overlapping genes between the gene list associated with sleep traits (1064 genes total) and the genes associated with longevity (367 genes total), indicating significantly more overlap than expected by chance. An overrepresentation analysis identified enriched pathways that suggest endocrine and epigenetic regulation as potential shared mechanisms between sleep traits and longevity. Concordantly, functional network analysis retrieved two clusters, being one associated with proteins of nuclear functions and the other, with extracellular proteins. This overlapping gene set, and the highlighted biological pathways may serve as preliminary findings for new functional investigations of sleep and longevity shared genetic mechanisms.

Toll-Like Receptor Evolution: Does Temperature Matter?

Toll-like receptors (TLRs) recognize conserved pathogen-associated molecular patterns (PAMPs) and are an ancient and well-conserved group of pattern recognition receptors (PRRs). The isolation of the Antarctic continent and its unique teleost fish and microbiota prompted the present investigation into Tlr evolution. Gene homologues of tlr members in teleosts from temperate regions were present in the genome of Antarctic Nototheniidae and the non-Antarctic sister lineage Bovichtidae. Overall, in Nototheniidae apart from D. mawsoni, no major tlr gene family expansion or contraction occurred. Instead, lineage and species-specific changes in the ectodomain and LRR of Tlrs occurred, particularly in the Tlr11 superfamily that is well represented in fish. Positive selective pressure and associated sequence modifications in the TLR ectodomain and within the leucine-rich repeats (LRR), important for pathogen recognition, occurred in Tlr5, Tlr8, Tlr13, Tlr21, Tlr22, and Tlr23 presumably associated with the unique Antarctic microbiota. Exposure to lipopolysaccharide (Escherichia coli O111:B4) Gram negative bacteria did not modify tlr gene expression in N. rossii head-kidney or anterior intestine, although increased water temperature (+4°C) had a significant effect.

Differential expression of PTHrP and its receptor in pituitary gland and gills in estradiol-treated gilthead sea bream (Sparus auratus, L.).

In the gilthead sea bream (Sparus auratus) 17ß-estradiol (E2) plays an important role in the synthesis of vitellogenin. During vitellogenesis, vitellogenin as a nutritional precursor protein is loaded with calcium, which requires elevated plasma calcium levels. This is accomplished via E2-dependent processes. Reports have shown that hypercalcemic effects of E2 are possibly mediated by another hypercalcemic factor, viz. parathyroid hormone related protein (PTHrP). To further investigate the possibility of PTHrP as a mediator of E2-induced hypercalcemia, we investigated the local expression levels of the pthrp mRNA and of the gene coding for the PTHrP receptor, PTH1R (pth1r) in two tissues involved in the calcium regulation (gills, pituitary gland) of the sea bream treated with E2. Compared to control, treatment with E2 resulted in: significantly increased total calcium and plasma PTHrP levels (P<0.01), a down-regulation of pthrp mRNA in the pituitary gland (P<0.01), and up-regulation of expression levels for both pthrp and pth1r in the branchial system (P<0.05). These findings provide direct evidence for a mediating role of PTHrP in E2 induced hypercalcemia, and in addition support the idea for the presence of two independent systems, an endocrine pituitary PTHrP system and a peripheral paracrine branchial PTHrP system.

Environmental Salinity Modifies Mucus Exudation and Energy Use in European Sea Bass Juveniles.

The European sea bass (Dicentrarchus labrax) is a euryhaline marine teleost that can often be found in brackish and freshwater or even in hypersaline environments. Here, we exposed sea bass juveniles to sustained salinity challenges for 15 days, simulating one hypoosmotic (3‰), one isosmotic (12‰) and one hyperosmotic (50‰) environment, in addition to control (35‰). We analyzed parameters of skin mucus exudation and mucus biomarkers, as a minimally invasive tool, and plasma biomarkers. Additionally, Na+/K+-ATPase activity was measured, as well as the gill mucous cell distribution, type and shape. The volume of exuded mucus increased significantly under all the salinity challenges, increasing by 130% at 50‰ condition. Significantly greater amounts of soluble protein (3.9 ± 0.6 mg at 50‰ vs. 1.1 ± 0.2 mg at 35‰, p < 0.05) and lactate (4.0 ± 1.0 µg at 50‰ vs. 1.2 ± 0.3 µg at 35‰, p < 0.05) were released, with clear energy expenditure. Gill ATPase activity was significantly higher at the extreme salinities, and the gill mucous cell distribution was rearranged, with more acid and neutral mucin mucous cells at 50‰. Skin mucus osmolality suggested an osmoregulatory function as an ion-trap layer in hypoosmotic conditions, retaining osmosis-related ions. Overall, when sea bass cope with different salinities, the hyperosmotic condition (50‰) demanded more energy than the extreme hypoosmotic condition.

Morphophenotypic Classification of Hepatocellular Carcinoma: the Biliary/Stem Cell Subgroup and Worst Outcome-Implications on Patient Selection.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and the third cause of cancer-related death. Current clinical/pathological criteria contribute to risk stratification, but are far from the desired on individualized medicine. Recently, HCC classifications have been published based on immunohistochemical and morphological features. METHODS: A retrospective review of patients submitted to surgical treatment-partial hepatectomy (PH) or liver transplantation (LT), with pathological diagnosis of HCC, in a 9-year period (2007-2015) was performed. RESULTS: Applying the classification of Srivastava et al. (#1), based on the expression of CD31, p53, AFP and CD44, tumour size and presence of vascular invasion, HCC were categorized as low- and high-risk HCC. With the classification of Tsujikawa et al. (#2), HCC were classified into biliary/stem cell marker positive, Wnt signalling positive and the "all negative" HCC, according to the expression of CK19, SALL4, ß-catenin glutamine synthetase, EpCAM and p53. There were sixty-six patients (53 males; 13 females), with median age of 64.5 ± 9.46 years (range 38-86), with solitary HCC, comprehending 37 PH (56.1%) and 29 LT (43.9%). The mean overall survival (OS) was 75.4 ± 6.9 months. Biliary/stem cell type of HCC was a predictive factor of worse OS on the overall population (24.4 versus 78.3 months, p = 0.032) and in PH cohort (11.5 versus 64.01 months, p = 0.016), on uni- and multivariate analyses. CONCLUSION: These results support the relevance of a risk stratification classification of HCC. Classification #2 seems adequate to our reality demonstrating OS impact, allowing its application in future biopsies, prompting individualized medicine.

Piscine PTHrP regulation of calcium and phosphate transport in winter flounder renal proximal tubule primary cultures.

Multiple factors control calcium (Ca(2+)) and inorganic phosphate (P(i)) transport in the fish nephron, and the recently discovered members of the piscine parathyroid hormone-like protein family are likely participants in such regulatory mechanisms. The effects of an NH(2)-terminal peptide (amino acids 1-34) of Takifugu rubripes parathyroid hormone-related protein, (1-34)PTHrP, on Ca(2+) and P(i) transport were investigated in winter flounder (Pseudopleuronectes americanus) proximal tubule cells in primary culture (fPTCs). RT-PCR performed on RNA extracted from fPTCs and from intact kidney tissue indicated that expression of PTHrP and types 1 and 3 PTH/PTHrP receptors occurred both in vivo and in vitro and that circulating levels of PTHrP measured by specific radioimmunoassay averaged 2.5 +/- 0.13 ng/ml. fPTC monolayers were mounted in Ussing chambers, and under neutral electrochemical conditions, addition of 10 nM (1-34)PTHrP to the basolateral side induced a slight increase in Ca(2+) transport rate from luminal to peritubular side, significantly stimulating net Ca(2+) reabsorption. (1-34)PTHrP also significantly increased the P(i) secretory flux, and slightly reduced P(i) reabsorption, evoking a significant increase in P(i) net secretion. This stimulatory effect was partially inhibited by bisindolylmaleimide, an inhibitor of protein kinase C. Incubation of ex vivo flounder renal tubules with (1-34)PTHrP resulted in apparent reduction of Na(+)-P(i) cotransporter type II (NaP(i)-II) protein in tubule membranes. PTHrP seems therefore to participate in the modulation of Ca(2+) and P(i) homeostasis by fish kidney.

PRL and GH synthesis and release from the sea bream (Sparus auratus L.) pituitary gland in vitro in response to osmotic challenge.

The endocrine factors prolactin (PRL) and growth hormone (GH) are believed to have counteracting effects in the adaption of fish to changes in environmental salinity. In order to further investigate this interaction sea bream were challenged with full seawater (SW) or freshwater (FW) for 7 days and the response of pituitary glands cultured in vitro to an osmotic challenge (230, 275 and 320 mOsm/kg) was assessed. In vitro PRL secretion from pituitaries of SW-adapted fish was unaltered in response to an osmotic challenge, while GH secretion increased in the lowest osmolality (230 mOsm/kg). In contrast, both GH and PRL secretion by pituitaries from FW challenged fish was significantly increased (p<0.01) over that of pituitaries from SW fish at the highest osmolality (320 mOsm/kg). After FW challenge pituitary PRL content and de novo synthesised and released PRL were significantly increased (p<0.01), while total PRL secretion was not different from SW animals. GH pituitary content decreased in FW animals while total secretion and secretion of de novo synthesised protein were significantly increased (p<0.01). In addition, after transfer of fish to FW expression of PRL and GH increased 3- and 2-fold, respectively. Despite the increase in PRL expression, no increase in total PRL secretion occurred and although in gills a 2-fold increase in the osmoregulatory marker, Na(+)/K(+)-ATPase activity was detected, profound haemodilution and a cumulative mortality of 40% occurred in sea bream placed in FW. Taken together the results suggest that the sea bream pituitary gland fails to respond appropriately to the osmotic challenge caused by low salinity and the physiological response evoked in vivo is not enough to allow this species to withstand and adapt to FW.