Hypo-osmotic Conditions Research Articles

Discovery of prolactin-like in lamprey: Role in osmoregulation and new insight into the evolution of the growth hormone/prolactin family

We used a representative of one of the oldest extant vertebrate lineages (jawless fish or agnathans) to investigate the early evolution and function of the growth hormone (GH)/prolactin (PRL) family. We identified a second member of the GH/PRL family in an agnathan, the sea lamprey (Petromyzon marinus). Structural, phylogenetic, and synteny analyses supported the identification of this hormone as prolactin-like (PRL-L), which has led to added insight into the evolution of the GH/PRL family. At least two ancestral genes were present in early vertebrates, which gave rise to distinct GH and PRL-L genes in lamprey. A series of gene duplications, gene losses, and chromosomal rearrangements account for the diversity of GH/PRL-family members in jawed vertebrates. Lamprey PRL-L is produced in the proximal pars distalis of the pituitary and is preferentially bound by the lamprey PRL receptor, whereas lamprey GH is preferentially bound by the lamprey GH receptor. Pituitary PRL-L messenger RNA (mRNA) levels were low in larvae, then increased significantly in mid-metamorphic transformers (stage 3); thereafter, levels subsided in final-stage transformers and metamorphosed juveniles. The abundance of PRL-L mRNA and immunoreactive protein increased in the pituitary of juveniles under hypoosmotic conditions, and treatment with PRL-L blocked seawater-associated inhibition of freshwater ion transporters. These findings clarify the origin and divergence of GH/PRL family genes in early vertebrates and reveal a function of PRL-L in osmoregulation of sea lamprey, comparable to a role of PRLs that is conserved in jawed vertebrates.

Fish Embryo Acute Toxicity Testing and the RTgill-W1 Cell Line as In Vitro Models for Whole-Effluent Toxicity (WET) Testing: An In Vitro/In Vivo Comparison of Chemicals Relevant for WET Testing.

The fathead minnow (Pimephales promelas) fish embryo acute toxicity (FET) test was compared to the fish gill cells (RTgill-W1) in vitro assay and to the fish larvae acute toxicity test to evaluate their sensitivity for whole-effluent toxicity (WET) testing. The toxicity of 12 chemicals relevant for WET testing was compared as proof of principle. The concentrations lethal to 50% of a population (LC50) of embryos were compared to those in fish larvae and to the 50% effect concentration (EC50) in RTgill-W1 cells from previous literature. Along with traditional FET endpoints (coagulation, somite development, tail detachment, and heartbeat), cardiotoxicity was evaluated for WET applicability. Heart rate was measured at LC20 and LC50 values of six subselected chemicals (Cd, Cu, Ni, ammonia, 3,4-dichloraniline, and benzalkonium chloride). In addition, the toxicity of Cd and Ni was evaluated in RTgill-W1 cells exposed in a hypoosmotic medium to evaluate the effect that osmolarity may have on metal toxicity. A significant correlation was found between the FET and larvae LC50 values but not between the RTgill-W1 EC50 and FET LC50 values. Although sensitivity to Ni and Cd was found to increase with hypoosmotic conditions for FET and RTgill-W1 cells, a correlation was only found with removal of Ni from the analysis. Hypoosmotic conditions increased sensitivity with a significant correlation between RTgill-W1 cells and larvae. Cardiotoxicity was shown in three of the five subselected chemicals (Cd, Cu, and 3,4-dichloroaniline). Overall, both in vitro alternative models have shown good predictability of toxicity in fish in vivo for WET chemicals of interest. Environ Toxicol Chem 2022;41:2721-2731. © 2022 SETAC.

Control of Ca2+ signals by astrocyte nanoscale morphology at tripartite synapses.

Much of the Ca2+ activity in astrocytes is spatially restricted to microdomains and occurs in fine processes that form a complex anatomical meshwork, the so-called spongiform domain. A growing body of literature indicates that those astrocytic Ca2+ signals can influence the activity of neuronal synapses and thus tune the flow of information through neuronal circuits. Because of technical difficulties in accessing the small spatial scale involved, the role of astrocyte morphology on Ca2+ microdomain activity remains poorly understood. Here, we use computational tools and idealized 3D geometries of fine processes based on recent super-resolution microscopy data to investigate the mechanistic link between astrocytic nanoscale morphology and local Ca2+ activity. Simulations demonstrate that the nano-morphology of astrocytic processes powerfully shapes the spatio-temporal properties of Ca2+ signals and promotes local Ca2+ activity. The model predicts that this effect is attenuated upon astrocytic swelling, hallmark of brain diseases, which we confirm experimentally in hypo-osmotic conditions. Upon repeated neurotransmitter release events, the model predicts that swelling hinders astrocytic signal propagation. Overall, this study highlights the influence of the complex morphology of astrocytes at the nanoscale and its remodeling in pathological conditions on neuron-astrocyte communication at so-called tripartite synapses, where astrocytic processes come into close contact with pre- and postsynaptic structures.

Compensation mechanism for membrane potential against hypoosmotic stress in the Onchidium neuron

The effect of acute hypoosmotic stress on the neural response was investigated using the neurons identified in the abdominal ganglion of the amphibious mollusk Onchidium. The membrane potential of an identified neuron (Ip-1/2) was not significantly altered in 50% hypoosmotic artificial sea water. In isotonic 50% artificial seawater (ASW) with osmolarity that was compensated for using glycerol or urea, the membrane potentials of Ip-1/2 were also not altered compared to those in 50% hypoosmotic ASW. However, hyperpolarization was induced in isotonic 50% ASW when osmolarity was compensated for using sucrose or mannose. In the presence of volume-regulated anion channel (VRAC) inhibitors (niflumic acid and glibenclamide), the Ip-1/2 membrane potentials were hyperpolarized in 50% hypoosmotic ASW. These results suggest that there is a compensatory mechanism involving aquaglyceroporin and VRAC-like channels that maintains membrane potential under hypoosmotic conditions. Here, we detected the expression of aquaglyceroporin mRNA in neural tissues of Onchidium.

Hypo-Osmotic Swelling Test (HOST) for Feline Spermatozoa: The Simplified Procedure and the Aspect of Sperm Morphology

Simple SummaryOne of the methods used to assess semen quality is the hypo-osmotic swelling test (HOST). Different protocols for this test are available, including a very simple one: swelling of spermatozoa is induced by incubation in distilled water for 5 min. Such a protocol could have easily been introduced in clinical practice, but it has not yet been evaluated for feline sperm cells. The objectives of this study were: (1) to check if a simplified HOST procedure can be applied for feline semen and (2) to check if sperm abnormalities may influence the results of this test. The results proved that the simplified HOST gives the same results as the standard one and that the presence of live, abnormal spermatozoa does not interfere with the test results.Hypo-osmotic swelling test (HOST) is used to assess the functional integrity of sperm plasma membranes in many species. The primary aim of this study was to test a simplified HOST procedure for the evaluation of feline semen. The second objective was to check if sperm abnormalities can influence the results of this test. Urethral semen was collected from 19 male, domestic cats. In Exp. 1, HOST was performed in different media (50 mOsm/kg fructose or distilled water), temperature (37 °C or room temperature) and time (5 and 30 min). In Exp. 2, the potential effect of sperm abnormalities on HOST results was assessed by observing individual normal and abnormal spermatozoa microinjected into droplets of distilled water. The results showed no differences between the HOST results performed in different media, temperature and time. Viable abnormal spermatozoa were able to swell under hypo-osmotic conditions in the same manner as normal ones, except spermatozoa with distal droplets, which showed a higher frequency of ‘despiralization’. In conclusion, HOST can be reliably performed at 0 mOsm/kg for 5 min at room temperature, which may contribute to a wider use of this test under clinical environments. Viable abnormal spermatozoa are able to swell under hypo-osmotic conditions; therefore, their presence in the ejaculate would not bias the results of HOST when total coiling is calculated.

Can hypoosmotic shock and calcium influx lead to translocation of aquaporin-1 in shrimp muscle cells?

The physiological variations during the crustacean molting cycle have intrigued researchers for many years. Maintaining osmotic homeostasis in the face of hemolymph dilution and dealing with dynamic intracellular and extracellular calcium fluctuations are challenges these animals continuously confront. It has recently been shown that water channels present in the cell membrane (aquaporins) are essential for water uptake during premolt and postmolt. This study aims to investigate whether hypoosmotic shock and intracellular and extracellular calcium variations can lead to translocation of Aquaporin 1 (AQP-1) from the intracellular region to the plasma membrane during premolt and postmolt, thus allowing increased water flow in these stages. For this, we investigate in vitro the rapid change of AQP-1 positions in the abdominal muscle cells in the freshwater shrimp, Palaemon argentinus. Using cell volume analysis and immunohistochemistry, we show that hypoosmotic conditions and an elevation of the intracellular and extracellular calcium concentrations are concurrent with the translocation of AQP-1 to the plasma membrane. These results indicate that calcium flux and hypoosmotic shock may be regulators of AQP 1 in the translocation process.

Physical properties of the cytoplasm modulate the rates of microtubule polymerization and depolymerization.

The cytoplasm is a crowded, visco-elastic environment whose physical properties change according to physiological or developmental states. How the physical properties of the cytoplasm impact cellular functions invivo remains poorly understood. Here, we probe the effects of cytoplasmic concentration on microtubules by applying osmotic shifts to fission yeast, moss, and mammalian cells. We show that the rates of both microtubule polymerization and depolymerization scale linearly and inversely with cytoplasmic concentration; an increase in cytoplasmic concentration decreases the rates of microtubule polymerization and depolymerization proportionally, whereas a decrease in cytoplasmic concentration leads to the opposite. Numerous lines of evidence indicate that these effects are due to changes in cytoplasmic viscosity rather than cellular stress responses or macromolecular crowding per se. We reconstituted these effects on microtubules invitro by tuning viscosity. Our findings indicate that, even in normal conditions, the viscosity of the cytoplasm modulates the reactions that underlie microtubule dynamic behaviors.

Ecological aspects of bivalve adaptation to salinity fluctuations on the example of Anadara Kagoshimensis

Impact of salinity stress on the ark clam (Anadara kagoshimensis (Tokunaga, 1906)) hemocyte functions were investigated using flow cytometry and light scattering technique. In control group water salinity was 18 ppm and experimental groups were carried at 14 ppm, 8 ppm, 35 ppm and 45 ppm. Hemolymph osmolarity decreased at hypoosmotic conditions and increased after hyperosmotic treatment. Osmotic stress induced changes in osmotic fragility of the ark clam hemocytes. Salinity 14 ppm did not affect the functional parameters of hemocytes. Incubation of ark clams at salinity and 35 ppm did not influence on the mitochondrial membrane potential of hemocytes but led to a decrease in hemocyte reactive oxygen species (ROS) production by 30 % compared to control. An increase in water salinity to 45 ppm and its decrease to 8 ppm induced substantial changes in the ROS production and mitochondrial membrane potential of hemocytes. Hyposalinity (8 ppm) led to an increase in ROS production by hemocytes (up to 2.4 times) and mitochondrial membrane potential (up to 1.3 times). An increase of salinity level from 18 ppm to 45 ppm decreased the total ability of hemocytes to produce ROS by 11% and increased mitochondrial potential of hemocytes by 150%.

The developing zebrafish kidney is impaired by Deepwater Horizon crude oil early-life stage exposure: A molecular to whole-organism perspective

Crude oil is known to induce developmental defects in teleost fish exposed during early life stages (ELSs). While most studies in recent years have focused on cardiac endpoints, evidence from whole-animal transcriptomic analyses and studies with individual polycyclic aromatic hydrocarbons (PAHs) indicate that the developing kidney (i.e., pronephros) is also at risk. Considering the role of the pronephros in osmoregulation, and the common observance of edema in oil-exposed ELS fish, surprisingly little is known regarding the effects of oil exposure on pronephros development and function. Using zebrafish (Danio rerio) ELSs, we assessed the transcriptional and morphological responses to two dilutions of high-energy water accommodated fractions (HEWAF) of oil from the Deepwater Horizon oil spill using a combination of qPCR and whole-mount in situ hybridization (WM-ISH) of candidate genes involved in pronephros development and function, and immunohistochemistry (WM-IHC). To assess potential functional impacts on the pronephros, three 24 h osmotic challenges (2 hypo-osmotic, 1 near iso‐osmotic) were implemented at two developmental time points (48 and 96 h post fertilization; hpf) following exposure to HEWAF. Changes in transcript expression level and location specific to different regions of the pronephros were observed by qPCR and WM-ISH. Further, pronephros morphology was altered in crude oil exposed larvae, characterized by failed glomerulus and neck segment formation, and straightening of the pronephric tubules. The osmotic challenges at 96 hpf greatly exacerbated edema in both HEWAF-exposed groups regardless of osmolarity. By contrast, larvae at 48 hpf exhibited no edema prior to the osmotic challenge, but previous HEWAF exposure elicited a concentration-response increase in edema at hypo-osmotic conditions that appeared to have been largely alleviated under near iso‐osmotic conditions. In summary, ELS HEWAF exposure impaired proper pronephros development in zebrafish, which coupled with cardiotoxic effects, most likely reduced or inhibited pronephros fluid clearance capacity and increased edema formation.

Периоперационная инфузионная терапия у детей младшего возраста с ургентной абдоминальной патологией

Безопасная инфузионная терапия у детей требует глубокого понимания основных принципов, основанных на особенностях водно-электролитного обмена и кислотно-основного состояния в детском возрасте. Содержание воды и электролитов в организме ребенка поддерживается в очень узких пределах, поэтому необходимо понимать потенциальные физиологические эффекты нарушений баланса воды и растворенных в ней веществ, так как эти изменения всегда сопровождают периоперационный период. Периоперационная инфузионная терапия имеет прямое влияние на клинический результат лечения, в связи с чем ее назначение должно соответствовать потребностям больного. Целью инфузионной терапии у больных детей в периоперационном периоде является поддержка эффективного циркулирующего объема и недопущение, где только возможно, интерстициальной перегрузки жидкостью. Кроме знаний физиологии и патофизиологии водно-электролитного обмена в детском возрасте, для адекватной инфузионной терапии необходимо понимание фармакологии инфузионных препаратов, их дозировки, техники применения, нарушение которых может негативно сказаться на прогнозе заболевания. Именно поэтому целью нашего исследования стало изучение изменений гемодинамических показателей, водно-электролитного, кислотно-щелочного обменов при применении различных инфузионных растворов в периоперационном периоде. Работа выполнена на основе анализа лечения 55 пациентов младшего возраста с ургентной абдоминальной патологией, которые в зависимости от вида проведения периоперационной инфузионной терапии были разделены на 3 группы: 1-я группа — дети, у которых в качестве инфузионной терапии в периоперационном периоде использовали гипотонические глюкозо-солевые растворы; 2-я группа — дети, у которых использовали раствор Рингера лактат; 3-я группа — дети, у которых использовали сбалансированный электролитный раствор Рингера малат. Исследование показало, что у детей с острой ургентной абдоминальной патологией отмечается обезвоживание организма и абсолютная гиповолемия, которая ухудшается развитием гипоосмолярного состояния, а для быстрой нормализации гемодинамических показателей, водно-электролитного обмена, кислотно-щелочного состояния и водных секторов организма оптимальной инфузионной средой должен быть сбалансированный солевой раствор с добавлением аспартата и малата.

The mutation G133D on Leishmania guyanensis AQP1 is highly destabilizing as revealed by molecular modeling and hypo-osmotic shock assay

The Leishmania aquaglyceroporin 1 (AQP1) plays an important role in osmoregulation and antimony (Sb) uptake, being determinant for resistance to antimony. We have previously demonstrated that G133D mutation on L. guyanensis AQP1 (LgAQP1) leads to reduced Sb uptake. Here, we investigated the effects of G133D mutation on LgAQP1 structure, associated with Sb uptake and alterations in osmoregulation capacity. High confidence molecular models of wild-type LgAQP1 as well as the LgAQP1::G133D mutant were constructed and optimized via comparative homology modeling. Computational methods from the mCSM platform were used to evaluate the effects on protein stability and on its ability to bind to glycerol. Functional validation of the disruptive effect of the mutation on LgAQP1 was done by challenging the parasites with hypo-osmotic chock. Glycine 133 is on transmembrane helix 3, buried in the membrane in both open and closed conformation. G133D mutation was predicted to be highly destabilizing, as it alters the helical bundling arrangement in order to accommodate the aspartic acid side chain. The shift in helices also resulted in fewer favorable contacts with glycerol in the channel, which would explain the reduced affinity for similar small molecules as SbO3. Under hypo-osmotic condition, L. guyanensis AQP1G133D presented a 3-fold increase in cellular volume and pronounced delay to recover osmosis homeostasis when compared to the wild-type, a profile that was enhanced in LgAQP1−/− mutants. In conclusion, G133D is a highly disruptive mutation that will destabilize the monomer, compromise tetramer formation and alter pore conformation, leading to reduced Sb uptake and deficient osmoregulation.

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

Simple SummaryChanges in skin mucus production and composition offer a new means to study how fish cope with changes in the environment. We explored the utility of skin mucus as an indicator of physiological responses and energy use in a reference fish species, the European sea bass. We evaluated the exudation volume of skin mucus and the main stress- and osmoregulation-related biomarkers in both mucus and plasma. We demonstrate the viability to study the exuded volume of skin mucus composition and its parameters as an informative tool of the fish energy waste at different environmental salinities. This study is of great interest for both aquaculture and ecological studies.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.

Refractive Index Sensing for Measuring Single Cell Growth.

Accessing cell growth on adhesive substrates is critical for identifying biophysical properties of cells and their therapeutic response to drug therapies. However, optical techniques have low sensitivity, and their reliability varies with cell type, whereas microfluidic technologies rely on cell suspension. In this paper, we introduced a plasmonic functional assay platform that can precisely measure cell weight and the dynamic change in real-time for adherent cells. Possessing this ability, our platform can determine growth rates of individual cells within only 10 min to map the growth profile of populations in short time intervals. The platform could successfully determine heterogeneity within the growth profile of populations and assess subpopulations exhibiting distinct growth profiles. As a proof of principle, we investigated the growth profile of MCF-7 cells and the effect of two intracellular metabolisms critical for their proliferation. We first investigated the negative effect of serum starvation on cell growth. We then studied ornithine decarboxylase (ODC) activity, a key enzyme which is involved in proliferation, and degraded under low osmolarity that inhibits cell growth. We successfully determined the significant distinction between growth profiles of MCF-7 cells and their ODC-overproducing variants that possess strong resistance to the negative effects of low osmolarity. We also demonstrated that an exogenous parameter, putrescine, could rescue cells from ODC inhibition under hypoosmotic conditions. In addition to the ability of accessing intracellular activities through ex vivo measurements, our platform could also determine therapeutic behaviors of cancer cells in response to drug treatments. Here, we investigated difluoromethylornithine (DFMO), which has antitumor effects on MCF-7 cells by inhibiting ODC activity. We successfully demonstrated the susceptibility of MCF-7 cells to such drug treatment, while its DFMO-resistant subpopulation could survive in the presence of this antigrowth agent. By rapidly determining cell growth kinetics in small samples, our plasmonic platform may be of broad use to basic research and clinical applications.

Comparative analysis of spreading depolarizations in brain slices exposed to osmotic or metabolic stress

BackgroundRecurrent spreading depolarizations (SDs) occur in stroke and traumatic brain injury and are considered as a hallmark of injury progression. The complexity of conditions associated with SD in the living brain encouraged researchers to study SD in live brain slice preparations, yet methodological differences among laboratories complicate integrative data interpretation. Here we provide a comparative evaluation of SD evolution in live brain slices, in response to selected SD triggers and in various media, under otherwise standardized experimental conditions.MethodsRat live coronal brain slices (350 μm) were prepared (n = 51). Hypo-osmotic medium (Na+ content reduced from 130 to 60 mM, HM) or oxygen-glucose deprivation (OGD) were applied to cause osmotic or ischemic challenge. Brain slices superfused with artificial cerebrospinal fluid (aCSF) served as control. SDs were evoked in the control condition with pressure injection of KCl or electric stimulation. Local field potential (LFP) was recorded via an intracortical glass capillary electrode, or intrinsic optical signal imaging was conducted at white light illumination to characterize SDs. TTC and hematoxylin-eosin staining were used to assess tissue damage.ResultsSevere osmotic stress or OGD provoked a spontaneous SD. In contrast with SDs triggered in aCSF, these spontaneous depolarizations were characterized by incomplete repolarization and prolonged duration. Further, cortical SDs under HM or OGD propagated over the entire cortex and occassionally invaded the striatum, while SDs in aCSF covered a significantly smaller cortical area before coming to a halt, and never spread to the striatum. SDs in HM displayed the greatest amplitude and the most rapid propagation velocity. Finally, spontaneous SD in HM and especially under OGD was followed by tissue injury.ConclusionsWhile the failure of Na+/K+ ATP-ase is thought to impair tissue recovery from OGD-related SD, the tissue swelling-related hyper excitability and the exhaustion of astrocyte buffering capacity are suggested to promote SD evolution under osmotic stress. In contrast with OGD, SD propagating under hypo-osmotic condition is not terminal, yet it is associated with irreversible tissue injury. Further investigation is required to understand the mechanistic similarities or differences between the evolution of SDs spontaneously occurring in HM and under OGD.

Modulation of LXR signaling altered the dynamic activity of human colon adenocarcinoma cancer stem cells in vitro

BackgroundThe expansion and metastasis of colorectal cancers are closely associated with the dynamic growth of cancer stem cells (CSCs). This study aimed to explore the possible effect of LXR (a regulator of glycolysis and lipid hemostasis) in the tumorgenicity of human colorectal CD133 cells.MethodsHuman HT-29 CD133+ cells were enriched by MACS and incubated with LXR agonist (T0901317) and antagonist (SR9243) for 72 h. Cell survival was evaluated using MTT assay and flow cytometric analysis of Annexin-V. The proliferation rate was measured by monitoring Ki-67 positive cells using IF imaging. The modulation of LXR was studied by monitoring the activity of all factors related to ABC transporters using real-time PCR assay and western blotting. Protein levels of metabolic enzymes such as PFKFB3, GSK3β, FASN, and SCD were also investigated upon treatment of CSCs with LXR modulators. The migration of CSCs was monitored after being exposed to LXR agonist using scratch and Transwell insert assays. The efflux capacity was measured using hypo-osmotic conditions. The intracellular content of reactive oxygen species was studied by DCFH-DA staining.ResultsData showed incubation of CSCs with T0901317 and SR9243 reduced the viability of CD133 cells in a dose-dependent manner compared to the control group. The activation of LXR up-regulated the expression and protein levels of ABC transporters (ABCA1, ABCG5, and ABCG8) compared to the non-treated cells (p < 0.05). Despite these effects, LXR activation suppressed the proliferation, clonogenicity, and migration of CD133 cells, and increased hypo-osmotic fragility (p < 0.05). We also showed that SR9243 inhibited the proliferation and clonogenicity of CD133 cells through down-regulating metabolic enzymes PFKFB3, GSK3β, FASN, and SCD as compared with the control cells (p < 0.05). Intracellular ROS levels were increased after the inhibition of LXR by SR9243 (p < 0.05).Calling attention, both T0901317 and SR9243 compounds induced apoptotic changes in cancer stem cells (p < 0.05).ConclusionsThe regulation of LXR activity can be considered as a selective targeting of survival, metabolism, and migration in CSCs to control the tumorigenesis and metastasis in patients with advanced colorectal cancers.

Effects of short-term and continuous exposure to reduced salinities on the biochemical composition of larval lobster, Homarus gammarus

In coastal areas with estuarine influence, exposure to hypo-osmotic conditions may affect larval survival, development and growth. Most knowledge about effects of reduced salinity on coastal organisms is based on keeping individuals under constant conditions in the laboratory. By contrast, little is known about the effects of more realistic situations where organisms are exposed to low salinity over short time scales. Such environmental short-term fluctuations are expected to increase due to climate change. Here, we experimentally evaluated the sublethal effects of both short-term and continuous exposure to moderately reduced salinities (salinity 20 and 25; compared to seawater, salinity 32) in larvae of European lobster Homarus gammarus. Total body dry mass and biochemical composition (measured as: protein and lipid contents) were measured as response variables in Mysis stages I to III. Short-term effects of low salinity were quantified in a group of larvae kept in seawater from hatching until the time of transfer to the test salinities. After ca. 40 % of each moult cycle in seawater (determined in preliminary experiments for Mysis I, II and III), larvae were assigned to a seawater control or reduced salinities lasting for 16 h (i.e. until ca. 50 % of the time spent within the moulting cycle). Effects of continuous exposure to low salinity were quantified when larvae were exposed to the different salinities from hatching, until they reached ca. 50 % of the successive moulting stage. Surprisingly, in the Mysis II and III stages, short-term exposure to low salinity had much stronger effects on accumulation of reserves than the continuous exposure. Such effects were manifested mostly as limited accumulation, or even losses, in the lipid content as compared to reductions in the amount of protein accumulated. The most sensitive stage to exposure to low salinity was the Mysis III; by contrast in Mysis I such effects were relative weak (not always significant). Chronic exposure to low salinity also led to an increase in developmental time especially at the advanced stages. Our results highlight the importance of quantifying effects of environmental fluctuations at different time scales in order to better understand how organisms cope with realistic environmental change in the coastal zones. For H. gammarus, our results suggest that larvae respond adaptively to low salinity by maintaining protein levels at expenses of reductions in lipid accumulation and by extending the developmental time, but the capacity to elicit a fully compensatory response varies ontogenetically.

Hemolymph composition, gene expressions in the gills, and thus the survival of euryhaline crabs are controlled by ambient minor cations according to osmotic condition-dependent manner.

Na+ and Cl− are the most abundant dissolved ions in seawater, constituting ~ 85% of total ions. They significantly affect the osmolality of body fluids of marine invertebrates. Seawater also contains minor ions such as Mg2+, Ca2+, K+, and SO4 2‐ , but their effects on marine organisms are unclear. This study analyzed the effects of Mg2+, Ca2+, and K+ (ambient minor cations) on survival, hemolymph ionic composition, and gene expression in the gills of three euryhaline crabs: Helice tridens, Macrophthalmus japonicus, and Chiromantes dehaani. Ambient minor cations were required for survival of H. tridens and M. japonicus under isosmotic conditions with seawater. The ambient minor cations also affected the osmolality and ionic composition of hemolymph by regulating expressions of specific genes in the gills required for Na+ uptake, such as Na+/K+ ATPase, cytoplasmic carbonic anhydrase, and Na+/H+ exchanger. Administration of carbonic anhydrase and Na+/H+ exchanger inhibitors increased the survival rate even if ambient minor cations did not exist. In contrast, under hypo‐osmotic conditions, ambient minor cations had different effects on crabs, a lethal effect on M. japonicus, and an increase of the hemolymph K+ concentration in H. tridens and M. japonicus. It is thus concluded that the effects of ambient minor cations are osmolality‐dependent. In contrast, in C. dehaani, the hemolymph ionic composition and survival rate were hardly affected by ambient minor cations, probably reflecting the habitat of this species. These results strongly indicated that C. dehaani is less susceptive to ambient minor cations compared to H. tridens and M. japonicus.

Parkin-dependent and -independent degradation of synaptotagmin-11 in neurons and astrocytes

Synaptotagmin-11 (Syt11) is associated with schizophrenia and Parkinson’s disease (PD) and is a critical substrate of parkin, an E3 ubiquitin ligase linked to PD. Previously we reported that Syt11 regulates multiple membrane trafficking pathways in neurons and glia. However, the regulation of Syt11 degradation remains largely unknown. As the ubiquitin-proteasome pathway (UPP) plays crucial roles in protein degradation and quality control, we investigated UPP-dependent Syt11 degradation in this study. We found that Syt11 is a short-lived protein with a half-life of 1.49 h in the presence of a protein synthesis inhibitor cycloheximide and is mainly degraded by UPP in neurons. The degradation was further accelerated under sustained neuronal activity and was parkin-dependent. Interestingly, Syt11 had a faster turnover in astrocytes with a half-life of 0.58 h, and UPP partially contributed to its degradation. Mechanical stress applied on astrocytes by hypoosmotic treatment led to reduced Syt11 protein level but increased parkin level. However, the degradation of Syt11 was parkin-independent under both isoosmotic and hypoosmotic condition. Altogether, our results revealed active and distinct proteolytic regulation of Syt11 in neurons and astrocytes.

Evaluation of hemolymph biochemical properties, clearance rate, bacterial microbiota and expression of HSP genes of gulf pearl oyster Pinctada radiata in response to salinity changes

Hypo-salinity exposure is a serious concern for bivalve aquaculture, with profound effects on the animals’ physiological condition. The present study aimed to evaluate the influence of low-salinity exposure on the clearance rate, total heterotrophic marine bacteria and total Vibrio count in flesh, as well as the biochemical parameters of hemolymph and heat shock protein (HSP) gene expression, in Pinctada radiata. To this end, the oysters were reared for 14 days at salinities of 40 (control group), 35, 30, 25 and 20 PSU. Results indicated that low salinity led to a significantly lower clearance rate, with the lowest clearance rate observed at 20 PSU. However, no significant differences were found among the groups in the total heterotrophic marine bacteria, calcium concentration, aspartate aminotransferase (AST), alanine aminotransferase (ALT) or alkaline phosphatase (ALP) levels. In addition, the results demonstrated that the total Vibrio count increased significantly in oysters exposed to the hypoosmotic condition. A reduction in glucose concentration was observed in the 25 and 20 PSU groups compared to the other experimental groups. In addition, the highest survival rate was found in the 40 and 35 PSU groups. Based on q-RT-PCR results, HSP70 and HSP90 mRNA levels were significantly up-regulated by low-salinity exposure, while the expression of HSP20 mRNA was remarkably down-regulated in the 35 and 30 PSU salinity groups. The present study shows that pearl oysters tolerate a wide range of salinities (40–20 PSU), whereas optimal salinity for oyster culture ranges from 30 to 40 PSU. These results also provide a better understanding of the feeding behavior and physiological changes of P. radiata in response to salinity changes.

Rabbitfish (Siganus guttatus) culture in floating net cage with different stocking densities

Rabbitfish (Siganus guttatus) is a schooling species which potential for being farmed in high density, however the information about stocking density of this species remain rare. This study was aimed to evaluate growth, survival rate, FCR, stress level and osmoregulation of Rabbitfish under different stocking densities. The tested fishes were the second generation (G2) of Rabbitfish that produced by hatchery outdoor of Research Institute of Coastal Aquaculture and Fisheries Extension, Maros, Indonesia. There were two stages of this study: the first stage was fingerling production. The seeds of Rabbitfish with average of length 6.2±0.8 cm and body weight 4.7±1.9 g/ind were reared in total of 12 units of 1 x 1 x 1 m3 floating net cage for 90 days. The stocking densities were 50, 100, 150 and 200 ind/m3. The second stage of this study was fish growing, where the tested fishes were cultivated with stocking density of 100, 150 and 200 ind/m3 in the same size of net as the first stage. The result of first stage showed that stocking density did not significantly affect growth rate of Rabbitfish fingerling. However, survival rate at stocking density of 50 (99.33±1.15%) and 100 ind/m3 (98,33±0,58%) were higher compared to stocking densities 150 and 200 ind/m3 (94.89±1.39 and 93.50±2.65%, respectively). The result of second stage showed that stocking density of 100 ind/m3 resulted significant growth, survival rate and FCR compared to that observed on 150 and 200 ind/m3. The fish stocked with density of 100 ind/m3 had length (18.6±0.3 cm), weigh (121.8±9.8 g/ind.), survival rate (92.0±2.6%) and FCR (4,41±0,43). Whereas, the result for fish stocked with 150 ind/m3 were 17.6±0.3 cm, 107.1±7.0 gram, 86.2±7.1 % and 5.15±0.59, respectively, and 200 ind/m3 were 16.9±0.2 cm, 96.5±5.9 gram, 82.2±2.3 % and 5,64±0,6, respectively. High stocking density might trigger stress on Rabbitfish and lower blood osmolality found on stocking density of 150 ind/m3 (490.00±59.77 mOsm/kg) and 200 ind/m3 (469.00±23.30 mOsm/kg) compared to that observed on density of 100 ind/m3 (501.67±23.50 mosm/kg). During hypo-osmotic condition, the osmoregulation was not regulated by stocking density as indicated by blood osmotic performance levels <1, which was 0.37 mOsm/kg (100 ind/m3) and 0.33 mOsm/kg (for 150 and 200 ind/m3).