Increase In Osmolality Research Articles

Enhancement of preimplantation mouse embryo development with optimized in vitro culture dish via stabilization of medium osmolarity.

We evaluated the efficacy of the newly developed optimized in vitro culture (OIVC) dish for cultivating preimplantation mouse embryos. This dish minimizes the need for mineral oil and incorporates microwells, providing a stable culture environment and enabling independent monitoring of individual embryos. Mouse pronuclear (PN) zygotes and two-cell-stage embryos were collected at 18 and 46 hours after human chorionic gonadotropin injection, respectively. These were cultured for 120 hours using potassium simplex optimized medium (KSOM) to reach the blastocyst stage. The embryos were randomly allocated into three groups, each cultured in one of three dishes: a 60-mm culture dish, a microdrop dish, and an OIVC dish that we developed. The OIVC dish effectively maintained the osmolarity of the KSOM culture medium over a 5-day period using only 2 mL of mineral oil. This contrasts with the significant osmolarity increase observed in the 60-mm culture dish. Additionally, the OIVC dish exhibited higher blastulation rates from two-cell embryos (100%) relative to the other dish types. Moreover, blastocysts derived from both PN zygotes and two-cell embryos in the OIVC dish group demonstrated significantly elevated mean cell numbers. Use of the OIVC dish markedly increased the number of cells in blastocysts derived from the in vitro culture of preimplantation mouse embryos. The capacity of this dish to maintain medium osmolarity with minimal mineral oil usage represents a breakthrough that may advance embryo culture techniques for various mammals, including human in vitro fertilization and embryo transfer programs.

A proposal to account for the stimulus, the mechanism, and the mediators released in exercise-induced bronchoconstriction.

Exercise induced bronchoconstriction (EIB) describes the transient narrowing of the airways that follows vigorous exercise. It commonly occurs in children and adults who have asthma and in elite athletes. The primary stimulus is proposed to be loss of water, by evaporation, from the airway surface due to conditioning inspired air. The mechanism, whereby this evaporative loss of water provokes contraction of the bronchial smooth muscle, is thought to be an increase in osmolarity of the airway surface liquid. The increase in osmolarity causes mast cells to release histamines, prostaglandins, and leukotrienes. It is these mediators that contract smooth muscle causing the airways to narrow.

Puerarin alleviates hyperosmotic stress-induced oxidative stress, inflammation, apoptosis and barrier damage of human corneal epithelial cells by targeting SIRT1/NLRP3 signaling

The increase of tear osmolarity caused by excessive evaporation of tear phase is the main pathological mechanism of dry eye disease (DED). Puerarin, the major bioactive ingredient isolated from the root of the Pueraria lobata (Willd.) Ohwi, has been reported to improve ophthalmic diseases in clinic. However, the effect and the potential regulatory mechanism related to silent information regulator sirtuin 1 (SIRT1)/NOD-like receptor family pyrin domain containing 3 (NLRP3) signaling of puerarin in DED has not been evaluated. In this study, we aimed to explore the effect and mechanism of hyperosmotic stress (Hyp)-induced human corneal epithelial cell line (HCE-2). The viability of HCE-2 cells induced by Hyp with or without puerarin treatment was assessed by a CCK-8 assay. Results indicated that puerarin treatment enhanced cell viability, reduced reactive oxygen species (ROS) content, increased CAT and SOD activities, and elevated the ratio of GSH/GSSG in HCE-2 cells exposed to Hyp. Besides, TNF-α, IL-1β and IL-6 contents were decreased by puerarin. Additionally, puerarin inhibited Hyp-induced apoptosis and barrier disruption of HCE-2 cells. Moreover, molecular docking method suggested that puerarin bound to SIRT1, and upregulated SIRT1 and downregulated NLRP3 inflammasome proteins after puerarin treatment was observed. Furthermore, SIRT1 silencing alleviated the protective effects of puerarin on Hyp-induced HCE-2 cell damage. Collectively, puerarin attenuates Hyp-induced injury of HCE-2 cells by targeting regulating SIRT1/NLRP3 signaling.

SAT624 Neurosarcoidosis Presenting With Panhypopituitarism

Abstract Disclosure: E.M. Niedzialkowska: None. T. Blazin: None. K. Strubchevska: None. M. Kozyk: None. N. Saini: None. Introduction: Neurosarcoidosis is estimated to occur in approximately 5-15% patients with sarcoidosis. Pituitary involvement may be present in up to 5% of those cases and can manifest as hypothalamus, adenohypophysis dysfunction or arginine vasopressin deficiency (AVP-D) in isolated fashion or variedly combined. Clinical case: 32 year old male presented with chief complaint of worsening bilateral lower extremity weakness, urinary incontinence, polyuria and polydipsia. 9 months prior to admission the patient was diagnosed with neurosarcoidosis. At that time brain MRI showed diffuse leptomeningeal enhancement, chest CT showed hilar lymphadenopathy, lumbar puncture with CSF analysis showed protein 227 mg/dl (N<38), ACE 6 U/l (N <2.5), WBC 10 (N<5) with 88% lymphocytes. Hilar lymph node biopsy was consistent with non-caseating granuloma. The patient was diagnosed with neurosarcoidosis and started on prednisone and methotrexate. The patient was referred to our institution for further evaluation. On examination gait ataxia and gynecomastia were noted. Laboratory results showed urine specific gravity of 1.001, LH 0.4 mIU/ml (N 0.6-1.2), FSH 1.1 mIU/ml (N 1-12), ACTH <5 pg/ml (N 6-46), cortisol 2.1 ug/dl (N 2.9-19.4), prolactin 10.5 ng/ml (N 2-18), total testosterone 4 ng/dl (N 240-1080), free testosterone 0.1 ng/dl (N 4.8-19), TSH 0.58 uIU/ml (N 0.4-4.5), free ft3 1.5 pg/ml (N 1.7-3.7), free ft4 0.6 ng/dl (N 0.7-1.5), IGF-1 52 ng/ml (N 71-234), Copeptin proAVP of <2.8 pmol/l( N>13.2), urine osmolality of 72 mOsmol/kg, blood osmolality of 297 mOsmol/kg (N <295), vit D 25-OH 9 ng/ml (N 30-100), vit D 1,25-OH 57.3 pg/ml (N 19.9-79.3). MRI of the spine showed extensive nodular enhancement of the leptomeninges of the cervical, thoracic spine and lumbar cauda equina nerve roots. Brain MRI was positive for numerous small nodular enhancement involving leptomeninges and pituitary stalk. The patient was started on desmopressin with significant improvement of symptoms and increase in urine osmolality to 709 mOsmol/kg. Due to risk of hypercalcemia and normal vit D 1,25-OH vitamin D supplementation was not initiated. For neurosarcoidosis the patient was started on pulse dose methylprednisolone and infliximab infusion. Upon discharge he was transitioned to oral hydrocortisone and started on levothyroxine and testosterone replacement therapy. Conclusion: We present a case of pituitary neurosarcoidosis presenting with AVP-D, secondary hypothyroidism, secondary adrenal insufficiency, hypogonadism and growth hormone deficiency. Panhypopituitarism with complete anterior and posterior pituitary dysfunction is extremely rare and might be found in up to 13% cases of pituitary sarcoidosis. The diagnosis should be considered in patients with sarcoidosis and endocrine dysfunctions. Presentation: Saturday, June 17, 2023

Mild dehydration effects on the murine kidney single-nucleus transcriptome and chromatin accessibility.

Daily, we may experience mild dehydration with a rise in plasma osmolality that triggers the release of vasopressin. Although the effect of dehydration is well characterized in collecting duct principal cells (CDPCs), we hypothesized that mild dehydration (<12 h) results in many kidney cell-specific changes in transcriptomes and chromatin accessibility. Single-nucleus (sn) multiome (RNA-assay for transposase-accessible chromatin) sequencing and bulk RNA sequencing of kidneys from male and female mice that were mildly water deprived or not were compared. Water-deprived mice had a significant increase in plasma osmolality. sn-multiome-seq resulted in 19,837 nuclei that were annotated into 33 clusters. In CDPCs, aquaporin 2 (Aqp2) and aquaporin 3 (Apq3) were greater in dehydrated mice, but there were novel genes like gremlin 2 (Grem2; a cytokine) that were increased compared with ad libitum mice. The transcription factor cAMP-responsive element modulator (Crem) was greater in CDPCs of dehydrated mice, and the Crem DNA motif was more accessible. There were hundreds of sex- and dehydration-specific differentially expressed genes (DEGs) throughout the kidney, especially in the proximal tubules and thin limbs. In male mice, DEGs were enriched in pathways related to lipid metabolism, whereas female DEGs were enriched in organic acid metabolism. Many highly expressed genes had a positive correlation with increased chromatin accessibility, and mild dehydration exerted many transcriptional changes that we detected at the chromatin level. Even with a rise in plasma osmolality, male and female kidneys have distinct transcriptomes suggesting that there may be diverse mechanisms used to remain in fluid balance.NEW & NOTEWORTHY The kidney consists of >30 cell types that work collectively to maintain fluid-electrolyte balance. Kidney single-nucleus transcriptomes and chromatin accessibility profiles from male and female control (ad libitum water and food) or mildly dehydrated mice (ad libitum food, water deprivation) were determined. Mild dehydration caused hundreds of cell- and sex-specific transcriptomic changes, even though the kidney function to conserve water was the same.

Renal dysfunction in general overheating (literature review)

Over the past 30 years, climate change has taken one of the leading places among the ten main causes of death due to natural disasters. Rising temperatures have been shown to increase emergency department admissions for a wide range of kidney diseases, including acute kidney injury (AKI), chronic kidney disease, kidney stones, and urinary tract infections. The occupational effect of heat stress is also associated with AKI, which can quickly progress to acute kidney failure with high mortality. The basis of the pathogenetic mechanisms of heat-induced AKI is a decrease in the circulating blood volume and electrolyte disturbances due to increased perspiration. Water evaporation from the surface of the skin contributes to dehydration with an increase in serum osmolarity. In response to this, vasopressin is activated, the specific gravity of urine increases, and the amount of urine decreases. The glomerular filtration rate progressively decreases. Hypokalemia develops, which changes to hyperkalemia within 12 hours. Tubular endothelium is damaged, which leads to microthrombosis of afferent and efferent renal arteries, the development of an inflammatory response, and exhaustion of the renal interstitium. Possible rhabdomyolysis and myoglobinemia with subsequent tubular obstruction worsen existing kidney damage. According to the leading mechanism of damage, there are two types of acute heat damage to the kidneys: classical rhabdomyolysis and acute interstitial nephritis. Although modern medical advances have contributed to the development of effective treatment and management strategies (rapid cooling, extracorporeal detoxification methods, etc.), mortality in kidney damage due to general overheating has decreased slightly over the past decades. For health care and industry researchers, it is necessary to identify the harmful occupational conditions that lead to heat stress nephropathy and to develop certain occupational safety strategies.

Chromatin conformations of HSP12 during transcriptional activation in the Saccharomyces cerevisiae stationary phase

During evolution, living cells have developed sophisticated molecular and physiological processes to cope with a variety of stressors. These mechanisms, which collectively constitute the Environmental Stress Response, involve the activation/repression of hundreds of genes that are regulated to respond rapidly and effectively to protect the cell. The main stressors include sudden increases in environmental temperature and osmolarity, exposure to heavy metals, nutrient limitation, ROS accumulation, and protein-damaging events. The growth stages of the yeast S. cerevisiae proceed from the exponential to the diauxic phase, finally reaching the stationary phase. It is in this latter phase that the main stressor events are more active. In the present work, we aim to understand whether the responses evoked by the sudden onset of a stressor, like what happens to cells going through the stationary phase, would be different or similar to those induced by a gradual increase in the same stimulus. To this aim, we studied the expression of the HSP12 gene of the HSP family of proteins, typically induced by stress conditions, with a focus on the role of chromatin in this regulation. Analyses of nucleosome occupancy and three-dimensional chromatin conformation suggest the activation of a different response pathway upon a sudden vs a gradual onset of a stress stimulus. Here we show that it is the three-dimensional chromatin structure of HSP12, rather than nucleosome remodeling, that becomes altered in HSP12 transcription during the stationary phase.

Suero salino hipertónico e insuficiencia cardiaca: ¿«sodio-centrista» o «cloro-centrista»?

Up to 50% of patients admitted for heart failure have congestion at discharge despite diagnostic and therapeutic advances. Both persistent congestion and diuretic resistance are associated with worse prognosis. The combination of hypertonic saline and loop diuretic has shown promising results in different studies. However, it has not yet achieved a standardized use, partly because of the great heterogeneity in the concentration of sodium chloride, the dose of diuretic or the amount of sodium in the diet. Classically, the movement of water from the intracellular space due to an increase in extracellular osmolarity has been postulated as the main mechanism involved. However, chloride deficit is postulated as the main up-regulator of plasma volume changes, and its correction may be the main mechanism involved. This ‘chloride centric’ approach to heart failure opens the door to therapeutic strategies that would include diuretics to correct hypochloremia, as well as sodium free chloride supplementation.

Impact of Preparticipating Hypohydration on Cardiopulmonary Exercise Capacity in Ambitious Recreational Athletes.

Heat induces a thermoregulatory strain that impairs cardiopulmonary exercise capacity. The aim of the current study is to elucidate the effect of isolated dehydration on cardiopulmonary exercise capacity in a model of preparticipating hypohydration. Healthy recreational athletes underwent a standardised fluid deprivation test. Hypohydration was assessed by bioelectrical impedance analysis (BIA) and laboratory testing of electrolytes and retention parameters in the blood and urine. The participants underwent cardiopulmonary exercise testing (CPET) with a cycle ramp protocol. Each participant served as their own control undergoing CPET in a hypohydrated [HYH] and euhydrated [EUH] state. Fluid deprivation caused a mild (2%) but significant reduction of body water (38.6 [36.6; 40.7] vs. 39.4 [37.4; 41.5] %; p < 0.01) and an increase of urine osmolality (767 [694; 839] vs. 537 [445; 629] mosm/kg; p < 0.01). Hypohydration was without alterations of electrolytes, serum osmolality or hematocrit. The oxygen uptake was significantly lower after hypohydration (-4.8%; p = 0.02 at ventilatory threshold1; -2.0%; p < 0.01 at maximum power), with a corresponding decrease of minute ventilation (-4% at ventilatory threshold1; p = 0.01, -3.3% at maximum power; p < 0.01). The power output was lower in hypohydration (-6.8%; p < 0.01 at ventilatory threshold1; -2.2%; p = 0.01 at maximum power). Isolated hypohydration causes impairment of workload as well as peak oxygen uptake in recreational athletes. Our findings might indicate an important role of hypohydration in the heat-induced reduction of exercise capacity.

Sodium chloride impacts glycosylation and N- and O-glycan site occupancy of an Fc-fusion protein.

Fc-fusion proteins are highly complex molecules, difficult to manufacture at scale. In this work, undesired proteoforms were detected during the manufacture of a therapeutic fusion protein produced in CHO cells. These species were characterized using gel electrophoresis, size exclusion chromatography and liquid chromatography-mass spectrometry leading to the identification of low molecular weight proteoforms presenting low N- and O-glycan site occupancy, as well as a low sialylation content. Upstream process parameters were investigated, and fusion protein quality was shown to be linked to the sodium chloride content of the medium. A mitigation strategy was developed to avoid formation of unwanted glyco-variants, resulting in an increased yield of highly glycosylated Fc-fusion protein. The effect of sodium chloride was shown to be independent of the osmolality increase and was hypothesized to be linked to a modulation of Golgi acidity, which is required for the correct localization and function of glycosyltransferases. Altogether, this study highlights the importance of the salt balance in cell culture media used to produce highly sialylated and occupied glycoproteins, helping to maximize the yield and increase robustness of processes aiming at producing biopharmaceutical complex therapeutic molecules.

P-309 High-viscosity oil overlay provides less protection against media evaporation in extended dry incubation conditions compared with conventional light oil but a reduced heat loss

Abstract Study question Does high-viscosity oil (HVO) improve stability on osmolality, temperature, pH, Na+ concentration, and mouse embryo development compared with conventional light oil (CLO)? Summary answer In dry incubation condition, osmolality and Na+ concentration were significantly higher in embryo culture media overlaid with either oils, but HVO was worst than CLO. What is known already Suboptimal in-vitro culture conditions affect embryo quality and viability. Overlaying culture media with oil is used to maintain a stable optimal culture environment, including reduction of evaporation. Despite dry incubators are known to increase evaporation, they have become more common, especially with the widespread use of time-lapse imaging. The risk of excessive evaporation with the consequent impact on osmolality is even more significant with the promoted usage of undisturbed extended embryo culturing. Novel products (HVO) therefore been developed and recently commercialised with the aim of reducing such effect. Nonetheless, no independent data are available to determine their superiority over CLO. Study design, size, duration Culture media dishes overlaid with CLO or HVO were incubated in dry and humidified conditions while mimicking extended culture conditions in four independent experimental groups. Changes in osmolality, pH and Na+ concentrations over three days were measured. Lipid peroxidation and mouse embryo development were recorded to assess toxicity. Additionally, the ability of different oils to maintain the temperature in the culture drops when culture dishes were removed from the incubator was compared. Participants/materials, setting, methods Standard embryo culture media drops overlayered with HVO or CLO were incubated in two equally calibrated, identical incubators providing dry or humid conditions. Osmolality (Day1, D3, D5 measured by freezing-point depression osmometer), temperature drop rate at room temperature (T-type thermocouple sensor probe), pH and [Na+] (hand-held blood gas analyser), mouse embryo assay (MEA, 75 embryos from naturally mated CD1 female mice (N = 5), and lipid peroxidation end-product levels (MDA, TBARS assay) were compared among groups. Main results and the role of chance The osmolality of the media incubated in humidified conditions did not show a significant change throughout the 5-day experiment, regardless of the type of oil used. However, in dry conditions, a significant daily increase in osmolality was evident; changes were more prominent under HVO (D1, 278.13 ± 0.82; D3, 286.00 ± 1.51; D5 289.86 ± 1.17 mOsm/kg H2O ± SEM. p ≤ 0.001). The osmolality of the media overlaid by the same type of oil raised between different incubators, and reached a significantly higher value on D5. [Na+] values were consistent with the osmolality changes. The recovery time of the temperature on the heated stage to 37 °C was similar in CLO- and HLO-overlaid media (1:07 ± 1:02 min and 2:00 ± 0.7 min, respectively). The temperature drop rate after media were kept at ambient temperature was similar in the first 4 min. By the end, the temperature difference reached a significance between CLO and HVO overlays (32.4 ± 0.4 °C and 32.7 ± 0.4 °C respectively, p = 0.045). No significant differences were found in pH, lipid peroxidation or mouse embryo development between the study experimental groups. Limitations, reasons for caution The embryos for MEA were obtained without hyperstimulation, resulting in a small sample size for the study. The exact timing of fertilisation was impossible to detect after mice mating, which caused inconsistency in embryo grading. Wider implications of the findings Dry incubators may determine suboptimal embryo culture conditions. A newly developed heavy oil proved to be inferior to conventional light oil in maintaining osmolality stable. Further research should be carried out in engineering systems that limit culture media evaporation or support undisturbed embryo culture in humidified conditions. Trial registration number not applicable

#5852 EFFECT OF DIET ON THE COLLECTOR DUCT BEHAVIOUR: RELEVANCE IN PRECLINICAL DRUG DEVELOPMENT

Abstract Background and Aims Drug induced syndrome of inadequate antidiuretic hormone secretion (SIADH) is a common problem in clinical practice. Early detection of this side effect at a preclinical level would improve safety and efficiency. We investigate the role of different diets in preclinical detection of SIADH. Method Animals were divided into two groups (n = 4 each), one receiving standard pellet based diet, and the other one a diluted hyposodic gelified liquid diet. After one week, both groups were administered daily intraperitoneal (i.p.) injections of desmopressin acetate (ddAVP; 0.4 μg/kg) and a volume of distilled water equivalent to 2.5% of body weight for 7 days. Animals were placed in metabolic cages for 24h at the end of each period to collect and analyse the urine, and a blood sample from the tail vein was obtained. Weight, water and food intake were measured on a daily basis. Results Differences in water and electrolyte balance are shown in Table 1. Pellet fed animals presented an increased water intake and produced a more concentrated urine compared to those on a hyposodic diet. When analysed in detail, urinary osmolality was mainly driven by ionic osmoles in pellet fed animals (42,25 ± 4,72 vs 266,30 ± 17,32 mOsm/kg, p&amp;lt;0,005). Of note, there seems to be an occult osmole, especially prominent in the pellet group (osmol gap 20,40 ± 3,18 vs 99,89 ± 9,81 mOsm/kg, p = 0,017). All of the above suggest there is a net retention of water, sodium and potassium in those animals fed with a conventional pellet diet. Daily administration of ddAVP/water induced a mild transient hyponatremia that was recovered 24h later in both groups; this situation was repeated over a 7 day period. Pellet fed animals did not modify water intake, diuresis, free water clearance or urinary osmolality after ddAVP/water treatment, suggesting that hyperosmolar diets significantly increase urinary osmolality fixating urinary water losses and impairing further urinary concentration. On the contrary, animals on a hyposodic diet did present antiaquaresis, as shown by an increase in water intake, a decrease in diuresis and free water clearance and an increase in urinary osmolality. Animals fed with hyposodic diet progressively lost weight, while pellet fed animals did not. However, this trend was reversed after 4 days of treatment with ddAVP/water, again suggesting a degree of water retention. It is remarkable that we have found signs of water retention despite the fact that the induced hyponatremia is mild and transient. Conclusion The antiaquaretic effect of ddAVP is overridden by the amount of urinary osmoles excreted when on a pellet diet, which seem to fixate the amount of water in the urine. Therefore, the use of hyposodic diets in preclinical drug development is essential when SIADH is likely to be expected, as its effects will be unnoticed if a standard diet is used.

Shortening of primary cilia length is associated with urine concentration in the kidneys.

The primary cilium, a microtubule-based cellular organelle present in certain kidney cells, functions as a mechano-sensor to monitor fluid flow in addition to various other biological functions. In kidneys, the primary cilia protrude into the tubular lumen and are directly exposed to pro-urine flow and components. However, their effects on urine concentration remain to be defined. Here, we investigated the association between primary cilia and urine concentration. Mice either had free access to water (normal water intake, NWI) or were not allowed access to water (water deprivation, WD). Some mice received tubastatin, an inhibitor of histone deacetylase 6 (HDAC6), which regulates the acetylation of α-tubulin, a core protein of microtubules. WD decreased urine output and increased urine osmolality, concomitant with apical plasma membrane localization of aquaporin 2 (AQP2) in the kidney. After WD, compared with after NWI, the lengths of primary cilia in renal tubular epithelial cells were shortened and HDAC6 activity increased. WD induced deacetylation of α-tubulin without altering α-tubulin levels in the kidney. Tubastatin prevented the shortening of cilia through increasing HDAC6 activity and consequently increasing acetylated α-tubulin expression. Furthermore, tubastatin prevented the WD-induced reduction of urine output, urine osmolality increase, and apical plasma membrane localization of AQP2. WD shortens primary cilia length through HDAC6 activation and α-tubulin deacetylation, while HDAC6 inhibition blocks the WD-induced changes in cilia length and urine output. This suggests that cilia length alterations are involved, at least in part, in the regulation of body water balance and urine concentration.

Optimization of a non-activating medium for short-term chilled storage of barramundi (Lates calcarifer) testicular spermatozoa

Reliable short-term chilled sperm storage is a critical prerequisite to using advanced reproductive techniques for captive breeding of barramundi (Asian sea bass; Lates calcarifer). Marine Ringer's solution (MRS) is a common non-activating medium (NAM) and has previously been used to store sperm from wild-caught barramundi. However, MRS-stored spermatozoa from captive-bred barramundi were observed to lyse within 30 min incubation. Therefore, this study aimed to optimize the composition of NAM for short-term chilled storage by characterizing and mimicking the biochemical profile of seminal and blood plasma of captive-bred barramundi. To further understand the effect of each component, osmolality was first examined to determine its effect on sperm viability. Thereafter, the effects of NaHCO3, pH, and Na+ and K+ concentrations on sperm motility were investigated. Optimization of the NAM formula was achieved through iterative adaptions. The increase in NAM osmolality from 260 to 400 mOsm/kg led to a significant improvement in sperm viability. Moreover, using HEPES instead of NaHCO3 as buffering agent significantly enhanced sperm motility and velocity. As a result, sperm samples diluted with optimized NAM (185 mM NaCl, 5.1 mM KCl, 1.6 mM CaCl2·2H2O, 1.1 mM MgSO4·7H2O, 10.0 mM HEPES, 5.6 mM D+ glucose, 400 mOsm/kg, pH 7.4) and stored at 4 °C showed no significant loss in total motility for up to 48 h and retained progressive motility for up to 72 h. The optimized NAM developed in this study significantly extended the functional lifespan of spermatozoa during chilled storage, permitting the ongoing development of advanced reproductive technologies for barramundi.

Graves' ophthalmopathy and dry eye syndrome

Graves ophthalmopathy present different signs and symptoms, in particular ocular surface disease that is the most frequent cause of ocular discomfort. This pathology can lead to increased ocular surface exposure and evaporative dry eye disease, tear-film changes and ocular surface expression of pro-inflammatory cytokines. Patients with ocular surface disease complain of several symptoms from foreign body sensation, blurred vision, dryness, or conjunctival injection, at an estimated prevalence of 65-95%. The most useful assessment is the Ocular Surface Disease Index, which has been shown to be significantly increased in active thyroid-related ophthalmopathy patients. Every increase in clinical activity score of one, double the index values. A careful ocular examination should be performed to assess the severity of the pathology. Superior limbic keratoconjunctivitis, superficial keratopathy and obstructive Meibomian gland disease represent the structural pathological changes; tear break-up time, increase in lacrimal osmolarity and presence of inflammatory cytokines represent the functional changes. Management of dry eye syndrome associated to Graves ’ophthalmopathy can be challenging. Moderate disease may be handled with artificial tears, ointments, and night-time moisture chambers. Topical or oral steroids can be considered to treat the inflammatory component. Surgical intervention consists of early punctual occlusion with temporary punctual plugs. If the patient progresses to sight-threatening ophthalmopathy, surgical intervention may become necessary to reduce exophthalmos and lagophthalmos. Standardized treatment guidelines are not available, and the management depends on patient’s disease activity and severity. A stepwise approach must be applied, ranging from conservative management to surgical intervention combined with topical or systemic therapy.

«Pressure diuresis» phenomenon: mechanisms and physiological significance in diving medical support practice: prospective cohort study

INTRODUCTION: When staying in hyperbaric conditions there is an increase in diuresis, developing a negative water balance, loss of electrolytes and tissue dehydration after diving different in depth, duration, intensity and other parameters, which can be conditionally combined into specific physiological symptom complex – “pressure diuresis” phenomenon (or “diver diuresis”). OBJECTIVE: To investigate the mechanisms of “pressure diuresis” under the action of high gaseous medium pressure and determine the physiological nature of this phenomenon in divers. MATERIALS AND METHODS: 44 men aged 19–23 were examined. In the first study all the subjects had initial sustainability to hyperbaric adverse factors (decompression gas formation – DG, hypoxic hypoxia – HH, nitrogen toxic effect – NTE and oxygen toxic effect – OTE). Hormonal status and body’s osmotic homeostasis rates were defined in all the subjects. The second study evaluated changes of the subjects’ water-electrolyte metabolism, kidney function and osmotic homeostasis during the oral loading renal test with the water load in the baseline and conditions of hyperbaric adverse factor exposure on divers. Statistics: To perform statistical analysis, application packages of Statistica for Windows 10.0 were used. RESULTS: 17 (38,6 %), 29 (65,9 %), 20 (45,5 %) and 35 (79,6 %) subjects had medium and low resistance to DG, HH, OTE and NTE, respectively; 27 (61,4 %), 15 (34,1 %), 24 (54,5 %) and 9 (20,4 %) subjects had high resistance to DG, HH, OTE and NTE. Increase in the concentration of antidiuretic hormone was identified in the diving group with low and medium resistance to NTE, HH, and OTE (by 146,8, 141,1 and 93,2 %, respectively, compared to the results before hyperbaric adverse factor exposure). The highest concentration increase of aldosterone was found among the divers with low and medium resistance to OTE, DG and HH (by 41,5, 39,1 and 36,2 %, respectively). The increase in blood plasma osmolality was observed in the subjects with low and medium resistance to OTE (an increase by 6,6 % compared to the divers with high resistance). A significant reduction in blood plasma osmolality was recorded in the subject group with low and medium resistance to NTE and HH (reduction by 5,2 and 4,2 %, compared to the group with high resistance). The most significant decrease in urine osmolality was identified in the diving group with low and medium resistance to DG and OTE (reduction by 14,5 and 17,7 %, respectively). A significant increase in urine osmolality under the action of hyperbaric factors was defined in the diving group with low and medium resistance to NTE and HH (by 19,8 and 19,3 %). DISCUSSION: The data obtained indicate the emergence of a new body hydration status during person’s staying in hyperbaric conditions. Body hydration status will be connected with its individual resistance to hyperbaric adverse factor effect. Thus, with the development of marked DG iso-osmotic (isotonic) hyperhydration is formed, with OTE hyperosmotic (hypertonic) hyperhydration occurs, with NTE and HH hypo-osmolar (hypotonic) hyperhydration develops. CONCLUSION: The study showed a certain connection (correlation) between types of hyperhydration in the tissues of a diver’s body, mechanisms of occurrence, low and medium initial resistance to a particular hyperbaric factor. “Pressure diuresis” phenomenon (“diver diuresis”), that occurs in divers with high resistance to hyperbaric adverse factors, will be a normal body’s physiological response, aimed at eliminating hypervolemia and reduction in the volume of circulating plasma.

Impact of high- and low-flow nebulised saline on airway hydration and mucociliary transport.

Nebulised drugs, including osmotic agents and saline, are increasingly used during noninvasive respiratory support, including nasal high-flow therapy. The authors conducted an in vitro study to compare the hydration effect of nebulised isotonic 0.9% and hypertonic 7.0% saline on mucociliary transport. In a perfused organ bath, 10 sheep tracheas were exposed to 7.5 mL nebulised 0.9% and 7.0% saline entrained into heated (38°C) and humidified air delivered at high and low flow (20 and 7 L·min-1, respectively). Simultaneous measurements of the airway surface liquid height, mucus transport velocity, cilia beat frequency and surface temperature were made over time. The data are presented as mean±sd. The airway surface liquid height increased significantly with both 0.9% and 7.0% saline: at low-flow by 37.2±10.0 µm and 152.7±10.9 µm, respectively, and at high-flow by 62.3±5.6 µm and 163.4±25.4 µm, respectively (p<0.001). Mucus velocity was increased by both 0.9% and 7.0% saline from a baseline of 8.2±0.8 mm·min-1 to 8.8±0.7 mm·min-1 and 17.1±0.5 mm·min-1, respectively, with low-flow and at high-flow to 9.8±0.02 mm·min-1 (p=0.04) and 16.9±0.5 mm·min-1 (p<0.05), respectively. Ciliary beating did not change with 0.9% saline, but declined from 13.1±0.6 Hz to 10.2±0.6 Hz and 11.1±0.6 Hz (p<0.05) with 7.0% saline at low- and high-flow, respectively. The findings demonstrate that nebulised isotonic 0.9% saline, like hypertonic 7.0% saline, significantly stimulates basal mucociliary transport, and the use of high-flow delivery had no significantly different hydration effects compared with low-flow delivery. Hypertonic 7.0% saline suppressed ciliary beating, indicating an increase in airway surface liquid osmolarity, which may have negative effects on the airway surface with frequent use.

Deciphering the adaption of bacterial cell wall mechanical integrity and turgor to different chemical or mechanical environments

Bacteria adapt the mechanical properties of their cell envelope, including cell wall stiffness, turgor, and cell wall tension and deformation, to grow and survive in harsh environments. However, it remains a technical challenge to simultaneously determine these mechanical properties at a single cell level. Here we combined theoretical modelling with an experimental approach to quantify the mechanical properties and turgor of Staphylococcus epidermidis. It was found that high osmolarity leads to a decrease in both cell wall stiffness and turgor. We also demonstrated that the turgor change is associated with a change in the viscosity of the bacterial cell. We predicted that the cell wall tension is much higher in deionized (DI) water and it decreases with an increase in osmolality. We also found that an external force increases the cell wall deformation to reinforce its adherence to a surface and this effect can be more significant in lower osmolarity. Overall, our work highlights how bacterial mechanics supports survival in harsh environments and uncovers the adaption of bacterial cell wall mechanical integrity and turgor to osmotic and mechanical challenges.

Osmotically evoked PLCδ1-dependent translocation of ΔN-TRPV1 channels in rat supraoptic neurons

Osmoregulation is an essential homeostatic process that requires constant release of vasopressin during sustained increases in plasma osmolality. The magnocellular neurosecretory cells (MNCs) respond to increases in external osmolality through increases in the activity of ΔN-TRPV1 channels, which leads to increased action potential firing and vasopressin release. We show that sustained exposure of acutely isolated rat and mouse MNCs to hypertonic solutions (90min) causes a reversible translocation of ΔN-TRPV1 channels from internal stores to the plasma membrane that depends on the activation of phospholipase C and on SNARE-dependent exocytosis. ΔN-TRPV1 channel translocation is absent in MNCs isolated from transgenic mice lacking the PLCδ1 isoform, suggesting that PLCδ1 is essential for triggering this process. The translocation of ΔN-TRPV1 channels to the cell surface could contribute to the maintenance of MNC excitability during sustained increases in osmolality. Our data therefore have important implications for the mechanisms underlying mammalian osmoregulation.

Limited Effect of Dehydrating via Active vs. Passive Heat Stress on Plasma Volume or Osmolality, Relative to the Effect of These Stressors per Se.

The physiological, perceptual, and functional effects of dehydration may depend on how it is incurred (e.g., intense exercise releases endogenous water via glycogenolysis) but this basic notion has rarely been examined. We investigated the effects of active (exercise) heat- vs. passive heat-induced dehydration, and the kinetics of ad libitum rehydration following each method. Twelve fit participants (five females and seven males) completed four trials in randomised order: DEHydration to -3% change in body mass (∆BM) under passive or active heat stress, and EUHydration to prevent ∆BM under passive or active heat stress. In all trials, participants then sat in a temperate-controlled environment, ate a standard snack and had free access to water and sports drink during their two-hour recovery. During mild dehydration (≤2% ∆BM), active and passive heating caused comparable increases in plasma osmolality (Posm: ~4 mOsmol/kg, interaction: p = 0.138) and reductions in plasma volume (PV: ~10%, interaction: p = 0.718), but heat stress per se was the main driver of hypovolaemia. Thirst in DEHydration was comparably stimulated by active than passive heat stress (p < 0.161) and shared the same relation to Posm (r ≥ 0.744) and ∆BM (r ≥ 0.882). Following heat exposures, at 3% gross ∆BM, PV reduction was approximately twice as large from passive versus active heating (p = 0.003), whereas Posm perturbations were approximately twice as large from EUHydration versus DEHydration (p < 0.001). Rehydrating ad libitum resulted in a similar net fluid balance between passive versus active heat stress and restored PV despite the incomplete replacement of ∆BM. In conclusion, dehydrating by 2% ∆BM via passive heat stress generally did not cause larger changes to PV or Posm than via active heat stress. The heat stressors themselves caused a greater reduction in PV than dehydration did, whereas ingesting water to maintain euhydration produced large reductions in Posm in recovery and therefore appears to be of more physiological significance.