Hypotonic Research Articles

Structure of the human volume regulated anion channel

SWELL1 (LRRC8A) is the only essential subunit of the Volume Regulated Anion Channel (VRAC), which regulates cellular volume homeostasis and is activated by hypotonic solutions. SWELL1, together with four other LRRC8 family members, potentially forms a vastly heterogeneous cohort of VRAC channels with different properties; however, SWELL1 alone is also functional. Here, we report a high-resolution cryo-electron microscopy structure of full-length human homo-hexameric SWELL1. The structure reveals a trimer of dimers assembly with symmetry mismatch between the pore-forming domain and the cytosolic leucine-rich repeat (LRR) domains. Importantly, mutational analysis demonstrates that a charged residue at the narrowest constriction of the homomeric channel is an important pore determinant of heteromeric VRAC. Additionally, a mutation in the flexible N-terminal portion of SWELL1 affects pore properties, suggesting a putative link between intracellular structures and channel regulation. This structure provides a scaffold for further dissecting the heterogeneity and mechanism of activation of VRAC.

Comparative Cytogenetic Study on Male and Female Captive Sumatran Elephant in Elephant Training Center, Way Kambas National Park

Sumatran elephant (Elephas maximus sumatranus) was one of five big mammals in Way Kambas National Park (WKNP). Cytogenetic study was a part of conservation effort for sumatran elephant, especially in conservation genetic field. Captive sumatran elephant’s blood in Elephant Training Center (ETC) WKNP was collected to karyotype analysis. Captive sumatran elephant’s chromosome was analyzed by squashing technique with pretreating cells in a hypotonic solution. The study of karyotypes were made by giemsa staining applied to captive elephant’s blood cell. The diploid number of captive sumatran elephant was 56 both in male and female. The karyotype analysis performed different fundamental number in male and female, both of total fundamental number (FN) and autosomal fundamental number (FNa). Chromosome of female sumatran elephant showed the FN = 8, and FNa = 6, but in male sumatran elephant FN = 7, and FNa = . The karyotypes of X chromosome was large submetacentric and Y chromosome was small acrocentric.. The karyotype presented here will compilled with DNA fingerprinting analysis of all captive sumatran elephant in ETC WKNP as references in future breeding policy.

Calcium signalling regulates the functions of the bZIP protein VIP1 in touch responses in Arabidopsis thaliana.

VIP1 is a bZIP transcription factor in Arabidopsis thaliana. VIP1 and its close homologues transiently accumulate in the nucleus when cells are exposed to hypo-osmotic and/or mechanical stress. Touch-induced root bending is enhanced in transgenic plants overexpressing a repression domain-fused form of VIP1 (VIP1-SRDXox), suggesting that VIP1, possibly with its close homologues, suppresses touch-induced root bending. The aim of this study was to identify regulators of these functions of VIP1 in mechanical stress responses. Co-immunoprecipitation analysis using VIP1-GFP fusion protein expressed in Arabidopsis plants identified calmodulins as VIP1-GFP interactors. In vitro crosslink analysis was performed using a hexahistidine-tagged calmodulin and glutathione S-transferase-fused forms of VIP1 and its close homologues. Plants expressing GFP-fused forms of VIP1 and its close homologues (bZIP59 and bZIP29) were submerged in hypotonic solutions containing divalent cation chelators, EDTA and EGTA, and a potential calmodulin inhibitor, chlorpromazine, to examine their effects on the nuclear-cytoplasmic shuttling of those proteins. VIP1-SRDXox plants were grown on medium containing 40 mm CaCl2, 40 mm MgCl2 or 80 mm NaCl. MCA1 and MCA2 are mechanosensitive calcium channels, and the hypo-osmotic stress-dependent nuclear-cytoplasmic shuttling of VIP1-GFP in the mca1 mca2 double knockout mutant background was examined. In vitro crosslink products were detected in the presence of CaCl2, but not in its absence. EDTA, EGTA and chlorpromazine all inhibited both the nuclear import and the nuclear export of VIP1-GFP, bZIP59-GFP and bZIP29-GFP. Either 40 mm CaCl2or 80 mm NaCl enhanced the VIP-SRDX-dependent root bending. The nuclear-cytoplasmic shuttling of VIP1 was observed even in the mca1 mca2 mutant. VIP1 and its close homologues can interact with calmodulins. Their nuclear-cytoplasmic shuttling requires neither MCA1 nor MCA2, but does require calcium signalling. Salt stress affects the VIP1-dependent regulation of root bending.

Phytochemical Screening and Anti-Inflammatory Properties of the Hydroethanolic Leaf Extract of Calotropis procera (Ait). R. Br. (Apocynaceae)

Aims: Inflammation has been implicated in many disorders and currently available therapies elicit adverse effects. The coarse shrub of Calotropis procera has been used in folk medicine to treat several ailments such as asthma, rheumatism and also fever. The anti-inflammatory potential of Calotropis procera was assessed in this study to evaluate its effectiveness. Place and Duration of Study: University of Cape Coast, Ghana between June 2016 and March 2017. Methodology: This study was carried out to screen for the phytochemical constituents and investigate the anti-inflammatory properties of Calotropis procera extract (CPE) using four in vitro assays (heat-induced haemolysis, hypotonic-induced haemolysis, albumin denaturation and the bovine serum albumin assay (BSA)) and, two in vivo models (carrageenan and formalin-induced paw oedema models). Three graded concentrations of the plant extract (CPE) and a standard anti-inflammatory agent, diclofenac: 100, 300 and 1000 μg/mL were used in the in vitro models while 30, 100 and 300 mg/kg were used in the in vivo experiments. Qualitative tests for tannins, alkaloids, flavonoids, carbohydrates, triterpenoids, steroids, proteins, reducing sugars, glycosides, saponins and amino acids were performed to determine the phytochemical constituents of the extract. Results: The phytochemical screening revealed presence of alkaloids, tannins and triterpenes, reducing sugars, sterols, amino acids, and glycosides. CPE 1000μg/mL demonstrated anti-inflammatory characteristics by significantly stabilizing RBCs exposed to heat and hypotonic solution and also inhibiting protein denaturation in heat-induced denaturation (69.24%); hypotonic-induced (85.09%); albumin denaturation (87.80%) and BSA (96.86% assays). Similar results were observed for the two lower doses of CPE as well as diclofenac. In the in vivo tests, CPE was able to significantly reduce both carrageenan-induced (P=0.0105) and formalin-induced inflammation (P<0.0001) in rats. Diclofenac the reference anti-inflammatory agent also showed similar effects in both tests. The percentage inhibition of paw oedema produced by CPE 30, 100, and 300 mg/kg in the formalin test were 19.61% 35.88% and 47.66% respectively while diclofenac (10 mg/kg) produced 85.16% inhibition of paw oedema. Conclusion: From the present study, it can be observed that CPE possessed anti-inflammatory properties and thus vindicates the folkloric use of this plant in inflammatory conditions.

Stem Bark Extract of Sterculia setig­era Delile Exhibits Anti-inflammatory Properties through Membrane Stabilization, Inhibition of Protein Denaturation and Prostaglandin E2 Activity

Aims: The stem bark of Sterculia setigera has been used in several African countries for the treatment of many inflammatory conditions. However, there is no scientific report to authenticate this usage. The current study, therefore, aims at assessing the in vitro and in vivo anti-inflammatory activities of the stem bark extract of Sterculia setigera (SSE). Place and Duration of Study: The study was conducted at the Laboratories of Department of Biomedical Sciences, University of Cape Coast, Ghana between October, 2016 and April, 2017. Methodology: In vitro anti-inflammatory activities were assessed using heat and hypotonic solution - induced red blood cell haemolysis assays, as well as egg and bovine serum albumin denaturation assays. In vivo anti-inflammatory effect of SSE (30, 100 and 300 mg/kg, p.o.) and diclofenac (10 mg/kg, p.o.) was assessed using carrageenan as well as prostaglandin E2 -induced paw oedema models in rats. Results: SSE inhibited heat and hypotonic solution-induced haemolysis by a maximum effect of 62.81 ± 1.18% and 76.05 ± 3.08% respectively as compared to diclofenac which was 76.63 ± 3.36% and 76.49 ± 1.67% respectively. Also, egg and bovine serum albumin denaturation were markedly inhibited by SSE with a maximum inhibitory effect of 74.84 ± 64% and 45.73 ± 8.41% respectively. Diclofenac had a maximum inhibitory effect of 72.23 ± 3.14% and 52.02 ± 0.72% respectively for egg and bovine serum albumin denaturation assays. Also, SSE (30, 100 and 300 mg/kg, p.o.) and diclofenac (10 mg/kg, p.o.) significantly and dose-dependently reduced rats’ paw oedema in the carrageenan and prostaglandin E2 – induced inflammation tests. Conclusion: SSE exhibited anti-inflammatory effects in both in vitro and in vivo models and this gives credence to its folkloric usage.

Syzygium aqueum: A Polyphenol- Rich Leaf Extract Exhibits Antioxidant, Hepatoprotective, Pain-Killing and Anti-inflammatory Activities in Animal Models.

Syzygium aqueum is widely used in folk medicine. A polyphenol-rich extract from its leaves demonstrated a plethora of substantial pharmacological properties. The extract showed solid antioxidant properties in vitro and protected human keratinocytes (HaCaT cells) against UVA damage. The extract also reduced the elevated levels of ALT, AST, total bilirubin (TB), total cholesterol (TC) and triglycerides (TG) in rats with acute CCl4 intoxication. In addition to reducing the high MDA level, the extract noticeably restored GSH and SOD to the normal control levels in liver tissue homogenates and counteracted the deleterious histopathologic changes in liver after CCl4 injection. Additionally, the extract exhibited promising anti-inflammatory activities in vitro where it inhibited LOX, COX-1, and COX-2 with a higher COX-2 selectivity than that of indomethacin and diclofenac and reduced the extent of lysis of erythrocytes upon incubation with hypotonic buffer solution. S. aqueum extract also markedly reduced leukocyte numbers with similar activities to diclofenac in rats challenged with carrageenan. Additionally, administration of the extract abolished writhes induced by acetic acid in mice and prolonged the response latency in hot plate test. Meanwhile, the identified polyphenolics from the extract showed a certain affinity for the active pockets of 5-lipoxygenase (5-LOX), cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) explaining the observed anti-inflammatory activities. Finally, 87 secondary metabolites (mostly phenolics) were tentatively identified in the extract based on LC-MS/MS analyses. Syzygium aqueum displays good protection against oxidative stress, free radicals, and could be a good candidate for treating oxidative stress related diseases.

Abstract P-326: HYPERCHLOREMIA IN CRITICALLY ILL PEDIATRIC PATIENTS, THE “HYCCIP” STUDY

Aims & Objectives: Guidelines have evolved from previously recommending hypotonic intravenous (IV) fluids, to isotonic fluids as the solutions of choice in children. We hypothesized that this change in practice is associated with the emergence of hyperchloremic metabolic acidosis (HCMA). The objective of this study was to evaluate current IV fluid prescription practices and determine the prevalence of HCMA in children within the first 72 hours of Pediatric Intensive Care Unit (PICU) admission. Methods Retrospective study at McMaster and Stollery Children’s Hospital PICUs. Children under 18 years admitted to the PICU between January 2015 – January 2016 who received over 50% of their maintenance fluids parenterally, were included. We defined HCMA as serum chloride >107 mmol/L associated with serum pH <7.35 and HCO3- <23. Results 541 of 771 admitted patients were eligible. Median age was 69 months and 56% were males. The commonest prescribed IV maintenance fluids were 0.9% NaCl (72.8%), followed by Ringer’s Lactate (19.7%), and hypotonic solutions (3.9%; Figure 1). The incidence of hyperchloremia and HCMA was 86.8% and 32.8%, respectively. Significant predictors of HCMA were volume of 0.9% NaCl intake (OR 1.2, 95% CI [1.1,1.3]) and increasing disease severity (OR 13.6, 95% CI [4.1,45.5])Conclusions 0.9% NaCl has replaced hypotonic solutions as the current IV maintenance fluid of choice. The risk of HMCA is not restricted to the use of 0.9% NaCl as a volume expander, but as a maintenance fluid. Future studies should focus on determining if balanced salt solutions are more appropriate in critically ill children.

Effects of aqueous extract of Nelsonia canescens leaf on the osmotic fragility of red blood cell and blood parameters of Wistar albino rats.

The herbaceous plant Nelsonia canescens is a medicinal plant used in Asian and African traditional medicine for various diseases of humans and animals. The plant has been used for the treatment of pains and inflammatory action related diseases, cancer, gout, cough, fever, cardiovascular diseases, chicken pox and even malaria. Decoction of it has also been used as immune booster in patients by the traditionalists. This study was aimed at evaluating the effects of the aqueous leaf extract of the plant on the osmotic fragility of rats’ red blood cells, and also to determine the changes that occur in haematology and serum chemistry of the rats exposed orally to the extract for 28 days. Three groups of rats were administered orally with 200, 400, and 800mg/kg of the plant extract respectively while the fourth group which is the control was administered also orally with distilled water and their blood were then analyzed. The 2, 4 and 6mg/ml concentrations of the extract inhibit hypotonic solution induced rats erythrocytes hemolysis in concentration dependent manner and the inhibition is comparable to that of Indomethacin. The blood analysis showed a significant increase (p&lt;0.05) in total white blood cells and the lymphocytes for the groups administered with 400 and 800 mg/kg of the extract while the neutrophils decreased significantly. It was then concluded that the aqueous extract of the plant inhibits red blood cell hemolysis and hence its anti-inflammatory activities. Also, the significant increase in the total white blood cells and the lymphocytes could be the reason why the plant is useful as immune booster by the traditionalists.

A Method to Simultaneously Detect Changes in Intracellular Ca2+ Concentration and Cell Volume

A method to simultaneously assess the changes in intracellular calcium concentration and cell volume in single cells was developed using the Ca2+-sensitive fluorescent probe Fura-2 and a three-dimensional image-surface reconstruction technique, respectively. Studies with this method showed that Fura-2 loading had no significant effect on the kinetics of A549 human epithelial cell swelling in a hypotonic solution, as well as the volume restoration kinetics. Significant changes in intracellular Ca2+ concentration were not observed in the examined volume modulation range. The results suggest that Ca2+-mediated signaling pathways are not involved in the autoregulation of the cell volume in A549 cells exposed to hypotonic conditions.

Screening of Antibacterial, Thrombolytic, Membrane Stabilizing, Anti-inflammatory and Antitumor Activity of &lt;i&gt;Citrus assamensis&lt;/i&gt; Leaf Extracts

The methanol, ethanol and chlorofom leaf extracts of Satkara, Citrus assamensis (family: Rutaceae), were subjected to in vitro anti-bacterial, thrombolytic, membrane stabilizing and in vivo anti-inflammatory and antitumor activity tests. The chloroform extract of C. assamensis showed the most important spectrum of activity against Bacillus subtilis, Bacillus cereus, Sarcina lutea among 6 gram positive and against 11 gram negative bacteria at the concentration of 1000 μg/disc, while the range of zones of inhibition were within 7-16 mm. Among the tested three extracts CHCl3 extract showed potent thrombolytic activity and hypotonic solution induced haemolytic activity where the percentages of inhibition were found to be 35% and 55% respectively. All the extracts established significant (p&lt;0.05) anti-inflammatory effect by regulating biphasic inflammatory process induced by carrageenan. The leaf extract dose-dependently and significantly decreases the number of EAC cell count and inhibition of cell growth in comparison to the EAC control and standard. The results obtained in the present study indicate that, C. assamensis leaf can be a potential source of anti-bacterial, thrombolytic, membrane stabilizing, anti-inflammatory and antitumor agents.

English

This study was made to investigate in-vitro membrane stabilizing, thrombolytic, antioxidant and antimicrobial activities of ethanolic leaf extract of Coccinia indica. Different extractives showed significant membrane stabilizing activity. The ethanol soluble fraction showed (45.36&plusmn;0.45 % and 64.05&plusmn;0.22 %) of Red blood cells haemolysis inhibition under heat and hypotonic solution induced conditions, respectively. Mention worthy that carbon tetrachloride soluble fraction of C. indica exhibited highest thrombolytic activity 57.94&plusmn;0.23% in a comparison with standard streptokinase (SK) (68.89&plusmn;0.35 %). The antioxidant activities were determined by 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) free radical scavenging. In this case, ethanol soluble fraction (ESF) of crude extract showed fairly significant scavenging (with IC50 value of 2.52 &mu;g/ml) as opposed to a well-known antioxidant, butylated hydroxytoluene (BHT) (IC50 value of 3.99 &mu;g/ml). Pet ether soluble fraction (PESF) and ESF showed mild inhibitory effects against some Gram-positive and Gram-negative bacteria (zone of inhibition was 12 and 13 mm). The overall findings of the studies demonstrated that the leaves of C. indica has fairly significant membrane stabilizing and thrombolytic activity, mild to moderate antioxidant and antimicrobial potential. Thus, may indicated the scientific basis of C. indica leaves as a remedy for traditional uses against fever, pain, oxidative stress, inflammation, infectious diseases, diabetes, etc. Key words: Coccinia indica, in vitro, thrombolytic, 1,1‐diphenyl‐2‐picrylhydrazyl, antimicrobial activities. &nbsp

Trans-epithelial corneal collagen cross-linking with iontophoresis for progressive keratoconus

To evaluate the efficacy of trans-epithelial corneal collagen cross-linking (CXL) with Iontophoresis among patients with progressive keratoconus. It is a prospective interventional study, which is based on 41 eyes of 23 patients, suffering from progressive keratoconus and treated with trans-epithelial corneal cross-linking, using iontophoresis with ETDA and trometamol-enriched riboflavin 5 phosphates 0.1% hypotonic solution (Ricrolin+, Soot Italia SpA, Italy). The mean of uncorrected distance visual acuity and best corrected distant visual acuity was improved at 6months with statistically significant differences from baseline (p < 0.05). There was no statistically significant difference in keratometric values, including K1, K2, Km, topographic astigmatism, and central corneal thickness. Patients, who had completed 1year (21 eyes of 12 patients) of the treatment, showed similar results. The data indicated that corneal collagens cross-linking with iontophoresis using Ricroli+ may be an effective method in halting the progression of keratoconus without the side effects of epithelial removal, which may be encountered in the standard epi-off CXL procedure.

Development of novel inhibitors of swelling‐activated LRRC8 anion channels

Cells critically regulate their volume in response to hypoosmotic swelling by transporting chloride and small organic osmolytes out of the cell through so‐called volume‐regulated anion channels (VRACs). The 5 genes encoding VRACs were recently identified in two independent genome‐wide siRNA screens and are named LRRC8 (for Leucine Rich Repeat Containing) A‐E. LRRC8‐containing VRACs have recently been implicated in drug uptake, adipocyte size, and insulin signaling. Unfortunately, the pharmacological tools available to study the physiology and therapeutic potential of LRRC8 channels is limited to low‐affinity small‐molecule inhibitors that exhibit promiscuous activity toward other anion channels, cation channels, and myriad other off‐targets. To circumvent this barrier, we are employing high‐throughput screening (HTS) of small‐molecule libraries and medicinal chemistry to develop more potent and specific inhibitors of LRRC8 channels. HTS will be performed using a fluorescence assay based on halide quenching of a YFP mutant (i.e. F46L, H148Q, I152L) termed Ozzy. Briefly, HEK‐293 cells stably transfected with Ozzy will be plated in black‐walled, clear‐bottomed 384‐well plates, treated with hypotonic solution containing compounds from the Vanderbilt Institute of Chemical Biology library, and then treated with sodium iodide (NaI). Cell swelling opens endogenously expressed VRAC channels, through which iodide enters the cell and quenches Ozzy fluorescence. Small‐molecule inhibitors should block the iodide‐induced quenching of Ozzy. Checkerboard analysis using the best‐in‐class VRAC inhibitor, DCPIB, yielded a Z′ value of 0.74, indicating the assay is reproducible and robust enough to ensure high‐confidence hit‐picking in a large‐scale screen. The assay also reports dose‐dependent inhibition of iodide influx by DCPIB with an IC50 of 11 micromolar, which is close to values derived from electrophysiological experiments. We are currently using this assay to interrogate an analog library based on the DCPIB scaffold, with the goal of identifying more potent and specific inhibitors of VRAC.Support or Funding InformationDepartment of Anesthesiology Faculty Development FundsThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Blockade of the swelling-induced chloride current attenuates the mouse neonatal hypoxic-ischemic brain injury in vivo.

Activation of swelling-induced Cl- current (ICl,swell) during neonatal hypoxia-ischemia (HI) may induce brain damage. Hypoxic-ischemic brain injury causes chronic neurological morbidity in neonates as well as acute mortality. In this study, we investigated the role of ICl,swell in hypoxic-ischemic brain injury using a selective blocker, 4-(2-butyl-6,7-dichloro-2-cyclopentylindan-1-on-5-yl) oxybutyric acid (DCPIB). In primary cultured cortical neurons perfusion of a 30% hypotonic solution activated ICl,swell, which was completely blocked by the application of DCPIB (10 μmol/L). The role of ICl,swell in neonatal hypoxic-ischemic brain injury in vivo was evaluated in a modified neonatal hypoxic-ischemic brain injury model. Before receiving the ischemic insult, the mouse pups were injected with DCPIB (10 mg/kg, ip). We found that pretreatment with DCPIB significantly reduced the brain damage assessed using TTC staining, Nissl staining and whole brain imaging, and improved the sensorimotor and vestibular recovery outcomes evaluated in neurobehavioural tests (i.e. geotaxis reflex, and cliff avoidance reflex). These results show that DCPIB has neuroprotective effects on neonatal hypoxic-ischemic brain injury, and that the ICl,swell may serve as a therapeutic target for treatment of hypoxic-ischemic encephalopathy.

Xanthium indicum koenig meyvelerinin metanolik ekstraktının fitokimyasal tarama ve farmakolojik araştırması

Objective: This study aimed to assess the thrombolytic, membrane stabilizing, and antinociceptive activities of the methanolic extract of Xanthium indicum Koenig fruits along with its phytochemical screening. Material and Methods: Qualitative analysis method is adopted to evaluate phytochemicals. In-vivo antinociceptive efficacy was assessed using acetic acid induced writhing method in mice model. In-vitro membrane stabilizing and thrombolytic test were done by % inhibition of hypotonic solution produced hemolysis and clot disruption assay respectively. Results: Crude methanolic extract was used for phytochemical screening which confirmed the presence of reducing sugars, tannin, saponin, protein, phenol and diterpenes. In antinociceptive activity test, 500 mg/kg dose of crude extract showed 40% inhibition of writhing, while 200mg/kg showed 22.64%. 10 mg/ml extract inhibit highest rate of hemolysis with a value of 51.79% in membrane stabilizing activity test; while for acetyl salicylic acid (0.10 mg/ml), this value was 71.35%. In thrombolytic activity test, 10 mg/ml plant extract showed 27.11% of clot-lysis, which was maximum among our tested concentration; however 40.08%lysiswas occurred by the standard drug streptokinase. In all cases, we found the dose dependant response. Tannin and flavonoid are known to be responsible for antinociceptive and thrombolytic response. Conclusion: As the methanolic extract of the fruits of X. indicum possess potential pharmacological effects, the plant should be scrutinized comprehensively to uncover the inherent bioactive components with their exact mechanism of action for the drug development program.

Spectrophotometric assessment of effect of aqueous extract of Aloe vera on rabbit erythrocytes in varying concentrations of saline

Background: Hemolytic disorders are one of the prime reasons for frequent blood transfusions which involves lots of costs and sufferings to the patient. This study was undertaken to determine the effect of water soluble extract of Aloe vera on rabbit erythrocytes in varying concentrations of NaCl from 0.9% (isotonic) to 0.15% (hypotonic).Methods: Aqueous extract of Aloe vera (AVE) 200mg/kg was orally administered to rabbits in the test group while control group was given 1ml of distilled water (DW). Blood was withdrawn from rabbits, centrifuged and suspension in 1ml of normal saline was made. 20 microliter of red blood cells suspension from both control and test groups was added to normal saline of varying concentrations from 0.9% to 0.15% NaCl which were quantitatively analysed for hemolysis by UV spectrophotometer. Data was analysed by unpaired t test and P &lt;0.05 was considered statistically significant.Results: The difference in percentage of hemolysis in both test and control groups was not statistically significant. Therefore, acute administration of water soluble extract of Aloe vera (200mg/kg) did not have protective effect on rabbit erythrocytes against hypotonic solution of normal saline.Conclusions: Aloe vera might be useful for the treatment of oxidative stress-related human disorders by virtue of its antioxidant activity and may have a role in prevention of hemolysis which needs to be explored by further studies.

Tadalafil attenuates hypotonicity-induced Ca2+ influx via TRPV2 and TRPV4 in primary rat bladder urothelial cell cultures.

To investigate the localization of phosphodiesterase 5 (PDE5) and the molecular mechanism underlying the effect of the PDE5 inhibitor tadalafil in signal transduction in the bladder urothelium. PDE5 expression in rat bladder tissues and cultured primary rat bladder urothelial cells was evaluated using immunochemistry and western blot assays. Ca2+ influx in cells exposed to isotonic solution, hypotonic solution, a selective transient receptor potential vanilloid 2 (TRPV2) channel agonist (cannabidiol), a selective TRPV4 channel agonist (GSK1016790A), a TRP cation channel melastatin 7 (TRPM7) channel agonist (PIP2), or a purinergic receptor agonist (ATP) in the presence or absence of 10 µM tadalafil was evaluated using calcium imaging techniques. We also evaluated stretch-induced changes in ATP concentration in the mouse bladder in the presence or absence of 100 µM tadalafil. Immunochemistry and western blot analyses demonstrated that PDE5 is abundantly expressed in the bladder urothelium and in primary rat urothelial cells. Ca2+ influx induced by hypotonic stimulation, GSK1016790A, or cannabidiol was significantly inhibited by tadalafil, whereas ATP-induced Ca2+ influx was unaffected by tadalafil. PIP2 did not induce Ca2+ influx. ATP release in tadalafil-pretreated bladders significantly decreased compared to control bladders. Tadalafil attenuates Ca2+ influx via TRPV4 and TRPV2, and inhibits ATP release in the bladder urothelium. These findings indicate that tadalafil functions as an inhibitor of urothelial signal transduction.

Epidemiological characteristics of cerebral palsy in twelve province in China

Objective To investigate the epidemiological characteristics of cerebral palsy (CP) in children aged 1-6 years in China, including the incidence, prevalence, type of CP, etiology, prevention and rehabilitation status. Methods The survey was carried out by standard questionnaires, multi-center collaboration, stratified-cluster random sampling method.The surveyed adopted the following principles: streets in the city and villages in the rural areas, and the number of the urban and rural children was the same, and the proportion of children in each age group was balanced.The investigation areas included provinces and autonomous regions, including Heilongjiang, Beijing, Henan, Shandong, Shanxi, Shaanxi, Anhui, Hunan, Guangxi, Guangdong, Chongqing and Qinghai, and 323 858 children were investigated. Results The incidence of CP was 2.48‰(155/62 591 cases), and the prevalence was 2.46‰(797/323 858 cases) (1-6 years old). The prevalence varied in different regions, in which the highest prevalence was 5.40‰(54/9 998 cases) in Qinghai province, and the lowest prevalence was 1.04‰(47/45 133 cases) in Shandong province.The prevalence of the males (2.64‰, 461/174 391 cases) was higher than that of the females (2.25‰, 336/149 467 cases), and the difference was statistically significant (P<0.05). The types of CP were spastic type (58.85%, 469/797 cases), mixed type (13.17%, 105/797 cases), dyskinetic (9.79%, 78/797 cases), hypotonic (8.28%, 66/797 cases), ataxia (6.25%, 52/797 cases) and rigid (3.39%, 27/797 cases) respectively in 797 CP children.The first three risk factors for CP were long-term exposure to harmful physical factors during pregnancy, whether there were birth defects among the three generations of relatives of the children, such as children′s peers, parents or grandparents, whether there were birth defects among the children′s peers, parents or grandparents, and neonatal jaundice or persistent jaundice.Among 797 CP children, 79.67% of the children with CP were timely detected and treated in the local hospitals, while the other 19.93% of them were not timely treated.The places which could give them timely detection and early diagnosis and treatment were general hospitals (42.97%), Maternity and Infant Hospitals (27.03%) and Children′s Hospitals (20.31%). The main rehabilitation methods for 797 children with CP were 34.58% in the hospitals or rehabilitation centers, 31.61% in the communities (including at home), 33.80% mainly in the medical institution, and in the communities they could also receive partially rehabilitation services. Conclusions The prevalence of CP in China is coincident with international levels.The prevalence rate of CP in males is higher than that in females.The types of CP distribution are accorded with international distribution characteristics.There were still some children with CP who could not receive timely detection and treatment.Rehabilitation at the medical institutions is the chief way and proper rehabilitation guidance should be carried out in the communities. Key words: Cerebral palsy; Epidemiology; Prevalence; Prevention; Treatment; Rehabilitation

TRPM7 Mediates Mechanosensitivity in Adult Rat Odontoblasts

Odontoblasts, with their strategic arrangement along the outermost compartment of the dentin-pulp complex, have been suggested to have sensory function. In addition to their primary role in dentin formation, growing evidence shows that odontoblasts are capable of sensing mechanical stimulation. Previously, we found that most odontoblasts express TRPM7, the nonselective mechanosensitive ion channel reported to be critical in Mg2+ homeostasis and dentin mineralization. In line with this finding, we sought to elucidate the functional expression of TRPM7 in odontoblasts by pharmacological approaches and mechanical stimulation. Naltriben, a TRPM7-specific agonist, induced calcium transient in the majority of odontoblasts, which was blocked by TRPM7 blockers such as extracellular Mg2+ and FTY720 in a dose-dependent manner. Mechanical stretch of the odontoblastic membrane with hypotonic solution also induced calcium transient, which was blocked by Gd3+, a nonselective mechanosensitive channel blocker. Calcium transient induced by hypotonic solution was also blocked by high extracellular Mg2+ or FTY720. When TRPM7-mediated calcium transients in odontoblasts were analyzed on the subcellular level, remarkably larger transients were detected in the distal odontoblastic process compared with the soma, which was further verified with comparable immunocytochemical analysis. Our results demonstrate that TRPM7 in odontoblasts can serve as a mechanical sensor, with its distribution to facilitate intracellular Ca2+ signaling in the odontoblastic process. These findings suggest TRPM7 as a mechanical transducer in odontoblasts to mediate intracellular calcium dynamics under diverse pathophysiological conditions of the dentin.

The erythrocyte osmotic resistance test as screening tool for cholesterol-related lysosomal storage diseases

BackgroundErythrocyte volume regulation and membrane elasticity are essential for adaptation to osmotic and mechanical stress, and life span. Here, we evaluated whether defective cholesterol trafficking caused by the rare lysosomal storages diseases (LSDs), Niemann-Pick type C (NPC) and Lysosomal acid lipase (LAL) deficiency (LALD) impairs these properties. Moreover, we tested whether measurements of cholesterol membrane content and osmotic resistance serve as a screening test for these LSDs. MethodsPatients were genotyped for mutations in NPC1, NPC2, or LIPA genes. We measured LSD plasma biomarkers and LAL activity. Red blood cells (RBC) membrane cholesterol content was evaluated in 73 subjects. Osmotic resistance tests (ORT) were conducted in 121 blood samples from LSD suspected patients and controls. ResultsWe did not find statistically significant differences between RBC cholesterol content between subjects and controls. However, the ORT, particularly at 0.49% (w/v) hypotonic sodium chloride solution, revealed a significant higher osmotic resistance in LSDs patients than in controls. We established a cut-off value of ≤51% of haemolysis with sensibility and specificity values of 80% and 70%, respectively. ConclusionsNPC and LALD do not alter cholesterol content in the RBC membrane but increase osmotic resistance. Therefore, ORT serves as screening test for the studied LSDs.