In vitro assessment of Ag and TiO2 nanoparticles cytotoxicity

Background:Silver (Ag) and titanium dioxide (TiO2) nanoparticles are the most eminent nanoproducts. Due to their antimicrobial and antifungal activity, they have been the well commercialized nanosubstances. The hazards associated with human exposure to Ag and TiO2 nanoparticles should be investigated, and hence both the nanoparticles were synthesized to facilitate the risk assessment process.  Methods:Prior to the cytotoxic studies, Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM) were carried out to ensure the particle size. Glutathione (GSH), Nitric Oxide (NO) and superoxide dismutase (SOD) estimated by ELISA method.Results:In the present study, the cytotoxicity of Ag and TiO2 were investigated by using the glutathione (GSH), Nitric Oxide (NO) and superoxide dismutase (SOD) by incubating various concentration of silver (0.25 to 76 mg/mL) and titanium dioxide (0.25 to 2 mg/mL) nanoparticles in different incubation periods (24, 48 and 74 hours at 37°C) in plasma.  Results observed that significant decrease (P <0.0001) in the concentration of GSH associated with increased concentration of NO (P <0.0001) and SOD (P <0.0001) after incubation with silver and titanium dioxide nanoparticles at 24hrs at 37°C, however at 48 hours and 74 hours there is not much change.  Conclusion:The results indicate that silver and titanium dioxide nanoparticles exhibits, nanoparticles mediated cytotoxicity by induction of Reactive Oxygen Species (ROS).

Dynamics of cyclic GMP signaling in neuronal cells

Cyclic GMP (cGMP) is a crucial intracellular messenger used by nearly all eukaryotic cells. In neurons, cGMP is particularly important in mediating intracellular responses to transmitters such as acetylcholine, bradykinin, and natriuretic peptides. In addition, most of the effects of nitric oxide (NO) have been attributed to activation of soluble guanylate cyclase (sGC) and production of cGMP. While there is a growing awareness about the physiological importance and biochemistry of signaling cascades, very little is known about the intracellular dynamics of cGMP. This study used the patch-cram detection method to obtain the first real-time measurements of cGMP in intact, living N1E-115 neuroblastoma cells. This method involves impaling a cell with an electrode holding an excised, inside-out membrane patch containing cyclic nucleotide gated (CNG) ion channels, used as a detector for cGMP. Detector patches were obtained from Xenopus oocytes expressing a chimeric CNG channel that exhibits high specificity and sensitivity for cGMP (K 1/2 = 4 muM). The conductance of the detector patch was precalibrated with known concentrations of cGMP. After cramming the detector patch into a recipient cell, changes in patch conductance indicated changes in free cytoplasmic cGMP.Patch-cramming experiments on neuroblastoma cells show that both muscarinic agonists and NO rapidly elevate cGMP. NO elicits cGMP responses repeatedly without decrement, whereas responses to muscarinic agonists exhibit a profound and prolonged desensitization. Remarkably, muscarinic agonists also cause long-term (up to 2 hrs) suppression (LTS) of cGMP responses to G-protein coupled receptor activation and NO or other NO-independent sGC activators. Biochemical measurements demonstrate that rat sympathetic neurons exhibit a similar prolonged suppression of cGMP, suggesting that LTS may also occur in primary neurons.The mechanism of LTS was examined using the patch cramming method for detection of cGMP, and pharmacological agents that affect the enzymes involved in cGMP metabolism. Direct injection of cGMP into recipient cells demonstrates that enhancement of phosphodiesterase (PDE) activity, rather than depression of sGC, is responsible for LTS. Biochemical measurements show that both cGMP and cAMP are suppressed, suggesting that a non-selective PDE is responsible. Ca2+ mobilization is necessary and sufficient for LTS induction, but LTS maintenance is Ca2+-independent. Protein phosphatase injection reverses LTS, suggesting that a phosphorylation event is required for LTS maintenance. Specific inhibitors of Ca2+/calmodulin kinase II (CaMKII) but not protein kinase C prevent induction and inhibit maintenance of LTS. The Ca2+-independence of LTS maintenance is consistent with CaMKII autophosphorylation, similar to proposed mechanisms of some hippocampal long-term potentiation. Since the molecular machinery underlying LTS is common to many cells, LTS may be a widespread new mechanism for long-term silencing of cyclic nucleotide signaling.</p

Expanding the clinical spectrum of biglycan-related Meester-Loeys syndrome

Pathogenic loss-of-function variants in BGN, an X-linked gene encoding biglycan, are associated with Meester-Loeys syndrome (MRLS), a thoracic aortic aneurysm/dissection syndrome. Since the initial publication of five probands in 2017, we have considerably expanded our MRLS cohort to a total of 18 probands (16 males and 2 females). Segregation analyses identified 36 additional BGN variant-harboring family members (9 males and 27 females). The identified BGN variants were shown to lead to loss-of-function by cDNA and Western Blot analyses of skin fibroblasts or were strongly predicted to lead to loss-of-function based on the nature of the variant. No (likely) pathogenic missense variants without additional (predicted) splice effects were identified. Interestingly, a male proband with a deletion spanning the coding sequence of BGN and the 5' untranslated region of the downstream gene (ATP2B3) presented with a more severe skeletal phenotype. This may possibly be explained by expressional activation of the downstream ATPase ATP2B3 (normally repressed in skin fibroblasts) driven by the remnant BGN promotor. This study highlights that aneurysms and dissections in MRLS extend beyond the thoracic aorta, affecting the entire arterial tree, and cardiovascular symptoms may coincide with non-specific connective tissue features. Furthermore, the clinical presentation is more severe and penetrant in males compared to females. Extensive analysis at RNA, cDNA, and/or protein level is recommended to prove a loss-of-function effect before determining the pathogenicity of identified BGN missense and non-canonical splice variants. In conclusion, distinct mechanisms may underlie the wide phenotypic spectrum of MRLS patients carrying loss-of-function variants in BGN.</p

Empagliflozin in Patients with Chronic Kidney Disease

Background The effects of empagliflozin in patients with chronic kidney disease who are at risk for disease progression are not well understood. The EMPA-KIDNEY trial was designed to assess the effects of treatment with empagliflozin in a broad range of such patients. Methods We enrolled patients with chronic kidney disease who had an estimated glomerular filtration rate (eGFR) of at least 20 but less than 45 ml per minute per 1.73 m(2) of body-surface area, or who had an eGFR of at least 45 but less than 90 ml per minute per 1.73 m(2) with a urinary albumin-to-creatinine ratio (with albumin measured in milligrams and creatinine measured in grams) of at least 200. Patients were randomly assigned to receive empagliflozin (10 mg once daily) or matching placebo. The primary outcome was a composite of progression of kidney disease (defined as end-stage kidney disease, a sustained decrease in eGFR to &lt; 10 ml per minute per 1.73 m(2), a sustained decrease in eGFR of &amp; GE;40% from baseline, or death from renal causes) or death from cardiovascular causes. Results A total of 6609 patients underwent randomization. During a median of 2.0 years of follow-up, progression of kidney disease or death from cardiovascular causes occurred in 432 of 3304 patients (13.1%) in the empagliflozin group and in 558 of 3305 patients (16.9%) in the placebo group (hazard ratio, 0.72; 95% confidence interval [CI], 0.64 to 0.82; P &lt; 0.001). Results were consistent among patients with or without diabetes and across subgroups defined according to eGFR ranges. The rate of hospitalization from any cause was lower in the empagliflozin group than in the placebo group (hazard ratio, 0.86; 95% CI, 0.78 to 0.95; P=0.003), but there were no significant between-group differences with respect to the composite outcome of hospitalization for heart failure or death from cardiovascular causes (which occurred in 4.0% in the empagliflozin group and 4.6% in the placebo group) or death from any cause (in 4.5% and 5.1%, respectively). The rates of serious adverse events were similar in the two groups. Conclusions Among a wide range of patients with chronic kidney disease who were at risk for disease progression, empagliflozin therapy led to a lower risk of progression of kidney disease or death from cardiovascular causes than placebo