Exosome Adherence and Internalization by Hepatic Stellate Cells Triggers Sphingosine 1-Phosphate-dependent Migration

Exosomes are cell-derived extracellular vesicles thought to promote intercellular communication by delivering specific content to target cells. The aim of this study was to determine whether endothelial cell (EC)-derived exosomes could regulate the phenotype of hepatic stellate cells (HSCs). Initial microarray studies showed that fibroblast growth factor 2 induced a 2.4-fold increase in mRNA levels of sphingosine kinase 1 (SK1). Exosomes derived from an SK1-overexpressing EC line increased HSC migration 3.2-fold. Migration was not conferred by the dominant negative SK1 exosome. Incubation of HSCs with exosomes was also associated with an 8.3-fold increase in phosphorylation of AKT and 2.5-fold increase in migration. Exosomes were found to express the matrix protein and integrin ligand fibronectin (FN) by Western blot analysis and transmission electron microscopy. Blockade of the FN-integrin interaction with a CD29 neutralizing antibody or the RGD peptide attenuated exosome-induced HSC AKT phosphorylation and migration. Inhibition of endocytosis with transfection of dynamin siRNA, the dominant negative dynamin GTPase construct Dyn2K44A, or the pharmacological inhibitor Dynasore significantly attenuated exosome-induced AKT phosphorylation. SK1 levels were increased in serum exosomes derived from mice with experimental liver fibrosis, and SK1 mRNA levels were up-regulated 2.5-fold in human liver cirrhosis patient samples. Finally, S1PR2 inhibition protected mice from CCl₄-induced liver fibrosis. Therefore, EC-derived SK1-containing exosomes regulate HSC signaling and migration through FN-integrin-dependent exosome adherence and dynamin-dependent exosome internalization. These findings advance our understanding of EC/HSC cross-talk and identify exosomes as a potential target to attenuate pathobiology signals.     

Flow-injection determination of carbaryl and carbofuran based on KMnO(4)-Na(2)SO(3) chemiluminescence detection.

A flow-injection method is described for the determination of carbaryl and carbofuran. It was found that a strong chemiluminescence (CL) signal was generated when these pesticides were mixed with Na(2)SO(3) and KMnO(4) in acidic medium. Under the optimum experimental conditions, the enhanced CL intensity was linear, with the concentrations in the range 0.1-2.0 microg/mL (r(2) = 0.9996 and 0.9993, n = 6) with relative standard deviation (n = 4) in the range 1.0-2.3%. The limits of detection (3sigma blank) were 10 and 50 ng/mL, respectively, with a sample throughput of 180/h. The proposed method was applied to determine carbaryl and carbofuran in freshwaters with satisfactory results. Most metal and non-metal ions and some pesticides, such as carbophenothion and aldicarb, do not interfere with the determination. Dinoseb, diazinon and malathion calibration graphs (in the range 0.2-2.0 microg/mL, r(2) = 0.9966-0.9988, n = 6) were also established with relative standard deviations (n = 4) in the range 1.2-2.0% with limits of detection (3sigma blank) in the range 100-300 ng/mL.     

The novel heterozygous Thr377Arg MYOC mutation causes severe Juvenile Open Angle Glaucoma in a large Pakistani family

Glaucoma is one of the major causes of blindness worldwide with characteristic optic disc changes and elevated intraocular pressure. It is subcategorized into Primary Open Angle Glaucoma (POAG) and Juvenile Open Angle Glaucoma (JOAG) depending upon age of the disease onset. Myocilin (MYOC) is the frequently mutated gene in familial cases of glaucoma. MYOC mutations show variable phenotype and penetrance. This study was aimed to identify disease causing mutation in 8 affected of a consanguineous family diagnosed with severe form of Juvenile Open Angle Glaucoma. Homozygosity mapping with four microsatellite markers and subsequent direct sequencing of MYOC revealed a novel heterozygous transition c.1130 C>G, substituting Threonine in to Arginine at codon 377 (p.Thr377Arg) of MYOC. This mutation was segregating with phenotype in all affected and was not found in control subjects. Ophthalmological findings revealed JOAG with severe and rapidly progressive phenotype. The age of onset was in the first decade of life and maximum Intra Ocular Pressure (IOP) recorded was 25 mm Hg. Bioinformatic tools predicted C to G transition at c.1130 as pathogenic and no structural changes were predicted in protein. This is the first report of novel MYOC mutation from Pakistan; segregating as autosomal dominant trait in large family diagnosed with JOAG. Identification of novel disease causing allele in MYOC indicates genetic heterogeneity of the population. This finding will help to provide genetic counseling to the affected family and carriers of this mutation may be advised for early therapeutic intervention to avoid irreversible visual loss.     

Acetylcholine receptor pathway mutations explain various fetal akinesia deformation sequence disorders

Impaired fetal movement causes malformations, summarized as fetal akinesia deformation sequence (FADS), and is triggered by environmental and genetic factors. Acetylcholine receptor (AChR) components are suspects because mutations in the fetally expressed gamma subunit (CHRNG) of AChR were found in two FADS disorders, lethal multiple pterygium syndrome (LMPS) and Escobar syndrome. Other AChR subunits alpha1, beta1, and delta (CHRNA1, CHRNB1, CHRND) as well as receptor-associated protein of the synapse (RAPSN) previously revealed missense or compound nonsense-missense mutations in viable congenital myasthenic syndrome; lethality of homozygous null mutations was predicted but never shown. We provide the first report to our knowledge of homozygous nonsense mutations in CHRNA1 and CHRND and show that they were lethal, whereas novel recessive missense mutations in RAPSN caused a severe but not necessarily lethal phenotype. To elucidate disease-associated malformations such as frequent abortions, fetal edema, cystic hygroma, or cardiac defects, we studied Chrna1, Chrnb1, Chrnd, Chrng, and Rapsn in mouse embryos and found expression in skeletal muscles but also in early somite development. This indicates that early developmental defects might be due to somite expression in addition to solely muscle-specific effects. We conclude that complete or severe functional disruption of fetal AChR causes lethal multiple pterygium syndrome whereas milder alterations result in fetal hypokinesia with inborn contractures or a myasthenic syndrome later in life.     

Ergocalciferol and Microcirculatory Function in Chronic Kidney Disease and Concomitant Vitamin D Deficiency: An Exploratory,Double Blind,Randomised Controlled Trial

Background and Objectives

Vitamin D deficiency and endothelial dysfunction are non-traditional risk factors for cardiovascular events in chronic kidney disease. Previous studies in chronic kidney disease have failed to demonstrate a beneficial effect of vitamin D on arterial stiffness, left ventricular mass and inflammation but none have assessed the effect of vitamin D on microcirculatory endothelial function.

Study Design

We conducted a randomised controlled trial of 38 patients with non diabetic chronic kidney disease stage 3–4 and concomitant vitamin D deficiency (<16 ng/dl) who received oral ergocalciferol (50,000 IU weekly for one month followed by 50,000 IU monthly) or placebo over 6 months. The primary outcome was change in microcirculatory function measured by laser Doppler flowmetry after iontophoresis of acetylcholine. Secondary endpoints were tissue advanced glycation end products, sublingual functional capillary density and flow index as well as macrovascular parameters. Parallel in vitro experiments were conducted to determine the effect of ergocalciferol on cultured human endothelial cells.

Results

Twenty patients received ergocalciferol and 18 patients received placebo. After 6 months, there was a significant improvement in the ergocalciferol group in both endothelium dependent microcirculatory vasodilatation after iontophoresis of acetylcholine (p = 0.03) and a reduction in tissue advanced glycation end products (p = 0.03). There were no changes in sublingual microcirculatory parameters. Pulse pressure (p = 0.01) but not aortic pulse wave velocity was reduced. There were no significant changes in bone mineral parameters, blood pressure or left ventricular mass index suggesting that ergocalciferol improved endothelial function independently of these parameters. In parallel experiments, expression of endothelial nitric oxide synthase and activity were increased in human endothelial cells in a dose dependent manner.

Conclusions

Ergocalciferol improved microcirculatory endothelial function in patients with chronic kidney disease and concomitant vitamin D deficiency. This process may be mediated through enhanced expression and activity of endothelial nitric oxide synthase.

Trial Registration

Clinical trials.gov {"type":"clinical-trial","attrs":{"text":"NCT00882401","term_id":"NCT00882401"}}NCT00882401     

Flow‐injection determination of cysteine in pharmaceuticals based on luminol–persulphate chemiluminescence detection

A flow injection (FI) method is reported for the determination of l‐ cysteine, based on its enhancement on chemiluminescence (CL) emission of luminol oxidized by sodium persulphate in alkaline solution. The calibration graph was linear over the range 1.0 × 10^–9–5.0 × 10^–7 mol/L (r² = 0.9992), with relative standard deviations (RSDs) in the range 1.1–2.3% (n = 4). The limit of detection (3σ blank) was 5.0 × 10^–10 mol/L with a sample throughput of 120/h. The method was applied to pharmaceuticals and the results obtained were in reasonable agreement with the amount labelled. The proposed method was also applied to cysteine in synthetic amino acid mixtures. Calibration graphs of N‐acetylcysteine and glutathione over the range 1.0–50 × 10^–8 and 0.5–7.5 × 10^–7 mol/L were also established (r² = 0.998 and 0.9986) with RSDs in the range 1.0–2.0% (n = 4), and the limits of detection (3σ blank) were 5.0 × 10^–9 and 1.0 × 10^–8 mol/L, respectively. Copyright © 2008 John Wiley & Sons, Ltd.     

Surface-Enhanced Raman Scattering (SERS) on 1D Nano-gratings

Surface-enhanced Raman scattering (SERS) enhancement factor (EF) is among the major applications of surface plasmon polaritons (SPP’s). In this work, the SERS EF of 1D rectangular and sinusoidal-shaped gold (Au) grating structures has been designed and optimized on Au film using COMSOL multiphysics (5.3a) RF module taking glass as substrate. The 1D grating models are simulated by variation in slit width ranging 200–600 nm while other parameters including periodicity of 700 nm and Au film thickness of 50 nm remained fixed. In order to study the several phenomena including enhanced optical transmission and SERS EF, the transmission and electric field spectra have been obtained from both types of grating structures. In agreement with fundamental plasmonic mode, the slit width of two-thirds of the periodicity found to be optimum for SERS EF. Remarkable value of SERS EF is obtained in the case of a sinusoidal Au grating device (6.4 × 10⁹) which is calculated to be five times that of the rectangular grating (1.2 × 10⁹). These devices are also the fingerprints of molecules, hence find applications in biosensing, pollution control, and chemical and food industry.

     

High-efficiency transfection of nucleic acids by the modified calcium phosphate precipitation method in chondrocytes

Extracellular matrix (ECM)-rich cartilage-derived chondrocytes are difficult to transfect with DNA/RNA. We modified the classical calcium phosphate transfection method by detaching adherent chondrocytes with trypsin and resuspending in CaPo4-nucleic acid precipitate followed by readherence for 24 h. Due to the absence of ECM, chondrocytes could be transfected with 80% efficiency. Potent gene silencing with several antisense oligonucleotides and small interfering RNAs and strong promoter-luciferase activity could be achieved. This approach is applicable to any adherent or suspended cells and has utility in gene knockdown, ectopic overexpression, promoter regulation studies, and gene delivery in tissue engineering and gene therapy applications.