Regulation of endothelial nitric oxide synthase: involvement of protein kinase G 1 beta, serine 116 phosphorylation and lipid structures

1. Endothelial nitric oxide synthase (NOS3) is important for vascular homeostasis. The role of protein kinase G (PKG) in regulation of NOS3 activity was studied in primary cultures of newborn lamb lung microvascular endothelial cells (LMVEC). 2. We determined the presence of PKG in fetal and neonatal LMVEC as well as subcellular localization of PKG isoforms in the neonatal cells by fluorescence immunohistochemistry. We used diaminofluorescein (DAF) fluorophore to measure nitric oxide (NO) production from neonatal LMVEC. We confirmed that NO measured was from constitutive NOS3 by inhibiting it with NOS inhibitors. 3. To identify a role for PKG in basal NO production, we measured NO release from LMVEC cells using 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM; 0.5-0.8 micromol/L) with and without prior stimulation with the PKG activator 8-bromo-cGMP (8-Br-cGMP; 0.3 and 3 micromol/L) or prior PKG inhibition with beta-phenyl-1,N2-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothionate (BPC; 0.3 and 3 micromol/L). With the same drugs, we determined the role of PKG on cellular expression of NOS3 and serine 116 phosphorylated NOS (pSer116-NOS) by qualitative and quantitative immunofluorescence assays, as well as western blotting. 4. Because PKG 1 beta was distributed throughout the cytosol in a punctate expression, we used 2 mmol/L cyclodextrin, a cholesterol extractor, to determine a role for lipid vesicles in PKG regulation of NO production. 5. Protein kinase G 1 beta gave a punctate appearance, indicating its presence in intracellular vesicles. Nitric oxide production decreased by approximately 20% with 300 nmol/L and 3 micromol/L 8-Br cGMP (P < 0.05) and increased by 20.8 +/- 3.7% with 3 micromol/L BPC (P < 0.001), indicating that both stimulated and basal PKG activity has inhibitory effects on basal NOS3 function. Nitric oxide synthase immunofluorescence and immunoblot expression were decreased and pSer116-NOS immunofluorescence was increased by 800 nmol/L 8-Br-cGMP and 170 micromol/L (Z)-1-[2-(2-aminoethyl)-N-(2-ammonio-ethyl)amino]diazen-1-ium-1, 2-diolate (DETANONOate). The effect of cyclodextrin indicated that cholesterol extraction interfered with PKG inhibition of NOS. Further examination of pSer116-NOS by immunohistochemistry showed it abundant in the endoplasmic reticulum and colocalized with PKG 1 beta, especially in nuclear vesicles. 6. We conclude that endothelial PKG is involved in endogenous regulation of basal NOS3 activity with the involvement of lipid structures, the endoplasmic reticulum and the nucleus. Protein kinase G 1 beta is colocalized with pSer116-NOS, indicating that PKG action may involve serine 116 phosphorylation on NOS.     

4‐Substituted thieno[2,3‐d]pyrimidines as potent antibacterial agents: Rational design,microwave‐assisted synthesis,biological evaluation and molecular docking studies

In an attempt to discover a new class of antibacterial agents with improved efficacy and to overcome the drug‐resistant problems, some novel 4‐substituted thieno[2,3‐d]pyrimidines have been synthesized via microwave‐assisted methodology and evaluated for their in vitro antibacterial activity against various pathogenic bacterial strains. Compounds 12 b and 13 c showed the promising inhibitory potencies against Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa and Escherichia coli with MICs ranging from 2 to 10 μg/ml. Compound 13 c was also found to be highly potent against methicillin‐resistant S. aureus (MRSA) with MIC value of 4 μg/ml. Docking simulation studies have been performed to unravel the mode of action and association study indicate the binding of potent compounds with DHPS enzyme. In silico ADME studies suggest the drug‐like characteristics of the potent compounds.     

Magnetic resonance enterography/enteroclysis in acquired small bowel diverticulitis and small bowel diverticulosis

Purpose

Small bowel (SB) diverticulosis is a rare disorder that may entail serious complications, including SB diverticulitis. Both are often missed in imaging. Magnetic resonance enterography/enteroclysis (MRE) is increasingly used to assess SB disease; awareness of the appearance of SB diverticulitis is essential to ensure appropriate management. Our aim was to systematically describe imaging characteristics of SB diverticulosis and diverticulitis in MRE.

Methods

This retrospective, HIPAA-compliant study identified 186 patients with suspected SB diverticulosis/diverticulitis in medical databases of two tertiary medical centres between 2005 and 2011. Patients with surgically confirmed diagnoses of SB diverticulosis/diverticulitis were included. Two observers analyzed MR images for the presence, location, number, and size of diverticula, wall thickness, and mural and extramural patterns of inflammation.

Results

Seven patients were recruited. MRI analysis showed multiple diverticula in all (100 %). Diverticular size ranged from 0.5 to 6 cm. Prevalence of diverticula was higher in the proximal than the distal SB (jejunum 86 %, ileum 57 %, distal ileum43%). Diverticulitis occurred in 3/7 patients (43 %) showing asymmetric bowel wall thickening and focal mesenteric inflammation.

Conclusion

SB diverticulitis demonstrates characteristic MRE imaging features to distinguish this rare disorder from more common diseases. Asymmetric, focal mesenteric and mural inflammation and presence of multiple diverticula are keys to diagnosis.

Key points

? Small bowel diverticulosis and diverticulitis is rare and often missed in imaging ? Acquired small bowel diverticula are variable in size and number ? Small bowel diverticulitis demonstrates characteristic features on MR enterography/enteroclysis ? A focal or segmental asymmetric small bowel inflammation should prompt the search for diverticula

     

Characteristics of atmospheric aerosol particles and their role in aerosol radiative forcing over the northwestern Indian Himalaya in particular and over India in general

Recent investigations suggest that the accumulation of polluted aerosols at mountain sites of the Indian Himalaya potentially may accelerate the seasonal warming. In view of the importance of aerosols, the present study was carried out over Mohal (1154 m) during April 2006 to March 2010. The investigation of the optical properties of aerosols such as single-scattering albedo (SSA), asymmetry parameter (AP), and their effect on aerosol radiative forcing (ARF) from this part of India are also reported in earlier studies. However, those studies did not fully focus on the radiative effect of aerosols. In this study, we focus on this important aspect; the ARF was computed using the atmospheric radiation transfer model (RTM). The radiative properties of aerosols used in RTM are retrieved from Optical Properties of Aerosol and Cloud (OPAC) model. The seasonal highest SSA at 0.5 μm was obtained in premonsoon season (0.875?±?0.03) and lowest in winter (0.754?±?0.07). The seasonal highest AP at 0.5 μm was obtained in premonsoon (0.678?±?0.01) and lowest in winter (0.654?±?0.01). The mean (period April 2006 to March 2010) values of ARF at the surface, top of the atmosphere, and the atmosphere are estimated to be ?19.1?±?1.9, +0.5?±?3.3, and +19.6?±?3.7 W m^?2, respectively. During the dust-laden period, this study reports strong response to atmospheric forcing; this increases the atmospheric heating rate by a factor 2.4 when compared to low aerosol-laden period. Based on review of the literature, it is found that in the northern and eastern subcontinent of India, the large decrease in surface reaching solar radiation is due to the abundance of desert dust aerosols. In the western and southern subcontinent of India, the large decrease in surface reaching solar radiation is mainly due to the abundance of anthropogenic aerosols.     

Nanoemulgel (NEG) of Ketoprofen with eugenol as oil phase for the treatment of ligature-induced experimental periodontitis in Wistar rats

Aim: The aim of the novel study was to check the efficacy of a locally applied 2%w/w nanoemulgel (NEG) of Ketoprofen (KP) in preventing the periodontitis, and was also checked NEG without KP to ensure the effect of eugenol in NEG as an oil phase.

Design: For experimentally induced periodontitis, sterile silk ligatures (3/0) were placed around the crevices of the first left lower molar teeth of the male Wistar rats. During 8 weeks, all rats were fed with 10%w/v sucrose solution. The experimental assessment was carried out at 11 d after treatment of experimental periodontal disease (EPD) rats by various clinical parameters like gingival index (GI), tooth mobility (TM), alveolar bone loss (ABL), histological analysis, detection of TNF-α, and IL-1β in gingival tissue by ELISA and the roughness were measured by atomic force microscopy (AFM) in tapping modes.

Results: After treatment, comparison studies with EPD were performed. NEG loaded with KP prevents significantly (p?<?0.05) various parameters (GI, TM, and ABL), which were responsible for periodontitis. The histopathology of the periodontium showed that Group 3 (NEG loaded with KP) had a more significant reduction in inflammatory cell infiltration, alveolar bones resorption, and cementum (p?<?0.05). In the topographical images, significant reduction in roughness of NEG loaded with KP was observed in comparison with EPD without treatment.

Conclusion: The study revealed the great synergistic potential of the combined NEG of an anti-inflammatory drug KP along with eugenol as the oil phase, which have potential antibacterial, analgesic, and anesthetic properties to combat periodontal disease.     

Genetic components in diabetic retinopathy

Diabetic retinopathy (DR) is a serious complication of diabetes, which is fast reaching epidemic proportions worldwide. While tight glycemic control remains the standard of care for preventing the progression of DR, better insights into DR etiology require understanding its genetic basis, which in turn may assist in the design of novel treatments. During the last decade, genomic medicine is increasingly being applied to common multifactorial diseases such as diabetes and age-related macular degeneration. The contribution of genetics to the initiation and progression of DR has been recognized for some time, but the involvement of specific genes and genetic variants remains elusive. Several investigations are currently underway for identifying DR susceptibility loci through linkage studies, candidate gene approaches, and genome-wide association studies. Advent of next generation sequencing and high throughput genomic technologies, development of novel bioinformatics tools and collaborations among research teams should facilitate such investigations. Here, we review the current state of genetic studies in DR and discuss reported findings in the context of biochemical, cell biological and therapeutic advances. We propose the development of a consortium in India for genetic studies with large cohorts of patients and controls from limited geographical areas to stratify the impact of the environment. Uniform guidelines should be established for clinical phenotyping and data collection. These studies would permit identification of genetic loci for DR susceptibility in the Indian population and should be valuable for better diagnosis and prognosis, and for clinical management of this blinding disease.     

Role of AIM2 inflammasome in inflammatory diseases,cancer and infection

AIM2 is a cytosolic innate immune receptor which recognizes double‐stranded DNA (dsDNA) released during cellular perturbation and pathogenic assault. AIM2 recognition of dsDNA leads to the assembly of a large multiprotein oligomeric complex termed the inflammasome. This inflammasome assembly leads to the secretion of bioactive interleukin‐1β (IL‐1β) and IL‐18 and induction of an inflammatory form of cell death called pyroptosis. Sensing of dsDNA by AIM2 in the cytosol is crucial to mediate protection against the invading pathogens including bacteria, virus, fungi and parasites. AIM2 also responds to dsDNA released from damaged host cells, resulting in the secretion of the effector cytokines thereby driving the progression of sterile inflammatory diseases such as skin disease, neuronal disease, chronic kidney disease, cardiovascular disease and diabetes. Additionally, the protection mediated by AIM2 in the development of colorectal cancer depends on its ability to regulate epithelial cell proliferation and gut microbiota in maintaining intestinal homeostasis independently of the effector cytokines. In this review, we will highlight the recent progress on the role of the AIM2 inflammasome as a guardian of cellular integrity in modulating chronic inflammatory diseases, cancer and infection.