<Emphasis Type="Italic">SUMO4</Emphasis> 163 G&gt;A variation is associated with kidney disease in Indian subjects with type 2 diabetes

Genetic susceptibility probably plays a role in the development and/or progression of diabetic kidney disease. Small ubiquitin-related modifier 4 (SUMO4) mRNA is expressed in human kidney. Substitution of methionine with valine at codon 55 (M55V) of SUMO4 gene induces higher nuclear factor-kB activity, which is known to mediate the development of kidney disease in individuals with diabetes. We investigated the association between the SUMO4 M55V (rs237025, c.163 G>A) and kidney disease in north Indian subjects with diabetes. A case–control analysis was performed using genomic DNA samples from 216 diabetic patients without nephropathy (DM) and 201 diabetic with nephropathy (DN). The SUMO4 c.163 G>A polymorphism was genotyped using polymerase chain reaction amplification followed by restriction digestion. The duration of diabetes was significantly greater in DN. The genotypic and allelic frequencies were different in DM and DN groups: GG genotype was significantly more frequent in DN as compared to DM (p = 0.018, OR 1.72, 95 % CI 1.1–2.7). Similarly the G allele was more frequent in DN compared to DM (p = 0.017, OR 1.4, 95 % CI 1.1–1.8). This study suggests that SUMO4 c.163 G>A polymorphism is associated with the susceptibility to diabetic nephropathy in north Indian subjects with type 2 diabetes.     

Restoration of β-GC trafficking improves the lysosome function in Gaucher disease

Lysosomes function as a primary site for catabolism and cellular signaling. These organelles digest a variety of substrates received through endocytosis, secretion and autophagy with the help of resident acid hydrolases. Lysosomal enzymes are folded in the endoplasmic reticulum (ER) and trafficked to lysosomes via Golgi and endocytic routes. The inability of hydrolase trafficking due to mutations or mutations in its receptor or cofactor leads to cargo accumulation (storage) in lysosomes, resulting in lysosome storage disorder (LSD). In Gaucher disease (GD), the lysosomes accumulate glucosylceramide because of low β-glucocerebrosidase (β-GC) activity that causes lysosome enlargement/dysfunction. We hypothesize that improving the trafficking of mutant β-GC to lysosomes may improve the lysosome function in GD. RNAi screen using high throughput based β-GC activity assay followed by reporter trafficking assay utilizing β-GC-mCherry led to the identification of nine potential phosphatases. Depletion of these phosphatases in HeLa cells enhanced the β-GC activity by increasing the folding and trafficking of Gaucher mutants to the lysosomes. Consistently, the lysosomes in primary fibroblasts from GD patients restored their β-GC activity upon the knockdown of these phosphatases. Thus, these studies provide evidence that altering phosphatome activity is an alternative therapeutic strategy to restore the lysosome function in GD.     

Polyaniline protected gold nanoparticles based mediator and label free electrochemical cortisol biosensor

Polyaniline protected gold nanoparticles (PPAuNPs) were electrophoretically deposited onto a gold electrode, and utilized to fabricate an electrochemical cortisol biosensor. Cortisol specific monoclonal antibody (C-Mab) was covalently immobilized onto the surface of a PPAuNP/Au electrode using N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (EDC/NHS) chemistry. BSA was employed for blocking nonspecific adsorption on the electrode surface. PPAuNP formation and BSA/C-Mab/PPAuNP/Au electrode fabrication were characterized using transmission electron microscopy, atomic force microscopy and electrochemical impedance techniques, respectively. Cyclic voltammetry and differential pulse voltammetric techniques were used to determine the cortisol concentration in a phosphate buffer saline (PBS) solution. Results confirmed that the PPAuNP based electrode was stable during repeated scans and exhibited repeatable redox peaks. Further, the BSA/C-Mab/PPAuNP/Au electrode in the PBS buffer accurately detected cortisol in the range of 1 pM-100 nM with a sensitivity of 1.63 μAM(-1). The biosensor was found to be selective against BSA and 17-α-hydroxy progesterone. This research establishes the feasibility of using a PPAuNP based matrix for a label and mediator free electrochemical biosensor for cortisol, a stress biomarker.     

Rapid multiplication of adventitious somatic embryos in peach and nectarine by secondary embryogenesis

Somatic embryos were multiplied by secondary embryogenesis in cotyledonary cultures of peach and nectarine (Prunus persica L.) using a simplified culture medium for immature seeds. A three-stage process with an initial callus phase was established in darkness on a medium containing basal salts (modified MS) supplemented with 2,4-D (5 mg/l), Kn (2 mg/l) and BAP (2 mg/l) and casein hydrolysate (500 mg/l). This was followed by a growth regulator-free medium with activated charcoal for the adventitious and direct multiplication of somatic embryos under continuous light. Somatic embryos (10–15) originated from the epidermal layer of primary somatic embryos of 4–6 mm size. The incidence of morphologically abnormal embryos was reduced by subculturing every 20 days. Calli which were isolated and grown on a 2,4-D medium were more embryogenic than those on NAA. These embryos multiplied continuously for more than 10 months by a repetitive somatic embryogenic process. A third stage medium, supplemented with BAP (2 mg/l), was required for axis elongation, germination and transfer to soil.     

Function of conserved residues of human glutathione synthetase: implications for the ATP-grasp enzymes

Glutathione synthetase is an enzyme that belongs to the glutathione synthetase ATP-binding domain-like superfamily. It catalyzes the second step in the biosynthesis of glutathione from gamma-glutamylcysteine and glycine in an ATP-dependent manner. Glutathione synthetase has been purified and sequenced from a variety of biological sources; still, its exact mechanism is not fully understood. A variety of structural alignment methods were applied and four highly conserved residues of human glutathione synthetase (Glu-144, Asn-146, Lys-305, and Lys-364) were identified in the binding site. The function of these was studied by experimental and computational site-directed mutagenesis. The three-dimensional coordinates for several human glutathione synthetase mutant enzymes were obtained using molecular mechanics and molecular dynamics simulation techniques, starting from the reported crystal structure of human glutathione synthetase. Consistent with circular dichroism spectroscopy, our results showed no major changes to overall enzyme structure upon residue mutation. However, semiempirical calculations revealed that ligand binding is affected by these mutations. The key interactions between conserved residues and ligands were detected and found to be essential for enzymatic activity. Particularly, the negatively charged Glu-144 residue plays a major role in catalysis.     

ACACβ gene (rs2268388) and AGTR1 gene (rs5186) polymorphism and the risk of nephropathy in Asian Indian patients with type 2 diabetes

Patients with type 2 diabetes (T2DM) are usually obese and concurrent obesity results into activation of the renin–angiotensin-system (RAS) which is a risk factor for diabetic nephropathy (DN). Gene–gene interaction between acetyl-coenzymeA carboxylase beta (ACACβ) gene, which is involved in fatty acid metabolism and angiotensin II receptors (AGTR1) gene, which mediates RAS proteins actions on renal tissue, polymorphism with DN have not been studied earlier. The present study was designed with the aim to examine the association of an ACACβ (rs2268388) and AGTR1 (rs5186) gene polymorphism with the risk of DN in Asian Indians. 1,158 patients with T2DM belonging to two independently ascertained North Indian and one South Indian cohorts were genotyped for ACACβ (rs2268388) and AGTR1 (rs5186) polymorphism using real time PCR-based Taq-man assay and PCR–RFLP assays. In all the three cohorts, a significantly higher frequency of T allele and TT genotypes of ACACβ and C allele and CC genotypes of AGTR1 were found in patients with DN as compared to patients without nephropathy. Further, T allele of ACACβ and C allele of AGTR1 were found to be significantly associated with proteinuria, a hallmark of DN. We also found significant epistatic interactions between these two genes. TT genotypes of ACACβ gene and CC genotype of AGTR1 gene confers the risk of DN and both genes had significant epistatic interaction in Asian Indian patients with T2DM.     

Fusarium falciforme Infection of Foot in a Patient with Type 2 Diabetes Mellitus: A Case Report and Review of the Literature

Fungal infections of foot in patients with diabetes are not uncommon; however, foot infection due to Fusarium species has been rarely reported. We report here a case of a 50-year-old male with type 2 diabetes who developed multiple spontaneous nodular lesions on right foot without any systemic symptoms and signs for 6 months. The lesions were unresponsive to broad-spectrum antibacterial treatment. Fine needle aspiration cytology of nodular lesions revealed the presence of fungal hyphae, and Fusarium species was isolated from the same sample which was identified as Fusarium solani species complex: Fusarium falciforme. Radiological investigations and blood culture ruled out any dissemination of the disease. The lesions healed after voriconazole therapy for 3 months. No relapse was noted at the end of the next 6-month follow-up. All reported cases of Fusarium infection of foot in patients with diabetes in English and non-English literature since 1970 have been reviewed.