Promising renoprotective effect of gold nanoparticles and dapagliflozin in diabetic nephropathy via targeting miR-192 and miR-21

Diabetic nephropathy (DN) is a worldwide issue that eventually leads to end-stage renal failure, with limited therapeutic options. Prior research has revealed that gold nanoparticles (AuNPs) have a substantial antidiabetic impact. In addition, sodium-glucose cotransporter2 (SGLT2) inhibitors, including dapagliflozin (DAPA), had renoprotective impact on DN. Therefore, this research attempted to determine the potential AuNPs and DAPA impacts in ameliorating experimentally DN induction and the underlying mechanisms focusing on miR-192 and miR-21, correlating them with autophagy, apoptosis, fibrosis, and oxidative stress. Diabetes induction was through a single intraperitoneal streptozotocin (55 mg/kg) injection, and rats with diabetes received AuNPs (2.5 mg/kg/day) as well as DAPA (2 mg/kg/day) for 7 weeks as a treatment. AuNPs and DAPA treatment for 7 weeks substantially alleviated DN. AuNPs and DAPA significantly increased catalase (CAT) activity as well as serum total antioxidant capacity (TAC), along with a substantial decline in malondialdehyde (MDA). AuNPs and DAPA treatment alleviated renal fibrosis as they decreased transforming growth factorß1(TGF-ß1) as well as matrix metalloproteinase-2 (MMP-2) renal expression, decreased apoptosis through alleviating the proapoptotic gene (caspase-3) renal expression and increased the antiapoptotic gene (Bcl-2) renal expression, and increased autophagy as they increased LC-3 as well as Beclin-1 renal expression. Autophagy activation, inhibition of apoptosis, and renal fibrosis could be due to their inhibitory impact on miR-192 and miR-21 renal expression. AuNPs and DAPA have a protective effect on DN in rats by targeting miR-192 and miR-21 and their downstream pathways, including fibrosis, apoptosis, autophagy, and oxidative stress.     

Molecular Modelling of a Multiphosphorylated Sequence Motif Bound to Hydroxyapatite Surfaces

Proteins and peptides containing the multiphosphorylated motif -Ser(P)-Ser(P)- Ser(P)--Glu-Glu- stabilise amorphous calcium phosphate (ACP) in body fluids and bind with high affinity to crystalline calcium phosphate phases such as hydroxyapatite (HA) regulating crystal growth. Binding of this motif to hydroxyapatite surfaces was investigated in this study using molecular modelling techniques. Using a three-step computational procedure, we have determined the relative binding energies of the motif Ser(P)-Ser(P)-Ser(P)-Glu-Glu to different crystalline surfaces of HA. This analysis revealed preferences of the motif for (100) and (010) surfaces of the crystal and preferences for particular orientations on a given surface. These preferences are principally governed by electrostatic interactions between the crystal lattice and the peptide with the most stable conformers adopting structures where alternate residues exhibit backbone angles characteristic of a -strand and values of an -helix or a distorted -helix, allowing maximal interaction between the acidic side groups and surface calciums. The results of this study are consistent with experimentally-derived data on the interaction of multiphosphorylated proteins/peptides with HA and have implications for the role of these proteins/peptides in calcium phosphate stabilisation and biomineralisation processes.Electronic Supplementary Material available.     

Cocaine-induced oxidative stress precedes cell death in human neuronal progenitor cells

By 2003, an estimated 34 million Americans had used cocaine according to the National Survey on Drug Use & Health. About 5.9 million of those had used in the past 12 months. Chronic cocaine users often develop addiction, dependency and tolerance to the drug. The psychological and physical effects of cocaine are due to the disruption of the limbic system in the central nervous system (CNS). Increased oxidative stress reported in the frontal cortex and the striatum of rats exposed to cocaine suggests that oxidative damage plays a significant role in cocaine-induced disruption of the CNS. Although it is evident that cocaine induces oxidative stress in the CNS, little has been learned about whether such increased oxidative stress is also relevant to apoptosis in cocaine-exposed models. To gain insight into the role of cocaine-induced oxidative stress in apoptosis, we hypothesized that oxidative stress precedes cell death when cocaine is administrated. To test this hypothesis, we have monitored the oxidative stress and apoptotic effects of acute cocaine exposure in human neuronal progenitor cells (HNPC). We found that oxidative stress was significantly increased at 48h after a 30min cocaine exposure compared to control cells, and that this was followed by cell death at 72h. Using the same experimental paradigm we have previously shown that pro-inflammatory genes are up-regulated in cocaine-exposed HNPC at 24h. Therefore, we suggest that the increased oxidative stress (possibly mediated by inflammatory responses) precedes cell death in cocaine-exposed HNPC. This may have implications for the consequences of cocaine abuse in situations where antioxidant capacity is compromised, as in the aging brain.     

ADP Ribosylation Factor 6 (ARF6) Promotes Acrosomal Exocytosis by Modulating Lipid Turnover and Rab3A Activation

Regulated secretion is a central issue for the specific function of many cells; for instance, mammalian sperm acrosomal exocytosis is essential for egg fertilization. ARF6 (ADP-ribosylation factor 6) is a small GTPase implicated in exocytosis, but its downstream effectors remain elusive in this process. We combined biochemical, functional, and microscopy-based methods to show that ARF6 is present in human sperm, localizes to the acrosomal region, and is required for calcium and diacylglycerol-induced exocytosis. Results from pulldown assays show that ARF6 exchanges GDP for GTP in sperm challenged with different exocytic stimuli. Myristoylated and guanosine 5′-3-O-(thio)triphosphate (GTPγS)-loaded ARF6 (active form) added to permeabilized sperm induces acrosome exocytosis even in the absence of extracellular calcium. We explore the ARF6 signaling cascade that promotes secretion. We demonstrate that ARF6 stimulates a sperm phospholipase D activity to produce phosphatidic acid and boosts the synthesis of phosphatidylinositol 4,5-bisphosphate. We present direct evidence showing that active ARF6 increases phospholipase C activity, causing phosphatidylinositol 4,5-bisphosphate hydrolysis and inositol 1,4,5-trisphosphate-dependent intra-acrosomal calcium release. We show that active ARF6 increases the exchange of GDP for GTP on Rab3A, a prerequisite for secretion. We propose that exocytic stimuli activate ARF6, which is required for acrosomal calcium efflux and the assembly of the membrane fusion machinery. This report highlights the physiological importance of ARF6 as a key factor for human sperm exocytosis and fertilization.     

Über die Bildung der Doppelembryonen

Ohne ZusammenfassungÜbersetzt von E.Schelcher, München.     

A Review of the Salivary Proteome and Peptidome and Saliva-derived Peptide Therapeutics

Saliva is a glandular secretion that is vital in the maintenance of healthy oral tissues. In this review we outline the high abundance salivary proteins, summarise the status of the salivary proteome and peptidome, the genetic origin and recognised functions of these proteins, the diseases associated with salivary disorders, and the emerging saliva-derived peptide therapeutics. Different proteomic approaches have reported the identification of over 1,300 proteins in saliva. However there are fewer than 100 high abundance proteins, identified by multiple methods including, two-dimensional polyacrylamide gel electrophoresis and HPLC combined with mass spectrometry. Analysis of the genes coding for the salivary proteins demonstrated a non-uniform chromosomal distribution with chromosome 4 having the largest proportion of genes expressed in salivary glands. Several diseases are associated with salivary disorders including Sjögren’s syndrome, Prader-Willi syndrome, dental caries and stress related disorders. Saliva as a diagnostic medium for various biochemical tests has provided a non-invasive and accessibility advantage over other more regularly tested body fluids such as blood and urine. To-date the emerging saliva-based therapeutics include artificial salivas and antimicrobial agents based on histatins and mucins.     

Physicochemical characterization of casein phosphopeptide-amorphous calcium phosphate nanocomplexes

Milk caseins stabilize calcium and phosphate ions and make them available to the neonate. Tryptic digestion of the caseins yields phosphopeptides from their polar N-terminal regions that contain clusters of phosphorylated seryl residues. These phosphoseryl clusters have been hypothesized to be responsible for the interaction between the caseins and calcium phosphate that lead to the formation of casein micelles. The casein phosphopeptides stabilize calcium and phosphate ions through the formation of complexes. The calcium phosphate in these complexes is biologically available for intestinal absorption and remineralization of subsurface lesions in tooth enamel. We have studied the structure of the complexes formed by the casein phosphopeptides with calcium phosphate using a range of physicochemical techniques including x-ray powder diffraction, scanning electron microscopy, transmission electron microscopy, and equilibrium binding analyses. The amorphous nature of the calcium phosphate phase was confirmed by two independent methods: x-ray powder diffraction and selected area diffraction. In solution, the ion activity product of a basic amorphous calcium phosphate phase was the only ion product that was a function of bound phosphate independent of pH, consistent with basic amorphous calcium phosphate being the phase stabilized by the casein phosphopeptides. Detailed investigations of calcium and calcium phosphate binding using a library of synthetic homologues and analogues of the casein phosphopeptides have revealed that although the fully phosphorylated seryl-cluster motif is pivotal for the interaction with calcium and phosphate, other factors are also important. In particular, calcium binding and calcium phosphate stabilization by the peptides was influenced by peptide net charge, length, and sequence.