Screening of salt stress in the overexpressed type of Arabidopsis thaliana (L.) Heynh. for the identification of significant hub genes using a systems biology approach

Worldwide, it is known that abiotic and biotic stresses can affect the production of crops by a declining trend. To control the situation, SnRK2 (a subfamily 2 of SNF1-related protein kinase) overexpression levels can induce salt tolerance. This study used a dataset for 2 types of Arabidopsis thaliana including the wild and PtSnRK2.7 overexpressed in mock and salt conditions to compare and identify the salt stress-responsive genes. A computational systems biology approach was employed to identify the differentially expressed genes and determine their mechanisms in terms of molecular functionalities, cellular components, KEGG enrichment pathways and plant ontology analyses. The results indicate that the 15 genes identified for PtSnRK2.7 overexpressed type in mock against salt conditions were upregulated (AT1G19180 and AT2G23150 were downregulated) and related to various environmental stresses. Furthermore, 8 out of 15 identified genes were downregulated for the wild type exposed to salt stress and the rest were upregulated. And, the only upregulated gene found differentially expressed between wild and overexpressed types in salt stress conditions was AT4G15110. In contrast, the other two AT1G15010 and AT4G19430 were downregulated and involved in transient stress and inactivation of chloroplast, respectively. Taken together, it has been shown that A. thaliana PtSnRK2.7 overexpressed type can resist salt stress. Finally, more experimental studies and computational systems biology methodologies are needed to reveal and confirm the responsive gene for salt stress in A. thaliana

Prediction of the Human EP1 Receptor Binding Site by Homology Modeling and Molecular Dynamics Simulation

The prostanoid receptor EP1 is a G-protein-coupled receptor (GPCR) known to be involved in a variety of pathological disorders such as pain, fever and inflammation. These receptors are important drug targets, but design of subtype specific agonists and antagonists has been partially hampered by the absence of three-dimensional structures for these receptors. To understand the molecular interactions of the PGE2, an endogen ligand, with the EP1 receptor, a homology model of the human EP1 receptor (hEP1R) with all connecting loops was constructed from the 2.6 Å resolution crystal structure (PDB code: 1L9H) of bovine rhodopsin. The initial model generated by MODELLER was subjected to molecular dynamics simulation to assess quality of the model. Also, a step by step ligand-supported model refinement was performed, including initial docking of PGE2 and iloprost in the putative binding site, followed by several rounds of energy minimizations and molecular dynamics simulations. Docking studies were performed for PGE2 and some other related compounds in the active site of the final hEP1 receptor model. The docking enabled us to identify key molecular interactions supported by the mutagenesis data. Also, the correlation of r2=0.81 was observed between the Ki values and the docking scores of 15 prostanoid compounds. The results obtained in this study may provide new insights toward understanding the active site conformation of the hEP1 receptor and can be used for the structure-based design of novel specific ligands

Production and Purification of a Novel Anti-TNF-α Single Chain Fragment Variable Antibody

Purpose: TNF-α is an inflammatory cytokine with a key role in initiation of inflammatory responses. Anti-TNF-α antibodies are being used in clinic for the purpose of diagnosis and treatment due to their high specificity. The objective of the current study was to express and purify an anti-TNF-α scFv antibody identified by phage display technology. Methods: The DNA coding sequence of the identified scFv was cloned into pET28a vector and the corresponding protein was expressed as 6×His tagged using E.coli BL21 (DE3) pLysS expression system followed by affinity purification on Ni-Sepharose affinity column. Results: The J44 scFv antibody was cloned into the expression vector and successfully expressed and purified. The purity of the scFv fraction was confirmed using SDS-PAGE analysis. Western blotting technique was used to detect expression of 6×His tagged protein. Conclusion: In the current study an anti-TNF-α scFv antibody was successfully expressed in bacterial expression system and purified on affinity column. The purified protein can be used in different in vitro and in vivo experiments in order to elucidate its functionality

Empagliflozin alleviates renal inflammation and oxidative stress in streptozotocin-induced diabetic rats partly by repressing HMGB1-TLR4 receptor axis

Objective(s): Empagliflozin, a sodium-glucose cotransporter-2 (SGLT-2) inhibitor, possesses verified anti-inflammatory and anti-oxidative stress effects against diabetic nephropathy. The present investigation aims to examine empagliflozin effects on the renal levels of high mobility group box-1 (HMGB1), a potent inflammatory cytokine, and its respective receptor toll-like receptor-4 (TLR-4) in STZ-induced diabetic rats.Materials and Methods: Empagliflozin at 10 mg/kg per os (p.o.) was administered for 4 weeks, starting 8 weeks after the induction of diabetes. Renal function, kidney inflammation, oxidative stress, and apoptosis markers as well as renal HMGB1, receptor for advanced glycation end products (RAGE), and TLR-4 levels were assessed.Results: In addition to down-regulating NF-κB activity in renal cortices, empagliflozin reduced renal levels of HMGB1, RAGE, and TLR-4. It alleviated renal inflammation as indicated by diminished renal expressions of inflammatory cytokines and chemokines like tumor necrosis factor-alpha (TNF-α) and monocyte chemoattractant protein-1 (MCP-1) and also decreased urinary levels of interleukin-6 (IL-6) and alpha-1 acid glycoprotein (AGP). Moreover, empagliflozin ameliorated renal oxidative stress as demonstrated by decreased renal malondialdehyde (MDA) and elevated renal activities of superoxide dismutase (SOD) and glutathione peroxidase (GPX). It also suppressed renal caspase-3, the marker of apoptosis; and furthermore, enhanced renal function noticed by the declined levels of serum urea and creatinine.Conclusion: These findings underline that empagliflozin is able to attenuate diabetes-related elevations in renal HMGB1 levels, an influential inflammatory cytokine released from the necrotic and activated cells, and its correspondent receptors, i.e., RAGE and TLR-4

Tangeretin protects renal tubular epithelial cells against experimental cisplatin toxicity

Objective(s): Cisplatin is an effective antineoplastic agent; its clinical utility, however, is limited by a few salient toxic side effects like nephrotoxicity. This study aimed to determine the potential protective effects of tangeretin, a citrus-derived flavonoid, against renal tubular cell injury in cisplatin-induced renal toxicity of rats.Materials and Methods: Tangeretin was injected intraperitoneally at 2.5 and 5 mg/kg doses for 10 days, and a single dose of cisplatin (8 mg/kg) was injected on the 7th day. Tests of kidney function and tubular injury in renal tissues and urine together with oxidative stress and inflammation markers were examined.Results: Tangeretin ameliorated cisplatin-induced elevations in serum creatinine, BUN, and histopathologic changes. It also attenuated kidney oxidative stress elicited by cisplatin as demonstrated by reduced MDA and increased GSH, CAT, and SOD activities, elevated Nrf2 expression and protein levels of its downstream effectors, HO-1 and NQO-1. Tangeretin further alleviated inflammation evoked by cisplatin as indicated by reduced NF-κB p65 subunit phosphorylation with a simultaneous decrement in its downstream effectors IL-1β and TNF-α expression and protein levels. Moreover, it declined caspase-3 protein levels and TUNEL positive cells in the kidneys, the markers of apoptosis and DNA fragmentation, thus improving renal endurance. Additionally, tangeretin mitigated renal levels of KIM-1 and NGAL, as well as urinary cystatin C and β2-microglobulin concentrations, the markers of renal tubular injury.Conclusion: Collectively, these data signify the binary profit of tangeretin: enhancement of renal protective mechanisms against cisplatin and attenuation of renal tubular cell injuries induced by the agent

Expression, purification and DNA-binding properties of zinc finger domains of DOF proteins from Arabidopsis thaliana

Introduction: DOF proteins are a family of plant-specific transcription factors with a conserved zinc finger (ZF) DNA-binding domain. Although several studies have demonstrated their specific DNA binding, quantitative affinity data is not available for the binding of DOF domains to their binding sites. Methods: ZF domains of DOF2.1, DOF3.4, and DOF5.8 from Arabidopsis thaliana were expressed and purified. Their DNA binding affinities were assessed using gel retardation assays and microscale thermophoresis with two different oligonucleotide probes containing one and two copies of recognition sequence AAAG. Results: DOF zinc finger domains (DOF-ZFs) were shown to form independently folded structures. Assessments using microscale thermophoresis demonstrated that DOF-ZFs interact more tightly (~ 100 fold) with double-motif probe than the single-motif probe. The overall Kd values for the DOF3.4-ZF and DOF5.8-ZF to the double-motif probe were ~2.3±1 and 2.5±1 µM, respectively. Conclusion: Studied DOF-ZF domains formed stable complexes with the double-motif probe. Although DOF3.4-ZF and DOF5.8-ZF do not dimerize with an appreciable affinity in the absence of DNA (judging from size-exclusion and multiangle laser light scattering data), it is possible that these ZFs form protein-protein contacts when bound to this oligonucleotide, consistent with previous reports that DOF proteins can homo- and hetero-dimerize