فهرست مطالب siavoush dastmalchi

 Medical usage of L-asparaginase (ASNase), the first-line of acute lymphoblastic leukemia treatment, is linked to allergic responses and toxicities, which necessitates the development of new bio-better ASNases. The aim of the current study was in silico design of a novel ASNase with predicted improved enzymatic properties using strategies encompassing sequence-function analysis of known ASNase mutants. Additionally, current study aimed to show that the new enzyme is active.

 Based on 21 experimentally reported mutations for ASNase, a virtual library of mutated enzymes with all 7546 possible combinations of up to 4 mutations was generated. Three-dimensional models of proposed mutant enzymes were built and their in silico stabilities were calculated. The most promising mutant was selected for preparing a genetic construct suitable for expression of the designed ASNase in bacterial cells.

 Computational study predicted that Y176F/S241C double mutation of Escherichia coli ASNase may increase its folding stability. The designed ASNase was expressed in two different E. coli strains (Origami B(DE3) and BL21(DE3)pLysS) and then the soluble fractions prepared from the cell lysates of the host cells were used in enzyme activity assay. Results showed that enzyme activity of soluble fraction from Origami (95.4±7.5 IU/0.1 mL) was four times higher than that of soluble fraction from pLysS (25.8±2.5 IU/0.1 mL).

 A novel functional double mutant ASNase with predicted improved enzymatic properties was designed and produced in E. coli. The results of the current study suggest a great commercial potential for the identified enzyme in pharmaceutical and industrial applications.

 Exosomes are natural nanoparticles that participate in intercellular communication through molecular transport. Recently, due to their membrane vesicular structure and surface proteins, exosomes have been used extensively in the research field of drug delivery. Osteoporosis is an inflammation in which the cellular balance of bone tissue is disturbed that reduces bone density and making bone prone to abnormal fractures with small amount of force. Utilizing estrogen is one of the main therapeutic strategies for osteoporosis. Despite the positive effects of estrogen on bone tissue, changes in the natural estrogen levels of the body can cause a number of diseases such as different types of cancer. Therefore, designing a therapeutic system which controls more accurate tissue targeting of estrogen seems to be a rational and promising practical approach.

 In this study, bone marrow mesenchymal stem cells (BMMSCs)-derived exosomes were loaded by estradiol using two different methods of drug loading, namely incubation and sonication methods and then the survival effects of the drug loaded exosomes on BMMSCs was investigated.

 Examination of size, shape, and surface factors of exosomes in different states (pure exosomes and drug-loaded exosomes) showed that the round morphology of exosomes was preserved in all conditions. However, the particles size increased significantly when loaded by sonication method. The increased survival of BMMSCs was noted with estradiol-loaded exosomes when compared to the control group.

 The results suggest that estradiol-loaded exosomes have potential to be used as nano-drug carriers in the treatment of osteoporosis.

Non-steroidal antiinflammatory drugs (NSAIDs) constitute an important class of pharmaceuticals acting on cyclooxygenase COX-1 and COX-2 enzymes. Due to their numerous severe side effects, it is necessary to search for new selective, safe, and effective anti-inflammatory drugs. In silico design of novel therapeutics plays an important role in nowadays drug discovery pipelines. In most cases, the design strategies require the use of molecular docking calculations. The docking procedure may require case-specific condition for a successful result. Additionally, many different docking programs are available, which highlights the importance of identifying the most proper docking method and condition for a given problem.

In the current work, the performances of five popular molecular docking programs, namely, GOLD, AutoDock, FlexX, Molegro Virtual Docker (MVD) and Glide to predict the binding mode of co- crystallized inhibitors in the structures of known complexes available for cyclooxygenases were evaluated. Furthermore, the best performers, Glide, AutoDock, GOLD and FlexX, were further evaluated in docking-based virtual screening of libraries consisted of active ligands and decoy molecules for cyclooxygenase enzymes and the obtained docking scores were assessed by receiver operating characteristics (ROC) analysis.

The results of docking experiments indicated that Glide program outperformed other docking programs by correctly predicting the binding poses (RMSD less than 2 Å) of all studied co-crystallized ligands of COX-1 and COX-2 enzymes (i.e., the performance was 100%). However, the performances of the other studied docking methods for correctly predicting the binding poses of the ligands were between 59% to 82%. Virtual screening results treated by ROC analysis revealed that all tested methods are useful tools for classification and enrichment of molecules targeting COX enzymes. The obtained AUCs range between 0.61-0.92 with enrichment factors of 8 – 40 folds.

The obtained results support the importance of choosing appropriate docking method for predicting ligand-receptor binding modes, and provide specific information about docking calculations on COXs ligands.

Teduglutide is the first and only FDA-approved drug for long-term treatment of short bowel syndrome (SBS). The current study aimed to present an approach for production of teduglutide using recombinant DNA technology.

The coding gene for teduglutide was cloned into pGEX-2T vector, where coding sequence for factor Xa cleavage site was added between GST and teduglutide coding genes. The GST-teduglutide protein was overexpressed in E. coli BL21 (DE3) strain and affinity purified using glutathione sepharose affinity column.

On-column proteolytic activity of factor Xa followed by size exclusion chromatography resulted in the pure teduglutide. Circular dichroism (CD) spectropolarimetry showed that the produced teduglutide folds into mainly α-helical structure ( > 50%), as expected. In mass spectroscopy analysis, the fragments of teduglutide resulted by cyanogen bromide cleavage as well as those expected theoretically due to mass fragmentation were identified. The functionality of the produced peptide was evaluated by measuring its proliferative effect on Caco2 intestinal epithelial cells, and the results indicated that produced teduglutide induces cell proliferation by 19 ± 0.30 and 33 ± 7.82 % at 1.21 and 3.64 μM concentrations, respectively, compared to untreated cells.

Teduglutide was successfully expressed and purified and its functionality and structural integrity were confirmed by in vitro experiments. We believe that the experimentalscale method presented in the current study can be useful for pilot-scale and also industrial-scale production of teduglutide.

 Drug repurposing is an approach successfully used for discovery of new therapeutic applications for the existing drugs. The current study was aimed to use the combination of in silico methods to identify FDA-approved drugs with possible S1P1 agonistic activity useful in multiple sclerosis (MS).

 For this, a 3D-QSAR model for the known 21 S1P1 agonists were generated based on 3D-QSAR approach and used to predict the possible S1P1 agonistic activity of FDA-approved drugs. Then, the selected compounds were screened by docking into S1P1 and S1P3 receptors to select the S1P1 potent and selective compounds. Further evaluation was carried out by molecular dynamics (MD) simulation studies where the S1P1 binding energies of selected compounds were calculated.

 The analyses resulted in identification of cobicistat, benzonatate and brigatinib as the selective and potent S1P1 agonists with the binding energies of -85.93, -69.77 and -67.44 kcal. mol-1, calculated using MM-GBSA algorithm based on 50 ns MD simulation trajectories. These values are better than that of siponimod (-59.35 kcal mol-1), an FDA approved S1P1 agonist indicated for MS treatment. Furthermore, similarity network analysis revealed that cobicistat and brigatinib are the most structurally favorable compounds to interact with S1P1 .

 The findings in this study revealed that cobicistat and brigatinib can be evaluated in experimental studies as potential S1P1 agonist candidates useful in the treatment of MS.

Today, the discovery of novel and effective chemotherapeutic compounds is the mainchallenge in cancer therapy. In recent years, the anti-tumoral activity of natural naphthoquinonejuglone (JUG), present in different parts of walnut trees, has received considerable interest. Thepurpose of the current study was to prepare and evaluate the in vitro antiproliferative activity ofJUG-loaded bovine serum albumin nanoparticles (JUG-BSA NPs).

BSA NPs and JUG-BSA NPs were prepared using the desolvation technique. The NPswere characterized for their particle size (PS), zeta potential (ZP), drug loading (DL) capacityand encapsulation efficiency (EE). The anti-proliferative activity of JUG-BSA NPs was evaluatedon A431 and HT29 cancer cell lines using cellular uptake and MTT assays.

The PS and ZP values of JUG-BSA NPs were 85 ± 6.55 nm and −29.6 mV, respectively.The DL capacity and EE were 3.7% to 5% and 50.4% to 94.6%, respectively. The cytotoxicityof JUG-BSA NPs was significantly less on both cultured A431 and HT29 cells at the studiedconcentrations when compared to free JUG. However, the effect was not very substantial,particularly at high levels.

In conclusion, BSA NPs can be used as a suitable and safe carrier for the deliveryof JUG, a cytotoxic hydrophobic natural compound.

Fibroblast growth factors (FGFs) are involved in angiogenesis, wound healing and embryonic development. However, one of the causes of cancer cell growth in fibroblast-dependent cancers is FGF7 secreted by fibroblasts. Therefore, antibodies against FGF7 can be used for the treatment of these types of cancers.

In the previous studies, a phage displaying single domain antibody, D53, against human FGF7 has been identified using the phage display technique. In the present study, D53 was produced and purified in its isolated form. ELISA experiment was performed to evaluate the binding of D53 to FGF7. The mode of interaction of D53-FGF7 was explored using docking study and molecular dynamics (MD) simulations.

The expression and purification processes were verified using western blotting and SDS-PAGE analyses. ELISA experiment showed that D53 is able to recognize and bind FGF7. Docking study and MD simulations indicated that compared to dummy VH, D53 has more affinity towards FGF7.

The findings in the current study can be useful for the generation and the development of FGF7 inhibitors with a potential use in fibroblast-dependent cancers.

This research introduces a polymeric nanosphere as a new dispersive solid phaseextraction (DSPE) adsorbent for the extraction of methylphenidate (MPH) from urine and itshigh performance liquid chromatography (HPLC) analysis.

Polymeric nanosphere is a kind of copolymeric network obtained by copolymerizationof an ionic liquid monomer and styrene in the presence of vinyltriethoxysilane and2-hydroxyethylmethacrylate. HPLC coupled with ultra violet detector was applied for thedetermination and quantification of MPH. Dominant parameters in extraction were modified bythe one-parameter-at-a-time method. The results are as follow: 10 mg of polymeric nanospheres(PNS), 400 μL of acetonitrile (ACT), 5 mL of urine with the pH value of 9, and the extractionand desorption times of 2 and 5 minutes, respectively, which can be selected as the optimumextraction conditions.

Calibration curve was plotted through optimized conditions, and the proposed methodwas validated. The results demonstrated that the method presented linearity in the concentrationrange of 30-1200 ng/mL. Selectivity, matrix effect and metabolites interference effect wereinvestigated and the method presented no obvious interference effect during the analysis runtime. Repeatability, limit of detection (LOD) and limit of quantification (LOQ) values of themethod can be reported in this section as well. The method showed satisfactory results with98.8% relative recovery in the analysis of positive urine samples.

The findings convinced the applicability of the introduced method for DSPE andHPLC analysis of the positive urine samples in different laboratories.

This paper established the application of synthesized graphitic carbon nitride nanosheets (GCNNs) as an influential dispersive solid phase extraction (DSPE) adsorbent in extracting methamphetamine from complicated urine media coupled with high performance liquid chromatography.

The GCNNs were synthesized easily and applied as adsorbent in the extraction process. The effective extraction parameters were investigated by one-parameter-at-a-time. Under optimized conditions the method was validated.

The calibration curve was plotted in the concentration range of 50-1500 ng/mL through the optimized conditions and the proposed method was validated. The method was used for the analysis of positive urine samples and showed satisfactory results with the average 99.7% relative recovery.

The results persuade the capability of this novel method in analyzing of the positive urine samples in diverse clinical and forensic laboratories.

Physicochemical properties play important role in fundamental issues likeabsorption and distribution of pharmaceuticals to the target tissue. This is particularly importantfor drugs acting in central nervous system (CNS). In this study, physicochemical properties ofpreviously synthesized thiazole-pyridinium derivatives with anti-acetylcholinesterase activityand possible anti-Alzheimer effect were studied.

Partition coefficient (n-octanol/water) and chromatographic Rf values for the studiedcompounds were determined using shake flask and high performance thin layer chromatography(HPTLC) methods, respectively. Different druglikeness properties of the compounds were alsocalculated using available software and web-servers.

The experimentally determined logarithm of partition coefficients (log P) for thestudied compounds were in the range of -1.00 to -0.38. The Rf values for the studied compoundsunder the applied chromatographic condition ranged between 0.38 to 0.58. Moreover, calculatedphysicochemical properties, and druglikeness scores of the studied thiazole-pyridiniumderivatives and matching piperidine analogues were predicted. Furthermore, some ADMETfeatures of studied compounds like toxicity and metabolism by CYP450 (2C9, 2D6, 3A4, 1A2and 2C19) enzymes were predicted.

The ranges of experimental and calculated LogP values for the studied thiazolepyridinumswere close. However, the determined Rf values showed relatively better correlationto the predicted LogP values indicating the suitability of used chromatographic method forcomparing the lipophilicity of the positively charged pyridinium derivatives. The studiedcompounds were predicted to pass GI membrane and reach the CNS where they can exerttheir effects. In silico studies indicate that the piperidine counterparts of the studied thiazolepyridiniumsmay represent anti-Alzheimer agents with improved druglikeness properties.

DOF (DNA-binding with One Finger) proteins are plant-specific transcription factors which mediate numerous biological processes. The purpose of the current study is to report new naturally occurring mutations in the gene encoding for one of the members of DOF proteins named DOF 4.2.

The expression of zinc finger domain of DOF 4.2 (DOF 4.2-ZF) was investigated by first synthesis of cDNA library using different parts of Arabidopsis thaliana plant. Then the coding sequence for zinc finger domain of DOF 4.2 protein was prepared using nested PCR experiment and cloned into pGEX-6P-1 expression vector. Finally, the prepared construct was used for protein expression. Furthermore, molecular dynamics (MD) simulation was carried out to predict DNA binding affinity of DOF 4.2-ZF using AMBER package.

For the first time a new variant of DOF 4.2-ZF protein with three mutations was detected. One of the mutations is silent while the other two mutations lead to amino acid replacement (S18G) as well as introduction of a stop codon ultimately resulting in a truncated protein production. In order to investigate whether the truncated form is able to recognize DNA binding motif, MD simulations were carried out and the results showed that the chance of binding of DOF 4.2-ZF protein to cognate DNA in its truncated form is very low.

The findings demonstrated that the observed mutations adversely affect the DNA binding ability of the truncated form of DOF4.2 if it is expressed in the mutant variant of A. thaliana used in this study.

Application of natural-based herbal medicine is on a growing trend in some countries and people prefer to use plant-originated drugs rather than chemical-based ones. The present study describes an interesting sample preparation method for extraction and determination of cyproheptadine in herbal supplements as appetizing stimulant by using carbon nitride nanosheets as dispersive solid phase extraction method coupled with HPLCUV.

Various techniques used for characterization of adsorbent such as: Infrared spectroscopy (IR), scanning electron microscopy (SEM), Zeta potential analysis and powder X-ray diffraction (XRD). Optimization of the important extraction parameters were conducted by one parameter-at-a time method. Next, method validation was carried out.

The optimized cyproheptadine extraction parameters were introduced and under optimized conditions the method presented a good linearity in the concentration range of 300-2000 ng/g. The limit of detection (LOD) was 100 ng/g for the introduced method.

Quantitative analysis of fifteen real samples (Tablets or capsules) by proposed method confirmed the illegal presence of cyproheptadine in herbal appetizing stimulants supplements of the markets.

Combination therapy is one of the new strategies that minimize resistance to chemotherapy and reduces drug toxicity. Here,we investigated the effect of combination therapy with 5-Fluorouracil and Gamma Tocopherol on cell survival and BAX/BCL2 gene expression ratio in HT29 colon cancer cells.

The proliferation of cancer cells was determined via colony formation assay.BAX/BCL2 ratio was evaluated after incubation with concentrations of 5-Fluorouracil and Gamma Tocopherol via real-time-PCR.

The average number of colonies in the cells treated with 5-Fluorouracil, Gamma Tocopherol and their combination of them was 63±4, 78±3, and 28±2, respectively which significantly decreased in the combination group. In contrast with the control group, the BAX/BCL2 ratio remarkably increased when the cell underwent combinational treatment (p<0.05).

5-Fluorouracil and Gamma Tocopherolreduced HT 29 cell proliferation. Our results suggest that combination therapy with 5-Fluorouracil and Gamma Tocopherol can be considered as a strategy for induction of apoptosis via increasing the BAX/BCL2 ratio.

Iron-regulated outer membrane virulence protein (IrgA) involved in the initial step of iron uptake by binding ferric- iron chelation siderophore that allows the bacterium to extract iron from the environment. IrgA homologue adhesion (Iha) revealed as a novel adherence conferring molecule. In this study, homology modeling, fold recognition and Ab-initio approaches along with their combination were invoked to determine the tertiary structure of Iha.

Specific bioinformatics methods were used to forecast their immunological, biochemical and functional properties.

The results showed that IrgA constitutes beta barrel structures. The immunological, biochemical and functional analyzes led us to pick a region of every antigen with the highest immunogenic properties. Comparison of antigenicity scores for selected regions and the whole proteins showed that the antigenicity of the selected regions is considerably higher than the whole antigen.

Our strategy for prediction of the 3D structure and epitopes could be deemed as an amenable approach for efficient vaccine design. These approaches used could provide the basis for future functional studies to design and development of a suitable vaccine. In this regard, a region includes residues 200-340, covering a part of barrel, was chosen as vaccine candidate against Iha protein in Escherichia Coli.

In the recent years, histamine H3 receptor (H3R) has been receiving increasing attention in pharmacotherapy of neurological disorders. The aim of the current study was to investigate structural requirements for the prediction of H3 antagonistic activity using quantitative structure-activity relationship (QSAR) and molecular docking techniques.

To this end, genetic algorithm coupled partial least square and stepwise multiple linear regression methods were employed for developing a QSAR model. The obtained QSAR model was stringently assessed using different validation criteria.

The generated model indicated that connectivity information and mean absolute charge are two important descriptors for the prediction of H3 antagonistic activity of the studied compounds. To gain insight into the mechanism of interaction between studied molecules and H3R, molecular docking was performed. The most important residues involved in the ligandreceptor interactions were identified.

The result of current study can be used for designing of new H3 antagonist and proposing structural modifications to improve H3 inhibitory potency.

Despite the discovery and synthesis of several anticancer drugs, cancer is still a major life threatening incident for human beings after cardiovascular diseases. Toxicity, severe side effects, and drug resistance are serious problems of available commercial anticancer drugs. Coumarins are synthetic and natural heterocycles that show promising antiproliferative activities against various tumors. The aim of this research is to computationally study the coumarin derivatives in order to develop reliable quantitative structure-activity relationship (QSAR) models for predicting their anticancer activities.

A data set of thirty one coumarin analogs with significant antiproliferative activities toward HepG2 cells were selected from the literature. The molecular descriptors for these compounds were calculated using Dragon, HyperChem, and ACD/Labs programs. Genetic algorithm (GA) accompanied by multiple linear regression (MLR) for simultaneous feature selection and model development was employed for generating the QSAR models.

Based on the obtained results, the developed linear QSAR models with three and four descriptors showed good predictive power with r2 values of 0.670 and 0.692, respectively. Moreover, the calculated validation parameters for the models confirmed the reliability of the QSAR models.

The findings of the current study could be useful for the design and synthesis of novel anticancer drugs based on coumarin structure.

Uncontrolled activity of tumor necrosis factor alpha (TNF-α) as pro-inflammatory cytokine has been linked with pathogenesis of autoimmune/inflammatory diseases. Therefore, modulating of TNF-α associated biological pathways is a promising strategy for alleviating of such diseases. In view of this, the use of antibody fragments such as single-chain variable fragments (scFv) in therapeutic applications has been gained much attention in terms of pharmacokinetic as well as production and therapeutic costs.

In the current investigation, the previously designed and humanized hD2 antibody was modeled and docked onto the TNF-α structure. The binding free energy was predicted for the complex of hD2-TNF-α using molecular dynamics calculation followed by per-residue energy decomposition for residues of hD2. In addition in silico mutations of important amino acids at the binding site of enzyme were performed and the binding free energy was calculated for mutant forms of scFv in complex with TNF-α.

The analyses of the results proposed Y27F mutation in heavy chain CDR1 of hD2 scFv antibody may be considered as a promising substitution.

The results may be used for designing new anti-TNF-α antibody with improved activity

Lawsone (2-hydroxy-1,4-naphtoquinone; LAW), as a naphthoquinone derivative, is the biologically active component of Henna leaves. In this study, the structural and functional effects of LAW on bovine liver catalase (BLC), has been studied utilizing ultraviolet-visible (UV-vis) absorption, fluorescence and ATR-FTIR spectroscopic techniques, and molecular docking approach. In vitro kinetic study showed that by adding gradual concentrations of LAW, catalase activity was significantly decreased through noncompetitive inhibition mechanism. UV–vis and ATR-FTIR spectroscopic results illustrated that additional concentration of LAW lead to significant change in secondary structure of the enzyme. The fluorescence spectroscopic results at different temperatures indicated that LAW quenches the intrinsic fluorescence of BLC by dynamic mechanism and there is just one binding site for LAW on BCL. Changing the micro- environment nearby two aromatic residues (tryptophan (Trp) and tyrosine (Tyr)) were resulted from synchronous fluorescence. The thermodynamic parameters were implied that the hydrophobic bindings have a significant impress in the organization of the LAW-catalase complex. Molecular docking data in agreement with experimental results, confirmed that hydrophobic interactions are dominant. Inhibition of enzyme activity by LAW, showed that along with its helpful effects as an anti-oxidant compounds, the side effects of LAW should not be overlooked.

Hypercholesterolemia is a common metabolic disorder in developing and developed countries and is associated with the increased rates of chronic kidney disease (CKD). Statin therapy could reduce cholesterol synthesis as well as progression of CKD. Diversity between statins causes variety in pharmacokinetics and pharmacodynamics and also their pleiotropic effects. In the present investigation we aimed to evaluate the protective potentials of both atorvastatin (Ator) (as lipid-soluble statin) and rosuvastatin (Ros) (as water-soluble statin) against renal histopathological damages in the high cholesterol diet induced hypercholesterolemic rats (HCDIHR).

Serum lipid profile, oxidized low density lipoprotein (OX-LDL), malondialdehyde (MDA), urea and creatinine levels, as well as renal histopathology were evaluated.

While Ros acted better than Ator to reduce serum low density lipoprotein cholesterol (LDL-C) (P<0.01), atherogenic index (AI) (P<0.01), MDA (P<0.01), and OX-LDL (P<0.01); no significant differences were noted in their cholesterol (P=0.72), triglyceride (TG) (P=0.79), and very low density lipoprotein cholesterol lowering (VLDL-C) (P=0.79) and high density lipoprotein cholesterol elevating effects (HDL-C) (P=0.72). Ator was more effective to reduce renal histopathologic indices compared to Ros, including accumulation of lipid droplet, glomerular foam cells, mesangial cell proliferation, renal hemorrhage, and tubulointerstitial damages in the kidneys of diet induced hypercholesterolemic rats.

The findings underline that the lipophilic Ator may performs better than Ros in attenuating renal damages in HCDIHR.

Glutamate-specific endopeptidase (GSE, EC 3.4.21.19) is belonging to the serine protease family enzymes. A glutamate-specific endopeptidase has the ability to cleavage peptide bonds for the protein structure analysis, solid phase synthesis of peptides and preparation of nanotubes. The purpose of this investigation was to produce glutamate endopeptidase enzyme from the Bacillus licheniformis SL-1from isolated from Aran-Bidgol salt lake in Iran.

In this study, the expression of glutamate-endopeptidase enzyme from the B. licheniformis SL-1 wasinvestigated in periplasmic spaces of E. coli BL21 in different temperature (16ᴼC, 20ᴼC, 37ᴼC) and different concentrations of inducer (0.4, 1, 2 mM).

Analysis the results demonstrated that periplasmic and soluble expression of glutamate endopeptidase was observed in E. coli BL21at 37ᴼC and 2 mM concentration of IPTG.

Qualitative investigation of the expression results revealed that the expression of the enzyme is temperature-dependant and requires higher concentrations of IPTG. This preliminary study provides the possibility of recombinant enzyme production in E. coli BL21 expression system. In order to produce large scale of this enzyme, other expression systems as well as alternative purification systems can be studied.

Tumor necrosis factor alpha (TNF-α) is an inflammatory cytokine which plays crucial roles in pathogenesis of inflammatory diseases. The current study aimed to investigate the binding abilities of I44 and I49 domain antibodies to TNF-α. The dAbs were expressed in bacterial expression system and purified by affinity chromatography using Ni-sepharose column. The expression and purity of the proteins were evaluated using western blotting and SDS-PAGE techniques, respectively. ELISA experiment showed that I44 and I49 dAbs bind to TNF-α with the binding constants (Kd) of 5.18 ± 1.41 and 2.42 ± 0.55 µM, respectively. The inhibitory effect of dAbs on TNF-α biological effect was determined in MTT assay in which I44 and I49 prevented TNF-α cell cytotoxicity with IC50 values of 6.61 and 3.64 µM, respectively. The identified anti-TNF-α dAbs could bind to and inhibit TNF-α activity. The dAbs activities can be attributed to their ability to establish hydrogen bonds as well as hydrophobic contacts with TNF-α. The results of the current study can pave the way for further structural studies in order to introduce new more potent anti-TNF-α antibodies.

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. 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. 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. 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.

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. 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. 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. 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.

The pro-inflammatory cytokine, TNF-α, which plays a major role in the development and persistence of inflammatory diseases, is the basis for the use of anti-TNF-α therapies. The neutralization of TNF-α or blockage of its binding to the corresponding receptor has mainly served as a therapeutic strategy against some diseases. This study aimed to investigate the production of a humanized single chain antibody (scFv) against TNF-α. Therefore, a murine monoclonal antibody, D2 mAb, was selected for humanizing by the complementarity determining region (CDR)-grafting method. Briefly, the replacement of the CDRs from D2 mAb with the specific human single chain scaffold led to the production of a novel humanized scFv mAb against human TNF-α (hD2). Cloning of hD2 into a suitable expression vector, pGEX-6P-1, resulted in the expression of a 52-kDa GST-fusion protein in E. coli, mostly in the form of inclusion bodies. The solubilization and refolding of GST-hD2 inclusion bodies was achieved with the addition of 4 M urea and subsequent dialysis to recover the fusion protein in soluble form. Then the soluble GST-hD2 was purified by affinity chromatography through immobilized glutathione. The GST pull-down experiment showed a positive interaction between GST-hD2 and TNF-α protein. Moreover, the results of an MTT assay showed that the purified GST-hD2 has TNF-α neutralizing activity (Kd of 1.03 nM) and hence hD2 has the potential to be developed into a therapeutic agent. However, more investigation is needed to elucidate the potential of in vivo TNF-α neutralizing activity of hD2 in comparison to other anti-TNF-α antibodies.

The bacterial enzyme has gained more attention in therapeutic application because of the higher substrate specificity and longer half-life. L-asparaginase is an important enzyme with known antineoplastic effect against acute lymphoblastic leukemia (ALL). Novel L-asparaginase genes were identified from a locally isolated halo-thermotolerant Bacillus strain and the recombinant enzymes were overexpressed in modified E. coli strains, OrigamiTM B and BL21. In addition, the biochemical properties of the purified enzymes were characterized, and the enzyme activity was evaluated at different temperatures, pH, and substrate concentrations. The concentration of pure soluble enzyme obtained from Origami strain was ~30 mg/L of bacterial culture, which indicates the significant improvement compared to L-asparaginase produced by E. coli BL21 strain. The catalytic activity assay on the identified L-asparaginases (ansA1 and ansA3 genes) from Bacillus sp. SL-1 demonstrated that only ansA1 gene codes an active and stable homologue (ASPase A1) with high substrate affinity toward L-asparagine. The Kcat and Km values for the purified ASPase A1 enzyme were 23.96s-1 and 10.66 µM, respectively. In addition, the recombinant ASPase A1 enzyme from Bacillus sp. SL-1 possessed higher specificity to L-asparagine than L-glutamine. The ASPase A1 enzyme was highly thermostable and resistant to the wide range of pH 4.5–10. The biochemical properties of the novel ASPase A1 derived from Bacillus sp. SL-l indicated a great potential for the identified enzyme in pharmaceutical and industrial applications.