Effects of Salvia mirzayanii extract administration on hyperglycemia improvement in diabetic rats: The role of GLUT4, PEPCK and G6Pase genes.

Diabetes is a dangerous metabolic disorder by increasing incidence in human societies worldwide. Recently, much attention has been focused on the development of hypoglycemic agents, particularly the derivatives of herbal drugs, in the treatment of diabetes. This research aimed to study the anti-diabetic effect of Salvia mirzayanii in the diabetic rat models. First, the plant material was extracted from the leaves, and orally administered to the rats. After treating the animals with the aqueous extract of S. mirzayanii at a dose of 600 mg/kg, animal body weight for 12 weeks, fasting blood glucose, oral glucose tolerance test (OGTT), and body weight changes were examined. To analyze the anti-diabetic function of S. mirzayanii, we measured the expression of glucose transporter-4 (GLUT4), phosphoenolpyruvate carboxykinase (PEPCK), and glucose 6-phosphatase (G6Pase) genes in healthy and streptozotocin (STZ)-diabetic rats. The expression levels of the genes of interest in muscle and liver tissues were determined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC). There were no significant differences in fasting blood glucose and OGTT between normal control (NC) group and the diabetic control (DC) group treated with S. mirzayanii. In contrast, there was a significant difference with the untreated DC (P < 0.05). The treatment of diabetic rats with S. mirzayanii significantly increased the expression of GLUT4 in the muscle and decreased the expression levels of PEPCK and G6Pase in the liver compared to the DC group (P < 0.05). These findings clearly show that S. mirzayanii can improve hyperglycemia by increasing the GLUT4 expression, and inhibiting the gluconeogenesis pathway in the liver. In general, the obtained results provided a new insight into the efficacy of S. mirzayanii aqueous extract as an anti-diabetic herbal medicine.

PASylated Urate Oxidase Enzyme: Enhancing Biocatalytic Activity, Physicochemical Properties, and Plasma Half-Life.

Recombinant urate oxidase (UOX, E.C.1.7.3.3) is an important therapeutic enzyme used in preventing and treating chemotherapy-induced hyperuricemia and severe gout. However, UOX use is limited due to the poor stability and short plasma half-life. To solve this problem, we designed three PASylated variants of Aspergillus flavus UOX with different PAS sequences at the C- or N-terminus. The genes of native and PASylated variants (UOX-PAS20, PAS24-UOX, and UOX-PAS100) were designed and produced in Escherichia coli strain BL21 (DE3). The expressed recombinant native and PASylated enzymes were compared in terms of biophysical properties, kinetics parameters, and pharmacokinetics behavior using standard methods. PASylation of UOX with PAS100 polymer caused a 1.24-fold reduction in K m to 52.61 µM, and a 3.87-fold increase in K cat/K m for uric acid compared to the native variant. UOX-PAS100 retained its activity in different temperatures (20-55 °C); however, other variants lost nearly 50% of their original activity at 55 °C. UOX-PAS100 exhibited a 1.78-fold increase in hydrodynamic radius and a 1.64-fold larger apparent molecular size in comparison to the native UOX. Circular dichroism (CD) spectroscopy demonstrated that the addition of the PAS tag does not change the secondary structure of the fusion enzyme. The tryptophan fluorescence emission spectra for PASylated enzymes showed a significant modification in the conformational state of UOX by the PAS polymer presence. UOX-PAS100 retained 89.0% of the original activity following 72 h incubation in the presence of plasma at 37 °C. However, only about 61.0%, 57.0%, 50.0%, and 52.0% of activity from PAS24-UOX, UOX-PAS20, native UOX, and rasburicase (Fasturtec, Italy) remained, respectively, at the identical time. UOX-PAS100 had an increased biological half-life (8.21 h) when compared with the rasburicase (3.12 h) and native UOX (2.87 h) after being injected into a rat. Having considering everything, our results suggest that the UOX-PAS100, an A. flavus UOX fused with a C-terminally 100 amino acid PAS-residue, is a proper candidate with enhanced biological activity and extended plasma half-life for clinical therapy in patients suffering from hyperuricemia.

In silico drug repurposing against SARS-CoV-2 using an integrative transcriptomic profiling approach: Hydrocortisone and Benzhydrocodone as potential drug candidates against COVID-19.

COVID-19 pathogenesis is mainly attributed to dysregulated antiviral immune response, the prominent hallmark of COVID-19. As no established drugs are available against SARS-CoV-2 and developing new ones would be a big challenge, repurposing of existing drugs holds promise against COVID-19. Here, we used a signature-based strategy to delve into cellular responses to SARS-CoV-2 infection in order to identify potential host contributors in COVID-19 pathogenesis and to find repurposable drugs using in silico approaches. We scrutinized transcriptomic profile of various human alveolar cell sources infected with SARS-CoV-2 to determine up-regulated genes specific to COVID-19. Enrichment analysis revealed that the up-regulated genes were involved mainly in viral infectious disease, immune system, and signal transduction pathways. Analysis of protein-protein interaction network and COVID-19 molecular pathway resulted in identifying several anti-viral proteins as well as 11 host pro-viral proteins, ADAR, HBEGF, MMP9, USP18, JUN, FOS, IRF2, ICAM1, IFI35, CASP1, and STAT3. Finally, molecular docking of up-regulated proteins and all FDA-approved drugs revealed that both Hydrocortisone and Benzhydrocodone possess high binding affinity for all pro-viral proteins. The suggested repurposed drugs should be subject to complementary in vitro and in vivo experiments in order to be evaluated in detail prior to clinical studies in potential management of COVID-19.

The Effective Control of Hyperuricemia in Cancer Patients: A New Recombinant Conjugated Variant of Urate Oxidase.

OBJECTIVE: Management of hyperuricemia is crucial to controlling tumor lysis syndrome (TLS) during cancer therapy. Urate oxidase (UOX) that catalyzes the enzymatic oxidation of uric acid into allantoin, is effective in lowering plasma uric acid levels and controlling hyperuricemia. Recently, we developed a new recombinant conjugate variant of UOX therapeutic drug using PASylation technology. This study was designed to evaluate the stability, plasma half-life and immunogencity of PASylated UOX. METHODS: A recombinant variant of PASylated UOX from the Aspergillus flavus was manufactured using bioinformatics and experimental techniques. Ex vivo evaluation of stability of PASylated UOX was done in 50% human serum. For half-life test, recombinant PASylated UOX and rasburicase were administered at 1.5 mg/kg to 10 rats in two different groups and samples were collected after injection Production of antibodies against PASylated drug was also assayed. RESULTS: Residual activity of PASylated UOX in 50% human serum was higher than rasburicase and native UOX. Stability of PASylated UOX at 25°C and 37°C was also higher than rasburicase and native UOX. The PASylated half-life was ~32.1 hours, whereas half-life for rasburicase and native UOX was ~25.1 and ~22.8 hours, respectively. In immunogenicity examination, there is 33% and 36% decrease in the absorbance of native UOX and rasburicase, respectively when compared with that of PASylated UOX. CONCLUSION: Our data confirmed the efficacy and stability of PASylated UOX in comparison to the rasburicase. In summary, the results indicated that PASylated UOX drug is effective at lowering plasma uric acid levels with prolonged plasma half-life and decreased cost.
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An amperometric biosensor for specific detection of glycated hemoglobin based on recombinant engineered fructosyl peptide oxidase.

Here, we present a specific biosensor based on the detection of glycated hemoglobin (HbA1c) proteolytic digestion product, fructosyl valyl histidine (Fru-ValHis). A recombinant engineered fructosyl peptide oxidase (FPOX) enzyme with improved specificity was immobilized on the electrode surface modified by chitosan (CHIT), graphene oxide (GO) and gold nanoparticles (AuNPs). The biosensor exhibited a linear response toward different concentrations of Fru-ValHis ranging from 0.1 to 2 mM with a sensitivity of 8.45 µA mM-1 cm-2. Detection limit of the current biosensor for Fru-ValHis was 0.3 µM as the lowest quantity required giving a signal to a background. Analytical recovery of added Fru-ValHis in whole blood was 95.1-98.35% for FPOX/AuNPs/GO/CHIT/FTO electrode. For Fru-ValHis determination by FPOX-AuNPs-GO-CHIT/FTO electrode, within-run coefficient of variation (CV) was between 1.3% and 2.4% and between run CV was between 2.1% and 3.5%. A significant change in electron transfer resistance after the incubation of FPOX-modified electrode with Fru-ValHis was observed, while no response was achieved with control, indicating specific measurement of Fru-ValHis. Moreover, designed biosensor measured HbA1c in human blood samples and the results were well agreed with that obtained with NORUDIA™ N HbA1c diagnostic kit. Overall, suitable specificity of the engineered FPOX made the bioelectrode responded well to the Fru-ValHis level, which demonstrates a promising application for specific detection of HbA1c biomarker.

Partitioning of Recombinant Pseudomonas putida POS-F84 Proline Dehydrogenase in Aqueous Two-Phase Systems: Optimization Using Response Surface Methodology.

Empirical modeling the partition behavior and recovery of a recombinant Pseudomonas putida POS-F84 proline dehydrogenase (ProDH) in aqueous two-phase systems (ATPS) was carried out by response surface methodology (RSM). Polyethylene glycol 1000 (PEG-1000) concentration, sodium carbonate concentration, and pH, which were the most important factors, were chosen for modeling the partition feature of enzyme. The adequacy of the models was investigated by means of variance analysis. Also, to confirm the efficiency of the ATPS in partition and purification of recombinant ProDH, purity and enzymatic activity was studied. After numerical optimization, an optimal ATPS composed of 14.33% PEG-1000 and 11.79% sodium carbonate at pH 7.48 was achieved. Yield, purification factor, and recovery were 81.41%, 60.82, and 270.82%, respectively. Purified recombinant ProDH was found as a single protein band into the upper PEG-rich phase and the specific activity was calculated to be 46.23 ± 2.1 U/mg. Collectively, our data showed that the RSM could be an appropriate and powerful tool to define the best ATPS system for recovery and purification of P. putida ProDH.

Engineering an efficient mutant of Eupenicillium terrenum fructosyl peptide oxidase for the specific determination of hemoglobin A1c.

Fructosyl peptide oxidase (FPOX, EC 1.5.3) belongs to the family of oxidoreductases, which is used as a diagnostic enzyme for diabetes mellitus. FPOX has activities toward Fru-ValHis and Fru-Lys as model compounds for hemoglobin A1c (HbA1c) and glycated albumin, respectively. However, when the concentration of HbA1c is measured, the activity toward Fru-Lys will cause interference. In this study, we focused on the substrate specificity engineering of FPOX from Eupenicillium terrenum through computational and experimental methods with characteristics more suitable for HbA1c measurement in the blood. Based on structural knowledge of E. terrenum FPOX (PDB ID 4RSL) and molecular modeling results, residues His-377, Arg-62, Lys-380, and Tyr-261 were selected as mutagenesis sites. The best mutant with lower binding energy, stronger hydrophobic interactions, and more hydrogen bonds with Fru-ValHis and higher binding energy toward Fru-Lys was selected for experimental studies. To investigate the conformational changes in FPOX due to the mutation, molecular dynamics simulation was also performed. The genes encoding of native and engineered variants were cloned into pET-22b(+) and produced in Escherichia coli strain BL21 (DE3). The expressed recombinant enzymes were purified and their kinetic properties were studied. Substitution of Tyr261 with Trp resulted in a mutant enzyme with improved specificity for Fru-ValHis, a model compound of HbA1c. The specific activity of mutant FPOX increased by 5.1-fold to 145.2 ± 3.2 U/mg for Fru-ValHis and decreased by 13.7-fold to 1.3 U/mg ± 0.9 for Fru-Lys compared to the native variant. Kinetics analysis indicated that Tyr261Trp FPOX mutant had 11.7-fold increase in Kcat/Km for Fru-ValHis compared to the wild-type enzyme, while the Kcat/Km for Fru-Lys diminished by 22.4-fold. In summary, our computational and experimental results suggested that the engineered FPOX is a good candidate to efficient determination of HbA1c.

Medium Optimization for Improved Production of Dihydrolipohyl Dehydrogenase from Bacillus sphaericus PAD-91 in Escherichia coli.

Dihydrolipohyl dehydrogenase (DLD) is a FAD-dependent enzyme that catalyzes the reversible oxidation of dihydrolipoamide. Herein, we report medium optimization for the production of a recombinant DLD with NADH-dependent diaphorase activity from a strain of Bacillus sphaericus PAD-91. The DLD gene that consisted of 1413 bp was expressed in Escherichia coli BL21 (DE3), and its enzymatic properties were studied. The composition of production medium was optimized using one-variable-at-a-time method followed by response surface methodology (RSM). B. sphaericus DLD catalyzed the reduction of lipoamide by NAD+ and exhibited diaphorase activity. The molecular weight of enzyme was about 50 kDa and determined to be a monomeric protein. Recombinant diaphorase showed its optimal activity at temperature of 30 °C and pH 8.5. K m and V max values with NADH were estimated to be 0.025 mM and 275.8 U/mL, respectively. Recombinant enzyme was optimally produced in fermentation medium containing 10 g/L sucrose, 25 g/L yeast extract, 5 g/L NaCl and 0.25 g/L MgSO4. At these concentrations, the actual diaphorase activity was calculated to be 345.0 ± 4.1 U/mL. By scaling up fermentation from flask to bioreactor, enzyme activity was increased to 486.3 ± 5.5 U/mL. Briefly, a DLD with diaphorase activity from a newly isolated B. sphaericus PAD-91 was characterized and the production of recombinant enzyme was optimized using RSM technique.

Purification and Characterization of Recombinant Darbepoetin Alfa from Leishmania tarentolae.

Darbepoetin alfa is a biopharmaceutical glycoprotein that stimulates erythropoiesis and is used to treat anemia, which associated with renal failure and cancer chemotherapy. We herein describe the structural characterization of recombinant darbepoetin alfa produced by Leishmania tarentolae T7-TR host. The DNA expression cassette was integrated into the L. tarentolae genome through homologous recombination. Transformed clones were selected by antibiotic resistance, diagnostic PCRs, and protein expression analysis. The structure of recombinant darbepoetin alfa was analyzed by isoelectric focusing, ultraviolet-visible spectrum, and circular dichroism (CD) spectroscopy. Expression analysis showed the presence of a protein band at 40 kDa, and its expression level was 51.2 mg/ml of culture medium. Darbepoetin alfa have 5 isoforms with varying degree of sialylation. The UV absorption and CD spectra were analogous to original drug (Aranesp), which confirmed that the produced protein was darbepoetin alfa. Potency test results revealed that the purified protein was biologically active. In brief, the structural and biological characteristics of expressed darbepoetin alfa were very similar to Aranesp which has been normally expressed in CHO. Our data also suggest that produced protein has potential to be developed for clinical use.

The Rapid and Sensitive Quantitative Determination of Galactose by Combined Enzymatic and Colorimetric Method: Application in Neonatal Screening.

The quantitative measurement of galactose in blood is essential for the early diagnosis, treatment, and dietary monitoring of galactosemia patients. In this communication, we aimed to develop a rapid, sensitive, and cost-effective combined method for galactose determination in dry blood spots. This procedure was based on the combination of enzymatic reactions of galactose dehydrogenase (GalDH), dihydrolipoyl dehydrogenase (DLD), and alkaline phosphates with a colorimetric system. The incubation time and the concentration of enzymes used in new method were also optimized. The analytical performance was studied by the precision, recovery, linearity, and sensitivity parameters. Statistical analysis was applied to method comparison experiment. The regression equation and correlation coefficient (R (2)) were Y = 0.0085x + 0.032 and R (2) = 0.998, respectively. This assay exhibited a recovery in the range of 91.7-114.3 % and had the limit detection of 0.5 mg/dl for galactose. The between-run coefficient of variation (CV) was between 2.6 and 11.1 %. The within-run CV was between 4.9 and 9.2 %. Our results indicated that the new and reference methods were in agreement because no significant biases exist between them. Briefly, a quick and reliable combined enzymatic and colorimetric assay was presented for application in newborn mass screening and monitoring of galactosemia patients.