Oxidized LDL-regulated microRNAs for evaluating vascular endothelial function: molecular mechanisms and potential biomarker roles in atherosclerosis.

As a simple monolayer, vascular endothelial cells can respond to physicochemical stimuli. In addition to promoting the formation of foam cells, oxidized low-density lipoprotein (ox-LDL) contributes to the atherosclerotic process through different mechanisms, including endothelial cell dysfunction. As conserved noncoding RNAs, microRNAs (miRNAs) naturally lie in different genomic positions and post-transcriptionally regulate the expression of many genes. They participate in integrated networks formed under stress to maintain cellular homeostasis, vascular inflammation, and metabolism. These small RNAs constitute therapeutic targets in different diseases, including atherosclerosis, and their role as biomarkers is crucial given their detectability even years before the emergence of diseases. This review was performed to investigate the role of ox-LDL-regulated miRNAs in atherosclerosis, their molecular mechanisms, and their application as biomarkers of vascular endothelial cell dysfunction.

Epigenetic alterations and genetic variations of angiotensin-converting enzyme 2 (ACE2) as a functional receptor for SARS-CoV-2: potential clinical implications.

Receptor recognition is a crucial step in viral infection and is a critical factor for cell entry and tissue tropism. In several recent studies, angiotensin-converting enzyme 2 (ACE2) has been demonstrated to be the cellular receptor of SARS-CoV-2 as it was previously well known as the receptor of SARS-CoV. SARS-CoV-2 can bind with high affinity to human ACE2 and engages it as an entry receptor. It seems that the genetic, notably epigenetic variations of ACE2 are less known in different populations, indicating the need for its further investigation. These variations have the potential to affect its contribution to the pathogenicity of COVID-19. The contribution of epigenetics in the interindividual variability of ACE2 merits more attention because epigenetic processes can play important roles in ACE2 alterations in various tissues and different people and populations. Analyzing different DNA methylation patterns and microRNAs, contributing to the ACE2 modulation in the lungs will have a high priority. The epigenetic and genetic variations of ACE2 become even more important when considering that some people have mild clinical symptoms despite having COVID-19. The pathogenicity of SARS-CoV-2 infection is complex; therefore, a better understanding of the underlying pathobiology, especially binding the virus to its receptors, could help improve therapeutic and preventive approaches. This review aims to highlight the importance of evaluating both the epigenetic and genetic variations of ACE2 as a receptor for the deadly SARS-CoV-2.

Enhanced nuclear translocation and activation of aryl hydrocarbon receptor (AhR) in THP-1 monocytic cell line by a novel niosomal formulation of indole-3-carbinol.

Although niosomes structurally resemble liposomes, they are composed of nonionic surfactants which result in less toxicity and more stability. Here, we developed a novel niosomal formulation of I3C and investigated the nuclear translocation and activation of AhR among human acute myeloid leukaemia (AML) monocytic THP-1 cell line. Niosomal vesicles comprised of nonionic surfactants, cholesterol and I3C were prepared using thin film hydration (TFH) method and characterized according to the entrapment efficiency (EE %), size and zeta potential, by Dynamic light scattering method (DLS), and the surface morphology visualized by Transmission electron microscopy (TEM). In vitro release of I3C was evaluated and MTS assay was used to evaluate the viability of THP-1 cells. The nuclear translocation of AhR was assessed by immunocytochemistry (ICC) and Real-time RT-PCR was conducted using AhR target genes. The ratio of Cholesterol:Span 60 (1:1) niosomal formulations with the highest significant EE% were selected. I3C exerted cytotoxic effects on THP-1 cells in a dose- and time-dependent manner, while administration of niosomal I3C reduced these effects. Both niosomal and free I3C formulations facilitated the nuclear translocation of AhR. CYP1A1 was overexpressed in response to both free and niosomal I3C treatments, while IL1ß was overexpressed merely in niosomal I3C-treated THP-1 cells. Niosomal formulation of I3C resulted in reduced cytotoxicity effects by enhancing the functional effects of I3C on AhR in THP-1 cells, including its nuclear translocation and overexpression of the target genes.

Enzymatic characterization of a NADH-dependent diaphorase from Lysinibacillus sp. strain PAD-91.

Diaphorases are flavin-containing enzymes with potential applications in biotransfomation reactions, biosensor design and in vitro diagnostic tests. In this paper, we present recombinant expression, characterization and medium optimization of a lipoamide dehydrogenase (DLD) with NADH-dependent diaphorase activity from a Lysinibacillus sp. strain. DLD encoding sequence showed an open reading frame of 1413-bp encoding a 470 amino acid chain. Lysinibacillus sp. DLD catalyzed the NADH-dependent reduction of electron acceptors and exhibited diaphorase activity. The molecular mass of the isolated enzyme was found to be about 50 kDa, and determined to be a monomeric protein. The optimum pH and temperature for the catalytic activity of the enzyme was about pH 7.5 and 30 °C. The Km and Vmax values were estimated to be 0.025 mM and 1.33 µmol/min, 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. By Scaling up fermentation from flask to bioreactor, enzyme activity was increased to 487.5 U/ml. This study provides data on the identification, characterization and medium optimization of a NADH-dependent diaphorase from a newly isolated Lysinibacillus sp. PAD-91.

Synergistic induction of cardiomyocyte differentiation from human bone marrow mesenchymal stem cells by interleukin 1ß and 5-azacytidine.

Interleukin-1ß (IL-1ß) is a cytokine protein expressed by cardiomyocyte in myocardial damage-associated inflammation. Although several methods are currently available for treatment of heart diseases none of them are completely successful. Recently, stem cells have gained enormous attention and are expected to play a significant role for treating heart diseases. 5-Azacytidine (5-aza) has recently been found to cause stimulation of stem cells to differentiate into cardiomyocytes. Here we present the determination of whether IL-1ß can induce the differentiation of mesenchymal stem cells (MSCs) to cardiomyocytes. MSCs were derived from bone marrow, propagated and then cultured in differentiation medium supplemented with 5-aza and IL-1ß (at two levels, 5 and 10 ng/ml). After 21 days, total RNA was extracted and cDNA synthesis was carried out. Quantitative polymerase chain reaction (Q-PCR) was performed for detecting cardiac-specific markers. Besides, to investigate the expression of cardiac markers in protein levels, immunocytochemistry was done by specific antibodies. Ultimately, cardiac markers expression suggested that IL-1ß and 5-aza synergistically induces the cardiomyocyte differentiation.

Cloning and expression of codon-optimized recombinant darbepoetin alfa in Leishmania tarentolae T7-TR.

Darbepoetin alfa is an engineered and hyperglycosylated analog of recombinant human erythropoietin (EPO) which is used as a drug in treating anemia in patients with chronic kidney failure and cancer. This study desribes the secretory expression of a codon-optimized recombinant form of darbepoetin alfa in Leishmania tarentolae T7-TR. Synthetic codon-optimized gene was amplified by PCR and cloned into the pLEXSY-I-blecherry3 vector. The resultant expression vector, pLEXSYDarbo, was purified, digested, and electroporated into the L. tarentolae. Expression of recombinant darbepoetin alfa was evaluated by ELISA, reverse-transcription PCR (RT-PCR), Western blotting, and biological activity. After codon optimization, codon adaptation index (CAI) of the gene raised from 0.50 to 0.99 and its GC% content changed from 56% to 58%. Expression analysis confirmed the presence of a protein band at 40 kDa. Furthermore, reticulocyte experiment results revealed that the activity of expressed darbepoetin alfa was similar to that of its equivalent expressed in Chinese hamster ovary (CHO) cells. These data suggested that the codon optimization and expression in L. tarentolae host provided an efficient approach for high level expression of darbepoetin alfa.

Response surface methodology to optimize partition and purification of two recombinant oxidoreductase enzymes, glucose dehydrogenase and d-galactose dehydrogenase in aqueous two-phase systems.

Oxidoreductases are an important family of enzymes that are used in many biotechnological processes. An experimental design was applied to optimize partition and purification of two recombinant oxidoreductases, glucose dehydrogenase (GDH) from Bacillus subtilis and d-galactose dehydrogenase (GalDH) from Pseudomonas fluorescens AK92 in aqueous two-phase systems (ATPS). Response surface methodology (RSM) with a central composite rotatable design (CCRD) was performed to optimize critical factors like polyethylene glycol (PEG) concentration, concentration of salt and pH value. The best partitioning conditions was achieved in an ATPS composed of 12% PEG-6000, 15% K2HPO4 with pH 7.5 at 25°C, which ensured partition coefficient (KE) of 66.6 and 45.7 for GDH and GalDH, respectively. Under these experimental conditions, the activity of GDH and GalDH was 569.5U/ml and 673.7U/ml, respectively. It was found that these enzymes preferentially partitioned into the top PEG-rich phase and appeared as single bands on SDS-PAGE gel. Meanwhile the validity of the response model was confirmed by a good agreement between predicted and experimental results. Collectively, according to the obtained data it can be inferred that the ATPS optimization using RSM approach can be applied for recovery and purification of any enzyme from oxidoreductase family.

Expression and response surface optimization of the recovery and purification of recombinant D-galactose dehydrogenase from Pseudomonas fluorescens.

The enzyme D-galactose dehydrogenase (GalDH) has been used in diagnostic kits to screen blood serum of neonates for galactosemia. It is also a significant tool for the measurement of ß-D-galactose, α-D-galactose and lactose as well. In this study, response surface methodology (RSM) was used to identify the suitable conditions for recovery of recombinant GalDH from Pseudomonas fluorescens in aqueous two-phase systems (ATPS). The identified GalDH gene was amplified by PCR and confirmed by further cloning and sequencing. E. coli BL-21 (DE3) containing the GalDH gene on a plasmid (pET28aGDH) was used to express and purify the recombinant enzyme. The polyethylene glycol (PEG) and ammonium sulfate concentrations and pH value were selected as variables to analyze purification of GalDH. To build mathematical models, RSM with a central composite design was applied based on the conditions for the highest separation. The recombinant GalDH enzyme was expressed after induction with IPTG. It showed NAD'-dependent dehydrogenase activity towards D-Galactose. According to the RSM modeling, an optimal ATPS was composed of PEG-2000 14.0% (w/w) and ammonium sulfate 12.0% (w/w) at pH 7.5. Under these conditions, GalDH preferentially concentrated in the top PEG-rich phase. The enzyme activity, purification factor (PF) and recovery (R) were 1400 U/ml, 60.0% and 270.0%, respectively. The PEG and salt concentrations were found to have significant effect on the recovery of enzyme. Briefly, our data showed that RSM could be an appropriate tool to define the best ATPS for recombinant P. fluorescens GalDH recovery.

Positive effect of miR-2392 on fibroblast to cardiomyocyte-like cell fate transition: An in silico and in vitro study.

INTRODUCTION: Somatic cell fate transition is now gained great importance in tissue regeneration. Currently, research is focused on heart tissue regeneration by reprogramming diverse cells into cardiomyocyte-like cells. Here, we examined the possible effect of miRNAs on the transdifferentiation of fibroblasts into cardiomyocyte-like cells. METHODS: First heart-specific miRNAs were identified by comparing the gene expression profiles of heart tissue to other body tissues using bioinformatic techniques. After identifying heart-specific miRNAs, their cellular and molecular functions were studied using the miRWalk and miRBase databases. Then the candidate miRNA was cloned into a lentiviral vector. Following, human dermal fibroblasts were cultured and treated with compounds forskolin, valproic acid, and CHIR99021. After 24 h, the lentivector harboring miRNA gene was transfected into the cells to initiate the transdifferentiation process. Finally, after a two-week treatment period, the efficiency of transdifferentiation was examined by inspecting the appearance of the cells and measuring the expression levels of cardiac genes and proteins using RT-qPCR and immunocytochemistry techniques. RESULTS: Nine miRNAs were identified with higher expression in the heart. The miR-2392 was nominated as the candidate miRNA due to its function and specific expression in the heart. This miRNA has a direct connection with genes involved in cell growth and differentiation; e.g., MAPK and Wnt signaling pathways. According to in vitro results cardiac genes and proteins demonstrated an increase in expression in the fibroblasts that simultaneously received the three chemicals and miR-2392. CONCLUSION: Considering the ability of miR-2392 to induce the expression of cardiac genes and proteins in fibroblast cells, it can induce fibroblasts to differentiate into cardiomyocyte-like cells. Therefore, miR-2392 could be further optimized for cardiomyocyte regeneration, tissue repair, and drug design studies.

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.

Importance of paraoxonase 1 (PON1) as an antioxidant and antiatherogenic enzyme in the cardiovascular complications of type 2 diabetes: Genotypic and phenotypic evaluation.

Oxidant-antioxidant imbalance is involved in the etiology of different diseases, including cardiovascular diseases (CVDs), liver disorders, kidney diseases, cancers and diabetes mellitus. Antioxidant enzymes play a key role in striking an oxidant-antioxidant balance. Moreover, paraoxonase 1 (PON1) is an antioxidant enzyme that binds with high-density lipoprotein (HDL) in the circulation, and antioxidant and antiaterogenic properties of this lipoprotein are significantly associated with PON1. Research suggests PON1 contributes to the pathogenesis of certain human diseases such as type 2 diabetes (T2D). The association between PON1 and T2D appear to be reciprocal so that the disease significantly decreases PON1 levels and in turn, the genetics of PON1 may have a role the risk of susceptibility to T2D. Several factors that reduce the activity and concentration of PON1 in patients with T2D include increased glycation and loss-of-function polymorphisms. The genotypic and phenotypic evaluations of PON1 are therefore crucial for assessing the risk of cardiovascular complications in these patients, and strategies for increasing or restoring PON1 levels are useful for reducing or preventing their cardiovascular complications as their main cause of mortality. The present review aimed at discussing and emphasizing the key role of PON1 in T2D as a silent and dangerous disease.

Extremely low frequency-pulsed electromagnetic fields affect proangiogenic-related gene expression in retinal pigment epithelial cells.

OBJECTIVES: It is known that extremely low frequency-pulsed electromagnetic fields (ELF-PEMF) influence multiple cellular and molecular processes. Retinal pigment epithelial (RPE) cells have a significant part in the emergence and pathophysiology of several ocular disorders, such as neovascularization. This study assessed the impact of ELF-PEMF on the proangiogenic features of RPE cells. MATERIALS AND METHODS: Primary cultured RPE cells were treated with ELF-PEMF (50 Hz) for three days. Using ELISA assay, we evaluated the effects of treatment on RPE cell proliferation and apoptosis. Also, RT-PCR was used to determine the gene expression of proangiogenic factors, such as matrix metalloproteinase-2 (MMP-2), MMP-9, vascular endothelial growth factors receptor 2 (VEGFR-2), hypoxia-inducible factor 1 (HIF-1α), VEGFA, cathepsin D, connective tissue growth factor (CTGF), E2F3, tissue inhibitors of metalloproteinases 1 (TIMP-1), and TIMP-2. RESULTS: No noticeable changes were observed in cell proliferation and cell death of ELF-PEMF-exposed RPE cells, while transcript levels of proangiogenic genes (HIF-1α, VEGFA, VEGFR-2, CTGF, cathepsin D, TIMP-1, E2F3, MMP-2, and MMP-9) increased significantly. CONCLUSION: RPE cells are important for homeostasis of the retina. ELF-PEMF increased the gene expression of proangiogenic factors in RPE cells, which highlights concerns about the impact of this treatment on human health.

Production of a germline-humanized cetuximab scFv and evaluation of its activity in recognizing EGFR- overexpressing cancer cells.

The aim of this study was to produce a humanized single chain antibody (scFv) as a potential improved product design to target EGFR (Epidermal Growth Factor Receptor) overexpressing cancer cells. To this end, CDR loops of cetuximab (an FDA-approved anti-EGFR antibody) were grafted on framework regions derived from type 3 (VH3 and VL3 kappa) human germline sequences to obtain recombinant VH and VL domainslinked together with a flexible linker [(Gly4Ser)3] to form a scFv. Codon optimized synthetic gene encoding the scFv (with NH2-VH-linker-VL-COOH orientation) was expressed in E. coli Origami™ 2(DE3) cells and the resultant scFv purified by using Ni-NTA affinity chromatography. The scFv, called cet.Hum scFv, was evaluated in ELISA and immunoblot to determine whether it can recognize EGFR. The scFv was able to recognize EGFR over-expressing cancer cells (A-431) but failed to detect cancer cells with low levels of EGFR (MCF-7 cells). Although the affinity of the scFv forA-431 cells was 9 fold lower than that of cetuximab, it was strong enough to recognize these cells. Considering its ability to bind EGFR molecules, the scFv may exhibit a potential application for the detection of EGFR-overexpressing cancer cells.

Paraoxonase 1 (PON1)-L55M among common variants in the coding region of the paraoxonase gene family may contribute to the glycemic control in type 2 diabetes.

OBJECTIVE: Genome studies have shown that the genes encoding paraoxonase 1 (PON1) and PON2 are associated with glucose metabolism. The goal of this study was to simultaneously evaluate the association between functional variants in PON1 and PON2 genes and susceptibility for type 2 diabetes (T2D) and determine whether they can affect glycemic control. METHODS: We performed a case-control study with 145 newly diagnosed patients with T2D and 148 controls. The common variants including PON1-Q192R, PON1-L55M and PON2-S311C were genotyped by PCR-based RFLP. A mismatch-PCR/RFLP was applied for genotyping the PON2-A148G variant. RESULTS: The variant PON1-Q192R in males (OR = 2.55, 95%CI 1.16-5.69, p = 0.023) and PON2-A148G in females (OR = 1.56, 95%CI 1.00-2.44, p = 0.059) were associated with T2D. Compared with the LL genotypes of PON1-L55M, HbA1c levels were significantly lower in the LM genotypes (p = 0.01) and MM genotypes (p = 0.032) in patients. Multiple linear regression analyses showed that among the study variants only the PON1-L55M variant as an independent variable significantly associated with glycemic control. This variant significantly influenced glycemic control in patients with poor glycemic control so that it was better with the following order: LL < LM < MM. Based on gamma correlation, there was a significant inverse association between the number of M alleles of the PON1-L55M and HbA1c levels (r = -0.261, p = 0.001). CONCLUSIONS: Sex should be considered a confounding variable in association studies on the variants PON1-Q192R and PON2-A148G in T2D. Patients sharing the 55 M allele were prone to having good glycemic control. Our findings provide genetic evidence that the PON1-L55M variant may be a factor contributing to glycemic control.

Recombinant expression, characterization and application of a dihydrolipoamide dehydrogenase with diaphorase activity from Bacillus sphaericus.

Diaphorases are flavin-containing enzymes with potential applications in biotransfomation reactions, biosensor design and in vitro diagnostic tests. In this communication, we describe recombinant expression, characterization and application of a lipoamide dehydrogenase (DLD) with diaphorase activity from a strain of Bacillus sphaericus. The DLD gene consisting of 1413 bp encoding a protein of 470 amino acids was expressed in Escherichia coli BL21 (DE3) and the recombinant enzyme was characterized. B. sphaericus DLD catalyzed the reduction of NAD+ by dihydrolipoamide and exhibited NADH-dependent diaphorase activity. The molecular weight of purified enzyme was about 50 kDa, and determined to be a monomeric protein. Diaphorase was active and stable from pH 7.0 to 9.0 with an optimal activity at pH 8.5. It showed its maximal activity at temperature of 30 °C and was almost stable at temperatures between 25 and 30 °C. Different metal ions and inhibitors showed no influence on the activity of target enzyme. The K m and V max values for NADH were estimated to be 0.33 mM and 200.0 U/ml, respectively. Moreover, recombinant B. sphaericus diaphorase exhibited considerable potential to be used as a component of diagnostic tests for the quantification of metabolites. In conclusion, considering the properties of diaphorase from B. sphaericus PAD-91, it can have potential application as a diagnostic enzyme.

The Role of Metformin Response in Lipid Metabolism in Patients with Recent-Onset Type 2 Diabetes: HbA1c Level as a Criterion for Designating Patients as Responders or Nonresponders to Metformin.

BACKGROUND: In this study, we investigated whether response to metformin, the most frequently drug for diabetes treatment, influences the therapeutic effects of antilipidemic medication in newly diagnosed patients with type 2 diabetes mellitus (T2DM). METHODS: A total of 150 patients with T2DM were classified into two groups following 3 months of metformin therapy (1000 mg twice daily): responders (patients showing ≥1% reduction in HbA1c from baseline) and nonresponders (patients showing <1% reduction in HbA1c from baseline). The patients received atorvastatin 20 mg, gemfibrozil 300 mg, or atorvastatin 20 mg and gemfibrozil 300 mg daily. PRINCIPAL FINDINGS: HbA1c and fasting glucose levels were significantly different between baseline and 3 months among responders receiving atorvastatin; however, these differences were not statistically significant in nonresponders. Atherogenic ratios of low-density lipoprotein cholesterol to high-density lipoprotein cholesterol (LDL-C/HDL-C; p = 0.002), total cholesterol to HDL-C (TC/HDL-C; p<0.001) and AIP (the atherogenic index of plasma; p = 0.004) decreased significantly in responders receiving atorvastatin than in nonresponders. Moreover, responders receiving atorvastatin showed a significant increase in HDL-C levels but nonresponders receiving atorvastatin did not (p = 0.007). The multivariate model identified a significant association between metformin response (as the independent variable) and TG, TC, HDL-C and LDL-C (dependent variables; Wilk's λ = 0.927, p = 0.036). CONCLUSIONS: Metformin response affects therapeutic outcomes of atorvastatin on atherogenic lipid markers in patients newly diagnosed with T2DM. Metformin has a greater impact on BMI in responders of metformin compared to nonresponders. Adoption of better therapeutic strategies for reducing atherogenic lipid markers may be necessary for metformin nonresponders.

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.

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.

Synthesis, characterization and immunological properties of Escherichia coli 0157:H7 lipopolysaccharide- diphtheria toxoid conjugate vaccine.

BACKGROUND AND OBJECTIVE: Escherichia coli O157:H7, an emerging pathogen, causes severe enteritis and the extraintestinal complication of hemolytic-uremic syndrome. The goal of this study was to evaluate the conjugate of E. coli O157: H7 lipopolysaccharide (LPS) with diphtheria toxoid (DT) as a candidate vaccine in mice model. MATERIAL AND METHODS: LPS from E. coli O157:H7 was extracted by hot phenol method and then detoxified. Purified LPS was coupled to DT with adipic acid dihydrazide (ADH) as a spacer and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) as a linker. The coupling molar ratio of LPS to DT was 3:1. Clinical evaluation of E. coli O157:H7 LPS-DT conjugate was also performed. RESULTS: The conjugate was devoid of endotoxin activity and indicated 0.125 U/ml of D-LPS. Mice immunization with D-LPS DT conjugate elicited fourfold higher IgG antibody in comparison to D-LPS. Also, in vivo protection of mice with conjugate provided high protection against the LD50 of E. coli O157:H7, which indicated a good correlation with the IgG titer. CONCLUSION: Our results showed that the suggested vaccine composed of E. coli O157:H7 LPS and DT had a significant potential to protect against E. coli infections.