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. © 2015 Elsevier Ltd. All rights reserved

Positive effect of Periostin on repair of Isopreternol induced ischemic damaged cardiomyocyte: an in vitro model

Introduction: One of the diseases in developed counties is myocardial infarction that causes the death of many people. A Problem of many new drugs usage concerns their assessment for applying therapeutically for heart disease. Previous studies used animal models but today many researchers tend to apply cellular models for feasibility of cellular model production and application. Methods: For this purpose, we differentiated human bone marrow mesenchymal stem cells (hBM-MDCs) into cardiomyocyte, then induced damage into cells by Isoproterenol, and finally we assayed repair of cardiomyocyte by Periostin. Damage induction and repair were confirmed by measurement of selected markers for cardiac damage and repair at mRNA level. In addition, we measured LDH activity in culture medium during damage and repair processes. Results: Our results showed LDHa and b mRNA levels increased and also cardiac markers decreased during damage of cardiomyocytes significantly. Reciprocally LDH isozymes decreased and cardiac markers increased during repair of cardiomyocytes. Discussion/conclusion: These alterations in cardiac markers after Periostin treatment demonstrate that Periostin is an effective factor on repair of cardiomyocytes. LDH activity in culture medium decreases after damage induction and increases during repair process. According to our data, Isoproterenol and Periostin are good inducer to produce damaged and repaired differentiated cardiomyocytes respectively. © 2022 The Japanese Society for Regenerative Medicin

An Overview of Chemical and Biological Materials lead to Damage and Repair of Heart Tissue

Background: Cardiovascular diseases (CVDs) are major causes of mortality in developing countries. One of the challenges during CVDs studies is the creation of a damaged model of the heart. Many injured models of cardiac diseases are created by using chemical and biological materials. Many approaches were applied to simulate heart injury for investigating CVDs. In previous years, animal models could be used as a useful pattern in many investigations about the pathogenesis of the heart. Nowadays it has been proven that there are many differences between human and animal models in terms of responses or reactions to treatments. For such reasons, researchers prefer to use cellular models alongside the animal models for studying heart diseases. Purpose: In this review, we collected information about some chemical and biological materials used to create damaged-heart models both in vitro and in vivo. After explaining the materials that induce cardiac damage, we explicate some methods for repairing the damage of heart. Finally, the role of extracellular vesicles as an important biological candidate for repairing heart damage is briefly discussed. Conclusion: This mini-review tried to explain some methods which can induce cardiac damage and repair of heart cells by use chemical and biological materials. We considered that various molecular pathways play a role in restoration and that most of these pathways are connected in a complex network and, to this end, different chemicals and drugs have been studied to date. Nonetheless, more studies are needed to ensure the performance and safety of the drugs and chemicals produced. © 2021, Biomedical Engineering Society

Association between promoter polymorphism (− 108C > T) of paraoxonase1 gene and it's paraoxonase activity in patients with Type2 diabetes in northern Iran

Type 2 diabetes mellitus (T2DM) is one of the diseases which depend on the obesity associated with insulin resistance. Various factors, such as a reduction in the activity of paraoxonase-1 enzyme (PON1), could affect T2DM. PON1 is a multifunctional enzyme with paraoxonase and arylesterase activities. The Activity of PON1 is influenced by various SNPs. The aim of presence study is to investigate the association between promoter polymorphism (− 108C > T) of PON1 gene and its paraoxonase activity in patients suffering from Type 2 diabetes in Golestan Province, north of Iran. To this end, genomic DNA was extracted from whole blood then genotyping was carried out using PCR-RFLP and enzymatic activity of paraoxonase was measured in the serum of 90 healthy subjects and 90 diabetic patients. Data was analyzed using SPSS version 16. The relationship between the level of paraoxonase activity with polymorphisms of CC, CT, and TT was statistically significant in patients with T2DM. There was a significant association between polymorphism C-108 > T of PON1 and type2 diabetes mellitus, with 24.4% of control group subjects and 15.5% of patients having CC genotype; p < 0.05. The ratio for CT genotype in target and control groups was 51.1% and 61.1% respectively; p < 0.05. TT genotype was 33.3% in patients and 14.4% in the control group; p < 0.05. In the present study, the highest frequency belonged to CT genotype in both the control and the target groups, followed by CC genotype in control group and TT genotype in target group. Our findings revealed that paraoxonase activity of PON1 in the control group was significantly higher in comparison with diabetic patients. © 2017 Elsevier B.V

Polymorphism of secretary PLA2G2A gene associated with its serum level in type2 diabetes mellitus patients in Northern Iran

Background: Inflammation may occur in Type2 diabetes mellitus. sPLA2 is among the factors that contribute to the activation of pathways involved in inflammation. Several agents affect serum sPLA2 level, one of which is genetic diversity. Objective: The current study was performed to determine whether there is a relationship between sPLA2 gene (�763C &gt; G) polymorphism and circulating sPLA2 level in patients with Type 2 diabetes. Methods: DNA was extracted from blood samples and used for the amplification of sPLA2 gene using ARMS-PCR. Results: A statistical analysis using SPSS (version 16) revealed a significant correlation between �763C &gt; G sPLA2 gene polymorphisms and the disease incidence in patients with T2DM. Among the three possible genotypes (GG, CC, and CG), CG genotype was found to have a higher frequency(53) in T2DM patients. GG and CC genotypes frequencies were 20 and 27, respectively. In healthy individuals, the frequencies of CC, GG, and GC genotypes were 77, 9.8 and 13.2, respectively). Patients with genotype GG had the highest level of sPLA2. We showed that C&gt;G polymorphism at position� 763 is associated with a high level of sPLA2 in both T2DM patients and healthy individuals. The average of sPLA2 circulating level was (170.4

Evaluation of Relationship Between Arylesterase-Based Activity and Genetic Variants of Paraoxonase1 in T2DM Patients within Golestan Province

Arylesterase activity of Paraoxonase-1 (PON1) enzyme may be play important role in initiation and progression of several diseases. Activity or serum level of Arylesterase can be affected by many genetic alterations such as SNPs. The reduction in the activity and serum level of Arylesterase could be involved in Type2 diabetes mellitus (T2DM). The aim of this investigation is to examine the association between Arylesterase activity and promoter polymorphism (� 108C > T) of PON1gene in patients with T2DM in Golestan Province, northern area of Iran. Achievement of this purpose was due to DNA obtaining from blood then SNP determination using PCR�RFLP and Arylesterase activity measurement in the serum of 90 normal individuals and 90patients suffering diabetes. Data was processed by SPSS software version 16. The significant association was observed between the Arylesterase activity and three genotypes of PON1 gene such as CC, CT, and TT in subjects with T2DM. In the present study, it has been shown level of Arylestrase activity of PON1 in patients (117.33 ± 63.96) is lower than it in control group (289.33 ± 68.38); P < 0.05. Our results declared that activity of Arylesterase in diabetic patients was significantly lower than the healthy individuals. © 2019, Association of Clinical Biochemists of India

Genetic polymorphisms of cytochrome p450 (2C9) enzyme in patients with type 2 diabetes mellitus in turkmen and fars ethnic groups

Background: Cytochrome P450 2C9 (CYP450 2C9) has an important role in metabolic processes. Mutations in CYP450 2C9 genes may affect the catalytic activity of this enzyme. The aim of the present study is to assess the genetic polymorphisms of Cytochrome P450 (2C9) enzyme in Turkmen and Fars ethnic groups with type 2 diabetes compared with controls. Methods: A total of 336 Turkmen and 336 Fars type 2 diabetic patients and 336 healthy Turkmen and Fars individuals were included in this study. Genomic DNA was extracted from whole blood samples and then the CYP2C9 genotyping was done using the Polymerase Chain Reaction-Restriction Fragment Length Polymorphism technique. Results: The CYP2C9*1, CYP2C9*2 and CYP2C9*3 allele frequencies in type 2 diabetic patients were 85.27, 11.68, and 3.05, and in control were 87.13, 8.56, and 4.31, respectively. We found significant differences between allele distribution of 2C9 in type 2 diabetic patients and controls. CYP2C9*2 and CYP2C9*3 allele frequency was significantly different in Turkmen and Fars type 2 diabetic compared to two ethnic controls. The CYP2C9*1/*1 and CYP2C9*1/*2 genotypes frequencies in type 2 diabetic Turkmen showed significant differences compared to Turkmen control. There were significant differences in the genotype frequency of CYP2C9*1/*1, CYP2C9*1/*2, CYP2C9*1/*3, CYP2C9*2/*2, and CYP2C9*2/*3 between type 2 diabetic Fars and Fars controls. Two diabetic ethnic groups showed statistically significant differences in frequencies of CYP2C9*2/*2 and CYP2C9*2/*3 genotypes. Conclusion: Our study suggests that diabetic patients with mutant CYP2C9 polymorphism may show different antidiabetic drug metabolism compared to the wild-type allele. In this regard, determination of CYP2C9 alleles and genotypes can be a useful tool for the treatment of diabetic patients with antidiabetic drugs because it may assist physicians’ to determine optimal dosage and efficiency of drugs metabolized by this polymorphic enzyme. © 2018 Bentham Science Publishers

Alzheimer’s disease and paraoxonase 1 (PON1) gene polymorphisms

Background: Some studies have indicated that human paraoxonase 1 (PON1) activity shows a polymorphic distribution. The aim of this study was to determine the distribution of PON1 polymorphism in patients with Alzheimer’s disease in Gorgan and compare it with a healthy control group. Method: The study included 100 healthy individuals and 50 patients. Enzyme activity and genetic polymorphism of PON1 were determined. Result: There were significant differences in distribution of genotypes and alleles among patients and control group. The most common genotype was CT in patients and control group, while the most frequent alleles were T and C in patients and controls, respectively. There was a statistically significant variation between serum PON1 activity and –108C> T polymorphism. The highest PON1 enzyme activities in the patients and controls were found in CC, while lower enzyme activities were seen in CT and TT genotypes in both genders and age groups. Conclusion: Onset of Alzheimer’s disease may depend on different polymorphisms of the PON1 enzyme. Late or early-onset of Alzheimer’s disease may also depend on age and gender distribution, especially for arylesterase enzyme. Further studies on polymorphism of the enzyme are necessary for interpretation of possible polymorphic effects of enzyme on PON1 activity in humans. © 2017 Saeidi et al

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

Interleukin-1β (IL-1β) 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-1β 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-1β (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-1β and 5-aza synergistically induces the cardiomyocyte differentiation. © 2016 Walter de Gruyter GmbH, Berlin/Boston

MicroRNA-146a as a prognostic biomarker for esophageal squamous cell carcinoma

Background and Aims: MicroRNAs including miR146a have a regulatory role on the expression of genes and act with binding to 3ʹ-UTR region of the genes. Cyclooxygenase-2 (COX-2) is involved in carcinogenesis as an inflammatory marker, and microRNA-146a (miR-146a) as a negative regulatory factor. We aimed to evaluate miR146a expression as a prognostic or diagnostic biomarker for esophageal squamous cell carcinoma (ESCC) and also an association between miR146a and COX2 expression. Materials and Methods: We quantified the level of miR-146a and COX-2 expression in cancerous and adjacent normal tissue samples obtained from 34 patients with ESCC, using real-time�PCR. Statistical analyses were conducted using one-sample t-test. Receiver-operating characteristic (ROC) curve and Kaplan�Meier analysis were applied to assay miR146a as a diagnostic and prognostic marker, respectively, during 4 years of the study. Furthermore, the Cox regression model was performed to assay the hazard ratio (HR). The association between miR-146a and COX2 expression level in ESCC patients was evaluated by nonparametric Spearman�s rho analysis. Results: The results revealed a reduction of miR-146a expression in 50 of cancerous tissue when compared with adjacent normal regions (P-value=0.127). COX-2 expression in 80 of ESCC patients was higher than in the controls (P-value=0.001). Overall, in 60 of cases, direct association was seen between microRNA-146a and COX-2 expression level (correlation coefficient= 0.438, P-value=0.011). COX2 can be considered as a diagnostic biomarker (AUC=0.834, sensitivity=72, specificity =83, P-value<0.0001) but miR146a cannot be considered as a diagnostic biomarker (AUC=0.553, sensitivity=88, specificity =28, P-value=0.453). Survival analysis by Kaplan�Meier method showed miR146a and COX2 expression can be probably considered as prognostic biomarkers for ESCC because patients with high expression of miR146a had 7 months shorter life span and patients with low expression of COX2 had 8 months shorter life span. Conclusion: COX2 expression is a diagnostic biomarker. MiR-146a and COX2 expression can probably be considered as prognostic biomarkers for survival in ESCC. © 2020 Sadegh Shesh Poli et al