The Oxidative Status and Na+/K+-ATPase Activity in Obsessive-Compulsive Disorder: A Case Control Study.

Background: Oxidative stress is involved in pathogenesis of some psychiatric disorders. To examine the role of oxidative stress in the etiopathogenesis of obsessive-compulsive disorder (OCD), we aimed to determine oxidative stress indices, including malondialdehyde (MDA) levels in serum and red blood cells (RBC) membrane, total antioxidant capacity (TAC), serum glutathione (GSH) levels, serum antioxidant vitamins (A and E), and Na+/K+-ATPase activity, in patients with the mentioned disorder vs. healthy controls. Method: 39 OCD patients diagnosed based on Diagnostic and Statistical Manual of Mental Disorders (DSM-V) and 39 volunteer healthy subjects were included in this study. MDA levels in serum and RBC membrane were measured using fluorometric method. Serum TAC level, serum GSH level, and Na+/K+-ATPase activity were also measured using spectrophotometric methods. Serum levels of vitamins were calculated by reversed-phase high-performance liquid chromatography (RP-HPLC). Result: There was a significantly higher MDA level in serum (p < 0.0001) and RBC membrane (p = 0.002) of OCD patients compared with those in controls. A significant reduction in vitamin A (p = 0.001) and vitamin E (p = 0.024) levels was found in OCD patients vs. controls. There was significantly lower activity of erythrocyte membrane Na+/K+-ATPase in RBC membrane of OCD patients vs. controls (p < 0.0001). Conclusion: Our findings indicate significantly higher levels MDA in both serum and RBC membrane, lower levels of serum vitamins A and E, and lower activity of membrane Na+/K+-ATPase in OCD patients compared to controls. These suggest an imbalance between oxidant and antioxidant factors in OCD patients that might play a fundamental role in the etiopathogenesis of OCD.

Liver disorders caused by inborn errors of metabolism.

Inborn errors of metabolism (IEMs) are a vast array of inherited/congenital disorders, affecting a wide variety of metabolic pathways and/or biochemical processes inside the cells. Although IEMs are usually rare, they can be represented as serious health problems. During the neonatal period, these inherited defects can give rise to almost all key signs of liver malfunction, including jaundice, coagulopathy, hepato- and splenomegaly, ascites, etc. Since the liver is a vital organ with multiple synthetic, metabolic, and excretory functions, IEM-related hepatic dysfunction could seriously be considered life-threatening. In this context, the identification of those hepatic manifestations and their associated characteristics may promote the differential diagnosis of IEMs immediately after birth, making therapeutic strategies more successful in preventing the occurrence of subsequent events. Among all possible liver defects caused by IEMs, cholestatic jaundice, hepatosplenomegaly, and liver failure have been shown to be manifested more frequently. Therefore, the current study aims to review substantial IEMs that mostly result in the aforementioned hepatic disorders, relying on clinical principles, especially through the first years of life. In this article, a group of uncommon hepatic manifestations linked to IEMs is also discussed briefly.

Effect of pretreatment with a synbiotic on Perfluorooctanoic acid-induced liver damage after sub-acute oral exposure in C57BL/6J mice.

BACKGROUND: Perfluorooctanoic acid (PFOA(is used in several industrial applications, and serves as a surfactant. It is persistent in the environment and is resistant to typical environmental degradation processes. Exposure to this contaminant has been shown to reduce the normal gastrointestinal flora, especially Lactobacillus and Bifidobacterium. Since exposure to this contaminant still occurs and it has been suggested that gut microbiota imbalance might accelerate the progression of liver disorders, we aimed to study the effect of synbiotics pretreatment on PFOA-induced hepatotoxicity. METHOD AND MATERIALS: Herein, C57BL/6 J mice were administered 1, 5, 10, and 20 mg PFOA per kg body weight orally by gavage once daily up to 28 days. Another group was pretreated with synbiotic 4 h before receiving 10 mg PFOA/kg. Also, a control group received 2% Tween 80 orally as a vehicle of PFOA during the study. Plasma ALT, AST, TNF-α, HGF, IL-6, and IFN-γ were measured every week. In addition, a liver histopathological assessment was performed at the end of exposure studies. RESULTS: It was observed that exposure to PFOA can trigger inflammatory markers such as TNF-α, HGF, IL-6, and IFN-γ as well as hepatic enzymes AST and ALT in comparison with the control group. Synbiotic pretreatment prevented or statistically significant reduced the release of the inflammatory markers and the liver enzymes compared to PFOA only treated group. CONCLUSION: It could be inferred that having intact gut flora or even using synbiotic complements containing Lactobacillus, Bifidobacterium, and Streptococcus plus fructooligosaccharides as prebiotic is an appropriate strategy to reduce the negative effects of PFOA exposure.

Evaluation of the gene encoding carnitine transporter (OCTN2/SLC22A5) expression in human breast cancer and its association with clinicopathological characteristics.

BACKGROUND: Fatty acid oxidation (FAO) is a major energy-generating process in the mitochondria and supports proliferation, growth, and survival of cancer cells. L-Carnitine is an essential co-factor for carrying long-chain fatty acids into the mitochondria. The entry of l-carnitine across cell membrane is regulated by OCTN2 (SLC22A5). Thus, it can plays a significant role in the mitochondrial fatty acid oxidation. This study aimed to evaluate the OCTN2 expression and its association with clinicopathological characteristics in breast cancer. METHODS: In this work, OCTN2 was examined in 54 pairs of fresh samples of breast cancer (BC) and adjacent noncancerous tissue using quantitative real-time polymerase chain reaction and immunohistochemistry (IHC). The IHC approach was also used to investigate the expression of additional clinicopathological features. RESULTS: The present research findings revealed that the relative expression of OCTN2 in BC tissues was substantially higher than the adjacent normal tissues. This up-regulation was correlated positively with tumor size and Ki-67 and negatively with the progesterone receptor (PR) status, providing evidence of the opposite effects of OCTN2 and PR on tumor development. CONCLUSION: The study shows that the OCTN2 expression in BC patients may be used as a prognostic biomarker and a tumor oncogene. As a result, it could be considered a possible therapeutic target. Nevertheless, the significance of the findings needs to be confirmed by further studies.

Investigation of the relationship between miR-33a, miR-122, erythrocyte membrane fatty acids profile, and serum lipids with components of metabolic syndrome in type 2 diabetic patients.

Background and purpose: MicroRNAs (miRNAs) are small non-coding RNA molecules acting as critical regulators of post-transcriptional gene expression. MiR-33a and miR-122 have a crucial role in cholesterol and lipid metabolism. Therefore, their dysregulation may contribute to metabolic abnormality and their inhibition may be a useful therapeutic strategy. The objective of the present study was to investigate the relationship between miR-33a, miR-122, erythrocyte membrane fatty acids profile, and serum lipids with components of metabolic syndrome in an Iranian population suffering from type 2 diabetes mellitus (T2DM). Experimental approach: Expression of miR-33a and miR-122 was measured by real-time polymerase chain reaction and erythrocyte membrane fatty acid profiles were analyzed by gas chromatography-mass spectrometry. Findings/Results: T2DM patients with and without metabolic syndrome had significantly higher miR-33a and miR-122 levels compared to controls. MiRNAs were significantly correlated with saturated fatty acid (SFAs), total SFAs/total polyunsaturated fatty acids (PUFAs) ratio, fasting plasma glucose, triacylglycerols, insulin and homeostatic model assessment of insulin resistance. In addition, there was a significant negative correlation between miR-33a and miR-122 levels and PUFAs, total PUFAs/total SFAs ratio and omega 6 fatty acids. Conclusion and implications: Considering the roles of miR-33a and miR-122 in cholesterol and lipids metabolism, it may be concluded that the measurement of their expression may be useful as a potential additional biomarker for cardiometabolic derangement in T2DM patients. In addition, these findings may suggest that the inhibition of these miRNAs by anti-miRNA therapies may be explored as a potential therapeutic strategy.

Effect of Selenium Supplementation on Lipid Profile, Anemia, and Inflammation Indices in Hemodialysis Patients.

Objective: Trace elements deficiency is common among end-stage renal disease (ESRD) patients due to excessive loss during dialysis and the lower intake secondary to loss of appetite. Selenium (Se) is a trace element that plays an important role in the radical scavenging system and helps the body defend against oxidative stress. This study aims to evaluate the effects of Se supplementation on lipid profile, anemia, and inflammation indices in ESRD patients. Methods: Fifty-nine hemodialysis patients enrolled and were randomly divided into two groups. Two hundred microgram Se capsules once daily for the case group and matching placebo for the control group were administered for three months. Demographic data were collected at the study beginning. Uric acid (UA), anemia and inflammation indices, and lipid profiles were recorded at the beginning and the end of the study. Findings: UA and UA-to-HDL (high-density lipoprotein) ratio decreased significantly in the case group (P < 0.001). The changes in lipid profile were not significant among both groups. Hemoglobin slightly increased in the case group, however, it decreased significantly in the control group (P = 0.031). High-sensitivity C-reactive protein (hs-CRP) decreased in the case group and increased in the control group, however, none of these changes were significant. Conclusion: According to the results of this study, selenium supplementation in ESRD patients could reduce some risk factors related to their mortality, such as the ratio of uric acid to HDL. However, the changes related to lipid profile, hemoglobin level and hs-CRP biomarker were not significant.

Effect of Omega-3 Fatty Acids Supplementation on Homocysteine Level in Patients Undergoing Continuous Ambulatory Peritoneal Dialysis.

Objective: One of the most common diseases with high morbidity and mortality rates is chronic kidney disease. Cardiovascular disease affects most patients with chronic kidney disorders, particularly patients undergoing dialysis; hence, appropriate prevention and management approaches are essential. This study aimed to evaluate the reduction of inflammatory biomarkers, especially homocysteine, by omega-3 fatty acids in peritoneal dialysis patients. Methods: This study enrolled 60 peritoneal dialysis patients who met specified inclusion and exclusion criteria and were randomized to intervention or placebo groups. Omega-3 capsules were given at a dose of 3 g/d for 8 weeks. Inflammatory markers, including high-sensitivity C-reactive protein (hs-CRP), homocysteine, albumin, and lipid profile measured before and after the study. Findings: Results of this trial revealed that the levels of homocysteine, hs-CRP, and albumin did not change significantly during the study. Analysis of lipid profiles before and after intervention showed omega-3 has no significant effect on the level of total cholesterol or low-density lipoprotein cholesterol; However, the level of triglyceride reduced remarkably (P = 0.002). In addition, serum levels of high-density lipoprotein cholesterol increased at the end of the study (P < 0.001). Conclusion: Omega-3 does not seem to be able to change the inflammatory markers significantly, particularly homocysteine. More extensive trials must be conducted to better understand the impact of omega-3 on inflammatory and nutritional markers, particularly in peritoneal dialysis patients.

Determination of plasma and erythrocyte levels of copper, magnesium and zinc by atomic absorption spectrometry in type-2 diabetes mellitus patients with metabolic syndrome.

BACKGROUND AND PURPOSE: Imbalance in blood levels of trace elements is independent risk factor for metabolic syndrome (MetS), type 2 diabetes mellitus (T2DM), and its complications. This study investigated plasma and erythrocyte levels of copper, magnesium, zinc, and their correlations with biochemical components of the MetS in T2DM patients compared to the healthy controls. EXPERIMENTAL APPROACH: Forty men recently diagnosed T2DM with MetS without complications and thirty six age-matched healthy controls were enrolled in this cross-sectional study. Plasma and erythrocyte levels of selected elements were measured by graphite furnace atomic absorption spectroscopy. FINDINGS/RESULTS: The results of the present study showed significantly lower plasma levels of copper, magnesium, and zinc and lower erythrocytes copper in the patients' group compared to the controls; while erythrocyte levels of magnesium and zinc were not significantly different between the two groups. Significant negative correlations were observed between plasma levels of copper with waist and hip circumferences, waist to hip ratio, systolic and diastolic blood pressures, fasting blood glucose, and glycated hemoglobin levels in all subjects; while erythrocyte copper levels showed significant negative correlation with triglyceride, and erythrocyte zinc was positively correlated with diastolic blood pressure and negatively with triglyceride. CONCLUSION AND IMPLICATIONS: Alterations of trace elements may have a significant role in the pathogenesis of MetS and T2DM patients. It is suggested that the body status of copper, magnesium, and zinc might be significantly correlated with components of MetS in T2DM patients; and plasma copper levels may be correlated with complications of type 2 diabetes mellitus.

Evaluation of the Effectiveness of Cinnamon Oil Soft Capsule in Patients with Functional Dyspepsia: A Randomized Double-Blind Placebo-Controlled Clinical Trial.

BACKGROUND: Different effects of cinnamon and its oil in traditional medicine in the treatment of diseases, including gastrointestinal diseases, were reported. The aim of this study is to evaluate the efficacy and safety of cinnamon oil (Cinnamomum zeylanicum) in patients with functional dyspepsia in a double-blind, randomized placebo-controlled trial. METHODS: Soft gelatin capsule was made using the rotary die process, and the final capsule was standardized based on its cinnamaldehyde amount and analyzed by high-performance liquid chromatography (HPLC) method. Sixty-four patients with symptomatic functional dyspepsia were randomized to receive cinnamon oil soft capsule (n = 29) or sesame oil soft capsule as placebo (n = 35) for 6 weeks. The primary efficacy variable was the sum score of the patient's gastrointestinal symptom (five-point scale). Secondary variables were the scores of each dyspeptic symptom including severity of vomiting, sickness, nausea, bloating, abdominal cramps, early satiety, acidic eructation/heartburn, loss of appetite, retrosternal discomfort, and epigastric pain/upper abdominal pain, as well as any reported adverse events. RESULTS: The results showed that, after 6 weeks of treatment, the cinnamon oil and placebo groups significantly decreased the total dyspepsia score compared to the baseline at the endpoint (P < 0.001). However, there was no significant difference between the cinnamon oil and placebo groups in terms of the baseline and endpoint values of the outcome variables (P=0.317 and P=0.174, respectively). Two patients in the cinnamon oil group complained of rashes, and three patients in the placebo group complained of nausea. CONCLUSION: This study showed significant improvements in gastrointestinal symptom score in both treatment and placebo groups. However, there was no significant difference between the cinnamon oil and sesame oil groups in terms of the baseline and endpoint values of the outcome variables. This study was registered as https://clinicaltrials.gov/ct2/show/IRCT20170802035460N2, 29 December 2017, in the Iranian Registry of Clinical Trials with https://www.IRCT.ir.

Oxidant/antioxidant status in Type-2 diabetes mellitus patients with metabolic syndrome.

BACKGROUND: The concurrence of metabolic syndrome (MS) and diabetes mellitus (DM) is increasing worldwide. The long-term complications of these chronic diseases are a threat to patients' well-being. Oxidative stress is involved in the pathogenesis of several diseases. To understand the basic pathophysiological mechanisms of Type-2 DM (T2DM) and its related complications, we aimed to investigate the oxidant/antioxidant status and Na+-K+ ATPase activity in T2DM with MS. MATERIALS AND METHODS: A population of ninety individuals including fifty patients diagnosed with T2DM and MS, but without overt diabetes complications, and forty individuals without T2DM or MS as control group participated in this study. Plasma malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) activities, total antioxidant capacity (TAC), and Na+-K+ ATPase activity were assessed by standard laboratory methods. RESULTS: Plasma MDA in patients group was statistically significantly higher than that of controls (P ≤ 0.05). Whereas, Na+-K+ ATPase activity was statistically significantly lower in patient group (P ≤ 0.05). TAC, CAT, SOD, and GPx enzyme activities were not statistically significantly different between two groups (P > 0.05). Results from the patient group showed positive correlations between CAT activity and triglyceride and positive correlations between GPx activity and weight, body mass index (BMI), and waist circumference. In addition, there was a positive correlation between MDA results with high-density lipoprotein-cholesterol (HDL-C) and total cholesterol and a negative correlation with TAC, BMI, and weight (P ≤ 0.05) in controls. CONCLUSION: Because T2DM patients were without any vascular complications, antioxidant defense results may reflect the lack of progression of diabetes complications in these patients. These results emphasize the need for initial and continued assessment of cardiovascular disease risks in diabetic individuals. Implementation of timely interventions may improve the management of diabetes and prevent the progression of diabetes complications.

Association of the serum apelin, but not ghrelin, with the presence and severity of coronary artery disease.

Increasing evidence suggests that apelin and ghrelin may participate in atherogenesis. We sought to investigate whether the serum levels of apelin and ghrelin are significantly different in patients with coronary artery disease (CAD) compared to patients with nonsignificant coronary stenosis and determine the correlation between these adipokines and the severity of coronary stenosis. The study population included 31 stable CAD patients, 38 unstable CAD patients, and 39 non-CAD subjects. Serum levels of apelin and ghrelin, fasting blood glucose, lipid parameters, hs-CRP and hematological indices were determined in all groups using routine standard laboratory procedures. Serum apelin levels were significantly lower in patient with unstable CAD (0.354 ± 0.063 ng/mL) compared to stable CAD patients (0.401 ± 0.045 ng/mL, p = 0.003) and non-CAD subjects (0.415 ± 0.055 ng/mL, p<0.001). In addition, serum apelin levels were inversely correlated with the severity of coronary stenosis in CAD patients (p<0.05). However, there was no significant difference in ghrelin levels among the 3 groups. This data may suggest that the presence of unstable CAD may be associated with lower serum apelin which may indicate the potential role of this peptide in the progression and destabilization of coronary plaques.

Homocysteine-Lowering Interventions in Chronic Kidney Disease.

The incidence of cardiovascular events and mortality is higher in patients with chronic kidney disease (CKD) compared to the general population. Homocysteine (Hcy) appears to be an independent risk factor for cardiovascular diseases in general populations and patients with CKD. Further, hyperhomocysteinemia can cause endothelial damage and increase the activity and production of coagulation factors, and its prevalence among patients with end-stage renal disease is approximately 85%-100%. Most treatments, which lower Hcy levels and have been considered in previous studies, include folic acid, B vitamins, omega-3 fatty acids, and N-acetylcysteine. However, the effect of therapies that can decrease Hcy levels and thus cardiovascular events in these patients is still unclear. The results are conflicting and require further investigation. To guide treatment decisions and improve patient outcomes, multiple databases were searched, including Web of Science, PubMed, and Medline to summarize the available evidence (i.e., clinical trial and meta-analyses) on Hcy-lowering interventions and cardiovascular events.

Potential Benefits of Selenium Supplementation in Patients with Kidney Disease.

Trace element deficiency is common among patients with end-stage renal disease (ESRD); the reason is that since these patients undergo dialysis, they lose these elements more than healthy people, and also the use of trace elements is restricted due to loss of appetite. Selenium (Se) is a trace element that is essential for the oxidative stress defense system. Se deficiency leads to some complications similar to those often seen in ESRD patients, such as all-cause mortality due to cardiovascular diseases, bone loss, uric acid elevation, and anemia. This article aims to review the evidence on consequences of Se deficiency in ESRD patients, as well as effects of Se supplementation in hemodialysis patients. Multiple databases were searched to summarize the available evidence on selenium's role in kidney diseases. Since the complications of ESRD and those of Se deficiency are mostly similar, this triggers the idea that Se deficiency may be considered as a cause of these problems, but it needs to be more assessed that Se deficiency is a single factor or there are other factors participated in. Also the role of Se supplementation on resolving the mentioned complications, needs to be more studied through welldesigned clinical studies.

A review of strategies for untargeted urinary metabolomic analysis using gas chromatography-mass spectrometry.

BACKGROUND: Human urine gives evidence of the metabolism in the body and contains different metabolites at various concentrations. A number of analytical techniques including mass spectrometry (MS) and nuclear magnetic resonance (NMR) have been used to obtain metabolites levels in urine samples. However, gas chromatography-mass spectrometry (GC-MS) is one of the most widely used techniques for urinary metabolomics studies due to its higher sensitivity, resolution, reproducibility, reliability, relatively low cost and ease of operation compared to liquid chromatography-mass spectrometry and NMR. AIM OF REVIEW: This review looks at various aspects of urine preparation prior to analysis by GC-MS including sample storage, urease pretreatment, derivatization, use of internal standard and quality control samples for data correction. In addition, most common types of inlet liners, ionization techniques and columns are discussed and a summary of mass analyzers are also highlighted. Lastly, the role of retention index in metabolite identification and data normalization methods are presented. KEY SCIENTIFIC CONCEPTS OF REVIEW: The purpose of this review is summarizing methods of sample storage, pretreatment, and GC-MS analysis that are mostly used in urine metabolomics studies. Specific emphasis is given to the critical steps within the GC-MS urine metabolomics that those new to this field need to be aware of and the remaining challenges that require further attention and studies.

Evaluation of long-chain acyl-coenzyme A synthetase 4 (ACSL4) expression in human breast cancer.

BACKGROUND AND PURPOSE: Breast cancer (BC) is one of the major causes of female cancer-related death. It has recently been demonstrated that metabolic reprogramming including alteration in lipid metabolism is indicated in various types of cancer. The enzymes of the acyl-coenzyme A synthetase long-chain family (ACSLs) are responsible for converting fatty acids to their corresponding fatty acyl-coenzyme A esters which are essential for some lipid metabolism pathways. ACSL4 is one of the isoforms of ACSLs and has a marked preference for arachidonic and eicosapentaenoic acids. The objective of this study was to evaluate ACSL4 expression, its prognostic significance, and its correlation with p53 tumor suppressor in BC patients. EXPERIMENTAL APPROACH: In this study 55 pairs of fresh samples of BC and adjacent non-cancerous tissue were used to analyze ACSL4 expression, using real-time polymerase chain reaction and immunohistochemistry (IHC) staining. The expression of other studied variables was also examined using the IHC technique. FINDINGS / RESULTS: ACSL4 expression was significantly higher in BC tissues compared to the adjacent normal tissue. This upregulation was negatively correlated with Ki-67 and age, and positively correlated with p53 status. The correlation between ACSL4 and p53 may indicate the role of p53 in the regulation of lipid metabolism in cancer cells, in addition to its role in the regulation of ferroptosis cell death. CONCLUSION AND IMPLICATIONS: Our results indicated that the expression of ACSL4 may be considered as a prognostic indicator and potential therapeutic target in BC. However, further studies are needed to confirm the significance of these findings.

Expression and clinicopathological significance of lipin-1 in human breast cancer and its association with p53 tumor suppressor gene.

Breast cancer (BC) is an important cause of female cancer-related death. It has recently been demonstrated that metabolic disorders including lipid metabolism are a hallmark of cancer cells. Lipin-1 is an enzyme that displays phosphatidate phosphatase activity and regulates the rate-limiting step in the pathway of triglycerides and phospholipids synthesis. The objective of this study was to evaluate lipin-1 expression, its prognostic significance, and its correlation with p53 tumor suppressor in patients with BC. In this study, 55 pairs of fresh samples of BC and adjacent noncancerous tissue were used to analyze lipin-1, using quantitative real-time polymerase chain reaction and immunohistochemistry (IHC) staining. The expression of other clinicopathological variables and p53 was also examined using IHC technique. The cell migration was studied in MCF-7 and MDA-MB231 cells following the inhibition of lipin-1 by propranolol. Our results show that the relative expression of lipin-1 messenger RNA was significantly higher in BC tissues compared with the adjacent normal tissue and its inhibition reduced cell migration in cancer cells. This upregulation was negatively correlated with histological grade of tumor and p53 status (p = .001 and p = .034) respectively and positively correlated with the tumor size (p = .006). Our results also seem to indicate that the high lipin-1 expression is related to a good prognosis in patients with BC. The expression of lipin-1 may be considered as a novel independent prognostic factor. The inhibition of lipin-1 may also have therapeutic significance for patients with BC. The correlation between lipin-1 and p53 confirms the role of p53 in the regulation of lipid metabolism in cancer cells.

Investigation of Supercharging as A Strategy to Enhance the Solubility and Plasminogen Cleavage Activity of Reteplase.

BACKGROUND: Reteplase, the recombinant form of tissue plasminogen activator, is a thrombolytic drug with outstanding characteristics, while demonstrating limited solubility and reduced plasminogen activation. Previously, we in silico designed a variant of Reteplase with positively supercharged surface, which showed promising stability, solubility and activity. This study was devoted to evaluation of the utility of supercharging technique for enhancing these characteristics in Reteplase. OBJECTIVE: To test the hypothesis that reinforced surface charge of a rationally-designed Reteplase variant will not compromise its stability, will increase its solubility, and will enhance its plasminogen cleavage activity. MATERIALS AND METHODS: Supercharged Reteplase coding sequence was cloned in pDest527 vector and expressed in E. coli BL21 (DE3). The expressed protein was extracted by cell disruption. Inclusion bodies were solubilized using guanidine hydrochloride, followed by dialysis for protein refolding. After confirmation with SDS-PAGE and western blotting, extracted proteins were assayed for solubility and tested for bioactivity. RESULTS: SDS-PAGE and western blot analysis confirmed the successful expression of Reteplase. Western blot experiments showed most of Reteplase expressed in the insoluble form. Plasminogen cleavage assay showed significantly higher activity of the supercharged variant than the wild type protein (P < 0.001). The stability of the supercharged variant was also comparable to the wild type. CONCLUSION: Our findings, i.e. the contribution of the surface supercharging technique to retained stability, enhanced plasminogen cleavage activity, while inefficiently changed solubility of Reteplase, contain implications for future designs of soluble variants of this fibrinolytic protein drug.

Improving the solubility, activity, and stability of reteplase using in silico design of new variants.

Reteplase (recombinant plasminogen activator, r-PA) is a thrombolytic agent recombined from tissue-type plasminogen activator (t-PA), which has several prominent features such as strong thrombolytic ability and E. coli expressibility. Despite these outstanding features, it demonstrates reduced fibrin binding affinity, reduced stimulation of protease activity, and lower solubility, hence higher aggregation propensity, compared to t-PA. The present study was devoted to design r-PA variants with comparable structural stability, enhanced biological activity, and high solubility. For this purpose, computational molecular modeling techniques were utilized. The supercharging technique was applied for r-PA to designing new species of the protein. Based on the results from in silico evaluation of selected mutations in comparison to the wild-type r-PA, the designed supercharged mutant (S7 variant) exhibited augmented stability, decreased solvation energy, as well as enhanced binding affinity to fibrin. The data also implied increased plasminogen cleavage activity of the new variant. These findings have implications to therapies which involve removal of intravascular blood clots, including the treatment of acute myocardial infarction.

Decreased Na+/K+-ATPase Activity and Altered Susceptibility to Peroxidation and Lipid Composition in the Erythrocytes of Metabolic Syndrome Patients with Coronary Artery Disease.

BACKGROUND: The increased generation of reactive oxygen species that occurs in the case of a metabolic syndrome (MetS) may be responsible for the increased oxidative injury to erythrocyte membranes in coronary artery disease (CAD). Therefore, we studied the effects of MetS on both indexes of oxidative damage and biochemical properties of erythrocyte membranes in CAD patients. METHODS: We analyzed the markers of oxidative stress, Na+/K+-ATPase activity, total cholesterol content of erythrocyte membranes (CEM), and fatty acid compositions of the erythrocyte membrane in 82 patients with stable CAD and 39 non-CAD subjects. RESULTS: The CAD patients had higher levels of CEM, membrane lipid peroxidation, erythrocyte superoxide dismutase (SOD) activity, and Na+/K+-ATPase activity compared with non-CAD subjects. The Na+/K+-ATPase activity was correlated negatively with membrane lipid peroxidation, and positively with the CEM. In CAD patients with MetS compared with those without MetS, we found that the membrane lipid peroxidation and CEM were increased, whereas the n-3 fatty acids content, SOD activity, Na+/K+-ATPase activity were decreased. CONCLUSION: These findings suggest an impairment of erythrocyte membrane biochemical properties in stable CAD patients as a consequence of oxidative injury that may contribute to the development of CAD. In addition, MetS may be related to increased oxidative injury to erythrocyte membranes.

The effect of FADS2 gene rs174583 polymorphism on desaturase activities, fatty acid profile, insulin resistance, biochemical indices, and incidence of type 2 diabetes.

BACKGROUND: In this study, we investigated the associations of erythrocytes fatty acid composition, activities of delta-5 desaturase (D5D) and delta-6 desaturase (D6D), and other metabolic risk factors, with type 2 diabetes (T2D) risk to determine if rs174583 polymorphism of FADS2 gene had any effect on these associations. MATERIALS AND METHODS: Fatty acid profile of erythrocytes was determined using gas chromatography-mass spectrometry in 95 T2D patients and 95 apparently healthy participants. The genotypes of single-nucleotide polymorphism (SNP) of FADS2 gene were determined using the polymerase chain reaction-restriction fragment length polymorphism technique. Other biochemical parameters were measured in the serum using standard analytical procedures. RESULTS: D6D activity was increased (P < 0.001) and D5D activity was decreased in T2D patients (P < 0.001) compared to controls. Homeostatic model assessment insulin resistance (HOMA-IR) index was positively correlated with D6D (r = 0.34, P < 0.001) and negatively correlated with D5D (r = -0.19, P = 0.02). Palmitic acid (P < 0.001) and dihomo-gamma-linolenic acid (P = 0.03) were higher and linoleic acid (P < 0.001) and arachidonic acid (AA) (P < 0.001) were lower in T2D patients. The distribution of rs174583 genotypes which includes C/T, C/C, and T/T was not different in the two groups (P = 0.63). CONCLUSION: In the population studied, there was a strong association in the erythrocytes fatty acid composition, D5D and D6D activities and other metabolic risk factors between non-T2D and T2D patients. In addition, there was a strong association in erythrocytes DGLA and AA contents and D5D activities between rs174583 genotypes in all participants. However, the distribution of rs174583 genotypes did not differ significantly between T2D patient and controls, and it did not appear to be an association between rs174583 SNP and incident of T2D.