Cloning, Expression and Purification of Full-length Recombinant Ecarin and Comparing Its Expression and Function with Its Truncated Form.

Ecarin is a metalloproteinase found in snake venom (SVMP) with an important role in coagulation and control of hemostasis. It can specifically produce active-thrombin from prethrombin-2 and does not differentiate between normal and abnormal prothrombin. It is used in diagnostic tests and to evaluate the treatment process of many diseases. There are many drawbacks associated with separating these compounds from snake venom. Therefore, in this study, full-length recombinant Ecarin (r-Ecarin) was cloned, expressed, and purified in eukaryotic host cells. To determine the most effective form of the enzyme, r-Ecarin was compared with the recombinant truncated form, which has only the metalloprotease domain of the protein (r-Ecamet) in terms of function and expression. Briefly, A DNA construct composed of sequence-encoding Ecarin was designed and cloned into pCAGGS expression vector and, subsequently, expressed in Chinese Hamster Ovary (CHO) cells. To identify the enzymatic activity of expressed protein, a bioactivity assay was performed. Blood coagulation time and expression levels of r-Ecarin and r-Ecamet proteins were compared. Also, a histopathological assessment was carried out on the liver of mice treated with these proteins. Comparison of r-Ecarin and r-Ecamet expression pattern demonstrated that full-length Ecarin expression has at least 2-fold higher expression in eukaryotic cells. Determination of r-Ecarin function proved that this protein is capable of prothrombin cleavage and producing thrombin. Comparison of PT test results between the r-Ecarin and r-Ecamet showed that there is a significant difference in the activity of the two enzymes and the full-length protein coagulates the blood in less time.

Prokaryotic expression, evaluation, and prediction of the structure and function of the ecarin metalloproteinase domain.

Ecarin is one of the most widely used drug compounds in blood clotting experiments and is used to monitor and treat many diseases such as cancer, liver, lupus, and cardiovascular disease. The metalloproteinase domain is known as the active site of ecarin. In this study, an ecarin metalloproteinase cassette was designed and synthesized in the pUC57 vector. The gene fragment was released and cloned into the pET-28a vector and expressed in Escherichia coli. The recombinant protein was confirmed by western blotting. Enzyme activity was estimated by a laboratory coagulation test, and prothrombin time and tertiary structure were determined by using the Iterative Threading ASSEmbly Refinement (I-TASSER) server. Data from blood clotting tests for the produced ecarin activity were analyzed using an independent t test. As per I-TASSER server prediction, model 1 with the highest confidence score 0.95, template modeling score (0.84 ± 0.08), and root mean square deviation (3.5 ± 2.4 Å) was considered as the best model, and the 2e3xA enzyme was more similar to the target protein. The predictive results helped to better understand the relationship between the structure and function of the ecarin metalloproteinase domain. Also, the production of this active site in the prokaryotic expression system, which is simpler and more cost-effective than the production of the eukaryotic system, showed that this recombinant ecarin could be used as a substitute for the raw snake venom of Echis carinatus because it converts prothrombin into thrombin, and its activity, as estimated using the prothrombin time test, was found to be faster than normal ecarin.

Heavy metals (Cr, Pb, Cd, Ni) in aerosols emitted from electronic cigarettes sold in Malaysia.

While past studies have detected heavy metals in aerosols emitted from electronic cigarettes (ECIG), they have provided little information detailing the practical implications of the findings to the Malaysian population due to variations between products. The aims of this study were to analyse heavy metals of interest (HMOI) in the aerosols emitted from selected ECIG and to evaluate potential health risks by referring to the permissible daily exposure (PDE) from inhalational medications defined by the United States Pharmacopeia Chapter 232. All four HMOI were detected in aerosols emitted from the selected ECIG in Sarawak. Among the four, Cr was present at the highest median levels (6.86 ng/m3), followed by Ni (0.30 ng/m3), Pb (0.19 ng/m3) and Cd (0.01 ng/m3). Five out of 100 combinations (5%) of ECIG and ECIG liquids were found to emit Cr that exceed the recommended PDE. Future studies examining more heavy metal variants, using a larger sample size and different analytical techniques to compare various ECIGs are recommended.

E-Cigarette Users' Attitudes on the Banning of Sales of Nicotine E-Liquid, Its Implication on E-Cigarette Use Behaviours and Alternative Sources of Nicotine E-Liquid.

The banning of sales of nicotine e-liquid in e-cigarette shops has been implemented in several states in Malaysia. The distribution of nicotine e-liquid can only be allowed by licensed pharmacies or registered medical practitioners. This study aimed to evaluate e-cigarette users' responses to the control policy in a cross-sectional survey of 851 e-cigarette users by utilizing a self-report questionnaire that assessed (1) attitudes on regulation policy of e-cigarette banning; (2) e-cigarette use behaviors; and (3) sources of e-liquid after the regulation policy has been implemented. Participants from the state of Selangor where the banning policy was implemented were surveyed. The majority (95.8%) opposed the banning and believed e-cigarettes should be sold to anyone aged 18 years or above as with tobacco cigarettes, only a minority believed that nicotine e-liquid should only be available for sale over the counter in pharmacy stores (14.6%) and in clinics with a doctor's prescription (11.8%). The majority (44.2%) reported that they would continue their e-cigarette use as before the banning policy, while 20% plan to completely stop e-cigarette usage without replacing it with any alternatives. The vast majority (87.9%) was still able to obtained nicotine e-liquid from e-cigarette shops in spite of the ban and the second most common source was from online purchase (63.1%). The sales of nicotine e-liquid from black-market were evidenced as many reported obtaining zero nicotine e-liquid from the black market (54.4%). Self- or home-made (30.8%) nicotine e-liquid was also reported. Majority of respondents that self-made e-liquid were from the average monthly income group (below MYR3000). Obtaining nicotine from the pharmacy was least preferred (21.4%). Provision of professional advice to nicotine e-liquid users along with the ban may lessen the likelihood of users switching to tobacco cigarettes or other nicotine alternatives. Banning of sales of nicotine e-liquid in e-cigarette shops resulted in a boom in the black market supplying nicotine e-liquid and, self- or home-made nicotine e-liquid. Enforcing regulations and monitoring black market sales is warranted. Efforts to educate e-cigarette users of the danger of sourcing nicotine e-liquid from the black market and self- or home-made nicotine e-liquid are essential.

Tobacco Use and its Relationship with Health Complaints Among Employees of Kermanshah Province, Iran.

BACKGROUND: Identifying the pattern of tobacco use and its related factors in employees is crucial. This study aimed to investigate the pattern of tobacco use and its related factors in employees of Kermanshah Province, Iran. METHODS: In 2012, 7129 employees were investigated in a cross-sectional study using the census method. Data on tobacco use and on several chronic diseases obtained using a standardized questionnaire on noncommunicable diseases risk factors of the World Health Organization through face-to-face interviews. Statistical analysis was performed based on the Chi-square test and multivariate logistic regression. RESULTS: In general, the prevalence of tobacco use, smoking cigarettes, and smoking waterpipe was 9.9%, 8.9%, and 1.2% among the employees, respectively. Tobacco use was significantly higher in the age group over 40 (14.0%), in male gender (13.3%), in married individuals (10.8%) and in those with diploma and lower degree (16.4%), (P < 0.001). At the individual level, the odds ratio of tobacco use was 1.5 (95% confidence interval [CI]: 1.2-1.8) in hypertensive, 1.8 (95% CI: 1.2-2.6) in diabetic employees and 1.7 (95% CI: 1.3-2.3) in those with heart diseases, compared to healthy individuals. After adjusting for age, gender, marital status and educational level, there was not any significant relationship between tobacco use and health complaints and only the demographic variables remained significant. CONCLUSIONS: Preventive public health policies are mandatory especially in younger ages and male employees to promote their knowledge on disadvantages of tobacco use.

G-CSF for mobilizing transplanted bone marrow stem cells in rat model of Parkinson's disease.

OBJECTIVES: Granulocyte-colony stimulating factor (G-CSF) is used in clinical practice for the treatment of neutropenia and to stimulate generation of hematopoietic stem cells in bone marrow donors. In the present study, the ability of G-CSF in mobilizing exogenous bone marrow stem cells (BMSCs) from peripheral blood into the brain was tested. We for the first time injected a small amount of BMSCs through the tail vein. MATERIALS AND METHODS: We choose 25 male Wistar rats (200-250 g) were lesioned by 6-OHDA injected into the left substantia nigra, pars compacta (SNpc). G-CSF (70 µg/kg/day) was given from the 7th day after lesion for five days. The BMSCs (2×105) were injected through the dorsal tail vein on the 7th day after lesion. RESULTS: The number of rotations was significantly lower in the stem cell therapy group than in the control group. In the third test in the received G-CSF and G-CSF+stem cells groups, animals displayed significant behavioral recovery compared with the control group (P<0.05). There was a significant difference in the average of dopaminergic neurons in SNpc between the control group and G-CSF and G-CS+stem cells groups. We didn't detect any labeling stem cells in SNpc. CONCLUSION: G-CSF can't mobilize low amounts of exogenous BMSCs from the blood stream to injured SNpc. But G-CSF (70 µg/kg) is more neuroprotective than BMSCs (2×105 number[w1] of BMSCs). Results of our study suggest that G-CSF alone is more neuroprotective than BMSCs.

Improving adherence and biomedical markers in hemodialysis patients: the effects of relaxation therapy.

OBJECTIVE: This study aimed to assess the effectiveness of Benson's relaxation technique in improving the hemodialysis patients' dietary and fluid adherence and biomedical markers. DESIGN: This randomized controlled trial with a pre-post test design was conducted on 86 hemodialysis patients randomly divided into an intervention (receiving Benson's relaxation technique) and a control group (usual care). SETTING: The setting of the study was two hemodialysis units affiliated to Shiraz University of Medical Sciences, Shiraz, Iran. INTERVENTION: The patients listened to the audiotape of Benson's relaxation technique twice a day each time for 20min for 8 weeks. MAIN OUTCOME MEASURES: Dietary and fluid adherence and some biomedical markers were measured in both the intervention and the control group at baseline and at the 8th week after the intervention. RESULTS: The results showed significant differences between the two groups regarding blood urea nitrogen and phosphate as dietary adherence and interdialytic weight gain as fluid adherence in the 8th week of the intervention (P<0.05). Also, a significant difference was found between the two groups concerning blood glucose level after the intervention (P<0.05). CONCLUSIONS: This study highlighted the importance of Benson's relaxation technique in improvement of adherence and some biomedical markers in hemodialysis patients. Thus, Benson's relaxation therapy could be used as a part of the nursing care practice for hemodialysis patients and those suffering from chronic diseases.

Evaluation of the effect of Benson's relaxation technique on pain and quality of life of haemodialysis patients: a randomized controlled trial.

BACKGROUND: Haemodialysis patients may suffer from pain and impairment of quality of life. Some complementary interventions, such as relaxation therapy, might affect the pain and quality of life. The present study aimed to identify the effectiveness of Benson's relaxation technique in relieving pain and improving the quality of life in haemodialysis patients. STUDY DESIGN: The study was a randomized controlled trial. SETTING AND PARTICIPANTS: The data were collected in two haemodialysis units affiliated to Shiraz University of Medical Sciences. A total of 86 haemodialysis patients were randomly assigned to either the intervention (receiving Benson's relaxation technique) or the control group (routine care) from 2011 to 2012. INTERVENTION: The patients in the intervention groups listened to the audiotape of relaxation technique twice a day each time for 20min for eight weeks. MEASUREMENTS AND OUTCOMES: The pain numeric rating scale and Ferrans and Powers Quality of Life Index-dialysis version questionnaire were completed at baseline and 8 weeks after the intervention. The data were analyzed using independent t-test and ANCOVA. RESULTS: The results of ANCOVA showed a significant difference between the intervention and the control group concerning the mean score of the intensity of pain (F=6.03, p=0.01). Moreover, a significant difference was found between the intervention and the control group regarding the total quality of life (F=10.20, p=0.002) and health-functioning (F=8.64, p=0.004), socioeconomic (F=12.45, p=0.001), and family (F=8.52, p=0.005) subscales of quality of life. CONCLUSION: These findings indicated that Benson's relaxation technique might relieve the intensity of pain and improve the quality of life in haemodialysis patients. Thus, Benson's relaxation technique could be used as part of the care practice for relieving the pain intensity and improvement of the quality of life in haemodialysis patients.

The effect of Benson's relaxation technique on the quality of sleep of Iranian hemodialysis patients: a randomized trial.

OBJECTIVE: This study was performed to evaluate the effectiveness of Benson's relaxation technique in the quality of sleep of hemodialysis patients. DESIGN: It was a randomized controlled trial with a pre-post-test design. A total of 86 hemodialysis patients referring to hemodialysis units were assigned to either the intervention (receiving Benson's relaxation technique) or the control group (routine care) through block randomization. SETTING: The study was performed in two hemodialysis units affiliated to Shiraz University of Medical Sciences, Shiraz, Iran. INTERVENTION: The patients in the intervention group listened to the audiotape of Benson's relaxation technique twice a day each time for twenty minutes for eight weeks. MAIN OUTCOME MEASURES: The global score of Pittsburgh Sleep Quality Index (PSQI) as well as its components was computed in both the intervention and the control group before and at the 8th week of the intervention. RESULTS: The results of ANCOVA indicated significant differences between the two groups regarding the scores of Pittsburgh Sleep Quality Index subscales, such as sleep disturbance, daytime dysfunction, the use of sleep medication, and subjective sleep quality and as well as its global scores at the 8th week of the intervention (p<0.05). CONCLUSIONS: This study highlighted the importance of Benson's relaxation technique in improvement of the sleep quality of the patients on hemodialysis. Thus, educational sessions are recommended to be planned on this cost effective and easy to use relaxation technique in order to improve hemodialysis patients' sleep quality. Further studies are needed to assess the effectiveness of this technique in other groups of patients.