Production and activity assay of recombinant micro-plasminogen in Pichia pastoris

Introduction: Plasmin is a trypsin like serine protease that can catalyze the hydrolytic cleavage of peptide bonds at the COOH sides of arginines and lysines in extracellular matrix (ECM) components including fibrin, laminin and fibronectin. Microplasminogen (µPlg) is the COOH-terminal portion of plasminogen from 531 to 791, and has molecular weight of about 30kDa. µPlg consists of the catalytic domain of plasminogen and can be converted by many plasminogen activators, for example, streptokinase (SK) into microplasmin, which shares the same catalytic properties as human plasmin. µPlg can be used as a therapeutic agent in vitreoretinopathies and thrombotic diseases. Methods and Results: The purpose of this study was production and activation of recombinant µPlg. After codon optimization and synthesis of the µPlg gene, it was inserted into expression vector pPICZα and this plasmid was transformed into Pichia pastoris X-33 cells by electroporation. The selected positive transforments were transferred into YPM (yeast extract-peptone-methanol) medium and were induced by methanol. The recombinant protein (33kDa) was successfully secreted into the supernatant of the induction medium and was purified by Ni-sepharose column. The presence of the recombinant µPlg was confirmed by SDS-PAGE. The activation of µPlg by streptokinase was performed at a 1:1 molar ratio of streptokinase to µPlg at 37°C for 2hr in Tris-HCl buffer, pH=7.8. Microplasminogen activity was measured by FDP (Fibrinogen Degradation Product) ELISA kit. In a blood sample the clot is broken down by plasmin. Some of These broken fibrin fragments are called d-dimers. In general, D-dimer elevation indicates increased fibrin turnover. In this procedure, 30 μL of active enzyme was added. And the rate of D-dimers was measured at 380 μg/mL. While the natural amount of D-dimers in the blood is less than 40 μg/mL. Conclusions: The availability of microplasminogen with lower molecular weight can be a valuable pharmaceutical tool for treatment of thrombotic diseases and vitreoretinopathies because the isolation of autologous plasmin is an expensive and time-consuming process. In this study we managed to produce microplasminogen and successfully turn it into active microplasmin. However more effort is needed to increase the yields of Protein Production

Recombinant production of Soluble Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (sTRAIL) as a therapeutic protein

Abstract:  Successfully cancer therapies aim to induce apoptosis in cancer cell lines. Recent advances in cancer therapy based on the use of some recombinant proteins such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). TRAIL is a new member of the TNF superfamily. In this paper, we report the expression, purification, and  preparation of a recombinant form of the extracelluar domain of the TRAIL (sTRAIL) in Escherichia coli rosetta gami under the control of T7 promoter; which  may selectively induce apoptosis of tumor cells in vitro. To obtain recombinant sTRAIL protein, the encoding region for sTRAIL was cloned between Xho1 and BamHI in pET28a expression vector. The results showed that the recombinant sTRAIL was efficiently produced in Ecoli rosetta gami strain.                               Introduction: Apoptosis is an evolutionarily conserved and essential for maintenance of  tissue homeostasis and removal of unwanted cells. TRAIL belongs to the group of therapeutic agents selectively targeting a wide variety of cancer cells without affecting the normal cells .The therapeutic potential of TRAIL is attributed to its receptor expression in a variety of tissues; which initiates apoptosis in cancer cells through interaction with the death receptors DR4 and DR5. Due to its selective nature, it is considered as a significant therapeutic agent in cancer therapy. The purpose of this study was to produce recombinant human sTRAIL in Rosetta Gami2 E.coli strain and its functions on cancerous cells in vitro. Methods and results:  we optimized the coding sequence of this protein. The recombinant plasmid was transformed into Rosetta Gami2 E.coli strain for expression. The transformed bacteria which contain recombinant plasmid were cultured in 37ºC with 250 rpm in LB and in 20ºC in TB medium for 18 hours. TRAIL was purified by Ni sepharose column, and the presence of the recombinant protein was confirmed by SDS-PAGE. The concentration of purified protein was measured by Bradford assay. Our finding showed that the recombinant protein (34kD) has been successfully produced for next experiments, the purified protein was desalted and applied toward cancerous cells. Conclusions: In summary, TRAIL can be considered as a promising therapeutic agent for effective, targeted and less toxic agents for treatment of cancers

Determination of the Nonlinear Muskingum Model Coefficients Using Genetic Algorithm and Numerical Solution of the Continuity Equation

The optimization method is an appropriate choice for determining optimal parameters in the Muskingum model, in order to increase the speed of computations; coefficients of this model have been computed optimally with assistance of the genetic algorithm. These coefficients were computed from the linear Muskingum and Muskingum-Cunge models using required data and the available relations. In order to evaluate efficiency of the procedure of optimizing coefficients of the nonlinear Muskingum model via the genetic algorithm method compared with the other two methods used for determining these coefficients, outflow hydrographs were computed using the optimal coefficients and solving the continuity equations according to the Runge-Kutta method order 4 and was compared with the two flood routing methods from the Muskingum and Muskingum-Cunge models as well. To study the precision of these three methods, square root of sum of squares of difference of discharges computed from each of the three methods and observational discharges obtained from the HEC-RAS RMSE software was used as the objective function and achieved results indicate more proximity of the computed hydrographs from the optimization coefficients in the Runge-Kutta order 4 to the outflow hydrographs obtained to the HEC-RAS software compared with the two Muskingum and Muskingum-Cunge models

The Quality of Maternity Services in Mashhad Educational Hospitals, Using SERVUSE Model

Introduction: Reducing maternal and neonatal mortality requires improved care quality; the aim of this study was to determine various dimensions of the quality of services in maternity wards in Iran from the patient’s perspective. Methods: In this cross-sectional study which was conducted from April to October 2017, 363 patients were selected from maternity wards of five hospitals affiliated to Mashhad University of Medical Sciences by convenience sampling method. Data collection tools consisted of the SERVUSE (Service Usability questionnaire), including 51 items in 6 dimensions of services quality; its validity and reliability were determined in previous studies. Data analysis was performed using SPSS, version 20. The analysis was performed using descriptive and inferential statistical methods including Wilcoxon, Kruskal Wallis and Mann-Whitney tests. The significance level in all the tests was considered 5%. Results: The total mean scores of the patients’ expectation and perception were 19.10±2.14 and 15.11±4.25, respectively. The highest expectation and perception were related to the usability dimension, and the lowest expectation and perception were related to reliability dimension. The differences between the median score of perception and expectation for all dimensions and total median score of perceptions and expectations were statistically significant (P<0.05, using Wilcoxon test). There were no statistically significant differences in the median score of the gap between the patient’s expectations and perceptions at the levels of demographic variables (P>0.05, using Mann-Whitney and Kruskal Wallis tests). Conclusion: It seems that the service quality of maternity ward of hospitals was approximately unsatisfactory from the patients’ perspective, and they had high expectations in maternity wards of hospitals. Since maternity service quality is critically important, improvement of quality requires management, concordant participation, and efforts of the hospital and staff at all levels of the medical facilities and convenience

Covid-19 mobility restrictions: impacts on urban air quality and health.

In 2020, Covid-19-related mobility restrictions resulted in the most extensive human-made air-quality changes ever recorded. The changes in mobility are quantified in terms of outdoor air pollution (concentrations of PM2.5 and NO2) and the associated health impacts in four UK cities (Greater London, Cardiff, Edinburgh and Belfast). After applying a weather-corrected machine learning (ML) technique, all four cities show NO2 and PM2.5 concentration anomalies in 2020 when compared with the ML-predicted values for that year. The NO2 anomalies are -21% for Greater London, -19% for Cardiff, -27% for Belfast and -41% for Edinburgh. The PM2.5 anomalies are 7% for Greater London, -1% for Cardiff, -15% for Edinburgh, -14% for Belfast. All the negative anomalies, which indicate air pollution at a lower level than expected from the weather conditions, are attributable to the mobility restrictions imposed by the Covid-19 lockdowns. Spearman rank-order correlations show a significant correlation between the lowering of NO2 levels and reduction in public transport (p < 0.05) and driving (p < 0.05), which is associated with a decline in NO2-attributable mortality. These positive effects of the mobility restrictions on public health can be used to evaluate policies for improved outdoor air quality. POLICY RELEVANCE: Finding the means to curb air pollution is very important for public health. Empirical evidence at a city scale reveals significant correlations between the reduction in vehicular transport and in ambient NO2 concentrations. The results provide justification for city-level initiatives to reduce vehicular traffic. Well-designed and effective policy interventions (e.g. the promotion of walking and cycling, remote working, local availability of services) can substantially reduce long-term air pollution and have positive health impacts

Electrochemical Alcohol Oxidation and Biological Properties of Mn3O4-Co3O4-rGO

A multi-component nanocomposite consisting of manganese oxide (Mn3O4), cobalt oxide (Co3O4), and reduced graphene oxide (rGO) in the form of Mn3O4-Co3O4-rGO was synthesized by the hydrothermal method. Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and linear sweep voltammetry (LSV) analyses were performed to investigate the synergistic effect of metal oxides on the surface of rGO nano-sheets in the methanol oxidation reaction (MOR) and ethanol oxidation reaction (EOR) process. The good electrochemical results show that Mn3O4-Co3O4-rGO can be a promising, inexpensive nano-catalyst for application in alcohol fuel cells. In addition, as nanoparticles inhibit cancer cells growth by producing reactive oxygen species (ROS), we explored the synergic effect of the three-component synthetic nanomaterial in gastric cancer cells (AGS). Results indicated that Mn3O4-Co3O4-rGO inhibited AGS cell growth by induction of ROS, upregulation of Mir-20a-5p, and downregulation of ZBTB4 gene. This might provide a novel molecular-targeted strategy of microRNA-based therapeutics for gastric cancer treatment

Epi-drug-Loaded Niosomes in Combination with Blepharis Persica Improves Spinal Cord Injury in Male Rat

1) Background: Taxol facilitates nerve regeneration but it has unwanted side-effects via the tumor suppressive function of miR-34a which induces XIST expression and affects paclitaxel administration. We aimed to examine the neuroprotective effect of Paclitaxel-loaded niosomes (PLNs) to enhance fertility by combining with the Belpharis Persica (B.P) extract in male Wistar rat model of spinal cord injury (SCI). 2) Methods: PLNs were prepared using the thin-film hydration method. After laminectomy at thoracic level (T9/T10) SCI was induced using the weight drop method. Rat treated with either PLNs (2.5mg/kg), PLNs (2.5mg/kg)+B.P extract (300mg/kg-14 days), B.P extract (300mg/kg-14 days), Normal, SCI (normal saline), and sham (laminectomy). 3) Results: The transmission electron microscopy TEM and laser diffraction methods were confirmed the existence of spherical vesicles with feature sizes about 200 nm. The various behavioral, pathological and molecular parameters (CATSPER gene expression) were distinguished. SCI resulted in a significant decrease (P<0.05) in sperm motility, behavioral skills, and CATSPER genes expression were all regenerated by treatment with combination therapy (PLNs+B.P extract) in comparison to the others. Besides, the increased in sperm motility, and CATSPER genes expression were significantly (P<0.05) demonstrated in PLNs and B.P treated groups. 4) Conclusion: Taken together, the results indicated that the PLNs and B.P groups showed a significant increase in sperm parameters, but combination therapy caused the greater recovery of both gene expression and motor functions to decrease the neurological deficits no serious adverse reactions and enhanced fertility

NiO–Co3O4–rGO as a Multicomponent Transition Metal Oxide Nanocatalyst for Ultra‐level Detection of Nitrite in Beef and Tap Water Samples

The detection of nitrite as a challenging compound in food industries has always been a necessity. To this purpose, herein, two transition metal oxide-based nanocatalysts, NiO–Co3O4 (NC) and its reduced graphene hybridized form, NiO–Co3O4–rGO (NCR), are synthesized by a hydrothermal method and characterized by X-ray diffraction, Raman spectroscopy, scanning electron microscope (SEM) images, and energy dispersive X-ray analysis (EDAX) mapping. The synthesized materials are exploited to modify a glassy carbon electrode and fabricate an effective sensor for nitrite detection in real samples through differential pulse voltammetry (DPV). NCR shows excellent sensitivity (40.44 μA per dec), very low limit of detection (20 × 10-9 m), significant accuracy, and good stability at the wide range of nitrite ions (100–500 × 10-9 m) in real sample