High-levelexpression of functional recombinant human coagulation factor VII in insect cells

Abstract: Recombinant coagulation factor VII (FVII) is used as a potential therapeutic intervention in hemophilia patients who produce antibodies against the coagulation factors. Mammalian cell lines provide low levels of expression, however, the Spodoptera frugiperda Sf9 cell line and baculovirus expression system are powerful systems for high-level expression of recombinant proteins, but due to the lack of endogenous vitamin K-dependent carboxylase, expression of functional FVII using this system is impossible. In the present study, we report a simple but versatile method to overcome the defect for high-level expression of the functional recombinant coagulation FVII in Sf9 cells. This method involves simultaneous expression of both human γ-carboxylase (hGC) and human FVII genes in the host. It may be possible to express other vitamin K-dependent coagulation factors using this method in the future. Keywords: Baculovirus; γ-carboxylase; Coagulation FVII; Factor VII; Insect cel

Assessment of Streamflow Drought Based on Truncation Level (TL) Using Permanent Streamflow Data in one of the Sub-Basin of Lut Desert, Iran

Drought is known as one of the main natural hazards especially in arid and semi-arid regions where there are considerable issues in regard to water resources management. The focus of the present study is mainly on hydrological aspects of drought. For hydrological drought analysis, streamflow data is used as the key variable to identify drought events with reference to a demand specific threshold level, termed as truncation level. Thus, the objective of the present study is to (a) investigate the hydrological drought characteristics in Nesa River using streamflow data; (b) determine independent drought events, their duration, and severity using the variable truncation level approach; and (c) derive streamflow drought severity index. Based on expedience probabilities, the monthly flow duration curves for Nesa River were derived. These were utilized to estimate different dependable flows, and the values of variable truncation levels were obtained for a 75% probability level for each month. These values were used to distinguish the deficit and surplus flow periods independent drought events identified using the pooling procedure. Since 10 daily flow data were utilized, the minimum deficit flow duration was 10 days. In the following, have been identified some short duration (one or two 10-daily time step) surplus and deficit events. To decide on independent drought despite the short duration inter-event surplus has been used for a pooling procedure known as inter-event time and volume criterion (IC). Eventually, identified independent drought events and also describe their duration, severity, intensity, and DSI. Analysis of independent drought Characteristics in Nesa River indicated that are prolonged dry period in the hydrological regime of this river. In addition, based on DSI, Nesa droughts mostly are in sever category. Hence, it is suggested more realistic reload occurs in management programs of this river including storage, distribution and assign to various resources

Modeling the Endothelial Glycocalyx Layer in the Human Conventional Aqueous Outflow Pathway

A layer of proteoglycans and glycoproteins known as glycocalyx covers the surface of the trabecular meshwork (TM), juxtacanalicular tissue (JCT), and Schlemm’s canal (SC) inner wall of the conventional aqueous outflow pathway in the eye. This has been shown to play a role in the mechanotransduction of fluid shear stress and in the regulation of the outflow resistance. The outflow resistance in the conventional outflow pathway is the main determinant of the intraocular pressure (IOP) through an active, two-way, fluid–structure interaction coupling between the outflow tissues and aqueous humor. A 3D microstructural finite element (FE) model of a healthy human eye TM/JCT/SC complex with interspersed aqueous humor was constructed. A very thin charged double layer that represents the endothelial glycocalyx layer covered the surface of the elastic outflow tissues. The aqueous humor was modeled as electroosmotic flow that is charged when it is in contact with the outflow tissues. The electrical–fluid–structure interaction (EFSI) method was used to couple the charged double layer (glycocalyx), fluid (aqueous humor), and solid (outflow tissues). When the IOP was elevated to 15 mmHg, the maximum aqueous humor velocity in the EFSI model was decreased by 2.35 mm/s (9%) compared to the fluid–structure interaction (FSI) model. The charge or electricity in the living human conventional outflow pathway generated by the charged endothelial glycocalyx layer plays a minor biomechanical role in the resultant stresses and strains as well as the hydrodynamics of the aqueous humor

Monitoring saliva compositions for non-invasive detection of diabetes using a colorimetric-based multiple sensor

Abstract The increasing population of diabetic patients, especially in developing countries, has posed a serious risk to the health sector, so that the lack of timely diagnosis and treatment process of diabetes can lead to threatening complications for the human lifestyle. Here, a multiple sensor was fabricated on a paper substrate for rapid detection and controlling the progress of the diabetes disease. The proposed sensor utilized the sensing ability of porphyrazines, pH-sensitive dyes and silver nanoparticles in order to detect the differences in saliva composition of diabetic and non-diabetic patients. A unique color map (sensor response) was obtained for each studied group, which can be monitored by a scanner. Moreover, a good correlation was observed between the colorimetric response resulting from the analysis of salivary composition and the fasting blood glucose (FBG) value measured by standard laboratory instruments. It was also possible to classify participants into two groups, including patients caused by diabetes and those were non-diabetic persons with a total accuracy of 88.9%. Statistical evaluations show that the multiple sensor can be employed as an effective and non-invasive device for continuous monitoring of diabetes, substantially in the elderly

Expression and purification of recombinant human coagulation factor VII fused to a histidine tag using Gateway technology

Background. Factor VII is a plasma glycoprotein that participates in the coagulation process leading to generation of fibrin. Construction, expression and purification of recombinant FVII fused to poly histidin tag through gateway technology were aimed in this study. Methods. To construct entry clone, blunt-end FVII cDNA and subsequent PCR product isolated from HepG2 cell line was TOPO cloned into pENTR TOPO vector. To construct expression clone, LR recombination reaction was carried out between entry clone and destination vector, pDEST26. CHO c ells were transfected with 1 g of DNA of PDEST26 FVII using the FuGENE HD transfection reagent. Two cell lines that permanently expressed recombinant factor VII were established. The expression of recombinant FVII was confirmed by RT-PCR and ELISA. Culture medium containing his-FVII was added to the nickel-nitrilotriacetic acid resin c olumn and bound protein was eluted. The purified protein was detected by SDS-PAGE and western blot analysis. Biological activity of the recombinant factor VII was determined by prothrombin time assay using factor FVII-depleted plasma. Results. The results showed that human recombinant FVII successfully was cloned and accuracy of the nucleotide sequence of the gene and its frame in the vector were confirmed by DNA sequencing. Stable clones transfected with the construct expressed FVII mRNA and related protein but any expression was not detected in the CHO cells containing empty vector. A protein of about 52KDa was detected in SDS-PAGE and was further confirmed by western blot analysis. A three-fold decrease in clotting time was observed by using this rFVII. Conclusion. As we are a ware, this is the first report of expression of recombinant FVII fused with his-tag through gateway technology. The next steps including large scale expression, purification, activation and stabilization are underwa y