A high-throughput platform for lentiviral overexpression screening of the human ORFeome

In response to the growing need for functional analysis of the human genome, we have developed a platform for high-throughput functional screening of genes overexpressed from lentiviral vectors. Protein-coding human open reading frames (ORFs) from the Mammalian Gene Collection were transferred into lentiviral expression vector using the highly efficient Gateway recombination cloning. Target ORFs were inserted into the vector downstream of a constitutive promoter and upstream of an IRES controlled GFP reporter, so that their transfection, transduction and expression could be monitored by fluorescence. The expression plasmids and viral packaging plasmids were combined and transfected into 293T cells to produce virus, which was then used to transduce the screening cell line. We have optimised the transfection and transduction procedures so that they can be performed using robotic liquid handling systems in arrayed 96-well microplate, one-gene-per-well format, without the need to concentrate the viral supernatant. Since lentiviruses can infect both dividing and non-dividing cells, this system can be used to overexpress human ORFs in a broad spectrum of experimental contexts. We tested the platform in a 1990 gene pilot screen for genes that can increase proliferation of the non-tumorigenic mammary epithelial cell line MCF-10A after removal of growth factors. Transduced cells were labelled with the nucleoside analogue 5-ethynyl-2'-deoxyuridine (EdU) to detect cells progressing through S phase. Hits were identified using high-content imaging and statistical analysis and confirmed with vectors using two different promoters (CMV and EF1α). The screen demonstrates the reliability, versatility and utility of our screening platform, and identifies novel cell cycle/proliferative activities for a number of genes.     

Shape optimization of stress concentration-free lattice for self-expandable Nitinol stent-grafts

In a mechanical component, stress-concentration is one of the factors contributing to reduce fatigue life. This paper presents a design methodology based on shape optimization to improve the fatigue safety factor and increase the radial stiffness of Nitinol self-expandable stent-grafts. A planar lattice free of stress concentrators is proposed for the synthesis of a stent with smooth cell shapes. Design optimization is systematically applied to minimize the curvature and reduce the bending strain of the elements defining the lattice cells. A novel cell geometry with improved fatigue life and radial supportive force is introduced for Nitinol self-expandable stent-grafts used for treating abdominal aortic aneurism. A parametric study comparing the optimized stent-graft to recent stent designs demonstrates that the former exhibits a superior anchoring performance and a reduction of the risk of fatigue failure.     

Comparison of different methods for the isolation of mesenchymal stem cells from human umbilical cord Wharton’s jelly

Several techniques have been devised for the dissociation of tissues for primary culture. These techniques can affect the quantity and quality of the isolated cells. The aim of our study was to develop the most appropriate method for the isolation of human umbilical cord-derived mesenchymal (hUCM) cells. In the present study, we compared four methods for the isolation of hUCM cells: three enzymatic methods; collagenase/hyaluronidase/trypsin (CHT), collagenase/trypsin (CT) and trypsin (Trp), and an explant culture (Exp) method. The trypan blue dye exclusion test, the water-soluble tetrazolium salt-1 (WST-1) assay, flow cytometry, alkaline phosphatase activity and histochemical staining were used to evaluate the results of the different methods. The hUCM cells were successfully isolated by all methods but the isolation method used profoundly altered the cell number and proliferation capacity of the isolated cells. The cells were successfully differentiated into adipogenic and osteogenic lineages and alkaline phosphatase activity was detected in the hUCM cell colonies of all groups. Flow cytometry analysis revealed that CD44, CD73, CD90 and CD105 were expressed in all groups, while CD34 and CD45 were not expressed. The expression of C-kit in the enzymatic groups was higher than in the explant group, while the expression of Oct-4 was higher in the CT group compared to the other groups. We concluded that the collagenase/trypsin method of cell isolation yields a higher cell density than the others. These cells expressed a higher rate of pluripotent cell markers such as C-kit and Oct-4, while the explant method of cell isolation resulted in a higher cell proliferation rate and activity compared to the other methods.     

Species of Adialytus F?rster, 1862 (Hymenoptera,Braconidae, Aphidiinae) in Iran: taxonomic notes and?tritrophic associations

The species of Adialytus Förster in Iran are taxonomically studied and new data on distribution and host associations are presented. The existence of a species complex, in the case of Adialytus ambiguus (Haliday), and the morphological variability in commonly used taxonomic characters has been discussed. In total, four valid species belonging to the genus Adialytus including Adialytus ambiguus (Haliday), Adialytus salicaphis (Fitch), Adialytus thelaxis (Starý) and Adialytus veronicaecola (Starý) have been identified and recorded from Iran. Also, we recognized two additional phenotypes: “Adialytus arvicola” (Starý) and “Adialytus cf. ambiguus” (Haliday). These phenotypes and Adialytus veronicaecola are newly recorded from Iran in association with Sipha and Aphis species, respectively. An illustrated key for identification of the species and two variable phenotypes is presented.     

How Transmembrane Inner Ear (TMIE) plays role in the auditory system: A mystery to us

Different cellular mechanisms contribute to the hearing sense, so it is obvious that any disruption in such processes leads to hearing impairment that greatly influences the global economy and quality of life of the patients and their relatives. In the past two decades, transmembrane inner ear (TMIE) protein has received a great deal of research interest because its impairments cause hereditary deafness in humans. This evolutionarily conserved membrane protein contributes to a fundamental complex that plays role in the maintenance and function of the sensory hair cells. Although the critical roles of the TMIE in mechanoelectrical transduction or hearing procedures have been discussed, there are little to no review papers summarizing the roles of the TMIE in the auditory system. In order to fill this gap, herein, we discuss the important roles of this protein in the auditory system including its role in mechanotransduction, olivocochlear synapse, morphology and different signalling pathways; we also review the genotype-phenotype correlation that can per se show the possible roles of this protein in the auditory system.     

Assessment of the Parameters Influencing Microbial Calcite Precipitation in Injection Experiments Using Taguchi Methodology

Soil improvement is one of the major concerns in civil engineering. Therefore, a variety of approaches have been employed for different soil types. The loose granular soils and sediments have always imposed challenges due to their low strength and bearing capacity as well as presenting difficulties in drilling and excavation. Biomediated soil improvement, i.e., utilizing some bacteria to precipitate calcite on soil particles, has recently been introduced as a novel link of biotechnology and civil engineering to improve the problematic soils. Biogrout as a branch of biomediated soil improvement is based upon microbial calcium carbonate precipitation (MICP). In the present study, the Taguchi method with the aim of optimizing the process was utilized to design the experiments (DOE). A standard L9 orthogonal array with four parameters comprising bacterial cell concentration, molar concentration ratio of nutrient solution, curing time, and flow rate, each assigned to three levels, was selected. In this regard, soil samples were stabilized in sandy soil columns. Two-phase injections were conducted by injecting the bacterium Sporosarcina pasteurii PTCC 1642 in the first phase and nutrient in the second phase. Specimens were subjected to an unconfined compressive strength (UCS) test. ANOVA pointed out how effectual each parameter was. The most effective parameter was curing time, which accounted for 45.97% of the overall variance of the experimental data followed by bacterial cell concentration (22.01%), nutrient strength (19.98%), and flow rate (12.04%). Predicted UCS values for the optimum condition were validated in a confirmation test. Indeed, the UCS of the soil increased from 85 kPa in the control sample to 930 kPa for the optimally treated specimen. It was concluded that rather than curing time, the other parameters are almost equally influential in the applied injection procedure.     

Cloning and High-Level Expression of the Enzymatic Region of Phytase in E. coli

Phytase is an important enzyme poses great nutritional significance in humans and monogastric animals diets. The phytase production yield using wild sources, including micro-organisms, plants, and animals is sorely low. Thus, recombinant expression of phytase has received increasing interest for achieving production rate. Escherichia coli is the most preferred host for expression of heterologous proteins but overexpression of recombinant phytase in E. coli, met with limited success due to the sequestration of the enzyme into inclusion bodies. In the present study, artificial phytases gene with excellent thermostability and activity were designed by detecting the enzymatic region of the E. coli phytase gene by employing bioinformatics tools. Then, the PCR amplified recombinant gene was expressed in E. coli and the active enzyme was recovered from inclusion bodies. Employing cysteine amino acid in the dialysis buffer succeed to the superior activity of the enzyme with a specific activity of 73.8 U/mg. The optimum temperature and pH for enzyme activity were determined at 60 °C and 4, respectively. The novel recombinant enzyme illustrated perfect thermostability up to 70 °C with maintenance 75% of its activity. The enzyme was stable at pH range of 2–10. Moreover, the effects of ions and chemical compounds on enzyme stability and activity were assessed.

     

Optical fluorescence imaging with shortwave infrared light emitter nanomaterials for in vivo cell tracking in regenerative medicine

In vivo tracking and monitoring of adoptive cell transfer has a distinct importance in cell‐based therapy. There are many imaging modalities for in vivo monitoring of biodistribution, viability and effectiveness of transferred cells. Some of these procedures are not applicable in the human body because of low sensitivity and high possibility of tissue damages. Shortwave infrared region (SWIR) imaging is a relatively new technique by which deep biological tissues can be potentially visualized with high resolution at cellular level. Indeed, scanning of the electromagnetic spectrum (beyond 1000 nm) of SWIR has a great potential to increase sensitivity and resolution of in vivo imaging for various human tissues. In this review, molecular imaging modalities used for monitoring of biodistribution and fate of administered cells with focusing on the application of non‐invasive optical imaging at shortwave infrared region are discussed in detail.     

Characterization and optimization of extracellular alkaline lipase production by Alcaligenes sp. Using stearic acid as carbon source

The aim of this study was to improve the production of an extracellular alkaline lipase from Alcaligenes sp. (ATCC 31371) by optimization of the culture medium, for economic production of biodiesel from waste vegetable oil. A number of carbon sources including different types of starch, sugar, sugar alcohol, organic acids, and surfactants were investigated. Polyoxyethylene (20) sorbitan tristearate, whose side chain is stearic acid, was the most effective carbon source for lipase production. Box-Behnken experimental design was used for three factors (soy protein, sodium nitrate, and stearic acid) and the optimal composition for maximum lipase production (1.7-fold enhancement) was established as soy protein 4.07%, sodium nitrate 0.17%, and stearic acid 0.28% at 28°C with an agitation rate of 220 rpm for 24 h. The enzyme was purified to homogeneity and the recovery of the lipase activity was 7.8% with a 30-fold purification. The estimated molecular size of the protein determined by SDS-PAGE was 33 kDa. The optimum pH and temperature of the purified lipase was 8.5 and 40°C, respectively. The purified enzyme was stable in the pH range of 6.0 and 9.5 and in the temperature range of 20 and 50°C.