Preparation and Characterization of Different Multilayer Alginate Microcapsules

Introduction: Microencapsulation technology is a valuable technique for protection and delivery of materials which cannot be administered alone due to their low solubility, volatility and etc. A biopolymer alginate has been used most commonly as microcapsule forming material. In order to increase the mechanical stability and safety of alginate microcapsules, multilayered microcapsules are prepared. Alginate-poly l-lysine alginate, is the oldest multilayered microcapsule. Introducing another polycationic polymer which has similar properties to poly l-lysine (PLL) and also is  much more cost effective , so it might be a valuable suggestion. The aim of this study was to compare linear (LPEI) and branched (BPEI) form of polyethylene imine, with the oldest and rather expensive one, PLL, and also, we have compared sodium cellulose sulfate (NCS) as an anionic layer with sodium alginate in outer layer of multilayered microcapsules to investigate the effect of different covering layers in microcapsule’s cytotoxicity. Methods and Results: In this study by using electrostatic bead generator different types of microcapsules, APA, ALA and ABA were produced. Shape, size, surface morphology, mechanical stability and cytotoxicity of microcapsules were evaluated using optical microscope, SEM, explosion test and MTT assay respectively. According to shape and size evaluation, multilayered microcapsules with different cationic layer concentrations (0.01, 0.03 and 0.06 W/V %) were spherical, with a diameter range of 500- 900 μm. SEM images showed uniform and smooth surfaces. Explosion test revealed that applying cationic solutions with 0.03% and 0.01% concentration resulted in higher mechanical stability for ALA and APA in comparison to ABA (P<0.05), while mechanical strength induced by cationic solutions with 0.06% concentration were not statistically different in all three groups of microcapsules (P>0.05). MTT assay on HepG2 cell line was performed using microcapsules ALA, ALN, APA and APN and showed no statistically significant difference in cell viability for the all types. Conclusions: According to our results, LPEI as a covering layer for alginate microcapsules showed the same properties as PLL. Therefore it could be introduced as a cost effective alternative to PLL in fabrication of multilayered microcapsules

Search for new particles in events with energetic jets and large missing transverse momentum in proton-proton collisions at root s=13 TeV

A search is presented for new particles produced at the LHC in proton-proton collisions at root s = 13 TeV, using events with energetic jets and large missing transverse momentum. The analysis is based on a data sample corresponding to an integrated luminosity of 101 fb(-1), collected in 2017-2018 with the CMS detector. Machine learning techniques are used to define separate categories for events with narrow jets from initial-state radiation and events with large-radius jets consistent with a hadronic decay of a W or Z boson. A statistical combination is made with an earlier search based on a data sample of 36 fb(-1), collected in 2016. No significant excess of events is observed with respect to the standard model background expectation determined from control samples in data. The results are interpreted in terms of limits on the branching fraction of an invisible decay of the Higgs boson, as well as constraints on simplified models of dark matter, on first-generation scalar leptoquarks decaying to quarks and neutrinos, and on models with large extra dimensions. Several of the new limits, specifically for spin-1 dark matter mediators, pseudoscalar mediators, colored mediators, and leptoquarks, are the most restrictive to date.Peer reviewe

Probing effective field theory operators in the associated production of top quarks with a Z boson in multilepton final states at root s=13 TeV

Peer reviewe

The improvement of anti-HER2 scFv soluble expression in Escherichia coli

The relationship between the expression of HER2 and malignity of breast tumors has led to the generation of antibodies targeting HER2+ tumors. In addition, the expression of scFvs, as the smallest antigen-binding region of antibody containing two disulfide bonds in Escherichia coli often results in accumulating non-functional protein in the cytoplasm. A redox-modified strain of E. coli such as Origami (DE3) may facilitate the formation of proper disulfide bond in cytoplasm. The present study aimed to optimize the expression of anti-HER2 scFv in Origami and evaluate the influence of induction temperature, and host strain on the solubility of the protein. To this aim, chemically synthesized anti-HER2 scFv of Trastuzumab was cloned in pET-22b (+). The results demonstrated that anti-HER2 scFv is expressed in Origami, purified by using Ni-NTA column, and detected by anti-His antibody in Western blot analysis. The highest anti-HER2 scFv expression in Origami was achieved 24 h after IPTG induction (1 mM) at 37 °C. Further, the total anti-HER2 scFv expression level was higher in BL21, compared to Origami strain. However, the ratio of soluble/insoluble forms of anti-HER2 scFv increased in Origami strain. Furthermore, higher soluble expression was achieved when the culture of recombinant Origami was conducted at lower temperature (25 °C)

Enhanced Solubility of Anti-HER2 scFv Using Bacterial Pelb Leader Sequence: Influence of leader sequence on anti-HRR2 scFv expression

Single chain Fragment variable (scFv) is an antibody fragment consisting variable regions of heavy and light chains. scFvs enhance their penetrability into tissues while maintaining specific affinity and having low immunogenicity. Insoluble inclusion bodies are formed when scFvs are expressed in reducing bacterial cytoplasm. One strategy for obtaining functionally active scFv is to translocate the scFv into the oxidized environment of the periplasm where the possibility for disulfide bond formation is increased. This can be achieved by cloning the gene in a vector containing N-terminal pelB leader peptide that exports foreign proteins to the periplasmic space. The aim of this study is to evaluate the influence of periplasmic localization using pelB leader peptide on the solubility of anti-HER2 scFv. Herein, anti-HER2 scFv gene was cloned between NcoI and XhoI sites of pET-22b (+) containing pelB leader peptide and in the same sites of pET-28b (+) (without pelB). The expression in BL21 (DE3) was induced using IPTG and was analyzed using SDS-PAGE and Western blot experiment. Then, the solubility of anti-HER2 scFv in BL21 (DE3) containing both pET-22 and pET-28 (anti-HER2 scFv) was determined. The results of the present study demonstrated that anti-HER2 scFv was expressed by both pET-22b (+) (+) and pET-28b (+) vectors in BL21 (DE3). The proper expression of anti-HER2 scFv was confirmed by appearance of a ~ 28 kDa band in Western blot analysis. The most anti-HER2 scFv expression from BL21 containing pET-28 (anti-HER2 scFv) was achieved when it was induced by 0.25 mM IPTG at 37 ᵒC, 24 h post-induction. The ratio of soluble/insoluble anti-HER2 scFv was significantly higher in BL21 containing pET22-(anti-HER2 scFv) than in that containing pET-28 (anti-HER2 scFv). Totally, the fusion of pelB signal sequence to anti-HER2 scFv resulted in solubility enhancement. Therefore, the production of functional anti-HER2 scFv with proper disulfide bond can be achieved by directing the recombinant protein to periplasmic space using pelB signal peptide in pET-22b (+) vector

Soluble Expression of Recombinant Human Bone Morphogenetic Protein-7 (rhBMP-7) in Escherichia coli Using SUMO Fusion System: Soluble expression of rhBMP-7 in E. coli using SUMO fusion

BMPs belong to transforming growth factor β superfamilies, which their principal role is inducing bone and cartilage formation at heterotopic and orthotopic sites. Since the formation of inclusion bodies is the main limitation of producing these proteins in Escherichia coli, in this study, the small ubiquitin-like modifiers (SUMO) fusion system was employed to improve solubility and expression of recombinant human BMP-7 (rhBMP-7) in E. coli. The SUMO fusion system has the ability to enhance protein expression, reduce target protein proteolytic degradation, and increase protein folding and solubility. In the current study, the SUMO protein gene was fused to the N-terminus of the BMP-7 gene, and cloned in the pET-28a vector. After purification of the expressed SUMO-BMP-7 protein by Ni-NTA chromatography, SUMO was removed from the BMP-7 protein using SUMO protease. In the second step of purification using Ni-NTA chromatography, the cleaved BMP-7 protein was purified and then identified by Western blot analysis. The results of the current study demonstrated that the SUMO fusion system is able to increase the soluble form of rhBMP-7. Furthermore, rhBMP-7 can be purified by a two-step purification strategy including: 1) purification of SUMO-BMP-7 and 2) purification of rBMP-7 after cleavage using Ni-NTA chromatography. Altogether, this research has provided a feasible approach for large-scale production of soluble rhBMP-7, to facilitate its further medical development. HIGHLIGHTS SUMO is a well characterized family of ubiquitin-like molecules. SUMO fusion led to increased expression and solubility of BMP-7. BMP-7 is involved in the process of bone formation

Cytoplasmic Expression of Human Bone Morphogenetic Protein-7 by a Genetically Engineered Strain of Escherichia coli, SHuffle® Strain: Human BMP-7 soluble expression by SHuffle® strain

Homodimeric bone morphogenetic protein-7 (BMP-7) plays a key role in bone metabolism. The functionality of human BMP-7 protein is dependent on its disulfide bond formation and proper folding. Therefore, the expression of soluble recombinant BMP-7 using Escherichia coli cells as the host remains a challenge. Given the need for these disulfide-bonded proteins for stabilized native conformation, the cytoplasm of SHuffle® T7 Express as an E. coli engineered strain can effectively fold disulfide-bonded proteins with a need for proper oxidative folding. These cellular features turn the SHuffle® expression system into an efficient host for the recombinant production of human BMP-7 protein. A soluble dimeric form of recombinant human BMP-7 (rhBMP-7) which has a wide range of applications in medicine and can be used in the treatment of bone defects was produced using the SHuffle® strain as the expression system. This study demonstrated the production of rhBMP-7 using E. coli SHuffle® T7 Express strain. Also, an effective protocol was proposed for the expression and purification of soluble human BMP-7. In addition, it was found that the genetically engineered SHuffle® strain can efficiently enhance the solubility of recombinant human BMP-7 as a therapeutic target. HIGHLIGHTS                                                              E. coli SHuffle® T7 Express strain is an effective host to express disulfide-bonded proteins. BMP-7 is involved in the process of bone formation. Expression of human BMP-7 in SHuffle® strain increased its solubility. &nbsp