Crucial role of non-hydrophobic residues in H-region signal peptide on secretory production of l-asparaginase II in Escherichia coli.

Protein secretion into the periplasmic space requires several interactions between the amino-terminal signal sequence of the protein and secretion machinery components. Therefore, modification of the components of amino-terminal sequence can be used as a powerful strategy to improve secretion efficiency. The hydrophobic region is an important domain for signal peptide function due to interaction with different components of the secretion apparatus. In this study, to evaluate the effect of hydrophobicity level and secondary structure of the h-domain signal peptide on the secretion efficiency, a series of missense mutations were constructed in the hydrophobic domain of the l-asparaginase II signal peptide. The h-region hydrophobicity level of mutants G8L, T16L, and G8L/T16L was increased compared with the wild-type. In addition, the amino acid glycine as a helix-breaker residue was substituted with leucine (G8L), forming a stable and extended α-helix structure in the h-domain. The effect of introducing an aromatic residue in this region was also investigated by mutant G8F. Our mutagenesis studies showed that increasing the hydrophobicity levels, extending the α-helical conformation, and the introduction of an aromatic residue within the h-region signal sequence reduced the secretion level of asparaginase. These results imply a vital role of non-hydrophobic residues in the H-region.

Immobilization of modular peptides on graphene cocktail for differentiation of human mesenchymal stem cells to hepatic-like cells.

In this study, two novel biomimetic modular peptide motifs based on the alpha-2 subunit of type IV collagen (CO4A2) were designed and immobilized on a graphene platform to imitate integrin and heparan sulfate- (HS-) binding proteins. The in silico study was used to design 9-mer K[KGDRGD]AG and 10-mer KK[SGDRGD]AG for testing designed Integrin-Binding Peptide (dIBP) and HS-Binding Peptide (dHBP). The virtual docking technique was used to optimize the peptide motifs and their relevant receptors. Molecular dynamic (MD) simulation was used to evaluate the stability of peptide-receptor complexes. The effect of the platform on the differentiation of human mesenchymal stem cells (hMSCs) to hepatic-like cells (HLCs) was evaluated. After differentiation, some hepatic cells' molecular markers such as albumin, AFP, CK-18, and CK-19 were successfully followed. Graphene-heparan sulfate binding peptide (G-HSBP) enhances the mature hepatic markers' expression instead of control (p ≤ 0.05). The pathological study showed that the designed platform is safe, and no adverse effects were seen till 21 days after implantation.

Appraisal of SARS-CoV-2 mutations and their impact on vaccination efficacy: an overview.

With the unexpected emergence of the novel 2019 Wuhan coronavirus, the world was faced with a sudden uproar that quickly shifted into a serious life-threatening pandemic. Affecting the lives of the global population and leaving drastic damage in various sections and systems, several measures have been constantly taken to tackle down this crisis. For instance, numerous vaccines have been developed in the past two years, some of which have been granted emergency use, thus providing sufficient immunity to the vaccinated individuals. However, the appearance of newly emerged SARS-CoV-2 variants with accelerated transmission and fatality has led the world towards another pandemic. Having undergone various mutations in genomic and/or amino acid profiles, some of the emerged variants of concern (VOCs) including Alpha, Beta, Gamma, and Delta have displayed immune evasion and pathogenicity even in the vaccinated population, hence raising concerns regarding the efficacy of current vaccines against new VOCs of COVID-19. Therefore, genomic investigations of SARS-CoV-2 mutations are expected to provide valuable insight into the evolution of SARS-CoV-2, while also determining the impact of different mutations on infection severity. This study was constructed with the aim of shining light on recent advances regarding mutations in major COVID-19 VOCs, as well as vaccination efficacy against those VOCs.

Phenotype Compensation in Reproductive ADAM Gene Family: A Case Study with ADAM27 Knockout Mouse.

Background: A disintegrin and metalloproteinase (ADAM) cell surface proteins are expressed in different cells and are involved in biological processes such as cell-cell interactions, cell differentiation, sperm attachment and fertilization. A significant number of ADAM isoforms are expressed in the reproductive tracts of male mice and other mammals, which shows the importance of this gene family in reproduction. Objectives: The role of ADAM27 protein in reproduction was investigated. Materials and Methods: ADAM27 knock-out mutant mice were generated using the blastocyst microinjection technique. The knock-out mice were analyzed genetically and phenotypically to discover any abnormalities. Results: The results of this study revealed that the homozygote mutant male mice were fertile and showed no significant differences compared to wild-type male mice. A histological exam, sperm analysis and in-vitro fertilization experiments showed no statistical differences. Conclusions: We can conclude that the role of deficient ADAM27 protein is probably compensated mainly by other ADAM isoforms which are expressed in the reproductive system.

Depinar, a drug that potentially inhibits the binding and entry of COVID-19 into host cells based on computer-aided studies.

BACKGROUND AND PURPOSE: The new coronavirus (Covid-19) has resulted in great global concerns. Due to the mortality of this virus, scientists from all over the world have been trying to employ different strategies to tackle down this concern. This virus enters cells via phagocytosis through binding to the angiotensin-converting enzyme II receptor. After invading the body, it can stay hidden in there for a period of up to 24 days (incubation period). EXPERIMENTAL APPROACH: In this report, by the use of in silico studies we selected several FDA-approved compounds that possess antiviral properties. We chose the viral Spike protein as the target of drug compounds and carried out the screening process for the FDA databank in order to find the most effective ligand. FINDINGS/RESULTS: The results from dock and MD revealed 10 compounds with high affinity to the receptor-binding domain motif of S protein. The best inhibitors were the ingredients of Depinar, which managed to effectively block the interactions between cells and virus. CONCLUSION AND IMPLICATION: The results of this study were approved by in silico studies and due to the lack of time; we did not test the efficiency of these compounds through in vitro and in vivo studies. However, the selected compounds are all FDA approved and some are supplements like vitamin B12 and don't cause any side effects for patients.

Bottom-up synthesis of nitrogen and oxygen co-decorated carbon quantum dots with enhanced DNA plasmid expression.

In this paper, a bottom-up hydrothermal route is reported for the synthesis of oxygen and nitrogen co-decorated carbon quantum dots (CQDs) using ammonium hydrogen citrate (AHC) as a single precursor. DLS data approved the formation of 4.0 nm (average size) CQDs. XRD pattern shows the interlayer spacing (002) of 3.5 Šfor CQDs, which is exactly the same as that of crystalline graphite. XPS and FTIR spectra verified the formation of oxygen and nitrogen functional groups on the CQDs surface. Co-decorated carboxyl, hydroxyl and amine groups on the CQDs surfaces make them as promising polyelectrolyte for gene delivery. Toxicity assay showed a survival rate of 70% under different incubation times and up to 500 µg/mL. The highly water-soluble, stable fluorescence and low toxic CQDs increased the gene expression of DNA plasmid in E. coli bacteria 4-fold more than the control group.

An Efficient Approach for Two Distal Point Site-Directed Mutagenesis from Randomly Ligated PCR Products.

Site-directed mutagenesis is one of the most important tools in molecular biology. The majority of the mutagenesis methods have been developed to mutate one region of target DNA in each cycle of mutagenesis, while in some cases there is a need to mutate several distal points. We used a new method to simultaneously mutate two distal points in the target DNA. Different regions of the target DNA were amplified in three separate PCR reactions. The PCR products were back-to-back and together they made the complete length of the template DNA. Mutations were introduced to PCR products by middle mutagenic primers. PCR products were mixed and ligated with random blunt ligation, and then the desired mutated DNA fragments were selected in two steps by flanking restriction enzyme digestion and size selection. Selected fragments were amplified in another PCR reaction using flanking primers and finally cloned into the plasmid vector. This mutagenesis process is simple, there is no need to use modified primers and long or difficult PCR reactions.

Enhanced Gene Delivery in Bacterial and Mammalian Cells Using PEGylated Calcium Doped Magnetic Nanograin.

BACKGROUND: Beyond viral carriers which have been widely used in gene delivery, non-viral carriers can further improve the delivery process. However, the high cytotoxicity and low efficiency impedes the clinical application of non-viral systems. Therefore, in this work, we fabricated polyethylene glycol (PEG) coated, calcium doped magnetic nanograin (PEG/Ca(II)/Fe3O4) as a genome expression enhancer. METHODS: Monodisperse magnetic nanograins (MNGs) with tunable size were synthesized by a solvothermal method. The citrate anions on the spherical surface of MNGs capture Ca2+ ions by an ion exchange process, which was followed by surface capping with PEG. The synthesized PEG/Ca(II)/Fe3O4 was characterized using Fourier-transform infrared spectroscopy (FTIR), dynamic light scattering (DLS) spectra, vibrating sample magnetometer (VSM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). MTT test was utilized to assess the toxicity of PEG/Ca(II)/Fe3O4. Real time qPCR was applied for quantification of gene expression. RESULTS: DLS spectra and TEM images confirmed a thin layer of PEG on the nanocarrier surface. Shifting the zeta potential in the biological pH window from -23.9 mV (for Fe3O4) to ≈ +11 mV (for PEG/Ca(II)/Fe3O4) confirms the MNGs surface protonation. Cytotoxicity results show that cell viability and proliferation were not hindered in a wide range of nanocarrier concentrations and different incubation times. CONCLUSION: PEGylated calcium doped magnetic nanograin enhanced PUC19 plasmid expression into E. Coli and GFP protein expression in HEK-293 T cells compared to control. A polymerase chain reaction of the NeoR test shows that the transformed plasmids are of high quality.

Effect of graphene oxide nanosheets on visible light-assisted antibacterial activity of vertically-aligned copper oxide nanowire arrays.

In the present work, the effect of graphene oxide (GO) nanosheets on the antibacterial activity of CuO nanowire arrays under visible light irradiation is shown. A combined thermal oxidation/electrophoretic deposition technique was employed to prepare three-dimensional networks of graphene oxide nanosheets hybridized with vertically aligned CuO nanowires. With the help of standard antibacterial assays and X-ray photoelectron spectroscopy, it is shown that the light-activated antibacterial response of the hybrid material against gram-negative Escherichia coli is significantly improved as the oxide functional groups of the GO nanosheets are reduced. In order to explore the physicochemical mechanism behind this behavior, ab-initio simulations based on density functional theory were performed and the effect of surface functional groups and hybridization were elucidated. Supported by the experiments, a three-step photo-antibacterial based mechanism is suggested: (i) injection of an electron from CuO into rGO, (ii) localization of the excess electron on rGO functional groups, and (iii) release of reactive oxygen species lethal to bacteria. Activation of new photoactive and physical mechanisms in the hybrid system makes rGO-modified CuO nanowire coatings as promising nanostructure devices for antimicrobial applications in particular for dry environments.

A Study on Merits and Demerits of Two Main Gene Silencing Techniques in Mice.

BACKGROUND: Transgenic mice are being considered as invaluable tool in biological sciences towards comprehension of the cause of the genetic diseases. Manipulated embryonic stem (ES) cells are used to produce loss-of-function mutant mice. Microinjection of manipulated ES cells into blastocoel cavity, and morula fusion are the two main techniques in producing transgenic mice. So far, no reports have dealt with the comparison of these two methodologies provide. OBJECTIVE: The object of this study was to determine advantages and disadvantages of knockout mouse creation protocols. MATERIALS AND METHODS: Both blastocyst microinjection and morula aggregation were implemented to produce chimeric mice and the advantages and disadvantages of each technique were evaluated. For this, embryonic stem cells were transfected with a GFP-expression vector. In blastocyst microinjection technique, first transfected ES cell were cultured and appropriate colonies were selected. The cells were microinjected to blastocoel cavity of the expanded blastocyst. In morula aggregation technique, the transfected ES cell colonies were sandwiched between two naked morulas. After 16 h incubation in a 5% CO2 at 37 °C the morulas and infected ES cell were aggregated to produce a new morula. All the injected blastocyst and aggregated morulas were transferred to uterus of foster mice. The new born mice were analyzed for chimera confirmation. RESULTS: Five chimeric mice (21.75%) from morula aggregation and eight chimeric mice (63%) from blastocyst microinjection were born. The results indicated that both techniques can be used to generate chimeric mouse, however the success rate was higher in blastocyst microinjection. CONCLUSION: Morula fusion stands out where the required instrumentations are in place. Furthermore, the quality of ES cells plays a prominent role in the success rate. When the cell quality is low the blastocoel microinjection is recommended. The microinjection technique is more effective than morula aggregation.

Molecular Engineering of the Geobacillus stearothermophilus α-Amylase and Cel5E from Chlostridium thermocellim; In Silico Approach.

BACKGROUND: Considering natural thermal stability, Geobacillus stearothermophilus amylase and Cel5E from Clostridium thermocellum are good candidates for industrial applications. To be compatible with the industrial applications, this enzyme should be stable in the high temperatures, so any improvement in their thermal stability is valuable. OBJECTIVES: Using in silico approach and identifying point mutations in the structure amylase of G. stearothermophilus and Cel5E from C. termocellum we tried to increase thermal stability of the enzymes along with their catalytic activity to reach a new industrial amylase with higher thermostability and an improved function. MATERIALS AND METHODS: In this study we predicted the 3D structure of the enzymes, then simulated the molecular docking study using MolDock, PLANTS, and Lamarkian genetic algorithm as scoring functions for the docking and in silico engineering of the protein aiming to increase the thermal stability and catalytic activity. RESULTS: A series of thermal stability increasing point mutations were exerted around the active site of the enzyme, then by docking procedure, the binding affinity was measured and finally a list of mutations which theoretically improved the increased thermal stability as well as catalytic activity were proposed. CONCLUSIONS: Based on the in silico results obtained the modified enzymes seems to be suitable candidates for considering in both laboratory and industrial scales.