Effective and economical column-based method for RNA isolation from animal cells Abstract Objectives To develop an efficient, economical, and low-toxicity method for the extraction of RNA from animal cells to meet a basic requirement of biological research: the isolation of high-quality RNA. Results Guanidine hydrochloride was used as a lysis buffer and Na-acetate was used as a wash buffer to extract RNA fragments from TM3 Leydig cells and ovarian granulosa cells efficiently. The functionality of the extracted RNA samples was verified through polymerase chain reaction (PCR) and real-time fluorescence quantitative PCR (RT-PCR). PCR results showed that the normal DNA column-based method could guarantee RNA integrity and could be used to amplify gene fragments successfully. RT-PCR analysis showed that the RNA samples isolated through the proposed method could be used to detect the expression levels of steroidogenic acute regulatory protein and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 mRNA in TM3 Leydig cells under induction by luteinizing hormone. The proposed method could be used to isolate RNA from mammalian cells and provided RNA yields of > 120 ng/5 × 106 cells. This method provided RNA with purities and yields that are sufficient for cDNA synthesis and PCR amplification in gene expression studies. Conclusions The proposed RNA extraction method has the advantages of low toxicity, safe handling, and low cost. Isolation can be completed in 20 min. The proposed method can be used to extract RNA from various animal cell samples and is worth promoting.

SpyTag/SpyCatcher cyclization enhances the thermostability and organic solvent tolerance of l -phenylalanine aldolase Abstract Objectives To improve the thermostability and organic solvent tolerance of L-phenylserine aldolase, the in vivo SpyTag/SpyCatcher cyclization strategy was applied in this work. Results The in vivo cyclization of L-phenylserine aldolase was achieved by fusing the tags of SpyCatcher and SpyTag to the N- and C-termini of the enzyme, respectively. The kcat values and the circular dichroism spectra of the linear and cyclized LPAs are very similar, indicating that the cyclized LPA can be folded appropriately like the wild type. The cyclized enzyme has better thermostability and organic solvent tolerance than does the wild type. The half-life of L-phenylserine aldolase after cyclization was increased by 8.3 times at 70 °C, and the T50 also increased from 56.8 to 67.1 °C. The cyclized enzyme showed a remarkably higher tolerance to organic solvents (e.g., methanol, ethanol and acetone). Conclusions These results suggest that the in vivo cyclization using SpyTag/SpyCatcher is an effective strategy to improve the stability of enzymes, which potentially could be applied in industrial bioconversion.

Identification of cis -regulatory regions responsible for developmental and hormonal regulation of HbHMGS1 in transgenic Arabidopsis thaliana Abstract Objectives 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase (HMGS) is an important enzyme in mevalonate (MVA) pathway of isoprenoid biosynthesis, which regulates the rubber biosynthetic pathway in rubber tree (Hevea brasiliensis) in coordination with HMG-CoA reductase (HMGR). However, little information is available about the regulation of HMGS gene expression. To understand the mechanism controlling the HbHMGS1 gene expression, we characterized the HbHMGS1 promoter sequence in transgenic plants with the β-glucuronidase (GUS) reporter gene. Results GUS activity analysis of the transgenic plants showed that the HbHMGS1 promoter is active in all organs of the transgenic Arabidopsis plants during various developmental stages (from 6 to 45-day-old). Deletion of different portions of the upstream HbHMGS1promoter identified sequences responsible for either positive or negative regulation of the GUS expression. Particularly, the − 454 bp HbHMGS1 promoter resulted in a 2.19-fold increase in promoter activity compared with the CaMV 35S promoter, suggesting that the − 454 bp HbHMGS1 promoter is a super-strong near-constitutive promoter. In addition, a number of promoter regions important for the responsiveness to ethylene, methyl jasmonate (MeJA) and gibberellic acid (GA) were identified. Conclusion The − 454 bp HbHMGS1 promoter has great application potential in plant transformation studies as an alternative to the CaMV 35S promoter. The HbHMGS1 promoter may play important roles in regulating ethylene-, MeJA- and GA-mediated gene expression. The functional complexity of cis-elements revealed by this study remains to be elucidated.

Preincubation with a low concentration of methyl-β-cyclodextrin enhances baculovirus expression system productivity Abstract Objectives To enhance the productivity of foreign protein in culture cells using baculovirus expression system. Results A low concentration of MβCD, with the optimal application concentration of 0.25 mM and the appropriate preincubation time range from 10 to 120 min, can efficiently enhance expression levels in both the AcMNPV and BmNPV expression systems. Conclusions Preincubation with a low concentration MβCD enhance baculovirus infection and foreign protein expression productivity.

An optimized yeast display strategy for efficient scFv antibody selection using ribosomal skipping system and thermo resistant yeast Abstract Objectives Establish a method to restrict unexpected fragments including stop codons in scFv library and generate a thermo resistant strain for screening of thermal stable scFv sequences. Results Here, we have constructed a T2A–Leu2 system for selection of yeast surface display libraries that blocks amplification of “stop codon” plasmids within the library, thereby increasing the quality of the library and efficiency of the selection screen. Also, we generated a temperature-resistant yeast strain, TR1, and validated its combined use with T2A–Leu2 for efficient screening. Thus, we developed a general approach for a fast and efficient screening of scFv libraries using a ribosomal skipping system and thermo-resistant yeast. Conclusions The method highlights the utility of the T2A–Leu2-based ribosomal skipping strategy for increasing the quality of the input library for selection, along with an optimized selection protocol based on thermo-resistant yeast cells.

Lactobacillus rhamnosus GG microcapsules inhibit Escherichia coli biofilm formation in coculture Abstract Objectives Microbial biofilms have become one of the most significant causes of nosocomial infections. The aim of this study was to examine the potential quorum sensing inhibitor activities of Lactobacillus rhamnosus GG microcapsules. Results Lactobacillus rhamnosus GG microcapsules effectively inhibited initial biofilm formation at a concentration of 2.5 × 108 CFU/mL. Furthermore, the inhibition rate was increased to 79% in the Lactobacillus rhamnosus GG microcapsules group, resulting in a reduction in the biofilm maturation stage. In addition, real-time PCR analysis revealed that the LGG microcapsules can act as effective inhibitors of transcriptional activators of the quorum sensing circuit in E.coli, luxS, lsrK, and lsrR. Conclusions Lactobacillus rhamnosus GG microcapsules can effectively inhibit biofilm formation and disturb mature biofilms.

Extracellular biosynthesis of silver nanoparticles from Penicillium italicum and its antioxidant, antimicrobial and cytotoxicity activities Abstract Objectives The synthesis of silver nanoparticles and its antioxidant, antimicrobial and cytotoxic activities was investigated and extracellularly biosynthesized using Penicillium italicum isolated from Iraqi lemon fruits. Results The formation of synthesized nanoparticles was observed after 72 h of incubation. Color changing, ultraviolet and visible spectrum, X-ray diffraction, Fourier transform infrared spectroscopy, scanning electron microscopy, transmission electron microscopy and energy dispersive analysis X- ray confirmed the biosynthesis and characterization of silver nanoparticles. Ultraviolet and visible spectroscopy showed silver surface plasmon resonance band at 415 nm. X- ray diffraction showed that the particles were crystalline with face-centered cubic lattice phase and a size of 39.5 nm. Fourier transform infrared analysis shows the possible interactions between silver and bioactive molecules, which may be responsible for synthesis and stabilization of silver nanoparticles. Scanning electron microscopy imaging revealed different morphologies of the AgNPs, some of nanoparticles were close to spherical in shape, while others had platelet like structure, in size range between 32 and 100 nm. The transmission electron microscopy image demonstrated that AgNPs were spherical with a size < 50 nm. The biosynthesized silver nanoparticles were proved to be potential antioxidants by showing effective radical scavenging activity against 2, 2-diphenyl-1-picrylhydrazyl, hydroxyl radical and resazurin. The nanoparticles also showed potent antimicrobial activity against various pathogens, including bacteria and fungi as well as significant concentration-dependent cytotoxic effects against human breast cancer cells. Conclusions The powerful bioactivity of the synthesized silver nanoparticles recommends their biomedical use as antioxidant, antimicrobial as well as cytotoxic agents.

Insights into the hydrolytic activity of Asclepias fruticosa L. protease Abstract Objective To determine the enzymatic properties of asclepain f, a plant cysteine protease isolated and purified from the latex of Asclepias fruticosa, and to investigate its potential application to hydrolyze soybean proteins. Results Kinetic parameters were determined by hydrolysis of p-Glu-Phe-Leu-p-nitroanilide (PFLNA). The Km value for asclepain f was 6 to 8 times higher than those achieved for papain, bromelain and ficin, the main plant cysteine proteases. Asclepain f showed 12 cut-off points toward the oxidized B chain insulin, revealing that the enzyme possesses broad substrate specificity. The cut specificity was governed by the presence of hydrophobic residues (F, L, V) in the P2 position. Asclepain f was able to selectively hydrolyze soybean proteins at pH 10, employing an enzyme/substrate ratio of 0.2% (w/w). The enzymatic hydrolysis allowed a strong increase in the solubility, water and oil holding capacity. Conclusions Asclepain f was revealed as a successful enzyme for biocatalysis of protein hydrolysis processes at alkaline pH. This new plant protease has a broad substrate specificity and is capable of selectively degrading the fractions of soy proteins and improving its functional properties.

Basic concepts, current evidence, and future potential for gene therapy in managing cutaneous wounds Abstract Objective Several studies have investigated the role of gene therapy in the healing process. The aim of this review is to explain the gene delivery systems in wound area. Results Ninety-two studies were included and comprehensively overviewed. We described the importance of viral vectors such as adenoviruses, adeno-associated viruses, and retroviruses, and conventional non-viral vectors such as naked DNA injections, liposomes, gene gun, electroporation, and nanoparticles in achieving high-level expression of genes. Application of viral transfection, liposomal vectors, and electroporation were the main gene delivery systems. Genes encoding for growth factors or cytokines have been shown to result in a better wound closure in comparison to application of the synthetic growth factors. In addition, a combination of stem cell and gene therapy has been found an effective approach in regeneration of cutaneous wounds. Conclusions This article gives an overview of the methods and investigations applied on gene therapy in wound healing. However, clinical investigations need to be undertaken to gain a better understanding of gene delivery technologies and their roles in stimulating wound repair.

Antibacterial mode of fibrauretine and synergistic effect with kanamycin against multi-drug resistant Escherichia coli Abstract Objective The present study evaluated the antibacterial activity and mode of action of fibrauretine on Escherichia coli (E. coli) and Staphylococcus aureus, and synergistic effect with kanamycin against multi-drug resistant E. coli. Results The fibrauretine exhibited inhibitory effect on the growth of the tested bacteria with minimum inhibitory concentration (MIC) and minimum bactericidal concentration of 2.5–5 and 5–10 mg/ml, respectively. Morphological changes of cell microstructure were observed after adding fibrauretine at MIC. The mode of action was further confirmed by measuring release of 260-nm absorbing materials and extracellular potassium ions. Checkerboard dilution test suggested that fibrauretine exhibited synergistic activity when combined with kanamycin (FICI ranging from 0.5625 to 0.625). Conclusions Our results indicated that fibrauretine exerted synergistic effect with kanamycin and its antibacterial mode of action mainly attributed to disruption of cell membrane integrity.