CRISPR/Cas9-mediated MSTN gene editing induced mitochondrial alterations in C2C12 myoblast cells

BackgroundMyostatin (MSTN) negatively regulates muscle mass and is a potent regulator of energy metabolism. However, MSTN knockout have affect mitochondrial function. This research assessed the mitochondrial energy metabolism of Mstn −/+ KO cells, and wondered whether the mitochondria biogenesis are affected.ResultsIn this study, we successfully achieved Mstn knockout in skeletal muscle C2C12 cells using a CRISPR/Cas9 system and measured proliferation and differentiation using the Cell-Counting Kit-8 assay and qPCR, respectively. We found that MSTN dysfunction could promote proliferation and differentiation compared with the behaviour of wild-type cells. Moreover, Mstn KO induced an increase in KIF5B expression. The mitochondrial content was significantly increased in Mstn KO C2C12 cells, apparently associated with the increases in PGC-1α, Cox1, Cox2, ND1 and ND2 expression. However, no differences were observed in glucose consumption and lactate production. Interestingly, Mstn KO C2C12 cells showed an increase in IL6 and a decrease in TNF-1α levels.ConclusionThese findings indicate that MSTN regulates mitochondrial biogenesis and metabolism. This gene-editing cells provided favourable evidence for animal breeding and metabolic diseases.How to cite: Wang L, Ding Q, Ma S, et al. CRISPR/Cas9-mediated MSTN gene editing Induced Mitochondrial Alterations in C2C12 myoblast Cells. Electron J Biotechnol 2019;40. https://doi.org/10.1016/j.ejbt.2019.03.009     

Cloning,characterization and expression of Peking duck fatty acid synthase during adipocyte differentiation

BackgroundFatty acid synthase (FAS) is a key enzyme of de novo lipogenesis (DNL), which has been cloned from several species: Gallus gallus, Mus musculus, Homo sapiens, but not from Anas platyrhynchos. The current study was conducted to obtain the full-length coding sequence of Peking duck FAS and investigate its expression during adipocyte differentiation.ResultsWe have isolated a 7654 bp fragment from Peking duck adipocytes that corresponds to the FAS gene. The cloned fragment contains an open reading frame of 7545 bp, encodes a 2515 amino acid protein, and displays high nucleotide and amino acid homology to avian FAS orthologs. Twelve hour treatment of oleic acid significantly up-regulated the expression of FAS in duck preadipocytes (P < 0.05). However, 1000 μM treatment of oleic acid exhibited lipotoxic effect on cell viability (P < 0.05). In addition, during the first 24 h of duck adipocyte differentiation FAS was induced; however, after 24 h its expression level declined (P < 0.05).ConclusionWe have successfully cloned and characterized Peking duck FAS. FAS was induced during adipocyte differentiation and by oleic acid treatment. These findings suggest that Peking duck FAS plays a similar role to mammalian FAS during adipocyte differentiation.     

Construction of an RNAi vector for knockdown of GM-ACS genes in the cotyledonary nodes of soybean

BackgroundEthylene plays an important role in the regulation of floral organ development in soybean, and 1-aminocyclopropane-1-carboxylate synthase (ACS) is a rate-limiting enzyme for ethylene biosynthesis. However, whether ACS also regulates floral organ differentiation in soybean remains unknown. To address this, we constructed an RNAi vector to inhibit ACS expression in cotyledonary nodes. Linear DNA cassettes of RNAi-ACS obtained by PCR were used to transform soybean cotyledonary nodes.ResultsIn total, 131 of 139 transiently transformed plants acquired herbicide resistance and displayed GUS activities in the new buds. In comparison to untransformed seedling controls, a greater number of flower buds were differentiated at the cotyledonary node; GM-ACS1 mRNA expression levels and ethylene emission in the transformed buds were reduced.ConclusionThese results indicate that the cotyledonary node transient transformation system may be suitable for stable transformation and that the inhibition of ACS expression may be an effective strategy for promoting floral organ differentiation in soybean.     

Inhibition of preadipocyte differentiation by Lycium barbarum polysaccharide treatment in 3T3-L1 cultures

BackgroundLycium barbarum (also called wolfberry), a famous Chinese traditional medicine and food ingredient, is well recognized for its significant role in preventing obesity; however, the molecular mechanisms underlying its preventive effects on fat accumulation are not well understood yet. The aim of this study was to determine the effects and mechanism of Lycium barbarum polysaccharides (LBP) on the proliferation and differentiation of 3T3-L1 preadipocytes. MTT was used to detect the proliferation of 3T3-Ll preadipocytes. Oil red O staining and colorimetric analysis were used to detect cytosolic lipid accumulation during 3T3-L1 preadipocyte differentiation. Real-time fluorescent quantitative PCR (qPCR) technology was used to detect peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα), adipocyte fatty-acid-binding protein (aP2), fatty acid synthase (FAS), and lipoprotein lipase (LPL) expression.ResultsThe concentration of LBP from 25 to 200 μg/mL showed a tendency to inhibit the growth of preadipocytes at 24 h, and it inhibited the differentiation of 3T3-L1 preadipocytes in a dose-dependent manner. In the preadipocytes treated with 200 μg/mL LBP, there were reduced lipid droplets in the cytoplasm, and its effect was opposite to that of rosiglitazone (ROS), which significantly reduced the PPARγ, C/EBPα, aP2, FAS, and LPL mRNA expression of adipocytes.ConclusionsLBP exerts inhibitive effects on the proliferation and differentiation of 3T3-L1 preadipocytes and decreases the cytoplasm accumulation of lipid droplets during induced differentiation of preadipocytes toward mature cells. Above phenomenon might link to lowered expression of PPARγ, C/EBPα, aP2, FAS, and LPL after LBP treatment. Thus, LBP could serve as a potential plant extract to treat human obesity or improve farm animal carcass quality via adjusting lipid metabolism.How to cite: Xu X, Chen W, Yu S, et al. Inhibition of preadipocyte differentiation by Lycium barbarum polysaccharide treatment in 3T3-L1 cultures. Electron J Biotechnol 2021;50. https://doi.org/10.1016/j.ejbt.2021.01.003     

Biochemical characterization of the recombinant schistosome tegumental protein SmALDH_312 produced in E. coli and baculovirus expression vector system

BackgroundThe heterologous expression of parasitic proteins is challenging because the sequence composition often differs significantly from host preferences. However, the production of such proteins is important because they are potential drug targets and can be screened for interactions with new lead compounds. Here we compared two expression systems for the production of an active recombinant aldehyde dehydrogenase (SmALDH_312) from Schistosoma mansoni, which causes the neglected tropical disease schistosomiasis.ResultsWe produced SmALDH_312 successfully in the bacterium Escherichia coli and in the baculovirus expression vector system (BEVS). Both versions of the recombinant protein were found to be active in vitro, but the BEVS-derived enzyme showed 3.7-fold higher specific activity and was selected for further characterization. We investigated the influence of Mg²⁺, Ca²⁺ and Mn²⁺, and found out that the specific activity of the enzyme increased 1.5-fold in the presence of 0.5 mM Mg²⁺. Finally, we characterized the kinetic properties of the enzyme using a design-of-experiment approach, revealing optimal activity at pH 7.6 and 41°C.ConclusionsAlthough, E. coli has many advantages, such as rapid expression, high yields and low costs, this system was outperformed by BEVS for the production of a schistosome ALDH. BEVS therefore provides an opportunity for the expression and subsequent evaluation of schistosome enzymes as drug targets.How to cite: Harnischfeger J, Beutler M, Salzig D, et al. Biochemical characterization of the recombinant schistosome tegumental protein SmALDH_312 produced in E. coli and baculovirus expression vector system. Electron J Biotechnol 2021;54. https://doi.org/10.1016/j.ejbt.2021.08.002     

Biomarker-free dielectrophoretic sorting of differentiating myoblast multipotent progenitor cells and their membrane analysis by Raman spectroscopy

Myoblasts are muscle derived mesenchymal stem cell progenitors that have great potential for use in regenerative medicine, especially for cardiomyogenesis grafts and intracardiac cell transplantation. To utilise such cells for pre-clinical and clinical applications, and especially for personalized medicine, it is essential to generate a synchronised, homogenous, population of cells that display phenotypic and genotypic homogeneity within a population of cells. We demonstrate that the biomarker-free technique of dielectrophoresis (DEP) can be used to discriminate cells between stages of differentiation in the C2C12 myoblast multipotent mouse model. Terminally differentiated myotubes were separated from C2C12 myoblasts to better than 96% purity, a result validated by flow cytometry and Western blotting. To determine the extent to which cell membrane capacitance, rather than cell size, determined the DEP response of a cell, C2C12 myoblasts were co-cultured with GFP-expressing MRC-5 fibroblasts of comparable size distributions (mean diameter ∼10 μm). A DEP sorting efficiency greater than 98% was achieved for these two cell types, a result concluded to arise from the fibroblasts possessing a larger membrane capacitance than the myoblasts. It is currently assumed that differences in membrane capacitance primarily reflect differences in the extent of folding or surface features of the membrane. However, our finding by Raman spectroscopy that the fibroblast membranes contained a smaller proportion of saturated lipids than those of the myoblasts suggests that the membrane chemistry should also be taken into account.     

Reactive aqueous two-phase systems for the production and purification of PEGylated proteins

BackgroundPEGylation, defined as the covalent attachment of polyethylene glycol, allows the synthesis of PEGylated therapeutic proteins with enhanced physicochemical properties. Traditional alkylating N-terminal PEGylation reactions on amine groups involve the use of modified linear mono-methoxy polyethylene glycol (mPEG) molecules looking for the synthesis of mono-PEGylated products. However, this approach requires different purification steps since inevitably undesired cross-linked products are synthesized. Herein, we propose the use of reactive aqueous two-phase systems (ATPS) to produce and purify PEGylated therapeutic conjugates using Ribonuclease A (RNase A) as a model protein.ResultsSelected linear 5 kDa and 20 kDa mPEG – potassium phosphate systems were produced according to equilibrium data obtained from constructed binodal curves. All reactive systems were able to generate biphasic systems and to PEGylate RNase A. Two 5 kDa and two 20 kDa systems were selected based on the reaction yield percentage and the feasibility of purifying the mono-PEGylated RNase A from the di-PEGylated and native RNase A by contrasting the differences in their partition behaviors. The remnant biological activity was of 94% and of 100% for the mono-PEGylated RNase A purified from the 5 kDa and 20 kDa mPEG systems when compared to the mono-PEGylated conjugate obtained by standard procurement methods.ConclusionsThis novel approach using reactive ATPS shows that it is feasible to simultaneously produce and purify PEGylated therapeutic proteins with conserved biological activity and presents another example where reactive ATPS can be successfully implemented.How to cite: Campos-García VR, Benavides J, González-Valdez J. Reactive aqueous two-phase systems for the production and purification of PEGylated proteins. Electron J Biotechnol 2021;54. https://doi.org/10.1016/j.ejbt.2021.09.003     

Therapeutic evaluation of galangin on cartilage protection and analgesic activity in a rat model of osteoarthritis

BackgroundOsteoarthritis (OA) is a form of arthritis due to degradation of articular cartilage. OA is associated with stiffness, joint pain, and dysfunction, affecting adults worldwide. Galangin is a bioactive flavonoid that exerts several therapeutic and biological activities. Anti-hyperglycemic, anti-inflammatory, anti-cancer, and anti-apoptotic activities of galangin have been reported in several studies. In the present study, rats were divided into normal control, OA (control), galangin 10 mg/kg (low-dose), galangin 100 mg/kg (high-dose), and celecoxib 30 mg/kg (positive control) groups. All doses were administered orally for 14 consecutive days. The urinary type II collagen (µCTX-II) level as well as reactive oxygen species, tumor necrosis factor-alpha, interleukin-1 beta, interleukin-6, superoxide dismutase, catalase, lipid peroxidation, reduced glutathione, and glutathione peroxidase levels were measured. In addition, the CTX-II mRNA and protein expression levels were measured.ResultsGalangin supplementation significantly reduced the µCTX-II level compared with controls. Galangin treatment significantly reduced reactive oxygen species, lipid peroxidation, interleukin-1 beta, interleukin-6, and tumor necrosis factor-alpha levels, but increased catalase, superoxide dismutase, glutathione peroxidase, and reduced glutathione levels. Galangin treatment significantly reduced the CTX-II mRNA and protein expression levels. The low CTX-II level in tissue indicated the inhibition of cartilage degradation.ConclusionsIn summary, supplementation with galangin was effective against OA. The identification of potential therapeutic agents that inhibit inflammation may be useful for the management and prevention of OA.How to cite: Su Y, Shen L, Xue J, et al. Therapeutic evaluation of galangin on cartilage protection and analgesic activity in a rat model of osteoarthritis. Electron J Biotechnol 2021;53. https://doi.org/10.1016/j.ejbt.2021.05.005     

High-level soluble expression of phospholipase D from Streptomyces chromofuscus in Escherichia coli by combinatorial optimization

BackgroundPhospholipase D (PLD) is used as the biocatalyst for phosphatidylserine (PS) production. In general, PLD was expressed in insoluble form in Escherichia coli. High-level soluble expression of PLD with high activity in E. coli is very important for industrial production of PLD.ResultsStreptomyces chromofuscus PLD coding gene was codon-optimized, cloned without signal peptide, and expressed in E. coli. The optimal recombinant E. coli pET-28a+PLD/BL21(DE3) was constructed with pET-28a without His-tag. The highest PLD activity reached 104.28 ± 2.67 U/mL in a 250-mL shake flask after systematical optimization. The highest PLD activity elevated to 122.94 ± 1.49 U/mL by feeding lactose and inducing at 20°C after scaling up to a 5.0-L fermenter. Substituting the mixed carbon source with 1.0 % (w/v) of cheap dextrin and adding a feeding medium could still attain a PLD activity of 105.81 ± 2.72 U/mL in a 5.0-L fermenter. Fish peptone from the waste of fish processing and dextrin from the starch are both very cheap, which were found to benefit the soluble PLD expression.ConclusionsAfter combinatorial optimization, the high-level soluble expression of PLD was fulfilled in E. coli. The high PLD activity along with cheap medium obtained at the fermenter level can completely meet the requirements of industrial production of PLD.How to cite: Wu R, Cao J, Liu F, et al. High-level soluble expression of phospholipase D from Streptomyces chromofuscus in Escherichia coli by combinatorial optimization. Electron J Biotechnol 2021;50.https://doi.org/10.1016/j.ejbt.2020.12.002     

Expression of Pinellia ternata leaf agglutinin under rolC promoter confers resistance against a phytophagous sap sucking aphid,Myzus persicae

BackgroundPiercing/sucking insect pests in the order Hemiptera causes substantial crop losses by removing photoassimilates and transmitting viruses to their host plants. Cloning and heterologous expression of plant-derived insect resistance genes is a promising approach to control aphids and other sap-sucking insect pests. While expression from the constitutive 35S promoter provides broad protection, the phloem-specific rolC promoter provides better defense against sap sucking insects. The selection of plant-derived insect resistance genes for expression in crop species will minimize bio-safety concerns.ResultsPinellia ternata leaf agglutinin gene (pta), encodes an insecticidal lectin, was isolated and cloned under the 35S and rolC promoters in the pGA482 plant transformation vector for Agrobacterium-mediated tobacco transformation. Integration and expression of the transgene was validated by Southern blotting and qRT-PCR, respectively. Insect bioassays data of transgenic tobacco plants showed that expression of pta under rolC promoter caused 100% aphid mortality and reduced aphid fecundity up to 70% in transgenic tobacco line LRP-9. These results highlight the better effectivity of pta under rolC promoter to control phloem feeders, aphids.ConclusionsThese findings suggested the potential of PTA against aphids and other sap sucking insect pests. Evaluation of gene in tobacco under two different promoters; 35S constitutive promoter and rolC phloem-specific promoter could be successfully use for other crop plants particularly in cotton. Development of transgenic cotton plants using plant-derived insecticidal, PTA, would be key step towards commercialization of environmentally safe insect-resistant crops.How to citeUmer N, Naqvi RZ, Rauf I, et al. Expression of Pinellia ternata leaf agglutinin under rolC promoter confers resistance against a phytophagous sap sucking aphid, Myzus persicae. Electron J Biotechnol 2020;47. https://doi.org/10.1016/j.ejbt.2020.07.004.     

Effect of miR-125b on dermal papilla cells of goat secondary hair follicle

BackgroundMicroRNAs (miRNAs) are endogenous noncoding RNAs that regulate various biological processes. miR-125b is a miRNA that has been reported to be critical for hair follicle (HF) morphogenesis and development. We identified that the expression of miR-125b varies during an individual hair cycle (anagen, catagen, and telogen) in the skin of cashmere goats. We constructed a gain model (by overexpressing miR-125b) and a loss model (by inhibiting endogenous miR-125b) based on dermal papilla cells (DPCs) to further investigate the role of miR-125b in HF cycle. In addition, we used a dual-luciferase system to highlight the predicated target genes of miR-125b.ResultsWe found that miR-125b affects the expression of FGF5, IGF-1, SHH, TNF-α, MSX2, LEF-1, FGF7, NOGGIN, BMP2, BMP4, TGF-β1, and β-catenin. The dual-luciferase assay further validated a direct interaction between miR-125b and FGF5 and TNF-α.ConclusionmiR-125b affects the expression levels of genes related to hair cycle and may also play a critical role in regulating the periodic development of HF.     

Identification of key genes and construction of CircRNA–miRNA–mRNA regulatory networks in osteoarthritis

BackgroundOsteoarthritis (OA) is one of the most frequent degenerative joint diseases with high rate of disability, but its mode of action remains largely unclear. The current study was aimed at identifying key genes and molecular mechanism in OA. Gene expression datasets (GSE1919, GSE55235, GSE55457) were downloaded from Gene Expression Omnibus for integrated bioinformatics analysis. Differentially expressed genes (DEGs) in OA synovial tissues were identified using GeoDiver and GEO2R. Gene ontology enrichment analyses were undertaken via FunRich and Metascape. Also, Gene Set Enrichment Analysis was performed using miRWalk3.0. Subsequently, pathways interrelation analysis of hub genes was carried out using plug-in ClueGO v2.3.3. Additionally, circRNA–miRNA–mRNA regulatory networks were visualized using Cytoscape.ResultsA total of 508 DEGs were obtained from three GSE datasets, of these five intersection DEGs (TNFAIP3, VEGFA, GADD45B, SIK1, KLF9) were shared by three GSE datasets. Intersection DEGs were significantly enriched in LKB1 signaling events, signaling events mediated by focal adhesion kinase, and PDGFR-beta signaling pathway. Enrichment analysis for all the DEGs showed that they mainly enriched in inflammatory response, cytokine production, blood vessel development, stress response, osteoclast differentiation, and MAPK signaling pathway. A total of 39 genes were regarded as hub genes and pathways interrelation analysis indicated that hub genes mainly enriched in TNF signaling pathway, IL-17 signaling pathway, and NF-kappa B signaling pathway.ConclusionsThe current study revealed the potential key genes, pathways, and circRNA–miRNA–mRNA regulatory networks in OA, which may contribute to a more comprehensive understanding of OA pathogenesis.How to cite: Li HZ, Xu XH, Lu HD. Identification of key genes and construction of CircRNA–miRNA–mRNA regulatory networks in osteoarthritis. Electron J Biotechnol 2019;37. https://doi.org/10.1016/j.ejbt.2018.11.004.