Electronic Journal of Biotechnology最新文献

Background

The lack of reference genes makes it difficult to conduct molecular biology research on the plant genus, Clematis L. Clematis lanuginosa belongs to Sect. Viticella DC of Clematis L. It is also an important ornamental cultivated variety parent of the early and late large-flowered groups. Studying the reference genes of C. lanuginosa in different tissues will provide a theoretical basis for the reference selection of early and late large-flowered groups of Clematis, which could promote research progress on molecular biology of ornamental Clematis.

Results

The roots, stems, leaves, sepals, stamens, and carpels of C. lanuginosa were used as research materials, and seven candidate reference genes were used for quantitative real-time PCR analysis. Comprehensive stability analysis using geNorm, NormFinder, BestKeeper, and RefFinder software showed that suitable reference genes in C. lanuginosa root, stem, and leaf were PP2A-2 and UBC34; and in floral tissue were UBC34, PP2A-2, and ARP7. These reference genes can be used as internal reference either alone or in combination. The pairwise variation value evaluated with geNorm software showed that two internal reference genes were needed for gene expression correction in the tissues. In the floral organs, three reference genes were required for gene expression correction.

Conclusions

Our results provide a foundation for future gene expression analysis of C. lanuginosa and guidance for the screening of reference genes in Clematis.

How to cite: Li Q, Wang S, Lv F, et al. Selection and validation of reference genes for quantitative real-time PCR in different tissues of Clematis lanuginosa. Electron J Biotechnol 2024;70. https://doi.org/10.1016/j.ejbt.2024.04.005.

Background

Cold damage of maize during germination is a global problem; it occurs frequently in northeast China, and leads to a large-scale reduction in yield. Low temperature tolerance of maize in germination is a complex quantitative trait controlled by multigenes, and no major QTLs or key genes have been identified.

Results

An F₂ isolation population with S319 and R144 as parents was constructed. The bulked segregant analysis (BSA) and specific-locus amplified fragment-sequencing (SLAF-seq) methods were applied to locate the chromosomal association regions related to low-temperature tolerance of maize during germination. Sequencing obtained 221.72 Gbp clean data, with an average sequencing depth of 25.96X. Four candidate regions associated with low-temperature tolerance trait of maize in germination were obtained, with a total length of 25.71 Mb and 1513 annotated genes, including 456 nonsynonymous mutant genes and 111 frameshift mutant genes.

Conclusions

This study aimed to lay the foundation for the mining of candidate genes of low-temperature tolerance in maize during germination, and accelerate the process of targeted improvement of maize low-temperature tolerance molecular marker-assisted breeding.

How to cite: Yu T, Zhang J, Cao J, et al. QTL analysis of low temperature tolerance in maize germination by SLAF-seq and BSA technique. Electron J Biotechnol 2024;70. https://doi.org/10.1016/j.ejbt.2024.04.003.

Background

Cancer continues to be one of the greatest challenges in modern medicine and is second only to cardiovascular disease as the main cause of death. Breast cancer in particular is responsible for 15% of deaths in women. In this study, Lactobacillus bulgaricus was microencapsulated using a chitosan/alginate mixture. Parameters such as chitosan, alginate, and L. bulgaricus populations were optimized using Design Expert software. The responses were loading efficiency, particle size, release, and ζ-potential. Subsequently, the cytotoxicity of the optimized ratio of chitosan/alginate nanoparticles was investigated on MCF-7 cancer cells.

Results

The research revealed that optimal conditions for the mentioned variables were a chitosan concentration of 1% w/w, an alginate concentration of 1% w/w, and a L. bulgaricus count of 8.15 CFU/ml. Following numerical optimization, the loading efficacy = 91.15%, the release = 71.55%, the polydispersity index = 0.11, and the ζ-potential = 61.94 based on numerical optimization. Findings revealed that miracle drinks with L. bulgaricus-loaded chitosan/alginate microcapsule ratios exhibited toxic and potential apoptotic effects on MCF-7 cancer cells. This study showed that a miracle drink prepared with the optimal ratio of probiotic nanoparticles stops cells in the S and G2/M phases.

Conclusions

The results show that Miracle drink supplemented with L. bulgaricus loaded-chitosan/alginate nanoparticles has a toxic and lethal effect on MCF-7 cancer cells. This compound can be suggested and used as an alternative candidate or complementary cancer therapy.

How to cite: Jovaini K, Mortazavian Farsani SAM, Aghaee-Bakhtiari SH, et al. Miracle drink supplemented with L.bulgaricus loaded-chitosan/alginate nanoparticles as a medicinal food for control of MCF7 cancer cells. Electron J Biotechnol 2024;69. https://doi.org/10.1016/j.ejbt.2024.04.002.

Background

Coffee fermentation process influences the final coffee composition and the sensory aspects which define the quality of the beverage. In this study, coffee fruits underwent spontaneous self-induced anaerobic fermentation using samples with two percentages of immature fruits in submerged and solid-state processing. The effects on the physicochemical composition and sensory quality of coffees were evaluated.

Results

The two percentages of immature fruits corresponded to 11.0 and 0.3% of unripe fruits. The percentage of immature fruits significantly altered the initial content of sugars (sucrose, glucose, and fructose), ash, and titratable acidity. The water addition during the fermentative process did not significantly influence final moisture, proteins, citric acid, and propionic acid concentrations. Compared to the solid-state, the submerged process gave rise to coffees with lower concentrations of ethanol, glycerol, ash, lipids, succinic, and acetic acids. Coffee fermented with 0.3% of immature fruits showed higher lactic acid production in submerged fermentation (67.44 mg/g), and higher concentrations of ethanol (42.84 mg/g) and glycerol (1.68 mg/g) in solid-state fermentation. All coffees produced were classified as specialty coffees with a score above 84 points. However, the submerged fermented coffee with 11% immature fruit stood out with notes of caramel, brown sugar, honey, orange, lemon, floral, nut, yellow and red fruits.

Conclusions

This study confirmed that spontaneous fermentation can be used to produce specialty coffees. Differentiation in sensory attributes can be achieved through the addition of water and varying the percentage of green fruits during the fermentation process. Up to 11% of immature fruits did not compromise coffee quality.

How to cite: Ferreira LJC, Casé IN, Bertarini PLL, et al. Impact of immature coffee fruits and water addition during spontaneous fermentation process: Chemical composition and sensory profile. Electron J Biotechnol 2024;69. https://doi.org/10.1016/j.ejbt.2024.04.001.

Over the past two decades, interest in pollutant removal by yeasts has grown substantially. Yeasts can remove high amounts of pollutants at low production costs under non-sterile conditions. This work presents a compilation of the studies carried out regarding the potential application of yeasts in the treatment of wastewater. For example, a summary is presented on data about various yeast strains that are used to treat wastewater. The study will help the decision-making process for the selection of yeast for a type of wastewater and support research efforts by acquiring an overview of advancements in this area. Yeast treatment is versatile and has outstanding adaptability to varying treatment conditions. The effectiveness of yeast in treating wastewater is influenced by multiple factors. Yeast technology could potentially be retrofitted to existing activated sludge processes or be used instead of bacteria. Within its characteristics, we can observe tolerance to low pH (3.0–5.0), high salinity, high organic loads, antibiotics, and survive in up to 12% v/v alcohol mixtures. In fact, using low pH for yeast cultivation reduces bacterial contamination and supports yeast domination under non-sterile conditions. Laboratory-scale trials for yeast wastewater treatment have shown improvement over the past two decades; however, efficiencies differ according to the type of wastewater. In general, yeast offers several benefits compared to traditional microbial treatment methods, especially in its capacity to effectively process diverse organic carbon sources. However, it still must be proven to be an effective technology at an industrial scale.

How to cite: Mohiuddin O, Harvey A, Orta Ledesma MT, et al. Bioremediation of waste by yeast strains. Electron J Biotechnol 2024;69. https://doi.org/10.1016/j.ejbt.2024.01.005.

Background

Ferredoxin 1 (Fd1) is the main electron donor to hydrogenase (HydA) for generating molecular hydrogen (H₂) in green microalgae. In order to obtain an increased H₂ production, the Fd1 of Chlamydomonas reinhardtii (CrFd1, encoded by crfd1) was therefore overexpressed in Chlorella sp. DT (DT) in this study.

Results

The coding region of crfd1 was constructed into the p121-crfd1 plasmid, which was also constructed with a resistance gene to the antibiotic geneticin (G418) as a selection marker. The p121-crfd1 plasmid was transformed into DT cells by electroporation. Observation of the crfd1 gene fragment in the genomic DNA of DT-crfd1 mutants by PCR indicated that the transgene was successfully transformed. Using western blotting, the overexpressed CrFd1 protein, with a molecular weight of about 14 kDa, was found in DT-crfd1 mutants of DT-crfd1-4, DT-crfd1-22, and DT-crfd1-23. Using an in vitro assay, the H₂ production of DT-crfd1-4, DT-crfd1-22, and DT-crfd1-23 mutants was about 4.4-, 5.0-, and 3.8-fold higher, respectively, than the DT wild type (DT-WT). Using an in vivo assay, the H₂ production of DT-crfd1-4, DT-crfd1-22, and DT-crfd1-23 mutants was about 1.3-, 1.4-, and 1.2-fold higher, respectively, than the DT-WT.

Conclusions

The results suggested that heterologous overexpression of CrFd1 could enhance H₂ production in DT-crfd1 mutants even though in vitro H₂ production of DT-crfd1-22 mutant was up to 5-fold higher than the DT-WT.

How to cite: Lin YJ, Chien LF. Hydrogen production in the Chlorella sp. DT mutants carrying heterologous electron donor ferredoxin 1 of Chlamydomonas reinhardtii. Electron J Biotechnol 2024;69. https://doi.org/10.1016/j.ejbt.2024.03.001.

Background

Complex graft bioengineering is an actual topic in bone defects’ repair. For those, different scaffolds may be seeded with mesenchymal stromal cells and growth / differentiation factors. The natural role of platelet factors in reparative processes justifies the possibility of its usage for mesenchymal stromal cell proliferation and differentiation into osteoblasts in vitro in terms of the scaffold-based bioengineering. To develop and evaluate in vitro biocompatibility and osteoconductivity of a complex biograft based on a bioorganic scaffold seeded with human bone marrow mesenchymal stromal cells and saturated with growth and differentiation factors of allogeneic platelet-rich plasma.

Results

The properties of viability and adhesion of human bone marrow mesenchymal stromal cells in four types of bioorganic scaffolds were evaluated with biochemical and immunomorphological methods. Scaffold with the least cytotoxicity was used as a basis for complex biograft formation, so as a carrier for cells and platelet-derived factors. Then, cell proliferation activity and osteogenic differentiation were estimated with biochemical, morphological, histochemical and molecular-biological methods. The study showed high viability of cells in all bioorganic scaffolds but the least cytotoxicity was the one based on xenogeneic collagen sponge. We also found that allogeneic platelet-rich plasma positively affects the proliferation and osteogenic differentiation of bone marrow mesenchymal stromal cells in a complex biograft in vitro.

Conclusions

The properties of the developed complex biograft characterize its biocompatibility and osteoconductivity and make it potentially suitable for regenerative medicine, particularly for reconstructive surgery of bone defects.

How to cite: Danilkovich NN, Kosmacheva SM, Ionova AG, et al. Formation of osteoconductive biograft with bioorganic scaffold, human mesenchymal stromal cells, and platelet rich plasma with its evaluationin vitro. Electron J Biotechnol 2024;69. https://doi.org/10.1016/j.ejbt.2024.01.004.

Background

Targeted insertion of the repair template into the genome is a common strategy for high-precision base replacements; however, the main challenge likely remains regarding the limited efficiency of homologous-directed repair (HDR). A precise genome cut achieved by CRISPR-Cas9 system combining with a single-stranded oligodeoxynucleotide (ssODN), as the donor template, improves significantly the rate of HDR. It is well-established that the spatial availability of the donor template to the repair system effectively enhances knock-in events in CRISPR-Cas9. PolyPurine Reverse Hoogsteen hairpins (PPRHs), as an alternative repairing strategy, benefits from a Triplex-forming oligonucleotide (TFO) for the repair template providing the ease of access. The main objective of the study was to evaluate the HDR frequency as a result of improvement of the spatial accessibility of the donor template adjacent to the cutting site. Hence, a flanking purine-rich hairpin complementary to the genomic DNA adjacent to the repairing site was fused to the ssODN with the incorporated bases for the alteration of EGFP to EBFP.

Results

Results from the comparison between the donor templates, ssODN and TFO-tailed ssODN, demonstrated an increased rate of knock-in from 18.2% ± 1.09 to 38.3% ± 4.54, respectively. From another perspective, findings indicated that the targeted Cas9-mediated DNA cleavage improves the efficiency of the repair-PPRH approach four-fold, as well.

Conclusions

The present study provides a viewpoint that highlights the significance of the designing of the donor template in terms of the structural features and positional access for the HDR-based repairing CRISPR-Cas9 systems.

How to cite: Ebrahimi Z, Kazemi B, Salehi M, et al. Successful CRISPR/Cas9-mediated HDR at individual DNA breakpoints using TFO-based targeted template design. Electron J Biotechnol 2024, 68. https://doi.org/10.1016/j.ejbt.2024.01.001.

Background

Naringin is one of the main flavonoids in citrus fruits and byproducts. This flavanone has been shown to be a good antioxidant nutraceutical component, and it also has potential as a gut microbiome modulator, although its applications in final formulations represent a challenge due to its low solubility, both in water and in organic solvents. This work addresses this problem by functionalizing naringin through enzymatic acylation.

Results

The enzymatic acylation catalyzed by the lipase Novozym® 435 and using acyl donors of different chain lengths, acetate (C2), propionate (C3), and laurate (C12), yielded in conversions of 95% at 24 h and 100% at 48 h, generating a monoacylated product. Both the aqueous and solvent solubility of acylated naringin products were improved while maintaining or even increasing their antioxidant activity.

Conclusions

This acylation process significantly enhanced both the water and solvent solubility of the acylated naringin products while preserving or even enhancing their antioxidant activity. In addition to the gut-modulating properties of flavonoids, acylating them with short- and medium-chain fatty acids could enhance their potential applications in the emerging field of research dedicated to understanding and modulating gut health.

How to cite: Gutiérrez-Navarro E, Padilla-de la Rosa JD, Macías A, et al. Enzymatically acylated naringin with gut modulation potential. Electron J Biotechnol 2024; 68. https://doi.org/10.1016/j.ejbt.2023.12.003.

Background

High chitosanase-producing microorganisms from natural sources have extensive applications in food and agriculture. This study aimed to optimal conditions for high-activity chitosanase production. A named CGMCC21422 chitosanase-producing strain -2T-2 was isolated from soil and identified named as Aspergillus fumigatus chitosanase (A. fumigatus chitosanase). This enzymatic activity was validated in various culture conditions. It is stored in the China General Microbiological Culture Collection Center. The efficacy of A. fumigatus chitosanase in the degradation of chitosan was validated.

Results

In this study, we determined that the optimal fermentation conditions of stain-2T-2 were 1.0% powered chitosan, 0.8% ammonium nitrate, 37°C culture temperature, initial pH 5.0, culture time 6 d, bottle volume 50 mL, and 2% inoculation dosage. Under these culture conditions, the highest enzyme activity of fermentation broth in the shaker flask reached 827.53 U/mL. The optimal reactive conditions of A. fumigatus-produced chitosanase are 55–60°C and pH 4.5. When the reactive temperature was over 60°C, the A. fumigatus chitosanase was easily inactivated. The chitosanase catalyzed substrate chitosan to produced ≈20% chito-oligosaccharide and ≥80% glucosamine salt samples in a variety of acidic solutions. These reactive products are not cytotoxic or mild to MH7A cells.

Conclusions

A. fumigatus chitosanase strain -2T-2 is a strain with high chitosanase and can catalyze chitosan into chito-oligosaccharide in acidic solutions.

How to cite: Yang, H., Wang, L., Xu, C, et al. Screening for Aspergillus fumigatus strain-2T-2 with high chitosanase production activity and its application in chitosan degradation. Electron J Biotechnol 2024; 68. https://doi.org/10.1016/j.ejbt.2024.01.003.