Biotechnology Letters最新文献

Microbial fuel cells (MFCs) utilize the metabolic activities of microorganisms, through which the chemical energy is directly converted into electrical energy. Bacteria produce electrons by means of oxidation of organic/inorganic substrates within the MFCs. Metal organic frameworks (MOFs) that are porous coordination polymers have gained much interest in the field of efficient catalysts due to their unique characteristics. The utilization of MOF catalysts for oxygen reduction reaction (ORR) in the MFC cathode is one of the most remarkable research areas in material science. MOF (zeolitic imidazole framework-leaf like, ZIF-L) decorated cathode system was employed for the first time in MFC to monitor the improvement in performance by taking advantages of both electrocatalytic activity and porosity of MOFs for the utilization of bioelectrons for ORR. Analysis of ORR performance of ZIF-L/carbon black (CB) composite cathode demonstrated that ZIF-L containing cathode system had an improved ORR activity compared to MFC cathode materials in the literature. The remarkable current density value of 2.1 mA cm^-2 and the maximum power density value of 1,462 mW m^-2 at room temperature revealed that ZIF-L decorated cathode is an excellent alternative for efficient reduction of oxygen in MFCs.

Background: Interleukin-21 (IL-21) is a cytokine produced by various cell types, including T cells, natural killer cells, myeloid cells, and B cells, and has a broad range of potential applications in cancer therapy. To improve the therapeutic index, we explored the use of fusion technologies that involved linking other anticancer peptides to the IL-21 gene using specific linkers.

Objectives: This study aimed to compare the anticancer potential of IL-21 and IL-21 fusion proteins.

Methods: Antimicrobial peptides possessing anticancer properties were fused with IL-21 gene using a flexible linker (-GGGGS-), and the resulting construct was inserted into the pSecTag2a mammalian expression vector. The cassette was transfected into several cancer cell lines including H1 HeLa, HepG2, MCF-7, MDA-MB-231, HCT-116, HCC-1954, HEK-293, and SF-767. The cytotoxic effects of IL-21 and fusion proteins were evaluated using MTT, Caspase-3, LDH, and scratch assays.

Results: The IL-21-Tachyplesin I fusion protein had the strongest antiproliferative activity against all tested cancer cells, followed by IL21-LPSBD2 and IL-21. In contrast, IL21-Cop A3, IL21-CSP I-Plus, and IL21-RGD Temporin-Las did not inhibit the viability of cancer cells.

Conclusion: Fusion technology is a promising therapeutic technique that can be used to enhance the cytotoxicity and antiproliferative activity of anticancer proteins such as IL-21.

Temperature-responsive elastin-like polypeptides (ELPs) exhibit a low critical solution temperature-type phase transition and offer potential as useful materials for the construction of nanoparticles. Herein, we developed a novel decoration method for ELP-based nanoparticles via isopeptide bond formation with the SnoopTag/SnoopCatcher system that is not affected by the heating process required for particle formation. A mixture of a fusion protein of ELP and poly(aspartic acid) (poly(D)), known as ELP-poly(D), and ELP-poly(D) fused with SnoopCatcher (ELP-poly(D)-SnC) formed protein nanoparticles as a result of the temperature responsiveness of ELP, with the resultant nanoparticles displaying the SnoopCatcher binding domain on their surfaces. In the present study, two model proteins fused to SnoopTag were displayed on the surfaces of protein nanoparticles constructed from ELP-poly(D)-SnC and ELP-poly(D). The model proteins are enhanced green fluorescent protein (EGFP) and Renilla luciferace (Rluc), which exhibits luminescent capability and weak thermostability, respectively. EGFP on the particle surface was found to retain 48.7% activity, while Rluc exhibited almost full activity, as calculated from the binding efficiency and nanoparticle activities recovered after purification. ELP-based nanoparticles containing the SnoopTag/SnoopCatcher system offer the opportunity for particle decoration with a wide range of functional proteins via isopeptide bond formation.

Purpose: To investigate the impact of Butea monosperma (BM) bark extract on the osteogenic differentiation potential of rat adipose-derived mesenchymal stem cells (rADMSCs) and to elucidate the involvement of Wnt/β-catenin pathway in mediating this osseous effect.

Methods: Characterizations (antioxidant assays, FTIR and LC/MS analyses) and docking studies (in silico) were performed to evaluate the presence of phytochemicals in the BM extract and their binding capacity to that of the frizzled receptor. rADMSCs were isolated and characterised for its differentiation potential of osteogenesis for stemness. Dose fixation, cytotoxicity, osteogenic differentiation (calcium, mineral deposition, alkaline phosphatase and osteocalcin) and gene expression (osteocalcin, Col1, osteonectin, Bmp2, Runx2, Wnt2, and β-catenin-14 and 28 days) of the extract were also evaluated in vitro.

Results: FTIR and LC/MS analyses unveiled the phytochemicals in the extract and with docking studies confirmed their interaction with the frizzled receptor of Wnt/β-catenin pathway. rADMSCs were isolated and differentiated in the presence of the osteogenic induction medium. Dose fixation studies, cytotoxicity and cell viability assessments demonstrated the phytochemicals concentration-dependent cytotoxicity. The presence of specific bone markers highlighted the osteogenic differentiation potential of the phytochemicals. Furthermore, gene expression studies of rADMSCs depicted a heightened bone-forming capacity potentially facilitated by the activation of Wnt/β-catenin pathway.

Conclusion:  The phytochemicals of BM promoted the osteogenic differentiation of rADMSCs through the activation of the signalling Wnt/β-Catenin pathway, as evidenced by the significant upregulation of early and late bone markers. The phytochemicals may therefore be positioned as promising therapeutic agents for enhancing bone regeneration, offering new avenues for regenerative medicine.

Evaluating the methicillin resistance of Staphylococcus aureus (S. aureus) is highly important for adapting nursing strategies. Nevertheless, the identification of methicillin-resistant S. aureus (MRSA) that is both sensitive and reliable continues to pose a significant obstacle. This study describes a method for detecting MRSA using a combination of fixed rolling circle amplification (RCA) and the exonuclease-iii (Exo-iii) assisted CRISPR-Cas12a system for signal amplification. When MRSA is present, the interaction between the "b" chain in the capture probe and MRSA allows the "a" chain to be exposed. This "a" chain acts as a primer to initiate the fixed RCA process. The H probe, which includes the crRNA segment, forms a bond with the RCA product and then releases the crRNA segment with the aid of Exo-iii. The Cas12a protein, when combined with the crRNA, generates an activated CRISPR-Cas12a system that cleaves the "Reporter" probe, resulting in the production of fluorescent signals. Furthermore, this fluorescent test has been utilized for the examination of clinical samples with a satisfactory rate of retrieval. Based on the elegant design, the proposed method exhibited a low detection limit of 4.6 cfu/mL, while maintaining a high specificity for MRSA even from a mixture of several interfering bacteria. Due to its cost-effectiveness, simplicity, and adaptability, the sensing system shows potential as a platform for detecting MRSA and evaluating postoperative nursing for stomach cancer patients.

Purpose: Guanarito mammarenavirus (GTOV) is a highly pathogenic virus that leads to Venezuelan hemorrhagic fever (VHF). Despite being a severe disease, there are currently no commercially available drugs or vaccines for its prevention.

Methods: Here we computationally formulated a mRNA vaccine construct (VC) from the genome of GTOV to produce immunity against its infections. Two proteins, namely zinc-finger motif protein (NP_899220.1), and nucleocapsid protein (NP_899211.1) were screened as potential candidates for downstream analysis.

Results: We determined the T and B cell epitopes of the candidate proteins. The resulting epitopes were analyzed, and the best epitopes were utilized in the formation of the peptide vaccine construct. The secondary and tertiary structures of the peptide construct were predicted and validated. Docking was conducted to check the binding energy of the designed peptide vaccine with the human immune receptors, namely TLR2 and TLR4. Our designed vaccine showed stable interactions with the HLA molecules, as verified through normal mode and MD simulation analysis. The immune simulation results indicated a positive immune response against the construct. A potentially stable mRNA vaccine was formulated by adding of sequences such as the Kozak, Goblin 5' UTR, tPA-signal peptide, MITD, 3' UTRs, and a poly(A) tail to the peptide vaccine construct. Lastly, the expression probability of the mRNA vaccine was confirmed in the expression system of E. coli strain K12.

Conclusion: The designed vaccine showed the potential to elicit an immune response against the GTOV infection; however, experimental validation is recommended to verify the in-silico findings of this study.

The monooxygenase activity of engineered CYP102A1 on α-terpineol was investigated. CYP102A1 M850 mutant (F11Y/R47L/D68G/F81I/F87V/E143G/L188Q/E267V/H408R) showed the highest catalytic activity toward α-terpineol among the engineered mutants produced by random mutagenesis. The major product (P1) of α-terpineol, p-menth-1-ene-3,8-diol, was characterized by high-performance liquid chromatography, gas-chromatography mass spectrometry, and nuclear magnetic resonance spectroscopy. Three minor products (P2-P4) of α-terpineol were considered as 6-hydroxy-α,α,4-trimethyl-3-cyclohexene-1-methanol (P2), trans-sobrerol (P3), and carvone hydrate (P4). Optimal conditions for product formation were determined as pH 7.0 and 30 °C. Production of p-menth-1-ene-3,8-diol was 0.87 mM at 1 h. Structure modeling using PyMOL and CAVER Web 1.2 server indicated that several mutations of CYP102A1 M850 were involved in access tunnels and active sites, resulting in increased activity toward α-terpineol. The major product, p-menth-1-ene-3,8-diol, of α-terpineol was produced by engineered CYP102A1 M850 via regioselective carbon hydroxylation. The engineered CYP102A1 could be a suitable biocatalyst for producing α-terpineol derivatives.

Chlorimuron-ethyl is currently the primary herbicide used for chemical weed control in a soybean field. In this study, a solid microbial inoculum (corn stalk-white rot fungus (W-1)) was prepared for the remediation of farmland soil contaminated by chlorimuron-ethyl. Firstly, the preparation method of the microbial inoculum was studied. Secondly, the degradation rate of the chlorimuron-ethyl in the ground by the solid microbial inoculum is improved by optimizing the proportion of the protective agent. Then the effects of applying solid microbial inoculum, free bacteria and corn straw on the degradation rate of chlorimuron-ethyl in soil were weighed. Finally, Illumina MiSeq sequencing was used to measure the composition and diversity of bacterial and fungal communities in the ground before and after using microbial inoculum. The degradation rate of chlorimuron-ethyl in soil by solid microbial inoculum was 84.87% after 20 d using corn straw as the support, room temperature drying, 4% Ca₃(PO₄)₂ as the protective drying agent, and 1%(w) dextrin as the ultraviolet protective agent. Inoculation of white rot fungi could significantly affect the community structure of bacteria and fungi in the soil, making the chlorimuron-ethyl degrading communities become the dominant communities and playing an essential role in the degradation of chlorimuron-ethyl. The results showed that using solid microbial inoculum was an effective way to repair farmland soil polluted by chlorimuron-ethyl.

Diclofenac is a hepatotoxic non-steroidal anti-inflammatory drug (NSAID) that affects liver histology and its protein expression levels. Here, we studied the effect of diclofenac on rat liver when co-administrated with either Yersinia enterocolitica strain 8081 serotype O:8 biovar 1B (D*Y) or Lactobacillus fermentum strain 9338 (D*L). Spectroscopic analysis of stool samples showed biotransformation of diclofenac. When compared with each other, D*Y rats lack peaks at 1709 and 1198 cm^-1, while D*L rats lack peaks at 1411 cm^-1. However, when compared to control, both groups lack peaks at 1379 and 1170 cm^-1. Assessment of serum biomarkers of hepatotoxicity indicated significantly altered activities of AST (D*Y: 185.65 ± 8.575 vs Control: 61.9 ± 2.607, D*L: 247.5 ± 5.717 vs Control: 61.9 ± 2.607), ALT (D*Y: 229.8 ± 6.920 vs Control: 70.7 ± 3.109, D*L: 123.75 ± 6.068 vs Control: 70.7 ± 3.109), and ALP (D*Y: 276.4 ± 18.154 vs Control: 320.6 ± 9.829, D*L: 298.5 ± 12.336 vs Control: 320.6 ± 9.829) in IU/L. The analysis of histological alterations showed hepatic sinusoidal dilation with vein congestion and cell infiltration exclusively in D*Y rats along with other histological changes that are common to both test groups, thereby suggesting more pronounced alterations in D*Y rats. Further, LC-MS/MS based label-free quantitation of proteins from liver tissues revealed 74.75% up-regulated, 25.25% down-regulated in D*Y rats and 51.16% up-regulated, 48.84% down-regulated in D*L experiments. The proteomics-identified proteins majorly belonged to metabolism, apoptosis, stress response and redox homeostasis, and detoxification and antioxidant defence that demonstrated the potential damage of rat liver, more pronounced in D*Y rats. Altogether the results are in favor that the administration of lactobacilli somewhat protected the rat hepatic cells against the diclofenac-induced toxicity.

Sucrose isomerase (SIase) catalyzes the hydrolysis and isomerization of sucrose to form isomaltulose, a valuable functional sugar widely used in the food industry. However, the lack of safe and efficient heterologous expression systems hinders SIase production and application. In this study, we achieved antibiotic-free SIase expression in Bacillus subtilis through genome integration. Using CRISPR/Cas9 system, SIase expression cassettes were integrated into various genomic loci, including amyE and ctc, both individually and in combination, resulting in single-copy and muti-copy integration strains. Engineered strains with a maltose-inducible promoter effectively expressed and secreted SIase. Notably, multi-copy strain exhibited enhanced SIase production, achieving 4.4 U/mL extracellular activity in shake flask cultivations. Furthermore, crude enzyme solution from engineered strain transformed high concentrations sucrose into high yields of isomaltulose, reaching a maximum yield of 94.6%. These findings demonstrate antibiotic-free SIase production in B. subtilis via genome integration, laying the foundation for its industrial production and application.