BMC Biotechnology最新文献

Background: It is mandatory to demonstrate the removal or inactivation of potential viral contaminants in the manufacturing processes of pharmaceuticals derived from biomaterials. Porcine-derived gelatin is used in various medical fields, including regenerative medicine, tissue engineering, and medical devices. However, the steps of virus inactivation in the gelatin manufacturing process are poorly defined. In this study we evaluated virus inactivation in two steps of the gelatin manufacturing process.

Methods: Pig skin (4.5 g), including solid pieces as intermediate products, was spiked with model viruses, including CPV (canine parvovirus), BAV (bovine adenovirus), BPIV3 (bovine parainfluenza type 3), PRV (pseudorabies virus), BReoV3 (bovine reovirus type 3), and PPV (porcine parvovirus), and underwent chemical treatment with alkaline ethanol or heat treatment at 62 °C followed by inoculation in relevant cell cultures. Viral titers in the samples were calculated based on the Behrens-Kärber method.

Results: Model viruses were inactivated at different rates; however, effective inactivation of all model viruses was demonstrated by an LRV (log reduction value) over 4 by both chemical and heat treatment, and chemical treatment demonstrated rapid inactivation compared to heat treatment.

Conclusion: The chemical and heat treatment steps exhibited meaningful viral inactivation capacity. They are integrated parts in the extraction and manufacturing process of porcine-derived gelatin, ensuring virus safety for use in medical applications.

Hyperhydricity is the most extensive physiological disorder during in vitro propagation. This disturbance can induce anatomical, morphological and physiological problems that cause serious damage. The factors that cause hyperhydricity are the composition of nutrient media and cultures conditions. To reduce the hyperhydricity of Pistacia khinjuk, ammonium nitrate (NH₄NO₃), calcium chloride (CaCl₂·2H₂O), cytokinins of meta-topolin (mT) and 6-benzylaminopurine (BAP) at different concentrations were investigated in Murashige and Skoog (MS) medium. The lowest percentage of hyperhydricity (34.30%) were obtained from the medium containing 1650 mg/L NH₄NO₃, 110 mg/L CaCl₂·2H₂O and1 mg/L mT; the highest percentage of hyperhydricity (68.42%) were obtained from the medium containing 206.25 mg/L NH₄NO₃, 440 mg/L CaCl₂·2H₂O and 0.5 mg/L BAP. The maximum average number of shoots per explant (2.45), average shoots length (18.47 mm) and proliferation rate (85%) were obtained from the medium containing 1650 mg/L NH4NO3, 110 mg/L CaCl2·2H2O of MS and 1 mg/L mT. In addition, when soluble protein (2.12 mg/g) and total chlorophyll a, b (0.96 mg/g) value of normal (non-hyperhydric) shoots were higher than hyperhydric shoots, carotenoid (11.75 µg /g) and water content (78.70%) value of normal shoots were lower than hyperhydric shoots. This study concludes that the hyperhydricity percentage of in vitro P. khinjuk shoots was reduced (12.8%) on modified MS medium with NH₄NO₃, CaCl₂·2H₂O and mT according to standard MS medium.

Objective: This study aims to investigate the biocompatibility and pharmacokinetic characteristics of polyvinyl pyrrolidone-modified selenium nanoparticles (PVP-Se NPs). Understanding the biosafety of PVP-Se NPs is crucial due to their potential applications in mitigating oxidative stress-related diseases and improving drug delivery systems.

Methods: Selenium nanoparticles were prepared using a sodium selenite solution, followed by PVP modification. Particle size analysis was conducted using dynamic light scattering (DLS), and particle morphology was observed using transmission electron microscopy (TEM). Different concentrations of PVP-Se NPs were intraperitoneally injected into SD rats, and the survival rate was observed. Liver and kidney tissues, urine, feces, and blood samples were collected at the highest safe dose, and the concentration of selenium ions was measured.

Results: The average particle size of PVP-Se NPs was 278.4 ± 124.8 nm, exhibiting a semi-spherical shape. The maximum safe dose of PVP-Se NPs for intraperitoneal injection in rats was approximately 320 µg/kg. At this dose, the content of PVP-Se NPs significantly increased in the liver and kidney tissues from day 1 to day 3, in urine and feces during the first 8 h, and in blood during the first 2 h, followed by a gradual decrease.

Conclusion: When administered at a safe dose, PVP-Se NPs do not damage liver and kidney tissues and can be eliminated from the body through liver and kidney metabolism without accumulation.

The rise in global temperature and accumulation of petroleum-based wastes in the environment forces the scientific focus towards renewable alternatives. In the present work, an under-exploited resource - spruce bark - is investigated as a raw material for production of bio-oil as a liquid energy carrier. To enhance the energy-content of the produced bio-crude, ultimately being produced through hydrothermal liquefaction, the polysaccharides were extracted through organosolv fractionation and converted to lipids by oleaginous microorganisms. The effect originating from tannins was also investigated by performing a pre-extraction before the organosolv fractionation. It was found that performing the organosolv fractionation and upgrading the isolated organosolv lignin to bio-oil greatly reduced the oxygen content of the oil fraction thereby improving its energy content, and introducing upgraded polysaccharides in the form of lipids, as well as pre-extracted tannins, caused clear changes in the product distribution of the final bio-oil and kept a final product with low oxygen content. The other factor largely influencing the product distribution originated from the various heating rates tested by altering operational mode of the HTL process between batch and semi-continuous. Ultimately, performing the organosolv fractionation and individual upgrading of the polysaccharides had a beneficial effect on reducing the final solids content and enhancing the liquid oil yield.

Background: The laccase Lcc9 from Coprinopsis cinerea has optimal catalytic activity at moderate to alkaline pH conditions, making it invaluable for industrial applications. However, C. cinerea naturally secretes Lcc9 at low expression levels, which limits the industrial application of Lcc9 on a large scale. Recombinant production of Lcc9 using Aspergillus niger would be an effective way to achieve its high production.

Results: This study achieved the secretory production of Lcc9 in A. niger and established an efficient transformation procedure for A. niger by optimizing its protoplast preparation system. The transformation efficiency of A. niger was increased 3.8-fold under the optimal system (cell wall digestion enzyme solution: 2% cellulase, 1% snailase, 1% lyticase, and 0.5% lysozyme; incubation time: 3 h; incubation temperature: 37 ℃; culture time: 48 h). The extracellular yield of Lcc9 was enhanced by optimizing gene expression cassette and bioprocess. First, the strain AnGgcL (containing P_gpdA) mediated by the SP_CAT, a signal peptide of the extracellular high abundance protein catalase, had an extracellular laccase activity of 10 U/L after shake flask fermentation. Then, by optimizing promoter and signal peptide combinations that regulate lcc9 expression, the strain AnGcgL mediated by P_citA-SP_GlaA had an extracellular laccase activity of 20 U/L. Subsequently, the strain AnRcgL1 (containing P_citA-SP_GlaA) obtained by random integration had an extracellular laccase activity of 86 U/L. Sequencing revealed that the lcc9 expression cassette was integrated into the citrate synthase gene locus in the AnRcgL1 genome in a 9-copy form. By optimizing the microparticle, osmolyte, and Cu²⁺ in the fermentation medium, the AnRcgL1 extracellular laccase activity was further increased to 1566.7 U/L, which was 156.7-fold higher than that of AnGgcL. Furthermore, its extracellular laccase activity was increased to 1961 U/L in a 1-L fermenter.

Conclusions: To our knowledge, this study is the first to report the recombinant extracellular production of the C. cinerea laccase Lcc9 in A. niger and to use SP_CAT in the A. niger expression system. The results of this study will help accelerate the industrial application of Lcc9. Moreover, the strategy used in this work provides methodological guidance for increasing other exogenous protein yields in A. niger.

Background: Dendrobium catenatum is a perennial herb of the genus Dendrobium orchidaceae. It has been known as "Golden Grass, Soft Gold" since ancient times with effects of strengthening the body, benefiting the stomach, generating body fluid, nourishing Yin and clearing internal heat. The flowers of D. catenatum have anti-oxidation, immune regulation and other biological activities. The composition analysis of flowers showed that flavonoid glycosides were significantly accumulated in floral tissue. However, in the flowers of D. catenatum, there was only one case of the UDP-glycosyltransferase (UGT) responsible for the glycosylation of flavonoids has been reported.

Result: In this study, a new UGT (named UGT708S6) was cloned from D. catenatum flowers rich in O-glycosides and C-glycosides, and its function and biochemical properties were characterized. Through homology comparison and molecular docking, we identified the key amino acid residues affecting the catalytic function of UGT708S6. The glycosyltransferase UGT708S6 was characterized and demonstrated C-glycosyltransferase (CGT) activity in vitro assay using phloretin and 2-hydroxynaringenin as sugar acceptors. The catalytic promiscuity assay revealed that UGT708S6 has a clear sugar donor preference, and displayed O-glycosyltransferase (OGT) activity towards luteolin, naringenin and liquiritigenin. Furthermore, the catalytic characteristics of UGT708S6 were explored, shedding light on the structural basis of substrate promiscuity and the catalytic mechanism involved in the formation of flavonoid C-glycosides. R271 was a key amino acid residue site that sustained the catalytic reaction. The smaller binding pocket resulted in the production of new O-glycosides and the reduction of C-glycosides. This highlighted the importance of the binding pocket in determining whether C-glycosides or O-glycosides were produced.

Conclusions: The findings suggest that UGT708S6 holds promise as a new glycosyltransferase for synthesizing flavonoid glycosides and offer valuable insights for further understanding the catalytic mechanisms of flavonoid glycosyltransferases.

Cold atmospheric pressure plasma (CAP) has garnered significant attention in recent years for its potential applications in biomedical, environmental, and agricultural fields. Cold plasma treatment exhibits a variety of effects in agricultural applications, including impacts on seed germination and seedling growth; however, further research is required. Soybean serves as a fundamental source of nutrients for both animals and humans. Soybean seeds possess impermeable and thick testae, which results in prolonged germination times and suboptimal germination rates. The soybeans exhibit low uniformity. As a result, poor crop establishment and yield reduction are inevitable outcomes. Therefore, the purpose of this study was to examine the effects of Iranian soybean cultivars, such as Sari, Saba, Arian, Katoul, and Williams, on seedling growth properties, seed germination, and antioxidant enzyme activity, using argon at time intervals of 30, 60, 180, 300, and 420 s. Cold plasma treatment significantly enhanced germination potential from 1.18 to 66.97%, germination index from 0.50 to 60.09%, germination rate from 1.78 to 32.17%, seedling length from 2.70 cm to 78.13 cm, root length from 2.87 cm to 56.13 cm, and seedling dry weight from 1.80 g to 36.63 g. Additionally, CAT activity increased from 0.88- to 4.40-fold, SOD activity from 0.86- to 5.89-fold, and APX activities from 0.40- to 4.01-fold compared to the control treatment. The findings indicated that the samples exhibited optimal results at treatment durations of 60 and 180 s. The influence of plasma on the antioxidant responses of seedlings, seed germination, and growth characteristics was contingent upon the duration of treatment. Cold plasma, when applied for an appropriate duration, may enhance soybean seedling growth characteristics and seed germination.

Pathogenic fungi employ numerous strategies to colonize plants, infect them, reduce crop yield and quality, and cause significant losses in agricultural production. The increasing use of chemical pesticides has led to various ecological and environmental issues, including the emergence of resistant weeds, soil compaction, and water pollution, all negatively impacting agricultural sustainability. Additionally, the extensive development of synthetic fungicides has adverse effects on animal and human health, prompting the exploration of alternative approaches and green strategies for phytopathogen control. Microorganisms living in sponges represent a promising source of novel bioactive secondary metabolites, potentially useful in developing new nematicidal and antimicrobial agents. This study focuses on extracting bioactive compounds from endosymbiotic bacteria associated with the marine sponge Hyrtios erect sp. (collected from NIOF Station, Hurghada, Red Sea, Egypt) using various organic solvents. Bacillus sp. was isolated and identified through 16 S rRNA gene sequencing. The biocidal activity of Bacillus gotheilii MSB1 extracts was screened against plant pathogenic bacteria, fungi, and nematodes. The n-butanol extract showed significant potential as a biological fungicide against Alternaria alternata and Fusarium oxysporum. Both n-hexane and ethyl acetate extracts exhibited negative impacts against the plant pathogenic bacteria Erwinia carotovora and Ralstonia solanacearum, whereas the n-butanol extract had a positive effect. Regarding nematicidal activity, ethyl acetate and n-butanol extracts demonstrated in-vitro activity against the root-knot nematode Meloidogyne incognita, which causes serious vegetable crop diseases, but the n-hexane extract showed no positive effects. The findings suggest that bioactive compounds from endosymbiotic bacteria associated with marine sponges, particularly B. gotheilii MSB1, hold significant potential as alternative biological control agents against plant pathogens. The n-butanol extract, in particular, displayed promising biocidal activities against various plant pathogenic fungi, bacteria, and nematodes. These results support further exploration and development of such bioactive compounds as sustainable, environmentally friendly alternatives to synthetic pesticides and fungicides in agricultural practices.

Background: Exploration of whether circRNAs in the skin of systemic sclerosis (SSc) model mice interact with 4E-BP1 protein to mediate the mTOR signaling pathway to regulate SSc fibrosis is crucial to identify homologous human circRNAs as markers to guide the diagnosis and treatment of SSc.

Methods: C57BL/6 mice aged 6-8 weeks and weighing approximately 20 g were subcutaneously injected with bleomycin (BLM) to establish an SSc model. High-throughput sequencing was used to screen the differentially expressed circRNA in the skin of SSc model mice and control mice. RNA immunoprecipitation and RNA pulldown confirmed the interaction between circRNA and 4E-BP1 protein. SSc model mice were treated with empty plasmid (OE-NC), overexpression plasmid of mmu_circ_0005372 (OE-circ_0005372), interference plasmid of mmu_circ_0005372 (sh-circ5372), mutant plasmid of mmu_circ_0005372 (circ5372-MT), mTOR activator (MHY1485), mTOR inhibitor (omipalisib), or JAK1/2 inhibitor (ruxolitinib). Sections of mouse skin tissue were stained with Hematoxylin and eosin and Masson's stain. The collagen volume fraction (CVF) was calculated as CVF = area of blue collagen/total area with ImageJ. The correlation between homologous human circRNAs and clinical data was analyzed.

Results: Compared to the control group, 21,839 circRNAs were upregulated and 27, 946 circRNAs were downregulated in the skin tissue of mice in the SSc model group. Among them was mmu_circ_0005372, which is derived from the FZD3 gene, is closely related to fibrosis, and is involved in the mTOR signaling pathway. Hsa_circ_0136256 was identified as the homologous human circRNA of mmu_circ_0005372. RT-qPCR confirmed that the expression of mmu_circ_0005372 was significantly reduced in the skin tissue of SSc mice, and the expression of hsa_circ_0136256 was significantly reduced in the peripheral blood mononuclear cells of patients with SSc. The interaction between mmu_circ_0005372 and 4E-BP1 protein was inhibited in the skin tissue of SSc model mice. The results showed that the CVF of OE-circ_0005372 group was significantly lower than that of the sh-circ5372, circ5372-MT, and MHY1485 groups, indicating that OE-circ5372 significantly improved skin fibrosis in the SSc mice. ROC curve analysis was performed on hsa_circ_0136256 (AUC = 0.719, P = 0.035). The expression of hsa_circ_0136256 was negatively correlated with COL IV, RDW-SD, and RDW-CV, and positively correlated with VC, PLT, and PCT. The results suggested that hsa_circ_0136256 may have important roles in the clinical diagnosis of SSc.

Conclusion: Mmu_circ_0005372 and homologous human hsa_circ_0136256 may be biomarkers and therapeutic targets for SSc fibrosis.

Background: Vitamin B₁₂ is a crucial B-group vitamin, first isolated from the liver due to its role in combating pernicious anemia. It is distinguished by its unique and complex structure, which makes its chemical synthesis challenging and expensive. Consequently, vitamin B₁₂ is alternatively obtained through microbial fermentations. Molasses, an affordable and safe agro-industrial waste, can be used as a carbon source for vitamin B₁₂ production, offering a cost-effective alternative to expensive sugars in the production medium.

Results: A total of 87 yeast, actinomycete, and bacterial isolates were screened for vitamin B₁₂ production, with 15 isolates showing high productivity. Bacillus isolates were selected for further analysis using MALDI-TOF and molecular identification. These isolates were identified as four strains of Bacillus subtilis (MZ08, JT10, BY11, and JT17), one strains of Bacillus sp. (CB09), and one strain of Peribacillus acanthi (MZ01). Genetic circuits associated with vitamin B₁₂ production were demonstrated in a closely related strain of Peribacillus acanthi MZ01 strain. Three strains (MZ01, MZ08, and JT17) were selected for further evaluation of vitamin B₁₂ productivity under different sugar types (glucose, sucrose, fructose, lactose, and galactose) and varying inoculum sizes. The inoculum size significantly impacted vitamin B₁₂ production, with an increase from 5 to 10% enhancing yields. The ability of the strains to produce vitamin B₁₂ varied depending on the type of sugar used. Peribacillus acanthi MZ01 strain showed the highest productivity and subsequently, selected for optimizing vitamin B₁₂ production conditions using response surface methodology. Furthermore, the optimized conditions were then applied to molasses-based medium to achieve high vitamin B₁₂ yields by MZ01 strain.

Conclusion: In this study, Peribacillus acanthi was characterized for the first time as a vitamin B₁₂ producer, demonstrating high productivity among various tested strains. The optimization of production conditions using response surface methodology, further enhanced vitamin B₁₂ yields, showcasing the strain's efficiency in microbial fermentations. This research also highlights the potential of using molasses as a cost-effective alternative carbon source, significantly reducing production costs.