BMC Biotechnology最新文献

This study aimed to optimize the production of carotenoid pigments from Micrococcus luteus (ATCC 9341) through the statistical screening of media components and the characterization of antimicrobial, antioxidant, cytogenetic and cytotoxic activities. A BOX-Behnken design was used to assess the effects of whey concentration, inoculum size, pH, temperature, and agitation speed on carotenoid yield. The optimum combination increased production to 2.19 g/L, with a productivity of 0.045 g L^-1 h^-1 and a productivity yield of 0.644 g/g, as confirmed by an observed carotene production of 2.19 g/L. The final response surface model fitting the data had an R² of 0.9461. High-performance liquid chromatography (HPLC) analysis identified 12 carotenoid pigment compounds produced by M. luteus. The extracts displayed moderate antimicrobial efficacy against Gram-positive bacteria such as Bacillus cereus (ATCC 11778), Staphylococcus aureus (ATCC 6538), and E. faecalis (ATCC 19433), with inhibition zone diameters (IZD) of 29.0, 14.0, and 37.0 mm, respectively, at 1000 μg/mL. However, its effectiveness against Gram-negative bacteria is limited. In comparison, tetracycline exhibited greater antimicrobial potency. The IC₅₀ value of carotenoids was used to indicate the antioxidant activity. IC₅₀ value from the DPPH assay was 152.80 mg/100mL. An IC₅₀ cytotoxicity value greater than 300 μg/mL was found against normal mouse liver cells, with over 68% cell viability even at 300 μg/mL, indicating low toxicity. Histological structure studies revealed normal myocardial muscle tissue, lung tissue, and kidney tissue sections, whereas liver tissue sections revealed ballooning degeneration of hepatocytes and disorganization of hepatic cords. Cytogenetic parameters revealed that the carotene treatment group had a mitotic index (70%) lower than that of the control but higher than that of the positive control, mitomycin, and did not substantially increase numerical (1.2%) or structural aberrations compared with those of the control, suggesting a lack of genotoxic effects under the experimental conditions. In conclusion, optimized culture conditions enhanced carotenoid yields from M. luteus, and the extracts displayed promising bioactivity as moderate antibiotics against certain gram-positive bacteria and as antioxidants. The high IC₅₀ values demonstrate biosafety. Overall, this bioprocess for enhanced carotenoid production coupled with bioactivity profiling and low cytotoxicity support the application of M. luteus carotenoids.

Background: Biotransformation of steroid compounds into therapeutic products using microorganisms offers an eco-friendly and economically sustainable approach to the pharmaceutical industry rather than a chemical synthesis way. The biotransformation efficiency of progesterone into the anticancer compound testololactone using Penicillium chrysogenum Ras3009 has been investigated. Besides, maximization of testololactone formation was achieved by studying the kinetic modelling and impact of some fermentation conditions on the biotransformation process.

Results: The fungal strain Ras3009 was selected among twelve fungal strains as the most runner for the transformation of 81.18% of progesterone into testololactone. Ras3009 was identified phenotypically and genotypically as Penicillium chrysogenum, its 18 S rRNA nucleotide sequence was deposited in the GenBank database by the accession number OR480104. Studying the impact of fermentation conditions on biotransformation efficiency indicated a positive correlation between substrate concentration and testololactone formation until reaching the maximum velocity v_max. Kinetic studies revealed that v_max was [Formula: see text] gL^- 1hr^- 1 with high accuracy, giving R² of 0.977. The progesterone transformation efficiency generally increased with time, reaching a maximum of 100% at 42 h with testololactone yield (Y_pt/s) 0.8700 mg/mg. Moreover, the study indicated that the enzymatic conversion by P. chrysogenum Ras3009 showed high affinity to the substrate, intracellularly expressed, and released during cell disruption, leading to higher efficiency when using whole microbial cell extract.

Conclusions: Fungi can be promising biocatalysts for steroid transformation into valuable chemicals and pharmaceutical compounds. The study revealed that the new fungal isolate P. chrysogenum Ras3009 possesses a great catalytic ability to convert progesterone into testololactone. Kinetic modelling analysis and optimization of the fermentation conditions lead to higher transformation efficiency and provide a better understanding of the transformation processes.

Background: Timothy grass (Phleum pratense) is a significant source of allergens, and recombinant allergens are increasingly used for diagnostic purposes. However, the performance of different recombinant allergen production systems in diagnostic assays needs further investigation to optimize their use in clinical settings.

Objective: The main objective of this study was to analyze and compare the diagnostic performance of recombinant timothy grass allergens produced in E. coli and N. benthamiana using a custom-made microarray chip.

Methods: Recombinant timothy grass allergens Phl p 1, Phl p 2, Phl p 5, Phl p 6, Phl p 11, and Phl p 12 were produced in E. coli and/or N. benthamiana. A total of 113 patient serum samples were tested to evaluate the diagnostic sensitivity, specificity, inter-assay variability, and correlation of allergen-specific IgE detection compared to commercial multiplex tests (ALEX and ISAC). Additionally, the prevalence of sIgE to these allergens was assessed.

Results: Phl p 1, Phl p 2, Phl p 5, Phl p 6 and Phl p 11 showed high or very high positive correlation in immunoreactivity with other commercial multiplex tests. Notably, Phl p 11 fused with maltose-binding protein (MBP) demonstrated high diagnostic specificity and sensitivity, with a 0.3 arbitrary cut-off value. However, a high intra-assay variation was observed. The study also assessed specific IgE prevalence to timothy grass allergens within the tested patient cohort.

Conclusions: Recombinant allergens from both E. coli and N. benthamiana demonstrated strong diagnostic potential on the microarray platform, with Phl p 11 (MBP-fused) showing particularly high performance. High intra-assay variation highlights the need for further optimization in allergen formulation and microarray storage conditions. These results highlight the potential of recombinant allergens for diagnostic applications, despite challenges with allergen stability in microarray formats. Specific IgE prevalence to timothy allergens revealed a sensitization profile consistent with findings from multiple studies.

This work aimed to fabricate a Cloisite 30B-incorporated carboxymethyl cellulose graft copolymer of acrylic acid and itaconic acid hydrogel (Hyd) via a free radical polymerization method for controlled release of Sunitinib malate anticancer drug. The synthesized samples were characterized by FTIR, XRD, TEM, and SEM-dot mapping analyses. The encapsulation efficiency of Hyd and Hyd/Cloisite 30B (6 wt%) was 81 and 93%, respectively, showing the effectiveness of Cloisite 30B in drug loading. An in vitro drug release study showed that drug release from all samples in a buffer solution with pH 7.4 was higher than in a buffer solution with pH 5.5. During 240 min, the cumulative drug release from Hyd/Cloisite 30B (94.97% at pH 7.4) is lower than Hyd (53.71% at pH 7.4). Also, drug-loaded Hyd/Cloisite 30B (6 wt%) demonstrated better antibacterial activity towards S. Aureus bacteria and E. Coli. High anticancer activity of Hyd/Cloisite 30B against MCF-7 human breast cancer cells was shown by the MTT assay, with a MCF-7 cell viability of 23.82 ± 1.23% after 72-hour incubation. Our results suggest that Hyd/Cloisite 30B could be used as a pH-controlled carrier to deliver anticancer Sunitinib malate.

Background: Human papillomavirus type 16 (HPV-16) infection is strongly associated with considerable parts of cervical, neck, and head cancers. Performed investigations have had moderate clinical success, so research to reach an efficient vaccine has been of great interest. In the present study, the immunization potential of a newly designed HPV-16 construct was evaluated in a mouse model.

Results: Initially, a construct containing HPV-16 mutant (m) E6/E7 fusion gene was designed and antigen produced in two platforms (i.e., DNA vaccine and recombinant protein). Subsequently, the immunogenicity of these platforms was investigated in five mice) C57BL/6 (groups based on several administration strategies. Three mice groups were immunized recombinant protein, DNA vaccine, and a combination of them, and two other groups were negative controls. The peripheral blood mononuclear cells (PBMCs) proliferation, Interleukin-5 (IL-5) and interferon-γ (IFN-γ) cytokines, IgG1 and IgG2a antibody levels were measured. After two weeks, TC-1 tumor cells were injected into all mice groups, and subsequently further analysis of tumor growth and metastasis and mice survival were performed according to the schedule. Overall, the results obtained from in vitro immunology and tumor cells challenging assays indicated the potential of the mE6/E7 construct as an HPV16 therapeutic vaccine candidate. The results demonstrated a significant increase in IFN-γ cytokine (P value < 0.05) in the Protein/Protein (D) and DNA/Protein (E) groups. This finding was in agreement with in vivo assays. Control groups show a 10.5-fold increase (P value < 0.001) and (C) DNA/DNA group shows a 2.5-fold increase (P value < 0.01) in tumor growth compared to D and E groups. Also, a significant increase in survival of D and E (P value < 0.001) and C (P value < 0.01) groups were observed.

Conclusions: So, according to the findings, the recombinant protein could induce stronger protection compared to the DNA vaccine form. Protein/Protein and DNA/Protein are promising administration strategies for presenting this construct to develop an HPV-16 therapeutic vaccine candidate.

Orange (Citrus sinensis L.) is a common fruit crop widely distributed worldwide with the peel of its fruits representing about 50% of fruit mass. In the current study, orange peel was employed to mediate the synthesis of silver nanoparticles (AgNPs) in a low-cost green approach. Aqueous extracts of suitably-processed peel were prepared using different extraction methods; and their phytochemical profile was identified. Based on phytochemical screening, amount of main phytochemicals, free radical-scavenging ability, reducing power and antioxidant activity, the peel extract prepared by boiling seemed to be the most promising. Thus, major compounds of this extract were identified by gas chromatography-mass spectrometry. Potency of the peel extract to mediate the synthesis of AgNPs was then monitored by visual observation, UV-visible spectroscopy, energy dispersive X-ray analysis, transmission electron microscopy and zetametry. Color change of the reaction mixture to brown and absorption peak at 450 nm indicated AgNPs formation. Characterization of AgNPs revealed spherical shape, size of 30-40 nm, zeta potential of -18.2 mV and yield conversion of 82%. The as-synthesized AgNPs had antioxidant capacity (free radical-scavenging ability, reducing power and antioxidant activity) lower than that of the orange peel extract. However, these biogenic AgNPs had antitumor activity (IC₅₀ of 16 ppm against HCT-116 and 1.6 ppm against HepG2 cell lines) much higher than the peel extract that was completely non-toxic to the considered cell lines.

The present study deals with the production of cellulase-free endoxylanase by Aspergillus niger ISL-9 using wheat bran as a solid substrate. Endoxylanase was produced under a solid-state fermentation. Various growth parameters were optimized for the improved production of the enzyme. The Substrate level of 15 g was optimized as it provided the fungus with balanced aeration and nutrition. Among the six moisture contents investigated, Moisture Content 5 (MC5) was optimized (g/l: malt extract, 10; (NH₄)₂HPO₄, 2.5; urea, 1.0) and 10 mL of MC5 was found to give the highest production of endoxylanase. The pH and time of incubation were optimized to 6.2 and 48 h respectively. The Inoculum size of 2 mL (1.4 × 10⁶ spores/mL) gave the maximum enzyme production. After optimization of these growth parameters, a significantly high endoxylanase activity of 21.87 U/g was achieved. Very negligible Carboxymethylcellulase (CMCase) activity was observed indicating the production of cellulase-free endoxylanase. The notable finding is that the endoxylanase activity was increased by 1.4-fold under optimized conditions (p ≤ 0.05). The overall comparison of kinetic parameters for enhanced production of endoxylanase by A. niger ISL-9 under Solid State Fermentation (SSF) was also studied. Different kinetic variables which included specific growth rate, product yield coefficients, volumetric rates and specific rates were observed at 48, 72 and 96 h incubation time and were compared for MC1 and MC5. Among the kinetic parameters, the most significant result was obtained with volumetric rate constant for product formation (Q_p) that was found to be optimum (1.89 U/h) at 72 h incubation period and a high value of Q_p i.e.1.68 U/h was also observed at 48 h incubation period. Thus, the study demonstrates a cost-effective and environmentally sustainable process for xylanase production and exhibits scope towards successful industrial applications.

Introduction: Developing somatic embryogenesis is one of the main steps in successful in vitro propagation and gene transformation in the carrot. However, somatic embryogenesis is influenced by different intrinsic (genetics, genotype, and explant) and extrinsic (e.g., plant growth regulators (PGRs), medium composition, and gelling agent) factors which cause challenges in developing the somatic embryogenesis protocol. Therefore, optimizing somatic embryogenesis is a tedious, time-consuming, and costly process. Novel data mining approaches through a hybrid of artificial neural networks (ANNs) and optimization algorithms can facilitate modeling and optimizing in vitro culture processes and thereby reduce large experimental treatments and combinations. Carrot is a model plant in genetic engineering works and recombinant drugs, and therefore it is an important plant in research works. Also, in this research, for the first time, embryogenesis in carrot (Daucus carota L.) using Genetic algorithm (GA) and data mining technology has been reviewed and analyzed.

Materials and methods: In the current study, data mining approach through multilayer perceptron (MLP) and radial basis function (RBF) as two well-known ANNs were employed to model and predict embryogenic callus production in carrot based on eight input variables including carrot cultivars, agar, magnesium sulfate (MgSO₄), calcium dichloride (CaCl₂), manganese (II) sulfate (MnSO₄), 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BAP), and kinetin (KIN). To confirm the reliability and accuracy of the developed model, the result obtained from RBF-GA model were tested in the laboratory.

Results: The results showed that RBF had better prediction efficiency than MLP. Then, the developed model was linked to a genetic algorithm (GA) to optimize the system. To confirm the reliability and accuracy of the developed model, the result of RBF-GA was experimentally tested in the lab as a validation experiment. The result showed that there was no significant difference between the predicted optimized result and the experimental result.

Conclutions: Generally, the results of this study suggest that data mining through RBF-GA can be considered as a robust approach, besides experimental methods, to model and optimize in vitro culture systems. According to the RBF-GA result, the highest somatic embryogenesis rate (62.5%) can be obtained from Nantes improved cultivar cultured on medium containing 195.23 mg/l MgSO₄, 330.07 mg/l CaCl₂, 18.3 mg/l MnSO₄, 0.46 mg/l 2,4- D, 0.03 mg/l BAP, and 0.88 mg/l KIN. These results were also confirmed in the laboratory.

Adenoviruses are commonly utilized as viral vectors for gene therapy, genetic vaccines, and recombinant protein expression. To generate replication-defective adenoviruses, E1-complementing cell lines such as HEK293A are utilized; however, limitations remain. Repeated passage of E1-deleted virus in HEK293A cells increases the occurrence of replication-competent adenoviruses (RCAs). In the present study, we developed a novel cell line originating from human primary cells. L132 cells were transduced two times with E1-encoded retrovirus and three times with E1A-encoded retrovirus. Finally, we selected the most productive L132 cell line for generation of RCA-free adenovirus, GT541. GT541 can serve as an alternative cell line to HEK293A and other adenovirus-producing cells.

This study introduces a novel diagnostic modality for the detection of feline panleukopenia virus (FPV) antibodies in feline serum by using fluorescent microsphere immunochromatographic test strips (FM-ICTS). Leveraging the inherent specificity of antigen-antibody interactions, the FM-ICTS approach demonstrates considerable potential for efficient and accurate FPV antibody detection within a short timeframe. The FM-ICTS method demonstrates strong diagnostic performance, with consistent accuracy and stability over time. PBS buffer dilution enables detection across the range of FPV antibody haemagglutination inhibition (HI) titres in both healthy and immunized or infected cats. A high correlation (R² = 0.9733) between the T/C ratio and FPV antibody titres confirms the method's effectiveness in quantifying these titres. Clinical validation with 84 samples supports its reliability by matching results with HI assays. Additionally, stability tests show that the test strips maintain performance during storage, with a coefficient of variation (CV) below 12% over three months at 25℃. This innovative FM-ICTS framework emerges as a promising avenue for expedient and dependable disease diagnosis within the realm of veterinary science, offering implications for timely disease management and surveillance.