BMC Biotechnology最新文献

Background: RNA degradation during freeze‒thaw cycles in cryopreserved tissues is a major challenge for biomedical research, particularly when tissues are stored without preservatives. While agents such as TRIzol and RNALater are effective for fresh tissues, their utility for archival frozen tissues remains unclear.

Results: We evaluated various RNA preservation strategies for frozen rabbit kidney tissues without preservatives, considering key variables such as thawing temperatures (ice vs. room temperature (RT)), preservatives (RNALater, TRIzol, and RL lysis buffers), processing delay (time before disruption), tissue aliquot sizes (ranging from 70-100 mg, 100-150 mg, to 250-300 mg), and freeze‒thaw cycles. Compared with RT-treated frozen rabbit tissues, preservative-treated tissues presented significantly greater RNA integrity when thawed on ice (p < 0.01). The RNALater group performed best in maintaining high-quality RNA (RIN ≥ 8). Although a significant difference in the RIN was observed between the 120-minute and 7-day processing delays (9.38 ± 0.10 vs. 8.45 ± 0.44), all the samples ≤ 30 mg maintained a RIN ≥ 8. For tissues ≤ 100 mg, thawing overnight on ice or at -20 °C maintained a marginally higher RIN (RIN ≥ 7). However, larger tissue aliquots (250-300 mg) presented significantly lower RINs with ice thawing than with thawing at -20 °C (5.25 ± 0.24 vs. 7.13 ± 0.69). After 3-5 freeze‒thaw cycles, tissues thawed at -20 °C presented notably greater variability in the RIN, particularly in larger tissue aliquots. In validation experiments involving cryopreserved human and murine kidney tissues, the RNALater-treated murine kidney tissues ≤ 30 mg consistently maintained high-quality RNA integrity (RIN ≥ 8), whereas the frozen human kidney tissues resulted in marginally reduced RINs compared with those of the LN grinding control (7.76 ± 0.54).

Conclusions: Preservatives, tissue aliquot sizes, and thawing methods significantly impact the RNA quality of frozen tissues originally stored without preservatives. Key recommendations include (1) adding RNALater during thawing, (2) thawing on ice for small aliquots (≤ 100 mg) or at -20 °C for larger samples, and (3) minimizing freeze‒thaw cycles, despite observed variations among species.

Adenovirus is one of the most attractive viral vectors for therapeutic vaccines and gene therapy with the caveat that replication-competent adenoviruses (RCA) can be produced. To remediate this problem, engineered A-549 adenoviral vector complementing cells (SF-BMAdR cells) were previously generated by our organization for the production of E1-deleted adenoviral vectors without RCA. However, the production process remained to be improved for high titer production and scalability, as cost-effective and scalable biomanufacturing processes are critical for commercializing adenovirus-based vaccines and gene therapy. In this study, we first explored the potential of batch and fed-batch culture to increase maximum cell density and virus productivity by evaluating four different commercially available serum-free media and their combinations, and several feeds. A mixture (1:1) of two culture media improved the maximum cell density from 2.8 × 10⁶ cells/mL obtained in the current batch culture to 4.2 × 10⁶ cells/mL, and increased the virus productivity by 70% at a titer of 1.5 × 10¹⁰ vp/mL. The fed-batch culture process, however, did not yield a significant improvement in either the maximum cell density or virus productivity. In contrast, batch culture with one medium replacement not only increased the cell growth but also resulted in an additional 70% improvement in the virus productivity at 2.6 × 10¹⁰ vp/mL. The virus productivity was further increased to 6.3 × 10¹⁰ vp/mL in a 3 L bioreactor perfusion culture infected at 7.0 × 10⁶ cells/mL. This titer is 7.5 folds of the titer obtained in the current process. This study demonstrated the potential for a drastic improvement in the productivity of RCA-free adenovirus in the SF-BMAdR culture process. Furthermore, various processes developed fulfill different operational needs in manufacturing of RCA-free adenovirus to meet the increasing demands for therapeutic vaccines and gene therapy.

In ocular drug delivery, increasing the retention time of the drug by using in situ forming hydrogels can be effective. In the current study, hydrogels with suitable gelling properties were obtained using oxidized gellan gum as a green crosslinker and gelatin via Schiff-base linkages. In the first step, oxidation of gellan gum was done to obtain aldehyde groups, and in situ gelling hydrogels were formed based on a simplistic Schiff-base reaction between oxidized gellan gum and amine groups of gelatin in the presence of β-glycerophosphate disodium salt as a pH adjustment agent and gelation accelerator. The calculated aldehyde content was about 64.5% by the titration method. By optimizing the concentration of gelatin, oxidized gellan gum, and β-glycerophosphate disodium salt, hydrogels with a fast gelation time were obtained. The synthesized hydrogels were investigated via Fourier-transform infrared (FT-IR) spectroscopy, scanning electron microscope (SEM), X-ray diffraction (XRD), swelling degree, and rheological properties. The effect of oxidized gellan gum and gelatin concentration on the physical and chemical characteristics of the obtained hydrogel system were studied. The results of in vitro and ex vivo research indicated that the prepared hydrogel with a high concentration of oxidized gellan gum and gelatin had a higher release rate, stronger network, and homogeneous morphological structure. The synthesized hydrogels have adequate porosity for drug loading (around 98-99%). Also, the MTT cytotoxicity test on bone marrow mesenchymal stem cells (BMSCs) confirmed that the developed hydrogels with green crosslinker do not have cytotoxic effects. In vitro and ex vivo drug release tests demonstrated a facile preparation strategy for fast-gelling hydrogels that can be considered as an appropriate candidate for timolol maleate delivery in glaucoma treatment.

Background: Mesenchymal stem cells (MSC) play a vital role in bone regeneration. Poly-(l-lactide-co-ε-caprolactone) scaffolds functionalised with modified rhamnogalacturonan-I (RG-I) potato pectin, can modulate inflammation and promote bone regeneration in-vitro and in-vivo by modulating the action of the galactoside-binding galectin-3. In this study, we determined the binding affinity to galectin-3 of potato unmodified RG-I (PU) and its arabinose-deficient form (PA) and investigated the transcriptomic effects of PA treatment on human MSCs evaluated through in-silico pathway analysis in relation to galectin-3.

Methods: The binding affinity of RG-I (PU and PA) to galectin-3 was assessed by tryptophan fluorescence spectroscopy. Human MSC (hMSC) isolated from the bone marrow of elderly patients (> 60yrs), were analysed by transcriptomic profiling. The effect of PA surface coating on gene expression was assessed in-silico by the ingenuity pathway analysis (IPA).

Results: RG-I binds to galectin-3 with a binding affinity of 8.66 × 10^- 6 M. Galactose sidechains of PA resulted in ~ 10-fold increased binding of RG-I to galectin-3 compared with PU. Forty-two genes were found to be differentially expressed (DE) in hMSCs cultured on PA surface coating, with a false-discovery rate (FDR) < 0.1. IPA of these DE genes including LGALS3, encoding for galectin-3, showed enrichment for organismal disorders-abnormalities, connective tissue and skeletal muscular disorders, immunological and inflammatory diseases, and identified three gene networks: infectious diseases, immune & inflammatory response and cell cycle control.

Conclusions: This study indicates that modified potato pectin (PA) can regulate the expression of a broad range of genes, including galectin-3, that are involved in cellular, inflammatory, and immunological functions of hMSCs and may potentially be used to promote bone regeneration via modulation of inflammation.

Collagen, the most abundant protein in the mammalian extracellular matrix, is critical for skin structure and function, with Type I and Type III collagens being particularly important. Collagen degradation in skin is accelerated by aging and UV exposure, leading to structural and functional impairments. Exogenous collagen supplementation has been shown to restore skin structure and function. Traditional collagen extraction from animal tissues is limited by safety and quality concerns, while recombinant human collagen offers improved safety but faces challenges in solubility and production. This study aimed to construct a chimeric collagen derived from both Type I and Type III collagens and achieve its soluble expression in E. coli. Through translational pausing technology, a recombinant chimeric human collagen containing functional domains of both collagen types was successfully constructed and expressed with a yield of 1.36 g/L. A cysteine-rich C-propeptide domain was fused to enhance assembly and stability. The "dual-function" collagen significantly promoted fibroblast proliferation, migration, and adhesion, while stimulating endogenous Type I and Type III collagen production. Using the C. elegans model, the recombinant protein extended lifespan and enhanced oxidative aging resistance. Skin imaging confirmed its penetration into the dermis, and human skin efficacy tests demonstrated its ability to reduce periorbital wrinkles and crow's feet. This recombinant "dual" human collagen promotes endogenous collagen synthesis, accelerates skin repair, reduces aging signs, and shows no observed side effects, offering promising potential for anti-aging applications.

During the fermentation of small-leaved Kuding tea by Aspergillus cristatus, significant fluctuations in environmental osmotic pressure can affect the growth and metabolism of A. cristatus, thereby influencing the quality of the fermented tea. To investigate osmotic pressure effects on its metabolism, this study used NaCl (0%, 4%, 8% and 12%) in small-leaved Kuding tea medium to establish osmotic gradients, followed by morphological observation, in vitro antioxidant activity assays, and liquid chromatography‒mass spectrometry (LC‒MS) nontargeted metabolomics analysis. The results demonstrated that the NaCl concentration had significant effects on colony morphology and metabolites. The colony diameters and antioxidant activities were significantly greater at 4% and 8% NaCl than at 0% or 12% NaCl. Nontargeted metabolomics identified 556 differential metabolites, 93 of which common differential metabolites covered seven categories, such as amino acids, organic acids and flavonoids. Under 4% NaCl, key antioxidants (melatonin, erythritol, and luteolin) significantly accumulated, which correlated with the activation of the core metabolic pathways, including tryptophan metabolism and flavonoid biosynthesis. Hypertonic stress (12% NaCl) suppressed these core pathways, leading to the induction of repair metabolites (e.g., methyl jasmonate). Low salt (0% NaCl) also inhibited core pathways, leading to a reduction in broad-spectrum antioxidant capacity and an accumulation of specific metabolites (e.g., xanthoxylin). Aspergillus cristatus dynamically regulates metabolism to adapt to different salt concentrations, with 4%-8% NaCl optimal for achieving maximal growth-antioxidant defense equilibrium in small-leaved Kuding tea. These findings provide a foundation for optimizing fermented tea processes and high-antioxidant products.

Background: Foot-and-mouth disease (FMD), caused by foot-and-mouth disease virus (FMDV), is an acute, highly contagious, and economically devastating disease affecting cloven-hoofed animals worldwide. Vaccination remains the cornerstone of FMD control strategies. To increase the production yield and structural stability of virus-like particles (VLPs), we investigated the effects of the N-terminal truncation of VP0 on the soluble co-expression of three capsid proteins, and the stability of the assembled particles.

Results: A series of VP0 variants with progressive N-terminal truncations (5-20 amino acids) were co-expressed with VP3 and VP1 via a single plasmid in the Escherichia coli (E. coli) expression system by fusing these proteins with a small ubiquitin-like modifier (SUMO) tag. The assembly characteristics of the co-expressed fusion proteins were systematically characterized. Our findings show that all engineered fusion proteins were successfully co-expressed, and truncations between residues 10 and 20 yielded significantly higher expression levels and purification efficiencies than those of full-length VP0. The 031-VP0 dN10, 031-VP0 dN15, and 031-VP0 dN20 exhibited increased stability of the assembly and retained the native morphology. An in vitro binding assay for neutralizing antibodies against FMDV indicated that all VLPs maintained wild-type-like antigenic properties, demonstrating preserved conformational epitopes. Pigs immunized with VLPs-based vaccines exhibited humoral and cellular immune responses, with the 031-VP0 dN20 variant eliciting significantly stronger cellular immunity.

Conclusions: Collectively, our findings establish a robust platform for the efficient production of structurally stable FMDV VLPs in E. coli, providing a valuable framework for developing soluble expression systems for other FMDV serotypes.

Background: Forkhead box K1 (FOXK1) is a newly discovered gene encoding a transcription factor in the FOX family. It plays important roles in the cell cycle, cell growth, proliferation, differentiation, apoptosis, metabolism, DNA damage, drug resistance, angiogenesis, and carcinogenesis by binding to DNA to function as a transcription factor. Many studies have confirmed that FOXK1 plays a role in promoting cancer in a variety of tumors. However, the pathogenic role of FOXK1 in diabetic retinopathy (DR) is still unclear. The present study was conducted to investigate the mechanism of FOXK1 in the vascular endothelial dysfunction of DR.

Methods: Human umbilical vein endothelial cells (HUVECs) were exposed to high glucose (HG) concentrations (25 mmol/L) to establish a vascular endothelial dysfunction model of DR. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the differential expression of FOXK1 in HUVECs treated with HG and low glucose (LG). CCK-8, Transwell, flow cytometry and tube formation assays were used to investigate the effect of FOXK1 on HUVECs function. To explore the downstream signaling pathway of FOXK1, Western blotting was subsequently used to detect the protein expression of p-AKT and AKT after FOXK1 was knocked down in HUVECs. The AKT inhibitor MK-2206 was used to treat HUVECs, and cell migration, apoptosis and angiogenesis were observed.

Results: The results showed that FOXK1 expression was significantly increased in HUVECs cultured with high glucose. In this endothelial dysfunction model, knockdown of FOXK1 increased HUVECs activity and migration, inhibited HUVECs apoptosis, angiogenesis and VEGF expression, and activated the p-AKT/AKT signaling pathway. All of these were reversed by the AKT inhibitor MK-2206.

Conclusions: FOXK1 may mediate vascular endothelial dysfunction in DR by inhibiting p-AKT/AKT pathway.