BMC Biotechnology最新文献

Spider silk is highly attractive material because of its superior mechanical properties and excellent biocompatibility, enabling it to self-assemble into a wide range of morphological structures for drug delivery system. However, most spider silk particles developed as drug carriers are based on complex repetitive domains of spider silk proteins and exhibit relatively large particle sizes (> 300 nm), which limits their biomedical applications. In this study, we engineered a novel recombinant spider silk protein (NC-iRGD) by integrating terminal domains derived from major ampullate silk and the tumor-penetrating peptide iRGD. The silk particles were generated by mixing with a high-concentration potassium phosphate buffer and exhibited an average particle size of approximately 170 nm, which is smaller than that of other reported spider silk particles. Under incubation of silk particles in the drug solution, a 90% loading efficiency for the peptide drug (ChMAP-28) were determined. The cytotoxicity result showed that NC-iRGD particles displayed excellent biocompatibility and high drug loading efficiency in the neutral pH and low ionic strength. The release of ChMAP-28 was shown to be dependent on the ionic strength and pH of the release buffer. Additionally, NC-iRGD demonstrated enhanced tumor penetration and greater cytotoxicity against cancer cells compared to NC particles due to its iRGD sequence. Overall, the high drug loading capacity, controlled-release capability, and improved tumor penetration of NC-iRGD particles make them a promising novel drug delivery system for targeting polypeptide therapeutics to tumor microenvironments.

Background: The discovery of novel DNA methyltransferase (DNMT) inhibitors as anticancer agents represents a significant milestone in pharmaceutical research. However, the absence of robust high-throughput screening methods for these compounds has substantially hindered their development.

Results: In this study, we found that the epithelial splicing regulatory protein 1 (ESRP1) was underexpressed in renal cell carcinoma (RCC) cells. ESRP1 overexpression induced G1-phase arrest and inhibited the proliferation of RCC cells by downregulating cyclin A2 expression. Furthermore, the ESRP1 promoter was hypermethylated in RCC cells, and treatment with 5-aza-2'-deoxycytidine (5-Aza-CdR), a DNMT inhibitor, effectively demethylated the CpG sites within the promoter region of ESRP1, thereby upregulating the transcriptional activity of the ESRP1 promoter and gene expression both in vitro and in vivo. Additionally, we constructed a bioluminescent reporter gene (designated ESRP1-P-Luc2) by fusing the promoter sequence of the ESRP1 gene with the luciferase gene using molecular cloning techniques. Bioluminescence imaging revealed that 5-Aza-CdR treatment could upregulate the expression of the reporter gene both in vitro and in vivo.

Conclusions: Our results demonstrate that in RCC cells, ESRP1 promoter hypermethylation is accompanied by downregulation of its expression level; restoring ESRP1 expression can induce cell cycle G1-arrest and inhibit RCC cell proliferation by downregulating cyclin A2 expression; ESRP1-P-Luc2 may serve as a useful tool for monitoring the effects of DNMT inhibitor anticancer drugs at both the cellular level and in living animals, thereby providing a potential tool for high-throughput screening (HTS) of such drugs.

Background: Natural killer (NK) cell immunotherapy is a promising approach for cancer treatment. However, its extensive clinical application was limited to the large-scale clinical-grade expansion of NK cells. In this study, we expanded NK cells from healthy donor's peripheral blood mononuclear cells (PBMCs) using a newly designed K562 feeder cell line.

Methods: The feeder cells were generated by transducing K562 cells with lentiviral particles carrying 4-1BBL and mbIL-21/-15. NK cells were expanded from PBMCs with these genetically modified, frozen-thawed and irradiated K562 feeder cells in the presence of IL-2. The purity, quantity, and receptors expression of the expanding NK cells were dynamically monitored. Furthermore, their anti-tumor efficacy was evaluated both in vitro and in vivo following a two-week expansion period.

Results: The K562-4-1BBL-mbIL-21/-15 feeder cells induced highly-efficient NK cells expansion from PBMCs (17902-fold) within two weeks. There was a notable upregulation in the expression of activating receptors including NKG2D, NKp30, NKp44, and NKp46 during the expansion process. Moreover, the expanded NK cells displayed enhanced cytotoxicity against a variety of hematological (K562, MOLM-13, OCI-AML-3, THP-1) and solid (Hep-G2, OVCAR3) cancer cell lines in vitro. In the humanized U937 xenograft mouse model, the NK cells extended the median survival time of the AML-bearing mice from 19.40 to 28.25 days.

Conclusions: We have successfully established a highly-efficient, cost-effective and rapid NK cell expansion platform from PBMCs utilizing K562-4-1BBL-mbIL-21/-15 feeder cells, which also significantly improved the cytotoxicity both in vitro and in vivo, presenting a significant advancement in the field of NK cell-based immunotherapy.

Accurately predicting the therapeutic response of non-small cell lung cancer (NSCLC) patients to tyrosine kinase inhibitors (TKIs) is of significant clinical importance. The use of TKIs in clinical is primarily guided by the detection of EGFR gene mutations. However, the current EGFR mutation assays face challenges such as inconsistent correlation with therapeutic outcomes, inconvenient sample availability and limited sensitivity. To address these, we have designed and synthesized a novel theranostic agent, OSA-Cy7, by conjugating the third-generation EGFR-TKI osimertinib with the near-infrared (NIR) fluorophore Cy7. This conjugate aims to enable fluorescence-based detection of mutant EGFR and targeted therapy of NSCLC. Our studies demonstrated that OSA-Cy7 selectively accumulates in EGFR-mutant NSCLC cell lines, such as PC9 (exon 19 deletion) and H1975 (L858R/T790M), exhibiting enhanced fluorescence signals, while showing minimal uptake in wild-type EGFR A549 cells. Western blot analysis confirmed that OSA-Cy7 effectively inhibits EGFR phosphorylation in mutant cell lines, with negligible effects on wild-type EGFR phosphorylation. Furthermore, OSA-Cy7 treatment resulted in significant suppression on cell proliferation and colony formation in EGFR-mutant cells, indicating its potent anticancer activity. These findings suggest that OSA-Cy7 holds promise as a theranostic agent for the selective imaging and treatment of EGFR-mutant NSCLC, potentially improving patient stratification and therapeutic monitoring.

Background: Alkaline proteases are hydrolytic enzymes that play a crucial role in various biological processes. Proteases produced by halophiles showed exceptional efficiency in breaking down the complex structures ranging from pigments to extracellular proteins, tissue proteins to tumors, and thrombin clots. There is a huge global demand for naturally occurring thrombolytic proteases to treat intravascular thrombosis, as they are cost-effective and have minimal detrimental effects, making them widely used in the biomedical applications.

Results: The production of thrombolytic protease from the rare halophile Brachybacterium paraconglomeratum strain M4 and its in-vitro thrombolytic activity is investigated for the first time. On skim milk agar, a distinct zone of casein hydrolysis was observed, in submerged fermentation a substantial protease production was noted. The enzyme was purified through a four-step purification process following an initial precipitation with 60% ammonium sulfate, subsequent ion-exchange chromatography using DEAE-cellulose, and final gel-filtration chromatography utilizing Sephadex G-100. As a result, a protein with a specific activity of 300 ± 32 U/mg was obtained with a purification fold of 19 and a recovery percentage of 38.2%. The molecular mass of the purified enzyme was 14.4 kDa via SDS-PAGE while MALDI/MS analysis further revealed a 131 amino acid sequence with an isoelectric point of 8.55 and on comparison, the strain M4 alkaline protease aligns with the proteasome subunit alpha. The protease is classified as a serine protease based on the inhibition by PMSF and its activity profile. The in-vitro thrombolytic assay revealed that the purified enzyme achieved a clot lysis rate of 65 ± 3%, performing effectively in comparison to the standard.

Conclusion: The findings indicate that strain M4 is an efficient producer of thrombolytic protease and the purified form effectively dissolves thrombin clots. Currently, the studies are underway to explore its potential for biomedical applications and industrial-scale production.

Biodiesel, a renewable and eco-friendly liquid biofuel, plays a crucial role in reducing greenhouse gas emissions. Initially, biodiesel production relied on vegetable oils, non-edible oils, and waste oils. However, these sources face challenges, including high costs, labor and land requirements, and insufficient supply to meet demand, especially in the case of waste oils. Recent research highlights the potential of lignocellulosic substrates for biodiesel production via oleaginous microorganisms, which can accumulate lipids similar to those in vegetable oils under stress conditions. This review investigates various biodiesel feedstocks from microorganisms such as microalgae, fungi, yeast, and bacteria. It details the biodiesel production process from lignocellulosic substrates, biological pretreatment and bioconversion. Additionally, the review underscores the role in biofuel and biorefinery development and briefly discusses the integration of biofuels within a circular economy framework.

This study aims to explore and optimize the conditions for in vivo transfection and gene editing, emphasizing their potential applications in the treatment of infertility in mice. Our findings indicate that physical transfection can be effective in the dynamic fluid environment of mouse seminiferous tubules, with electroporation achieving transfection in multilayered cell tissues. Using the mTmG fluorescence reporter system, we visually assessed the efficiency of electroporation-based transfection and observed stable gene editing outcomes across different individuals. Additionally, we achieved effective transfection of germ cells in vivo for the clinical application of gene tools. We further investigated the impact of various delivery methods and molecular methods on transfection efficacy, revealing that RNP technology is adaptable and efficient in vivo, particularly in the context of treating hereditary diseases. We attempted to leverage gene editing techniques to address spermatogenesis blockage at different stages in Ythdc2-KO and CK137956-KO mice. While we did not succeed in rescuing spermatogenic blockage in Ythdc2 KO mice, the treatment of CK137956 KO mice yielded significant physiological responses. These results could be beneficial for the optimization of in vivo gene editing technologies for clinical applications.

Novel antifungal compounds effective against phytopathogenic fungiwere identified by evaluating an n-butanol extract obtained from the fermentation broth of Streptomyces diastatochromogenes strain No.1628. The extract exhibited had strong antifungal activity against Botrytis cinerea, Fusarium oxysporum, and Rhizoctonia solani, markedly reducing the spore germination rates of F. oxysporum and B. cinerea to 25.65% and 28.23%, respectively, at a concentration of 35 mg/L. In vivo efficacy assays further demonstated that the extract achieved disease control efficiencies of 53.42% and 55.68% against Rhizoctonia rot following irrigation at 10 mg/L for 14 and 21 days, respectively. Subsequent chemical investigation led to the isolation of five antifungal compounds from the n-butanol extract: the novel tetraene macrolide, which was structurally elucidated through spectroscopic analysis as (7E,12Z,13E,15E,17E,19E)-21- ((4-amino-3,5-dihydroxy-6-methyltetrahydro-2 H-pyran-2-yl)oxy -)-12-ethylidene-1,5,6,25-tetrahydroxy-11-methyl-9-oxo-10,27-dioxabi-cyclo[21.3.1] -heptacosa-7,13,15,17,19-pentaene-24-carboxylic acid (compound 1), and four other already known antifungal agents, namely tetrin B (2), tetramycin A (3), toyocamycin (4) and anisomycin (5). Compound 1 exhibited potent inhibitory activity against the hyphal growth of R. solani, F. oxysporum, and B. cinerea, with IC₅₀ values of 0.20, 1.28, and 1.53 µg/mL, respectively. These fundings underscore S. diastatochromogenes as a promising microbial source for the discovery of natural antifungal agents.

Background: Tuberculosis (TB) and lung cancer (LC) are among the leading causes of death worldwide and present serious challenges in diagnosis and treatment. Therefore, developing new strategies for their treatment is crucial. MicroRNAs (miRNAs) are biological molecules that play a critical role in regulating essential processes, such as apoptosis and autophagy, in TB and LC by targeting specific genes. Recently, carbon nanotubes functionalized with Polyethyleneimine (CNT-PEI) to deliver miRNAs to target cells have been investigated to enhance therapeutic effects.

Methods: In this study, miR-146a was transfected into LC (A549), macrophages infected with TB (THP1), and healthy lung cells (MRC5) using CNT-PEI. Then, the expression of miR-146a and its target gene, TNF receptor-associated factor-6 (TRAF6), and other genes involved in apoptosis and autophagy pathways including BCL-2, IL-6, tumor necrosis factor-alpha (TNFα), were measured using Real-Time PCR. Finally, the effect of overexpression of miR-146a on these genes was investigated in all three cell lines.

Result: The results showed successful transfection of miR-146a using the CNT-PEI nano delivery system in LC and TB cell models. Then, increased expression of miR-146 increased apoptosis and autophagy by targeting the TRAF6 gene and affecting other genes such as BCL-2, IL-6, and TNFα through the NF-kB signaling pathway.

Conclusion: The findings suggest an important role for miR-146a in TB and LC, which regulates inflammatory responses and treats these diseases. However, further studies are needed on using CNT-PEI in vivo, as well as the balance between local anti-inflammatory and non-inflammatory factors.