Applied Biochemistry and Biotechnology最新文献

MicroRNA (miR)-328-3p is believed to have anti-tumor impacts in various human cancers. However, its role in prostate cancer (PCa) is uncertain. In this research, miR-328-3p expression in PCa was reduced. Meanwhile, it was discovered that miR-328-3p directly targeted profilin-1 (PFN1) 3'-untranslated region to negatively modulate PFN1. Elevating miR-328-3p or reducing PFN1 suppressed cell growth, migration, and invasion, and epithelial-mesenchymal transition; overexpression of miR-328-3p or inhibition of PFN1 delayed tumor growth in vivo. Further studies found that PCa patients with advanced T stage or high Gleason score had significantly lower miR-328-3p compared to PCa patients with early stage or low score. In addition, PCa patients with high miR-328-3p had a better prognosis than those with low miR-328-3p. Briefly, this study highlights the clinical and biological role of miR-328-3p as a tumor suppressor miRNA in PCa and explores the downstream mechanisms of miR-328-3p.

This study aimed to determine the effects of novel N-{3-[(pyridin-4-yl)carbamoyl] phenyl} thiophene-2-carboxamide or PCPTC chemical moiety loaded Poly(lactic-co-glycolic acid)-Poly (Ethylene glycol) or (PLGA-PEGylated) NP as an anti-metastatic Ran GTPase therapeutic agent on MDA-MB231 triple-negative human breast cancer cells. Molecular docking and MD simulation was done to determine the binding potential of novel carboxamide PCPTC with Ran GTPase. PLGA and PLGA-PEG based NP encapsulating PCPTC were fabricated using the Modified Double Emulsion Solvent Evaporation Technique and characterized for size, zeta potential, polydispersity and morphology. In vitro evaluation of loaded nanoparticles such as cellular localization study, cell proliferation, cell migration, cell invasion and Ran Pull Down assay were carried out on MDA-MB231 breast cancer cells. Ran downregulation was determined by pull down assay. PCPTC with Ran GTPase exhibited strong structural stability based on in silico analysis. The average sizes of PCPTC loaded NP ranged between 166.3 nm to 257.5 nm and were all negatively charged. Scanning electron microscopy data showed that loaded NP were smooth and spherical. Fluorescence microscopy data confirmed the intracellular localization of loaded nanoparticles inside the MDA-MB231 cells. Cell proliferation assay (MTT assay) confirmed the cytotoxic effect of the loaded-NP when compared to blank nanoparticles. PCPTC-loaded NP inhibited metastasis and invasion of MDA-MB231 cells. This anti-metastatic and the anti-invasive effect was due to the Ran GTPase cycle blockage, which was confirmed by performing Ran Pull down assay. we propose that PCPTC is a promising compound to inhibit Ran GTPase and may act as a potential therapeutic agent against breast cancer. PCPTC-loaded NP successfully stopped the metastasis of MDA-MB231 cells by disrupting the Ran cycle.

A probiotic is a live bacterium that, when given in sufficient proportions, helps to improve the host's gut health. Kimchi and pearl millet, two traditional foods, were used to isolate probiotic bacterial strains. This study's primary goals were to isolate, identify and analyse the microorganisms for potential probiotic traits, tolerance to gastrointestinal environments, and antimicrobial activity, and produce probiotic capsule. The present investigation resulted with identification of two probiotic strains (KAC1 and PAC1) from conventional foods, such as kimchi and pearl millet porridge. The isolated probiotics were identified as Enterobacteriaceae family by 16S rRNA sequencing and are deposited in GenBank (NCBI), accession numbers OQ629827 (KAC1) and OQ629828 (PAC1), respectively. These strains exhibited the characteristics of possible probiotic traits, such as the ability to tolerate simulated gastric juice, inhibits the growth of pathogenic bacteria, auto-aggregation, co-aggregation, and hydrophobicity. Furthermore, spectroscopic analysis divulges some critical findings which corroborate the results obtained. Finally, capsules containing freeze-dried  probiotics was successfully produced.

Non-alcoholic steatohepatitis (NASH) is a potential serious disease, which almost has no available medicine for effective treatment today. Efruxifermin is a bivalent Fc-FGF21 candidate drug developed by Akero Therapeutics that has shown promising results in preclinical and clinical trials for NASH and may be approved in future. However, it is produced by Escherichia coli (E. coli) expressing system, which has no glycosylation modifications and is hard to purify for inclusion body. Suspension mammalian cell expression systems, human embryonic kidney 293 (HEK293), and Chinese hamster ovary (CHO) are good choice for protein expression of biopharmaceutical use. In this report, the objective was to produce Fc-FGF21 by mammalian cell expression systems, which enabled necessary glycosylation modifications to occur on the Fc-FGF21 protein and was relatively easy for future large-scale production. We observed that the target protein Fc-FGF21 could be easily degraded in CHO system, such as CHOK1SV or CHOZN, and it was hard to purify, whereas it was more stable in the HEK293 expressing system. Then, similarity between Fc-FGF21 from E. coli and Fc-FGF21 from HEK293 was focused by in vitro and in vivo studies, and we observed no significant difference between the proteins expressed from the two different expressing systems, indicating that a biosimilar of Efruxifermin has been developed successfully. Proteomics analysis from in vivo study samples further identified some potential biomarkers or FGF21 related signaling pathways. Taken together, this study demonstrates a good example of how to develop and validate a biosimilar for therapeutic purposes. In future, more efforts, such as mutation to FGF21 or linking FGF21 with effective antibody to form dual targets, could be done to obtain more effective FGF21 analogs.

A dual lipase system has been developed to convert soybean oil into biodiesel through synergistic catalysis of Thermomyces lanuginosus lipase (TLL) and Yarrowia lipolytica lipase 2 (YLL) in this study. Pichia pastoris recombinant strains expressing lipases were successfully constructed, and the activities of TLL and YLL in the fermentation supernatant reached 23,142.71 ± 280.54 U/mL and 895.44 ± 27.31 U/mL, respectively. Immediately thereafter, free lipase was used to catalyze the preparation of biodiesel from soybean oil. After optimizing reaction conditions, 80 U/g oil TLL and 20 U/g oil YLL were used to catalyze the production of biodiesel, and a 95.56% biodiesel yield was obtained at 40 °C, 40% moisture content (water/oil, w/w), and stepwise addition of five molar equivalents of methanol. Double lipase plasmids (tll gene and yll gene in different proportions) were constructed in vitro and introduced into P. pastoris to construct a recombinant strain with optimal activity. Under the reaction conditions of 40% moisture content, 8% whole-cell biocatalyst dosage, and stepwise addition of five molar equivalents of methanol, the biodiesel yield reached 95.35% after 24 h at 40 °C. These results show that synergistic catalysis is an effective strategy for biodiesel synthesis and can not only improve the biodiesel yield but also shorten the reaction time. This study provides a scientific basis for biodiesel production by multi-enzyme compounding, with potential industrial applications.

The purpose of this study was to utilize a two-photon microscope excitation Airy beam to achieve three-dimensional imaging of intrahepatic bile ducts in BDL mice and patients with biliary atresia (BA). Ten male Balb/c mice aged 6-8 weeks underwent extrahepatic bile duct ligation (BDL), and 10 underwent sham operation as control. After the operation, the mice resulted in symptoms such as jaundice, darkened urine, and weight loss. Taken liver tissues from BDL and control mice and trimmed to 5*5*3 mm³ after 10 days. Sixteen patients with BA were included in this study. Liver transplantation was performed in 12 cases of them; liver hilar and liver margin tissues were taken during the operation. Kasai portoenterostomy (KPE) was performed in 4 cases, and liver margin tissues were taken. Intraoperative liver tissue samples were trimmed to a size of 5*5*5 mm³. The specimens were subjected to tissue fixation, antigen retrieval, antibody incubation, and subsequent tissue hyalinization following the principles of immunofluorescence staining. Subsequently, light-sheet fluorescence microscopy (LSFM) was followed, and intrahepatic bile ducts of the specimen were imaged utilizing Airy beam which was excited with high imaging depth attenuation-compensated two-photon. Deconvolution was applied to image processing to construct a three-dimensional model of intrahepatic bile ducts. Three-dimensional imaging of liver tissue was conducted in both BDL mice and BA patients, and the distribution of intrahepatic bile ducts was visualized. BDL mice exhibited notable widening of intrahepatic bile ducts, accompanied by bile duct hyperplasia. There was no obvious hyperplasia of intrahepatic bile duct in the control group. Significant small bile duct hyperplasia was seen on imaging of the intrahepatic bile ducts in patients with BA. The intrahepatic bile duct was disorganized and hyperplasia especially in patients who performed liver transplantation. The technique of Airy beam three-dimensional reconstruction can effectively image the intrahepatic bile ducts in Balb/c mice and BA patients in three dimensions. This approach contributes to a better understanding of the distribution of intrahepatic bile ducts in BA patients. Moreover, it facilitates the exploration of models that more accurately simulate BA disease by elucidating the distribution of intrahepatic bile ducts in animal models. Understanding the distribution characteristics of intrahepatic bile duct will facilitate the formulation of hilar bile duct microstructure classification, which can guide the operation and evaluate the prognosis better.

Uterine corpus endometrial carcinoma (UCEC) is one of the most common gynecologic tumors. Due to the high recurrence and metastasis of UCEC, it is crucial for patients to find new biomarkers for diagnosis and therapy. In this study, R software and the TCGA database were used to screen candidate UCEC predictive markers. Western blot and RT-qPCR were performed to detect protein and mRNA expression of EXT1 in UCEC cell lines. In addition, MTT assay, flow cytometry, transwell assay, and wound healing assay were conducted to assess the cell viability, apoptosis, invasion, and migration in UCEC cells. Overlap-extension PCR technique was employed to construct the vector targeting the deletion of the methylated segment of EXT1. The results showed that a total of 11 candidate genes were obtained and EXT1 was identified as a potential target. The expression and methylation levels of EXT1 were both increased in UCEC tissues and cell lines, as well as elevated EXT1 was closely related to the poor prognosis of patients. Besides, the knockdown of EXT1 significantly inhibited the malignant biological behaviors in UCEC cells. Additionally, the current study also found that the deletion of 1559-2146 bp CpG island segment upregulated EXT1 expression and promoted malignant biological behaviors in UCEC cells. Furthermore, the presence of m7G RNA methylation in UCEC cells also was found. In conclusion, the methylation of EXT1 influenced the gene expression, thereby affecting the malignant biological behaviors in UCEC cells and regulating the pathological progression of UCEC.

Nasopharyngeal carcinoma (NPC) is a malignant tumor originating from nasopharyngeal mucosa. Anoikis, a form of programmed cell death induced by detachment from the extracellular matrix, normally prevents metastasis. Resistance to anoikis in cancer cells can enhance their metastatic potential. This study identifies anoikis-related genes (ARGs) associated with NPC to elucidate tumorigenesis mechanisms. Analysis of the GSE12452 dataset from GEO revealed 77 differentially expressed ARGs in NPC tissues. GO and KEGG analyses highlighted significant enrichment in apoptosis-related pathways. A PPI network identified MYC, FN1, BRCA1, and FGF2 as Hub genes. Correlation analysis showed MYC positively correlated with activated dendritic cells (p < 0.01) but negatively with naive CD4 T cells (p < 0.001). FN1 was positively correlated with activated dendritic cells (p < 0.01) and negatively with M1 macrophages (p < 0.05). FGF2 negatively correlated with naive CD4 T cells (p < 0.001), while BRCA1 was positively correlated with eosinophils (p < 0.01). GSVA and GSEA indicated that MYC, FN1, BRCA1, and FGF2 were significantly enriched in cell cycle and DNA replication pathways. Immunohistochemistry and qPCR of 50 NPC samples confirmed the overexpression of these genes. Knockdown of MYC, FN1, BRCA1, and FGF2 led to increased tumor cell malignancy, with statistical significance (p < 0.05). This study identifies MYC, FN1, BRCA1, and FGF2 as anoikis-related genes (ARGs) with significant regulatory roles in nasopharyngeal carcinoma (NPC). These ARGs are found to be involved in the development and progression of NPC, suggesting their potential as therapeutic targets for this cancer.

MARCH5 is a key regulatory factor in mitochondria. However, the expression and function of MARCH5 in thyroid cancer (TC) are not yet clear. The research explores the role and the potential mechanism of MARCH5 in the tumorigenesis of TC. MARCH5 expression were measured by qRT-PCR and Western blot. CCK-8 kits were used to measure the cell viability. Cell scratch assay and Tanswell assay were used to measure cell migration and invasion, respectively. The pyroptosis related proteins (NLRP3, caspase-1, GSDMD) and mitochondrial autophagy related proteins (LC3-II, p62, parkin, pink1) were detected. The mitochondrial ROS GSH, MDA, and SOD were detected using commercial kits. Finally, a TC mouse model was constructed to detect the role of MARCH5 in tumor growth in vivo. The results displayed that the expression of MARCH5 was increased in TC patients and cells, and was significantly correlated with prognosis. Functional studies have found that MARCH5 inhibits oxidative stress levels and mitochondrial autophagy in TPC-1 cells. Further research has found that MARCH5 promotes the progression of thyroid cancer by degrading FUNDC1 and inhibiting the mitochondrial autophagy mediated pyroptosis pathway, regulating cell proliferation, migration, and invasion in TPC-1 cells. More importantly, interference with MARCH5 inhibits tumor growth and further development of TC in vivo. In conclusion, MARCH5 promotes the progression of thyroid cancer by degrading FUNDC1 and inhibiting the mitochondrial autophagy mediated pyroptosis, regulating cell proliferation, migration, and invasion. This study provides new theoretical basis for the treatment and prevention of TC in clinical practice.

Tamoxifen (TAM) is vital in breast cancer (BC) treatment, yet its resistance significantly impairs its efficacy. While miR-592 is known for its suppressive role in BC, its effect on chemotherapy resistance remains unclear. In this study, we observed a significant reduction in miR-592 levels in TAM-resistant BC tissues and cell lines. Low miR-592 expression was significantly associated with advanced TNM stage, lymph node metastasis, and poorer patient survival. Dual-luciferase assay confirmed miR-592 binding to the predicted gene PIK3CA. Increasing miR-592 levels decreased the IC50 of TAM, inhibited cell viability, migration, and invasion, and enhanced apoptosis in vitro, which was mimicked by PIK3CA knockdown and reversed by PIK3CA overexpression. Moreover, miR-592 upregulation suppressed tumor growth and improved TAM responsiveness in vivo. Molecularly, both si-PIK3CA and miR-592 mimics decreased the expression ratios of p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR, while increasing cleaved caspase-3 and E-cadherin expression in MCF-7/TAM cells. PIK3CA overexpression partially reversed these reductions. In conclusion, our study demonstrates that miR-592 attenuates TAM resistance by inhibiting the PIK3CA-driven PI3K/AKT/mTOR signaling pathway, representing a promising strategy to address chemoresistance in BC.