Development and verification of a reversed phase high-performance liquid chromatography method for purity determination of recombinant Mycobacterium tuberculosis Ag85b protein stock solution / 中国生物制品学杂志

@#Objective To develop and verify a reversed phase high-performance liquid chromatography method for the determination of the purity of recombinant Mycobacterium tuberculosis(Mtb)Ag85b protein stock solution.Methods Fourfactor,three-level orthogonal test was designed,with the area,trailing factor,peak area and peak area RSD as the evaluation indexes to explore the optimal detection conditions. The methodology verification of specificity,linear range,precision and durability was conducted in accordance with the general principles of Chinese Pharmacopoeia(Volume Ⅳ,2020 edition)9101.Results The results of all the evaluation indexes were good when the elution ratio of organic phase was30% ～ 95%,the detection temperature was 35 ℃,the sample volume was 3 μg,and the elution time of 95% organic phase was 15 min. The method had the linear correlation coefficient(R2)of 0. 998 5,the linear range of 1. 8 ～ 4. 2 μg,the reproducibility RSD of 0. 01%,and the intermediate precision RSD of 0. 16%,with good durability under slight changes of column temperature and flow rate.Conclusion The reversed phase high-performance liquid chromatography method for the purity determination of recombinant Mtb Ag85b protein stock solution was developed,which has good specificity,precision and durability,and can be used for the quality control of recombinant Mtb Ag85b protein stock solution.

Characterization of gut dominant microbiota in obese patients with nonalcoholic fatty liver disease.

In obese patients, non-alcoholic fatty liver (NAFLD) is common. However, whether there is a connection between the gut microbiota and the onset of NAFLD in obese people is yet unknown. Using quantitative real-time PCR, the microbiota of feces of the eligible 181 obese individuals was identified to compare the differences in gut microbiota between obesity with NAFLD and simple obesity. According to the findings, the gut dominant microbiota was similar between obesity with NAFLD and simple obesity. Nonetheless, compared to the simple obesity group, the quantity of Faecalibacterium prausnitzii colonies was much lower in the obesity with the NAFLD group. Bacteroides were present in greater than 65% of both groups. Bacteroides, Clostridium leptum, and Clostridium butyricum accounted for more than 80% of the cases in the obesity with NAFLD group, whereas Bacteroides, Clostridium butyricum, and F. prausnitzii accounted for more than 80% of the cases in the simple obesity group. We look for potential contributing variables to obesity-related NAFLD and potential prevention measures for obese people. Based on a multi-factor logistic regression analysis, lymphocytes may be a risk factor for obesity with NAFLD while F. prausnitzii may be a protective factor. Additionally, F. prausnitzii is positively impacted by Bacteroides, Clostridium leptum, Clostridium butyricum, and Eubacterium rectale, yet adversely impacted by Enterobacteriaceae. Notably, lymphocytes and F. prausnitzii may help determine whether obese patients would develop NAFLD.

Added value of systemic inflammation markers for monitoring response to neoadjuvant chemotherapy in breast cancer patients.

BACKGROUND: Complete response after neoadjuvant chemotherapy (rNACT) elevates the surgical outcomes of patients with breast cancer, however, non-rNACT have a higher risk of death and recurrence. AIM: To establish novel machine learning (ML)-based predictive models for predicting probability of rNACT in breast cancer patients who intends to receive NACT. METHODS: A retrospective analysis of 487 breast cancer patients who underwent mastectomy or breast-conserving surgery and axillary lymph node dissection following neoadjuvant chemotherapy at the Hubei Cancer Hospital between January 1, 2013, and October 1, 2021. The study cohort was divided into internal training and testing datasets in a 70:30 ratio for further analysis. A total of twenty-four variables were included to develop predictive models for rNACT by multiple ML-based algorithms. A feature selection approach was used to identify optimal predictive factors. These models were evaluated by the receiver operating characteristic (ROC) curve for predictive performance. RESULTS: Analysis identified several significant differences between the rNACT and non-rNACT groups, including total cholesterol, low-density lipoprotein, neutrophil-to-lymphocyte ratio, body mass index, platelet count, albumin-to-globulin ratio, platelet-to-lymphocyte ratio, and lymphocyte-to-monocyte ratio. The areas under the curve of the six models ranged from 0.81 to 0.96. Some ML-based models performed better than models using conventional statistical methods in both ROC curves. The support vector machine (SVM) model with twelve variables introduced was identified as the best predictive model. CONCLUSION: By incorporating pretreatment serum lipids and serum inflammation markers, it is feasible to develop ML-based models for the preoperative prediction of rNACT and therefore facilitate the choice of treatment, particularly the SVM, which can improve the prediction of rNACT in patients with breast cancer.

LncRNA-HAGLR motivates triple negative breast cancer progression by regulation of WNT2 via sponging miR-335-3p.

BACKGROUND: Triple negative breast cancer (TNBC) is a group of highly heterogeneous mixed breast cancer at the level of gene expression profile. Therefore, it is of great clinical significance to explore the molecular mechanism of TNBC and find a targeted therapeutic approach from the molecular level. METHODS: Long non-coding RNA (lncRNA) HAGLR expression level was measured by and qRT-PCR in TNBC tissues and cell lines. EdU, MTT, wound healing and Transwell assays were performed to explore the role of HAGLR on the malignancy of TNBC cells. Luciferase assay was used to clarify the binding between miR-335-3p with HAGLR and WNT2. The tumor formation experiment in nude mice was used to explore the function of HAGLR in vivo. RESULTS: HAGLR was increased in TNBC tissues and cell lines. Silencing of HAGLR inhibited viability, proliferation, migration, and invasion of BT549 cells. Furthermore, HAGLR acted as a sponge of miR-335-3p and inhibited its expression. And miR-335-3p directly targeted WNT2. Functionally, forced expression of miR-335-3p or knockdown of WNT2 removed the promoted effects of lncRNA HAGLR on TNBC development. In vivo tumorigenesis experiments indicated HAGLR accelerated tumor growth via miR-335-3p/WNT2 axis. CONCLUSION: Our study revealed that HAGLR promoted the growth of TNBC, which was mediated by miR-335-3p/WNT2 axis.

Adjacent skin rotation flap for large defect in primary breast tumor.

BACKGROUNDS: Surgical resection of large primary breast tumor often results in large chest wall defects. The purpose of this study is to evaluate the feasibility of using adjacent skin rotation (ASR) flap in patients with giant primary breast tumor. METHODS: A total of 26 giant primary breast tumor patients treated with ASR flap were included in this study. The postoperative conditions, including operating time, blood loss, length of hospital stay, and clinical complications were observed. Meanwhile, the information on 17 breast tumor patients treated with transverse rectus abdominis myocutaneous (TRAM) flap were collected and assigned to a control group. RESULTS: The mean defect size after mastectomy was 16.7 × 13.4 cm, while the median follow-up period was 13 months after surgery. A total of 15.4% patients had developed with local complications, and one of them had more than one complication. When comparing the postoperative outcomes, statistically significant differences were found between the two groups with respect to operating time, blood loss, and length of hospital stay (P < 0.001). CONCLUSIONS: ASR flap is a reliable technique for immediate reconstruction of massive chest wall defects in patients with giant primary breast tumor.

Identification of potential core genes in triple negative breast cancer using bioinformatics analysis.

BACKGROUND: Triple-negative breast cancer (TNBC) is a subtype of breast cancer with poor clinical outcome and limited treatment options. Lacking molecular targets, chemotherapy is the main adjuvant treatment for TNBC patients. MATERIALS AND METHODS: To explore potential therapeutic targets for TNBC, we analyzed three microarray datasets (GSE38959, GSE45827, and GSE65194) derived from the Gene Expression Omnibus (GEO) database. The GEO2R tool was used to screen out differentially expressed genes (DEGs) between TNBC and normal tissue. Gene Ontology function and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis were performed using the Database for Annotation, Visualization and Integrated Discovery to identify the pathways and functional annotation of DEGs. Protein-protein interaction of these DEGs was analyzed based on the Search Tool for the Retrieval of Interacting Genes database and visualized by Cytoscape software. In addition, we used the online Kaplan-Meier plotter survival analysis tool to evaluate the prognostic value of hub genes expression in breast cancer patients. RESULTS: A total of 278 upregulated DEGs and 173 downregulated DEGs were identified. Among them, ten hub genes with a high degree of connectivity were picked out. Overexpression of these hub genes was associated with unfavorable prognosis of breast cancer, especially, CCNB1 overexpression was observed and indicated poor outcome of TNBC. CONCLUSION: Our study suggests that CCNB1 was overexpressed in TNBC compared with normal breast tissue, and overexpression of CCNB1 was an unfavorable prognostic factor of TNBC patients. Further study is needed to explore the value of CCNB1 in the treatment of TNBC.

Rab34 regulates adhesion, migration, and invasion of breast cancer cells.

The small GTPase Rab34 regulates spatial distribution of the lysosomes, secretion, and macropinocytosis. In this study, we found that Rab34 is over-expressed in aggressive breast cancer cells, implying a potential role of Rab34 in breast cancer. Silencing Rab34 by shRNA inhibits cell migration, invasion, and adhesion of breast cancer cells. Rab34 specifically binds to the cytoplasmic tail of integrin ß3, and depletion of Rab34 promotes the degradation of integrin ß3. Interestingly, EGF induces the translocation of Rab34 to the membrane ruffle, which is greatly enhanced by the expression of Src kinase. Accordingly, Rab34 is tyrosine phosphorylated by Src at Y247 residue. A mutant mimicking phosphorylated form of Rab34 (Rab34Y247D) promotes cell migration and invasion. Importantly, the tyrosine phosphorylation of Rab34 is inhibited in cells in suspension, and increased with the cells re-adhesion. In addition, Rab34Y247D promotes cell adhesion, and enhances integrin ß3 endocytosis and recycling. The results uncover a role of Rab34 in migration and invasion of breast cancer cells and its involvement in cancer metastasis, and provide a novel mechanism of tyrosine phosphorylation of Rab34 in regulating cell migration, invasion, and adhesion through modulating the endocytosis, stability, and recycling of integrin ß3.

Breast cancer lung metastasis: Molecular biology and therapeutic implications.

Distant metastasis accounts for the vast majority of deaths in patients with cancer. Breast cancer exhibits a distinct metastatic pattern commonly involving bone, liver, lung, and brain. Breast cancer can be divided into different subtypes based on gene expression profiles, and different breast cancer subtypes show preference to distinct organ sites of metastasis. Luminal breast tumors tend to metastasize to bone while basal-like breast cancer (BLBC) displays a lung tropism of metastasis. However, the mechanisms underlying this organ-specific pattern of metastasis still remain to be elucidated. In this review, we will summarize the recent advances regarding the molecular signaling pathways as well as the therapeutic strategies for treating breast cancer lung metastasis.

Characterization of primary human mammary epithelial cells isolated and propagated by conditional reprogrammed cell culture.

PURPOSE: Conditional reprogramming methods allow for the inexhaustible in vitro proliferation of primary epithelial cells from human tissue specimens. This methodology has the potential to enhance the utility of primary cell culture as a model for mammary gland research. However, few studies have systematically characterized this method in generating in vitro normal human mammary epithelial cell models. RESULTS: We show that cells derived from fresh normal breast tissues can be propagated and exhibit heterogeneous morphologic features. The cultures are composed of CK18, desmoglein 3, and CK19-positive luminal cells and vimentin, p63, and CK14-positive myoepithelial cells, suggesting the maintenance of in vivo heterogeneity. In addition, the cultures contain subpopulations with different CD49f and EpCAM expression profiles. When grown in 3D conditions, cells self-organize into distinct structures that express either luminal or basal cell markers. Among these structures, CK8-positive cells enclosing a lumen are capable of differentiation into milk-producing cells in the presence of lactogenic stimulus. Furthermore, our short-term cultures retain the expression of ERα, as well as its ability to respond to estrogen stimulation. MATERIALS AND METHODS: We have investigated conditionally reprogrammed normal epithelial cells in terms of cell type heterogeneity, cellular marker expression, and structural arrangement in two-dimensional (2D) and three-dimensional (3D) systems. CONCLUSIONS: The conditional reprogramming methodology allows generation of a heterogeneous culture from normal human mammary tissue in vitro. We believe that this cell culture model will provide a valuable tool to study mammary cell function and malignant transformation.

Inhibition of lobuloalveolar development by FOXC1 overexpression in the mouse mammary gland.

The forkhead box transcription factor FOXC1 plays a critical role in embryogenesis and the development of many organs. Its mutations and high expression are associated with many human diseases including breast cancer. Although FOXC1 knockout mouse studies showed that it is not required for mammary gland development during puberty, it is not clear whether its overexpression alters normal mammary development in vivo. To address this question, we generated transgenic mice with mammary-specific FOXC1 overexpression. We report that transgenic FOXC1 overexpression suppresses lobuloalveologenesis and lactation in mice. This phenotype is associated with higher percentages of estrogen receptor-, progesterone receptor-, or ki67-positive mammary epithelial cells in the transgenic mice at the lactation stage. We also show that expression of the Elf5 transcription factor, a master regulator of mammary alveologenesis and luminal cell differentiation, is markedly reduced in mammary epithelial cells of transgenic mice. Likewise, levels of activated Stat5, another inducer of alveolar expansion and a known mediator of the Elf5 effect, are also lowered in those cells. In contrast, the cytokeratin 8-positive mammary cell population with progenitor properties is elevated in the transgenic mice at the lactation stage, suggesting inhibition of mammary cell differentiation. These results may implicate FOXC1 as a new important regulator of mammary gland development.

Quantum dot-based immunofluorescent imaging and quantitative detection of TOP2A and prognostic value in triple-negative breast cancer.

BACKGROUND: Topoisomerase 2 alpha (TOP2A) is a key enzyme in DNA replication and a target of various cytotoxic agents including anthracyclines. Previous studies evaluating the predictive and prognostic values of TOP2A in breast cancer are contradictory, likely secondary to the use of both different detection methods and different cutoff thresholds for positive status. Our own studies have previously confirmed the advantages of quantum dot-based nanotechnology for quantitative analysis of biomarkers relative to conventional immunohistochemistry (IHC). This study was designed to 1) assess the expression of TOP2A, 2) investigate the relationship between TOP2A expression and major clinical pathological parameters, and 3) evaluate the prognostic value of TOP2A by quantum dot-based immunofluorescent imaging and quantitative analytical system (QD-IIQAS) in triple-negative breast cancer (TNBC). PATIENTS AND METHODS: TOP2A expression in 145 TNBC specimens was detected using IHC and QD-IIQAS, and a comparative analysis of the two methods was conducted, including an exploration of the relationship between TOP2A expression and major clinical pathological parameters in TNBC. The prognostic value of TOP2A in TNBC was assessed. RESULTS: A similar antigen localization, a high correlation of staining rates (r=0.79), and a high agreement of measurements (κ=0.763) of TOP2A expression in TNBC were found by QD-IIQAS and conventional IHC (cutoff: 45.0 and 0.45, respectively). TOP2A was significantly higher in larger tumors (P=0.002), higher grade tumors (P=0.005), and lymph node positive patients (P<0.001). The 5-year disease-free survival (5-DFS) of the high and low TOP2A subgroups was significantly different for both QD-IIQAS and IHC (P<0.001, log-rank test for both). TOP2A expression was an independent predictor of survival in TNBC (P=0.001). CONCLUSION: QD-IIQAS was an easy and accurate method for detecting and assessing TOP2A. The TOP2A expression was an independent prognostic indicator of 5-DFS in TNBC. Our study provides a good foundation for future studies exploring the relationship between TOP2A expression and response to anthracyclines.

CO2 adsorption of three isostructural metal-organic frameworks depending on the incorporated highly polarized heterocyclic moieties.

A systematic investigation of CO2 adsorption behavior in three metal-organic frameworks was executed. The three MOFs adopted the same NbO-type structure, except that the organic ligands were grafted with different highly polarized heterocyclic moieties, namely, oxadiazole, thiadiazole, and selenadiazole, respectively. After activation, the three MOF materials, ZJNU-41a showed different surface areas and pore volumes depending on the incorporated heterocyclic rings attached to the organic ligands as well as the MOF's stabilities. Among the three MOF materials, exhibited an impressive CO2 uptake capacity of 97.4 cm(3) (STP) g(-1) at 298 K and 1 atm, which is comparable and even superior to those reported in NbO-type MOFs. In particular, when the molecular dipole of the attached heterocyclic moieties increases, the CO2 uptake also increases, which was further supported by comprehensive quantum chemical calculations. This work demonstrates that the introduction of highly polarized heterocyclic functional groups into frameworks is a promising approach to target porous metal-organic framework materials with improved CO2 adsorption performance.

Bilateral breast cancer with a unilateral carcinoma within a fibroadenoma: A case report.

Fibroadenomas are a type of benign tumor that occur in young women below the age of 35 years old. The tumors are the second most common type of tumor after fibrocystic disease. The chance of carcinoma arising in a fibroadenoma is extremely low. To date, <130 such cases have been reported. Previous studies have reported that fibroadenomas can evolve into a number of different types of malignancy. The present study is the first to describe a case of bilateral primary breast cancer with a unilateral invasive ductal carcinoma within a fibroadenoma. The current study presents a case of a 48-year-old female who presented with 2 stiff lumps on bilateral breasts, diagnosed as bilateral breast carcinoma, with a unilateral invasive ductal carcinoma within a fibroadenoma in the right breast. The patient underwent a bilateral mastectomy and subsequently received 4 cycles of chemotherapy (epirubicin, 60 mg/m2 and cyclophosphamide, 600 mg/m2) every 21 days, followed by 4 cycles of docetaxel chemotherapy (100 mg/m2) every 21 days. The patient then received maintenance endocrine therapy (tamoxifen, 20 mg, twice daily) for 19 months. The patient was followed up every 3 months, and at the last follow-up examination in May 2015, the patient exhibited no signs of recurrence.

Fourier transform infrared spectroscopy for the distinction of MCF-7 cells treated with different concentrations of 5-fluorouracil.

BACKGROUND: In order to provide personalized treatment to patients with breast cancer, an accurate, reliable and cost-efficient analytical technique is needed for drug screening and evaluation of tumor response to chemotherapy. METHODS: Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) was used as a tool to assess cancer cell response to chemotherapy. MCF-7 cells (human breast adenocarcinoma cell line) were treated with different concentrations of 5-fluorouracil (5-FU). The inhibition of cell proliferation was monitored by MTT, and apoptosis rates were determined by flow cytometry. Finally, spectra of the cell populations were acquired by ATR-FTIR. RESULTS: The cell response to 5-FU was detectable at different concentrations by ATR-FTIR. First, a band observed at 1741 cm(-1), representing membrane phospholipids, was enhanced with increasing 5-FU concentrations. In addition, the MCF-7 cell spectrum shifted progressively from 1153 to 1170 cm(-1) with increasing drug doses. Finally, the normalized band intensity of 1741 cm(-1)/Amide I was highly correlated with the percentage of apoptotic cells as assessed by partial correlation analysis. CONCLUSIONS: These findings suggest that the effects of different concentrations of drugs can be monitored by ATR-FTIR, which may help evaluate the response to chemotherapy and improve treatment strategies.