Electric Field Promoted Click Surface-Enhanced Raman Spectroscopy for Rapid and Specific Detection of DNA 2-Deoxyribose 5'-Aldehyde Oxidation Products in Plasma.

Rapid identification of DNA oxidative damage sites is of great significance for disease diagnosis. In this work, electric field-regulated click reaction surface-enhanced Raman spectroscopy (e-Click-SERS) was developed aiming at the rapid and specific analysis of furfural, the biomarker of oxidative damage to the 5-carbon site of DNA deoxyribose. In e-Click-SERS, cysteamine-modified porous Ag filaments (cys@p-Ag) were prepared and used as electrodes, amine-aldehyde click reaction sites, and SERS substrates. Cysteamine was controlled as an "end-on" conformation by setting the voltage of cys@p-Ag at -0.1 V, which ensures its activity in participating in the amine-aldehyde click reaction during the detection of furfural. Benefiting from this, the proposed e-Click-SERS method was found to be sensitive, rapid-responding, and interference-resistant in analyzing furfural from plasma. The method detection limits of furfural were 5 ng mL-1 in plasma, and the whole "extraction and detection" procedure was completed within 30 min with satisfactory recovery. Interference from 13 kinds of common plasma metabolites was investigated and found to not interfere with the analysis, according to the exclusive adaptation of the amine-aldehyde click reaction. Notably, the e-Click-SERS technique allows in situ analysis of biological samples, which offers great potential to be a point-of-care testing tool for detecting DNA oxidative damage.

The Effect of GPX2 on the Prognosis of Lung Adenocarcinoma Diagnosis and Proliferation, Migration, and Epithelial Mesenchymal Transition.

Objective: To investigate the expression of glutathione peroxidase 2 (GPX2) in human lung adenocarcinoma tissues and its effect on the biological function of lung adenocarcinoma A549 cells. Methods: The expression of GPX2 in lung adenocarcinoma and its effect on survival were analyzed by the TCGA database and the GEPIA 2 database. A total of 45 cases of primary lung adenocarcinoma tissue specimens and 45 cases of their paracancerous tissue specimens were collected, and the expression of GPX2 in the two types of tissues was detected by immunohistochemistry. Lung adenocarcinoma A549 cells were divided into the GPX2 overexpression group (GPX2), the GPX2 knockdown group (si-GPX2), the empty vector group (Vector), the siRNA negative control group (si-NC), and the WT group; the mRNA level and protein expression of GPX2 in each group of A549 cells were detected by real-time fluorescence quantitative PCR and Western blotting; the proliferation activity of each group of cells was detected by the CCK-8 assay; the effect of GPX2 on cell migration and invasion ability was detected by the scratch assay and the Transwell invasion assay; the apoptosis of each group of cells was detected by flow cytometry; Western blotting was performed to detect the expression levels of Bax, Bcl-2, E-cadherin, vimentin, and MMP2 and MMP9 proteins in each group of cells. Results: Bioinformatics analysis showed that the expression of GPX2 was strongly correlated with the prognosis of lung adenocarcinoma patients (P < 0.01). The positive expression rates of GPX2 in lung adenocarcinoma and its paracancerous tissues were 66.0% and 15.7%, respectively (P < 0.05). The results of RT-qPCR and Western blotting showed that the expression level of GPX2 mRNA and protein in A549 cells in the GPX2 group increased, which was significantly higher than that in the WT group (P < 0.05); the expression levels of GPX2 mRNA and protein in A549 cells in the si-GPX2 group were the same, that is, significantly lower than the WT group (P < 0.05). GPX2 overexpression promoted the proliferation, migration, and invasion of A549 cells and inhibited their apoptosis; the results in the si-GPX2 group were opposite to those in the GPX2 group. Compared with the WT group, the expression of Bcl-2, vimentin, and MMP2 and MMP9 protein in the GPX2 group increased (P < 0.05), while the expression of Bax and E-cadherin protein decreased in the GPX2 group (P < 0.05); the results in the si-GPX2 group were opposite to those in the GPX2 group. Conclusion: The expression of GPX2 in lung adenocarcinoma is related to the prognosis of patients. It is proved that GPX2 can promote the migration and invasion of lung adenocarcinoma cells and is related to the EMT/ß-catenin pathway. Thus, GPX2 is expected to be an important target for the diagnosis and treatment of lung adenocarcinoma.

The development of autoverification system of lymphocyte subset assays on the flow cytometry platform.

OBJECTIVES: Peripheral blood lymphocyte subsets are important parameters for monitoring immune status; however, lymphocyte subset detection is time-consuming and error-prone. This study aimed to explore a highly efficient and clinically useful autoverification system for lymphocyte subset assays performed on the flow cytometry platform. METHODS: A total of 94,402 lymphocyte subset test results were collected. To establish the limited-range rules, 80,427 results were first used (69,135 T lymphocyte subset tests and 11,292 NK, B, T lymphocyte tests), of which 15,000 T lymphocyte subset tests from human immunodeficiency virus (HIV) infected patients were used to set customized limited-range rules for HIV infected patients. Subsequently, 13,975 results were used for historical data validation and online test validation. RESULTS: Three key autoverification rules were established, including limited-range, delta-check, and logical rules. Guidelines for addressing the issues that trigger these rules were summarized. The historical data during the validation phase showed that the total autoverification passing rate of lymphocyte subset assays was 69.65% (6,941/9,966), with a 67.93% (5,268/7,755) passing rate for T lymphocyte subset tests and 75.67% (1,673/2,211) for NK, B, T lymphocyte tests. For online test validation, the total autoverification passing rate was 75.26% (3,017/4,009), with 73.23% (2,191/2,992) for the T lymphocyte subset test and 81.22% (826/1,017) for the NK, B, T lymphocyte test. The turnaround time (TAT) was reduced from 228 to 167 min using the autoverification system. CONCLUSIONS: The autoverification system based on the laboratory information system for lymphocyte subset assays reduced TAT and the number of error reports and helped in the identification of abnormal cell populations that may offer clues for clinical interventions.

RNAi-mediated gene silencing of vascular endothelial growth factor C suppresses growth and induces apoptosis in mouse breast cancer in vitro and in vivo.

Vascular endothelial cell growth factor (VEGF)-C promotes tumorigenesis by allowing lymph node metastasis and lymphangiogenesis, among other actions. RNA interference (RNAi) is a novel technique for suppressing target gene expression and may increase the effectiveness of cancer treatments. The present study assessed the influence of VEGF-C RNAi on the apoptosis and proliferation of mouse breast cancer cells in vitro and in vivo. A total of three pairs of small interfering RNA (siRNA) targeting mouse VEGF-C were designed and synthesized prior to transfection into 4T1 cells via a liposomal approach. Reverse transcription polymerase chain reaction, western blot analysis, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, Hoechst 33258 staining and flow cytometry were performed in vitro to analyze VEGF-C expression, cleaved caspase-3 protein expression and 4T1 cell proliferation and apoptosis. Experiments were also conducted in vivo on BALB/c mice with breast cancer. Tumor weight and volume were measured and the number of apoptotic cells in tumor tissues was assessed by a TUNEL assay. Immunohistochemical assays and an enzyme-linked immunosorbent assay were used to measure the expression of VEGF-C in tumor tissues. The results demonstrated that the three pairs of siRNA, particularly siV2, significantly reduced VEGF-C mRNA and protein levels in 4T1 cells. siV2 was deemed to be the most efficient siRNA and therefore was selected to be used in subsequent experiments. Furthermore, in vitro studies indicated that VEGF-C RNAi significantly decreased cell growth, induced apoptosis and upregulated the expression of cleaved caspase-3 protein. Tumor weight and volume in breast cancer in vivo models was reduced by the intratumoral injection of siV2. Antitumor efficacy was associated with decreased VEGF-C expression and increased induction of apoptosis. The present study therefore indicated that VEGF-C RNAi inhibited mouse breast cancer growth in vitro and in vivo and that it may be a novel targeted therapy for breast cancer.

The effect of thalassemia on erythrocyte reference intervals in a representative Han Chinese adult population.

BACKGROUND: The data from apparently healthy individuals with thalassemia has been demonstrated to have an effect on the reference intervals for the erythrocyte indices in areas with a high incidence of thalassemia. METHODS: Six clinical centers screened apparently healthy individuals using a questionnaire and a physical examination. Then, the qualified reference individuals were selected by hematological indices and a genotypic thalassemia diagnosis. Statistical comparisons were conducted for the erythrocyte reference intervals in the Chinese population with and without thalassemia. The constituent ratios and the mean (SD) of erythrocyte indices according to the thalassemia genotype were calculated. The relationship between the MCV values and the thalassemia genotype was also estimated. RESULTS: 4,636 reference individuals were included using hematological indices and genotypic thalassemia screening. The results of the erythrocyte reference intervals for individuals in Guangzhou with thalassemia demonstrated that the RBC, MCV, and MCH values significantly differed by gender compared with other regions (p < 0.01). In contrast, for individuals without thalassemia, the results tended to be similar and clinically acceptable. In addition, the results of the erythrocyte indices revealed significant differences among α-thalassemia patients, ß-thalassemia patients, and the control group. CONCLUSIONS: Apparently healthy individuals with thalassemia in the high prevalence zone of thalassemia could not be excluded by the questionnaire, physical examination or laboratory indices (Fe < 6 µmol/L, Hb < 90 g/L). The screening of genotypic thalassemia based on the MCV or MCH values to exclude unqualified individuals is the most effective way to obtain accurate and reliable reference intervals for the erythrocyte indices.

Correlation of IL-1F genetic polymorphisms with the risk of colorectal cancer among Chinese populations.

Inflammatory/immune cells have the power of infiltrating almost all human solid tumors and influencing all stages of carcinogenesis because of their stimulation of various cytokine subsets. This study aims to determine the correlation of single nucleotide polymorphisms in the IL-17F gene and the risk of colorectal cancer (CRC). One thousand patients diagnosed with CRC and a control group of 354 healthy controls were involved. Peripheral blood samples were collected. The PCR-RFLP method was used to detect the 7383A>G (rs2397084) and 7488T>C (rs763780) in the IL-17F gene. Statistical analyses were conducted with version 12.0 STATA statistical software. We found that the allele model suggested that patients carrying C allele were 1.67 times more likely to develop CRC than healthy controls (odds ratio (OR) = 1.67, 95% confidence interval (CI) = 1.22-2.27, P = 0.001). Similarly, the homozygous and dominant models also revealed that the minor IL-17F 7488C allele conferred an increased CRC risk compared to the major T allele among our study participants (CC vs. TT: OR = 4.15, 95% CI = 1.26-13.36, P = 0.011; TC+CC vs. TT: OR = 1.46, 95% CI = 1.04-2.05, P = 0.027). However, all genetic models indicated that the IL-17F 7383A>G (rs2397084) polymorphism was not associated with CRC risk (all P > 0.05). The results of this study indicate that the 7488T>C (rs763780) in the IL-17F gene may be correlated with increased risk of CRC.

Type IV secretion system in Helicobacter pylori: a new insight into pathogenicity.

OBJECTIVE: To review the research progress on Type IV secretion system (T4SS) in Helicobacter pylori. DATA SOURCES: The data used in this review were identified by searching of PUBMED (1995 - 2007) online resources using the key terms 'Type IV secretion system' and 'Helicobacter pylori'. STUDY SELECTION: Mainly original articles and critical reviews written by major pioneer investigators of this field were selected. RESULTS: The research progress on T4SS in Helicobacter pylori was summarized. The structure and function was discussed. CONCLUSIONS: T4SS is not only involved in toxin secretion and injection of virulence factors into eukaryotic host target cells, but also involved in horizontal DNA transfer to other bacteria and eukaryotic cells, through DNA uptake from or release into the extracellular milieu. It provides a new insight into the pathogenicity of Helicobacter pylori and a novel target for antimicrobials development. However, many challenges remain for us in understanding the biological role of T4SS in Helicobacter pylori.