Single probe PCR melting curve analysis MTHFR C677T SNP sites.

BACKGROUND: The detection and analysis of methylene tetrahydrofolate reductase (MTHFR) C677T single nucleotide polymorphism (SNP) from blood samples is time-consuming and costly. We aimed to establish a method to detect these SNPs by direct whole blood PCR and without DNA extraction. METHODS: Probes modified by different fluorescent groups on the same sequence were designed. Various MTHFR genotypes from direct blood PCR experiments were used to verify the similarity of the obtained and sequencing results. The SNP sites adjacent to the MTHFR C677T SNP were used to verify whether the method can accurately distinguish these sites. RESULTS: The ROX probe was found to be the most suitable for this study. We tested 291 samples with 1 µL whole blood as a template, and obtained 126, 43, and 122 cases of C677C, C677T, and C677 C/T genotypes, respectively. The melting curve was consistent with the sequencing results. The detection limit was approximately 1000 white blood cells/µL. Through PCR and the melting curve method, the adjacent sites were accurately distinguished. CONCLUSION: We established a reliable, simple, rapid, and low-cost direct blood PCR method for the detection of MTHFR C677T SNPs. This could also be used as a potential diagnostic tool for a variety of diseases.

Exploring the value of multi-sensory aids in co-designing assistive home devices for older adults with cognitive impairment.

In this study, we aimed to investigate the benefits of co-design prompts/aids in the development of assistive devices for and with older adults who have cognitive impairment (CI), with the goal of improving their ability to live independently at home. We conducted a series of co-design workshops and utilized eight sets of multi-sensory aids to explore their values and effectiveness in engaging older adults with CI in co-design processes. Our findings revealed that the co-design aids had several benefits, including: (1) increasing the exchange of knowledge and awareness between older adults and designers; (2) eliciting insightful information through multi-sensorial aids, and (3) generating novel assistive design solutions to support seniors' independent living at home. We discuss our findings in relation to the multi-sensorial attributes of co-design aids, which empower older adults with CI to express their opinions and actively participate in co-designing assistive devices that meet their needs/expectations.

Quantitative Chemical Proteomics Reveals Triptolide Selectively Inhibits HCT116 Human Colon Cancer Cell Viability and Migration Through Binding to Peroxiredoxin 1 and Annexin A1.

Triptolide (TPL), a natural product extracted from Tripterygium wilfordii Hook F, exerts potential anti-cancer activity. Studies have shown that TPL is involved in multiple cellular processes and signal pathways; however, its pharmaceutical activity in human colorectal cancer (CRC) as well as the underlying molecular mechanism remain elusive. In this study, the effects of TPL on HCT116 human colon cancer cells and CCD841 human colon epithelial cells are first evaluated. Next, the protein targets of TPL in HCT116 cells are identified through an activity-based protein profiling approach. With subsequent in vitro experiments, the mode of action of TPL in HCT116 cells is elucidated. As a result, TPL is found to selectively inhibit HCT116 cell viability and migration. A total of 54 proteins are identified as the targets of TPL in HCT116 cells, among which, Annexin A1 (ANXA1) and Peroxiredoxin I/II (Prdx I/II) are picked out for further investigation due to their important role in CRC. The interaction between TPL and ANXA1 or Prdx I is confirmed, and it is discovered that TPL exerts inhibitory effect against HCT116 cells through binding to ANXA1 and Prdx I. The study reinforces the potential of TPL in the CRC therapy, and provides novel therapeutic targets for the treatment of CRC.

Triptolide inhibits colon cancer cell proliferation and induces cleavage and translocation of 14-3-3 epsilon.

Triptolide is a diterpenoid triepoxide derived from the traditional Chinese medical herb Tripterygium wilfordii. In the present study, we demonstrated that this phytochemical attenuated colon cancer growth in vitro and in vivo. Using a proteomic approach, we found that 14-3-3 epsilon, a cell cycle- and apoptosis-related protein, was altered in colon cancer cells treated with triptolide. In this regard, triptolide induced cleavage and perinuclear translocation of 14-3-3 epsilon. Taken together, our findings suggest that triptolide may merit investigation as a potential therapeutic agent for colon cancer, and its anticancer action may be associated with alteration of 14-3-3 epsilon.

[Differential expressions of the receptor for advanced glycation end products in prostate cancer and normal prostate].

OBJECTIVE: To study the differential expressions of the receptor for advanced glycation end products (RAGE) in the tissues of prostate cancer and normal prostate, and to find the role of RAGE in the pathogenesis of prostate cancer. METHODS: We collected the tissue of prostate cancer and that of normal prostate from the same patient, and compared the differential expressions of RAGE at the tissue, protein and mRNA levels between prostate cancer and normal prostate tissues of 10 patients by immunohistochemistry, Western blot and real-time quantitative PCR. RESULTS: Immunohistochemistry exhibited a significantly higher expression of RAGE in the prostate cancer tissue than in the normal prostate tissue; Western blot showed that the RAGE protein expression was 2.13 times higher in the former than in the latter (P < 0.05); and real-time quantitative PCR revealed the RAGE mRNA expression of the former to be 4.2 times that of the latter (P < 0.05). CONCLUSION: RAGE may play an important role in the pathogenesis and progression of prostate cancer.

Establishment and Evaluation of a Novel Method Based on Loop-Mediated Isothermal Amplification for the Rapid Diagnosis of Thalassemia Genes.

PURPOSE: Currently, thalassemia is commonly detected using gap-polymerase chain reaction (PCR) and deoxyribonucleic acid (DNA) reverse dot blot, which have high requirements of space, instruments, and personnel. Therefore, it is necessary to develop a new method for thalassemia detection with high sensitivity, low cost, and simple and fast operation. In this study, we aimed to design and evaluate a new method for detecting three α-thalassemia genes including -Southeast Asian (SEA), -α3.7, and -α4.2 and five ß-thalassemia genes including 654M, 41/42M, -28M, 17M, and 27/28M based on loop-mediated isothermal amplification (LAMP). METHODS: Primer sequences were designed using Primer Explorer V4 software. Blood samples (5 mL) were collected from all participants in EDTA. DNA was extracted using Chelex 100 and was subjected to LAMP. LAMP products were detected by fluorescence development in ultraviolet light. RESULTS: We found that LAMP assays for positive samples of thalassemia reached a plateau before 60 minutes, whereas the negative control samples entered the plateau after 70 minutes or showed no amplification. The concentration range of positive reactions was between 20-60 pg/µL and 20-60 ng/µL. Additionally, there were no cross-reactivities among 8 thalassemia subtypes. For clinical samples, the positive sample tube showed strong green fluorescence, whereas the negative tube showed light green fluorescence. According to these results, the LAMP method has high sensitivity for detecting thalassemia (252/254). However, 43 false-positive results were obtained in the LAMP test. The LAMP assay was also of low cost and with simple and fast operation. CONCLUSION: The novel LAMP assay can be completed within 60 min using a heating block or a water bath, and the result can be read visually based on color change to detect thalassemia. The LAMP assay fulfills the requirements of field application and resource-limited areas, especially those with primary hospitals and rural areas.

Design and Evaluation of a Novel Multiplex Real-Time PCR Melting Curve Assay for the Simultaneous Detection of Nine Sexually Transmitted Disease Pathogens in Genitourinary Secretions.

Background: Sexually transmitted diseases (STD) are a major cause of infertility, long-term disability, ectopic pregnancy, and premature birth. Therefore, the development of fast and low-cost laboratory STD diagnostic screening methods will contribute to reducing STD-induced reproductive tract damage and improve women's health worldwide. In this study, we evaluated a novel multiplex real-time PCR melting curve assay method for the simultaneous detection of 9 STD pathogens, including Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Trichomonas vaginalis, Mycoplasma hominis, Ureaplasma urealyticum, Ureaplasma parvum, and herpes simplex virus. Methods: The analytical performance of the method, including its limit of detection (LOD), specificity, repeatability, and effect on different DNA extraction kits were evaluated. Additionally, we obtained 1,328 clinical specimens from 3 hospitals to detect the 9 STD pathogens using multiplex real-time PCR melting curve and Sanger sequencing, to evaluate the sensitivity, specificity, and consistency of the assay method. Results: The results showed that the analytical sensitivity of the novel multiplex real-time PCR melting curve assay is very excellent, with LOD of DNA corresponding to <200 copies/µL for the DNA of the 9 STDs and 1.00 × 104 color change unit /ml for those of UU and UP. Additionally, this assay demonstrated excellent analytical specificity, excellent repeatability, and its results had no effect of different DNA extraction kits. The performance, in terms of sensitivity (91.06-100%) and specificity (99.14-100%), was remarkable, since the consistency between it and Sanger sequencing was more than 0.85 in the clinic. Conclusion: The novel multiplex real-time PCR melting curve assay method has high sensitivity and specificity, relatively low cost, and simple to use for the simultaneous detection of 9 STD pathogens in genitourinary secretions.

[Construction, expression and location of eukaryotic expression vector CMV-FLAG-RB in human prostate cancer PC-3 cells].

OBJECTIVE: To construct an eukaryotic recombinant expression vector for retinoblastoma 1 gene (RB-1) and investigate the role of RB-1 in prostate cancer. METHODS: The coding sequence of RB-1 gene tagged with FLAG was amplified from the plasmid CMV-RB by PCR method. The fragment was cloned into CMV expression vector and identified by restriction enzyme digestion and sequence analysis. Western Blotting was used to detect RB-1 expression and immunofluorescence was used to observe RB-1 distribution in PC-3 cells transfected with the recombinant. RESULTS: The expression vector CMV-FLAG-RB was successfully constructed as confirmed by PCR, endonuclease digestion and DNA sequence analysis. RB-1 protein was highly expressed and showed a nuclear distribution in PC-3 cells transfected with the recombinant. CONCLUSIONS: The eukaryotic expression vector for RB-1 has been successfully constructed and can be efficiently expressed in PC-3 cells. The expression of RB-1 is located in the cell nuclei.