Circulating tumor DNAs (ctDNAs) are DNA fragments released from tumor cells into bloodstream, containing genetic mutations and epigenetic variations related to cancer. DNA methylation variation is a kind of epigenetic variation which happens in early carcinogenesis and dynamically changes with cancer development. Liquid biopsy of ctDNA methylation has the advantages of non-invasiveness, target molecule stableness, considerable cost-effectiveness, high diagnostic performance and wide application expansion, detection of whose level is conducive to early diagnosis and prognostic evaluation of cancer. The pre-analytical procedures and methylation detection methodologies significantly influence after test results, and should be standardized to obtain high quality results. Up to now, a large amount of literature covering the utility of ctDNA methylation in cancer diagnosis and prognosis have been published. It is believed that in the near future, the detection process of ctDNA methylation would be standardized, and the large-scale clinical application of ctDNA methylation as a liquid biopsy project would be promoted.

Objective:Explore the establishment of a fast, stable and sensitive high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for detecting the level of m6A modification in RNA and its application.Methods:The degree of m6A in RNA can be expressed as the ratio of m6A and adenosine (A) in concentration, which can be determined by ESI source positive ion multiple reaction monitoring (MRM) mode. The established method was verified by analyzing three quality control samples (m6A: 4, 40, 400 nmol/L; A: 40, 400, 4 000 nmol/L) with three different concentrations of low, medium, and high. The method was used to detect the degree of m6A in RNA from mouse spleen T cells treated in different ways. The t test was used to compare the differences between the two groups of data. Results:The established method had a good Linearity ( R2>0.99) in a range of 1-500 nmol/L for m6A and 10-5 000 nmol/L for A. The limit of detection (LOD) was 1 nmol/L for m6A and 10 nmol/L for A. The recoveries were between 98.9% and 116.5%. The intra-day ( n=5) RSDs and the inter-day ( n=15, 5 days) RSDs were 2.4%-9.5% and 4.4%-9.6%, respectively. And this method was used to detect the degree of m6A in the RNA from mouse spleen T cells cultured in different conditions. The results showed that the m6A modification level in the RNA of primary CD8 +T cell was 0.271 5±0.017 9, and the m6A modification level in the RNA of primary CD8 +T cell with IL-27 was 0.251 7±0.015 0, indicating that primary CD8 +T cells have a higher level of RNA methylation. Conclusion:This research has established a fast, simplemethylation degree in RNA with HPLC-MS/MS. This method is easy to be popularized and is suitable for the detection of large quantity of samples, and of great significance in analyzing the relationship between methylation and diseases.

Objective:Explore the establishment of a fast, stable and sensitive high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for detecting the level of m6A modification in RNA and its application.Methods:The degree of m6A in RNA can be expressed as the ratio of m6A and adenosine (A) in concentration, which can be determined by ESI source positive ion multiple reaction monitoring (MRM) mode. The established method was verified by analyzing three quality control samples (m6A: 4, 40, 400 nmol/L; A: 40, 400, 4 000 nmol/L) with three different concentrations of low, medium, and high. The method was used to detect the degree of m6A in RNA from mouse spleen T cells treated in different ways. The t test was used to compare the differences between the two groups of data. Results:The established method had a good Linearity ( R2>0.99) in a range of 1-500 nmol/L for m6A and 10-5 000 nmol/L for A. The limit of detection (LOD) was 1 nmol/L for m6A and 10 nmol/L for A. The recoveries were between 98.9% and 116.5%. The intra-day ( n=5) RSDs and the inter-day ( n=15, 5 days) RSDs were 2.4%-9.5% and 4.4%-9.6%, respectively. And this method was used to detect the degree of m6A in the RNA from mouse spleen T cells cultured in different conditions. The results showed that the m6A modification level in the RNA of primary CD8 +T cell was 0.271 5±0.017 9, and the m6A modification level in the RNA of primary CD8 +T cell with IL-27 was 0.251 7±0.015 0, indicating that primary CD8 +T cells have a higher level of RNA methylation. Conclusion:This research has established a fast, simplemethylation degree in RNA with HPLC-MS/MS. This method is easy to be popularized and is suitable for the detection of large quantity of samples, and of great significance in analyzing the relationship between methylation and diseases.

In recent years , the potential clinical value of circulating tumor cells ( CTCs ) is more obvious, and a myriad of detection methods for CTCs have been developed .Among them, a series of microfluidic devices are particularly promising as they uniquely offer micro-scale analytical systems that are highlighted by low consumption of samples and reagents , high flexibility to accommodate other cutting-edge technologies , precise and well-defined flow behaviors , and automation capability , presenting themselves significant advantages over the conventional larger systems .Here, a brief review of the recent advances in the CTC detection by microfluidic devices and their challenges in clinical application will be given .

Hepatocellular carcinoma (HCC) is a malignant tumor with high morbidity and high mortality.Early diagnosis is the key for HCC treatment.DNA methylation is one of the important mechanisms of epigenetic genetics.To investigate the mechanism of DNA methylation can provide a theoretical basis for the diagnosis of HCC,and the methylation level of the specific genes can also be used as the biomarkers of HCC for the early diagnosis and prognosis evaluation.

BACKGROUND:General y considered bone marrow cells obtained by adherent method are mesenchymal stem cells, and they can differentiate into osteoblasts, adipocytes and chondrocytes.
OBJECTIVE:To explore the differentiation capacity of bone marrow adherent cells into osteoclasts.
METHODS:Primary mouse mesenchymal stem cells were obtained using adherent method, and bone marrow cells were obtained through adherence 1-5 days. Both of them were cultured with normal medium and inducing medium containing m-csf and RANKL. After 9 days, cells were stained by alkaline phosphatase and tartrate-resistant acid phosphatase. The passage 2 mesenchymal stem cells were divided into four groups, which were induced by mock, m-csf, RANKL and m-csf+RANKL, respectively. After 9 days of culture, the cells were stained by alkaline phosphatase and tartrate-resistant acid phosphatase.
RESULTS AND CONCLUSION:Primary culture of adhered cells was uniform. Alkaline phosphatase and tartrate-resistant acid phosphatase staining of primary and passaged bone marrow adherent cells induced with m-csf+RANKL were positive. This result showed that there were cells that could be induced into osteoclasts in the primary and passaged bone marrow adherent cells. Alkaline phosphatase and tartrate-resistant acid phosphatase staining showed differences of adherent cells at different times after the induction, indicating that adherent cells at different times had different differentiation capacity.

BACKGROUND: The transcription factor Runx2 is the key factor that regulates osteogenic differention and bone development. It has been reported that the C2C12 mesenchymal cells can be induced to differentiate into osteoblasts by Runx2 overexpression, but the molecular mechanism of induction is stil largely unclear. OBJECTIVE: To investigate the role of the members of the miR-376 family during Runx2-induced osteogenic differentiation in C2C12 cells. METHODS: The expression of the members of the miR-376 family was detected by real-time quantitative PCR at different time points using C2C12/Runx2Dox sub-line with conditional Runx2 expression. In miR-376b-3p-transfected C2C12/Runx2Dox cells, the expression of osteoblast markers, such as alkaline phosphatase and osteocalcin, was detected by real-time quantitative PCR, and the alkaline phosphatase activity was also examined by alkaline phosphatase staining. The putative miR-376b-3p targets were commonly predicted by online tools (miRanda, miRWalk and TargetScan). The functional classification of these putative targets was performed by DAVID Bioinformatics Resources database. RESULTS AND CONCLUSION: The expression of miR-376b-3p was significantly increased during Runx2-induced osteogenic differentiation of C2C12 cells, but the expression of other members was not changed. Transfection of miR-376b-3p mimic upregulated alkaline phosphatase expression, but had no effect on osteocalcin expression. The alkaline phosphatase activity was also increased by transfection of miR-376b-3p. The functional classification of miR-376b-3p putative targets showed that miR-376b-3p is involved in the skeleton development, indicating the role of miR-376b-3p in osteoblast differentiation. Taken together, these results suggest that Runx2 promotes early osteogenic differentiation in C2C12 cells by regulating the expression of genes related to osteogenic differentiation through upregulation of miR-376b-3p.

Coding sequences of BMP-2 and BMP-7 were amplified using PCR and ligated with a DNA sequence encoding a flexible peptide (Gly4Ser)5. The fusion gene was inserted into plasmid pIRESneo3. The expression level of BMP2/7 heterodimers in the transfected CHO-K1 cells was 230.75+/-13.34 ng/mL. Culture medium of stably tansfected clone pool was collected as conditional medium to treat osteoblast MC3T3 cells. Staining of Alkalin phosphatase and Alizarin red demonstrated that the conditional medium significantly promoted osteogenic differentiation to a higher extent than BMP-2 homodimers expressed in either CHO-K1 cells or E. coli. Transcriptional levels of Osteogenic phenotype-related molecular markers such as OC, ALP, Runx2 and Osx were increased (P<0.05), and BMP/Smad signal activities were significantly enhanced by BMP-2/7 heterodimers, comparing with BMP-2 homodimers (P<0.05). The results demonstrate that BMP-2/7 heterodimers expressed in CHO-K1 cells have potent ability to stimulate osteogenic differentiation.
3T3 Cells ; Animals ; Artificial Gene Fusion ; Bone Morphogenetic Protein 2 ; biosynthesis ; genetics ; Bone Morphogenetic Protein 7 ; biosynthesis ; genetics ; CHO Cells ; Cell Differentiation ; drug effects ; Cloning, Molecular ; Cricetinae ; Cricetulus ; Dimerization ; Escherichia coli ; genetics ; metabolism ; Gene Fusion ; genetics ; Humans ; Mice ; Osteoblasts ; cytology ; Osteogenesis ; drug effects ; Transfection

AIM: To obtain a high and stable expression analog of human basic fibroblast growth factor by genetic engineering. METHODS: The cysteins 78 and 96 of natural hbFGF polypeptide was substituted with serines by means of site-directed mutagenesis. Using pET- 3c as vector, the mutated polynucleotide was cloned and then transferred into BL21 (DE3)plysS. After induction by IPTG, the analog was obtained and analyzed by SDS - PAGE. RESULTS: After purification the form of soluble mutant increased remarkably but the forms of dimmer and higher multimer were reduced greatly to no more than 8% of the total recombinant protein. By MTT assay, the analog showed the same biological activity. This new analog represented a desirable complementation for native hbFGF to develop pharmaceutical drug in clinical use. CONCLUSION: Substitution of certain amino acids of polypeptide without altering native protein' s bioactivity to get the analog is an effective means to increase stability of foreign protein and its solubility in E. coli.

AIM: To get high biological activity of recombinant human bone morphogenetic protein -2 (rhBMP-2) expressed in Escherichia coli by the methods of refolding. METHODS: The rhBMP-2,expressed in Escherichia coli, was washed by Triton X- 100 and further purified by DEAE chromatography.The inclusion bodies were resolved in 8 mol/L urea, and were refolded and dimerized in the redox systems (reduced and oxidized glutathione). Finally, a one - step purification procedure based on the heparin affinity chromatography was implemented. The biological activity of purified rhBMP - 2 were tested by induction of the alkaline phosphatase activity in C2C12 cells. RESULTS: The rhBMP - 2 was expressed in Escherichia coli in a non - active aggregated form of inclusion bodies using a temperature - inducible expression system. The high - purified rhBMP - 2 was obtained in the form of inclusion bodies by several purification courses. The rhBMP - 2 was refolded and dimerized in the redox systems (reduced and oxidized glutathione) and a one - step purification procedure based on the heparin affinity chromatography was implemented to isolate the rhBMP - 2 dimers and monomers. The purified rhBMP - 2 dimers showed the higher biological activity than the commercial rhBMP - 2. CONCLUSIONS: The method achieved the refolding of rhBMP - 2 would be applied to the whole TGF - β superfamily because the BMP - 2 belongs to the superfamily. Meanwhile, the inexpensive,high yield rhBMP - 2 is suitable for clinic application.