To construct the eukaryotic vector that expresses the fusion protein of Axud1 and influenza virus hemagglutin HA epitope tag, the total RNA was isolated from the peripheral blood lymphocytes, and reverse transcription reaction was used to amplify the full length of human Axud1 cDNA. PCR product of Axud1 was then amplified using specific primers containing HA epitope sequence, and inserted into eukaryotic expression plasmid pcDNA3.1(+)digested with BamH Ⅰand Xba Ⅰ. The recombinant plasmid was identified by polymerase chain reaction, restriction endonuclease mapping and sequencing, and then transfected into human lung adenocarcinoma SPC-A1 cell lines.The fusion HA-Axud1 protein expression in anti-G418 clones was verified by Western blot. This study might be instrumental in further study of the function of Axud1 protein in tumor cells.

Cell Differentiation ; Humans ; Stem Cells ; Tooth ; cytology

Objective To evaluate the performance of domestic matrix-assisted laser desorption/ionization time-of-flight mass spectrometry system Clin-TOF-Ⅱ MS with BioExplorer V2.3 database ( Clin-TOF MS system) on gram-negative bacteria identification.Methods This was a methodological comparison study.A total of 1 025 gram-negative strains of 32 genus, 56 species or species complex were included in this study from 1999 to 2000 and 2014 to 2016 in Peking Union Medical College Hospital.The Bruker Biotyper MS system ( Bruker MS system ) , Bruker Autoflex Speed with Biotyper v 3.1 database were used as control.Identification by both MALDI-TOF MS systems were parallel conducted by direct smear method.The 16S rDNA sequencing based identification was performed when either MALDI-TOF MS system gave“unbelievable result” or results from two systems were not consistent.Results Amongst the isolates studied, 98.05% (1 005/1 025) was correctly identified to species or species complex level by Clin-TOF MS system.Comparatively, 99.22%(1 017/1 025) was correctly identified by Bruker MS system.There were 17 isolates just identified to genus level and 2 isolates were “no identification” by Clin-TOF MS system, meanwhile 1 Pseudomonas monteilii misidentified as P.putida.There were only two 2 isolates identified to genus level and 3 isolates were“no identification” by Bruker MS system.But it misidentified all 3 Aeromonas hydrophila (2 isolates as A.caviae and 1 isolate as A.media).It′s noted that both MS systems identified 1 Chryseobacterium gleum and 1 C. bernardetii to genus level.Conclusion The identification capability of domestic Clin-TOF MS system was good on gram-negative bacteria.

OBJECTIVE:To screen and identify endophytic fungi from Schisandra chinensis with antioxidant activity. METH-ODS:The tissue isolation skill was used to isolate endophytic fungi from roots,leaves,stems and fruits of S. chinensis. And anti-oxidant activity of endophytic fungi were screened by DPPH radical scavenging assay and hydroxyl radical scavenging assay. The to-tal DNA were extracted;the 18S rDNA ITS were amplified and sequenced with primer ITS1 and ITS4;the results of sequencing were analyzed comparatively based on homology to confirm the classification of active strains. RESULTS:23 strains were isolated from S. chinensis. GSR-12,isolated from roots of S. chinensis,had strong antioxidant activities. The scavenging rate on DPPH and the hydroxyl radical were 87.96% and 82.31% respectively. GSR-12 strain was identified as Clonostachys rosea by analyzed com-paratively. CONCLUSIONS:1 strain of C. rosea,isolated from roots of S. chinensis,has strong antioxidant activity.

Bisphosphonate-related osteonecrosis of jaw(BRONJ)is characterized by impaired osteogenic differentiation of orofacial bone marrow stromal cells(BMSCs).Corin has recently been demonstrated to act as a key regulator in bone development and orthopedic disorders.However,the role of corin in BRONJ-related BMSCs dysfunction remains unclarified.A m6A epitranscriptomic microarray study from our group shows that the CORIN gene is significantly upregulated and m6A hypermethylated during orofacial BMSCs osteogenic differentiation.Corin knockdown inhibits BMSCs osteogenic differentiation,whereas corin overexpression or soluble corin(sCorin)exerts a promotion effect.Furthermore,corin expression is negatively regulated by bisphosphonates(BPs).Corin overexpression or sCorin reverses BPs-impaired BMSCs differentiation ability.Mechanistically,we find altered expression of phos-ERK in corin knockdown/overexpression BMSCs and BMSCs under sCorin stimulation.PD98059(a selective ERK inhibitor)blocks the corin-mediated promotion effect.With regard to the high methylation level of corin during osteogenic differentiation,we apply a non-selective m6A methylase inhibitor,Cycloleucine,which also blocks the corin-mediated promotion effect.Furthermore,we demonstrate that METTL7A modulates corin m6A modification and reverses BPs-impaired BMSCs function,indicating that METTL7A regulates corin expression and thus contributes to orofacial BMSCs differentiation ability.To conclude,our study reveals that corin reverses BPs-induced BMSCs dysfunction,and METTL7A-mediated corin m6A modification underlies corin promotion of osteogenic differentiation via the ERK pathway.We hope this brings new insights into future clinical treatments for BRONJ.

The morbidity of tooth missing is the highest one among all the human organ diseases. The present restorations used in clinic, including fixed bridges, removable dentures and implant prosthetics, all exhibit their own defects, and hardly to restore the whole tooth structure and function. With the development of stem cells and tissue engineering, as an alternative, tooth regeneration, aiming at the generation of a structure like nature tooth, will be the therapeutic orientation to restore the lost tooth. The dental root, which supports the crown and occlusal force, is the fundamental part for tooth function. Based on the theory of tissue engineering, bio-roots were successfully generated by using mesenchymal stem cells (MSC) in miniature pigs. But the success rate of bio-root is not too high, is urgent to be improved for future clinic application. MSC mediated bio-root regeneration is depended on the dentinogenic differentiation regulation of MSC. Up to now, many factors affect the directed differentiation of MSC and further for the success rate of bio-root, including seeding cells, scaffold, growth factors and microenvironmental niche, etc. Microenvironmental niche is the key factor for affecting the MSC characteristics and special tissue regeneration. Basically, the bio-root is regenerated in jaw, while the jaw microenvironmental niche is prone to induce MSC for osteogenic differentiation, instead of dentinogenic differentiation. How to improve the dentinogenic differentiation of MSC in jaw microenvironmental niche is the key issue for increasing the success rate of bio-root.

Objective:To explore the changes of neuron specific enolase (NSE) concentration in serum samples of patients with silicosis.Methods:In January 2020, 455 cases of silicosis diagnosed in Yantaishan Hospital from January 2018 to December 2019 were collected, and 60 healthy cases and 120 cases of lung cancer were selected as the healthy group and the lung cancer group. The serum levels of NSE were detected by chemical immunofluorescence assay and compared.Results:The serum NSE level of silicosis patients was [(22.88±7.86) ng/ml], higher than that of healthy group [(17.96±4.42) ng/ml] ( P<0.05) . Serum NSE levels in the first, second and third stage silicosis groups were higher than those in the healthy group ( P<0.05) , but there was no statistically significant difference between the silicosis groups at different periods ( P>0.05) . The serum NSE level of silicosis patients was lower than that of the small cell lung cancer group ( P<0.05) , but there was no statistically significant difference between silicosis group and non-small cell lung cancer group ( P>0.05) . The area under curve of silicosis NSE was 0.718 ( P<0.01) when the cut-off was 17.49 ng/ml, the sensitivity and specificity of NSE was 78% and 57% respectively. Conclusion:The serum NSE level of patients with silicosis is significantly increased, which can be used as an important reference index for the diagnosis and the differential diagnosis of silicosis.

Objective:To explore the changes of neuron specific enolase (NSE) concentration in serum samples of patients with silicosis.Methods:In January 2020, 455 cases of silicosis diagnosed in Yantaishan Hospital from January 2018 to December 2019 were collected, and 60 healthy cases and 120 cases of lung cancer were selected as the healthy group and the lung cancer group. The serum levels of NSE were detected by chemical immunofluorescence assay and compared.Results:The serum NSE level of silicosis patients was [(22.88±7.86) ng/ml], higher than that of healthy group [(17.96±4.42) ng/ml] ( P<0.05) . Serum NSE levels in the first, second and third stage silicosis groups were higher than those in the healthy group ( P<0.05) , but there was no statistically significant difference between the silicosis groups at different periods ( P>0.05) . The serum NSE level of silicosis patients was lower than that of the small cell lung cancer group ( P<0.05) , but there was no statistically significant difference between silicosis group and non-small cell lung cancer group ( P>0.05) . The area under curve of silicosis NSE was 0.718 ( P<0.01) when the cut-off was 17.49 ng/ml, the sensitivity and specificity of NSE was 78% and 57% respectively. Conclusion:The serum NSE level of patients with silicosis is significantly increased, which can be used as an important reference index for the diagnosis and the differential diagnosis of silicosis.

Objective To map the specific gene responsible for primary erythromelalgia and identify gene mutations in a Chinese family and one sporadic patient with primary erythromelalgia. Methods Geno-mic DNA was extracted from peripheral lymphocytes of the family members of the pedigree and the sporadic patient. Scanning the genes on chromosome 2q that had been identified was performed by using 6 microsatellite markers for the family members with primary erythromelalgia. Then linkage analysis and haplotype analysis were conducted. All exons of SCN9A gene were analyzed by PCR-DNA sequencing. The mutation identification was also confirmed by restriction fragment length polymorphism(RFLP). Results A maximum 2-point LOD score of 2.11 was found at a recombination fraction (? = 0.00) with markers D2S2370 and D2S2330. Recombination events were detected by markers D2S1353 and D2S2345 in this family by the haplotype analysis. There were two missense heterozygous point mutations in the 15th exon of SCN9A gene both in the family(T2573A) and the sporadic patient(T2543C), leading to the substitution of the amino acid leucine to histidine(L858H) and isoleucine to threonine(I848T), respectively. The above mutations were not found in 400 normal alleles. Conclusion It is proved that primary erythromelalgia is caused by mutations in SCN9A gene.

Insulin resistance has been regarded as a hallmark of diabetes heart disease (DHD). Numerous studies have shown that insulin resistance can affect blood circulation and myocardium, which indirectly cause cardiac hypertrophy and ventricular remodeling, participating in the pathogenesis of DHD. Meanwhile, hyperinsulinemia, hyperglycemia, and hyperlipidemia associated with insulin resistance can directly impair the metabolism and function of the heart. Targeting insulin resistance is a potential therapeutic strategy for the prevention of DHD. Currently, the role of insulin resistance in the pathogenic development of DHD is still under active research, as the pathological roles involved are complex and not yet fully understood, and the related therapeutic approaches are not well developed. In this review, we describe insulin resistance and add recent advances in the major pathological and physiological changes and underlying mechanisms by which insulin resistance leads to myocardial remodeling and dysfunction in the diabetic heart, including exosomal dysfunction, ferroptosis, and epigenetic factors. In addition, we discuss potential therapeutic approaches to improve insulin resistance and accelerate the development of cardiovascular protection drugs.