Objective To investigate and optimize the condition of purification and crystallization of oncogenic protein MDM2.Methods MDM2 was expressed in E.coli expression system, and purified by Ni-NTA chelating affinity chromatography and molecular sieve chromatography.The secondary structure of purified protein was analyzed by circular dichroism spectroscopy (CD).Then the crystallization condition of MDM2 was screened and optimized by sitting-drop vapor-diffusion method.Results High purity of MDM2 was obtained by Ni-NTA chelating affinity chromatography and molecular sieve chromatography purification.CD analysis indicated the secondary structure of MDM2 was ordered.Protein-crystallisation experiments illustrated that MDM2 was prone to crystallization under lower pH.Conclusion The optimum pH of MDM2 protein crystallization is 5.5, the optimum protein concentration is 10 mg/mL.

BACKGROUND:Intervertebral disc degeneration is clinically considered to be the main cause of low back pain,but due to the unclear pathogenesis of intervertebral disc degeneration,there is still a lack of effective means to delay the progression of the disease.Single-cell RNA sequencing technology can amplify and sequence mRNA at the single-cell level,reveal the gene expression intensity of a single cell,discover different cell subsets in tissues according to the heterogeneity of cells,study the pathogenesis of intervertebral disc degeneration at the molecular level,and provide a new theoretical basis for its early diagnosis and treatment. OBJECTIVE:To introduce the basic principles of single-cell RNA sequencing technology and review the research progress of single-cell RNA sequencing technology in intervertebral disc degeneration in recent years. METHODS:A computer was used to search PubMed,Web of Science,CNKI and WanFang databases for the literature published from 2012 to 2022.Key words were"single-cell RNA sequencing,intervertebral disc degeneration,sequencing Technology"in Chinese and English.Duplicate,poor-quality and irrelevant articles were excluded;a total of 70 articles were eventually included. RESULTS AND CONCLUSION:(1)We identified new cell subsets such as homeostatic chondrocytes,hypertrophy chondrocyte-like nucleus pulposus cells and fibrous nucleus pulposus cells,identified the marker genes and transcription factors of these cell subsets,and described the functions,differentiation paths and cell fate of these cell subsets during the development and progression of intervertebral disc degeneration,and proposed the concept of progenitor nucleus pulposus cells.A cell subpopulation with progenitor nucleus pulposus cells properties was identified and its effectiveness in treating intervertebral disc degeneration was verified in mice.(2)Fibro chondrocyte-like annulus fibrosus cells and annulus fibrosus stem cells with both cartilage and fiber properties were identified,and a new type of composite hydrogel was prepared by combining fibrous cartilage inducers silk fibroin and hyaluronic acid in vitro.Experiments in mice demonstrated that this hydrogel could repair both annulus fibrosus tissue and cartilage matrix,and was remarkably effective in the treatment of intervertebral disc degeneration.(3)Regulatory chondrocytes were found in endplate cartilage.Two distinct fates in the progression of intervertebral disc degeneration were analyzed and the differential genes in the two fates were identified.Intercellular communication analysis indicated that regulatory chondrocytes interact with endothelial cells to promote angiogenesis.(4)Immune cells such as macrophages,T cells,myeloid progenitor cells and neutrophils were identified in the degenerated intervertebral disc tissues,demonstrating the existence of immune response during intervertebral disc degeneration.It was found that apolipoprotein induced the polarization of macrophages M1 and M2 subtypes,and this polarization process affected the activity of progenitor nucleus pulposus cells by amplifying the inflammatory response through the MIF signaling pathway.

Objective:To assess the association between blood pressure and the risks of diabetes mellitus.Methods:Screening and intervention were conducted from 2015 to 2019 for high-risk subjects of cardiovascular diseases in eight counties of Henan. Information on demographic characteristics, lifestyle behaviors, and anthropometric measurements were obtained via a questionnaire. Fasting blood samples were collected for blood glucose and serum lipids. The R 3.6.3 software was used to analyze the relationship between blood pressure and diabetes mellitus.Results:The detection rate of diabetes mellitus was 23.5% among 120 040 participants aged 35-75 years. The mean fasting blood glucose level was significantly different among normotensive, prehypertensive, and hypertensive patients. Compared to normotensive patients, prehypertension and hypertension had adjusted ORs of 34%( OR=1.34, 95 CI%: 1.30-1.37) and 85%( OR=1.85, 95 CI%: 1.81-1.89). The corresponding ORs were 1.81(1.77-1.85) in controlled and 2.17(2.06-2.28) in uncontrolled patients. A subgroup analysis showed the same trend, where the risk of diabetes increased with blood pressure ( P<0.05). Conclusions:People with elevated BP may increase their risk of diabetes, while the risk declines when BP is under control. Therefore, targeted measures should be taken to reduce the risk.

Objective:To investigate the effects of methyltransferases like 3(METTL3)mediated m6A modification of double homology cassette A pseudogene8(DUXAP8)on the proliferation,migration and invasion of salivary adenoid cystic carcinoma SACC-LM cells and its potential molecular mechanisms.Methods:Whole-transcriptome sequencing showed that DUXAP8 was highly ex-pressed in SACC than in para-cancerous tissues(P＜0.05).The m6A modification sites on DUXAP8 were predicted using the SRAMP website,and the mRNA and protein expression of m6A-modified genes and the genes associated with the epithelial-mesen-chymal transition(EMT)was measured by qRT-PCR and Western blot,respectively.METTL3 and DUXAP8 was knocked down or overexpressed in SACC-LM cells,and the proliferation,migration,and invasion of the cells were assessed by CCK-8,scratch and Transwell assays.The correlation between METTL3 and DUXAP8 was evaluated using MeRIP-qPCR.Results:The expression of DUXAP8 in SACC tumor was higher than that in para-cancerous tissues(P＜0.05).Knockdown of DUXAP8 reduced proliferation,migration and invasion of SACC-LM cells,as well as the expression of EMT-related genes(P＜0.05).Multiple m6A modification sites of high confidence were found on DUXAP8.METTL3 was highly expressed in tumor tissues,more than other related genes(P＜0.05)and enzyme-encoding genes in SACC-LM cells(P＜0.05).METTL3 was found to function as a methyltransferase to regulate the expression of DUX-AP8,and downregulation of METTL3 inhibited prolifera-tion,migration and invasion of SACC-LM cells and partially reversed the promotion of these activities induced by DUX-AP8 overexpression(P＜0.05).Conclusion:METTL3-me-diated m6A modification upregulated DUXAP8 expression,which promotes the proliferation,migration and invasion of SACC cells.

Objective:To study the effects and the related molecular mechanisms of miR-148a-3p on the proliferation,invasion and migration of salivary adenoid cystic carcinoma SACC-LM cells.Methods:miR-148a-3p mimics and inhibitors,siRNA targeting EG-FR and their corresponding controls were transfected into SACC-LM cells.Bioinformatics was used to predict the potential target genes of miR-148a-3p.EGFR and miR-148a-3p mRNA expression levels were examined by qRT-PCR and the protein levels of EG-FR were detected by Western blotting.CCK-8,scratch,and Transwell assays were used to study the proliferation,migration,and invasion of SACC-LM cells,respectively.The direct targeting relationship between miR-148a-3p and EGFR was examined by using the double luciferase reporter gene assay.Statistical analysis of the data was performed by SPSS 22.0 software.Results:Overexpres-sion or inhibition of miR-148a-3p significantly inhibited or promoted the proliferation,invasion and metastasis of SACC-LM cells re-spectively(P＜0.05).Bioinformatics and double luciferase assay showed that miR-148a-3p directly targeted and regulated the expres-sion of EGFR(P＜0.001).Downregulation of EGFR inhibited the proliferation,migration and invasion of SACC-LM cells(P＜0.05)and partially reversed the promoting effect of miR-148a-3p inhibition(P＜0.05).Conclusion:The downregulation of miR-148a-3p leads to the abnormally high expression of its target gene EGFR,and promotes the proliferation,invasion,and migration of salivary adenoid cystic carcinoma cells.

Objective To explore the influence of the iodine disinfection on nasal bacterial colonization through the transsphenoidal approach. Methods Totally 133 pituitary adenoma patients who underwent transsphenoidal surgery in our department from January to August 2017 were enrolled in this study. Before disinfection,pharyngeal swabs of inferior turbinate root secretions were taken for bacterial culture. After iodine disinfection,pharyngeal swabs were taken again at the same site. Changes in the nasal bacterial spectrum before and after disinfection were compared. Patients were followed up for three months after the surgery,during which any intracranial infection/bacteraemia was recorded,and its correlation with nasal bacteria colonization was analyzed. Results Nasal bacterial colonization was detected in 45 (33.8%) of 133 patients before iodine disinfection and in only 6 cases (4.5%) after iodine disinfection (χ=34.5,P=0.000). Thus,iodine disinfection eliminated 86.7%(39/45) of the colonized bacteria. The most common nasal bacterium was Staphylococcus aureus (24.4%,11/45),followed by Klebsiella pneumoniae (24.4%,11/45),and Staphylococcus epidermidis (13.3%,6/45). One patient had high fever and chills 2 days after surgery,but blood culture and cerebrospinal fluid culture showed negative Results . After the administration of third-generation cephalosporins,the symptoms disappeared after two days. Conclusion sThere are colonized bacteria in nasal cavity. Iodine disinfection of nasal cavity can effectively clear most of the nasal bacteria. The possibility of intracranial infection/bacteremia after transsphenoidal approach is low.

Astrocytes are the largest glial population in the mammalian brain. However, we have a minimal understanding of astrocyte development, especially fate specification in different regions of the brain. Through lineage tracing of the progenitors of the third ventricle (3V) wall via in-utero electroporation in the embryonic mouse brain, we show the fate specification and migration pattern of astrocytes derived from radial glia along the 3V wall. Unexpectedly, radial glia located in different regions along the 3V wall of the diencephalon produce distinct cell types: radial glia in the upper region produce astrocytes and those in the lower region produce neurons in the diencephalon. With genetic fate mapping analysis, we reveal that the first population of astrocytes appears along the zona incerta in the diencephalon. Astrogenesis occurs at an early time point in the dorsal region relative to that in the ventral region of the developing diencephalon. With transcriptomic analysis of the region-specific 3V wall and lateral ventricle (LV) wall, we identified cohorts of differentially-expressed genes in the dorsal 3V wall compared to the ventral 3V wall and LV wall that may regulate astrogenesis in the dorsal diencephalon. Together, these results demonstrate that the generation of astrocytes shows a spatiotemporal pattern in the developing mouse diencephalon.
Mice ; Animals ; Astrocytes ; Neuroglia/physiology* ; Diencephalon ; Brain ; Neurons ; Mammals