Objective:To screen the differentially expressed immune-related genes(DEIRGs)in in-stent restenosis(ISR),and to analyze the immune cell infiltration in ISR,and to clarify the mechanism of occurrence and development of ISR.Methods:The mRNA gene expression data of GSE46560 dataset samples were downloaded from the Gene Expression Omnibus(GEO),and divided into ISR group and non-ISR group.The"Limma"package in R software was used to identify the differentially expressed genes(DEGs)which were then intersected with immune-related genes(IRGs)to identify the DEIRGs in ISR;R software was used for Gene Ontology(GO)functional enrichment andalysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)signaling pathway enrichment analysis on DEIRGs;the STRING database was used to construct the protein-protein interaction(PPI)network,which was visualized and analyzed for Hub genes by Cytoscape software;the receiver operating characteristic(ROC)curve of the Hub genes were plotted,and the area under the curve(AUC)was calculated and the diagnostic value was evaluated;CIBERSORT software was used to analyze the immune cell infiltration in ISR;Pearson correlation analysis was used to analyze the relationships between the immune cells and the relationships between the immune cells and key genes.Results:A total of 331 DEGs were identified(P<0.05,|log2FC|>1),including 176 upregulated genes and 155 downregulated genes,and 38 DEIRGs were obstained.The GO functional enrichment analysis results showed that the DEIRGs were mainly enriched in biological processes(BP)such as defense response,immune response,and immune system;in cellular components(CC),the DEIRGs were located primarily in the extracellular region and cytoplasmic membrane;and in molecular functions(MF),the DEIRGs were mainly involved in regulating signaling receptor binding and cytokine receptor activity.The KEGG signaling pathway enrichment analysis results indicated that the DEIRGs in ISR were primarily enriched in the phosphatidylinositol 3-kinase/protein kinase B(PI3K-AKT)and transforming growth factor-β(TGF-β)signaling pathways.In the PPI network,CD19 had the highest node among the top 10 Hub genes.Compared with non-ISR group,the expression level of the CD19 gene in the samples in ISR group was increased(P<0.05).The AUC value in the ROC curve of CD19 gene expression was 0.92(P<0.05).The immune cell infiltration analysis results showed that compared with non-ISR group,the infiltration level of T lymphocyte follicular helper(Tfh)cells in the patients in ISR group were increased(P<0.05),the infiltration levels of immature B lymphocytes,CD8+T lymphocytes,naive CD4+T lymphocytes,and M0 macrophages were increased,but the differences were not statistically significant(P>0.05),while the infiltration levels of memory B lymphocytes,activated memory CD4+T lymphocytes,regulatory T cells,resting natural killer(NK)cells,activated NK cells,monocytes,resting mast cells,and neutrophils were decreased,but the differences were not statistically significant(P>0.05).There were positive correlations between Tfh cells and M0 macrophages and resting mast cells(r=0.88,P<0.05;r=0.68,P<0.05),and there were negative correlations between Tfh cells and monocytes and neutrophils(r=-0.49,P<0.05;r=-0.42,P<0.05).Conclusion:CD19 may influence the occurrence and development of ISR by regulating the activation of the PI3K-AKT signaling pathway to affect the Tfh and B lymphocytes.CD19 can serve as a biomarker for the diagnosis of ISR.

Objective To study the diagnostic efficacy of magnifying endoscopy with narrow-band imaging(ME-NBI)in the diagnosis of early gastric cancer and intraepithelial neoplasia,and their manifestations under ME-NBI.Methods In this retrospective research,we enrolled 131 cases of early gastric cancer and intraepithelial neoplasia.Then analyze the ME-NBI manifestations of lesions,compare the diagnostic efficacy of ME-NBI and white light endoscopy(WLI)+biopsy methods in early gastric cancer and intraepithelial neoplasia and study the ME-NBI manifestations of different degrees of lesions.Results The diagnostic accuracy of ME-NBI for diagnosing early gastric cancer,high grade intraepithelial neoplasia(HGIN)and low grade intraepithelial neoplasia(LGIN)was 77.96%,77.96%and 77.96%,respectively.The diagnostic accuracy of WLI+biopsy for above lesions was 60.53%,58.47%and 69.70%,respectively.There was no statistically significant difference in the accuracy of two diagnostic methods for LGIN(P>0.05),while there was a statistically significant difference in the accuracy of two diagnostic methods for early gastric cancer and HGIN(P<0.05).In LGIN,the highest rate of cerebral gyrus glandular duct emergence was 60.46%.The incidence of papillary and villous ducts is highest in HGIN and early gastric cancer,with rates of 57.14%and 52.00%,respectively.Conclusion ME-NBI has more diagnostic efficiency in early gastric cancer than WLI+biopsy.The demarcation line has better sensitivity in differentiated early gastric cancer and intraepithelial neoplasia.Micro-surface tube like villous,papillary structures has higher occurrence rates in early gastric cancer.The cerebral gyrus microglandular duct appeares significantly higher in LGIN than other groups.

Cytosine methylation is one of the major types of DNA epigenetic modifications and plays an important role in maintaining normal cell function and regulating gene expression. Bisulfite sequencing PCR (BSP) based cloning and sequencing is a general method for detecting DNA methylation at specific sites, which can clarify the methylation status of each CpG site in the target fragment. However, this method requires large amounts of single-clonal sequencing, which is complicated to operate, time consuming and expensive. Therefore, the development of an accurate, efficient and convenient DNA methylation detection technology is of great significance to improve the efficiency of epigenetic research. Based on the high-throughput mutation detection platform Hi-TOM (high-throughput tracking of mutations) developed by our group, we further established a site-specific DNA methylation high-throughput detection platform Hi-Meth (High-throughput Detection of DNA Methylation). After bisulfite treatment of DNA samples, the specific site-specific DNA methylation analysis results could be obtained through the Hi-Meth platform by performing only one round of PCR amplification. Using the Hi-Meth platform, the DNA methylation status of two promoter regions of rice were detected. The DNA methylation results from Hi-Meth were consistent with the results from BSP-based method. Thus, site-specific DNA methylation analysis results could be obtained accurately and conveniently through the Hi-Meth platform. In conclusion, Hi-Meth provides an important methylation detection platform for specific DNA regions, which has important significance for epigenetic research.
DNA Methylation ; Epigenesis, Genetic ; Epigenomics ; High-Throughput Nucleotide Sequencing/methods* ; Polymerase Chain Reaction ; Sequence Analysis, DNA/methods*

OBJECTIVE To establish the quality standard of Onosma echioides . METHODS Sixteen batches of O. echioides from different sources were collected to observe their appearance ，microscopic powder identification ，and TLC identification ；the impurity，moisture，total ash and acid -insoluble ash of the medicinal materials were examined . The total pigment content of hydroxynaphthoquinone in O. echioides was determined by UV -visible spectrophotometry ；the contents of alkannin and β，β′- dimethylacrylalkannin in O. echioides were determined by HPLC . RESULTS Medicinal material and powder of O. echioides were purplish red ；non-glandular single cells ，embolized cells ，parenchyma cells ，reticulate ducts could be seen microscopically . TLC results showed that the color and change of the spots in the chromatogram of test sample were consistent with that of the control . The contents of moisture ，total ash and acid insoluble ashshall not exceed 13.0%，18.0%，6.0%. The total pigment content of hydroxynaphthoquinone should not be less than 0.80%. The content of alkannin was recommended to be no less than 0.06 mg/g. The content of β，β′-dimethylacrylalkannin was recommended to be no less than 0.60 mg/g. CONCLUSIONS The established standard can provide reference for the scientific evaluation of the quality of the medicinal materials of O. echioides.

Objective:To investigate the effect of extracellular vesicles derived from lung tissue on intrapulmonary inflammation and the formation of neutrophil extracellular traps (NETs) in sepsis rats.Methods:Sepsis rat model was established by cecal ligation and puncture (CLP). Collagenase D and DNase I were used to dissociate the lung tissue, the impurities were removed by centrifugation, and finally, the extracellular vesicles (Ti-EVs) derived from lung tissue were separated and extracted by differential ultracentrifugation. Eighteen male SD rats were randomly divided into the sham group, sepsis group and Ti-EVs group: in the Ti-sEV group, a sepsis model was established by CLP, and Ti-EVs suspension was instilled through the airway; rats in the CLP group received CLP, and an equal volume of PBS was instilled through the airway; and rats in the sham group was treated with sham operation. The pathological changes of lung tissue were detected by hematoxylin-eosin (HE) staining after 24 h. The content of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the bronchoalveolar lavage fluid (BALF) was measured by enzyme-linked immunosorbent assay (ELISA). Immunofluorescence was used to detect the NETs content in lung tissue.Results:The isolated extracellular vesicles derived from rat lung tissue were observed by transmission electron microscopy as double-layer circular cystic vesicles with particle diameter mainly distributed at 150 nm. Western blot showed positive expression of EVs markers CD9, CD63, and Tsg101. HE staining of lung tissue showed alveolar integrity damage and a large number of inflammatory cells infiltrated in the lung of sepsis rats. Compared with the CLP group, the degree of lung tissue damage was more serious in the Ti-EVs group and the levels of IL-1β, TNF-α and IL-6 in the BALF of rats were significantly increased ( P<0.01). The formation of NETs in the lungs of the rats in the sepsis group and the Ti-EVs group was observed under the laser confocal microscope. Compared with the sepsis group, the fluorescence intensity of NETs in the lung tissues of the Ti-EVs group increased significantly. Conclusions:After enzymatic digestion, differential ultracentrifugation and other treatments, the extracellular vesicles derived from rat lung tissue with high purity can be successfully isolated and extracted. In the process of septic lung injury, extracellular vesicles in lung tissue can aggravate the inflammatory response in the lung and promote the formation of NETs.

Background: Brucella infection induces brucellosis, a zoonotic disease. The intracellular circulation process and virulence of Brucella mainly depend on its type IV secretion system (T4SS) expressing secretory effectors. Secreted protein BspJ is a nucleomodulin of Brucella that invades the host cell nucleus. BspJ mediates host energy synthesis and apoptosis through interaction with proteins. However, the mechanism of BspJ as it affects the intracellular survival of Brucella remains to be clarified. Objectives: To verify the functions of nucleomodulin BspJ in Brucella's intracellular infection cycles. Methods: Constructed Brucella abortus BspJ gene deletion strain (B. abortus ΔBspJ) and complement strain (B. abortus pBspJ) and studied their roles in the proliferation of Brucella both in vivo and in vitro. Results: BspJ gene deletion reduced the survival and intracellular proliferation of Brucellaat the replicating Brucella-containing vacuoles (rBCV) stage. Compared with the parent strain, the colonization ability of the bacteria in mice was significantly reduced, causing less inflammatory infiltration and pathological damage. We also found that the knockout of BspJ altered the secretion of cytokines (interleukin [IL]-6, IL-1β, IL-10, tumor necrosis factor-α, interferon-γ) in host cells and in mice to affect the intracellular survival of Brucella. Conclusions BspJ is extremely important for the circulatory proliferation of Brucella in the host, and it may be involved in a previously unknown mechanism of Brucella's intracellular survival.

Objective:To use an isokinetic test to objectively evaluate the recovery of muscle strength and endurance after treatment of acute Achilles tendon rupture using a tunnel beneath paratenon.Methods:A retrospective study was conducted of the 23 patients who had been treated at Department of Orthopedics, The Forth Medical Center, General Hospital of Chinese PLA by a tunnel beneath paratenon for acute Achilles tendon rupture from January 2017 to January 2018. They were 22 males and one female, aged from 26 to 60 years (average, 35.7 years), with 11 right and 12 left sides involved. Surgery was performed 0.5 to 7.0 days (average, 2.7 days) after injury. Length of incision, skin necrosis, infection, re-rupture, ankle-hindfoot score of American Orthopedic Foot & Ankle Society (AOFAS) and Achilles tendon total rupture scores (ATRS) were followed up for 18 months. Surgical outcomes were objectively evaluated by an isokinetic test to compare the recovery of muscle strength and endurance between the affected and normal sides.Results:Skin necrosis, infection or re-rupture occurred in none of the patients. Incision length averaged 1.4 cm (from 1 to 2 cm), AOFAS 99.1 (from 93 to 100, giving an excellent and good rate of 100%), and ATRS 97.0 (from 88 to 100). Isokinetic evaluation showed that the peak torques of ankle plantar flexion and dorsal extension at 5 test speeds (30°/s, 60°/s, 90°/s, 120°/s and 240°/s) were not significantly different between the affected and normal sides ( P>0.05). In the endurance test, the total work of ankle plantar flexion was (691.2±258.8) J on the normal side and (670.6±304.2) J on the affected side, showing no significant difference between the 2 sides ( P>0.05); the total work of ankle dorsal extension at the normal side was (407.3±119.2) J, significantly larger than that at the affected side [(362.2±117.5) J] ( P=0.001). Conclusion:An isokinetic test can be used to objectively evaluate the recovery of muscle strength and endurance after treatment of acute Achilles tendon rupture using a tunnel beneath paratenon.

Background: Patients with acute gouty arthritis experienc severe pain and often have concomitant limited joint movement. High-dose intake of anti-inflammatory analgesics often results in adverse reactions, such as nausea, vomiting, and diarrhea, and might increase the risk of gastrointestinal bleeding. In order to reduce the adverse reactions by oral intake, local use of externally useddrugs with the same active ingredients as the oral intake is an option in clinical practice. However, externally useddrugs take effect slowly and efficacy to reduce painful feelingis unsatisfactory. Therefore, it is demanding to develop a safe and effective treatment plan. In this regard, researchers have put up a hypothesis. The skin of the human body has a dense stratum corneum, which is difficult for externally used drugs to penetrate effectively; if the keratin barrier of the skin around affected joints can be broken without further damaging the skin, externally applied drugs may safely and effectively alleviate the local symptoms of acute gouty arthritis. The nano plum blossom needle is made of a modern new material. The needle body is thin and the diameter of the needle tip is only 300 nm. The needle is used clinically to break through the keratin barrier of the human skin and promote the absorption of external medicine. Therefore, this trial was designed to preliminarily evaluate the safety and effectiveness of using the nano plum blossom needle for the introduction of diclofenac diethylamine emulgel in the treatment of acute gouty arthritis. Methods: A randomized controlled trial including 83 patients with acute gouty arthritis was conducted, who was assigned to three groups, the intervention group, control group 1, and control group 2 in a ratio of 2:2:1, respectively. The nano plum blossom needle was used for the introduction of diclofenac diethylamine emulgel in the intervention group; the nano plum blossom needle was used as a placebo along with the local use of diclofenac diethylamine emulgel in control group 1; and the nano plum blossom needle was used alone in control group 2. The treatments were applied once a day until the pain was completely relieved and the course of treatment lasted up to 7 days. The primary outcome measurement was the visual analog scale for evaluating the degree of pain. The secondary outcome indicators included scoring of the symptoms and signs with comprehensive consideration of the joint skin color, local tenderness, and degree of joint motion, the time and dose of emergency medication usage during trial, and adverse events. Discussion: This trial couldprovide preliminary evidence for evidence-based practice of using nano plum blossom needle transdermal drug delivery technology for diclofenac diethylamine emulgel for the treatment of acute gouty arthritis, and provide a reference for sample size calculation and experimental design of future clinical trials verifying the effectiveness and safety of such a technical scheme in a larger target population. Registration information This study has been registered in the China Clinical Trial Registry Centerwith the registration code of ChiCTR-IOR-17012154.

Animals ; CRISPR-Cas Systems ; Gene Editing ; Humans

Objective To explore the clinical efficacy of using a tunnel beneath the paratenon to repair chronic Achilles tendon rupture of Myerson type Ⅱ.Methods From August 2008 through January 2018,19 patients with chronic Achilles tendon rupture were treated with a self-designed minimally invasive suture technique at Department of Orthopaedics,The Fourth Medical Center,General Hospital of PLA.They were all male,aged from 25 to 64 years(average,40.4 years).The left side was injured in 12 patients and the right side in 7.The duration from injury to surgery ranged from 28 to 120 days,averaging 60 days.The Achilles tendon defects averaged 3.84 cm.Their clinical diagnoses were confirmed by positive results of clinical examination and magnetic resonance imaging(MRI) scans.After a 2 cm transverse incision was made at the proximal side,the proximal stump beneath the paratenon was adequately released with a periosteum elevator.The proximal stump was sutured by the Bunnell method with an Ethicon-X519 non-absorbable suture under direct vision.After a 1.0-1.5 cm transverse incision was made,percutaneous Bunnell suture was performed at the distal side.The proximal stump was then introduced into the distal incision through the paratenon tunnel.After the affected foot was fully flexed,the 2 stumps were tied closely together and buried under direct vision.Early rehabilitation was encouraged after surgery.The clinical efficacy was assessed according to the ankle-hindfoot scores of American Orthopaedic Foot&Ankle Society(AOFAS) and the Arner-Lindholm evaluation criteria.Results The 19 patients were followed up for 10 months to 9.5 years(average,2.45 years).All the wounds healed at the first stage without any complications related to incision,sural nerve injury or tendon re-rupture.Their AOFAS ankle-hindfoot scores averaged 98.4,with 19 excellent cases;according to the Arner-Lindholm criteria,13 cases were excellent and 6 cases good.Both of the evaluation systems yielded a good to excellent rate of 100%.In the 3 patients who underwent isokinetic testing,there was no significant difference between the normal and affected sides in the peak value of flexion or in the total work of fatigue.Conclusion Using a tunnel beneath the paratenon is a good self-designed minimally invasive suture technique for chronic Achilles tendon rupture of Myerson type Ⅱ because it is simple and reliable,and leads to limited tissue damage,adhesion or complications.