[The expression profile and potential regulatory mechanism of ACE2 in chronic rhinosinusitis with nasal polyps].

Objective: To preliminarily analyze the expression of angiotensin-converting enzyme 2 (ACE2) and to investigate its potential regulatory mechanism in chronic rhinosinusitis with nasal polyps (CRSwNP). Methods: Patients underwent nasal endoscopic surgery in the Third Affiliated Hospital of Sun Yat-sen University from February 2020 to May 2021 were selected, including 17 males and 6 females, aging from 23 to 66 years old. Expression of ACE2 was evaluated via immunohistochemical staining in controls with non-chronic rhinosinusitis, non-eosinophilic CRSwNP (non-ECRSwNP), and eosinophilic CRSwNP (ECRSwNP) tissue, respectively. Correlations between ACE2 and the indicated Th1/Th2-related cytokines (IFN-γ, IL-4, IL-5, IL-13, IL-25, IL-33, TSLP and periostin) were analyzed based on GSE72713 dataset. Protein-protein interaction (PPI) network was constructed via string database, immune infiltration of GSE72713 dataset was evaluated using cibersort algorithm. ACE2 was comprehensively analyzed by microRNA regulatory network, gene set enrichment analysis (GSEA) and pharmacological analysis. Statistical analysis was performed using GraphPad 7.0 and SPSS 20.0 software. Results: ACE2 was up-regulated in non-ECRSwNP compared with ECRSwNP. Microarray analysis showed that ACE2 was positively correlated with IFN-γ while inversely correlated with IL-5, IL-13 and periostin significantly. Analysis of immune infiltration suggested that ACE2 expression correlated positively with the number of M1 macrophage while negatively with M2 macrophage. GSEA demonstrated that interferon-related signaling pathways were up-regulated in non-ECRSwNP, and miRNA-200B/miRNA-200C/miRNA-429 pathways targeting ACE2 were enriched in ECRSwNP. Results of pharmacological analysis indicated that ampicillin was able to promote the expression of ACE2 whereas acetaminophen could down regulated the expression of ACE2. Conclusion: Expression pattern of ACE2 is varied in non-ECRSwNP and ECRSwNP, which may be related to the different infiltration of indicated cytokines and different regulatory pathways of miRNA.

[miR-18a enhances the radiosensitivity of nasopharyngeal carcinoma cells through inducing autophagy].

Objective: To explore the impacts of miR-18a overexpression or depression on the radiosensitivities of nasopharyngeal carcinoma cell line CNE1 and CNE2 and underlying mechanisms. Methods: CNE1 and CNE2 were transfected with miR-18a mimics, inhibitor and the corresponding control vectors. qRT-PCR and western blot were used to determine the ataxia telangiectasia mutated (ATM) expressions in CNE1 and CNE2. CNE1 and CNE2 with stably expressing miR-18a and miR-18a siRNA were constructed. Methyl thiazolyl tetrazolium (MTT) assay was used to detect the impacts of the miR-18a overexpression or depression combined with irradiation on the cell growth. Flow cytometry was used to detect the cell apoptosis and cell cycle. Colony formation assay was used to evaluate the raodiosensitivities of cells. Acridine orange (AO) staining and western blot were used respectively to test the autophagy and the expressions of related proteins. Independent samples t test was used to compare the mean value between groups by using SPSS 16.0. Results: ATM mRNA was decreased significantly in CNE1 and CNE2 cells transfected with 100 or 200 nmol/L miR-18a mimics for 48 hours (CNE1: RQ=0.174±0.139 and 0.003±0.001, t=9.939 and 19 470.783;CNE2: RQ=0.024±0.008 and 0.019±0.012, t=270.230 and 137.746, respectively, all P<0.001). ATM proteins were also decreased after transfected with 100 or 200 nmol/L miR-18a mimics for 72 hours. While in the cells transfected with 100 and 200 nmol/L miR-18a inhibitor for 48 hours, the expressions of ATM mRNA were upregulated significantly (CNE1: RQ=9.419±2.495 and 2.500±1.063, t=-4.427 and -41.241; CNE2: RQ=7.210±0.171 and 115.875±15.805, t=-62.789 and -12.589, all P<0.05), and the expressions of ATM proteins increased after transfected for 72 hours. The growth of cells with miR-18a overexpression plus 4 Gy irradiation were obviously inhibited compared to that of cells with the 4Gy irradiation alone; while the growth of miR-18a-inhibited cells increased compared to that of cells with 4 Gy irradiation alone (all P<0.05). CNE1 transfected with 100 nmol/L miR-18a mimics plus 4 Gy irradiation showed the higher apoptosis rate than the cells with 4 Gy irradiation alone ((22.9±2.1)% vs. (16.3±1.0)%, t=-4.870, P<0.01). Compared to the cells with 4 Gy irradiation alone, miR-18a-overexpressed cells plus 4 Gy irradiation decreased their percentages in G1 phases ((20.2±3.0)% vs. (29.8±4.4)%, t=3.119) and G2/M phases ((21.5±0.9)% vs. (33.4±3.1)%, t=6.410, P<0.05 for both), and increased their percentages in S phases ((56.7±4.9)% vs. (36.8±6.4)%, t=-4.246, P<0.05), and these cells possessed less colony number after exposure to different doses of irradiation, more autophagy-lysosome number, and more expressions of LC3 proteins (all P<0.05). There were no significant differences in the expressions of p62 expressions between different groups of cells. Conclusion: Overexpression of miR-18a can enhance the radiosensitivities of NPC cells by targeting ATM to abrogate G1/S, G2/M arrest and to induce autophagy and apoptosis.

[The expression of Notch pathway in nasal polyps and its correlation with the decreased Treg percentage and eosinophil infiltration].

Objective: To investigate the correlation between Notch pathway expression in nasal polyps and Treg percentage and Eos infiltration. Methods: Patients with chronic sinusitis and simple nasal septum deviation who received nasal endoscopic surgery in the Third Affiliated Hospital of Sun Yat-Sen University between November 2012 and August 2018 were selected and enrolled in CRS group and control group respectively. Nasal mucosa tissues were collected from 30 CRSsNP patients (14 males and 16 females aged from 18 to 63), 58 CRSwNP patients (38 males and 20 females aged from 18 to 65) and 29 patients (19 males and 10 females aged from 20 to 57), who underwent nasal endoscopic surgery for correction of simple nasal septum deviation. Hematoxylin-eosin(HE) staining was used to observe the infiltration of eosinophilic granulocytes in the tissues and to classify chronic sinusitis with polyps (CRSwNP) into eosinophilic chronic rhinosinusitis with nasal polyps (Eos-CRSwNP)and non-eosinophilic chronic rhinosinusitis with nasal polyps (Eos-CRSwNP). Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of Notch pathway receptors (Notch-l, 2, 3, 4) and their ligands (Jagded-l, Jagded-2, Delta-l, Delta-3and Delta-4) in the nasal mucosa of each group, as well as the expression of Th2 cytokines (IL-4, IL-5, IL-13), eosinophilic cationic protein (ECP)and the key transcription factor Foxp3 in Treg cells. Finally, flow cytometry was used to detect CD4+CD25+Foxp3+ Treg cells in nasal mucosa of each group. Results: Compared with controls, the expression of Th2 cytokines (IL-4, IL-5, IL-13) in CRSsNP and non-Eos-CRSwNP patients was the highest in Eos-CRSwNP (F=16.930,9.197,9.116, all P<0.05). Foxp3 had the lowest expression in Eos-CRSwNP patients and was lower than non-Eos-CRSwNP patients (F=2.780,P<0.05), and was negatively correlated with ECP (r=-0.326,P<0.05). Compared with controls, Eos-CRSwNP patients in CRSsNP patients and non-Eos-CRSwNP patients exhibited a significantly lower frequency of CD4+CD25+Foxp3+Treg cells (F=13.140, all P<0.01). The expression of Notch-l and Jagged-l in Eos-CRSwNP was significantly higher than that of the controls, CRSsNP patients and non-Eos-CRSwNP patients (F=5.953/F=6.380, P<0.05). In the nasal polyp group, the expression of Notch-l and Jagged-l showed significantly negative correlation with Foxp3 (r=-0.611/-0.346, all P<0.05), and positive correlation with Th2 cytokines (IL-4, IL-5, IL-13) and ECP, respectively (r=0.781/0.459,0.621/0.601,0.605/0.490,0.464/0.668, all P<0.05). There was no significant difference in the expression of receptor and ligand of the other Notch pathway among the groups. Conclusion: Abnormal activation of Notch-l/Jagged-l pathway may be involved in decreasing Treg ratio in Eos-CRSwNP, thereby promoting Th2 inflammatory response and Eosinophil infiltration.

[Expression and cellular provenance of interleukin 17A in non-eosinophilic chronic rhinosinusitis with nasal polyps].

Objective: To investigate the expression and cellular provenance of interleukin 17A (IL-17A) in non-eosinophilic chronic rhinosinusitis with nasal polyps (nECRSwNP), and to analyze the possible reasons for its different expression. Methods: Samples were collected from 14 patients with eosinophilic chronic rhinosinusitis with nasal polyps (ECRSwNP) and 28 patients with nECRSwNP, who underwent functional endoscopic sinus surgery in the Third Affiliated Hospital of Sun Yat-sen University from January 2017 to May 2018, including 33 males and 9 females, with the age ranging from 18 to 65 years old. Enzyme linked immune sorbent assay (ELISA) and flow cytometry were used to investigate the expression and cellular origins of IL-17A in the nasal tissue of ECRSwNP and nECRSwNP groups. Then the difference of quantity and differentiation ability of the major cells producing IL-17A between ECRSwNP and nECRSwNP groups were analyzed by flow cytometry. Finally, the expressions of IL-6, transforming growth factor-ß(TGF-ß), and IL-23, which were considered as the important factors in promoting Th17/Tc17 differentiation in CRSwNP and their correlation with IL-17A, were analyzed by ELISA. Statistical analysis was performed using IBM SPSS 20. Results: The IL-17A protein levels and IL-17A(+)lymphocyte percentages were higher in nECRSwNP group compared with that of the ECRSwNP group (158.56 (167.76) pg/ml (M(QR)) vs. 9.42 (11.33) pg/ml, 10.21%±1.54% (x±s) vs. 3.93%±0.80%, Z=2.95, t=3.62, all P<0.01). Tc17 cells (CD8(+)T cells producing IL-17A) and Th17 cells (CD4(+)T cells producing IL-17A) were major IL-17A producers in both ECRSwNP and nECRSwNP group. Further analysis revealed that there was no significant difference in quantity of CD8(+)and CD4(+)T cells between ECRSwNP and nECRSwNP group, but the differentiation ability about CD8(+)and CD4(+)T cells differentiating into Tc17 and Th17 in nECRSwNP group was stronger than that in ECRSwNP. The high expressions of IL-6 and TGF-ß, which were considered as the important factors in promoting Th17/Tc17 differentiation were also found in nECRSwNP group compared with ECRSwNP (56.07 (234.25) pg/ml vs. 8.27 (12.51) pg/ml, (5.44±0.34) pg/ml vs. (4.17±0.22) pg/ml, Z=2.426, t=2.29, all P<0.05). But the difference in expression of IL-23 was not significant difference between the two groups. Moreover, the expression of IL-17A showed significantly positive correlation with IL-6 (r=0.615, P=0.009). No positive correlation between IL-17A and TGF-ß or IL-23 was observed. Conclusions: The expression of IL-17A in nasal mucosa of nECRSwNP patients is significantly higher than that of ECRSwNP, which is due to the increase of expression and differentiation of Tc17/Th17 cells. IL-17A shows positive correlation with IL-6 in CRSwNP, which is the important factor in promoting Th17/Tc17 differentiation.

The effects of nasal irrigation with various solutions after endoscopic sinus surgery: systematic review and meta-analysis.

BACKGROUND: Nasal irrigation is commonly performed in patients with chronic rhinosinusitis after functional endoscopic sinus surgery. This study systematically assessed the clinical efficacy of nasal irrigation from the medical literature. METHODS: The PubMed, Embase and Cochrane Central Register of Controlled Trials databases were searched using a comprehensive strategy, limited to English-language articles, published from October 1971 to March 2017, and comprising human subjects. RESULTS: A total of 824 trials were identified, 5 of which, involving 331 participants, were included in this systematic review. After selection, only three trials were eligible for inclusion in a meta-analysis. Nasal irrigation using normal saline and various solutions was found to be effective in reducing symptom scores and endoscopic scores for chronic rhinosinusitis patients after functional endoscopic sinus surgery. Comparison of outcome measures, such as eosinophil count reduction, revealed that various solutions are more effective than normal saline alone; however, no statistical significance was found in terms of reduced symptom or endoscopic scores. CONCLUSION: Based on the current limited evidence, nasal irrigation is an effective therapy for chronic rhinosinusitis patients after functional endoscopic sinus surgery. However, when comparing various solutions with normal saline, no significant difference was found in symptom scores or endoscopic scores.

[Gene Analysis for the Sudden Death of Hypertrophic Cardiomyopathy by Whole Exome Sequencing.]

OBJECTIVES: To analyze the related pathogenicity gene mutations in a sudden death of hypertrophic cardiomyopathy （HCM） on whole exome level. METHODS: Whole exome sequencing （WES） was been performed on a sudden death case sample with pathological features of HCM by Illumina® Hiseq 2500 platform. Using hg19 as the reference sequences, the sequencing data were analyzed. Suspicious single nucleotide variants （SNV） were screened, and the conservatism and function were analyzed by the software such as PhyloP, PolyPhen-2, SIFT, etc. RESULTS: After screening, a heterozygous mutation C719R was finally identified in the gene MYBPC3 of this case. CONCLUSIONS: The molecular anatomy on whole exome level by second generation sequencing technology can help to define the molecular mechanism of HCM and provide a new mothed and thought for analysis of death cause.

The impact of plasma Epstein-Barr virus DNA and fibrinogen on nasopharyngeal carcinoma prognosis: an observational study.

BACKGROUND: The impact of combining plasma fibrinogen levels with Epstein-Barr Virus DNA (EBV DNA) levels on the prognosis for patients with nasopharyngeal carcinoma (NPC) was evaluated. METHODS: In this observational study, 2563 patients with non-metastatic NPC were evaluated for the effects of circulating plasma fibrinogen and EBV DNA levels on disease-free survival (DFS), distant metastasis-free survival (DMFS), and overall survival (OS). RESULTS: Compared with the bottom biomarker tertiles, TNM stage-adjusted hazard ratios (HR, 95% confidence intervals (CIs)) for predicting DFS in fibrinogen tertiles 2 to 3 were 1.26 (1.00 to 1.60) and 1.81 (1.45 to 2.26), respectively; HR for EBV DNA tertiles 2 to 3 were 1.49 (1.12 to 1.98) and 4.24 (3.27 to 5.49), respectively. After additional adjustment for established risk factors, both biomarkers were still associated (P for trend <0.001) with reduced DFS (HR: 1.79, 95% CI, 1.43 to 2.25 for top fibrinogen tertiles; HR: 4.04, 95% CI: 3.10 to 5.27 for top EBV DNA tertiles compared with the bottom tertiles). For patients with advanced-stage disease, those with high fibrinogen levels (3.34 g l(-1)) presented with worse DFS, regardless of EBV DNA 4000 or <4000 copies ml(-1) subgroup. Similar findings were observed for DMFS and OS. CONCLUSIONS: Circulating fibrinogen and EBV DNA significantly correlate with NPC patients survival. Combined fibrinogen and EBV DNA data lead to improved prognostic prediction in advanced-stage disease.

RAPD fingerprint construction and genetic similarity of Mesona chinensis (Lamiaceae) in China.

Mesona chinensis is an economically important agricultural crop, primarily cultivated for making grass jelly. It was originally discovered in South China. We examined 18 cultivars, including cultivars from Guangdong, Fujian, and Guangxi, China, Taiwan, and Indonesia, and a hybrid (a cross between cultivars from Indonesia and Guangdong), based on RAPD markers. The genetic similarity coefficient was calculated by NTSYS 2.10 and the clustering analysis was made by UPGMA. PCR amplification with 10 primers produced 163 bands; 94% of the amplified loci were polymorphic. The primers S208, S206, and S253 could completely distinguish all 19 samples by constructing a DNA fingerprint. Cluster analysis divided the 19 cultivars into five groups, with an overall genetic similarity coefficient of 0.68. Correlations were found among regional distributions, parental sources, and RAPD markers, demonstrating the rich genetic diversity of these 19 cultivars of M. chinensis. This study provides useful information for the classification, identification, and breeding of M. chinensis.

[Simultaneous determination of aspirin and phenobarbital in infantile co-phenobarbital powder by dual wavelength K-ratio spectrophotometry].

Aspirin (I) and phenobarbital (II) in infantile co-phenobarbital powder were determined simultaneously using dual wavelength K-ratio spectrophotometry. I and II were analyzed with dual wavelength K-ratio spectrophotometry at 255, 244 and 236 nm. This method showed a mean recovery of 101.3% and a coefficient of variation of 1.6% for (I). The mean recovery and CV of II were 99.21% and 1.90% (n = 6) respectively. The method is simple, rapid and accurate.