AIM: To investigate the regulatory effects of nuclear factor-κB ( NF-κB) and activator protein-1 (AP-1) on the expression of ectopic trypsin and proinflammatory cytokines in influenza A virus (IAV)-induced myocardi-tis.METHODS:Male BALB/c mice of 8 weeks old ( n=40) were randomly divided into 4 groups:normal control group ( NC) , infection control group ( IC) , NF-κB inhibitor group ( NI) and AP-1 inhibitor group ( AI) .The mice in NC group and IC group were instilled intranasally with 15μL saline and 40 plaque forming units ( PFU) IAV, respectively.The mice in NI group and AI group were infected intranasally with 40 PFU IAV and injected intraperitoneally with 10 mg/kg NF-κB inhibitor pyrrolidine dithiocarbamate ( PDTC) or 2.5 mg/kg AP-1 inhibitor nordihydroguaiaretic acid ( NDGA) once daily. The mice were euthanized at day 9 after instillation, and the hearts were removed for pathological and biochemical analysis. RESULTS:IAV infection induced significant up-regulation of ectopic trypsin, and proinflammatory cytokines interleukin 6 (IL-6), IL-1βand tumor necrosis factor-α(TNF-α) in the myocardium, and triggered acute myocarditis.PDTC signifi-cantly inhibited NF-κB activation and up-regulation of ectopic trypsin and proinflammatory cytokines, and effectively sup-pressed IAV replication and myocardial inflammatory response (P<0.01).NDGA effectively inhibited AP-1 activity (P<0.01) and mildly suppressed up-regulation of proinflammatory cytokines ( P<0.05) , but had no effects on the expression of ectopic trypsin, IAV replication and the extent of myocarditis ( P>0.05) .CONCLUSION:IAV infection induces up-regulation of ectopic trypsin and proinflammatory cytokines in myocardium predominantly by the activation of NF-κB.AP-1 signaling pathway might be only partially involved in the regulation of proinflammatory cytokines.

Objective To explore the impact of recombinant human hepatocyte growth factor (rhHGF) in Celsior (CS) solution on the expression of INF-γ, IL-4 and IL-10 in a rat liver transplan-tation model. Methods After flushed with CS solution with addition of rhHGF (experimental group) or saline (control group), NHBD livers were stored at 4℃; for 16 h.then they were transplanted using the two-cuff technique with arterial reconstruction. The serum levels of INF-γ, IL-4 and IL-10 at lh after reperfusion were detected using ELISA. The INF-γ, IL-4 and IL-10 mRNA in the corresponding liver tissue were determined by RT-PCR. The 7-day survival rate was calculated and the histopatho-logical examination results were analyzed by hematoxylin and eosin staining. Results Compared with the control group, the experimental group showed lower INF-γ level and higher IL-4 and IL-10 levels in serum at 1 h after reperfusion (P<0. 05). The level of INF-γ mRNA in liver tissue was significant decreased at 1 h after reperfusion (P<0. 05) , and the level of IL-4 and IL-10 mRNA was significantly increased in the experimental group (P<0. 05). In experimental group, recipients got a better survival rate and histopathological examination showed a well-preserved hepatic architecture without hepatocyte necrosis, milder sinusoidal and portal congestion. Conclusion Adding exogenous rhHGF in CS solu-tion can protect NHBD livers from ischemia-reperfusion injury and prolong the survival in rats, which might be due to down-regulation of TNF-γ and up-regulation of IL-4 and IL-10.

ObjectiveTo investigate an improved technique of orthotopic liver transplantation with the ("two-cuff method") in rats, and to observe allograft rejection after transplantation. Methods30 Wistar rats were grafted with livers of SD rats and 30 SD rats were grafted with livers of SD rats by using a modified "two-cuff technique" and examined for postoperative rejection. ResultsThe anhepatic period was about 15 minutes. The 24-hour survival rate was 100%. The Wistar rats grafted with SD livers died 8 to 15 days (after) liver transplantation, and the histopathologic examination showed various degrees of allograft rejection. The SD rats grafted with livers of SD rats had a survival rate of 97% at 3 weeks postoperatively. ConclusionsThe modified "two-cuff technique" can effectively reduce operative time, decrease postoperative (complications) and increase the survival rate of othortopic liver transplantation in rats. The transplantation model of Wistar rats grafted with livers of SD rats can be used to study allograft rejection.

Objective To evaluate the effect of sustained silencing Forkhead box Ml (F0XM1) gene by short-hairpin RNA (shRNA) expression vector on cell growth of hepatocelluar carcinoma (HCC) in vitro.Methods Four shRNA expression vectors targeting different sequences of human F0XM1 mRNA were constructed.The expression vector with the best interfering effect and the negative control plasmid were used to transfect HCC cell line QGY-7703, stably transfected cell clones were selected by neomycin (G418).Cell growth was evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and colony formation was assessed by clonogenic assay.Cell apoptosis was detected by double staining with APC conjugated Annexin V and PI.Results F0XM1 protein was detected with different levels in all these studied human cell lines.The expression vector shRNA-1026 exhibited excellent interference effect after transient transfection, which showed 38.5% and 53.2% reduction of FOXM1 mRNA and protein level respectively.The growth of QGY-7703 cells was inhibited after stable inhibition of FOXM1 expression by shRNA-1026, which was indicated by decreased absorbance value of the test group after culture for 48, 72 and 96 h compared to control group (t = 10.830,3.578 and 5.734 respectively, P ＜ 0.05).Stable inhibition of F0XM1 also led to reduced colony formation ( t = 5.336, P ＜ 0.05 ) and increased apoptosis of QGY-7703 cells in comparison to control cells (t = 6.827, P ＜ 0.05 ).Conclusions Stable silencing F0XM1 gene by shRNA suppresses the growth of HCC cells in vitro.

Objective: To explore the nursing experience of growth hormone provocative test in pediatric clinic. Methods: Five thousand and thirty-six children with short stature or slowing growth received combined simultaneous Levodopa Pyridostigmine stimulation test from June 2008 to October 2018 in the Child Growth Center of the First Affiliated Hospital of SUN Yat-sen University. Comprehensive nursing intervention was conducted to ensure the test carry through successfully before, during and after the test. Results: All children completed the five collections in the 120-minute growth hormone provocative test without cannula obstruction and blurt out. Some (986 out of 5 036 children, 19.58%) had different degrees of adverse reactions including nausea, vomiting, abdominal pain, sweating, salivation, dizziness, pallor, etc., most of which disappeared or alleviated after nursing, except 11 patients (0.22%) needed atropine muscular injection and 3 of whom needed intravenous fluids to release the marked symptoms. Conclusion Combined simultaneous Levodopa Pyridostigmine stimulation test is safe and practicable in pediatric clinics with nursing experience.

OBJECTIVETo investigate the effect of estrogen on cell proliferation and expression of proteins of C-type natriuretic peptide (CNP), natriuretic peptides B receptor (NPR-B) and natriuretic peptides C receptor (NPR-C) in ATDC5 cells during chondrogenesis.

METHODATDC5 cells were induced for differentiation with insulin 10 µg/ml (day 0), and were started to be investigated on day 6. They were incubated with: (1) Estradiol (E2) at different concentrations (10(-11)-10(-5) mol/L) for 24 hours (for studying cell proliferation), or for 48 hours (for studying CNP, NPR-B and NPR-C protein expression); (2) E2 (10(-8) mol/L) for 24, 48, 72, 96 and 120 h (for studying cell proliferation), or for 24, 48, 72 and 96 hours (for studying CNP, NPR-B and NPR-C protein expression); (3) E2 (10(-8) mol/L) , and/or ICI 182782 (estrogen receptor antagonist ) (10(-7) mol/L) for 24 hours (for studying cell proliferation). ATDC5 cells proliferation were determined by MTT (OD value). Western-blotting was performed to identify the protein levels of CNP, NPR-B and NPR-C.

RESULT(1) After incubation with E2 (10(-11)-10(-5) mol/L) for 24 h, ATD5 cell number increased with the increasing E2 concentration, peak in E2 concentrations of 10(-9) and 10(-8) mol/L (0.56 ± 0.06 and 0.52 ± 0.02, P < 0.05 and <0.01, respectively) , while significantly decreased in E2 (10(-5) mol/L) (0.30 ± 0.02) compared with DMSO-control (0.38 ± 0.02) (P < 0.05). After incubation with E2 (10(-11)-10(-5) mol/L) for 48 h, the protein level of CNP, NPR-B and NPR-C increased significantly, with the greatest effect seen at a concentration of 10(-10) mol/L E2 for CNP and NPR-B, 10(-9) mol/L E2 for NPR-C (P < 0.05). (2) After incubation with E2 (10(-8) mol/L) for 24 to 96 hours: (1) The cell number in each of the four time points was significantly increased compared with DMSO-control, with the greatest effect in 48 h (0.030 ± 0.003) (P < 0.05 or <0.01, respectively). While the cell number at 120 h was similar to that in DMSO-control. (2) The protein level of CNP increased significantly at 24 h (P < 0.05), seemed to be increased at 48 h and 72 h and decreased at 96 h. Both NPR-B and NPR-C level seemed to be increased at 24 h (P = 0.060 and 0.055, respectively) and seemed to decrease at 48 h, with decreasing significantly at both 72 h and 96 h (P < 0.05). (3) After incubation for 24 h, there was significant difference among the cell number of the four groups (P < 0.05). Cell number of group E2 (0.470 ± 0.032) was increased compared with group (E2+ICI) (0.410 ± 0.018), both being increased compared with group DMSO-control (0.370 ± 0.011, P < 0.05, respectively). There was no difference in cell number between group ICI 182782(0.360 ± 0.035) and group DMSO-control.

CONCLUSIONE2 promotes the proliferation of ATDC5 cells i.e. chondrogenesis via estrogen receptor mediated mechanism, in both concentration-dependent and time-dependent manner. E2 (10(-11)-10(-8) mol/L) up-regulates protein expression of CNP, NPR-B and NPR-C of ATDC5 cells during chondrogenesis, and regulate the expression of the three proteins mentioned above positively or negatively at different time point, which implied that estrogen is one of the regulators of CNP signaling pathway.

Animals ; Blotting, Western ; Cell Differentiation ; drug effects ; Cell Line ; Cell Proliferation ; drug effects ; Chondrogenesis ; Dose-Response Relationship, Drug ; Estradiol ; pharmacology ; Gene Expression Regulation ; drug effects ; Mice ; Natriuretic Peptide, C-Type ; metabolism ; Receptors, Atrial Natriuretic Factor ; metabolism ; Signal Transduction ; drug effects ; Time Factors

Objective: To analyze guanine nucleotide-binding protein subunit beta-2-like 1 (GNB2L1) expression based on bioinformatics, so as to evaluate its role and its relationship with survival rate during the occurrence and development of hepatocellular carcinoma. Methods: GEPIA, UALCAN and HPA databases were used to analyze the expression level of GNB2L1 and its relationship with HCC survival rate. Mutations in the GNB2L1 gene and their impact on survival were analyzed using the cBioPortal database. LinkedOmics database was used to analyze GNB2L1-related genes in HCC. Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis were performed simultaneously. STEING database was used to construct the GNB2L1 protein interaction network. TIMER database was used to analyze the relationship between GNB2L1 gene expression and immune infiltration in hepatocellular carcinoma. Differential expression of GNB2L1 in plasma platelets of HCC patients and healthy controls was analyzed using mRNA-based sequencing technology. Data between groups were compared using an independent-samples t-test. Results: GNB2L1 expression level was significantly increased in HCC tissues (P<0.05), and its expression was significantly correlated with body weight, classification and stage (P<0.05). The overall survival rate was higher in GNB2L1 low expression group (P<0.001). GNB2L1 and its related genes were related to biological process regulation, metabolic process, protein binding, oxidative phosphorylation, JAK-STAT signaling pathway, Ras signaling pathway and so on. GNB2L1 had interaction with RPS12, RPS11 and RPL19, and participated in multiple biological processes such as liver regeneration and positive regulation of endogenous apoptotic signaling pathway. GNB2L1 expression was significantly positively correlated with the infiltration degree of various immune cells in HCC (P<0.05). Cox regression analysis showed that GNB2L1 was an independent risk factor for lower survival rate in patients with HCC [Hazard ratio (95% confidence interval)=1.456 (1.034~2.051), P=0.031]. GNB2L1expression levels were significantly higher in platelets of HCC patients than that of healthy controls (10.40±1.36 vs. 9.58±0.51, t=2.194, P=0.037). Conclusion: GNB2L1 has high expression and close relationship to survival rate in HCC. Therefore, GNB2L1 may be a potential biomarker of HCC.
Humans ; Carcinoma, Hepatocellular/pathology* ; Computational Biology ; Liver Neoplasms/pathology* ; Protein Subunits/metabolism* ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; RNA, Messenger ; Guanine Nucleotides ; Gene Expression ; Biomarkers, Tumor/genetics*

The emergence of single-cell sequencing technology enables people to observe cells with unprecedented precision. However, it is difficult to capture the information on all cells and genes in one single-cell RNA sequencing (scRNA-seq) experiment. Single-cell data of a single modality cannot explain cell state and system changes in detail. The integrative analysis of single-cell data aims to address these two types of problems. Integrating multiple scRNA-seq data can collect complete cell types and provide a powerful boost for the construction of cell atlases. Integrating single-cell multimodal data can be used to study the causal relationship and gene regulation mechanism across modalities. The development and application of data integration methods helps fully explore the richness and relevance of single-cell data and discover meaningful biological changes. Based on this, this article reviews the basic principles, methods and applications of multiple scRNA-seq data integration and single-cell multimodal data integration. Moreover, the advantages and disadvantages of existing methods are discussed. Finally, the future development is prospected.
Base Sequence ; Gene Expression Profiling ; Gene Expression Regulation ; Humans ; Sequence Analysis, RNA ; Single-Cell Analysis

ObjectiveTo explore the effect of precocious puberty on glucose metabolism and lipid metabolism in female rats. MethodsSixty two-day-old female rats were randomly divided into 2 groups. When aged 5 days， the precocious puberty group and normal group were given a single subcutaneous injection of danazol and solvent soybean oil respectively. The vaginal opening of rats was monitored from their 21 days of age. After 12 hours of fasting， all successful modeling rats were randomly executed within 3 days after vaginal opening， when aged 7 and 12 weeks. Then we measured the rats’ body weight and length， determined the concentrations of glucose， insulin， blood lipids， estradiol， leptin and adiponectin with enzyme-linked immunosorbent assay and observed the pathological changes of perirenal fat， uterus and ovary. ResultsFor body weight and length， rats in the precocious puberty group were smaller than those in the normal group within 3 days after vaginal opening， but which did not affect their subsequent growth and development， and there was no significant difference between the two groups at 7 and 12 weeks of age. Within 3 days after vaginal opening， insulin levels had significant difference between the two groups （P = 0.001）， the precocious group showed hyperinsulinemia and increased number of perirenal adipocytes. At three execution times， no significant difference was noted in estradiol， leptin and adiponectin levels between the two groups. The same was true in the ratios of ovary or uterus to body weight between the two groups. ConclusionsPrecocious puberty makes earlier onset of pubertal development and allows body maladaptation to the sudden changes of the internal environment. However， the changes due to precocious puberty are temporary and reversible， and they may become normal in adulthood.

To evaluate the efficacy and safety of aromatase inhibitor letrozole in treatment of male adolescents with idiopathic short stature (ISS). Seventy five boys with height less than 2 standard deviation (SD) below the mean who had entered puberty were enrolled in our study from 2004 to 2017, in the Pediatric Department of the First Affiliated Hospital, Sun Yat-Sen University. Among 75 patients, 28 in letrozole group received letrozole and spironolactone, 30 in gonadotrophin releasing hormone analogue (GnRHa) group received GnRHa injection and 17 had no intervention. Height velocity (HV), increment of bone age/chronological age (ΔBA/ΔCA), the final adult height (FAH) were compared among groups and the safety of letrozole treatment was evaluated. HV maintained faster during letrozole treatment when compared with other groups. HV during GnRHa treatment showed slightly decline in the first 6 months, but decreased remarkably after 6 months, and was significantly lower than that in letrozole group ( < 0.05). The maturation of BA slowed down in both letrozole and GnRHa groups. But the ΔBA/ΔCA in letrozole group during the first and the second year of treatment were significantly higher (0.67±0.09, 0.50±0.15, respectively) when compared with GnRHa group (0.59±0.16, 0.44±0.13, respectively) ( =2.78 and 2.20, all < 0.05). FAH in letrozole group and GnRHa group were (170±4) cm and (170±6)cm, there was no significant differences between the two groups ( >0.05), and both were higher than that in no intervention group (162±4 cm, < 0.01). After 6 months of letrozole treatment, testicular volumes and serum testerone levels increased; 39.2% (11/28) boys had clinical manifestations of hyperandrogenemia, and 82.1% (23/28) boys had decreased serum high-density lipoprotein (HDL) levels. Serum levels of HDL and testerone returned normal and the hyperandrogenemia disappeared after the cessation of letrozole treatment. No significant changes in serum triglyceride, serum low-density lipoprotein (LDL), fating serum levels of insulin and glucose, HOMA-IR were observed. No abnormal liver function, myalgia, scoliosis or aggravations of scoliosis was found. Long term letrozole therapy during puberty in boys with ISS can delay bone maturation without significant decrease of linear growth, and thus can improve the final adult height. No severe adverse reactions were found.
Adolescent ; Body Height ; Bone Development ; Child ; Gonadotropin-Releasing Hormone ; Growth Disorders ; Humans ; Letrozole ; therapeutic use ; Male