This study was designed to explore the RNA interference technique in inhibition of the expression of the mouse fibrinogen like protein 2 (mfgl2), which has been reported to be involved in the development a variety of diseases including fulminant viral hepatitis. A plasmid named p-mfgl2shRNA,complementary to the sequence of mfgl2 was constructed, while another short hairpin RNA (shRNA)which was a mutated form of the mfgl2shRNA sequences was used as a control. A plasmid named pEGFP-mfgl2 expressing the mfgl2-EGFP fusion protein was also constructed for the screening of the effect of p-mfgl2shRNA on mfgl2 expression. By cotransfection of p-mfgl2shRNA and pEGFP-mfgl2 or pcDNA3.1-mfgl2 expression construct into CHO cells or HeLa cells, the inhibition of mfgl2 expression by mfgl2shRNA was analyzed by direct observation through fluorescent microscopy, FACS, RT-PCR and immunohistochemistry staining. The experiments showed the significant inhibitory effect of p-mfgl2shRNA on mfgl2 expression at 48h post-transfection in both CHO and Hela cell lines with the inhibitory efficiency as high as 80.1%. The study demonstrated that the construct of p-mfgl2shRNA successfully interfered with the mfgl2 expression in vitro.

Objective: To investigate the roles of N-acetyl-L-cysteine (NAC) against binge drinking-induced fatty liver in mice. Methods: SPF male C57BL/6 mice were randomly divided into 3 groups, i.e. control group, model group, and NAC/ethanol group (n=10). Mice in model and NAC/ethanol groups were exposed to 3 doses of ethanol (6 g/kg bw) to induced fatty liver, while mice in control group received equal volume and equal energy of maltodextrin solution. NAC was administered to mice at 1 h before ethanol exposure (100 mg/kg bw, i.p.). The mice were sacrificed at 6 h after the last ethanol exposure. The liver and epididymal adipose tissues were collected. Histopathological examination and biochemical assay kit were used to evaluate the fat accumulation, while Western-blot was performed to detect the protein levels of some key factors involved in fat metabolism in liver and adipose tissues. Results: Compored with control group mice, the liver index and liver weight were significantly increased compared with model group, the liver index and TG level in NAC/ethanol group mice were all significantly decreased (P<0.05). Histological examination showed NAC effectively suppressed binge drinking-induced fat accumulation in mice liver. In addition, NAC had no significant effects on the protein levels of peroxisome proliferator-activated receptor-α (PPAR-α), Acy-CoA oxidase (ACOX), sterol regulatory element binding protein 1 c (SREBP-1c) and fatty acid synthase (FAS). Furthermore, the protein levels of hormone sensitive lipase (HSL) did not significantly differ among 3 groups, whereas NAC prevented binge drinking-induced increase of HSL phosphorylation at ser563 and ser660. Conclusion NAC could effectively attenuate binge drinking-induced fatty liver, which might be associated with the inhibition of lipid mobilization by suppressing the phosphorylation of HSL.

BACKGROUNDThe genome of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) includes sequences encoding the putative protein X4 (ORF8, ORF7a), consisting of 122 amino acids. The deduced sequence contains a probable cleaved signal peptide sequence and a C-terminal transmembrane helix, indicating that protein X4 is likely to be a type I membrane protein. This study was conducted to demonstrate whether the protein X4 was expressed and its essential function in the process of SARS-CoV infection.

METHODSThe prokaryotic and eukaryotic protein X4-expressing plasmids were constructed. Recombinant soluble protein X4 was purified from E. coli using ion exchange chromatography, and the preparation was injected into chicken for rising specific polyclonal antibodies. The expression of protein X4 in SARS-CoV-infected Vero E6 cells and lung tissues from patients with SARS was performed using immunofluorescence assay and immunohistochemistry technique. The preliminary function of protein X4 was evaluated by treatment with and over-expression of protein X4 in cell lines. Western blot was employed to evaluate the expression of protein X4 in SARS-CoV particles.

RESULTSWe expressed and purified soluble recombinant protein X4 from E.coli, and generated specific antibodies against protein X4. Western blot proved that the protein X4 was not assembled in the SARS-CoV particles. Indirect immunofluorescence assays revealed that the expression of protein X4 was detected at 8 hours after infection in SARS-CoV-infected Vero E6 cells. It was also detected in the lung tissues from patients with SARS. Treatment with and overexpression of protein X4 inhibited the growth of Balb/c 3T3 cells as determined by cell counting and MTT assays.

CONCLUSIONThe results provide the evidence of protein X4 expression following SARS-CoV infection, and may facilitate further investigation of the immunopathological mechanism of SARS.

Amino Acid Sequence ; Animals ; BALB 3T3 Cells ; Cercopithecus aethiops ; Growth Inhibitors ; analysis ; physiology ; HeLa Cells ; Humans ; Immunohistochemistry ; Lung ; chemistry ; Mice ; Molecular Sequence Data ; SARS Virus ; chemistry ; Severe Acute Respiratory Syndrome ; metabolism ; Vero Cells ; Viral Structural Proteins ; analysis ; physiology

OBJECTIVETo construct the siRNA plasmid for mfgl2 gene, which has been reported to be involved in a variety of disease developments including fulminant viral hepatitis, acute rejection of allo/zero transplantation and fetal loss syndrome, and to investigate its inhibitory effects on mfgl2 expression in vitro.

METHODSA plasmid p-mfgl2shRNA complimentary to the sequence responsible for the functional domain of mouse fgl2 (mfgl2) was constructed. The pcDNA3.1 mfgl2 expression construct was able to show a satisfactory fgl2 protein expression. The plasmid expression pEGFP and a construct expressing irrelevant shRNA with a random combination of the p-mfgl2shRNA sequence were used as controls. A pEGFP-mfgl2 expressing mfgl2-EGFP fusion protein was also constructed for screening of the effect of p-mfgl2shRNA on the mfgl2 expression.

RESULTSCotransfection of p-mfgl2shRNA with pEGFP-mfgl2 decreased green fluorescent cells and the lightness of fluorescence within the cells at the 24 h, 48 h and 72 h post-transfection when compared with that in the control groups which were solely transfected with pEGFP-mfgl2. Furthermore the mfgl2 expression was significantly reduced when the pcDNA3.1 mfgl2 expression construct was cotransfected with p-mfgl2shRNA both at mRNA level by RT-PCR and protein level by RT-PCR, immunohistochemistry staining and FACS in both CHO cell and Hela cell lines.

CONCLUSIONSThe study demonstrated that the construct of p-mfgl2shRNA successfully interfered in the mfgl2 expression in vitro. It provides a basis for a further investigation of effect in vivo.

Animals ; Fibrinogen ; biosynthesis ; genetics ; Gene Expression ; Mice ; Plasmids ; genetics ; RNA Interference ; RNA, Small Interfering ; genetics

OBJECTIVETo observe the specific cellular and humoral immune responses after immunization with recombinant adenovirus Ad5F35-LMP2 in rhesus monkeys.

METHODSSixteen rhesuses were immunized with Ad5F35-LMP2 through intra-muscular injection in three groups: high dosage group (1.5 x 10(10) TCID(50)/rhesus), medium dosage group (1.5 x 10(9)TCID(50)/rhesus), low dosage group (1.5 x 10(8)TCID50/rhesus) and the last group was control (PBS 4 ml/rhesus). They were totally immunized three times at intervals of one month. The EBV-LMP2 specific cellular immune responses were tested during the 0, 4, 8, 12 weeks by Elispot after immunization respectively. And the titers of anti-LMP2 antibody were tested by EIA at the same time.

RESULTSEBV-LMP2 specific cellular and humoral immune responses which were induced by recombinant adenovirus Ad5F35-LMP2 can be found in all the three dosage groups. The potency of immune responses was related with the dosage of immunization. Higher dosage elicited more potent immune response.

CONCLUSIONThe recombinant adenovirus Ad5F35-LMP2 could elicit LMP2 specific cellular and humoral immune responses in rhesus.

Adenoviruses, Human ; genetics ; Animals ; Cell Differentiation ; Herpesvirus 4, Human ; genetics ; Immunity, Cellular ; immunology ; Immunization ; methods ; Macaca mulatta ; Recombinant Fusion Proteins ; genetics ; immunology ; Viral Matrix Proteins ; genetics ; immunology

To study the molecular epidemiological characteristics of norovirus gastroenteritis outbreaks in Guangdong, we collected fecal and anal swabs specimens from 24 outbreaks of acute gastroenteritis from 2005 to 2008 to detect norovirus. Specimens were detected by RT-PCR and then sequenced. The descriptive data were also collected. According to our research, 19 of 24 outbreaks of gastroenteritis were positive for norovirus. The occurrence time was from October to next February mainly. The strains in 2005 belonged to G II-3 genotype and all outbreaks occurred in kindergarten and school. But from autumn of 2006, the outbreaks were all caused by G II-4/2006b variant and occurred in universities and community. The number of outbreaks in 2007 increased greatly and covered all over province. The nucleotide sequences of Guangdong strains in some sites showed high regional identity. Our results showed that with the shift of genotype from G II-3 to G II-4, occurrence of norovirus outbreaks increased greatly. The outbreaks of norovirus caused by G II-4/2006b variant spreaded widely and the involved population covered children and adult, indicating the strong invasiveness of this variant.
Adolescent ; Adult ; Base Sequence ; Child ; China ; epidemiology ; DNA-Directed RNA Polymerases ; genetics ; Disease Outbreaks ; Gastroenteritis ; epidemiology ; Humans ; Molecular Sequence Data ; Norovirus ; classification ; enzymology ; genetics ; Phylogeny ; Reverse Transcriptase Polymerase Chain Reaction ; Young Adult

Objective To observe the effects of melatonin on autophagy in cortical neurons of neonatal rats with hypoxic-ischemic brain damage(HIBD)and to explore its mechanisms via the PI3K/AKT signaling pathway,aiming to provide a basis for the clinical application of melatonin.Methods Seven-day-old Sprague-Dawley neonatal rats were randomly divided into a sham operation group,an HIBD group,and a melatonin group(n=9 each).The neonatal rat HIBD model was established using the classic Rice-Vannucci method.Neuronal morphology in the neonatal rat cerebral cortex was observed with hematoxylin-eosin staining and Nissl staining.Autophagy-related protein levels of microtubule-associated protein 1 light chain 3(LC3)and Beclin-1 were detected by immunofluorescence staining and Western blot analysis.Phosphorylated phosphoinositide 3-kinase(p-PI3K)and phosphorylated protein kinase B(p-AKT)protein expression levels were measured by immunohistochemistry and Western blot.The correlation between autophagy and the PI3K pathway in the melatonin group and the HIBD group was analyzed using Pearson correlation analysis.Results Twenty-four hours post-modeling,neurons in the sham operation group displayed normal size and orderly arrangement.In contrast,neurons in the HIBD group showed swelling and disorderly arrangement,while those in the melatonin group had relatively normal morphology and more orderly arrangement.Nissl bodies were normal in the sham operation group but distorted in the HIBD group;however,they remained relatively intact in the melatonin group.The average fluorescence intensity of LC3 and Beclin-1 was higher in the HIBD group compared to the sham operation group,but was reduced in the melatonin group compared to the HIBD group(P<0.05).The number of p-PI3K+and p-AKT+cells decreased in the HIBD group compared to the sham operation group but increased in the melatonin group compared to the HIBD group(P<0.05).LC3 and Beclin-1 protein expression levels were higher,and p-PI3K and p-AKT levels were lower in the HIBD group compared to the sham operation group(P<0.05);however,in the melatonin group,LC3 and Beclin-1 levels decreased,and p-PI3K and p-AKT increased compared to the HIBD group(P<0.05).The correlation analysis results showed that the difference of the mean fluorescence intensity of LC3 and Beclin-1 protein in the injured cerebral cortex between the melatonin and HIBD groups was negatively correlated with the difference of the number of p-PI3K+and p-AKT+cells between the two groups(P<0.05).Conclusions Melatonin can inhibit excessive autophagy in cortical neurons of neonatal rats with HIBD,thereby alleviating HIBD.This mechanism is associated with the PI3K/AKT pathway.

OBJECTIVETo probe blood serum Ab-IgG characteristics of severe acute respiratory syndrome (SARS) patients in Guangzhou and investigate the related factors.

METHODSThe serum of such population diagnosed as SARS convalescent patients, non-SARS patients, family consanguineous contraction persons, wild animal and vegetable salesman and community common people was collected. The lab detective method of ELISA was adopted for these serum samples. And the epidemic investigations for the SARS patients were also carried out.

RESULTSOf these populations, the detective rate of Ab-IgG for the clinic diagnosed SARS patients, which was 53.7%; That for the wild animal salesman and community common people were 16.7% and 0.9%, respectively. Among the clinic diagnosed SARS patients, the positive antibody detective rate was 90.4% for those which had specific contact history or infectivity, which was higher than that for other population. Among the specific contact history or infectivity cases, the antibody positive rate for the young and the old was lower than that for the adult. Meanwhile the difference did not exist among other cases. The antibody positive rate was identical between the male and the female. And the antibody detective rate was decreased by the month.

CONCLUSIONAs a whole SARS-CoV Ab-IgG detective rate for the clinic diagnosed SARS patients was 53.7% only. The reasons for that mainly lie in the wrong clinic diagnosis besides these factors such as age, hormone use and reagent and so on. The combination of lab detection results and epidemic investigation was propitious to the diagnosis veracity. It was impossible for the sub-clinic infection of SARS-CoV virus. The importance in the virus transmitting course need to be further studied.

Adult ; Antibodies, Viral ; blood ; Child ; China ; epidemiology ; Female ; Humans ; Immunoglobulin G ; blood ; Male ; Middle Aged ; SARS Virus ; immunology ; Seroepidemiologic Studies ; Severe Acute Respiratory Syndrome ; epidemiology ; immunology

Objective: To explore the occurrence of cognitive impairment in Chinese heart failure (HF) patients and it's impact on prognosis. Methods: In this prospective observational study, a total of 990 HF patients were enrolled from 24 hospitals in China during December 2012 to November 2014. All patients were administrated with the interview-format Montreal Cognitive Assessment (MoCA), according to which they were divided into MoCA<26 (with cognitive impairment) group and MoCA≥26 (without cognitive impairment) group. Baseline data were collected and a 1-year follow up was carried out. Univariate and multivariate logistic or Cox regression were performed for 1-year outcomes. Results: Cognitive impairment was evidenced in 628 patients (63.4%) and they were more likely to be older, female, and with higher proportion of New York Heart Association(NYHA) class Ⅲ-Ⅳ, chronic obstructive pulmonary disease (COPD), ischemic heart disease, while body mass index (BMI), education level, and medical insurance rate were lower (all P<0.05) as compared to patients in MoCA≥26 group. The rate of percutaneous intervention, device implantation, cardiac surgery and evidence-based medications were significantly lower in MoCA<26 group than in MoCA≥26 group (all P<0.05). During the 1-year follow up, patients in the MoCA<26 group had higher all-cause mortality (10.2%(64/628) vs. 2.2%(8/362), P<0.01), cardiovascular mortality (5.9%(37/628) vs. 0.8%(3/362), P<0.01) and major adverse cardiac and cerebrovascular events (MACCE) (9.6%(60/628) vs. 2.5%(8/362), P<0.01) than patients in the MoCA≥26 group. In univariate regression, MoCA<26 was associated with increased all-cause mortality (HR(95%CI):4.739(2.272-9.885), P<0.01), cardiovascular mortality (HR(95%CI):7.258(2.237-23.548), P=0.001) and MACCE (OR(95%CI):4.143(2.031-8.453), P<0.01). After adjustment by multivariate regression, MoCA<26 was indicated as an independent risk factor for all-cause mortality (HR(95%CI): 6.387(2.533-16.104), P<0.01), cardiovascular mortality (HR(95%CI): 10.848(2.586-45.506), P=0.001) and MACCE (OR(95%CI): 4.081(1.299-12.816), P=0.016), while not for re-hospitalization for HF (OR(95%CI):1.010(0.700-1.457), P=0.957). Conclusions: Cognitive impairment is common in HF patients,and it is an independent prognostic factor for 1-year outcomes. Routine cognitive function assessment and active intervention are thus recommended for HF patients.
China ; Female ; Heart Failure ; Humans ; Mental Status and Dementia Tests ; Prognosis ; Prospective Studies

Esophageal cancer is one of the malignant tumors with a high morbidity and mortality in China. According to China's latest cancer report released by the National Cancer Center in 2019, the number of people suffering from esophageal cancer reached 246 000 in 2015, and the death toll reached 188 000. How to effectively treat esophageal cancer and improve the survival rate of patients is one of the most urgent problems in the field of medicine. Phosphatidylinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway is one of the most important signaling pathway for regulating cell survival, differentiation and apoptosis in the body. It also plays an important role in the occurrence and mechanism of various cancers. Recent studies have shown that the activation of PI3K/Akt signaling pathway is an important factor in regulating proliferation, apoptosis, cycle arrest, migration and invasion of esophageal cancer cells. The long-term clinical observation found that traditional Chinese medicine has a stable effect in the treatment of esophageal cancer and little side effects, especially in improving the quality of life of cancer patients and prolonging the survival period of patients. At present, it is a research hotspot to intervene this signal pathway with traditional Chinese medicine in the treatment of esophageal cancer, so as to explore its mechanism of action on esophageal cancer. This paper focused on literatures in CNKI and PubMed databases from 2009 to 2019, with PI3K/Akt signaling pathway, esophageal cancer and miRNA as the key words. A total of 226 literatures were retrieved, and 61 literatures relating to traditional Chinese medicine, esophageal cancer, miRNA and PI3K/Akt signaling pathway were sorted out and summarized. This paper reviewed the mechanism of PI3K/Akt signaling pathway in esophageal cancer, the relationship between miRNA and PI3K/Akt signaling pathway and esophageal cancer, and how traditional Chinese medicine can regulate the expressions of relevant proteins in PI3K/Akt signaling pathway to inhibit cell proliferation, affect cell growth cycle, induce cell apoptosis, inducing cell autophagy, inhibit tumor invasion and metastasis, inhibit angiogenesis. Finally, it can improve esophageal cancer to provide theoretical basis and scientific basis for the treatment of esophageal cancer with traditional Chinese medicine.