A simple and sensitive assay for rapid detection of human coronavirus NL63 (HCoV-NL63) was developed by colorimetic reverse transcription loop-mediated isothermal amplification (RT-LAMP). The method employed six specially designed primers that recognized eight distinct regions of the HCoV-NL63 nucleocapsid protein gene for amplification of target sequences under isothermal conditions at 63 degrees C for 1 h Amplification of RT-LAMP was monitored by addition of calcein before amplification. A positive reaction was confirmed by change from light-brown to yellow-green under visual detection. Specificity of the RT-LAMP assay was validated by cross-reaction with different human coronaviruses, norovirus, influenza A virus, and influenza B virus. Sensitivity was evaluated by serial dilution of HCoV-NL63 RNA from 1.6 x 10(9) to 1.6 x 10(1) per reaction. The RT-LAMP assay could achieve 1,600 RNA copies per reaction with high specificity. Hence, our colorimetric RT-LAMP assay could be used for rapid detection of human coronavirus NL63.
Colorimetry ; methods ; Coronavirus Infections ; diagnosis ; virology ; Coronavirus NL63, Human ; genetics ; isolation & purification ; DNA Primers ; genetics ; Humans ; Nucleic Acid Amplification Techniques ; methods ; Reverse Transcription ; Sensitivity and Specificity

Objective To investigate the clinical significance of serum cytokines concentrations and A- PACHE scores in evaluating the illness state for critical trauma patients.Methods A clinical prospective self-control trial was performed,in which 36 patients admitted to ICU by SIRS were enrolled.Objects were divided into mild and severe trauma group according to APACHE score.The TNF-?and IL-6 concentrations were determined on the 1st, 3rd and 5th day of admission,the APACHE score were assessed at the same time.Statistic analysis was performed according to this group.Results The TNF-?concentrations decreased continuously in the following days while IL-6 decreased from the 7th day in the mild trauma group.In the severe trauma group the TNF-?and IL-6,APACHE score concentrations kept increasing.There was a significant difference of TNF-?and IL-6 concentrations between severe trauma and mild trauma group.Conclusion Dynamic measurement of TNF-?and IL-6 concentrations with APACHE score provide great help to evaluate the illness state and predict the prognosis.

Objective - To analyze the relationship between cobalt level of post shift urine and individual exposure level of ， cobalt and its compounds in cobalt exposed workers and to explore the feasibility of using urine cobalt as a biomarker. Methods - A total of 148 occupational cobalt exposed workers from a new material company were selected as the exposed ， - - group and 44 non occupational cobalt exposed workers from the company were selected as the control group using the typical sampling method. The exposure concentration of time weighted average of cobalt and its compounds in the workplace air of the - two groups was determined by inductively coupled plasma mass spectrometry as the individual exposure level. The cobalt levels - - of pre shift and post shift urinary samples of the two groups were detected by this method. The linear relationship between the - cobalt level of post shift urine and the individual exposure level of cobalt and its compounds in the air of the workplace was Results - 3 analyzed. The individual exposure level of cobalt and its compounds in the exposed group was 1.10 131.71 μg/m with （M） 3 the median of 12.23 μg/m. No cobalt and its compounds were detected in the workplace air in the control group. The cobalt - - levels of pre shift and post shift urines in exposed group were higher than those in the control group at the same time point （M： vs ， vs ， P ） - - 1.54 0.56 μg/L 8.77 0.83 μg/L all <0.01 . The cobalt level of post shift urine was higher than that in pre shift （M： vs ，P ）， urine in the exposed group 8.77 1.54 μg/L <0.01 and it was positively correlated with the individual exposure level （ ，P ） ， of cobalt and its compounds Spearman correlation coefficient=0.86 <0.01 . After common logarithm conversion the linear regression equation of the cobalt level of post shift urine and the common logarithm of individual exposure level of cobalt and （x） ：ŷ x（ ；F ， its compounds in the exposed group was as follows = −0.178 + 0.988 coefficient of determination=0.72 =374.75 P ；t ， P ） Conclusion - <0.01 = - 19.36 <0.01 . There was a linear correlation between cobalt level of post shift urine and occupational cobalt exposure level of cobalt exposed workers. Urinary cobalt can be used as a biomarker of occupational cobalt

OBJECTIVETo investigate the effects of astragalus on tubulointerstitial lesions in rats with IgA nephropathy (IgAN) and to explore the possible mechanism.

METHODSTwenty-eight Sprague-Dawley rats were randomly assigned to three groups. The rat model of IgA nephropathy was induced by intragastric administration of bovine serum albumin and injections of LPS and CC14. Six weeks later, the rats with IgAN were randomly treated with oral astragalus (3 g/kg/d, for 6 weeks) or normal saline. Normal control rats which were not subjected to IgAN were treated with normal saline. The number of urinary erythrocytes and urinary protein and B-D-N-Acetyl glucosaminidase (NAG) contents were determined by Pan-automatic biochemistry analyzing meter. Expression of monocyte chemotactic protein-1 (MCP-1) and nuclear factor-kappa B (NF-kappaB) in tubulointerstitial tissues were analyzed by immunohistochemistry. A semiquantitative score was used to evaluate the degree of renal pathologic lesions.

RESULTSThe number of urinary erythrocytes (74.02+/-16.58 / microL vs 383.23+/-4.94 /microL) and urinary protein (13.88+/-4.94 vs 59.82+/-14.73 mg/L) and NAG contents (2.84+/-0.31 vs 5.24+/-0.80 U/L) in the astragalus-treated IgAN rats decreased remarkably compared with those in the IgAN rats without astragalus treatment (P<0.01). Expression of the NF-kappaB and MCP-1 in the renal tissues in the IgAN rats without astragalus treatment was significantly higher than that in the astragalus-treated IgAN rats and normal control rats (P<0.01). There were significant differences in the scores of renal pathologic lesions between the IgAN rats with or without astragalus treatment (6.03+/-0.46 vs 10.57+/-1.23; P<0.01).

CONCLUSIONSAstragalus can decrease the number of urinary erythrocytes and urinary protein and NAG contents, and relieves tubulointerstitial lesions, possibly through the down-regulation of NF-kappaB and MCP-1 expression in rats with IgAN.

Animals ; Astragalus Plant ; Chemokine CCL2 ; analysis ; Glomerulonephritis, IGA ; drug therapy ; metabolism ; pathology ; Immunohistochemistry ; Kidney Tubules ; pathology ; Rats ; Rats, Sprague-Dawley ; Transcription Factor RelA ; analysis

Objective To analyze the data of road traffic accidents, deaths and injuries in 31 provinces and municipalities in China from 2010 to 2017, and to predict that the number of deaths and injuries caused by road accidents set in the sustainable development goals (SDGs) will be halved by 2020 compared with the target set in 2015. Methods Describing and analyzing the current situation of road traffic accidents in 31 provinces and municipalities in China, and using the trend extrapolation method to predict whether the SDGs target can be achieved by 2020. Results The numbers of traffic accidents, deaths and injuries in Guangdong, Jiangsu, Zhejiang and Shandong Provinces showed a downward trend from 2010 to 2017, but still ranked the front in China. In 2017, Beijing had the highest death rate followed by Guizhou as the second, and Guizhou had the highest injury rate followed by Tianjin as the second. The projected results showed that the numbers of deaths and injuries caused by road traffic accidents in China would be increased by 17% and 1.3% respectively in 2020 indicating that SDGs target could not be met. Among the 31 provinces/municipalities, it was predicted that the numbers of deaths and injuries in Hunan would be reduced to 50.7% and 65.3% in 2015, respectively by 2020, and the target could be achieved; the number of injuries in Shanghai was decreased by 83.3%, but the deaths was only decreased by 34.5%, and there was still a gap with the target; the rest 29 provinces/municipalities could not meet SDGs target. It was expected that the deaths in 11 provinces and municipalities would increase by 2020, with Beijing, Jilin, Jiangxi, Hubei and Guizhou Provinces showing the most significant growth. The number of injured people in 8 provinces and municipalities shows an increasing trend, with Jilin, Jiangxi, Hubei and Guizhou provinces showing significant growth. Conclusions Except for Hunan Province, it was difficult for the whole country and the rest provinces and municipalities to reach the SDGs target. According to the forecast results and the actual situation, a targeted scientific prevention and control strategy can be formulated. The measures taken by Hunan and Shanghai in traffic accident prevention and control were effective and worth learning.

Objective:To investigate the effect of Astragalus polysaccharides on apoptosis,transdifferentiation and ROS content in renal tubular epithelial cells of diabetic nephropathy.Methods:HK-2 cells were divided into low glucose group,high glucose group and astragalus polysaccharide+high glucose group,cell proliferation was detected by CCK-8 assay after 48 h;cell apoptosis and ROS content was detected by flow cytometry;the expression of E-cadherin,α-SMA,STAT1,STAT3,p-STAT1,p-STAT3 protein were detected by Western blot.Results:The survival rate of cells in high glucose group was significantly lower than low sugar group (P<0.01),cell apoptosis rate,ROS content and E-cadherin,α-SMA,p-STAT1 and p-STAT3 protein expression was significantly higher than low sugar group (P<0.01),the cell survival rate in high glucose+astragalus polysaccharide group was significantly higher than high glucose group,cell apoptosis rate,ROS content and E-cadherin,α-SMA,p-STAT1 and p-STAT3 protein expression was significantly lower than high glucose group (P<0.01).Conclusion:Astragalus polysaccharides can promote the proliferation of renal tubular epithelial cells induced by high glucose,inhibit apoptosis and transdifferentiation,and the mechanism is related to down regulation of JAK/STAT signaling pathway.

Objective To measure the concentration of intracellular free Ca2+ ([Ca2+]i) in neuron-like cells resulted from rat bone mesenchymal stem cell (BMSCs) differentiation induced by salvia miltiorrhiza injection and provide some theoretical basis for the BMSCs transplantation. Methods The rat BMSCs were separated from rat bone marrow and cultured in vitro. After induced by basic fibroblast growth factor and 10mL/L salvia miltiorrhiza injection, the cells were identified with immunofluorescence staining against NeuN. The same procedure was performed on primarily cultured hippocampal neurons. Then, the [Ca2+]i of the differentiated neuron-like cells was determined and compared with primarily cultured hippocampal neurons. Results The BMSCs after induced by basic fibroblast growth factor and salvia miltiorrhiza injection expressed neuronal phenotypes similar to the cell appearance of neurons with NeuN. The average fluorescence intensity of the neuron-like cells derived from BMSCs was 984.75±79.51, while the average fluorescence intensity of the primarily cultured hippocampal neurons was 769.42±60.93. No significant difference was found between them (P>0.05). Conclusion The neuron-like cells from rat BMSCs differentiation induced by salvia miltiorrhiza injection possess certain neuronal properties.

OBJECTIVETo study the regulattory effect of Astragalus membranaceus on immune disturbance of the rats with IgA nephropathy.

METHODSRats IgA nephropathy (IgAN) model was duplicated by oral feeding of bovine serum albumin (BSA), subcutaneous injection of carbon tetrachloride (CCl4) and injection of lipopolysaccharide (LSP) into vena caudalis. The rats were divided into three groups randomly for the normal, IgAN model group and the group treated with Astragalus membranaceus (treatment group). The treatment group was given the Astragalus membranaceus granules via intragastric administratsion, the normal group and the IgAN model group were given the equal amount of aqua destillata by gastric perfusion. The rats were examined for albuminuria, hematuria and pathological changes of renal tissue and the distribution of TGF-beta and interleukin-5 in renal tissue was determined by immunohistochemistry and the IFN-gamma and IL-4 of cytokine of Th1 and Th2 types were detected in rats IgA nephropathy model by sandwich enzyme linked immunosorbent assay (ELISA).

RESULTS(1) The hematuria in rats with IgA nephropathy significantly increased compared with normal control group and Astragalus treatment group (P < 0.05). There was significant increase in albuminuria in rats with IgA nephropathy, compared with normal control group and astragalus treatment group (P < 0.01). (2) The pathological change of glomerular mesangium, renal tubules and renal interstitia became serious in rats IgA nephropathy model when compared with normal control group and astragalus treatment group. Immumofluorescence showed renal IgA density in rats IgA nephropathy model was significantly higher than that in the normal control group (P < 0.001) and astragalus treatment group (P < 0.001). (3) The result of immuno histochemistry showed that there was only weak expression of TGF-beta and interleukin 5 in normal renal tissue. The expression of TGF-beta and interleukin 5 in IgA nephropathy model was significantly stronger than those in normal control group (P < 0.05) and astragalus treatment group (P < 0.05). (4) The serum IL-4 levels were (33.74 +/- 7.52) pg/ml in rats IgA nephropathy model, significantly higher than that in normal control group (2.36 +/- 0.85) pg/ml and astragalus treatment group (3.24 +/- 1.13) pg/ml. The IFN-gamma level in serum of rats IgA nephropathy model was (18.79 +/- 3.80) pg/ml, which was significantly higher than that in normal control group (46.53 +/- 5.56) pg/ml and astragalus treatment group (41.28 +/- 2.95) pg/ml.

CONCLUSIONSThe astragalus could lower the level of hematuria and 24 hours-albuminuria of the IgAN model, and amelioratse the change of the renal pathology and reduce the deposit of IgA in glomerular mesangium. The possible mechanism of the effect is that astragalus could regulate the derangement of Th1, Th2, accordingly could improve the level of IL-4 and IFN-gamma in the serum and diminish the expression of cytokine Th2 TGF-beta1 and IL-5 of the renal tissue, and thereby could postpone the development of IgAN.

Animals ; Astragalus membranaceus ; chemistry ; immunology ; Cattle ; Drugs, Chinese Herbal ; pharmacology ; Glomerulonephritis, IGA ; immunology ; Interleukin-4 ; pharmacology ; Interleukin-5 ; pharmacology ; Kidney Tubules ; drug effects ; Rats ; Transforming Growth Factor beta ; immunology ; Transforming Growth Factor beta1 ; pharmacology

OBJECTIVETo investigate the aberrant promoter methylation of hMLH1 gene promoter and its clinical significance in papillary thyroid cancer (PTC).

METHODSmethylation of hMLH1 gene promoter in the cancer tissue and matched tumor-adjacent normal tissue of 152 PTC patients were detected by real-time methylation specific PCR (qMSP). The relationship between the methylation of hMLH1 gene promoter and clinicopathological features was analyzed.

RESULTSThe methylation rate of hMLH1 gene promoter in cancer tissues was 37.5% (57/152), of which 33 cases were totally methylated and 24 cases were partially methylated. The methylation rate of adjacent normal tissues was 5.3% (8/152)(all were partially methylated). The methylation rate of PTC tissues was significantly higher than that in the tumor-adjacent normal tissue (P < 0.01). The promoter methylation of hMLH1 gene in PTC was significantly correlated with age, size and number of the primary lesion, local invasion, T stage and lymph node metastasis (P < 0.05) , but not correlated with gender and clinical stage (P > 0.05).

CONCLUSIONPromoter methylation of hMLH1 gene is a common molecular event in PTC tissue, and it is significantly correlated with the progression of PTC.

Adaptor Proteins, Signal Transducing ; genetics ; metabolism ; Adolescent ; Adult ; Age Factors ; Aged ; Carcinoma ; genetics ; metabolism ; pathology ; Carcinoma, Papillary ; DNA Methylation ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Lymphatic Metastasis ; Male ; Middle Aged ; MutL Protein Homolog 1 ; Neoplasm Invasiveness ; Neoplasm Staging ; Nuclear Proteins ; genetics ; metabolism ; Promoter Regions, Genetic ; genetics ; Thyroid Neoplasms ; genetics ; metabolism ; pathology ; Tumor Burden ; Young Adult

OBJECTIVETo get the cDNA clones which cover the whole genome of SARS-CoV PUMC2 strain.

METHODSUsing the SARS-CoV PUMC2 strain genomic RNA as the template, the cDNA fragments were amplified by RT-PCR, the PCR products were further purified and ligated into the pGEM-T vector, and all the clones obtained were sequenced.

RESULTSThe cDNA clones which cover the whole genome of SARS-CoV PUMC2 strain were obtained.

CONCLUSIONSThese cDNAs can be provided for the function study of SARS-CoV proteins and the construction of full-length infectious cDNA clone of SARS-CoV.

Amino Acid Sequence ; Base Sequence ; Cloning, Molecular ; DNA, Complementary ; genetics ; DNA, Viral ; genetics ; Genome, Viral ; Molecular Sequence Data ; Nucleic Acid Amplification Techniques ; RNA, Viral ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; SARS Virus ; genetics ; isolation & purification ; Sequence Analysis, DNA ; Viral Proteins ; genetics