A recombinant vaccinia virus (rWR-SARS-S)expressing spike protein of severe acute respiratory syndrome coronavirus was constructed. The expression of full length recombinant SARS spike protein (rSS) in HeLa cells possessing specific reaction ability to chicken anti-sera was confirmed by SDS-PAGE and Western-blot (190 kD). HeLa cells infected with rWR-SARS-S also showed high sensitivity in detecting specific serum antibody by indirect immunofluoresence assay (IFA). The results above indicated that the availability of such a faithful model system offers particular advantages for the study of SARS in that it reduces the need for direct manipulation of an exotic pathogen. In the absence of infectious SARS, we may safely carry out detailed biochemical and genetic manipulations to investigate features of viral replication and gene function, as well as explore new avenue for vaccine development.
Animals ; Antibodies, Viral ; immunology ; Antigens, Viral ; immunology ; Blotting, Western ; Chickens ; Enzyme-Linked Immunosorbent Assay ; Fluorescent Antibody Technique, Indirect ; Gene Expression ; HeLa Cells ; Humans ; Immune Sera ; immunology ; Immunization ; methods ; Membrane Glycoproteins ; genetics ; immunology ; Mice ; Recombinant Proteins ; immunology ; SARS Virus ; genetics ; immunology ; Spike Glycoprotein, Coronavirus ; Vaccinia virus ; genetics ; Viral Envelope Proteins ; genetics ; immunology

The aim of the present study was to gain an insight into the thiopurine methytransferase (TPMT) genotyping assay, which was based on polymerase chain reaction (PCR), allele-specific PCR, restriction digestion of PCR products, denaturing high-performance liquid chromatography (DHPLC) and SNaPshot sequencing and in combination with direct DNA sequencing. Among the f our methods to test TPMT genetic SNPs based on PCR, allele specific PCR was not able to differentiate wild type from varied type. BsiYI, MwoI and AccI to digest PCR products were used so that SNP in TPMT exon 5, 7 and 10 tested. It showed that there were no differences between the results of digestion of PCR products and those of DNA sequence analysis. Therefore, this method was reliable. But some other methods were still needed to look for a compensation, because no restriction map changing resulted from the 2 SNPs in TPMT promotor was found. As to the results of DHPLC, those for the screening of TPMT exon-5 and -10 for SNPs were the same as restriction analysis of PCR products and direct DNA sequencing. But the variation of the heterozygotes in exon-7 was high, which was different from the results of direct DNA sequencing. After changing the Tm of DNA step by step, It was found that all the samples showed single peak when the temperature was 54 degrees C. But this result was unbelievable because a heterozygote in exon 7 as positive control could not be found. Therefore, it was necessary to test the sensitivity and accuracy of DHPLC, though DHPLC could be used as an effective method of SNPs screening. The results of the SNaPshot sequencing were also same as those of restriction analysis of PCR products and direct DNA sequencing. And the results showed that the bases of TPMT promoter -91 and -168 were G, instead of A and T. The results of the four methods to detect TPMT genetic SNPs based on PCR showed that SNPs analysis technique should be a combination of the techniques above-mentioned. One technique alone could not satisfy the need in clinics and research. The compensation of each other was very important.
Chromatography, High Pressure Liquid ; Exons ; Humans ; Methyltransferases ; genetics ; Polymerase Chain Reaction ; Polymorphism, Single Nucleotide

Objective To apply PDCA circle management method to eliminate redundant medical consumables items and reduce the burden of consumables item information system,so as to realize fine management of medical consumables.Methods The consumables items un-mobilized during 2013 to 2015 were summarized,and the causes were analyzed.The redundant consumables items were sealed up in the materials management system.Results The utilization rate of consumables items were increased from 61％ to 82％,and the target value was 71％,that is,fine management was realized.Conclusion PDCA method can optimize the management of medical consumables items,and enhance the efficiency of materials information system.

Objective:To observe the effects of acupuncture on the characteristics of neuro-electrophysiological activity in hippocampal CA1 and CA3 areas of rats with post-traumatic stress disorder (PTSD).Methods:Fifty Sprague-Dawley (SD) rats were randomly divided into a blank group,a model group,a grasping group,a Western medicine group and an acupuncture group,with 10 rats in each group.Except for the blank group,rats in the other 4 groups all received the combined stress modeling method.Rats in the Western medicine group were intragastrically administrated with paroxetine hydrochloride,those in the acupuncture group received acupuncture intervention,those in the grasping group received grasping fixation,and those in the model group and the blank group did not receive any interventions.After 14 d of intervention,the interspike interval (ISI) and power spectral densities (PSD) were analyzed and mapped by in vivo multiple channels to record the neuron clusters discharge in the hippocampal CA1 and CA3 areas.Results:Compared with the blank group,ISI was prolonged in the CA1 and CA3 areas of the model group and the grasping group,and the concentrated PSD distribution area moved down (P＜0.05 or P＜0.01).Compared with the grasping group,the ISI of the CA1 and CA3 areas in the Western medicine group and the acupuncture group was shortened,and the concentrated PSD distribution area moved up (P＜0.05 or P＜0.01).The ISI and PSD distributions in the CA1 and CA3 areas of the acupuncture group were not statistically different from those in the Western medicine group (both P＞0.05).Conclusion:Both acupuncture and paroxetine hydrochloride can significantly regulate the neuro-electrophysiology activity of hippocampal CA1 and CA3 areas in PTSD rats,which may be one of the mechanisms of acupuncture intervention to promote PTSD recovery.

OBJECTIVETo compare the characterization of coronary atherosclerotic plaques in patients with unstable angina pectoris (UAP) and stable angina pectoris (SAP) by optical coherence tomography (OCT).

METHODSOCT was performed in 47 patients (23 UAP and 24 SAP) undergoing coronary angiography. Lipid-rich plaque (defined by > or = 2 quadrants of the cross-section area), thin cap fibroatheroma (TCFA), thickness of fibrous cap, plaque rupture, calcification and thrombus visualized by OCT were compared between UAP and SAP patients.

RESULTSOCT imaging was successfully in 44 out of 47 patients (22 UAP, 22 SAP). Proportion of lipid-rich plaques was similar between UAP and SAP groups [91% (20/22) vs. 73% (16/22), P = 0.741]. The minimum thickness of fibrous cap in the UAP group was significantly thinner than that in SAP group [(69.5 +/- 34.7) microm vs. (141.1 +/- 68.5) microm, P = 0.000] and the rate of fibrous cap erosion in the UAP group was significantly higher than that in the SAP group [59% (13/22) vs. 9% (2/22), P = 0.000]. Percents of TCFA [73% (16/22) vs. 14% (3/22), P = 0.000] and plaque rupture [50% (11/22) vs. 9% (2/22), P = 0.003] were significantly higher in UAP group compared those in SAP group. Incidence of thrombus and calcification were similar between two groups.

CONCLUSIONSOCT imaging can clearly define plaque characterization of coronary atherosclerosis. UAP patients have thinner fibrous cap, higher incidences of fibrous cap erosion, plaque rupture and TCFA compared patients with SAP.

Aged ; Angina Pectoris ; diagnostic imaging ; Angina, Unstable ; diagnostic imaging ; Coronary Artery Disease ; diagnostic imaging ; Female ; Humans ; Male ; Middle Aged ; Radiography ; Retrospective Studies ; Tomography, Optical Coherence

OBJECTIVETo evaluate coronary artery atherosclerotic plaque characteristics and changes post coronary stenting by optical coherence tomography (OCT).

METHODSOCT images were obtained in 22 diseased coronary vessels after coronary angiography or percutaneous coronary interventions (PCI) in 20 patients and in 23 stents [7 sirolimus-eluting stents (SES) follow up at 4-29 months post stenting and 8 bare mental stents (BMS) at 4-35 months post stenting, 8 stents immediately after PCI].

RESULTSAll 22 vessels and 23 stents OCT images were successfully acquired. Two thromboses, 8 fibrous, 9 lipid-rich and 3 calcium plaques as well as 3 plaque ruptures were visualized by OCT. No significant neointimal proliferation and restenosis were found in SES stents and some struts were not covered with neointima even at 29 months post stenting. Significant neointimal proliferation on surfaces of stent struts were visualized in all 8 BMS stents and restenosis was detected in 3 BMS stents. OCT images obtained immediately after PCI showed that 3 stents were well positioned, tissue prolapse between coronary stent struts occurred in 4 stents and stent dissociation with vessel wall could be seen in 1 stent.

CONCLUSIONSOCT imaging can clearly visualize different types of atherosclerotic plaques. By providing detailed information on plaque characteristics, this technique might help cardiologists in choosing suitable stents and guiding preventive therapy for patients with coronary heart disease.

Adult ; Aged ; Coronary Disease ; diagnostic imaging ; therapy ; Drug-Eluting Stents ; Female ; Follow-Up Studies ; Humans ; Male ; Middle Aged ; Radiography ; Sirolimus ; administration & dosage ; Stents ; Tomography, Optical Coherence

China ; Color ; Color Vision ; History, 20th Century ; History, 21st Century ; Humans ; Psychology ; history ; Skin Pigmentation

OBJECTIVETo establish multiplex PCR-based assays for detecting H.influenzae and H.parainfluenzae. And the PCR-based assays were applied to detect the carriage rates of H.influenzae and H.parainfluenzae in nasopharyngeal swab specimens which were collected from healthy children.

METHODSMultiplex primers for species-specific PCR were designed by using DNAstar soft based on the sequences of 16S rRNA genes from genus Haemophilus to detect H.influenzae and H.parainfluenzae.

RESULTSThe sensitivity of the 16S rRNA PCR assay for detecting H.influenzae and H.parainfluenzae was 97.53% and 100% respectively, and the specificity was 95.89% and 96.63% respectively. Youden's Index on the ability to detect H.influenzae and H.parainfluenzae was 0.9342 and 0.9663 respectively. 666 nasopharyngeal swab specimens were collected from healthy children. The detection rates of H.influenzae and H.parainfluenzae were 14.11% and 16.07% respectively by using isolation and culture methods. The detection rates of H.influenzae and H.parainfluenzae were 43.54% and 57.96% respectively by 16S rRNA PCR assays. The carriage rates of serotypes a, b, c, d, e, f and non-typeable isolates were 0% (0/666), 0.15% (1/666), 1.20% (8/666), 0.15% (1/666), 1.20% (8/666), 1.80% (12/666), 95.50% (636/666) respectively.

CONCLUSIONThe multiplex PCR assays were very rapid, reliable and feasible methods for detection of H.influenzae and H.parainfluenzae in pharyngeal swab specimens which were compared to conventional isolation and culture methods. 95.5% of H.influenzae strains in healthy children were nontypeable. The encapsulated or typable strains were mainly three serotypes which was c, e, and f serotype.

Haemophilus influenzae ; classification ; genetics ; isolation & purification ; Haemophilus parainfluenzae ; classification ; genetics ; isolation & purification ; Humans ; Multiplex Polymerase Chain Reaction ; methods ; Nasopharynx ; microbiology ; RNA, Bacterial ; genetics ; RNA, Ribosomal, 16S ; genetics ; Sensitivity and Specificity

OBJECTIVETo conduct a molecular epidemiological survey on the mitochondrial DNA C1494T mutation in non-syndromic hearing loss patients in Chinese population.

METHODSPolymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) were used to screen the mitochondrial DNA 12S rRNA C1494T mutation in 20 patients with aminoglycoside antibiotic induced hearing loss, 136 sporadic non-syndromic hearing loss patients and 50 probands of pedigrees with non-syndromic hearing loss.

RESULTSThe C1494T mutation did not appear in all cases except for the positive control.

CONCLUSIONIncidence of mitochondrial DNA C1494T mutation is much lower than that of mitochondrial DNA A1555G mutation in non-syndromic hearing loss of Chinese population. Mitochondrial DNA C1494T mutation may be a rare variation in non-syndromic hearing loss and is not the main cause of aminoglycoside antibiotic induced-deafness.

Adolescent ; Aminoglycosides ; adverse effects ; Anti-Bacterial Agents ; adverse effects ; Asian Continental Ancestry Group ; genetics ; Child ; China ; DNA, Mitochondrial ; genetics ; Female ; Hearing Loss ; chemically induced ; ethnology ; genetics ; Humans ; Male ; Point Mutation ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; RNA, Ribosomal ; genetics

OBJECTIVETo study the prevalence of severe acute respiratory syndrome coronavirus (SARS-CoV) like virus in animals at a live animal market of Guanzhou in 2004 before and after culling of wild animal action taken by the local authority, in order to predict the re-emerging of SARS from animal originals in this region.

METHODSAnimals at live animal market were sampled for rectal and throat swabs in triplicate. A single step realtime reverse transcription-polymerase chain reaction (RT-PCR) diagnostic kit was performed for screening SARS-CoV like virus, the manual nested RT- PCR and DNA sequencing were performed for confirmation. Only specimens which tested positive for both of the N and P genes by nested RT-PCR were scored as positive.

RESULTSIn 31 animals sampled in January 5 2004 before culling of wild animals at Guangdong Province, including 20 cats (Felis catus), 5 red fox (Vulpes vulpes) and 6 Lesser rice field rats (Rattus losea), 8 (25.8%) animals were tested positive for SARS-CoV like virus by RT-PCR methods, of which 4 cats, 3 red fox and one Lesser rice field rats were included. However, two weeks after culling of animals and disinfection of the market were implemented, in 119 animals sampled in January 20 2004, including 6 rabbits (Oryctolagus cuniculus), 13 cats, 46 red jungle fowl (Gallus gallus), 13 spotbill duck (Anas platyrhynchos), 10 greylag goose (Anser anser), 31 Chinese francolin (Franclinus pintadeanus), only rectal swab from one greylag goose was tested positive for SARS-CoV like virus. Furthermore, in 102 animals that including 14 greylag gooses, 3 cats, 5 rabbits, 9 spotbill duck (Anaspoecilorhyncha), 2 Chinese francolin (Franclinus pintadeanus), 8 common pheasant (Phasianus colchicus), 6 pigeons, 9 Chinese muntjac (Muntiacus reevesi), 19 wild boar (Sus scrofa), 16 Lesser rice field rats, 5 dogs, 1 mink (Mustela vison), 3 goats, 2 green peafowl (Pavo muticus) sampled in April, May, June, July, August and November, only rectal swab from one pig was tested positive. However, of 12 and 10 palm civets sampled in November and December including five of which had been at the live animals market for 2 days, none of them was tested positive.

CONCLUSIONThis findings revealed that animals being sampled in April, May, June, July, August and November of 2004, only one rectal swab from a pig was tested positive as SARS-CoV like virus, much lower than the results from the previous year, suggesting that the possibility of re-emerging of human infection from animal origins is low for the winter of 2004-2005.

Animals ; Animals, Wild ; virology ; China ; DNA, Viral ; analysis ; Felidae ; virology ; Reverse Transcriptase Polymerase Chain Reaction ; SARS Virus ; isolation & purification