A simple and sensitive Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) method was established to provide a new alternative for clinical diagnosis of Avian influenza A H5N1 virus. The method employed a set of six specially designed primers that recognized eight distinct sequences of the target for amplification of nucleic acid under isothermal conditions. In current study, fifty-one experimentally infected animal specimens and viral cultures that had been tested were analyzed by RT-LAMP for NA gene and HA gene, respectively. The amplification process of LAMP was monitored in real-time by the addition of SYBR Green dye. Meanwhile, the result showed high correlation between nested PCR and RT-LAMP. The specificity of the RT-LAMP assay was confirmed by restriction enzyme digestion analysis and sequencing of the amplified product. When the sensitivity of this assay was tested by serial 10-fold dilutions of RNA molecules from specimens, it was found that the RT-LAMP method achieved theoretically a sensitivity of 10 RNA molecules. Thus, we concluded that the RT-LAMP assay has potential usefulness for rapid detection of the Avian influenza A H5N1 virus.
Animals ; Birds ; Influenza A Virus, H5N1 Subtype ; genetics ; isolation & purification ; Influenza in Birds ; diagnosis ; virology ; Nucleic Acid Amplification Techniques ; methods ; Reproducibility of Results ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sensitivity and Specificity

OBJECTIVETo introduce the experience in the treatment of lower eyelid pouches orbital rim.

METHODSAn incision was made along the margin of lower eyelid and dissection was performed under the orbicularis muscle to expose the orbital septum and periosteum of lower orbital rim. The fat released from orbital septum was transposed just below the lower orbital rim and fixed on the periosteum. If lacrimal groove deformity was not corrected completely, the musculocutaneous flap, which may be excised beside the incision, was kept to correct the deformities further with only the muscle portion.

RESULTS72 cases with lower eyelid pouches complicated with lacrimal groove deformities were treated with transposition of orbital fat and orbicularis muscular flaps. Satisfactory results were achieved in all the patients after a follow-up period of 3-6 months.

CONCLUSIONIt is an effective and feasible technique to correct lacrimal groove deformities with transposition of orbital fat and orbicularis muscular flaps.

Adipose Tissue ; transplantation ; Aged ; Blepharoplasty ; methods ; Eyelids ; surgery ; Humans ; Orbit ; Periosteum ; surgery

OBJECTIVETo study the activity of esterases, including butyrylcholinesterase (BchE), carboxylesterase (CarbE), paraoxonase (PonE) and acetylcholinesterase (AChE), and to explore the effect of genetic polymorphism on the activity of esterase for workers exposed to organophosphorus pesticides (OPs).

METHODSTwo hundred and forty-one long term OPs directly exposed workers and 151 indirectly exposed workers in the same factory were taken as study group. One hundred and sixty unexposed persons were taken as control group. The activity of serum enzymes was measured and the polymorphic distribution was detected using 7900 genotype detecting system and CMOS Chip technique. The effect of long-term exposure to organophosphorus pesticides was analyzed.

RESULTSThe activities of BchE, CarbE and PonE were independent on the gender or age in control group. Average values of Carb and BchE activities of directly and indirectly exposed workers were lower than those in control group respectively. PonE activity in directly exposed group was lower than that in control group. AChE activity in directly exposed group was lower than that in indirectly exposed group. All the differences were significant (P < 0.01). In the direct exposure group, the frequency of three variants of butyrylcholinesterase gene K (BCHE-K) polymorphism was 74.3%, 24.1% and 1.6% for UU, UK and KK respectively. Frequency of allele U and K was 0.863 and 0.137 respectively in the same group. Frequency of three variants of PON192 polymorphism was 15.0%, 45.5% and 39.5% for AA, AB and BB respectively in direct exposure group. Gene frequency of low activity (PON*A) and high activity (PON*B) was 0.378 and 0.622 respectively. Frequency of three variants of PON55 polymorphism was 96.2%, 3.8% and 0% for MM, LM and LL respectively in direct exposure group. Frequency of allele M and L was 0.981 and 0.019 respectively in the same group. The activity of PON was different in various genotypes of PON192 and PON55.

CONCLUSIONThe long-term exposure to OPs could inhibit the activities of CarbE, BchE, PonE and ACh E in different level. The genetic polymorphisms of PON192 and PON55 affect the activity of PonE, which is related to the detoxification of OPs and health impact.

Acetylcholinesterase ; metabolism ; Adult ; Alleles ; Aryldialkylphosphatase ; genetics ; metabolism ; Butyrylcholinesterase ; genetics ; metabolism ; Carboxylesterase ; metabolism ; Female ; Gene Frequency ; Genotype ; Humans ; Male ; Middle Aged ; Occupational Exposure ; Organophosphorus Compounds ; adverse effects ; Pesticides ; adverse effects ; Polymorphism, Single Nucleotide

AIM:To study of hypertensive retinopathy and brachial ankle pulse wave velocity (baPWV) correlation.METHODS:Totally 89 cases 140 eyes of patients with hypertensive retinopathy in our hospital from June 2015 to December 2016 and 30 healthy subjects (60 eyes) were selected as the research objects,and were divided into the observation group and the control group,respectively.All of the subjects were examined by direct ophthalmoscope,and the results were classified according to Keith-Wagener four classification.Application of OMRON health care (China) Co.,Ltd.production of BP-203RPE network arteriosclerosis detection device for selected objects of baPWV detection.The levels of baPWV and the results of fundus examination were compared between the two groups,and the correlation between the two methods was analyzed.RESULTS:Detection of baPWV in patients was higher as the hypertension severity increased,there was statistically significant differences between groups (P＜ 0.05).The retinopathy grade was higher,the baPWV detection value was larger,the detection value of every grade was different statistically with others (P＜0.05).CONCLUSION:Elevated blood pressure is closely related to retinopathy and accelerated pulse wave velocity,relationship between the two values is notable,brachial ankle pulse wave velocity can be used to reflect the level of hypertensive retinopathy.

The full-length genome of one human bocavirus (HBoV) and the VP1 sequences of nine HBoV were amplified from patients' samples by PCR, cloned into pGEM-T vector separately, and sequenced. In this study, the one full length gemome and nine VP1 sequences of HBoV were aligened with 14 sequences of Parvoviruses which were canonical exemplars in Parvovirinae. Phylogenetic analysis showed that HBoV capsid sequences positioned closely to B19 parvovirus, although they positioned far in phylogenetic tree based on full length genome. Many similarities were found between HBoV and B19 in capsid by alignment on secondary structural elements. Because both B19 and HBoV are the only Parvoviruses that infect mankind, so study on HBoV may be used for reference to B19 which had been studied for about 30 years. By analysis of mutational sites, HBoV capsid protein showed a highly conserved secondary structural elements, but highly active in VP1-U, leading end of VP2 and insertions between the strands of the betaG-H. This cued that HBoV inclined to immune evasion and infectant adaptive faculty.
Amino Acid Sequence ; Base Sequence ; Bocavirus ; classification ; genetics ; Capsid Proteins ; chemistry ; genetics ; Cloning, Molecular ; Conserved Sequence ; Genome, Viral ; Humans ; Molecular Sequence Data ; Phylogeny ; Polymerase Chain Reaction

OBJECTIVETo explore the effects of bisphenol A (BPA) exposure on toxicity characteristic and OCT4 and SOX2 gene expression of mouse embryonic stem cells (mESC).

METHODSmESC were cultured, and treated with the doses of 10(-8), 10(-7), 10(-6), 10(-5), 10(-4) mol/L respectively of BPA and DMSO (the solvent control group)for 24 hours, and three groups of cells were treated with the same method. The morphological changes of mESC in the control and exposure groups were observed through an inverted microscope. Cell counting kit 8 (CCK8) was used to detect the effects of BPA on proliferation of mESC, and based on the results, the half inhibitory concentration (IC50) was calculated. Real-time fluorescent quantitative polymerase chain reaction (RT-QPCR) and western blotting were used to detect the expression of OCT4 and SOX2.

RESULTSBPA had certain toxicity on mESC, the treatment of BPA significantly increased cell toxicity in a concentration-dependent manner, and the IC50 was 4.3×10(-4) mol/L, combined with the BPA exposure concentration of the environment and the related literature, eventually taking the five concentrations of 10(-8), 10(-7), 10(-6), 10(-5), 10(-4) mol/L as the experimental groups. The mESC morphology were effected after the treatment of BPA for 24 h, compared with the control group, the number of cells decreased, appearing some floating cells, and the cell cloning became irregular and differentiation in the higher concentration groups. The OCT4 mRNA expression level in the 10(-7) mol/L (1.146 ± 0.087), 10(-6) mol/L (1.156 ± 0.030), 10(-5) mol/L (1.158 ± 0.103) and the 10(-4) mol/L (1.374 ± 0.053) dose group were all significantly higher than the control group (1.000 ± 0.000) (t values were -2.384, -2.953, -3.203, -4.021 respectively, P value all < 0.05). Meanwhile, the SOX2 mRNA expression level in the 10(-4) mol/L (1.113 ± 0.052) were higher than the control group (1.000 ± 0.000) (t value was -2.765, P value < 0.05). Moreover, the OCT4 protein expression level in the 10(-5) mol/L (1.360 ± 0.168) and 10(-4) mol/L (1.602 ± 0.151) were all significantly higher than the control group (1.000 ± 0.000) (t values were -3.538, -4.002 respectively, P value all < 0.05), while no obvious change of the SOX2 protein expression level was detected in all treated groups.

CONCLUSIONBPA in a certain dose range could upregulate the expression of OCT4 gene in mouse embryonic stem cells while had no significant effect on the expression of SOX2 gene.

Animals ; Benzhydryl Compounds ; toxicity ; Cells, Cultured ; Embryonic Stem Cells ; drug effects ; metabolism ; Gene Expression ; Mice ; Octamer Transcription Factor-3 ; genetics ; Phenols ; toxicity ; SOXB1 Transcription Factors ; genetics ; Signal Transduction ; drug effects

OBJECTIVEStudying on the routes of vas deferens to provide anatomy basis for surgical operation, especially, reconstruction of long segment loss of vas deferens.

METHODSThe routes of vas deferens were observed and anatomic distances along epididymal, infrainguinal, inguinal, retroperitoneal and ampullar segments of vas deferens, the distances from external ring to extremity of vas deferens were measured respectively in 18 formalin fixed adult cadavers.

RESULTSThe vas deferens have a large curve from external ring to extremity in its route, draw it out from the external ring. Eliminating this curve will allow to shorten this segment of vas deferens for vasovasostomy by 6.1 - 12.9 (9.31 +/- 1.30) cm. The length of each segment of vas deferens, respectively, is epididymal: 3.2 - 5.6 (4.53 +/- 0.79) cm, infrainguinal: 4.5 - 9.5 (7.31 +/- 1.78) cm, inguinal: 4.4 - 7.5 (5.52 +/- 0.74) cm, retroperitoneal: 12.5 - 19.5 (16.75 +/- 1.87) cm and ampullar: 2.9 - 3.8 (3.63 +/- 0.23) cm. There was no significant differences in segment length and the distances from external ring to extremity of vas deferens between the right and left.

CONCLUSIONReconstruction of long segment loss of vas deferens can be performed by mobilization retroperitoneal vas deferens and draw it out from external ring. There were no significant differences in lengths of vas deferens and the distances from external ring to vassal extremity between the left and right in adults. The surgical operations of vas deferens are closely related each segment of vasa.

Adult ; Autopsy ; Humans ; Male ; Vas Deferens ; anatomy & histology

OBJECTIVETo understand the genotypes of human metapneumovirus (hMPV) and the genetic character of hMPV attachment protein G sequence in Hunan, China.

METHODS232 nasopharyngeal aspirates (NPA) samples from hospitalized children with acute respiratory infections were collected from Hunan, China in 2005. HMPV was detected. The full length of G glycoprotein genes were amplified and sequenced. Bioinformatics soft-wares were employed to analyze the sequences.

RESULTS17/232 (7.3%) were showed hMPV positive. And co-infection rate with other viruses is 35%. The diagnoses of these hMPV positive cases are pneumonia, bronchiolitis and bronchopneumonia. Phylogenetic analysis for G genes from 13 hMPVs revealed the existence of four major subgroups: A1, A2, B1, B2 in Hunan, China in 2005. There are four types of sequence lengths of hMPV G glycoprotein, which are 711, 675, 660, 696nt. It is different in potential N-linked glycosylation sites and number of cysteine residues among these hMPVs of Hunan, China and Beijing, China. Also it is different from those in Japan and North America.

CONCLUSIONThe investigation of hMPV from Hunan, China in 2005 revealed the high speed of genetic variation and the marked character of geographic epidemic differences.

Amino Acid Sequence ; Child ; China ; epidemiology ; Genotype ; Glycoproteins ; classification ; genetics ; Humans ; Metapneumovirus ; classification ; genetics ; isolation & purification ; Molecular Sequence Data ; Phylogeny ; RNA, Viral ; genetics ; Respiratory Syncytial Virus Infections ; epidemiology ; virology ; Sequence Homology, Amino Acid ; Viral Proteins ; classification ; genetics

Purpose To investigate the effect of DAPPER1 on the malignant biological behavior in esophageal carcinoma cells,and further to analyze the expression and the possible regulated mechanism of DAPPER1 in esophageal squamous cell cancer (ESCC) samples.Methods MTT assay,colony formation assay and wound healing were used to examine the effect of DAPPER1 on malignant biological behavior in esophageal carcinoma cells,RT-PCR and MSP methods were applied respectively to examine the expression and the methylation status of DAP-PER1 in three ESCC cell lines (TE13,T.Tn,Eca109).Immunohistochemistry was used to examine the DAPPER1 protein expression.Results The negative or weak expression of DAP-PER1 was detected in ESCC cell lines.After treated with 5-Aza2'-deoxycytidine (5-Aza-dC,a demethylation agent),the expression level of DAPPER1 was obviously increased.Meanwhile,over expression of DAPPER1 or treated with 5-Aza-Dc could obviously inhibit proliferation and immigration abilities of TEl3 cell line.The level of DAPPER1 expression had no obviously change after treated with trichostatin A (TSA).Decreased protein expression of DAPPER1 was observed in ESCC tumor tissues compared with non-cancerous tissues (P ＜ 0.01),and associated with the methylation status of this gene (P ＜ 0.01).Conclusion DAPPER1 plays an important role of tumor suppressor gene in esophageal carcinoma,and the aberrant hypermethylation may be one of the main mechanisms in abnormal expression of this gene.

OBJECTIVETo observe the therapeutic effect of eye-needling combined with medication for treatment of ophthalmoplegia and explore the possible mechanism.

METHODSOne hundred and twenty cases were randomly divided into a treatment group and a control group. According to etiological factors, the control group were treated with medication and the treatment group with the medication plus eye-acupuncture at main point ocular muscles. Changes of the rima oculi, the range of ocular movement and the dialopia angle after treatment were recorded and statistically analyzed in the two groups.

RESULTSThe total effective rate was 93.4% and the cured rate was 54.1% in the treatment group, and 74.6% and 18.6% in the control group, with significant difference between the two groups (P < 0. 01).

CONCLUSIONEye-needling combined with medication has an obvious therapeutic effect which is better than simple medication for ophthalmoplegia.

Acupuncture Therapy ; methods ; Adolescent ; Adult ; Aged ; Child ; Combined Modality Therapy ; Female ; Humans ; Male ; Middle Aged ; Ophthalmoplegia ; therapy