A simple, rapid and sensitive colorimetric loop mediated isothermal amplification (LAMP) method was established to detect HPV6 and HPV 16 respectively. The method employed a set of four specially designed primers that recognized six distinct sequences of HPV6-E6 or HPV16-E7 for amplification of nucleic acid under isothermal conditions at 63 degrees C for one hour. The amplification process of LAMP was monitored by the addition of HNB (hydroxy naphthol blue) dye prior to amplification. A positive reaction was indicated by a color change from violet to sky blue and confirmed by real-time turbidimeter and agarose electrophoresis. Thirteen cervical swab samples having single infection with 13 different HPV genotypes were examined to evaluate the specificity. A serial dilution of a cloned plasmid containing HPV-E6 or HPV-E7 gene was examined to evaluate the sensitivity. The results showed that no cross-reaction with other HPV genotypes was observed. The colorimetric LAMP assay could achieve a sensitivity of 1000 copies, 10-20 times lower than that of real-time PCR. The assay was further evaluated with 62 clinical specimens and consistent results were obtained compared with the detection using Kai Pu HPV Genotyping Kit. We concluded that this colorimetric LAMP assay had potential usefulness for the rapid screening of the HPV6 or HPV16 infection in the laboratories and hospitals of provincial and municipal region in China.
Colorimetry ; methods ; DNA Primers ; chemistry ; genetics ; Genotype ; Human papillomavirus 16 ; genetics ; isolation & purification ; Human papillomavirus 6 ; genetics ; isolation & purification ; Humans ; Nucleic Acid Amplification Techniques ; instrumentation ; methods ; Papillomavirus Infections ; virology

OBJECTIVETo determine the origin of Rana temporaria for quality Oviduetus Ranae in the light of historical documents and modern researches on the classification of Rana temporaria chensinensis.

METHODWorks of Chinese meteria medica of all ages, related historical documents and reports from home and abroad on researches of R. temporaria chensinensis were consulted, sorted out, analyzed and summarized.

RESULTThe original Shange recorded in the works of Chinese meteria medica is R. temporaria chensinensis, which is the independent species, not one of species of European forest frogs. R. temporaria chensinensis is divided into 4 subspecies: R. temporaria chensinensis, Lanzhou, Kangding, and Changbaishan. The origin of R. temporaria is Changbaishan subspecies of R. temporaria chensinensis.

CONCLUSIONChangbaishan subspecies of R. temporaria chensinensis is determined as the origin for quality Oviduetus Ranae.

Animals ; Female ; History, 18th Century ; History, 19th Century ; History, 20th Century ; History, Ancient ; Materia Medica ; history ; isolation & purification ; Oviducts ; chemistry ; Rana temporaria ; anatomy & histology ; Ranidae ; anatomy & histology ; classification ; Species Specificity ; Terminology as Topic

Child, Preschool ; Female ; Humans ; Male ; Mucormycosis ; drug therapy ; etiology ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; complications ; Remission Induction

OBJECTIVETo evaluate the clinical efficacy of sufentanil and fentanyl at equivalent dose for patient-controlled intravenous analgesia (PCIA) after thoracotomy.

METHODSSixty ASA I-II patients (20-60 years of age) undergoing radical operation for lung or esophageal cancer were randomly divided into sufentanil intravenous analgesia group (group S, with sufentanil 1 microg/ml) and fentanyl intravenous analgesia group (group F, fentanyl 10 microg/ml). PCIA was administered with background infusion of 2.5 ml/h, bolus injection of 2.5 ml and lockout time of 15 min. The pain intensity according to visual analogue scale (VAS), cumulative analgesic consumption (CAC), sedative scores and side effects at 24 and 48 h after administration were recorded. SpO(2), respiratory rate (RR), blood pressure (BP) and ECG were continuously monitored.

RESULTSThere were no significant differences in CAC between the two groups, but he VAS was lower in group S than in group F (P<0.05) and the sedative efficacy was superior in group S (P<0.05). The incidence of nausea and vomiting in group S was lower than that in group F (P<0.05). No significant differences were observed in SpO(2), RR, heart rate and mean arterial pressure between the two groups.

CONCLUSIONPCIA with sufentanil provides better efficacy of analgesia and sedation with lower incidence of nausea and vomiting than with fentanyl in postoperative patients with thoracotomy.

Adult ; Analgesia, Patient-Controlled ; Esophageal Neoplasms ; surgery ; Female ; Fentanyl ; administration & dosage ; adverse effects ; Humans ; Infusions, Intravenous ; Lung Neoplasms ; surgery ; Male ; Middle Aged ; Nausea ; chemically induced ; Pain, Postoperative ; drug therapy ; Sufentanil ; administration & dosage ; adverse effects ; Thoracotomy ; Vomiting ; chemically induced

OBJECTIVETo establish a new and rapid GeXP based multiplex PCR assay for the detection and typing of human papillomavirus 6, 11, 31, 33 and 52.

METHODSNucleotide sequences of HPV6, HPV11, HPV31, HPV33 and HPV52 from NCBI were obtained and compared. Genotype-specific primers were then designed and the sensitivity and specificity of multiple PCR assay was evaluated. Optimized assay was further validated with 30 clinical specimens collected from the cervical secretions of patients.

RESULTSA GeXP based multiplex PCR was developed for sensitive detection and reliable differentiation of five HPV genotypes (HPV6, 11, 31, 33 and 52),

CONCLUSIONA GeXP based multiplex PCR assay is demonstrated to be a new and rapid technique for simultaneous detection and typing of 5 different human papillomaviruses.

Adult ; Female ; Humans ; Middle Aged ; Papillomaviridae ; classification ; genetics ; isolation & purification ; Polymerase Chain Reaction ; methods

Purpose: To evaluate the use of signal intensity index (SII) of skull-base invasion in nasopharyngeal carcinoma (NPC) using magnetic resonance imaging (MRI), select a best cut-off SII value to predict the outcome of NPC. Materials and Methods: One hundred and twenty-two NPC patients (92 men, 30 women) with skull-base invasion were included. All patients underwent MRI, signal intensities on T1-weighted imaging (T1WI) were measured for each invaded site and its contralateral normal counterpart. The SIIs were calculated, receiver operating characteristic curves were constructed. The optimal cut-off values were extracted. The overall survival (OS) rates of 5-year follow-up were performed. Results: Sensitivities for differentiating skull-base invasion from normal contralateral anatomy were 98.9%, 88.5% and 70.0%, and specificities were 98.9%, 96.0% and 74.4%, respectively. There were three cut-off values for differentiating invasion from normal anatomy of skull-base, 49%, 98%, and 60%. Significant difference in OS rates (84.2% vs. 57.1%, p=0.007) was seen for SII threshold values > 60% and those ≤ 60%. Conclusions: The SII might be a useful means of differentiating invasion from normal tissue at the skull-base in NPC. The cut-off value of quantitative SII at the skull-base may aid in monitoring the response to treatment of NPC patients.

Objective We aimed to assess the association between urinary bisphenol A(BPA)concentrations and gestational age in pregnant women. Methods A total of 248 pregnant women were recruited from a maternal and child care hospital in Shanghai. A questionnaire survey was completed to collect socio-demographic information and spot urine samples were collected during pregnancy. Gas chromatography-tandem mass spectrometry(GC-MS/MS)was used to measure BPA concentrations in urine samples. Linear relationship between urinary BPA level and gestational age was assessed by using generalized additive models. Multivariate regression model was used to evaluate associations of prenatal BPA exposure with gestational age. Results BPA was detected in all the urine samples. Median value and geometric mean of urinary BPA levels were 0.85 μg/L and 1.21 μg/L, respectively. Linear relationship between urinary BPA concentration and gestational weeks was confirmed(non-linear P > 0.05). Positive association between urinary BPA level and gestational age was indicated(regression coefficient, β = 0.19;95%CI:0.04-0.35;P = 0.016). However, it was only observed in girls, stratified by sex of newborns(β = 0.18;95%CI:0.03-0.34;P = 0.020). After stratification by trimester, no significant association was found in the second or the third trimesters. Conclusion Pregnant women are extensively exposed to BPA. Urinary BPA exposure during pregnancy may extend gestational age, especially in girls.

OBJECTIVETo analyze the relationship between cutaneous glycometabolic disorders and cutaneous neuropathy in diabetic rats, and to look for the mechanism of neuropathy and impaired wound healing.

METHODSEighty male SD rats were randomly divided into the normal control group (NC, n = 20), diabetic group (D, n = 20), aminoguanidine-interfered group (AI, n = 20), and insulin-interfered group (II, n = 20) by drawing lots. Diabetes was reproduced in rats of D, AI, and II groups with intraperitoneal injection of streptozotocin (STZ). Then, rats in AI group were fed with 100 mg×kg(-1)×d(-1) aminoguanidine, while rats in II group were subcutaneously injected with insulin for satisfactory control of blood glucose. Changes in mechanical and heat pain thresholds of pad of hind limb were measured at post injection week (PIW) 2, 4, 8. Skin specimens were collected during PIW 2-8 from pads for determination of contents of glucose, advanced glycation end product (AGE), substance P (SP), calcitonin gene-related peptide (CGRP), and observation of distribution and ultrastructure of skin nerve fibers. Data were processed with t test.

RESULTSThe mechanical and heat pain thresholds in D group at PIW 2 [(6.3 ± 1.5) g, (6.0 ± 0.9) s, respectively ] were obviously lower than those in NC group [(13.0 ± 3.2) g, (10.3 ± 1.2) s, with t value respectively 2.71, 3.42, P values all below 0.05]. Contents of glucose and AGE in skin tissue in D group were significantly increased when compared with those in NC group, especially at PIW 8 [(2.85 ± 0.33) mg/g, (31.7 ± 3.2) U/mg of hydroxyproline vs. (0.82 ± 0.22) mg/g, (22.2 ± 1.9) U/mg of hydroxyproline, with t value respectively 1.65, 6.47, P values all below 0.01]. The myelinated nerve fibers were edematous and degenerated, with axons compressed, while the unmyelinated nerve fibers were vacuolated, with microfilament and microtubule disorderly arranged. Content of SP in skin tissue in D group was lower as compared with that in NC group, especially at PIW 2 [(16.8 ± 3.4) pg/g vs. (28.5 ± 5.0) pg/g, t = 2.42, P < 0.01]. There was no obvious difference in content of CGRP between NC and D groups, and also in content of glucose in skin between D and AI groups. Compared with those in D group, content of AGE in AI group at PIW 8 was decreased markedly [(27.2 ± 1.4) U/mg of hydroxyproline, t = 3.38, P < 0.05]; contents of glucose and AGE in II group at PIW 8 were significantly decreased [(1.42 ± 0.38) mg/g, (23.6 ± 1.3) U/mg of hydroxyproline, with t value respectively 1.74, 8.17, P < 0.05 or P < 0.01]. Compared with that in D group, contents of SP in AI and II groups were increased, with a delay in time of trough value. Content of CGRP showed no obvious difference among D, AI, and II groups.

CONCLUSIONSHigh glucose and accumulation of AGE are key mediators of cutaneous neuropathy and impaired wound healing in diabetes mellitus, which confirms that diabetic wound takes an atypical footing during wound repairing. Aminoguanidine and insulin can reduce contents of glucose and AGE in diabetic skin tissue, and ameliorate diabetic cutaneous neuropathy.

Animals ; Diabetes Mellitus, Experimental ; complications ; metabolism ; Glucose ; metabolism ; Glycation End Products, Advanced ; metabolism ; Male ; Peripheral Nervous System Diseases ; etiology ; Rats ; Rats, Sprague-Dawley ; Skin ; metabolism ; pathology ; Skin Diseases ; etiology ; Wound Healing

MUC1 mucin is a high molecular weight, type I transmembrane glycoprotein. High and aberrant expression of MUC1 is observed in various types of tumors, which make it an ideal target for tumor biotherapy as well as a biomarker for tumor diagnosis and prognosis. MUC1/Y is an isoform of MUC1 generated by alternative splicing. Specific expression of MUC1/Y in breast cancer as well as its involvement in tumor cell signal transduction have been reported. In order to purify peptides containing MUC1/Y-specific epitope in E. coli and prepare MUC1/Y-specific antibody, DNA fragment encoding the MUC1/Y-specific peptide was amplified by PCR using MUC1/Y full length cDNA as the template and cloned into fusion expression vector pGEX-2T, resulting pGEX-Y30. DNA sequencing was performed to confirm the correct amplification and orientation of the target sequence. Competent E. coli DH5alpha was transformed with pGEX-Y30 and the expression was induced for 4-5 hours in 0.2 mmol/L IPTG at 30 degrees C and 37 degrees C. Expressed proteins were released from the cells by ultrasonication or B-PER II reagent treatments. The fusion protein GST-Y30 were purified by affinity and anion exchange columns and identified by SDS-PAGE and Western-blotting. Polyclonal antibody was prepared by immunizing rabbits with the GST-Y30 protein for 4 times with intervals of 3 weeks and purified by GST column. Western blotting, ELISA and immunohistochemistry analysis were carried out using the purified antibody to confirm its MUC1/Y-binding capacity and specificity. The expressed fusion protein GST-Y30 is about 31 kD in size and represented about 20% of total cellular proteins. The majority of the GST-Y30 protein existed as soluble form when the induction was carried out at both 30 degrees C and 37 degrees C. After the two-step purification, the purity of GST-Y30 was about 94%. The titer of polyserum generated by GST-Y30 immunization was 1:320,000 by ELISA. The antiserum showed MUC1/Y specificity and can recognize MUC1/Y on MCF7 cell. The MUC1/Y-specific polyclonal antibody can be used for studying the role of MUC1/Y in carcinogenesis.
Animals ; Antibodies ; metabolism ; Antibody Specificity ; Cell Line, Tumor ; metabolism ; Epitopes ; chemistry ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Humans ; Immunohistochemistry ; Models, Genetic ; Mucin-1 ; chemistry ; genetics ; metabolism ; Peptides ; chemistry ; genetics ; metabolism ; Rabbits

OBJECTIVETo investigate the cerebral distribution of propofol during continued infusion at a constant rate when the cerebral propofol uptake reaches equilibrium in dogs.

METHODSSix healthy 1-year-old male dogs were used in this study. The venous channel was established in the great saphenous vein of the right posterior limb. Anesthesia was induced with a single bolus injection of propofol (7 mg/kg), followed by propofol infusion at a constant rate of 70 mg/(kg.h) using a microinfusion pump. The blood samples were taken from the right internal carotid and internal jugular vein at 30 min (T30) and 50 min (T50) during propofol infusion for measurement of plasma propofol concentrations with high performance liquid chromatography (HPLC). At T50, the frontal lobe, parietal lobe, temporal lobe, hippocampus, cingulate gyrus, thalamus, midbrain, pons, and cerebellum were dissected respectively for determination of propofol concentrations.

RESULTSPropofol concentrations in the internal carotid artery and internal jugular vein blood plasma were 3.107-/+1.067, 3.095-/+1.085 microg/ml at T30 and 3.091-/+1.101, 3.117-/+1.091 microg/ml at T50, respectively, showing no significant differences (P>0.05). Propofol concentrations in the frontal lobe, parietal lobe, temporal lobe, hippocampus, cingulate gyrus, thalamus, midbrain, pons, cerebellum at T50 were 3.085-/+1.123, 3.116-/+1.125, 3.073-/+1.159, 3.117-/+1.090, 3.075-/+1.178, 3.073-/+1.146, 3.075-/+1.151, 3.102-/+1.174, and 3.072-/+1.192 microg/g respectively, suggesting homogeneous propofol distribution in these cerebral tissues (P>0.05).

CONCLUSIONAt T50, the cerebral uptake of propofol reached equilibrium when propofol is distributed homogeneously in the cerebral tissues in dogs.

Anesthetics, Intravenous ; administration & dosage ; blood ; pharmacokinetics ; Animals ; Brain ; metabolism ; Carotid Artery, Internal ; metabolism ; Chromatography, High Pressure Liquid ; Dogs ; Infusions, Intravenous ; Jugular Veins ; metabolism ; Male ; Propofol ; administration & dosage ; blood ; pharmacokinetics ; Tissue Distribution