Purpose To investigate the occurrence of echinoderm microtubule-associated protein-like 4 (EML4) gene and the anaplas-tic lymphoma kinase (ALK) gene rearrangements in lung squamous cell carcinomas (SCC), and to provide with a test for targeted therapy of SCC. Methods In this study, we analyzed ALK protein expression with a specific rabbit monoclonal Ig antibody ( D5F3 clone) in 219 cases of lung SCC. The positive cases were confirmed with ALK fluorescence in situ hybridization ( FISH) . Results 4 out of 219 (1. 8%) cases of lung SCC were ALK positive detected by immunohistochemistry (IHC) staining, which were confirmed by ALK FISH. Conclusions There are SCC having EML4-ALK gene rearrangement. The response of SCC patients with ALK expression to targeted therapy of Crizotinib should be explored. Strong positive expression of ALK protein can be interpreted as EML4-ALK gene rearrangement.

To establish HPLC specific chromatogram and its correlation with the protection effect of Shuanghuanglian on MDCK (Madin-Darby canine kidney) cell injury induced by influenza A virus( H1N1). Nine recipes of Shuanghuanglian based on the official prescription were prepared according to orthogonal test for HPLC analysis and MDCK cells protection experiment separately (cytopathic effect (CPE) method was used for observing the virus infectivity and MTT staining results were used as the determining indexes for drug concentration selection and analyzing cell viability). The results suggested that all the other Shuang-Huang-Lian recipes except recipe1 demonstrate protecting effect on MDCK cell injury induced by influenza A virus (P < 0.01, P < 0.001). Stepwise regression analysis was used for analyzing the relationships between HPLC fingerprint and the protecting effect of Shuanghuanglian on influenza A virus induced MDCK cell injury. Peak 2, 3, 6, 8 and 12 were found to be strongly related with anti-influenza A virus efficacy. Stepwise regression analysis of recipes data and efficacy data showed that Lonicerae Japonicae Flos and Forsythiae Fructus were positively associated with the protecting effect of cells injury. From HPLC fingerprints, we found that peak 2, 3, 12 were from Lonicerae Japonicae Flos and peak 6, 8 were from Forsythiae Fructus. Four peaks were identified through comparing the retention time between the standard and Shuanghuanglian recipes, and they were chlorogenicacid, cryptochlorogenic acid, forsythoside B and 3,4-dicaffeoylquinic acid respectively. Caffeic acid derivatives in Lonicerae Japonicae Flos and Forsythiae Fructus were found to be greatly correlated with anti-influenza A virus efficacy and maybe the substance basis of Shuanghuanglian.
Animals ; Antiviral Agents ; analysis ; pharmacology ; Chromatography, High Pressure Liquid ; methods ; Dogs ; Drugs, Chinese Herbal ; analysis ; pharmacology ; Forsythia ; chemistry ; Influenza A Virus, H1N1 Subtype ; drug effects ; physiology ; Lonicera ; chemistry ; Madin Darby Canine Kidney Cells ; Scutellaria baicalensis ; chemistry

The present experiment was designed to observe the genetic variation of equine infectious anemia virus (EIAV) envelop gp 90 gene in infected horse. One horse was infected experimentally with P337-V70 strain and showed no clinical signs after being infected at twice with the same virus strain. Seventeen proviral sequences covering principal neutralizing domain (PND) of EIAV gp 90 gene were obtained from the buffy coat and liver of the horse through PCR amplification and cloning. Comparative analysis of the sequences revealed that some sequences contained the nucleotide insertions in the PND region. The insertions might be generated by direct repeat and strand displacement of sequence segment in their PND gene, showing different lenghts.

OBJECTIVEOver the last few decades, diabetic cardiomyopathy has been identified as a significant contributor in cardiac morbidity. However, the mechanisms of diabetic cardiomyopathy have not been clarified.

METHODSIn the present study, a diabetic rat model was induced by the intraperitoneal injection of streptozotocin. The myocardial CD147 expression and extent of glycosylation, as well as thematrixmetalloproteinases(MMPs) expression and activity, were observed in the diabetic and synchronous rats.

RESULTSThe results showed that CD147 located on sarcolemma of cardiomyocytes. The myocardial CD147 expression and glycosylation were significantly increased in the diabetic rats as compared with the control. Expression of MMP-2 protein, MMP-2 and MMP-9 activity were also increased in left ventricular myocardium in the diabetic rats. Tamoxifen only inhibited the enhanced expression of myocardial CD147 in the diabetic rats, but not in synchronous control rats. Tamoxifen inhibited glycosylation of myocardial CD147 in both diabetic and control rats. The inhibition of tamoxifen on CD147 glycosylation was stronger than on the expression in the myocardium. The extent of myocardial CD147glycosylation was positively related toMMP-2 and MMP-9 activity. Tamoxifen induced an inhibition of myocardial MMP-2 and MMP-9 activity in the control and diabetic rats.

CONCLUSIONThese results indicate that myocardial CD147 expression, especially the extent of glycosylation, regulates MMP-2 and MMP-9 activity, then accelerates cardiac pathological remodeling inducing diabetic cardiomyopathy. Tamoxifen inhibits myocardial CD147 glycosylation and further depress the activity of MMPs. Therefore, tamoxifen may protect the diabetic rats against diabetic myocardium.

Animals ; Basigin ; metabolism ; Diabetes Mellitus, Experimental ; complications ; Diabetic Cardiomyopathies ; drug therapy ; Glycosylation ; Heart ; drug effects ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Myocardium ; metabolism ; Myocytes, Cardiac ; cytology ; Rats ; Sarcolemma ; metabolism ; Tamoxifen ; pharmacology

Objective Congenital muscular torticollis (CMT) and developmental dysplasia of the hip (DDH) are common congenital problems in infants. Association between CMT and DDH has been reported in literatures. The aim of this study was to assess coexistence of DDH in infants with CMT and curative effects of its rehabilitative therapy. Methods In total, 187 infants less than two months old with CMT were recruited in the study. DDH was diagnosed and graded by Graf's bilateral hip type B ultrasonography and pelvic X-ray films. Massage manipulation, magnetic strapping, postural orthosis at home and heat compress were instituted for CMT and frog spica device and hip-flexion abduction plaster immobilization were instituted for DDH. Their early rehabilitative effects were evaluated. Results DDH was coexisted in 24 of 187 infants with CMT, with incidence of 12. 8%. The hips of 22 infants were graded as type Ⅱ b (91.7%) and two as type Ⅲ a (8.3%), and eight at the left side and seven at the right side.Comorbid hips completely recovered normal with early frog spica device and hip-flexion abduction therapy in 24 infants. Conclusions Coexistence of CMT and DDH are relatively common in infants. Type B ultrasonography can be used as a measure for screening DDH in neonates with CMT. Early rehabilitative therapy is effective for those coexisted with DDH and CMT at the same time.

The aims of the study were to investigate the incidence of C-shaped root canal systems in mandibular second molars in a native Chinese population using radiography and clinical examination under microscope and to compare the relative efficacies of these methods. For the recognition of C-shaped root canal system, 1 146 mandibular second molars were selected and examined. Teeth with C-shaped canal systems were categorized by using the radiographic classification criteria and the modified Melton's method. C-shaped canals were identified in 397 (34.64%) mandibular second molars by radiography (type I, 31.23%; type II, 38.29%; type III, 30.48%). Clinical examination showed that 449 (39.18%) cases exhibited C-shaped canal systems (C1, 22.94%; C2, 48.11%; C3a, 15.59%; C3b, 13.36%). As for the result of the radiographic and clinical combined examination, C-shaped root canals were found in 473 (41.27%) mandibular second molars (C1, 21.78%; C2, 45.67%; C3a, 16.70%; C3b, 15.86%). The incidence of C-shaped root canal diagnosed by radiographic method was statistically different from that by clinical examination and the combined examination (P<0.05). The study indicated a high incidence of C-shaped canal system in a Chinese population. The combination of microscopic and radiographic examination is an effective method in identifying the C-shaped root canal system.
Adult ; Aged ; Anatomic Variation ; Asian Continental Ancestry Group ; Chi-Square Distribution ; China ; Dental Pulp Cavity ; anatomy & histology ; diagnostic imaging ; Female ; Humans ; Male ; Mandible ; Microscopy ; Middle Aged ; Molar ; anatomy & histology ; Radiography, Dental ; Tooth Root ; anatomy & histology ; Young Adult

OBJECTIVETo explore the role of autophagy in doxorubicin (DOX)-induced resistance of human myeloma cell line RPMI8226.

METHODSWe established doxorubicin induced resistant subline of myeloma cell line RPMI8226/DOX by drug concentration step-elevation method. Resistant index of DOX was measured by MTT assay. Autophagy of myeloma cell lines RPMI8226/s and RPMI8226/DOX was detected by transmission electron microscopy, immunofluorescence (LC3-FITC) and western blot respectively. Apoptosis of RPMI8226/DOX cells induced by DOX combined with autophagic inhibitor hydroxychloroquine or 3-MA was identified by AnnexinV-FITC/PI double fluorescence dyeing.

RESULTSResistant index of RPMI8226/DOX was approximately 10.8 fold of that of RPMI8226/S. Electron microscopic studies revealed that most of RPMI8226/DOX cells displayed viable attributes and contained numerous autophagic vacuoles. Fluorescent images of RPMI8226/DOX cells showed a punctuate distribution in LC3 protein. Increased LC3-II protein in RPMI8226/DOX cells was determined by immunoblotting. There were no differences among 8 μmol/L HCQ (3.24±1.08)%, 10 mmol/L 3-MA (2.81±0.80)% or control \[(2.12±1.24)%\] (P>0.05) in terms of AnnexinV-FITC/PI double fluorescence dyeing; Compared with apoptosis of (9.75±2.15)%, (24.36±2.16)% and (40.51±3.14)% of RPMI8226/DOX cells under 2, 4 and 6 μmol/L DOX, apoptosis increased significantly after 24 h incubation under 2, 4 and 6 μmol/L DOX combined with 8 μmol/L HCQ as of \[(16.56±1.89)%, (36.44±2.91)% and (62.68±3.75)%, respectively\], or under 2, 4 and 6 μmol/L DOX combined with 10 mmol/L 3-MA as of \[(15.47±1.85)%, (39.28±3.06)% and (55.46±4.07)%, respectively\] (P<0.05).

CONCLUSIONAutophagy was involved in doxorubicin-induced resistance of myeloma cell line RPMI8226, DOX resistance in myeloma cells was reversed partly by autophagy inhibitor hydroxychloroquine or 3-MA, and autophagy may be one of mechanisms for drug resistance.

Autophagy ; drug effects ; Cell Line, Tumor ; Doxorubicin ; pharmacology ; Drug Resistance, Neoplasm ; Humans ; Multiple Myeloma

BackgroundAn ideal animal model is very important for the investigation of the immune mechanism of high-risk rejection corneal transplantation.ObjectiveThis study was to compare three methods of creating a high-risk corneal transplantation model in rabbits to study high-risk rejection corneal transplantation.MethodsForty-five New Zealand white rabbits were utilized and assigned randomly to three groups of different modeling methods,with 15 rabbits for each group.The high-risk corneal transplantation models were created by suturing with 5-0 silk thread in 4 quadrants,inducing alkali burn with 1 mol/L NaOH or corneal xenotransplantation.In the suturing group and alkali burning group,the rabbits received a unilateral 7.25 mm diameter corneal allograft after corneal neovascularization was induced,and in the xenotransplantation group,corneas from cats were used as donors.Rabbits were followed-up for 4 weeks in all groups.Corneal neovascular area was calculated and compared among the three groups.The amount of rejection,inflammatory index ( IF),neovascularization and histology of grafts were clinically scored to calculate the reject index (RI).ResultsThere were 14,15 and 15 rabbits that survived the high-risk penetrating corneal transplantation,respectively,in the suturing group,alkali burning group and xenotransplantation group.Two weeks after operation,the IF scores were 0.543 ± 0.103,0.811 ± 0.054 and 0.191 ±0.087,and the RI were 2.111±0.928,7.0±0.816 and 3.182±0.751 in the suturing group,alkali burning group and xenotransplantationgroup,respectively,showingstatisticallysignificantdifferencesamongthethreegroups (x2 =25.736,22.432,P =0.000).The IF value was lower in the xenotransplantation group compared with the suturing group and alkali burning group (Z =3.841,3.993,P =0.000),and that of the suturing group was lower than the alkali burning group (Z =3.568,P =0.000).The RI value of the xenotransplantation group was significantly raised in comparison with the suturing group and declined in comparison with the alkali burning group (Z =2.373,P =0.018;Z =3.936,P =0.000),and that of the suturing group was lower than the alkali burning group (Z =3.729,P =0.000 ).The survival times of the grafts were ( 17.9±2.0 ) days,( 13.4 ±2.4) days and ( 15.5 ±2.0 ) days in these three groups with a significant difference among them ( F =9.474,P =0.001 ).The neovascularization area in the xenotransplantation group was smaller than the suturing group and alkali burning group (P＜ 0.05 ).Histological examination revealed a large number of inflammatory cells infiltration in the grafts 2 and 4 weeks after transplantation in the suturing group and alkali burning group,but less inflammatory cells were seen in the xenotransplantaion group.Immunofluorescence staining showed abundant CD4+ T positive cells in the grafts in the three groups.Conclusions The cat-rabbit corneal xenotransplantation can induce stable and moderate immune rejection.This animal model has milder inflammatory response and less corneal neovascularization than the suture and alkali burn models.This method therefore is an ideal model for high-risk corneal transplantation.

Ligustrazine, one of the major effective components of the Chinese traditional medicinal herb Ligusticum Chuanxiong Hort, has been reported plenty of biological activities, such as protect cardiovascular and cerebrovascular, neuroprotection and anti-tumor, et al. Because of its remarkable effects, studies on structural modification of ligustrazine have attracted much attention. Ligustrazine synthetic derivatives reported in recent decades are mainly derived from four primary intermediates (TMP-COOH, TMP-OH, TMP-NH2, HO-TMP-OH). To explore the neuroprotection activitiy of ligustrazine intermediates, six ligustrazine intermediates (2, 5, 8, 11, 12, 13) were synthesized and their protective effects against CoCl2-induced neurotoxicity in differentiated PC12 cells were studied. The target compounds were prepared via different chemical methods, including oxidation, substitution, esterification and amidation without changing the structure nucleus of ligustrazine. Compared with TMP (EC50 = 56.03 micromol x L(-1)), four compounds (2, 5, 12 and 13) exhibited higher activity (EC50 < 50 micromol x L(-1)) respectively, of which, compound 2 displayed the highest protective effect against the damaged PC12 cells (EC50 = 32.86 micromol x L(-1)), but target compounds 8 and 11 appeared lower activity (EC50 > 70 micromol x L(-1)). By structure-activity relationships analysis, the introduction of carboxyl, amino to the side chain of ligustrazine and appropriately increase the proportion of ligustrazine may contribute to enhance its neuroprotective activity, which provides a reference for the design, synthesis and activity screening of relevant series of ligustrazine derivatives in the future.
Animals ; Cell Differentiation ; drug effects ; Chemistry Techniques, Synthetic ; Cobalt ; toxicity ; Drugs, Chinese Herbal ; chemistry ; Neuroprotective Agents ; chemical synthesis ; chemistry ; pharmacology ; Neurotoxins ; toxicity ; PC12 Cells ; Pyrazines ; chemical synthesis ; chemistry ; pharmacology ; Rats

BACKGROUND: Altered mental status (AMS) is a very common emergency case, but the exact etiology of many AMS patients is unknown. Patients often manifest vague symptoms, thus, AMS diagnosis and treatment are highly challenging for emergency physicians. The aim of this study is to provide a framework for the assessment of AMS patients. This assessment should allow providers to better understand the etiology of mental status changes and therefore improve diagnostic skills and management. METHODS: This is a prospective cohort observational study. We recruited all adult patients with undifferentiated AMS at a single center tertiary care academic emergency department over 24 months (June 2009 to June 2011). Demographic characteristics, clinical manifestations, assessment approaches, causative factors, emergency treatments and outcomes were collected prospectively. RESULTS: In 1934 patients with AMS recruited, accounting for 0.93％ of all emergency department (ED) patients, 1026 (53.1％) were male, and 908 (46.9％) female. Their average age was 51.95±15.71 years. Etiologic factors were neurological (n=641; 35.0％), pharmacological and toxicological (n=421; 23.0％), systemic and organic (n=266; 14.5％), infectious (n=167; 9.1％), endocrine/metabolic (n=145; 7.9％), psychiatric (n=71; 3.9％), traumatic (n=38; 2.1％), and gynecologic and obstetric (n=35; 1.9％). Total mortality rate was 8.1％ (n=156). The death rate was higher in elderly patients (≥60) than in younger patients (10.8％ vs. 6.9％,P=0.003). CONCLUSIONS: Patients with AMS pose a challenge for ED physicians. The most frequently encountered diagnostic categories causing AMS were primary CNS disorders, intoxication, organ system dysfunction, and endocrine/metabolic diseases. AMS has a high fatality rate in the ED. AMS is an important warning signal for ED patients because of its potentially fatal and reversible effects. Prompt evaluation and treatment are essential to decreasing morbidity and mortality associated with AMS.