Objective: To establish a multiplex PCR-microarray method for detecting important enteropahogens.Methods: Uniplex and multiplex PCR were performed to obtain the best primer sets for identifying the target bacteria at species and multi-species level.Fluorescent dyes were mixed into PCR reaction to determine whether it can affect the efficiency of amplification.To improve the efficiency of microarray,a 35 pairs primer-labeling system was optimized based on the hybridization results to find the best combination to avoid false negative results.Results: Specific PCR products were all obtained using species-specific primer sets.More preferential amplification may happen when more primer pairs were added to the reaction.The hybridization results showed a positive association between the efficiency of multiplex-PCR and signal intensity.Conventional PCR yielded more products than fluorescent dyes labeled PCR.Thirty-five primers were divided into three different combinations to label target respectively,hybridization results showed a high specificity.Conclusion: Mixing fluorescent dyes into PCR may reduce the efficiency of amplification and hybridization,but may have no effect on the analysis of hybridization results.The hybridization efficiency of microarray depends on the amplification efficiency of multiplex PCR.For microarray target labeling,three primer sets could be used to avoid negative hybridization led by preferential amplification of multiplex-PCR.It indicates that the multiplex PCR-microarray method is an attractive diagnosis tool for the high-throughput identification of enteropathogenic organisms especially for multiple causative agents and epidemiological investigations.

OBJECTIVETo investigate the impact of all-trans retinoic acid (ATRA) on MDM2 gene expression in astrocytoma cell line SHG-44, and to provide basic data for further research on the progression mechanism and gene therapy of human astrocytoma.

METHODSThe differential expressions of MDM2 gene and protein in SHG-44 cells were detected by cDNA microarray and Western blot, respectively, before and after treatment of ATRA. The expressions of MDM2 protein in WHO grade II and grade IV astrocytomas were determined by immunohistochemical streptavidin-peroxidase method. Some differentially expressed genes were selected randomly for Northern blot analysis.

RESULTSThe intensity ratio of ATRA-treated to untreated SHG-44 cell was 0.37 in the cDNA microarray, suggesting that the expression of MDM2 gene was down-regulated in SHG-44 cells after treatment with ATRA. Some genes differentially expressed in the microarray were confirmed by Northern blot. Western blot demonstrated that the optical density ratios of MDM2 to beta-actin in ATRA-treated and untreated SHG-44 were 14.02+/-0.35 and 21.40+/-0.58 (t = 24.728, P = 0.000), respectively, suggesting that the expression of MDM2 protein was inhibited in ATRA-treated SHG-44 cells. Moreover, the percentages of MDM2-positive protein were 24.00% (6/25) and 56.52% (13/23) (chi(2) = 5.298, P = 0.021) in WHO grade II and grade IV astrocytomas, respectively, suggesting that the expression of MDM2 protein may increase along with the elevation of astrocytoma malignancy.

CONCLUSIONATRA can inhibit MDM2 gene expression in SHG-44 cells, and MDM2 is related to astrocytoma progression.

Antineoplastic Agents ; pharmacology ; Astrocytoma ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Size ; drug effects ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Oligonucleotide Array Sequence Analysis ; methods ; Proto-Oncogene Proteins c-mdm2 ; genetics ; metabolism ; Time Factors ; Tretinoin ; pharmacology

P300/CBP-associated factor(PCAF),an important member of histone acetyltransferase family(HATs) within eukaryotic cells,is capable of inducing the acetylation of histone,promoting the transcription of specific genes and involving in many biological effects.In the present study,full-length cDNA of PCAF was inserted into plasmid pGEX-5x-1,then the soluble protein GST-PCAF was expressed in E.coli BL21(DE3) after the optimization of inducing conditions.The recombinant protein was further purified with affinity chromatography and tested the activity by in vitro acetylation assays.High efficient PCAF protein produced by this method could serve for the study on the role of PCAF in gene regulation and the interaction between PCAF and other proteins.

OBJECTIVETo investigate the features and causes of complications of unilateral pedicle screw fixation combined with contralateral percutaneous translaminar facet screw fixation and lumbar interbody fusion in treating lower lumbar diseases.

METHODSThe clinical data of 166 patients with lower lumbar diseases who underwent unilateral pedicle screw fixation combined with contralateral percutaneous translaminar facet screw fixation and lumbar interbody fusion with intervertebral cages from January 2008 to December 2013 were retrospectively analyzed. There were 64 males and 102 females, aged from 24 to 74 years with a mean of 51.9 years old, suffered from lower lumbar lesions for 47.5 months on average (ranged, 8 months to 30 years). Among these patients, lumbar intervertebral disc degeneration was found in 49 patients, recurred lumbar intervertebral disc protrusion in 17 patients, massive lumbar intervertebral disc protrusion in 23 patients, lumbar intervertebral disc protrusion accompany with spinal canal stenosis in 27 patients, lumbar degenerative spondylolisthesis with degree I (Meyerding grade) in 21 patients, far lateral lumbar intervertebral disc protrusion in 5 patients. Single segmental diseases occurred in 124 patients and two segmental diseases in 42 patients. The diseases occurred at L(3,4) segment in 6 patients, at L(4,5) segment in 97 patients, at L5S1 segment in 21 patients, at L(2,3), and L(3,4) segments in 1 patient, at L(3,4) and L4,5) segments in 26 patients, and at L(4,5), and L5S1 segments in 15 patients.

RESULTSThere was no abnormal bleeding in the patients and no patient received blood transfusion. During the surgery, spinal dura mater injury with cerebrospinal fluid leakage complicated in 1 patient, a fracture of vertebral pedicle in 4 patients, and end plate injury in 2 patients. No postoperative cerebrospinal fluid, incision infection and skin necrosis were found after operation. Nerve root injury was found in 1 patient. According to the position of pedicles crew, 371 screws of 163 patients were in degree I and 3 screws of 3 patients were in degree II; position of translaminar facet screw, 199 screws of 157 patients were type I, 8 screws of 8 patients were type II, 1 screw of 1 patient was III. Translaminar facet screw was slightly short in 2 patients. Five patients were lost to follow-up, two patients were died. The remaining patients were followed up for 35.4 months on average (ranged, 12 to 60 months). During the follow-up period , end plate was cut off and intervertebral cages were embedded in 14 segments of 14 patients. Abnormal pain of both lower extremities was found in 1 patient. With the exception of 11 unidentified segments in 11 patients, 189 segments of 148 patients obtained intervertebral fusion. No loosening, displacement, breakage of pedicle screw or translaminar facet screw, displacement of intervertebral cages or obvious degeneration of adjacent segments were found. The coronal and sagittal planes balance of lumbar vertebra were obviously improved. Postoperative JOA score was significantly increased than that of preoperative.

CONCLUSIONUnilateral pedicle screw fixation combined with contralateral percutaneous translaminar facet screw fixation and lumbar interbody fusion with intervertebral cages is a good choice for the treatment of lower lumbar diseases, but it has a risk of complications. Abundant surgeon's surgical experience, careful operation, and rational use of imaging technique can effectively reduce the incidence of complications.

Adult ; Aged ; Bone Plates ; Female ; Humans ; Internal Fixators ; Intervertebral Disc Degeneration ; surgery ; Intervertebral Disc Displacement ; surgery ; Lumbar Vertebrae ; surgery ; Male ; Middle Aged ; Pedicle Screws ; Retrospective Studies ; Spinal Fusion ; Spondylolisthesis ; surgery ; Treatment Outcome ; Young Adult

Objective To investigate the differential expression of P57kip2 and CDK5 in neural tube defects t(NTD) from the normal ,and provide the clue for the research of the molecular mechanism of the normal neurula formation .Methods A cDNA mi-croarray containing 1 100 known genes was used to compare differences in P57kip2 and CDK5 gene expression between the normal control group and the retinoic acid(RA)-induced NTD group on embryonic(E) day 9 .5 and 10 .5 .Two differentially expressed genes were randomly selected from the two groups for Northern blotting to verify the results of the cDNA microarray .Results Compared the differences of between P57kip2 and CDK5 in normal and E9 .5 d ,E10 .5 d ,E9 .5 d-NTD ,E10 .5 d-NTD ,P57kip2 and CDK5 expression was significantly up-regulated in the before and after the formation of the normal neurulation ,but them showed a downward trend in retinoic acid (RA)-induced NTD(including two phase E9 .5 d and E10 .5 d) .Conclusion P57kip2 and CDK5 in-volved in the physiological process of NTD ,and provide the useful clue for the research of the molecular mechanism of the normal neurula formation .

OBJECTIVEIdiopathic nephrotic syndrome (INS) is a common glomerular disease. The pathogenesis of the disease remains unclear. Recent studies indicate that transforming growth factor beta (TGF beta) is the main cytokine involved in glomerular disease. It plays an important role in the development of INS and in occurrence of glomerulosclerosis. The present study aimed to study changes and significance of TGF beta in children with idiopathic nephrotic syndrome (INS).

METHODSTotally 35 cases with INS (13 males, 22 females) were studied. The age of onset was between 2 years and 1 months and 14 years with an average of 8 years and 3 months. The active stage group had 35 cases and the remission stage groups had 25 cases. The cases in active stage group had first onset of the disease with obvious clinical symptoms and abnormal laboratory findings without use of corticosteroids. The cases in remission stage group were asymptomatic without abnormal laboratory findings. Protein in urine was negative over 4 weeks after oral administration of prednisone for 8 weeks. Twenty five cases were steroid responsive and 10 cases were steroid non-responsive among the 35 cases. Thirty healthy young children were enrolled as control. TGF beta was detected by ELISA in peripheral blood mononuclear cell (PBMC) culture medium. The TGF beta mRNA gene expression was measured by in situ PCR in PBMC.

RESULTS(1) Concentration of TGF beta(247 +/- 26) ng/L and TGF beta mRNA expression (0.57 +/- 0.18) in active stage of simple type or nephritis type INS were higher than those of remission stage and control (P < 0.01). Concentration of TGF beta[(125 +/- 16) ng/L] and TGF beta mRNA expression (0.30 +/- 0.12) in remission stage were higher than that of control (P < 0.05). (2) The level of TGF beta protein in nephritis type [(275 +/- 26) ng/L] was significantly higher than that in simple type [(220 +/- 18) ng/L] in active stage INS (t = 6.45, P < 0.01). No significant difference in TGF beta mRNA expression was found between the nephritis type (0.58 +/- 0.15) and simple type (0.55 +/- 0.16) in active stage INS, either (P > 0.05). But these two types were different from the control (P < 0.01). (3) Concentration of TGF beta and TGF beta mRNA expression after therapy was clearly lower than that before therapy in steroid responsive group (P < 0.01). Whereas no significant change was seen in steroid non-responsive group. Both indicators were higher in steroid non-responsive group than in steroid responsive group whether before or after therapy.

CONCLUSIONTGF beta may play an important role in the mechanism of INS and its level in PBMC can be used as an immunological indicator for the illness state, therefore, determination of TGF beta level and mRNA may be of some clinical significance.

Adolescent ; Child ; Child, Preschool ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Leukocytes, Mononuclear ; drug effects ; metabolism ; Male ; Nephrotic Syndrome ; blood ; drug therapy ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Transforming Growth Factor beta ; genetics ; metabolism

OBJECTIVETo investigate the feasibility of utilizing self-designed score system for lower lumbar vertebral burst fractures to select surgical approach.

METHODSFrom January 2006 to December 2011, the clinical data of 56 patients with lower lumbar vertebra burst fractures who underwent surgical treatment were retrospectively analyzed. There were 42 males and 14 females with an average age of 43.1 years old (ranged, 19 to 65). Causes of injury included falling down (40 cases), traffic accidents (12 cases), and crashing injury by heavy objects(4 cases). Injury site was L3 in 37 cases, L4 in 16 cases, and L5 in 3 cases. According to the AO classification, 17 cases were type A3.1, 14 cases were type A3.2, 25 cases were type A3.3. According to Frankel grade of nerous function, 2 cases were grade B, 5 cases were grade C, 9 cases were grade D and 40 cases were grade E. Surgical methods and approaches were chosen based on the comprehensive evaluation of AO classification, condition of posterior column injury and spinal canal encroachment. Surgical methods and approaches included trans-vertebra fixation (15 cases), intra-vertebra pedicle screw fixation (21 cases), combination of anterior and posterior approaches (11 cases), one-stage posterior approaches (9 cases). Cobb angles, restorations of the affected vertebral anterior border height, and conditions of spinal canal encroachment were compared before and after surgery. Conditions of bone graft fusion and internal fixation (if bending, loosening or breakage existed) were observed. Spinal cord functions were assessed according to Frankel grade. Localized pain and working status of patients were also assessed at the last follow-up.

RESULTSNo incision infection was found and no spinal nerve symptoms improved in all of 56 patients. All patients were followed up for 12 to 60 months with a mean of 28.5 months, without internal fixation loosening or breakage. There was significant differences in Cobb angle, vertebral anterior border height and recovery of spinal canal encroachment between preoperative and postoperative instantly (P < 0.05), however, there was no significant difference between postoperative instantly and final follow-up (P > 0.05). Thirteen cases obtained fusion by trans-vertebra fixation, 20 cases obtained fusion by intra-vertebra fixation, and 20 cases were treated by the combination of anterior and posterior approaches or one-stage posterior approaches all of patients obtained fusion. Spinal nervous function recovered I to II grade, 1 case was grade C, 3 cases were grade D, 52 cases were grade E. Localized pain was assessed as P1 in 52 cases, P2 in 3 cases, and P3 in 1 case. Working status was classified into W1 in 12 cases, W2 in 39 cases, and W3 in 5 cases.

CONCLUSIONThe lower lumbar vertebra and thoracolumbar junction exhibit different injury characteristics due to variations in anatomy and biomechanics. A comprehensive score of the AO classification, posterior column injury and degree of spinal canal encroachment will guide the selection of surgical method and approach for the treatment of lower lumbar vertebra burst fractures.

Adult ; Aged ; Biomechanical Phenomena ; Female ; Follow-Up Studies ; Fracture Fixation, Internal ; methods ; Humans ; Lumbar Vertebrae ; injuries ; surgery ; Male ; Middle Aged ; Retrospective Studies ; Spinal Fractures ; surgery ; Spinal Fusion ; methods

OBJECTIVE: To study the genetic polymorphisms of 7 Y chromosome single nucleotide polymorphisms (Y-SNPs) among unrelated Mongolian males from Inner Mongolia Region. METHODS: Seven Y-SNPs with expected allelic frequencies close to 0.50:0.50 in Mongolian population were selected from databases including HapMap and relevant literature. The Y-SNPs were then analyzed among 95 unrelated male Mongolian DNA samples with ligase detection reaction (LDR) technique. Statistical analysis was carried out with Arlequin 3.5. RESULTS: All of the 7 Y-SNPs had 2 alleles. Seven haplotypes were identified among the 95 samples, with the haplotype diversity (HD) being 0.7990. Except for rs17316007, whose allelic frequencies was 0.832:0.168, each of the remaining Y-SNPs had a allelic frequency close to 0.50:0.50. The gene diversity (GD) for rs17316007 was 0.2825, while those of the remaining Y-SNPs were all greater than 0.4375. CONCLUSION Except for rs17316007, the other 6 Y-SNPs showed good diversity and genetic polymorphism, and may be used for individual identification and paternity testing for the Inner Mongolia region.
Asian Continental Ancestry Group ; China ; Chromosomes, Human, Y ; genetics ; Gene Frequency ; Genetics, Population ; Haplotypes ; Humans ; Male ; Polymorphism, Single Nucleotide

Objective To determine the prevalence and distribution of type 2 diabetes mellitus (T2DM) and the relationship between maximum body mass index (MAXBMI) and T2DM. Methods From June to August, 2005, a stratified cluster sampling of 1071 permanent residents in communities, over 20 years old, from 4 districts and 1 county of Mudanjiang was chosen. The prevalence of T2DM, and the association between T2DM and different levels of the MAXBMI, current BMI were studied. Results The prevalence in the communities was 7.09% and in those with past maximum BMI≥28 kg/m~2, it was 12.10%. With the increase of past MAXBMI levels, the risk of T2DM patients also increased significantly(trend X~2=17.387 23, P<0.0001). Data from multifactor analysis showed that MAXBMI in the past was positively related to T2DM (OR=3.06, P=0.0013). In T2DM patients, the group with MAXBMI≥27.4 kg/m~2 had higher 2-hour postprandial blood glucose than those with lower MAXBMI (P=0.0408). When compared with low maximum BMI group in normal blood glucose population, the group with higher MAXBMI (≥ 25.4 kg/m~2) had higher blood glucose and greater change of BMI. Conclusion In both groups that patients with T2DM and with normal glucose, in order to control blood glucose better, researchers should not only concern about the influence of the MAXBMI in the past, but also pay attention to constantly keep BMI at the normal range.

OBJECTIVESTo detect the expressions of Tec tyrosine kinase in hepatocellular carcinoma and the levels of phosphorylation of tyrosine kinase in liver cancer tissues, paracancerous tissues and normal liver tissues and to find the significance of their differences.

METHODS200 specimens of tissues, including liver cancer tissues, surrounding liver tissues not more than 1.5 cm from the cancers, and normal liver tissues were investigated for Tec protein expression and Tec phosphorylation by tissue microarrays and immunohistochemistry (SP method).

RESULTSThe positive immunohistochemical stainings of Tec in cancerous tissues and non-cancerous tissues showed no obvious differences, nevertheless, the immunostaining levels in liver cancer tissues were much higher than in non-cancerous tissues and they correlated with the grading of tumors (P < 0.05). The phosphorylation of Tec was significantly expressed in liver cancer tissues (73%) in comparison with other tissues (42%, 10% both P < 0.05), but it did not correlate with any clinicopathological characteristics.

CONCLUSIONOverexpression of Tec is associated with the tumorigenesis and development of liver cancer; inhibiting Tec or degrading Tec phosphorylation directly might affect the progression of liver cancer.

Adult ; Aged ; Aged, 80 and over ; Carcinoma, Hepatocellular ; metabolism ; Female ; Humans ; Liver Neoplasms ; metabolism ; Male ; Middle Aged ; Neoplasm Proteins ; genetics ; metabolism ; Phosphorylation ; Protein-Tyrosine Kinases ; genetics ; metabolism