Objective To test the possibility of reciprocal pathways between medullary visceral zone (MVZ) and hypothalamic paraventricular nucleus (PVN) or supraoptic nucleus (SON) following hyperosmotic stimulation. Methods Hyperosmotic pressure animal model was established by administering3% sodium chloride as drinking water to rats. The distributions and expressions ofHRP retrogradely labeled neurons, Fos, tyrosine hydroxylase (TH) or vasopressin (VP) positive neuron and lial fibrillary acidic protein (GFAP) positive astrocytes (AST) in MVZ, SON and PVN were observed by quadruple labeling methods of WGA-HRP retrograde tracing combined with anti-Fos, TH (or VP) and GFAP immunohistochemical technique. Results Fos positive neurons within the MVZ, PVN and SON increased markedly. There were also a large number of GFAP positive structures in the brain and their distribution pattern was fundamentally similar or analogous to Fos positive neurons in the above-mentioned areas. The augmented GFAP reactivities took on hypertrophic cell bodies, thicker and longer processes.Quadruplicate immunohistochemical staining showed that a neuron could be closely surrounded by many AST and they formed neuron-astrocytic complex (N-ASC). Conclusion The neurons and AST might be very active following hyperosmotic pressure and N-ASC as a functional unit might serve to modulate the osmotic pressure. There was reciprocal osmoregulation pathways between the MVZ and SON or PVN in the brain.

Transcriptions are regulated by transcription factors. Natural transcription factors usually consist of at least two functional domains: a DNA-binding domain and an effector domain. According to this, novel artificial transcription factors are designed to up or down regulate transcription and expression of a target gene. The Cys2-His2 zinc finger domain is a DNA-binding module that has been widely used as the DNA-binding domain in artificial transcription factors. Each zinc finger domain, which comprises about 30 amino acids that adopt a compact structure by chelating a zinc ion, typically functions by binding 3 base pairs of DNA sequence. Several zinc fingers linked together would bind proportionally longer DNA sequences. According to the "bipartite complementary" library strategy, a pair of zinc finger phage display libraries were constructed. After construction of the libraries, a 9bp sequence (5'-GCAGAGGCC-3') on the promoter of SV40 was chosen as a target for next step. After parallel selection, PCR amplification, desired fragments recovery, re-ligation, and additional rounds of selection, phage enzyme-linked ELISA experiments were performed to identify specific binding clones displaying the zinc fingers with predetermined sequence-specificity to our target sequence. Then two clones with strong ELISA signals were chosen to be tested for binding both to its full target site (5'-GCAGAGGCC-3') and to sites containing single transition mutations. The binding specificity of one of the two clones (clone 3) was shown to be fairly good. The three-finger DNA-binding domain targeted to SV40 promoter, that is, zinc finger sequences on clone 3, was fused to KOX1 suppression domain KRAB and cloned into pcDNA3.1 (+) (which expression product was artificial transcription factor). The zinc fingers (which expression product was the DNA-binding domain of artificial transcription factor) and KRAB domain only (which expression product was effector domain of artificial transcription factor) were also cloned separately into the same expression vector. All constructs contained an N-terminal nuclear localization signal. Every of the vectors (including pcDNA3.1 (+) without inserting sequences) were cotransfected with pGL3-Control and pRL-TK and the activity of luciferase was used to indicate the function of product from transfected expression vectors. Our artificial transcription factor was proved to repress the expression of reporter gene efficiently,while with only DNA-binding domain or effector domain the repression was not remarkable. By adding different effector domains and changing the DNA-binding domain, artificial transcription factor would have a wide range of potential applications.
Enzyme-Linked Immunosorbent Assay ; Genes, Synthetic ; genetics ; physiology ; Models, Theoretical ; Peptide Library ; Polymerase Chain Reaction ; Promoter Regions, Genetic ; genetics ; Transcription Factors ; chemical synthesis ; chemistry ; metabolism ; Zinc Fingers ; genetics ; physiology

OBJECTIVETo compare the diagnostic value of (18)F-FDG PET-CT with abdomen contrast CT in the diagnosis of peritoneal metastases.

METHODSBetween January 2008 and May 2011, imaging results of 97 patients with suspicious peritoneal metastases were retrospectively reviewed, and all the patients underwent both abdomen contrast CT and (18)F-FDG PET-CT imaging. Final diagnosis was made by histopathology or follow up.

RESULTSSeventy-seven patients were verified as peritoneal metastases after pathological examination(n=88) or follow up(n=9), while the other 20 patients were absent. The sensitivity of (18)F-FDG PET-CT was 90.9%(70/77), the specificity 85.0%(17/20), and the accuracy 89.7%(87/97). There were 3 false positive and 7 false negative. The sensitivity of contrast CT was 66.2%(51/77), the specificity 80.0%(16/20), and the accuracy 69.1%(67/97). There were 4 false positive and 26 false negative. The difference in diagnostic accuracy was statistically significantly between these two methods(P<0.05).

CONCLUSIONThe diagnostic value of (18)F-FDG PET-CT is significantly higher than that of abdominal enhanced CT for peritoneal metastases.

Adult ; Aged ; Aged, 80 and over ; Female ; Humans ; Male ; Middle Aged ; Peritoneal Neoplasms ; diagnostic imaging ; secondary ; Positron-Emission Tomography ; Retrospective Studies ; Sensitivity and Specificity ; Tomography, X-Ray Computed ; Young Adult

Objective To observe the expression of stromal cell-derived factor-1α(SDF-1α)and glial cell line-derived neurotrophic factor(GDNF)genes in bone marrow stromal cells(BMSCs)of rhesus monkey. Methods With gene recombination technique,SDF-1α and GDNF genes obtained from cDNA were subcloned into pBudce4.1 vector to get pBudCE4.1-SDF-1-GDNF, evaluated by restriction enzyme analysis and sequencing analysis.The pBudCE4.1-SDF-1α-GDNF was transfected into BMSCs with lipofectamine2000.After 48 h,the expression of SDF-1α and GDNF was measured by RT-PCIL,Western blotting and immunohistochemistry. Results Correct construction of pBudCE4.1-SDF-1α-GDNF was identified by enzyme restriction analysis and sequencing analysis.Western blotting and immunohistochemistry confirmed the expressions Of SDF-1α and GDNF genes in the transfected cells.The protein expressions of GDNF and SDF-1α in the GDNF and SDF-1α transfected BMSCs were 6 times higher than those in the negative control cells. Conclusion The pBudCE4.1-SDF-1α-GDNF can successfully express SDF-1α and GDNF in BMSCs of rhesus monkey,which may be used in gene therapy for Parkinson's disease.

The amino acid sequence of the fusion protein FP3 was measured by two types of LC-MS/MS and its primary structure was confirmed. After reduction and alkylation, the protein was digested with trypsin and glycosyl groups in glycopeptide were removed by PNGase F. The mixed peptides were separated by LC, then Q-TOF and Ion trap tandem mass spectrometry were used to measure b, y fragment ions of each peptide to analyze the amino acid sequence of fusion protein FP3. Seventy-six percent of full amino acid sequence of the fusion protein FP3 was measured by LC-ESI-Q-TOF with the remaining 24% completed by LC-ESI-Trap. As LC-MS and tandem mass spectrometry are rapid, sensitive, accurate to measure the protein amino acid sequence, they are important approach to structure analysis and identification of recombinant protein.
Amino Acid Sequence ; Chromatography, High Pressure Liquid ; Molecular Sequence Data ; Peptide Mapping ; Recombinant Fusion Proteins ; chemistry ; Spectrometry, Mass, Electrospray Ionization ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Tandem Mass Spectrometry ; Vascular Endothelial Growth Factor A ; antagonists & inhibitors ; chemistry

OBJECTIVETo evaluate the ability of multidetector computed tomography (MDCT) in differentiating ovarian tumors from non-ovarian masses.

METHODSForty-two cases with pelvic masses were examined with 16-row MDCT. All source image of each case was put into workstation for multi-planar reconstruction (MPR) and curved planar reconstruction(CPR). Axial image combined with 2D image was used for determining the relationship of the mass to ovarian vascular pedicle and identifying the normal ovary, which was compared with postoperative pathologic result and the finding during operation. All the data was compared using Fisher's exact test.

RESULTSThere were 28 ovarian tumors and 14 non-ovarian tumors in this series. If the ovarian vascular pedicle sign was used for determining whether the tumor was from the ovary or not, the sensitivity, specificity, positive predictive value, negative predictive value and accuracy was 89.3%, 85.7%, 92.6%, 80.0% and 88.1%, respectively, with a significant difference in differentiating the tumor from the ovary or non-ovarian organs (P <0.05). If the identification of full normal ovary was used to determine non-ovarian origin of the tumor, the sensitivity, specificity, positive predictive value, negative predictive value and accuracy was 50.0%, 100.0%, 100.0%, 80.0% and 83.3%, respectively, also with a significant difference in differentiating the tumors from the ovary or non-ovarian organs (P <0.05).

CONCLUSIONMDCT can clearly show the relationship of the tumor to the normal ovary and its vascular pedicle, which is very helpful in differentiating the ovarian tumors from a non-ovarian masses.

Adult ; Aged ; Cystadenocarcinoma, Mucinous ; diagnostic imaging ; Cystadenocarcinoma, Serous ; diagnostic imaging ; Cystadenoma, Mucinous ; diagnostic imaging ; Cystadenoma, Serous ; diagnostic imaging ; Diagnosis, Differential ; Female ; Gastrointestinal Stromal Tumors ; diagnostic imaging ; Humans ; Leiomyoma ; diagnostic imaging ; Middle Aged ; Ovarian Neoplasms ; diagnostic imaging ; Ovary ; blood supply ; diagnostic imaging ; Retroperitoneal Neoplasms ; diagnostic imaging ; Teratoma ; diagnostic imaging ; Tomography, X-Ray Computed ; methods ; Uterine Neoplasms ; diagnostic imaging ; Young Adult

Objective To investigate the oncolytic effect of E1B mutant adenovirus (d11520) on human malignant gliomas. Methods Ad-βgal vector was used to investigate the infectibility of dl1520.U251,Hep3B (positive control) and T24 (negative control) cell lines were infected with dl1520respectively at 50,5,0.5,0.005 and 0 pfu of multiplicity of infection (MOI).The replication efficiency of d11520 in host cells was assessed by plaque assay.The cytopathic effect (CPE) was assessed by crystal violet staining in a panel of tumor cells. Results Crystal violet staining showed the Hep3B cell line was the most sensitive to dl1520 and had the fastest CPE,followed by the U251 cell line,while the T24cell line had no CEP.The replication and infection rates ofdl1520 in the U251 cell line were lower than in the Hep3B cell line but significantly higher than in the T24 cell line (P＜0.05). Conclusion The E1B mutant adenovirus (dl1520) has a significant oncolytic effect on human malignant gliomas.

Objective To analyze the contact failure of monolithic lithium disilicate CAD/CAM crowns by experiment and numerical simulation, and explore the influences of adhesives aging in water on load-bearing capacity of the crowns. Methods The specimens of sectioned monolithic lithium disilicate crowns were designed and manufactured, and evenly divided into two groups and stored in the air and in the distilled water for 30 days, respectively. The specimens were then subjected to monotonic contact loads to compare and analyze their load-bearing capacity. The fractured surfaces and adhesive interfaces of the specimens were observed by scanning electronic microscope. Meanwhile, the stress distribution on the crowns was calculated by numerical simulation to analyze the adhesives aging influence on load-bearing capacity of the crowns. Results The fracture loads on crowns stored in the air and in the water were (561.51 ± 65.66) N and (398.09 ± 90.20) N, respectively, indicating a significant difference. The tensile stress increased considerably at lower surface of the ceramic crown due to the reduction of adhesive strength at the interface of ceramic crown and substrate, which could increase the propensity of contact failure. Conclusions The adhesives aging in water reduces the bonding strength, and accordingly changes the tensile stress distributions, which can lower the load bearing capacity of the lithium disilicate crowns. The research finding provides references for the design and manufacturing of all-ceramic CAD/CAM restored crowns in clinic.

Objective To explore the possibility of adipose-derived stem cells (ADSCs) to differentiate into fibroblasts, and to provide an effective way for the effective solution of skin tissue engineering seed cells. Methods Primary ADSCs were obtained from inguinal fat of ten healthy adult SD rats,weighing 280-320 g,and cultured in vitro and purified. When primary ADSCs expansion to the 3rd passages,the following experiments were performed alkaline phosphatase test on the 16th day after osteogenesis induction,staining of alizarin red mineralized nodules on day 23 after osteogenesis induction, oil red O staining on day 12 after adipogenic induction; Flow cytometry detection of cell surface markers; Addition of conditioned medium to induce differentiation into fibroblasts,Photograph the changes of cell morphology on the 2nd, 4th, 6th, and 8th days after induction,MTT to detect cell viability at various time points;Scanning electron microscopy on day 6 and day 8 after induction;Immunocytochemical staining on day 8 after induction,detect the expression of vimentin, the main marker of fibroblasts. Results Primary ADSCs grew in long spindles, showed strong positive expression of alkaline phosphatase (ALP) after osteogenesis induction,and alizarin red staining showed red mineralized nodules;Aggregation of intracellular red-stained lipid droplets after adipogenic induction were found;Flow cytometry showed positive expression of mesenchymal stem cell-related marker CD90,and hematopoietic stem cell marker CD45 was negative. Morphology of ADSCs started to change on day 2 after induction into fibroblasts. On the 4th day after induction, the cells were in the shape of water droplets or short rods. On the 6th day after induction, the cells were protruded polygonal or triangular. Cells crowded and covered the bottom of the bottle on day 8 after induction,becoming slender fibrous. MTT test showed that the cell viability was significantly lower on the second day after induction than in the control group. There were no significant differences in cell viability on the 4th, 6th, and 8th days after induction compared with the control group. Scanning electron microscopy showed that the cells were triangular on the 6th day after induction, and the surface had more cilia. On the 8th day after induction, the cells were slender and fibrous, with small protrusions, and the surface cilia were dense. Vimentin was positively expressed in most cells on the 8th day after induction. Conclusion ADSCs can have the morphological characteristics of fibroblasts after induced differentiation in vitro; that can express fibroblasts marker protein.

BACKGROUNDGastric cancer ranks high among the most common causes of cancer-related death worldwide. This study was designed to explore key genes involved in the progression of normal gastric epithelial cells to moderate gastric epithelial dysplasia (mGED) and to gastric cancer.

METHODSTwelve pairs of mGED tissues, gastric cancer tissues, and normal gastric tissues were collected by gastroscopy. Total RNA was then extracted and purified. After the addition of fluorescent tags, hybridization was carried out on a Gene chip microarray slide. Significance analysis of microarrays was performed to determine significant differences in gene expression between the different tissue types.

RESULTSMicroarray data analysis revealed totally 34 genes that were expressed differently: 18 highly expressed (fold change > 2; P < 0.01) and 16 down-regulated (fold change > 2; P < 0.01). Of the 34 genes, 24 belonged to several different functional categories such as structural molecule activity, extracellular regions, structural formation, cell death, biological adhesion, developmental processes, locomotion, and biological regulation that were associated with cancer. The remaining 10 genes were not involved in cancer research. Of these genes, the expression levels of Matrix metalloproteinase-12 (MMP12), Caspase-associated recruitment domain 14 (CARD14), and Chitinase 3-like 1 (CHI3L1) were confirmed by semi-quantitative RT-PCR. A two-way clustering algorithm divided the 36 samples into three categories and the overall correct classification efficiency was 80.6% (29/36). Almost all of these genes (31/34) showed constant changes in the process of normal gastric epithelial cells to mGED to gastric cancer.

CONCLUSIONSThe results of this study provided global gene expression profiles during the development and progression from normal gastric epithelial cells to mGED to gastric cancer. These data may provide new insights into the molecular pathology of gastric cancer which may be useful for the detection, diagnosis, and treatment.

Adult ; Aged ; Epithelial Cells ; metabolism ; Gastric Mucosa ; metabolism ; pathology ; Humans ; Middle Aged ; Reverse Transcriptase Polymerase Chain Reaction ; Stomach ; metabolism ; pathology ; Stomach Neoplasms ; genetics ; Transcriptome ; genetics