Objective Quantitative analysis and comparison of the expression of ribonucleic acid （RNA） from frozen organs and formaldehyde-fixed and paraffin-embedded （FFPE） tissues. Methods Frozen specimens of human brain, myocardium and liver tissues as well as FFPE samples at different postmortem intervals were collected and mass concentration of RNA was extracted and detected. Reverse transcription-quantitative polymerase chain reaction （RT-qPCR） technology was used to analyze the amplification efficiency and relative expression of each RNA marker. Results The mass concentration and integrity of RNA extracted from FFPE samples were relatively low compared with frozen specimens. The amplification efficiency of RNA markers was related with RNA species and the length of amplification products. Among them, glyceraldehyde-3-phosphate dehydrogenase （GAPDH） and β-actin （ACTB） with relatively long amplification products failed to achieve optimal amplification efficiency, whereas 5S ribosomal RNA （5S rRNA） achieved ideal amplification efficiency and showed quite stable expression across various tissues, therefore it was chosen as internal reference marker. The expression quantity of GAPDH and ACTB in frozen specimens with longer postmortem intervals and in FFPE samples with relatively long amplification products was decreased. The expressions of tissue-specific microRNAs （miRNAs）, GAPDH and ACTB with relatively short amplification products had consistency in the same tissues and FFPE samples. Conclusion Through standardizing the RT-qPCR experiment, selecting the appropriate RNA marker and designing primers of appropriate product length, RNA expression levels of FFPE samples can be accurately quantified.
DNA Primers ; Formaldehyde ; Gene Expression Profiling ; Humans ; MicroRNAs/analysis* ; Myocardium ; Paraffin Embedding ; RNA/analysis* ; Real-Time Polymerase Chain Reaction/standards*

OBJECTIVES: To explore the correlation between the expression levels of several RNA markers in human brain tissue and early postmortem interval （PMI）. METHODS: Twelve individuals with known PMI （range from 4.3 to 22.5 h） were selected and total RNA was extracted from brain tissue. Eight commonly used RNA markers were chosen including β-actin, GAPDH, RPS29, 18S rRNA, 5S rRNA, U6 snRNA, miRNA-9 and miRNA-125b, and the expression levels were detected in brain tissue by real-time fluorescent quantitative PCR. The internal reference markers with stable expression in early PMI were screened using geNorm software and the relationship between its expression level and some relevant factors such as age, gender and cause of death were analyzed. RNA markers normalized by internal reference were inserted into the mathematic model established by previous research for PMI estimation using R software. Model quality was judged by the error rate calculated with estimated PMI. RESULTS: 5S rRNA, miRNA-9 and miRNA-125b showed quite stable expression and their expression levels had no relation with age, gender and cause of death. The error rate of estimated PMI using β-actin was 24.6%, while GAPDH was 41.0%. CONCLUSIONS 5S rRNA, miRNA-9 and miRNA-125b are suitable as internal reference markers of human brain tissue owing to their stable expression in early PMI. The expression level of β-actin correlates well with PMI, which can be used as an additional index for early PMI estimation.
Actins/analysis* ; Autopsy ; Brain/metabolism* ; Humans ; MicroRNAs/analysis* ; Models, Theoretical ; Postmortem Changes ; RNA Stability ; RNA, Ribosomal, 18S/analysis* ; RNA, Ribosomal, 5S/analysis* ; RNA, Small Nuclear/analysis* ; Real-Time Polymerase Chain Reaction ; Software

OBJECTIVETo investigate the capability of semi-quantitative and quantitative parameters of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) to predict the response to concurrent chemoradiotherapy( CCRT) in patients with non-small cell lung cancer (NSCLC).

METHODSA total of 24 patients with stage III or IIIB NSCLC, who underwent 3.0T DCE-MRI before CCRT, were enrolled in this study. Semi-quantitative and quantitative parameters were calculated by Funtool and Omnikinetics software. The relationship between these obtained parameters and tumor response was evaluated by Spearmen' s correlation analysis. The patients were classified into two groups according to the tumor regression rate after treatment, as response group (group A) and non-response group ( group B). Mann-Whitney U test was used to compare the parameters of responders and non-responders. The value of the parameters on predicting response was calculated by receiver operating characteristic curve (ROC).

RESULTSThe tumor regression rate after treatment was negatively correlated with time to peak (TTP) and the extravascular-extracellular volume fraction (Ve), and was positively correlated with signal enhancement ratio (SERmax) and volume transfer constant (Ktrans) (P < 0.05 for all). Statistical significant differences were found between group A and group B both in semi-quantitative and quantitative parameters (P < 0.05). Group A had a lower TTP value [(34.66 ± 16.37) s vs. (44.09 ± 17.41) s] and Ve value [(0.19 ± 0.03) vs. (0.25 ± 0.05)] than group B, whereas group A had a higher SERmax [(166.50 ± 44.95)% vs. (113.57 ± 46.62)%] and Ktrans [(0.41 ± 0.17) min(-1) vs. (0.28 ± 0.12) min(-1)] than group B (P < 0.05 for all). The ROC analysis indicated that when setting the threshold of Ve on ≤ 0.21 for predicting response, the specificity, sensitivity and accuracy were 85.7%, 80.0% and 83.3%, respectively, with an area under curve of 0.875 (P < 0.001).

CONCLUSIONSBoth the semi-quantitative and quantitative DCE-MRI parameters are helpful for predicting the response after CCRT of NSCLC. Quantitative parameters seem to be more meaningful than semi-quantitative parameters.

Carcinoma, Non-Small-Cell Lung ; pathology ; therapy ; Chemoradiotherapy ; methods ; Contrast Media ; Humans ; Lung Neoplasms ; pathology ; therapy ; Magnetic Resonance Imaging ; methods ; ROC Curve ; Remission Induction ; Sensitivity and Specificity ; Time Factors

Objective To investigate the capability of semi-quantitative and quantitative parameters of dynamic contrast-enhanced magnetic resonance imaging ( DCE-MRI) to predict the response to concurrent chemoradiotherapy( CCRT) in patients with non-small cell lung cancer ( NSCLC) .Methods A total of 24 patients with stageⅢA orⅢB NSCLC, who underwent 3.0T DCE-MRI before CCRT, were enrolled in this study.Semi-quantitative and quantitative parameters were calculated by Funtool and Omnikinetics software. The relationship between these obtained parameters and tumor response was evaluated by Spearmen′s correlation analysis.The patients were classified into two groups according to the tumor regression rate after treatment, as response group ( group A) and non-response group ( group B) .Mann-Whitney U test was used to compare the parameters of responders and non-responders.The value of the parameters on predicting response was calculated by receiver operating characteristic curve (ROC).Results The tumor regression rate after treatment was negatively correlated with time to peak ( TTP ) and the extravascular-extracellular volume fraction ( Ve ) , and was positively correlated with signal enhancement ratio ( SERmax ) and volume transfer constant (Ktrans)(P<0.05 for all).Statistical significant differences were found between group A and group B both in semi-quantitative and quantitative parameters ( P<0.05) .Group A had a lower TTP value [(34.66±16.37)s vs.(44.09±17.41)s] and Ve value [(0.19±0.03) vs.(0.25±0.05)] than group B, whereas group A had a higher SERmax[(166.50±44.95)%vs.(113.57±46.62)%] and Ktrans[(0.41±0.17) min-1 vs.(0.28±0.12) min-1] than group B (P<0.05 for all).The ROC analysis indicated that when setting the threshold of Ve on≤0.21 for predicting response, the specificity, sensitivity and accuracy were 85 .7%, 8 0 .0%and 8 3 .3%, respectively , with an area under curve of 0 .8 7 5 ( P<0 .0 0 1 ) .Conclusions Both the semi-quantitative and quantitative DCE-MRI parameters are helpful for predicting the response after CCRT of NSCLC.Quantitative parameters seem to be more meaningful than semi-quantitative parameters.

Objective To investigate the capability of semi-quantitative and quantitative parameters of dynamic contrast-enhanced magnetic resonance imaging ( DCE-MRI) to predict the response to concurrent chemoradiotherapy( CCRT) in patients with non-small cell lung cancer ( NSCLC) .Methods A total of 24 patients with stageⅢA orⅢB NSCLC, who underwent 3.0T DCE-MRI before CCRT, were enrolled in this study.Semi-quantitative and quantitative parameters were calculated by Funtool and Omnikinetics software. The relationship between these obtained parameters and tumor response was evaluated by Spearmen′s correlation analysis.The patients were classified into two groups according to the tumor regression rate after treatment, as response group ( group A) and non-response group ( group B) .Mann-Whitney U test was used to compare the parameters of responders and non-responders.The value of the parameters on predicting response was calculated by receiver operating characteristic curve (ROC).Results The tumor regression rate after treatment was negatively correlated with time to peak ( TTP ) and the extravascular-extracellular volume fraction ( Ve ) , and was positively correlated with signal enhancement ratio ( SERmax ) and volume transfer constant (Ktrans)(P<0.05 for all).Statistical significant differences were found between group A and group B both in semi-quantitative and quantitative parameters ( P<0.05) .Group A had a lower TTP value [(34.66±16.37)s vs.(44.09±17.41)s] and Ve value [(0.19±0.03) vs.(0.25±0.05)] than group B, whereas group A had a higher SERmax[(166.50±44.95)%vs.(113.57±46.62)%] and Ktrans[(0.41±0.17) min-1 vs.(0.28±0.12) min-1] than group B (P<0.05 for all).The ROC analysis indicated that when setting the threshold of Ve on≤0.21 for predicting response, the specificity, sensitivity and accuracy were 85 .7%, 8 0 .0%and 8 3 .3%, respectively , with an area under curve of 0 .8 7 5 ( P<0 .0 0 1 ) .Conclusions Both the semi-quantitative and quantitative DCE-MRI parameters are helpful for predicting the response after CCRT of NSCLC.Quantitative parameters seem to be more meaningful than semi-quantitative parameters.

We cloned the lipoxygenase gene (ana-LOX) from Anabaena sp. PCC 7120 and expressed it in Escherichia coli BL21 (DE3) pLysS. We determined the active site of the recombinant ana-LOX through site-directed gene mutagenesis and obtained the shortest length of the functional gene. Meanwhile, we studied the properties of recombinant ana-LOX after purification. The C-terminal of the Aos (allene oxide synthase)-LOX fusion gene in Anabaena sp. PCC 7120 genome was found belonging to LOXs family by bioinformatics analysis. Further results of site-directed gene mutagenesis confirmed that the active sites of ana-LOX were His197, His202, His369, Asn373and Ile455. The shortest length of functional gene was identified to be 1 254 bp based on the strategy of shortening the gene length gradually. The highest activity of recombinant ana-LOX of 6 750 U/mL could be achieved when constructed to pET-32a vector and expressed at low temperature 16 degrees C. We purified the enzyme by Ni-NTA chelating affinity chromatography, with 60.89% yield and specific activity of 11.4 x 10(4) U/mg. The optimum reaction temperature and pH for ana-LOX were 45 degrees C and 6.0, respectively. Furthermore, the obtained ana-LOX was stable at room temperature. The effect of metal ions on ana-LOX was determined also. Fe2+, Mg2+ Ca2+ could markedly promote the activity of this enzyme whereas Fe3+ and Cu2+ had a strong inhibitory effect on it. Finally, the ana-LOX could improve the microscopical structure of dough. The results of this study will provide a basis for future improvements and food industrial applications of ana-LOX.
Anabaena ; enzymology ; genetics ; Catalytic Domain ; Cloning, Molecular ; Enzyme Stability ; Escherichia coli ; metabolism ; Lipoxygenase ; chemistry ; genetics ; Metals, Heavy ; chemistry ; Mutagenesis, Site-Directed ; Recombinant Proteins ; chemistry ; genetics

OBJECTIVETo study the clinical and laboratory features and diagnosis of the patient with anti-N-methyl-D-aspartate receptor(NMDAR)encephalitis in children.

METHODThe data of clinical feature, laboratory findings, and radiological manifestation were reviewed and analyzed.

RESULTOf the 7 patients, 4 were female and 3 were male. The age of onset was from 6.6 to 15.5 years (average 9.5 years). The onset of 4 cases started with convulsion. Six cases had seizures which was difficult to control by antiepileptic drugs. All patients had psychiatric symptoms and speech disorder. Six cases had different levels of decreased consciousness and dyskinesias. 6 cases had autonomic nerve instability, and 7 cases developed sleep disorders. The results of MRI examination were normal in all patients. The EEG of most patients showed focal or diffuse slow waves. Six cases had oligoclonal bands. All cases were confirmed to have the disease by detection of anti-NMDA receptor antibodies. No tumor was detected in any of the patients. All patients received immunotherapy.

CONCLUSIONAnti-NMDAR encephalitis is a severe but treatable disorder that frequently affects children and adolescents. Pediatric patients had clinical manifestations similar to those of adult patients. But children have a lower incidence of tumors and hypoventilation also occurs less frequently in children. Most of children had a good prognosis.

Adolescent ; Anti-N-Methyl-D-Aspartate Receptor Encephalitis ; complications ; diagnosis ; therapy ; Autoantibodies ; blood ; cerebrospinal fluid ; Autonomic Nervous System ; physiopathology ; Brain ; diagnostic imaging ; pathology ; Child ; Electroencephalography ; Female ; Humans ; Immunotherapy ; methods ; Magnetic Resonance Imaging ; Male ; Movement Disorders ; etiology ; Radiography ; Receptors, N-Methyl-D-Aspartate ; immunology ; Retrospective Studies ; Seizures ; etiology

Objective To explore the correlation between the expression of protease activated receptors-2 (PAR-2) and intestinal mucosal barrier injury of acute necrotizing pancreatitis (ANP) in rats.Methods The ANP rat model was created.The expression of PAR-2 in rat's intestinal mucosa of sham-operated group and ANP group at six,12 and 24 hours after model established was detected by immunohistochemistry (IHC),reverse transcription-polymerase chain reaction (RT-PCR) and Western blot.The difference between groups was analyzed by one way analysis of variance.Results The results of IHC indicated that PAR-2 expression in rat's intestinal mucosa of sham-operated group was weak.The number of PAR-2 expression positive cells and immunostaining intensity increased significantly after ANP model established.The IHC score was 4.88±0.33,5.87±0.32 and 11.17±0.27 at six,12 and 24 hours after model established respectively.Compared with those of shamoperated group (2.86 ± 0.31),the differences were statistically significant (F=747.08,P＜0.01).The expression of PAR-2 at mRNA and protein level in intestinal mucosa of sham-operated group was very low.As time extended after ANP model established,both expression increased gradually.The PAR-2 mRNA was 0.56±0.03,0.69±0.03,1.05±0.05,and the protein was 0.28±0.02,0.35±0.03,0.69±0.04 at six,12 and 24 hours after model established respectively.Compared with shamoperated group,the differences were statistically significant at each time point (F=785.69,1177.82,both P＜0.01).Conclusions PAR-2 is activated in the inflammatory progress of ANP,and may play an important role in the pathogenesis and development of intestinal mucosa barrier injury in ANP.

OBJECTIVEThe morphological characteristics of posterior tooth in the Beagle dog were investigated in order to provide reference to mini-screw implant (MSI) implantation in the root furcation site.

METHODSSeventy posterior teeth of five Beagle dogs were collected and measured as follows: First, the greatest mesio-distal diameter AB of the crown was measured, which was perpendicular to the tooth long axis. Second, mesial cusp of maxillary posterior tooth or central cusp of mandibular was marked as the piont C, and CD which was equal and perpendicular to AB was drawn. The piont D was planned to be the reference point for MSI implantation. Finally, Dm, Dd and Df were measured, which were the distance from the point D to the mesial and distal root surface and root furcation.

RESULTSThe difference between the left and right sides teeth with the same name had no statistical significant (P > 0.05). The measurement results of all indices assumed a Gaussian distribution. The mean Dd was basically equal to Dm. All the average Df were more than 4 mm.

CONCLUSIONThe point D could be used as a reference point for MSI implantation in the root furcation site of posterior tooth in the Beagle dog. The data of this study can provide certain reference for MSI implantation in the study of the biomechanics of anchorage stability, and provide certain reference for MSI implantation in the study of root repair after injury.

Animals ; Bone Screws ; Dogs ; Mandible ; Maxilla ; Tooth ; Tooth Movement Techniques ; Tooth Root

The fused tooth is the union of two dental enamel or dentin formed together. In the maxillary, the fusion usually occurred within the lateral incisor and canine and very rarely occurred in the upper third molar and supernumerary tooth. This paper reported a fused tooth occurred in the left maxillary impacted third molar with supernumerary tooth.
Fused Teeth ; Humans ; Incisor ; Male ; Maxilla ; Molar ; Molar, Third ; Tooth, Impacted ; Tooth, Supernumerary