NF-κB plays a key role in inducing CD274 expression in human monocytes after lipopolysaccharide treatment.

CD274, one of two co-stimulatory ligands for programmed death 1 and widely expressed in the mononuclear phagocyte system (MPS), may co-stimulate T cells and regulates inflammatory responses. However, changes in CD274 gene expression and the underlying molecular mechanism are poorly understood during inflammatory responses. Therefore, delineation of the complex mechanisms regulating CD274 expression is critical to understand this immunoregulatory system during inflammatory responses. The purpose of this study was to assess the molecular mechanisms regulating CD274 expression in an in vitro monocyte model of inflammatory response. Firstly, CD274 expression levels in human primary monocytes after lipopolysaccharide (LPS) treatment were observed and correlated with NF-κB activation. Secondly, based on the distribution of putative NF-κB binding sites, 5' truncated human CD274 promoter reporters were constructed, transfected into U937 cells and critical promoter regions for basal (nt -570 to +94) and LPS-induced (nt -1735 to -570) transcription were identified by dual luciferase assays. Finally, a key NF-κB binding site (nt -610 to -601) for LPS-inducible CD274 transcriptional activity was characterized by point mutation analysis and chromatin immunoprecipitation analysis assays (ChIP). Thus, the present study establishes a molecular basis to understand the mechanisms governing CD274 expression in certain infections and inflammatory disorders.

An efficient and rapid method for cDNA cloning from difficult templates using codon optimization and SOE-PCR: with human RANK and TIMP2 gene as examples.

As gene cloning from difficult templates with regionalized high GC content is a long recognized problem, we have developed a novel and reliable method to clone such genes. Firstly, the high GC content region of the target cDNA was synthesized directly after codon optimization and the remaining cDNA fragment without high GC content was generated by routine RT-PCR. Then the entire redesigned coding sequence of the target gene was obtained by fusing the above available two cDNA fragments with SOE-PCR (splicing by overlapping extension-PCR). We have cloned the human RANK gene (ten exons; CDS 1851 bp) using this strategy. The redesigned cDNA was transfected into an eukaryotic expression system (A459 cells) to verify its expression. RT-PCR and western blotting confirmed this. To validate our method, we also successfully cloned human TIMP2 gene (five exons; CDS 660 bp) also having a regionalized high GC content. Our strategy for combining codon optimization and SOE-PCR to clone difficult genes is thus feasible and potentially universally applicable.

FASLG polymorphism is associated with cancer risk.

Many studies have reported the association between the FASLG -844T/C polymorphism and cancer risk, but the data are remaining controversial. A pooled analysis was performed to assess this relationship comprehensively. Medline, PubMed, Embase and Web of Science were searched, and data were extracted and cross-checked independently by three authors. A total of 18 published studies including 22389 subjects were involved in this analysis. Overall, the -844C allele was associated with a significantly increased cancer risk (for CC versus TT: OR=1.23, 95% confidence interval (CI)=1.04-1.45; for CC+TC versus TT: OR=1.15, 95% CI=1.01-1.30; for CC versus TT+TC: OR=1.20, 95% CI=1.05-1.38). In the subgroup analysis by ethnicity, significantly elevated risks were found among Asians (for CC versus TT: OR=1.61, 95% CI=1.37-1.89; for CC+TC versus TT: OR=1.36, 95% CI=1.16-1.60; for CC versus TT+TC: OR=1.44, 95% CI=1.22-1.70). In the subgroup analysis by study design, significantly increased risks were found among population-based case-control studies (for CC versus TT: OR=1.40, 95% CI=1.06-1.84; for CC+TC versus TT: OR=1.25, 95% CI=1.01-1.55; for CC versus TT+TC: OR=1.31, 95% CI=1.06-1.61). These findings indicate that the FASLG -844C allele is emerging as a low-penetrant cancer susceptibility allele for cancer development. However, more comprehensive understanding of the association would certainly have an immense prospect in the promising field of individualised preventive care.

Rapid and simultaneous quantification of 4 urinary proteins by piezoelectric quartz crystal microbalance immunosensor array.

BACKGROUND: Urinary proteins are predictive and prognostic markers for diabetes nephropathy. Conventional methods for the quantification of urinary proteins, however, are time-consuming, and most require radioactive labeling. We designed a label-free piezoelectric quartz crystal microbalance (QCM) immunosensor array to simultaneously quantify 4 urinary proteins. METHODS: We constructed a 2 x 5 model piezoelectric immunosensor array fabricated with disposable quartz crystals for quantification of microalbumin, alpha1-microglobulin, beta2-microglobulin, and IgG in urine. We made calibration curves after immobilization of antibodies at an optimal concentration and then evaluated the performance characteristics of the immunosensor with a series of tests. In addition, we measured 124 urine samples with both QCM immunosensor array and immunonephelometry to assess the correlation between the 2 methods. RESULTS: With the QCM immunosensor array, we were able to quantify 4 urinary proteins within 15 min. This method had an analytical interval of 0.01-60 mg/L. The intraassay and interassay imprecisions (CVs) were <10%, and the relative recovery rates were 90.3%-109.1%. Nonspecificity of the immunosensor was insignificant (frequency shifts <20 Hz). ROC analyses indicated sensitivities were > or =95.8% and, specificities were > or =76.3%. Bland-Altman difference plots showed the immunosensor array to be highly comparable to immunonephelometry. CONCLUSIONS: The QCM system we designed has the advantages of being rapid, label free, and highly sensitive and thus can be a useful supplement to commercial assay methods in clinical chemistry.

Loss of ryanodine receptor calcium-release channel expression associated with overactive urinary bladder smooth muscle contractions in a detrusor instability model.

OBJECTIVE: To investigate the changes in spontaneous bladder smooth muscle contractions that occur during detrusor instability (DI), and to test the possibility that altered function or expression of ryanodine receptors (RyRs) could account for the increased bladder contractions. MATERIALS AND METHODS: After 8 weeks of partial bladder outlet obstruction, DI was confirmed in female experimental rats by filling cystometry. Muscle strips were dissected from freshly isolated bladders, and isometric tension recorded in strips from DI and normal bladders. The contractions were recorded during electrical stimulation or exposure to various agents. Western blot analysis was used to determine RyR expression in DI and normal bladder muscle. RESULTS: In DI bladder muscle, spontaneous contractile activity persisted in the presence of blockers for known neurotransmitter receptors in the bladder wall. The RyR blocker ryanodine significantly increased the spontaneous contractile frequency in normal bladder strips, but failed to affect spontaneous contractions in DI muscle. Caffeine inhibited spontaneous contractile activity in both the DI and normal strips. After administering the l-type Ca(2+) channel antagonist nimodipine, the myogenic contractile activity was abolished in normal strips; in contrast, in DI strips, the amplitude of contractions was reduced but the frequency of contractions was unchanged. Western blot analysis showed that RyR expression was lower in DI muscle than in normal bladder muscle. CONCLUSION: These results provide the first characterization of a loss of regulation of spontaneous contractile activity by RyRs in DI muscle associated with a significant decrease in RyR expression. RyRs in normal detrusor muscle act as negative-feedback regulators of spontaneous contractile activity, presumably by releasing Ca(2+) that activates Ca(2+)-dependent K(+) channels to decrease contractility. This mechanism might be weakened in DI muscle, resulting in spontaneous contractile overactivity.

[Comparison of the apical sealability of two canal obturation methods.].

PURPOSE: To compare the apical sealability of canal obturation with lateral condensation (LC) and combined LC with vertical condensation (hybrid condensation, HC). METHODS: 79 single canal extracted anterior teeth were instrumented with step-back technique and then randomly divided into three group. Group A was obturated only with LC; group B was obturated with HC. group C was as control group. All teeth were dyed in 1% india ink for 72 hours, then split into two parts longitudinally. The linear length of dye was measured as the result of microleakage. RESULTS: The linear length of microleakage between group A and group B had significant difference (P<0.01). The microleakage of HC was lower than LC. CONCLUSION: The vertical condensation to gutta-percha at crown third after canal obturation with LC could reduce the apical microleakage.