[Effect of RORC inhibitor on HIF-1α and VEGF in nasal mucosa of allergic rhinitis of mice].

Objective: To investigate the effects of the retinoic acid receptor related orphan C (RORC) inhibitor (SR1001) on the expression changes of proteins of hypoxia induced factor (HIF-1α) and vascular endothelial growth factor (VEGF) in the nasal mucosa of mice with allergic rhinitis (AR) model. Methods: Thirty BALB/c were randomly divided into normal group, AR model group and RORC inhibitor group, 10 mice each group. AR model of mice was established by ovalbumin (OVA) sensitization method. RORC inhibitor group was given intraperitoneal injection of SR1001 (25 mg/kg), while AR model group intraperitoneal injection of the same volume of 0.9% normal saline. The symptom score of the mice was determined every weekend after administration. The pathological morphological changes in the nasal mucosa tissue obtained from anesthetized mice were observed by light microscope. The expression of HIF-1α and VEGF protein were detected by immunohistochemistry. IFN-γ, IL-17, and sIgE in the serum were detected by ELISA and the expression of HIF-1α and VEGF in the nasal mucosal tissue of the mice were measured by Western blot. One-way ANOVA was used for inter-group comparison. LSD method was used for inter-group comparison with equal variance, and Dunnett T3 method for inter-group comparison with unequal variance. P<0.05 was considered statistically significant. Results: The AR model was successfully established. Compared with the model group, the RORC inhibitor group significantly reduced the symptom score of AR mice (4.02±0.97 vs 8.50±1.76, t=7.050, P<0.01). The damaged mucosal epithelium appeared to be improved, the glands and dilated ducts tended to be normal, the mast goblet cells significantly reduced, and the infiltration of inflammatory cells in the inherent mucosa reduced. Meanwhile, the content of IL-17 and sIgE in serum decreased [(25.10±4.11) ng/ml vs (42.56±5.98) ng/ml, (0.875±0.244) ng/ml vs (1.982±0.365) ng/ml, t value was 14.141, 10.275, respectively, all P<0.01] and the content of IFN-γ increased [(61.32±8.83) pg/ml vs (38.94±5.97) pg/ml, t=8.133, P<0.01]. The expression of HIF-1α and VEGF protein in the nasal mucosal tissues of AR mice significantly reduced (0.92±0.08 vs 1.67±0.31, 1.12±0.21 vs 2.54±0.46, t value was 7.408, 8.880, respectively, all P<0.01). Conclusion: The RORC inhibitor has the therapeutic effect on AR by changing the content of inflammatory factors in AR mice and reducing the expression level of HIF-1α and VEGF in the nasal mucosa.

A simplified genomic DNA extraction protocol for pre-germination genotyping in rice.

Genotyping is a critical step for molecular marker-assisted selection in rice. Rice genomic DNA samples for genotyping are typically isolated from living tissues such as seedlings. This requires the germination of all candidate seeds and extraction of DNA from the seedlings. Currently, an ideal individual is selected from a very large number of plants, which is time- and labor-consuming, requiring several transplantations of materials and sampling processes. In this study, we developed a simplified genomic DNA extraction protocol in rice by using amylase to treat half-seeds. The yields of genomic DNA from a half-seed of Indica and Japonica rice were greater than 203.8 ± 32.5 and 143.2 ± 25.5 ng, respectively, and the 260/280 nm absorbance ratio was 1.75-2.10. The DNA was confirmed to be sufficient for polymerase chain reaction amplification and can be used in a marker-assisted selection program.

A novel synthetic Cry1Ab gene resists rice insect pests.

A few insect control genes of Bacillus thuringiensis have been modified successfully to increase the expression in plants by replacing rare codons, increasing GC content, and avoiding the DNA elements that could cause premature transcription termination, mRNA instability, and potential methylation. However, the modification process was intricate and often confused researchers. In this study, we adopted a simple method to modify Cry1Ab only by individually replacing its amino acid sequence with corresponding rice-preferred codons based on analysis of 92,188 coding DNA sequences. Unexpectedly, all elements of A+T richness, which terminate or destabilize transcription in plants, were avoided in the newly designed mCry1Ab. However, mCry1Ab had 2 notable features: less synonymous codons and high GC content. mCry1Ab only employed 22 of the 61 codons to encode protein and had an enhanced GC content of 65%. The increase in GC content caused abundant potential methylation signals to emerge in mCry1Ab. To test whether mCry1Ab could be expressed in rice, we transferred it into Oryza japonica variety Wanjing97. Insect bioassays revealed that transgenic plants harboring this gene driven by 2 promoters, CaMV35S and OsTSP I, were highly resistant to rice leaffolder (Cnaphalocrocis medinalis). Analysis of R0 to R2 generation plants indicated that the mCry1Ab was inherited stably by the progeny. Our study provided a simple modified method for expressing exogenous genes in rice and confirmed that less synonymous codons and high GC content do not affect transgene expression in rice.

High-throughput, low-cost, and event-specific polymerase chain reaction detection of herbicide tolerance in genetically modified soybean A2704-12.

The aim of this study was to develop an event-specific qualitative and real-time quantitative polymerase chain reaction (PCR) method for detection of herbicide-tolerance genetically modified (GM) soybean A2704-12. The event-specific PCR primers were designed, based on the 5'-flanking integration sequence in the soybean genome, to amplify the 239-bp target fragment. Employing the same event-specific primers, qualitative PCR and real-time quantitative PCR detection methods were successfully developed. The results showed that the A2704-12 event could be specifically distinguished from other GM soybean events. In the qualitative PCR assay, the limit of detection was 0.05%, and in the real-time quantitative PCR assay, the limit of detection was less than 0.01%. Moreover, our genomic DNA (gDNA) extraction protocol is high-throughput, safe, and low-cost. The event-specific PCR assay system is cost-efficient by using SYBR Green I in real-time PCR, and by using the same primers in both the qualitative and quantitative PCR assays. We therefore developed a high-throughput, low-cost, and event-specific qualitative and quantitative PCR detection method for GM soybean A2704-12. The method would be useful for market supervision and management of GM soybean A2704-12 due to its high specificity and sensitivity.

Overexpression of an alternative oxidase gene, OsAOX1a, improves cold tolerance in Oryza sativa L.

Low temperature is a major environmental stress in rice cultivating and production. The alternative oxidase 1 (AOX1) gene is potentially important for genetic engineering to increase cold adaptation. However, previous studies related to this effect have mostly focused on the dicot plants Arabidopsis and tobacco, whereas functional research on rice is limited. In this study, we cloned a rice predominant cold-response AOX1 gene, OsAOX1a. Transgenic rice plants with overexpression of OsAOX1a were obtained. We found that OsAOX1a overexpression could strongly enhance the cold growth of seedlings, especially with respect to root extension. However, growth between transgenic and control plants did not differ under normal conditions. Furthermore, the lipid peroxidation and ion leakage rate were determined after cold treatment in transgenic plants. Both factors were reduced by OsAOX1a overexpression, which revealed that OsAOX1a could reduce oxidative damage under cold stress. Taken together, our results suggested that overexpressing OsAOX1a could improve growth performance of rice under cold stress, which might be closely related to the reduction of reactive oxygen species generation and oxidative damage.

A high-throughput, high-quality plant genomic DNA extraction protocol.

The isolation of high-quality genomic DNA (gDNA) is a crucial technique in plant molecular biology. The quality of gDNA determines the reliability of real-time polymerase chain reaction (PCR) analysis. In this paper, we reported a high-quality gDNA extraction protocol optimized for real-time PCR in a variety of plant species. Performed in a 96-well block, our protocol provides high throughput. Without the need for phenol-chloroform and liquid nitrogen or dry ice, our protocol is safer and more cost-efficient than traditional DNA extraction methods. The method takes 10 mg leaf tissue to yield 5-10 µg high-quality gDNA. Spectral measurement and electrophoresis were used to demonstrate gDNA purity. The extracted DNA was qualified in a restriction enzyme digestion assay and conventional PCR. The real-time PCR amplification was sufficiently sensitive to detect gDNA at very low concentrations (3 pg/µL). The standard curve of gDNA dilutions from our phenol-chloroform-free protocol showed better linearity (R(2) = 0.9967) than the phenol-chloroform protocol (R(2) = 0.9876). The results indicate that the gDNA was of high quality and fit for real-time PCR. This safe, high-throughput plant gDNA extraction protocol could be used to isolate high-quality gDNA for real-time PCR and other downstream molecular applications.

Quantitative detection of the rice false smut pathogen Ustilaginoidea virens by real-time PCR.

Rice false smut (RFS) is an important rice disease that is caused by the pathogen Ustilaginoidea virens. In this study, we developed a real-time polymerase chain reaction (PCR) assay to detect U. virens and to estimate the level of disease. The genomic DNAs of U. virens and rice were extracted together from the rice samples. Real-time PCR assays were performed and compared to conventional nested-PCR assays. The real-time PCR assay presented a consistent linearity of the standard curve (R(2) = 0.9999). The detection limit could be as low as 40 fg U. virens DNA with a rice genomic DNA background on using the real-time PCR assay, which showed significantly higher sensitivity than the conventional nested-PCR assay. We conclude that the real-time PCR quantitative assay is a useful tool for detecting U. virens and for early defense and control of RFS.

Regulation of ovarian cancer progression by microRNA-187 through targeting Disabled homolog-2.

MicroRNAs (miRNAs) play important roles in tumorigenesis by regulating oncogenes and tumor-suppressor genes. In this study, miR-187 and miR-200a were found to be expressed at higher levels in ovarian cancers than in benign tumors. In patients with ovarian cancer, however, higher levels of miR-187 and miR-200a expression were paradoxically associated with better OS and recurrence-free survival. Further, multivariate analysis showed that miR-187 served as an independent prognostic factor for patients with ovarian cancer (n=176). Computational prediction and microarray results indicated that miR-187 directly targeted Disabled homolog-2 (Dab2), and luciferase reporter assays confirmed that the target site of miR-187 was located at the 3'-UTR of the Dab2 gene. Generally considered as a tumor-suppressor gene, Dab2 may actually promote tumor progression in advanced cancers through epithelial-to-mesenchymal transition (EMT). Ectopic expression of miR-187 in cancer cells promoted cell proliferation, but continued overexpression of miR-187 suppressed Dab2 and inhibited migration. Suppression of miR-187 upregulated Dab2, which, by inhibiting E-cadherin levels while stimulating vimentin and phospho-FAK levels, promoted EMT. Reduced ovarian cancer Dab2 histoscores correlated with high miR-187 levels and improved outcomes of patients. Collectively, these results demonstrate distinct dual roles of Dab2 in cell proliferation and tumor progression. In the initial steps of tumorigenesis, upregulated miR-187 suppresses Dab2, promoting cell proliferation. During the later stages, however, continued increased levels of miR-187 inhibits the Dab2-dependent EMT that is associated with tumor invasiveness, which is presumed to be the reason why cancers with high miR-187 levels were associated with better survivals.

Matrix metalloproteinase-12 leads to elastin degradation in BALB/c mice with eosinophilic meningitis caused by Angiostrongylus cantonensis.

The rat lugworm Angiostrongylus cantonensis can cause eosinophilic meningitis. The purpose of this study was to determine whether matrix metalloproteinase (MMP)-12 and its substrate elastin participate in this inflammatory response. We showed that the MMP-12/tissue inhibitor of metalloproteinase-1 ratio was significantly increased in the CSF of A. cantonensis-infected mice from day 10 p.i., and reached high levels on days 20 and 25 p.i. MMP-12 production was correlated with elastin degradation, eosinophil count, blood-CSF barrier permeability and pathological changes in the subarachnoid space. Also, MMP-12 might contribute to elastin degradation in the meningeal vessel of the subarachnoid space. Simultaneous administration of albendazole and doxycycline significantly reduced the levels of MMP-12, elastin and Evans blue in mice with meningitis. These results imply that MMP-12 contributes to the elastin degradation that occurs in angiostrongyliasis meningitis, and doxycycline can reverse related inflammatory events by inhibition of MMP-12.

miR-495 is upregulated by E12/E47 in breast cancer stem cells, and promotes oncogenesis and hypoxia resistance via downregulation of E-cadherin and REDD1.

MicroRNAs (miRNAs) are involved in tumorigenecity by regulating specific oncogenes and tumor suppressor genes, and their roles in breast cancer stem cells (BCSCs) are becoming apparent. Distinct from the CD44(+)/CD24(-/low) sub-population, we have isolated a novel PROCR(+)/ESA(+) BCSC sub-population. To explore miRNA-regulatory mechanisms in this sub-population, we performed miRNA expression profiling and found miR-495 as the most highly upegulated miRNA in PROCR(+)/ESA(+) cells. Coincidently, high upregulation of miR-495 was also found in CD44(+)/CD24(-/low) BCSCs, reflecting its potential importance in maintaining common BCSC properties. Ectopic expression of miR-495 in breast cancer cells promoted their colony formation in vitro and tumorigenesis in mice. miR-495 directly suppressed E-cadherin expression to promote cell invasion and inhibited REDD1 expression to enhance cell proliferation in hypoxia through post-transcriptional mechanism. miR-495 expression was directly modulated by transcription factor E12/E47, which itself is highly expressed in BCSCs. These findings reveal a novel regulatory pathway centered on miR-495 that contributes to BCSC properties and hypoxia resistance.

Epitaxial growth of InN films by molecular-beam epitaxy using hydrazoic acid (HN3) as an efficient nitrogen source.

Epitaxial InN films have been successfully grown on c-plane GaN template by gas-source molecular-beam epitaxy with hydrazoic acid (HN3) as an efficient nitrogen source. Results in residual-gas analyzer show that the HN3 is highly dissociated to produce nitrogen radicals and can be controlled in the amounts of active nitrogen species by tuning HN3 pressure. A flat and high-purity InN epifilm has been realized at the temperature near 550 degrees C, and a growth rate of 200 nm/hr is also achieved. Moreover, the epitaxial relationship of the InN(002) on the GaN(002) is reflected in the X-ray diffraction, and the full-width at half-maximum of the InN(002) peak as narrow as 0.05 degrees is related to a high-quality crystallinity. An infrared photoluminescence (PL) emission peak at 0.705 eV and the integrated intensity increasing linearly with excitation power suggest that the observed PL can be attributed to a free-to-bound recombination.

Acellular dermal matrix allografts to achieve increased attached gingiva. Part 1. A clinical study.

BACKGROUND: Freeze-dried acellular dermal matrix (ADM) allograft, originally used for full-thickness burn wounds, was recently introduced as an alternative to the autogenous free gingival graft (FGG) in achieving increased attached keratinized tissue. The aim of part 1 of this study was to investigate the clinical efficacy of the ADM allograft for this particular purpose. METHODS: Twelve patients, 7 males and 5 females, with attached gingiva < or =1 mm on the facial aspect of mandibular anterior teeth demonstrating a tendency of progressive marginal tissue recession, were randomly assigned to either test or control treatment. Six patients received ADM graft (test) and 6 patients received an autogenous FGG harvested from the hard palate (control). Clinical variables including plaque index (PI), gingival index (GI), probing depth (PD), attached tissue width (AT), and gingival recession (GR) were recorded immediately before surgery and at the 6-month postoperative visit. Patients were seen at 2, 4, 6, 8, and 12 weeks to monitor wound healing and oral hygiene performance (PI and GI). Graft width was also measured, in corono-apical direction, on individually involved teeth during the surgery. RESULTS: When values between baseline and 6 months were compared in both groups, there was no statistically significant difference in changes of PI, GI, PD, and GR (P>0.05) with the exception of PD in the FGG group (1.01 +/- 0.03 versus 1.27 +/- 0.20 mm, P= 0.042). There was a statistically significant (P <0.05) increase in AT in both groups. Although the ADM group received wider grafts than the FGG group (8.81 +/- 0.46 versus 6.70 +/- 0.89 mm), the AT gain was significantly smaller (2.59 +/- 0.92 versus 5.57 +/- 0.44 mm) and the graft shrinkage significantly greater (71 +/- 10% versus 16 +/- 12%) in the ADM group than in the FGG group (P<0.01). CONCLUSIONS: The results of this study suggest that in procedures aiming at increasing the width of attached gingiva: 1) the ADM allograft was less effective and less predictable than the autogenous FGG in terms of increasing attached keratinized tissue due to considerable shrinkage and inconsistent quality of the attached tissue gained and 2) the esthetic results using the ADM allograft might be better than those using the autogenous FGG.

Successful treatment of pulpal-periodontal combined lesion in a birooted maxillary lateral incisor with concomitant palato-radicular groove. A case report.

Palato-radicular groove (PRG) is a common developmental anomaly of maxillary incisors, whereas PRG associated with a birooted maxillary incisor is relatively infrequent. The clinical significance of PRG is related to the incidence of localized periodontitis with or without pulpal pathosis, depending on the depth, extent, and complexity of the groove. Successful treatments of PRG in single-rooted incisors have been reported in the literature. However, treatment of PRG in birooted incisors has often been ineffective. This case report describes a pulpal-periodontal combined lesion occurring on a birooted maxillary left lateral incisor with concomitant PRG in a 13-year-old boy which was successfully treated by conventional endodontic therapy in combination with periodontal treatment including accessory root resection, radiculoplasty and bone grafting. Seven-year follow-up is included in this report. The basis of a successful result is accurate diagnosis and elimination of inflammatory irritants and contributory factors. Awareness of the existence of this abnormality by the clinician is important.

[Vertical root fracture--case report and clinical evaluation].

It is observed at the Dental OPD that some of the patients suffer from toothache are due to tooth fracture. Of the various types of tooth fracture, it is noted that vertical root fracture of molars which have never undergone endodontic treatment has rarely been reported. It is suggested that x-ray examination is the best diagnostic tool as it would show a certain section of the abnormally large root canal. This will indicate a fracture in the root. As the fractured root is mostly very poor in prognosis, the tooth in question should be extracted or it can be treated with root amputation or hemisection to eradicate the fractured root. Three cases of vertical root fracture are reported. The intent of this paper is to probe such cases, the means of diagnosis, and their treatments in clinical practices. Dental colleagues are thus advised to take into consideration the possibility of vertical root fracture whenever patients complain about toothache without any apparent cause.