Activation of PI3K/Akt/IKK-α/NF-κB signaling pathway is required for the apoptosis-evasion in human salivary adenoid cystic carcinoma: its inhibition by quercetin

Quercetin, one of the most common natural flavonoids, has been reported to possess significant anti-tumor activities both in vitro and in vivo. The present study was to investigate the effects of quercetin on growth and apoptosis in human salivary adenoid cystic carcinoma (ACC). The result from MTT assay showed that quercetin decreased cell viability of both low metastatic cell line ACC-2 and high metastatic cell line ACC-M in a concentration- and time-dependent manner. Moreover, treatment with quercetin resulted in significantly increased apoptosis in ACC cells. Our data also revealed that the apoptosis induced by quercetin treatment was through a mitochondria-dependent pathway which showed close correlation with the down-regulation of the PI3K/Akt/IKK-α/NF-κB pathway. Most importantly, quercetin significantly prevented in vivo growth of ACC xenografts in nude mice, accompanied by induction of tumor cell apoptosis, suppression of NF-κB nuclear translocation, as well as down-regulation of Akt and IKK-α activation. In addition, we explored the clinical significance of the PI3K/Akt/IKK-α/NF-κB signaling axis in ACC by immunohistochemical analysis of tissue specimens followed by the clustering analyses. We determined that the PI3K/Akt/IKK-α/NF-κB pathway is ubiquitously activated in ACC and plays an essential role in the evasion of apoptosis. Taken together, the results from our study implicated that quercetin would be a promising chemotherapeutic agent against ACC through its function of down-regulating the PI3K/Akt/IKK-α/NF-κB signaling pathway.     

Identification of two tungstate-sensitive molybdenum cofactor mutants, chl2 and chl7, of Arabidopsis thaliana.

Summary The characterization of mutants that are resistant to the herbicide chlorate has greatly increased our understanding of the structure and function of the genes required for the assimilation of nitrate. Hundreds of chlorate-resistant mutants have been identified in plants, and almost all have been found to be defective in nitrate reduction due to mutations in either nitrate reductase (NR) structural genes or genes required for the synthesis of the NR cofactor molybdenum-pterin (MoCo). The chlorate-resistant mutant ofArabidopsis thaliana, ch12, is also impaired in nitrate reduction, but the defect responsible for this phenotype has yet to be explained.chl2 plants have low levels of NR activity, yet the map position of thechl2 mutation is clearly distinct from that of the two NR structural genes that have been identified inArabidopsis. In addition,chl2 plants are not thought to be defective in MoCo, as they have near wild-type levels of xanthine dehydrogenase activity, which has been used as a measure of MoCo in other organisms. These results suggest thatchl2 may be a NR regulatory mutant. We have examinedchl2 plants and have found that they have as much NR (NIA2) mRNA as wild type a variable but often reduced level of NR protein, and one-eighth the NR activity of wild-type plants. It is difficult to explain these results by a simple regulatory model; therefore, we reexamined the MoCo levels inchl2 plants using a sensitive, specific assay for MoCo: complementation ofNeurospora MoCo mutant extracts. We found thatchl2 has low levels of MoCo — about one-eighth the wild-type level and less than the level in anotherArabidopsis MoCo mutantchl6 (B73). To confirm this result we developed a new diagnostic assay for MoCo mutants, growth inhibition by tungstate. Bothchl2 andchl6 are sensitive to tungstate at concentrations that have no effect on wildtype plants. The tungstate sensitivity as well as the chlorate resistance, low NR activity and low MoCo levels all cosegregate, indicating that all are due to a single mutation that maps to thechl2 locus, 10 centimorgans fromerecta on chromosome 2. We also report on the isolation of a new chlorate-resistant mutant ofArabidopsis, ch17, which is a MoCo mutant with the same phenotypes aschl2 andchl6.     

Mapping and Isolation of Quantitative Trait Loci Controlling Plant Height and Heading Date in Rice

Quantitative trait loci (QTL) were identified for heading date and plant height in rice ( Oryza sativa L.) using a recombinant inbred line population consisting of 241 lines. Totally 4 QTLs for heading date and 4 QTLs for plant height were detected in three years. The QTL with large effects located in the interval C1023-R1440 on chromosome 7 was simultaneously detected in three years for both traits. In order to distinguished the interval whether contained one QTL with pleiotropy effect or two close linked QTLs, a recombinant line RIL50, whose genetic background was high similar to Zhenshan 97 except the regions covered the major QTL from Minghui 63, was selected to cross with Zhenshan 97. A BC1F2 population from the cross, which could be regarded as near isogenic lines (NIL) with the targeted QTL (QTL-NIL), was used to collect heading date and plant height data. The frequency distribution of the two traits in the BC1F2 population was bimodal, and their segregation ratios were in accordance with the expected Mendelian inheritance ratios. Normally, the short plants flowered early in the population, the high plants with late heading date, but the relationships between the plant height and the heading date of 6 plants conflicted with the case. The above results clearly demonstrated that QTL could be treated as single Mendelian factor. Moreover, there are two close linked genes controlling the heading date and the plant height on chromosome 7, respectively.     

Y-27632, a ROCK Inhibitor,Promoted Limbal Epithelial Cell Proliferation and Corneal Wound Healing

Transplantation of ex vivo cultured limbal epithelial cells is proven effective in restoring limbal stem cell deficiency. The present study aimed to investigate the promoting effect of Y-27632 on limbal epithelial cell proliferation. Limbal explants isolated from human donor eyes were expanded three weeks on culture dishes and outgrowth of epithelial cells was subsequently subcultured for in vitro experiments. In the presence of Y-27632, the ex vivo limbal outgrowth was accelerated, particularly the cells with epithelial cell-like morphology. Y-27632 dose-dependently promoted the proliferation of in vitro cultured human limbal epithelial cells as examined by phase contrast microscopy and luminescent cell-viability assay 30 hours after the treatment. The colony forming efficacy determined 7 days after the treatment was enhanced by Y-27632 also in a dose-dependent manner. The number of p63- or Ki67-positive cells was dose-dependently increased in Y-27632-treated cultures as detected by immunofluorescent staining and western blotanalysis. Cell cycle analysis by flow cytometric method revealed an increase in S-phase proliferating cells. The epithelial woundclosure rate was shown to be faster in experimental group received topical treatment withY-27632 than the sham control using a rat corneal wounding model. These resultsdemonstrate that Y-27632 can promote both the ex vivo and in vitro proliferation oflimbal epithelial cell proliferation. The in vivo enhanced epithelial wound healingfurther implies that the Y-27632 may act as a new strategy for treating limbal stem cell deficiency.     

Factors Associated with Blood Culture Contamination in the Emergency Department: Critical Illness,End-Stage Renal Disease,and Old Age

Background

Blood culture contamination in emergency departments (ED) that experience a high volume of patients has negative impacts on optimal patient care. It is therefore important to identify risk factors associated with blood culture contamination in EDs.

Methodology/Principal Findings

A prospectively observational study in a university-affiliated hospital were conducted between August 2011 and December 2012. Positive monomicrobial and negative blood cultures drawn from adult patients in the ED were analyzed to evaluate the possible risk factors for contamination. A total of 1,148 positive monomicrobial cases, 391 contamination cases, and 13,689 cases of negative blood culture were identified. Compared to patients with negative blood cultures, patients in triage levels 1 and 2 (Incidence Rate Ratio, IRR = 2.24), patients with end-stage renal disease (ESRD) (IRR = 2.05), and older patients (IRR: 1.02 per year) were more likely to be associated with ED blood culture contamination.

Conclusions/Significance

Critical patients (triage levels 1 and 2), ESRD patients, and older patients were more commonly associated with blood culture contamination in the ED. Further studies to evaluate whether the characteristics of skin commensals contribute to blood culture contamination is warranted, especially in hospitals populated with high-risk patients.     

Characterization of an Insecticidal Toxin and Pathogenicity of Pseudomonas taiwanensis against Insects

Pseudomonas taiwanensis is a broad-host-range entomopathogenic bacterium that exhibits insecticidal activity toward agricultural pests Plutella xylostella, Spodoptera exigua, Spodoptera litura, Trichoplusia ni and Drosophila melanogaster. Oral infection with different concentrations (OD = 0.5 to 2) of wild-type P. taiwanensis resulted in insect mortality rates that were not significantly different (92.7%, 96.4% and 94.5%). The TccC protein, a component of the toxin complex (Tc), plays an essential role in the insecticidal activity of P. taiwanensis. The ΔtccC mutant strain of P. taiwanensis, which has a knockout mutation in the tccC gene, only induced 42.2% mortality in P. xylostella, even at a high bacterial dose (OD = 2.0). TccC protein was cleaved into two fragments, an N-terminal fragment containing an Rhs-like domain and a C-terminal fragment containing a Glt symporter domain and a TraT domain, which might contribute to antioxidative stress activity and defense against macrophagosis, respectively. Interestingly, the primary structure of the C-terminal region of TccC in P. taiwanensis is unique among pathogens. Membrane localization of the C-terminal fragment of TccC was proven by flow cytometry. Sonicated pellets of P. taiwanensis ΔtccC strain had lower toxicity against the Sf9 insect cell line and P. xylostella larvae than the wild type. We also found that infection of Sf9 and LD652Y-5d cell lines with P. taiwanensis induced apoptotic cell death. Further, natural oral infection by P. taiwanensis triggered expression of host programmed cell death-related genes JNK-2 and caspase-3.     

Association between Phosphorylated AMP-Activated Protein Kinase and Acetyl-CoA Carboxylase Expression and Outcome in Patients with Squamous Cell Carcinoma of the Head and Neck

Background

Epidemiological studies have indicated that impaired glucose metabolism may increase the risk of squamous cell carcinoma of the head and neck (SCCHN). AMP-activated protein kinase (AMPK) regulates glucose and lipid metabolism via the phosphorylation and subsequent inactivation of its downstream target acetyl-CoA carboxylase (ACC).Thus, we analyzed the expression of pAMPK and its downstream target phosphorylated acetyl-CoA carboxylase (pACC), as well as their impact on the survival of patients with resected SCCHN.

Methods

One hundred eighteen patients with surgically resected SCCHN were enrolled. Immunohistochemical (IHC) staining for pAMPK and pACC was performed using tissue microarrays of operative specimens of SCCHN. The expression was divided into two or three groups according to the IHC score [pAMPK: negative (0), positive (1–3); pACC: negative (0), low expression (1, 2), and high expression (3)]. Statistical analysis was performed to determine the association of pAMPK expression with clinicopathological features and pACC and pErk expression.

Results

The positive rates of pAMPK and pACC expression were 64.4% (76/118) and 68.6% (81/118), respectively. pAMPK was significantly higher in patients aged younger than 60 years (P = 0.024; χ²test) and those with early-stage (T1/T2; P = 0.02; χ² test) and oral cavity (P = 0.026; Fisher’s exact test) tumors. In multivariate analysis, pAMPK expression was not significantly correlated with overall survival (OS) (adjusted hazard ratio [HR]: 0.66; 95% confidence interval [CI]: 0.35–1.23), whereas high pACC expression was independently associated with worse OS in node-positive patients (adjusted HR: 17.58; 95% CI: 3.50–88.18).

Conclusions

Strong expression of pACC was found to be an independent prognostic marker for patients with node-positive SCCHN. Our results suggest that pACC may play a role in tumor progression of SCCHN and may help to identify patient subgroups at high risk for poor disease outcome.