Specific recognition of cyclic oligonucleotides by Cap4 for phage infection.

Under selective pressure, bacteria have evolved diverse defense systems against phage infections. The SMODS-associated and fused to various effector domains (SAVED)-domain containing proteins were identified as major downstream effectors in cyclic oligonucleotide-based antiphage signaling system (CBASS) for bacterial defense. Recent study structurally characterizes a cGAS/DncV-like nucleotidyltransferase (CD-NTase)-associated protein 4 from Acinetobacter baumannii (AbCap4) in complex with 2'3'3'-cyclic AMP-AMP-AMP (cAAA). However, the homologue Cap4 from Enterobacter cloacae (EcCap4) is activated by 3'3'3'-cyclic AMP-AMP-GMP (cAAG). To elucidate the ligand specificity of Cap4 proteins, we determined the crystal structures of full-length wild-type and K74A mutant of EcCap4 to 2.18 and 2.42 Å resolution, respectively. The DNA endonuclease domain of EcCap4 shares similar catalytic mechanism with type II restriction endonuclease. Mutating the key residue K74 in the conserved DXn(D/E)XK motif completely abolishes its DNA degradation activity. The potential ligand-binding cavity of EcCap4 SAVED domain is located adjacent to its N-terminal domain, significantly differing from the centrally located cavity of AbCap4 SAVED domain which recognizes cAAA. Based on structural and bioinformatic analysis, we found that Cap4 proteins can be classified into two types: the type I Cap4, like AbCap4, recognize cAAA and the type II Cap4, like EcCap4, bind cAAG. Several conserved residues identified at the surface of potential ligand-binding pocket of EcCap4 SAVED domain are confirmed by ITC experiment for their direct binding roles for cAAG. Changing Q351, T391 and R392 to alanine abolished the binding of cAAG by EcCap4 and significantly reduced the anti-phage ability of the E. cloacae CBASS system constituting EcCdnD (CD-NTase in clade D) and EcCap4. In summary, we revealed the molecular basis for specific cAAG recognition by the C-terminal SAVED domain of EcCap4 and demonstrates the structural differences for ligand discrimination among different SAVED-domain containing proteins.

Identification of Key Pathways Involved in White Strain of Hypsizygus marmoreus Extracts-Induced Cell Death of Human Hepatoma Hep3B Cells by Next Generation Sequencing.

White strain of Hypsizygus marmoreus is named as white genius mushroom (WGM) and is a popular food in Taiwan. We have confirmed the cytotoxicity of WGM extracts on human Hep3B liver cancer cells. A total of 8711 significantly differential genes were identified through large-scale transcriptome sequencing. According to the KEGG pathway enrichment analysis, autophagy, mitophagy and apoptosis pathways were identified as significant in WGM extracts-treated cells. WGM extracts induced a dose-dependent generation of reactive oxygen species (ROS) and membrane-enclosed vacuoles in Hep3B cells. The inhibition of ROS by the ROS scavengers blocked the induction of cell death and vacuoles formation. We suggested that the cell death and membrane-enclosed vacuoles induced by WGM extracts are dependent on ROS production in Hep3B cells. (2E,6E)-3,7,11,15,19,23,27,31,35-Nonamethylhexatriaconta-2,6,34-triene-1,11,15,19,23,27,31-heptol and (18:2) lysophosphatidylcholine were identified in WGM extracts. In addition to being a very popular edible mushrooms, WGM may be developed into a dietary supplement or dietary chemopreventive agent for the cancer treatment.

Repeated centrifuging and washing concentrates bacterial samples in peritoneal dialysis for optimal culture: an original article.

BACKGROUND: Bacterial cultures allow the identification of infectious disease pathogens. However, obtaining the results of conventional culture methods is time-consuming, taking at least two days. A more efficient alternative is the use of concentrated bacterial samples to accelerate culture growth. Our study focuses on the development of a high-yield sample concentrating technique. RESULTS: A total of 71 paired samples were obtained from patients on peritoneal dialysis (PD). The peritoneal dialysates were repeat-centrifuged and then washed with saline, namely the centrifuging and washing method (C&W method). The concentrated samples were Gram-stained and inoculated into culture plates. The equivalent unprocessed dialysates were cultured as the reference method. The times until culture results for the two methods were compared. The reference method yielded no positive Gram stain results, but the C&W method immediately gave positive Gram stain results for 28 samples (p < 0.001). The culture-negative rate was lower in the C&W method (5/71) than in the reference method (13/71) (p = 0.044). The average time for bacterial identification achieved with the C&W method (22.0 h) was shorter compared to using the reference method (72.5 h) (p < 0.001). CONCLUSIONS: The C&W method successfully concentrated bacterial samples and superseded blood culture bottles for developing adequate bacterial cultures. The C&W method may decrease the culture report time, thus improving the treatment of infectious diseases.

Brown Root Rot Disease of Phyllanthus myrtifolius: The Causal Agent and Two Potential Biological Control Agents.

Brown root rot (BRR), caused by Phellinus noxius (Corner) G. Cunningham, occurs on over 200 species of plants, especially woody trees and shrubs. Ceylon myrtle (Phyllanthus myrtifolius [Wight] Müll.Arg.), a common hedge plant, was recently observed to be infected with BRR. Disease diagnosis was performed by completing Koch's postulates, and Ceylon myrtle was confirmed to be a new host of P. noxius. Typical symptoms of BRR were observed, including reduction in leaf size, dieback of branches, and suspended growth of young leaves. A disease severity index was used to quantify BRR in this study. Compared with Malabar chestnut, Ceylon myrtle was relatively resistant to BRR. Surprisingly, phylogenetic analysis of the ITS and 28S sequences revealed that isolates identified as P. noxius from Taiwan and many other countries were clustered in the same clade but separate from the clade comprising isolates from China, which were designated Pyrrhoderma noxium based on P. noxius. Therefore, to temporarily distinguish these pathogens, the former clade was designated GPN (global P. noxius), whereas the latter clade was designated CPN (China Py. noxium). In biocontrol assays, Streptomyces padanus and Bacillus sp. were selected for BRR control of Ceylon myrtle. Disease severity was reduced from 0.51 to 0.37 by S. padanus and to 0.14 by Bacillus sp. in greenhouse trials. In addition, the two biocontrol agents, especially S. padanus, exhibited good growth-promoting effects on cuttings of Ceylon myrtle. With these double advantages, S. padanus and Bacillus sp. have great potential to control BRR in practical applications.

Polyketide Synthase Gene Expression in Relation to Chloromonilicin and Melanin Production in Monilinia fructicola.

Monilinia fructicola is a fungal pathogen of worldwide significance that causes brown rot of stone fruits. There are only few reports related to the production of biologically active polyketides by this pathogen. In this study, we examined an atypical M. fructicola strain TW5-4 that shows strong antimicrobial activity against various plant pathogens. TW5-4 also displays sparse growth in culture, low virulence, and higher levels of melanin compared with its albino mutant, TW5-4WM, and a wild-type strain Mf13-81. Antifungal compounds were extracted from TW5-4 and purified by thin-layer chromatography following visualization with an on-the-chromatogram inhibition assay. The principal antifungal compound was identified by linear ion trap mass spectrometry, high-resolution electro-spray ionization mass spectrometry, and proton nuclear magnetic resonance analyses as the polyketide chloromonilicin. Multiple M. fructicola polyketide synthase (PKS) sequences were then cloned by degenerate PCR and inverse PCR. Sequence analyses support presence of a 10-member PKS gene family in the M. fructicola genome. Analyses of PKS gene expression found no strong correlation between chloromonilicin production in culture and transcript levels of any of the PKS gene family members in mycelium of strains TW5-4, TW5-4WM, and Mf13-81. However, MfPKS12, a homolog of BcPKS12 involved in biosynthesis of 1,8-dihydroxynaphthalene (DHN)-melanin in Botrytis cinerea, was strongly expressed in mycelia of TW5-4 and Mf13-81. An MfPKS12-silenced mutant accumulated significantly less melanin in mycelia, had lower resistance to polyethylene glycol-induced osmotic stress, and displayed reduced virulence on nectarine fruit. The results suggest that DHN-melanin is required for tolerance to osmotic stress and full virulence in M. fructicola.

Fenofibrate induces human hepatoma Hep3B cells apoptosis and necroptosis through inhibition of thioesterase domain of fatty acid synthase.

This study demonstrated that fenofibrate, a lipid-lowering drug, induced a significant time-dependent cytotoxicity of hepatoma Hep3B cells. Hep3B cells are significantly more sensitive to cell killing by fenofibrate than hepatoma HepG2, lung cancer CH27 and oral cancer HSC-3 cells. From the result of docking simulation, fenofibrate can bind excellently to the thioesterase domain of fatty acid synthase (FASN) binding site as orlistat, a FASN inhibitor, acts. The fenofibrate-induced cell cytotoxicity was protected by addition of palmitate, indicating that the cytotoxic effect of fenofibrate is due to starvation of Hep3B cells by inhibiting the formation of end product in the FASN reaction. Inhibition of lipid metabolism-related proteins expression, such as proteins containing thioesterase domain and fatty acid transport proteins, was involved in the fenofibrate-induced Hep3B cell death. Fenofibrate caused S and G2/M cell cycle arrest by inducing cyclin A/Cdk2 and reducing cyclin D1 and E protein levels in Hep3B cells. The anti-tumor roles of fenofibrate on Hep3B cells by inducing apoptosis and necroptosis were dependent on the expression of Bcl-2/caspase family members and RIP1/RIP3 proteins, respectively. These results suggest that fenofibrate has an anti-cancer effect in Hep3B cells and inhibition of lipid metabolism may be involved in fenofibrate-induced Hep3B cells apoptosis and necroptosis.

Orlistat Displays Antitumor Activity and Enhances the Efficacy of Paclitaxel in Human Hepatoma Hep3B Cells.

Orlistat has been proved to be an effective fatty acid synthase inhibitor that is able to inhibit the proliferation and induce apoptosis in many cancer cell types. However, the anticancer effects of orlistat on hepatocellular carcinoma are undefined. We found that orlistat inhibited cell growth and induced G0/G1 cell cycle arrest with increased cyclin D, cyclin E, and p21 expression in human hepatoma Hep3B cells. Furthermore, protein expression of cyclin A, cyclin B, Cdk1, Cdk2, and Cdk4 was reduced by orlistat. This study investigated the role of lipid metabolism on orlistat-induced human hepatoma Hep3B cell death. The decrease in the expression of key enzymes in fatty acid metabolism, including FASN, ACOT8, PPT1, FABP1, CPT1 and CPT2, was observed after orlistat treatment. We also demonstrated that peroxisomal activity was involved in the orlistat-induced Hep3B cell death. In this study, we established an in vitro model to investigate the effect of orlistat on lipid accumulation. We found that orlistat significantly inhibited the cellular lipid content when administered in fatty acid overload conditions in Hep3B cells. Combination treatment of orlistat and paclitaxel was able to induce a synergistic effect on growth inhibition and cell apoptosis in Hep3B cells. Our data suggested that orlistat displays antitumor activity and enhances the efficacy of paclitaxel in Hep3B cells.

Lepista sordida Water Extract Enhances the Maturation of Mouse Dendritic Cells in Vitro and in Vivo.

BACKGROUND: Lepista sordida (LS) extract has been shown to possess anti-oxidant, anti-aging, and anti-tumor activities. However, the immunostimulatory effect of LS extract has not been elucidated. OBJECTIVE: To characterize the impact of a water extract of LS (WE-LS) on the maturation and function of mouse dendritic cell (DC) in vitro and in vivo. METHODS: Mouse bone marrow-derived DCs (BMDCs) were generated. Next, DC maturation was determined by flow cytometry, and cytokine production was measured by ELISA after WE-LS treatment. In addition, DC-induced OVA-specific T cell activation was assayed by [3H]-thymidine incorporation assay. Furthermore, the in vivo effects of WE-LS on DC maturation and Th1 responses in the spleens of mice were assessed by flow cytometry. RESULTS: WE-LS treatment up-regulated co-stimulatory (CD40 and CD80) and MHC class II molecules, increased the production of tumor necrosis factor-alpha (TNF-α), IL-6 and IL-12, and enhanced both the proliferation and IFN-γ secretion of allogenic T cells in BMDCs, partially mediated by the TLR2 and TLR4 signaling pathways. Moreover, the in vivo administration of WE-LS to mice enhanced the up-regulation of CD40, CD80 and MHC class II molecules in spleen DCs. WE-LS also increased the generation of T helper type 1 (Th1) cells in vivo. CONCLUSION: These results suggest that WE-LS might have the potential to promote immunity against infection and cancer or to serve as an adjuvant in vaccines and immunotherapies.

Induction of apoptosis and ganoderic acid biosynthesis by cAMP signaling in Ganoderma lucidum.

Apoptosis is an essential physiological process that controls many important biological functions. However, apoptosis signaling in relation to secondary metabolite biosynthesis in plants and fungi remains a mystery. The fungus Ganoderma lucidum is a popular herbal medicine worldwide, but the biosynthetic regulation of its active ingredients (ganoderic acids, GAs) is poorly understood. We investigated the role of 3',5'-cyclic adenosine monophosphate (cAMP) signaling in fungal apoptosis and GA biosynthesis in G. lucidum. Two phosphodiesterase inhibitors (caffeine and 3-isobutyl-1-methylxanthine, IBMX) and an adenylate cyclase activator (sodium fluoride, NaF) were used to increase intracellular cAMP levels. Fungal apoptosis was identified by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay and a condensed nuclear morphology. Our results showed that GA production and fungal apoptosis were induced when the mycelium was treated with NaF, caffeine, or cAMP/IBMX. Downregulation of squalene synthase and lanosterol synthase gene expression by cAMP was detected in the presence of these chemicals, which indicates that these two genes are not critical for GA induction. Transcriptome analysis indicated that mitochondria might play an important role in cAMP-induced apoptosis and GA biosynthesis. To the best of our knowledge, this is the first report to reveal that cAMP signaling induces apoptosis and secondary metabolite production in fungi.

Diagnosis of bacterial pathogens in the dialysate of peritoneal dialysis patients with peritonitis using surface-enhanced Raman spectroscopy.

BACKGROUND: Bacterial peritonitis is the most common cause of peritoneal dialysis (PD) therapy drop-out. A quick and accurate diagnosis of the bacterial pathogen can reduce the PD drop-out rate. Surface-enhanced Raman spectroscopy (SERS) can rapidly identify bacteria using chips coated with nano-sized metal particles. METHODS: Known bacteria were loaded in the SERS-chips and illuminated with laser light to establish a reference Raman spectra library. Dialysate from PD peritonitis patients was concentrated by centrifuge and examined with the same SERS, and the resulting Raman spectra were compared with library spectra for bacteria identification. Principal component analysis was used for further confirmation. The same batches of dialysate were sent to routine culture as a reference bacteria identification method. The results of the 2 identification methods were compared. RESULTS: A total of 43 paired-samples were sent for study. There were 37 samples with bacteria identified but 6 were culture-negative by the reference method. 31 bacteria were identified in paired-samples by SERS, among which, 29 bacteria were exactly the same as those identified by the reference method. Bacteria not included in the reference library spectra cannot be identified. CONCLUSIONS: SERS techniques can rapidly identify bacterial pathogens in the dialysate of PD peritonitis patients.

The Roles of 4ß-Hydroxywithanolide E from Physalis peruviana on the Nrf2-Anti-Oxidant System and the Cell Cycle in Breast Cancer Cells.

4[Formula: see text]-Hydroxywithanolide E is an active component of the extract of Physalis peruviana that has been reported to exhibit antitumor effects. Although the involvement of reactive oxygen species (ROS) production and the ataxia-telangiectasia mutated protein (ATM)-dependent DNA damage signaling pathway in 4[Formula: see text]-hydroxywithanolide E-induced apoptosis of breast cancer MCF-7 cells was demonstrated in our previous study, the relationship between ROS production and the cellular defense system response in 4[Formula: see text]-hydroxywithanolide E-induced cell death requires further verification. The present study suggests that ROS play an important role in 4[Formula: see text]-hydroxywithanolide E-induced MCF-7 cell death in which anti-oxidants, such as glutathione or N-acetylcysteine, can resist the 4[Formula: see text]-hydroxywithanolide E-induced accumulation of ROS and cell death. Furthermore, N-acetylcysteine or glutathione can reverse the 4[Formula: see text]-hydroxywithanolide E-induced changes in the cell cycle distribution and the expression of cell cycle regulators. We found that the 4[Formula: see text]-hydroxywithanolide E-induced ROS accumulation was correlated with the upregulation of Nrf2 and Nrf2-downstream genes, such as antioxidative defense enzymes. In general, the activity of Nrf2 is regulated by the Ras signalling pathway. However, we demonstrated that Nrf2 was activated during 4[Formula: see text]-hydroxywithanolide E-induced MCF-7 cell death in spite of the 4[Formula: see text]-hydroxywithanolide E-induced inhibition of the Ras/Raf/ERK pathway. The activity and protein expression of superoxide dismutase and catalase were involved in the 4[Formula: see text]-hydroxywithanolide E-induced ROS production in MCF-7 cells. Furthermore, 4[Formula: see text]-hydroxywithanolide E was demonstrated to significantly reduce the sizes of the tumor nodules in the human breast cancer MDA-MB231 xenograft tumor model.

Raf/ERK/Nrf2 signaling pathway and MMP-7 expression involvement in the trigonelline-mediated inhibition of hepatocarcinoma cell migration.

BACKGROUND: Trigonelline occurs in many dietary food plants and has been found to have anti-carcinogenic activity. Trigonelline is also found in coffee which is one of the most widely consumed beverages. Many epidemiological studies have reported that coffee consumption has an inverse relationship with the risk of cirrhosis or hepatocellular carcinoma. It would be interesting to investigate whether trigonelline is an ideal chemoprevent agent to prevent cancer progression. METHODS: The protein expression was performed by western blotting. The trigonelline content in snow pea (Pisum sativum) was analyzed by high-performance liquid chromatography (HPLC). The migratory activity of human hepatocarcinoma cells (Hep3B) was assessed by using a wound migration assay. The percentage of each phase in the cell cycle was analyzed on a FACScan flow cytometer. Gene expression was detected by real-time reverse transcriptase-polymerase chain reaction techniques. Native gel analysis was performed to analyze the activity of superoxide dismutase (SOD), catalase and glutathione peroxidase. RESULTS: According to the data of HPLC analysis, P. sativum, which is a popular vegetable, has relatively high content of trigonelline. Our findings suggest that trigonelline is an efficient compound for inhibiting Hep3B cell migration. Trigonelline inhibited the migration of hepatoma cells at concentrations of 75-100 µM without affecting proliferation. Raf/ERK/Nrf2 protein levels and further downstream antioxidative enzymes activity, such as SOD, catalase, and glutathione peroxidase, significantly decreased after treatment with 100 µM of trigonelline for 24 h. The migration inhibition of trigonelline is also related to its ability to regulate the matrix metalloproteinases 7 (MMP-7) gene expression. CONCLUSIONS: In this study, protein kinase Cα (PKCα) and Raf/ERK/Nrf2 signaling pathway and MMP-7 gene expression were involved in the trigonelline-mediated migration inhibition of Hep3B cells. We also demonstrated that trigonelline inhibits Hep3B cell migration through downregulation of nuclear factor E2-related factor 2-dependent antioxidant enzymes activity. This study analyzed the trigonelline content in a popular vegetable, snow pea, as a representative proof to prove that trigonelline is often found in the daily intake of food. Our finding suggested that trigonelline should be a useful chemopreventive agent derived from the daily intake of food to prevent cancer progression.

The medical diagnostic approaches with phylogenetic analysis for rare Brucella spp. diagnosis in Taiwan.

Brucellosis is a bacterial zoonotic disease which can be easy to misdiagnose in clinical microbiology laboratories. In the present study, we have tried to improve the current clinical method for detecting Brucella spp. and its antibiotic characteristics. Our method begins with detecting the clinical isolate through traditional biochemical methods and automatic identification systems. Then, we move on to editing the sequence for BLAST allows us to compare 16s rRNA sequences with sequences from other species, allowing the gene level to be determined. Next, the phylogenetic analysis of multiple genetic loci is able to determine the evolutionary relationships between our bacteria strain and those from other locations. Finally, an anti-microbial susceptibility test hones in on the level of antibacterial activity that the bacteria displays. Employing these four steps in concert is extremely effective in identifying rare bacteria. Thus, when attempting to determine the identity of rare bacteria such as Brucella, utilizing these four steps from our research should be highly effective and ultimately prevent further identification errors and misdiagnoses. The standards we have suggested to identify rare bacteria strains is applicable not only to Brucella, but also to other rarely encountered bacteria.

Nucleotide sequence-homology-independent breakdown of transgenic resistance by more virulent virus strains and a potential solution.

Controlling plant viruses by genetic engineering, including the globally important Papaya ringspot virus (PRSV), mainly involves coat protein (CP) gene mediated resistance via post-transcriptional gene silencing (PTGS). However, the breakdown of single- or double-virus resistance in CP-gene-transgenic papaya by more virulent PRSV strains has been noted in repeated field trials. Recombination analysis revealed that the gene silencing suppressor HC-Pro or CP of the virulent PRSV strain 5-19 is responsible for overcoming CP-transgenic resistance in a sequence-homology-independent manner. Transient expression assays using agro-infiltration in Nicotiana benthamiana plants indicated that 5-19 HC-Pro exhibits stronger PTGS suppression than the transgene donor strain. To disarm the suppressor from the virulent strain, transgenic papaya lines were generated carrying untranslatable 5-19 HC-Pro, which conferred complete resistance to 5-19 and other geographic PRSV strains. Our study suggested the potential risk of the emergence of more virulent virus strains, spurred by the deployment of CP-gene-transgenic crops, and provides a strategy to combat such strains.

Impact of interleukin-10 gene polymorphisms on survival in patients with colorectal cancer.

Chronic inflammation is thought to be the leading cause of colorectal cancer, and interleukin-10 (IL10) has been identified as a potent immunomodulatory cytokine that regulates inflammatory responses in the gastrointestinal tract. Although several single nucleotide polymorphisms (SNPs) in IL10 have been associated with the risk of colorectal cancer, their prognostic significance has not been determined. Two hundred and eighty-two colorectal cancer patients were genotyped for two candidate cancer-associated SNPs in IL10. The associations of these SNPs with distant metastasis-free survival and overall survival were evaluated by Kaplan-Meier analysis and Cox regression model. The minor homozygote GG genotype of IL10 rs3021094 was significantly associated with a 3.30-fold higher risk of death compared with the TT+TG genotypes (P=0.011). The patients with IL10 rs3021094 GG genotype also had a poorer overall survival in Kaplan-Meier analysis (log-rank P=0.007) and in multivariate Cox regression model (P=0.044) adjusting for age, gender, carcinoembryonic antigen levels, tumor differentiation, stage, lymphovascular invasion, and perineural invasion. In conclusion, our results suggest that IL10 rs3021094 might be a valuable prognostic biomarker for colorectal cancer patients.

Genetic polymorphisms of matrix metalloproteinases and clinical outcomes in colorectal cancer patients.

BACKGROUND: Colorectal cancer metastasis is a multistep process involving degradation of extracellular matrix components by proteolytic enzymes. Among them, matrix metalloproteinases (MMPs) are the principal degrading enzymes and their expressions/activities are also correlated with survival. Much research has showed the associations between genetic polymorphisms in MMPs and risk of colorectal cancer; however, their prognostic significance has not been well determined. METHODS: We selected and genotyped 4 cancer-associated single nucleotide polymorphisms (SNPs) in a cohort of 282 colorectal cancer patients. The associations of these SNPs with distant metastasis-free survival and overall survival were evaluated by Kaplan-Meier analysis, Cox regression model, and survival tree analysis. RESULTS: The relative risks of developing distant metastasis after curative surgery were higher in individuals with minor homozygote AA genotype than in those with GG/GA genotypes at MMP2 rs243866 (P = 0.012). Survival tree analysis also identified a higher-order genetic interaction profile consisting of MMP2 rs243866 and MMP2 rs2285053 that was significantly associated with distant metastasis-free survival (P trend = 0.016). After adjusting for possible confounders, the genetic interaction profile remained significant (P trend = 0.050). CONCLUSIONS: These results suggest that genetic variations in the MMP2 might be potential predictors of distant metastasis-free survival after curative surgery.

Common variants in IGF1 pathway genes and clinical outcomes after radical prostatectomy.

BACKGROUND: Insulin-like growth factor-1 (IGF1) pathway plays a critical role in malignant transformation, and epidemiology studies have also shown that single nucleotide polymorphisms (SNPs) in IGF1 pathway genes are associated with prostate cancer risk. However, the clinical significance of these SNPs on prostate cancer aggressiveness and prognosis after radical prostatectomy (RP) has not been determined. METHODS: We evaluated the associations of 4 common SNPs in IGF1 and IGF1R with age at diagnosis, preoperative prostate-specific antigen (PSA) level, pathologic Gleason score, pathologic stage, surgical margin, lymph node metastasis, and PSA recurrence in a cohort of 320 localized prostate cancer patients receiving RP. The prognostic significance on time to PSA recurrence was also assessed by Cox proportional hazards model. RESULTS: IGF1 rs2946834 alleles/genotypes and an IGF1 specific haplotype AT, containing the minor allele of rs2946834, were associated (P ≤ 0.028) with a 1.49- to 2.22-fold higher risk of having advanced-stage prostate cancer. In addition, a genetic interaction profile consisting of IGF1 rs2946834 and IGF1R rs2016347 was significantly associated with PSA recurrence (P = 0.033). CONCLUSIONS: Our study is the first to evaluate the impact of SNPs in IGF1 pathway genes on PSA recurrence. A genetic interaction between IGF1 rs2946834 and IGF1R rs2016347 might be a predictor of outcomes following RP.

Common genetic variants in Wnt signaling pathway genes as potential prognostic biomarkers for colorectal cancer.

Compelling evidence has implicated the Wnt signaling pathway in the pathogenesis of colorectal cancer. We assessed the use of tag single nucleotide polymorphisms (tSNPs) in adenomatous polyposis coli (APC)/ß-catenin (CTNNB1) genes to predict outcomes in patients with colorectal cancer. We selected and genotyped 10 tSNP to predict common variants across entire APC and CTNNB1 genes in 282 colorectal cancer patients. The associations of these tSNPs with distant metastasis-free survival and overall survival were evaluated by Kaplan-Meier analysis, Cox regression model, and survival tree analysis. The 5-year overall survival rate was 68.3%. Survival tree analysis identified a higher-order genetic interaction profile consisting of the APC rs565453, CTNNB1 2293303, and APC rs1816769 that was significantly associated with overall survival. The 5-year survival overall rates were 89.2%, 66.1%, and 58.8% for the low-, medium-, and high-risk genetic profiles, respectively (log-rank P = 0.001). After adjusting for possible confounders, including age, gender, carcinoembryonic antigen levels, tumor differentiation, stage, lymphovascular invasion, perineural invasion, and lymph node involvement, the genetic interaction profile remained significant. None of the studied SNPs were individually associated with distant metastasis-free survival and overall survival. Our results suggest that the genetic interaction profile among Wnt pathway SNPs might potentially increase the prognostic value in outcome prediction for colorectal cancer.

Diterpenoids with anti-inflammatory activity from the wood of Cunninghamia konishii.

Two new diterpenoids, konishone (1) and 3b-hydroxy-5,6-dehydrosugiol (2), along with three known diterpenoids--hinokiol (3), sugiol (4), and 12-hydroxy-6,7-secoabieta-8,11,13-triene-6,7-dial (5)--were isolated from the wood of Cunninghamia konishii. Compound 1 is a novel skeleton of the 7,20-dinorabietane-type diterpene. In addition, when RAW264.7 macrophages were treated with different concentrations of compounds 1, 3, and 5 together with LPS, a significant concentration-dependent inhibition of NO production was detected. The IC50 values for inhibition of nitrite production of compounds 1, 3, and 5 were about 9.8 ± 0.7, 7.9 ± 0.9, and 9.3 ± 1.3 µg/mL, respectively. This study presents the potential utilization of compounds 1, 3, and 5, as lead compounds for the development of anti-inflammatory drugs.

A novel approach to enhancing ganoderic acid production by Ganoderma lucidum using apoptosis induction.

Ganoderma lucidum is one of most widely used herbal medicine and functional food in Asia, and ganoderic acids (GAs) are its active ingredients. Regulation of GA biosynthesis and enhancing GA production are critical to using G. lucidum as a medicine. However, regulation of GA biosynthesis by various signaling remains poorly understood. This study investigated the role of apoptosis signaling on GA biosynthesis and presented a novel approach, namely apoptosis induction, to increasing GA production. Aspirin was able to induce cell apoptosis in G. lucidum, which was identified by terminal deoxynucleotidyl transferase mediated dUPT nick end labeling assay positive staining and a condensed nuclear morphology. The maximum induction of lanosta-7,9(11), 24-trien-3α-01-26-oic acid (ganoderic acid 24, GA24) production and total GA production by aspirin were 2.7-fold and 2.8-fold, respectively, after 1 day. Significantly lower levels of GA 24 and total GAs were obtained after regular fungal culture for 1.5 months. ROS accumulation and phosphorylation of Hog-1 kinase, a putative homolog of MAPK p38 in mammals, occurred after aspirin treatment indicating that both factors may be involved in GA biosynthetic regulation. However, aspirin also reduced expression of the squalene synthase and lanosterol synthase coding genes, suggesting that these genes are not critical for GA induction. To the best of our knowledge, this is the first report showing that GA biosynthesis is linked to fungal apoptosis and provides a new approach to enhancing secondary metabolite production in fungi.