PW06 Triggered Fas-FADD to Induce Apoptotic Cell Death In Human Pancreatic Carcinoma MIA PaCa-2 Cells through the Activation of the Caspase-Mediated Pathway.

Pancreatic cancer has higher incidence and mortality rates worldwide. PW06 [(E)-3-(9-ethyl-9H-carbazol-3-yl)-1-(2,5-dimethoxyphenyl) prop-2-en-1-one] is a carbazole derivative containing chalcone moiety which was designed for inhibiting tumorigenesis in human pancreatic cancer. This study is aimed at investigating PW06-induced anticancer effects in human pancreatic cancer MIA PaCa-2 cells in vitro. The results showed PW06 potent antiproliferative/cytotoxic activities and induced cell morphological changes in a human pancreatic cancer cell line (MIA PaCa-2), and these effects are concentration-dependent (IC50 is 0.43 µM). Annexin V and DAPI staining assays indicated that PW06 induced apoptotic cell death and DNA condensation. Western blotting indicated that PW06 increased the proapoptotic proteins such as Bak and Bad but decreased the antiapoptotic protein such as Bcl-2 and Bcl-xL. Moreover, PW06 increased the active form of caspase-8, caspase-9, and caspase-3, PARP, releasing cytochrome c, AIF, and Endo G from mitochondria in MIA PaCa-2 cells. Confocal laser microscopy assay also confirmed that PW06 increased Bak and decreased Bcl-xL. Also, the cells were pretreated with inhibitors of caspase-3, caspase-8, and caspase-9 and then were treated with PW06, resulting in increased viable cell number compared to PW06 treated only. Furthermore, PW06 showed a potent binding ability with hydrophobic interactions in the core site of the Fas-Fas death domains (FADD). In conclusion, PW06 can potent binding ability to the Fas-FADD which led to antiproliferative, cytotoxic activities, and apoptosis induction accompanied by the caspase-dependent and mitochondria-dependent pathways in human pancreatic cancer MIA PaCa-2 cells.

Dissecting the Transcriptomes of Multiple Metronidazole-Resistant and Sensitive Trichomonas vaginalis Strains Identified Distinct Genes and Pathways Associated with Drug Resistance and Cell Death.

Trichomonas vaginalis is the causative agent of trichomoniasis, the most prevalent non-viral sexually transmitted infection worldwide. Metronidazole (MTZ) is the mainstay of anti-trichomonal chemotherapy; however, drug resistance has become an increasingly worrying issue. Additionally, the molecular events of MTZ-induced cell death in T. vaginalis remain elusive. To gain insight into the differential expression of genes related to MTZ resistance and cell death, we conducted RNA-sequencing of three paired MTZ-resistant (MTZ-R) and MTZ-sensitive (MTZ-S) T. vaginalis strains treated with or without MTZ. Comparative transcriptomes analysis identified that several putative drug-resistant genes were exclusively upregulated in different MTZ-R strains, such as ATP-binding cassette (ABC) transporters and multidrug resistance pumps. Additionally, several shared upregulated genes among all the MTZ-R transcriptomes were not previously identified in T. vaginalis, such as 5'-nucleotidase surE and Na+-driven multidrug efflux pump, which are a potential stress response protein and a multidrug and toxic compound extrusion (MATE)-like protein, respectively. Functional enrichment analysis revealed that purine and pyrimidine metabolisms were suppressed in MTZ-S parasites upon drug treatment, whereas the endoplasmic reticulum-associated degradation (ERAD) pathway, proteasome, and ubiquitin-mediated proteolysis were strikingly activated, highlighting the novel pathways responsible for drug-induced stress. Our work presents the most detailed analysis of the transcriptional changes and the regulatory networks associated with MTZ resistance and MTZ-induced signaling, providing insights into MTZ resistance and cell death mechanisms in trichomonads.

Ergosta-7, 9 (11), 22-trien-3ß-ol Interferes with LPS Docking to LBP, CD14, and TLR4/MD-2 Co-Receptors to Attenuate the NF-κB Inflammatory Pathway In Vitro and Drosophila.

Ergosta-7, 9 (11), 22-trien-3ß-ol (EK100) was isolated from Cordyceps militaris, which has been used as a traditional anti-inflammatory medicine. EK100 has been reported to attenuate inflammatory diseases, but its anti-inflammatory mechanism is still unclear. We were the first to investigate the effect of EK100 on the Toll-like receptor 4 (TLR4)/nuclear factor of the κ light chain enhancer of B cells (NF-κB) signaling in the lipopolysaccharide (LPS)-stimulated RAW264.7 cells and the green fluorescent protein (GFP)-labeled NF-κB reporter gene of Drosophila. EK100 suppressed the release of the cytokine and attenuated the mRNA and protein expression of pro-inflammatory mediators. EK100 inhibited the inhibitor kappa B (IκB)/NF-κB signaling pathway. EK100 also inhibited phosphatidylinositol-3-kinase (PI3K)/Protein kinase B (Akt) signal transduction. Moreover, EK100 interfered with LPS docking to the LPS-binding protein (LBP), transferred to the cluster of differentiation 14 (CD14), and bonded to TLR4/myeloid differentiation-2 (MD-2) co-receptors. Compared with the TLR4 antagonist, resatorvid (CLI-095), and dexamethasone (Dexa), EK100 suppressed the TLR4/AKT signaling pathway. In addition, we also confirmed that EK100 attenuated the GFP-labeled NF-κB reporter gene expression in Drosophila. In summary, EK100 might alter LPS docking to LBP, CD14, and TLR4/MD-2 co-receptors, and then it suppresses the TLR4/NF-κB inflammatory pathway in LPS-stimulated RAW264.7 cells and Drosophila.

LINC01348 suppresses hepatocellular carcinoma metastasis through inhibition of SF3B3-mediated EZH2 pre-mRNA splicing.

Long non-coding RNAs (lncRNA) play crucial roles in hepatocellular carcinoma (HCC) progression. However, the specific functions of lncRNAs in alternative splicing (AS) and the metastatic cascade in liver cancer remain largely unclear. In this study, we identified a novel lncRNA, LINC01348, which was significantly downregulated in HCC and correlated with survival functions in HCC patients. Ectopic expression of LINC01348 induced marked inhibition of cell growth, and metastasis in vitro and in vivo. Conversely, these phenotypes were reversed upon knockdown of LINC01348. Mechanistically, LINC01348 complexed with splicing factor 3b subunit 3 (SF3B3) acted as a modulator of EZH2 pre-mRNA AS, and induced alterations in JNK/c-Jun activity and expression of Snail. Notably, C-terminal truncated HBx (Ct-HBx) negatively regulated LINC01348 through c-Jun signaling. Our data collectively highlight those novel regulatory associations involving LINC01348/SF3B3/EZH2/JNK/c-Jun/Snail are an important determinant of metastasis in HCC cells and support the potential utility of targeting LINC01348 as a therapeutic strategy for HCC.

Imperatorin Interferes with LPS Binding to the TLR4 Co-Receptor and Activates the Nrf2 Antioxidative Pathway in RAW264.7 Murine Macrophage Cells.

Imperatorin (IMP) could downregulate several inflammatory transcription factor signaling pathways. Some studies have pointed out that IMP could interfere with toll-like receptor 4 (TLR4) signaling. This study evaluates how IMP interferes with the TLR4 co-receptors signaling through the protein-ligand docking model, Western blotting, immunofluorescence (IF), and atomic force microscopy (AFM) assays in lipopolysaccharide (LPS) stimulated macrophage-like RAW264.7 cells in vitro. The results of the protein-ligand docking demonstrate that IMP interferes with LPS binding to the LPS-binding protein (LBP), the cluster of differentiation 14 (CD14), and the toll-like receptor 4/myeloid differentiation factor 2 (TLR4/MD-2) co-receptors in LPS-stimulated RAW264.7 cells. Compared with TLR4 antagonist CLI-095 or dexamethasone, IMP could suppress the protein expressions of LBP, CD14, and TLR4/MD-2 in LPS-stimulated cells. Furthermore, the three-dimensional (3D) image assay of the AFM showed IMP could prevent the LPS-induced morphological change in RAW264.7 cells. Additionally, IMP could activate the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway, and it increased the antioxidative protein expression of heme oxygenase-1 (HO-1), superoxidase dismutase (SOD), and catalase (CAT). Our results are the first to reveal that the anti-inflammatory effect of IMP interferes with LPS binding to TLR4 co-receptor signaling and activates the antioxidative Nrf2 signaling pathway.

An essential role of acetyl coenzyme A in the catalytic cycle of insect arylalkylamine N-acetyltransferase.

Acetyl coenzyme A (Ac-CoA)-dependent N-acetylation is performed by arylalkylamine N-acetyltransferase (AANAT) and is important in many biofunctions. AANAT catalyzes N-acetylation through an ordered sequential mechanism in which cofactor (Ac-CoA) binds first, with substrate binding afterward. No ternary structure containing AANAT, cofactor, and substrate was determined, meaning the details of substrate binding and product release remain unclear. Here, two ternary complexes of dopamine N-acetyltransferase (Dat) before and after N-acetylation were solved at 1.28 Å and 1.36 Å resolution, respectively. Combined with the structures of Dat in apo form and Ac-CoA bound form, we addressed each stage in the catalytic cycle. Isothermal titration calorimetry (ITC), crystallography, and nuclear magnetic resonance spectroscopy (NMR) were utilized to analyze the product release. Our data revealed that Ac-CoA regulates the conformational properties of Dat to form the catalytic site and substrate binding pocket, while the release of products is facilitated by the binding of new Ac-CoA.

Antimicrobial Peptide TP4 Targets Mitochondrial Adenine Nucleotide Translocator 2.

Tilapia piscidin (TP) 4 is an antimicrobial peptide derived from Nile tilapia (Oreochromis niloticus), which shows broad-spectrum antibacterial activity and excellent cancer-killing ability in vitro and in vivo. Like many other antimicrobial peptides, TP4 treatment causes mitochondrial toxicity in cancer cells. However, the molecular mechanisms underlying TP4 targeting of mitochondria remain unclear. In this study, we used a pull-down assay on A549 cell lysates combined with LC-MS/MS to discover that TP4 targets adenine nucleotide translocator (ANT) 2, a protein essential for adenine nucleotide exchange across the inner membrane. We further showed that TP4 accumulates in mitochondria and colocalizes with ANT2. Moreover, molecular docking studies showed that the interaction requires Phe1, Ile2, His3, His4, Ser11, Lys14, His17, Arg21, Arg24 and Arg25 residues in TP4 and key residues within the cavity of ANT2. These findings suggest a mechanism by which TP4 may induce mitochondrial dysfunction to disrupt cellular energy metabolism.

Cafestol Inhibits High-Glucose-Induced Cardiac Fibrosis in Cardiac Fibroblasts and Type 1-Like Diabetic Rats.

Diabetes is associated with the development of myocardial fibrosis, which is related to various cardiac diseases. Cafestol, one of the active ingredients in coffee, has been reported to exert biological effects. However, whether cafestol can ameliorate diabetes-induced cardiac fibrosis remains unknown. The aim of this study was to evaluate the effects of cafestol on cardiac fibrosis in high-glucose-treated cardiac fibroblasts and streptozocin- (STZ-) induced diabetic rats. Rat cardiac fibroblasts were cultured in high-glucose (25 mM) media in the absence or presence of cafestol, and the changes in collagen synthesis, transforming growth factor-ß1 (TGF-ß1) production, and related signaling molecules were assessed on the basis of 3H-proline incorporation, enzyme-linked immunosorbent assay, and western blotting. Cardiac fibroblasts exposed to high-glucose conditions exhibited increased collagen synthesis, TGF-ß1 production, and Smad2/3 phosphorylation, and these effects were mitigated by cafestol treatment. Furthermore, cafestol increased the translocation of nuclear factor erythroid 2-related factor 2 and increased the expression of heme oxygenase-1. The results of molecular docking analysis suggested a selective interaction of cafestol with Kelch-like ECH-associated protein 1. The rats with untreated STZ-induced diabetes exhibited considerable collagen accumulation, which was ameliorated by cafestol. Moreover, activities of catalase, superoxide dismutase, general matrix metalloproteinase, and reduced glutathione concentration were upregulated, whereas malondialdehyde level was downregulated by treatment with cafestol in rats with cardiac fibrosis. These findings highlight the effects of cafestol, which may be useful in treating diabetes-related cardiac fibrosis.

MAP4K3/GLK Promotes Lung Cancer Metastasis by Phosphorylating and Activating IQGAP1.

Overexpression of the serine/threonine kinase GLK/MAP4K3 in human lung cancer is associated with poor prognosis and recurrence, however, the role of GLK in cancer recurrence remains unclear. Here, we report that transgenic GLK promotes tumor metastasis and cell migration through the scaffold protein IQ motif-containing GTPase-activating protein 1(IQGAP1). GLK transgenic mice displayed enhanced distant metastasis. IQGAP1 was identified as a GLK-interacting protein; two proline-rich regions of GLK and the WW domain of IQGAP1 mediated this interaction. GLK and IQGAP1 colocalized at the leading edge including filopodia and lamellipodia of migrating cells. GLK directly phosphorylated IQGAP1 at Ser-480 enhancing Cdc42 activation and subsequent cell migration. GLK-induced cell migration and lung cancer metastasis were abolished by IQGAP1 depletion. Consistently, human NSCLC patient tissues displayed increased phospho-IQGAP1, which correlated with poor survival. Collectively, GLK promotes lung cancer metastasis by binding to, phosphorylating, and activating IQGAP1. SIGNIFICANCE: These findings show the critical role of the GLK-IQGAP cascade in cell migration and tumor metastasis, suggesting it as a potential biomarker and therapeutic target for lung cancer recurrence.

Nile Tilapia Derived Antimicrobial Peptide TP4 Exerts Antineoplastic Activity Through Microtubule Disruption.

Some antimicrobial peptides (AMPs) exhibit anti-cancer activity, acting on cancer cells either by causing membrane lysis or via intracellular effects. While intracellular penetration of AMPs has been shown to cause cancer cell death, the mechanisms of toxicity remain largely unknown. Here we show that a tilapia-derived AMP, Tilapia piscidin (TP) 4, penetrates intracellularly and targets the microtubule network. A pull-down assay identified α-Tubulin as a major interaction partner for TP4, and molecular docking analysis suggested that Phe1, Ile16, and Arg23 on TP4 are required for the interaction. TP4 treatment in A549 cells was found to disrupt the microtubule network in cells, and mutation of the essential TP4 residues prevented microtubule depolymerization in vitro. Importantly, the TP4 mutants also showed decreased cytotoxicity in A549 cells, suggesting that microtubule disruption is a major mechanistic component of TP4-mediated death in lung carcinoma cells.

Role of the RAD51-SWI5-SFR1 Ensemble in homologous recombination.

During DNA double-strand break and replication fork repair by homologous recombination, the RAD51 recombinase catalyzes the DNA strand exchange reaction via a helical polymer assembled on single-stranded DNA, termed the presynaptic filament. Our published work has demonstrated a dual function of the SWI5-SFR1 complex in RAD51-mediated DNA strand exchange, namely, by stabilizing the presynaptic filament and maintaining the catalytically active ATP-bound state of the filament via enhancement of ADP release. In this study, we have strived to determine the basis for physical and functional interactions between Mus musculus SWI5-SFR1 and RAD51. We found that SWI5-SFR1 preferentially associates with the oligomeric form of RAD51. Specifically, a C-terminal domain within SWI5 contributes to RAD51 interaction. With specific RAD51 interaction defective mutants of SWI5-SFR1 that we have isolated, we show that the physical interaction is indispensable for the stimulation of the recombinase activity of RAD51. Our results thus help establish the functional relevance of the trimeric RAD51-SWI5-SFR1 complex and provide insights into the mechanistic underpinnings of homology-directed DNA repair in mammalian cells.

MMP-13 is involved in oral cancer cell metastasis.

The oral cancer cell line OC3-I5 with a highly invasive ability was selected and derived from an established OSCC line OC3. In this study, we demonstrated that matrix metalloproteinases protein MMP-13 was up-regulated in OC3-I5 than in OC3 cells. We also observed that expression of epithelial-mesenchymal transition (EMT) markers including Twist, p-Src, Snail1, SIP1, JAM-A, and vinculin were increased in OC3-I5 compared to OC3 cells, whereas E-cadherin expression was decreased in the OC3-I5 cells. Using siMMP-13 knockdown techniques, we showed that siMMP-13 not only reduced the invasion and migration, but also the adhesion abilities of oral cancer cells. In support of the role of MMP-13 in metastasis, we used MMP-13 expressing plasmid-transfected 293T cells to enhance MMP-13 expression in the OC3 cells, transplanting the MMP-13 over expressing OC3 cells into nude mice led to enhanced lung metastasis. In summary, our findings show that MMP-13 promotes invasion and metastasis in oral cancer cells, suggesting altered expression of MMP-13 may be utilized to impede the process of metastasis.

Complete Genome Sequence of Xanthomonas campestris pv. campestris Strain 17 from Taiwan.

Xanthomonas campestris pv. campestris 17 is a Gram-negative bacterium that is phytopathogenic to cruciferous plants in Taiwan. The 4,994,426-bp-long genome consists of 24 contigs with 4,050 protein-coding genes, 1 noncoding RNA (ncRNA) gene, 6 rRNA genes, and 55 tRNA genes.

Analysis of differentially expressed novel post-translational modifications of plasma apolipoprotein E in Taiwanese females with breast cancer.

APOE ε2 or ε4 alleles being used as indicators of breast cancer risk are controversial in Taiwanese females. We provide a concept for relative comparisons of post-translational modifications (PTMs) of plasma apolipoprotein E (ApoE) between normal controls and breast cancer patients to investigate the association of ApoE with breast cancer risk. APOE polymorphisms (ApoE isoforms) were not assessed in this study. The relative modification ratio (%) of 15 targeted and 21 modified peptides were evaluated by 1D SDS-PAGE, in-gel digestion, and label-free nano-LC/MS to compare normal controls with breast cancer patients. Plasma levels of the ApoE protein did not significantly differ between normal controls and breast cancer patients. Eleven sites with novel PTMs were identified from 7 pairs of differentially expressed targeted and modified peptides according to the relative modification ratio including methylation at the E3 (↑1.45-fold), E7 (↑1.45-fold), E11 (↑1.19-fold), E77 (↑2.02-fold), E87 (↑2.02-fold), and Q98 (↑1.62-fold) residues; dimethylation at the Q187 (↑1.44-fold) residue; dihydroxylation at the R92 (↑1.25-fold), K95 (↑1.25-fold), and R103 (↑1.25-fold) residues; and glycosylation at the S129 (↑1.14-fold) residue. The clustered methylation and dihydroxylation of plasma ApoE proteins may play a role in breast cancer.

Relations among depression, self-efficacy and optimism in a sample of nurses in Taiwan.

AIMS: The present study investigated the level of depression among hospital nurses, to examine personality contributions to depression and to offer managers relevant organizational strategies to reduce levels of depression. BACKGROUND: The World Health Organization's Global Burden of Disease Study estimates that major depression is the leading cause of disability among women in the world today. It is surprising that there is a relative dearth of research investigating depression among nursing staff. METHOD: A cross-sectional survey of 314 staff nurses in a general hospital in Taiwan. Participants completed a set of questionnaires and a demographic information form. A number of statistical methods were used including descriptive statistics, product-moment correlations and multiple regression analysis. RESULTS: In all, 52.5% of nurses reported mild-to-moderate depressive symptoms. Self-efficacy and optimism were significant buffers against depression. CONCLUSIONS: The results of the present study confirm the importance of self-efficacy and optimism. Nurses with positive evaluation and expectation towards their self and others tend to report lower depression levels. IMPLICATIONS FOR NURSING MANAGEMENT: The results of the present study indicate that there is an immediate need to pay further attention to nurses' depression issues. It is therefore suggested that Nursing Managers take an empowering approach to strengthen nurses' self-efficacy and optimism levels to prevent depression in this profession.

DSAP: deep-sequencing small RNA analysis pipeline.

DSAP is an automated multiple-task web service designed to provide a total solution to analyzing deep-sequencing small RNA datasets generated by next-generation sequencing technology. DSAP uses a tab-delimited file as an input format, which holds the unique sequence reads (tags) and their corresponding number of copies generated by the Solexa sequencing platform. The input data will go through four analysis steps in DSAP: (i) cleanup: removal of adaptors and poly-A/T/C/G/N nucleotides; (ii) clustering: grouping of cleaned sequence tags into unique sequence clusters; (iii) non-coding RNA (ncRNA) matching: sequence homology mapping against a transcribed sequence library from the ncRNA database Rfam (http://rfam.sanger.ac.uk/); and (iv) known miRNA matching: detection of known miRNAs in miRBase (http://www.mirbase.org/) based on sequence homology. The expression levels corresponding to matched ncRNAs and miRNAs are summarized in multi-color clickable bar charts linked to external databases. DSAP is also capable of displaying miRNA expression levels from different jobs using a log(2)-scaled color matrix. Furthermore, a cross-species comparative function is also provided to show the distribution of identified miRNAs in different species as deposited in miRBase. DSAP is available at http://dsap.cgu.edu.tw.

Botulinum toxin type a in the prophylactic treatment of transformed migraine in Taiwanese patients: a review of 30 consecutive cases.

BACKGROUND: Botulinum toxin type A (BoNT-A) for the treatment of patients with various forms of migraine has been studied, but there is a paucity of data regarding the use of BoNT-A in Asian headache patients. Our study was designed to evaluate the efficacy of BoNT-A in the treatment of transformed migraine (TM) in a population of Taiwanese patients. METHODS: We retrospectively analyzed 30 patients who underwent BoNT-A treatment for TM from July 2003 to May 2004. Of 30 patients, 14 had palpable muscle tenderness (or tender points) in the pericranial region and 16 did not. All patients received injections into the corrugator, procerus, frontalis, and temporalis muscles (a total of 30 U), while a subset of TM patients with tender points (6 of 14 patients) also received injections to additional muscles based on a follow-the-tenderness approach (mean dose, 45 U). RESULTS: Twenty-seven of the 30 patients (90%) surveyed reported effective relief of their symptoms with BoNT-A treatment (at least a 50% reduction in the number of headache days or in headache intensity). The greatest reduction in headache days per month and headache intensity was found in TM patients with tender points who received a mean dose of 45 U compared to those who received fixed-site dosing of 30 U. CONCLUSION: Our results suggest that BoNT-A may be an effective prophylactic treatment for TM in Taiwanese patients. Interestingly, similar efficacy was demonstrated in TM patients with tender points compared to those without tender points when an additional dose of BoNT-A was injected into the tender muscles in the former.