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The mitochondrial gene cytochrome c oxidase I (COI) is commonly used for DNA barcoding in animals. However, most of the COI barcode nucleotides are conserved and sequences longer than about 650 base pairs increase the computational burden for species identification. To solve this problem, we propose a decision theory-based COI SNP tagging (DCST) approach that focuses on the discrimination of species using single nucleotide polymorphisms (SNPs) as the variable nucleotides of the sequences of a group of species. Using the example of 126 teleost mackerel fish species (order: Scombriformes), we identified 281 SNPs by alignment and trimming of their COI sequences. After decision rule making, 49 SNPs in 126 fish species were determined using the scoring system of the DCST approach. These COI-SNP barcodes were finally transformed into one-dimensional barcode images. Our proposed DCST approach simplifies the computational complexity and identifies the most effective and fewest SNPs to resolve or discriminate species for species tagging.

IntroductionGenetic polymorphisms and social factors (alcohol consumption, betel quid (BQ) usage, and cigarette consumption), both separately or jointly, play a crucial role in the occurrence of oral malignant disorders such as oral and pharyngeal cancers and oral potentially malignant disorders (OPMD).Material and methodsSimultaneous analyses of multiple single nucleotide polymorphisms (SNPs) and environmental effects on oral malignant disorders are essential to examine, albeit challenging. Thus, we conducted a case-control study (N = 576) to analyze the risk of occurrence of oral malignant disorders by using binary particle swarm optimization (BPSO) with an odds ratio (OR)-based method.ResultsWe demonstrated that a combination of SNPs (CYP26B1 rs887844 and CYP26C1 rs12256889) and socio-demographic factors (age, ethnicity, and BQ chewing), referred to as the combined effects of SNP-environment, correlated with maximal risk diversity of occurrence observed between the oral malignant disorder group and the control group. The risks were more prominent in the oral and pharyngeal cancers group (OR = 10.30; 95% confidence interval (CI) = 4.58-23.15) than in the OPMD group (OR = 5.42; 95% CI = 1.94-15.12).ConclusionsSimulation-based "SNP-environment barcodes" may be used to predict the risk of occurrence of oral malignant disorders. Applying simulation-based "SNP-environment barcodes" may provide insight into the importance of screening tests in preventing oral and pharyngeal cancers and OPMD.

BackgroundSecondary prevention therapy for patients with coronary artery disease using an antiplatelet agent, β-blocker, renin-angiotensin system blocker (RASB), or statin plays an important role in the reduction of coronary events after coronary artery bypass grafting (CABG) surgery or percutaneous coronary intervention (PCI). We analyzed the status and effects of secondary prevention after coronary revascularization in Taiwan.MethodsThis national population-based cohort study was conducted by analyzing the Longitudinal Health Insurance Database 2000 from the National Health Insurance Research Database of Taiwan. Patients who underwent CABG or PCI from 2004 to 2009 were included in the analysis. The baseline characteristics of the patients and ACC/AHA class I medication use at 12 months were analyzed. The primary endpoints were a composite of major adverse cardiac and cerebrovascular events.ResultsA total of 5544 patients comprising 895 CABG and 4649 PCI patients were evaluated. CABG patients had more comorbidities and a higher rate of major adverse event during the follow-up period. However, use of antiplatelet agents and RASB at 12 months was significantly lower in CABG patients than in PCI patients (44.2% vs. 50.9% and 38.6% vs. 48.9%, both p < 0.01). Age, diabetes, and chronic kidney disease were independent risk factors while statin use was a protective factor for the primary endpoints in both PCI and CABG groups.ConclusionThere is still much room to improve class I medication use in secondary prevention for patients after revascularization in Taiwan. Statin could be an effective treatment to improve the outcomes.

Breast cancer expresses clinically heterogeneous characteristics and requires multipurpose drug development for curing the different tumor subtypes. Many withanolides have been isolated from Physalis species showing anticancer effects, but the anticancer function of physapruin A (PHA) has rarely been investigated. In this study, the anticancer properties of PHA in breast cancer cells were examined by concentration and time-course experiments. In terms of cellular ATP content, PHA inhibited the proliferation of three kinds of breast cancer cells: MCF7 (estrogen receptor (ER)+, progesterone receptor (PR)+/−, human epidermal growth factor receptor 2 (HER2)−), SKBR3 (ER−/PR−/HER2+), and MDA-MB-231 (triple-negative). Moreover, PHA induced G2/M arrest in MCF7 and MDA-MB-231 cells. In terms of flow cytometry, PHA induced the generation of reactive oxygen species (ROS), the generation of mitochondrial superoxide, mitochondrial membrane potential depletion, and γH2AX-detected DNA damage in breast cancer MCF7 and MDA-MB-231 cells, which were suppressed by the ROS inhibitor N-acetylcysteine (NAC). In terms of flow cytometry and Western blotting, PHA induced apoptotic expression (annexin V, and intrinsic and extrinsic apoptotic signaling), which was suppressed by NAC and an apoptosis inhibitor (Z-VAD-FMK), in breast cancer cells. Therefore, PHA is a potential anti-breast-cancer natural product that modulates the oxidative-stress response, cell-cycle disturbance, apoptosis, and γH2AX-detected DNA damage.

Application of LC-MS/MS-based molecular networking indicated the ethanol extract of octocoral Asterospicularia laurae is a potential source for the discovery of new xenicane derivatives. A natural product investigation of this soft coral resulted in the isolation of four new xenicane diterpenoids, asterolaurins O–R (1–4), together with six known compounds, xeniolide-A (5), isoxeniolide-A (6), xeniolide-B (7), 7,8-epoxyxeniolide-B (8), 7,8-oxido-isoxeniolide-A (9), and 9-hydroxyxeniolide-F (10). The structures of isolated compounds were characterized by employing spectroscopic analyses, including 2D-NMR (COSY, HMQC, HMBC, and NOESY) and high-resolution electrospray ionization mass spectrometry (HRESIMS). Asterolaurin O is the first case of brominated tricarbocyclic type floridicin in the family Xeniidae. Concerning bioactivity, the cytotoxic activity of those isolates was evaluated. As a result, compounds 1 and 2 demonstrated a selective cytotoxic effect against the MCF-7 cell line at IC50 of 14.7 and 25.1 μM, respectively.

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Withaferin A (WFA), a Withania somnifera-derived triterpenoid, is an anticancer natural product. The anticancer effect of nonionizing radiation such as ultraviolet-C (UVC) as well as the combined treatment of UVC and WFA is rarely investigated. Low dose UVC and/or WFA treatments (12 J/m2 and/or 1 μM) were chosen to evaluate antioral cancer cell line effects by examining cytotoxicity, cell cycle disruption, apoptosis induction, and DNA damage. For two cancer cell lines (Ca9-22 and HSC-3), single treatment (UVC or WFA) showed about 80% viability, while a combined treatment of UVC/WFA showed about 40% viability. In contrast, there was noncytotoxicity to normal oral cell lines (HGF-1). Compared to single treatment and control, low dose UVC/WFA shows high inductions of apoptosis in terms of flow cytometric detections for subG1, annexin V, pancaspase changes as well as Western blotting for detecting cleaved poly (ADP-ribose) polymerase (c-PARP) and caspase 3 (c-Cas 3) and luciferase assay for detecting Cas 3/7 activity. Low dose UVC/WFA also showed high inductions of oxidative stress and DNA damage in terms of flow cytometric detections of reactive oxygen species (ROS), mitochondrial superoxide (MitoSOX) generation, and membrane potential (MitoMP) destruction, γH2AX and 8-oxo-2’deoxyguanosine (8-oxodG) types of DNA damages. For comparison, low dose UVC/WFA show rare inductions of annexin V, Cas 3/7 activity, ROS, MitoSOX, and MitoMP changes to normal oral HGF-1 cells. Therefore, low dose UVC/WFA provides a novel selectively killing mechanism to oral cancer cells, suggesting that WFA is a UVC sensitizer to inhibit the proliferation of oral cancer cells.

In protein engineering and synthetic biology, Methanosarcina mazei pyrrolysyl-tRNA synthetase (MmPylRS), with its cognate tRNAPyl, is one of the most popular tools for site-specific incorporation of non-canonical amino acids (ncAAs). Numerous orthogonal pairs based on engineered MmPylRS variants have been developed during the last decade, enabling a substantial genetic code expansion, mainly with aliphatic pyrrolysine analogs. However, comparatively less progress has been made to expand the substrate range of MmPylRS towards aromatic amino acid residues. Therefore, we set to further expand the substrate scope of orthogonal translation by a semi-rational approach; redesigning the MmPylRS efficiency. Based on the randomization of residues from the binding pocket and tRNA binding domain, we identify three positions (V401, W417 and S193) crucial for ncAA specificity and enzyme activity. Their systematic mutagenesis enabled us to generate MmPylRS variants dedicated to tryptophan (such as β-(1-Azulenyl)-l-alanine or 1-methyl-l-tryptophan) and tyrosine (mainly halogenated) analogs. Moreover, our strategy also significantly improves the orthogonal translation efficiency with the previously activated analog 3-benzothienyl-l-alanine. Our study revealed the engineering of both first shell and distant residues to modify substrate specificity as an important strategy to further expand our ability to discover and recruit new ncAAs for orthogonal translation

Some withanolides, particularly the family of steroidal lactones, show anticancer effects, but this is rarely reported for withanolide C (WHC)—especially anti-breast cancer effects. The subject of this study is to evaluate the ability of WHC to regulate the proliferation of breast cancer cells, using both time and concentration in treatment with WHC. In terms of ATP depletion, WHC induced more antiproliferation to three breast cancer cell lines, SKBR3, MCF7, and MDA-MB-231, than to normal breast M10 cell lines. SKBR3 and MCF7 cells showing higher sensitivity to WHC were used to explore the antiproliferation mechanism. Flow cytometric apoptosis analyses showed that subG1 phase and annexin V population were increased in breast cancer cells after WHC treatment. Western blotting showed that cleaved forms of the apoptotic proteins poly (ADP-ribose) polymerase (c-PARP) and cleaved caspase 3 (c-Cas 3) were increased in breast cancer cells. Flow cytometric oxidative stress analyses showed that WHC triggered reactive oxygen species (ROS) and mitochondrial superoxide (MitoSOX) production as well as glutathione depletion. In contrast, normal breast M10 cells showed lower levels of ROS and annexin V expression than breast cancer cells. Flow cytometric DNA damage analyses showed that WHC triggered γH2AX and 8-oxo-2′-deoxyguanosine (8-oxodG) expression in breast cancer cells. Moreover, N-acetylcysteine (NAC) pretreatment reverted oxidative stress-mediated ATP depletion, apoptosis, and DNA damage. Therefore, WHC kills breast cancer cells depending on oxidative stress-associated mechanisms.

The sensitizing effect of chromone-derived compounds on UVC-induced proliferation inhibition has not been comprehensively investigated so far. The subject of this study was to examine the proliferation change of oral cancer cells while using the combined treatment of UVC (254 nm) with our previously developed sulfonyl chromen-4-ones (CHW09), namely UVC/CHW09. Cell viability, apoptosis, oxidative stress, and DNA damage for the individual and combined treatments for UVC and/or CHW09 were examined in oral cancer Ca9-22 cells. In 24 h MTS assay, UVC (30 J/m2; UVC30), or CHW09 (25 and 50 µg/mL; namely, CHW09-25 and CHW09-50) show 54%, 59%, and 45% viability. The combined treatment (UVC30/CHW09-25 and UVC30/CHW09-50) show lower cell viability (45% and 35%). Mechanistically, UVC/CHW09 induced higher apoptosis than individual treatments and untreated control, which were supported by the evidence of flow cytometry for subG1, annexin V/7-aminoactinomycin D, pancaspase and caspases 3/7 activity, and western blotting for cleaved poly(ADP-ribose) polymerase. Moreover, this cleaved PARP expression was downregulated by pancaspase inhibitor Z-VAD-FMK. UVC/CHW09 showed higher oxidative stress than individual treatments and untreated control in terms of flow cytometry for reactive oxygen species, mitochondrial membrane potential, and mitochondrial mass. Furthermore, UVC/CHW09 showed higher DNA damage than individual treatments and untreated control in terms of flow cytometry for H2A histone family member X and 8-oxo-2’-deoxyguanosine. In conclusion, combined treatment UVC/CHW09 suppresses proliferation, and promotes apoptosis, oxidative stress, and DNA damage against oral cancer cells, providing a novel application of sulfonyl chromen-4-ones in order to sensitize UVC induced proliferation inhibition for oral cancer therapy.

Ultraviolet-C (UVC) irradiation provides an alternative radiotherapy to X-ray. UVC sensitizer from natural products may improve radiotherapy at low cytotoxic side effects. The aim of this study is to assess the regulation for oral cancer cell proliferation by a combined treatment of UVC and our previously reported anti-oral cancer natural product (ethyl acetate extract of Nepenthes adrianii × clipeata; EANA). The detailed possible UVC sensitizing mechanisms of EANA such as effects on cell proliferation, cell cycle, apoptosis, and DNA damage are investigated individually and in combination using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTS) assay, flow cytometry, and western blotting at low dose conditions. In a 24 h MTS assay, the low dose EANA (5 μg/mL) and low dose UVC (12 J/m2) individually show 80% and combinedly 57% cell proliferation in oral cancer Ca9-22 cells; but no cytotoxicity to normal oral HGF-1 cells. Mechanistically, low dose EANA and low dose UVC individually induce apoptosis (subG1 accumulation, pancaspase activation, and caspases 3, 8, 9), oxidative stress (reactive oxygen species, mitochondrial superoxide, and mitochondrial membrane potential depletion), and DNA damage (γH2AX and 8-hydroxy-2′-deoxyguanosine). Moreover, the combined treatment (UVC/EANA) synergistically induces these changes. Combined low dose treatment-induced antiproliferation, apoptosis, oxidative stress, and DNA damage were suppressed by the ROS scavenger N-acetylcysteine. In conclusion, UVC/EANA shows synergistic antiproliferation, oxidative stress, apoptosis, and DNA damage to oral cancer cells in an oxidative stress-dependent manner. With the selective killing properties of low dose EANA and low dose UVC, EANA provides a novel UVC sensitizing agent to improve the anti-oral cancer therapy.

Some lichens provide the resources of common traditional medicines and show anticancer effects. However, the anticancer effect of Usnproliea barbata (U. barbata) is rarely investigated, especially for oral cancer cells. The aim of this study was to investigate the cell killing function of methanol extracts of U. barbata (MEUB) against oral cancer cells. MEUB shows preferential killing against a number of oral cancer cell lines (Ca9-22, OECM-1, CAL 27, HSC3, and SCC9) but rarely affects normal oral cell lines (HGF-1). Ca9-22 and OECM-1 cells display the highest sensitivity to MEUB and were chosen for concentration effect and time course experiments to address its cytotoxic mechanisms. MEUB induces apoptosis of oral cancer cells in terms of the findings from flow cytometric assays and Western blotting, such as subG1 accumulation, annexin V detection, and pancaspase activation as well as poly (ADP-ribose) polymerase (PARP) cleavage. MEUB induces oxidative stress and DNA damage of oral cancer cells following flow cytometric assays, such as reactive oxygen species (ROS)/mitochondrial superoxide (MitoSOX) production, mitochondrial membrane potential (MMP) depletion as well as overexpression of γH2AX and 8-oxo-2′deoxyguanosine (8-oxodG). All MEUB-induced changes in oral cancer cells were triggered by oxidative stress which was validated by pretreatment with antioxidant N-acetylcysteine (NAC). In conclusion, MEUB causes preferential killing of oral cancer cells and is associated with oxidative stress, apoptosis, and DNA damage.

Upstroke time (UT), measured from the foot-to-peak peripheral pulse wave, is a merged parameter used to assess arterial stiffness and target vascular injuries. In this study, we aimed to investigate UT for the prediction of cardiovascular and all-cause mortality in patients with chronic kidney disease (CKD). This longitudinal study enrolled 472 patients with CKD. Blood pressure, brachial pulse wave velocity (baPWV), and UT were automatically measured by a Colin VP-1000 instrument. During a median follow-up of 91 months, 73 cardiovascular and 183 all-cause mortality instances were recorded. Multivariable Cox analyses indicated that UT was significantly associated with cardiovascular mortality (hazard ratio (HR) = 1.010, p = 0.007) and all-cause mortality (HR = 1.009, p < 0.001). The addition of UT into the clinical models including traditional risk factors and baPWV further increased the value in predicting cardiovascular and all-cause mortality (both p < 0.001). In the Kaplan–Meier analyses, UT ≥ 180 ms could predict cardiovascular and all-cause mortality (both log-rank p < 0.001). Our study found that UT was a useful parameter in predicting cardiovascular and all-cause mortality in CKD patients. Additional consideration of the UT might provide an extra benefit in predicting cardiovascular and all-cause mortality beyond the traditional risk factors and baPWV.

Withaferin A (WFA) has been reported to inhibit cancer cell proliferation based on high cytotoxic concentrations. However, the low cytotoxic effect of WFA in regulating cancer cell migration is rarely investigated. The purpose of this study is to investigate the changes in migration and mechanisms of oral cancer Ca9-22 cells after low concentrations of WFA treatment. WFA under 0.5 μM at 24 h treatment shows no cytotoxicity to oral cancer Ca9-22 cells (~95% viability). Under this condition, WFA triggers reactive oxygen species (ROS) production and inhibits 2D (wound healing) and 3D cell migration (transwell) and Matrigel invasion. Mechanically, WFA inhibits matrix metalloproteinase (MMP)-2 and MMP-9 activities but induces mRNA expression for a group of antioxidant genes, such as nuclear factor, erythroid 2-like 2 (NFE2L2), heme oxygenase 1 (HMOX1), glutathione-disulfide reductase (GSR), and NAD(P)H quinone dehydrogenase 1 (NQO1)) in Ca9-22 cells. Moreover, WFA induces mild phosphorylation of the mitogen-activated protein kinase (MAPK) family, including extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 expression. All WFA-induced changes were suppressed by the presence of ROS scavenger N-acetylcysteine (NAC). Therefore, these results suggest that low concentration of WFA retains potent ROS-mediated anti-migration and -invasion abilities for oral cancer cells.

DNA barcode sequences are accumulating in large data sets. A barcode is generally a sequence larger than 1000 base pairs and generates a computational burden. Although the DNA barcode was originally envisioned as straightforward species tags, the identification usage of barcode sequences is rarely emphasized currently. Single-nucleotide polymorphism (SNP) association studies provide us an idea that the SNPs may be the ideal target of feature selection to discriminate between different species. We hypothesize that SNP-based barcodes may be more effective than the full length of DNA barcode sequences for species discrimination. To address this issue, we tested a r ibulose diphosphate carboxylase ( rbcL ) S NP b arcoding (RSB) strategy using a decision tree algorithm. After alignment and trimming, 31 SNPs were discovered in the rbcL sequences from 38 Brassicaceae plant species. In the decision tree construction, these SNPs were computed to set up the decision rule to assign the sequences into 2 groups level by level. After algorithm processing, 37 nodes and 31 loci were required for discriminating 38 species. Finally, the sequence tags consisting of 31 rbcL SNP barcodes were identified for discriminating 38 Brassicaceae species based on the decision tree–selected SNP pattern using RSB method. Taken together, this study provides the rational that the SNP aspect of DNA barcode for rbcL gene is a useful and effective sequence for tagging 38 Brassicaceae species.

BACKGROUND:Several randomized control trials have established that drugs can decrease the heart failure (HF) rehospitalization in patients with HF. However, limited studies have investigated the duration of medicine use to decrease the rehospitalization period in the real world. Hence, this study aims to investigate whether the evidence-based medicine decreases the HF rehospitalization in different treatment intervals in the clinical practice. METHOD:We examined patients admitted with acute HF from the National Health Insurance Research Database in Taiwan. In addition, the major adverse cardiovascular events (MACE) were the composite endpoints of the in-hospital mortality and rehospitalization after 1 year. Furthermore, we analyzed the medicine use to decrease 14 days and 1, 6, and 12 months' HF rehospitalization. RESULTS:Overall, we examined 11,012 patients. The use of the renin-angiotensin system (RAS) blockers [hazard ratio (HR), 0.58; P < 0.01], β-blocker (HR, 0.67; P < 0.01), spironolactone (HR, 0.63; P < 0.01), and digitalis (HR, 0.67; P < 0.01) associated with the lower in-hospital mortality rate. The Cox regression analysis revealed that RAS blocker (HR, 0.86; P < 0.01) and β-blocker (HR, 0.71; P < 0.01) were independent predictors for MACE. Although RAS blockers declined rehospitalization to 6 months, β-blocker decreased the rehospitalization rate after 1 month use and the benefit persisted till 12 months. Furthermore, digitalis only lowered rehospitalization to 14 days. CONCLUSION:This study suggests that the use of evidence-based medicine is associated with lower MACE for patients with HF, and these drugs could play vital roles in different periods to decrease the rehospitalization in the clinical setting.