Grass carp (Ctenopharyngodon idella) deacetylase SIRT1 targets p53 to suppress apoptosis in a KAT8 dependent or independent manner.

Sirtuin1 (SIRT1) is known as a deacetylase to control various physiological processes. In mammals, SIRT1 inhibits apoptotic process, but the detailed mechanism is not very clear. Here, our study revealed that grass carp (Ctenopharyngodon idella) SIRT1 (CiSIRT1, MN125614.1) inhibits apoptosis through targeting p53 in a KAT8-dependent or a KAT8-independent manner. In CIK cells, CiSIRT1 over-expression results in significant decrease of some apoptotic gene expressions, including Bax/Bcl2, caspase3 and caspase9, whereas CiKAT8 or Cip53 facilitates the induction of apoptosis. Because CiSIRT1 separately interacted with CiKAT8 and Cip53, we speculated that CiSIRT1 blocked apoptosis may be by virtue of KAT8-p53 axis or directly by p53. In a KAT8-dependent manner, CiSIRT1 interacted with CiKAT8, then reduced the acetylation of CiKAT8 and subsequently promoted its degradation. Then, CiKAT8 acetylated p53 and induced p53-mediated apoptosis. MYST domain of CiKAT8 was critical in this pathway. In a KAT8-independent manner, CiSIRT1 also inhibited p53-induced apoptosis by directly deacetylating p53 and promoting the degradation of p53. Generally, these findings uncovered two pathways in which CiSIRT1 decreases the acetylation of p53 via a KAT8-dependent or a KAT8-independent manner.

Performance evaluation of E-nose and E-tongue combined with machine learning for qualitative and quantitative assessment of bear bile powder.

Bear bile powder (BBP) is a valuable animal-derived product with a huge adulteration problem on market. It is a crucially important task to identify BBP and its counterfeit. Electronic sensory technologies are the inheritance and development of traditional empirical identification. Considering that each drug has its own specific odor and taste characteristics, electronic tongue (E-tongue), electronic nose (E-nose) and GC-MS were used to evaluate the aroma and taste of BBP and its common counterfeit. Two active components of BBP, namely tauroursodeoxycholic acid (TUDCA) and taurochenodeoxycholic acid (TCDCA) were measured and linked with the electronic sensory data. The results showed that bitterness was the main flavor of TUDCA in BBP, saltiness and umami were the main flavor of TCDCA. The volatiles detected by E-nose and GC-MS were mainly aldehydes, ketones, alcohols, hydrocarbons, carboxylic acids, heterocyclic, lipids, and amines, mainly earthy, musty, coffee, bitter almond, burnt, pungent odor descriptions. Four different machine learning algorithms (backpropagation neural network, support vector machine, K-nearest neighbor, and random forest) were used to identify BBP and its counterfeit, and the regression performance of these four algorithms was also evaluated. For qualitative identification, the algorithm of random forest has shown the best performance, with 100% accuracy, precision, recall and F1-score. Also, the random forest algorithm has the best R2 and the lowest RMSE in terms of quantitative prediction.

Rapid discrimination of the authenticity and geographical origin of bear bile powder using stable isotope ratio and elemental analysis.

Bear bile powder (BBP) is one of the most famous traditional Chinese medicines derived from animals. It has a long history of medicinal use and is widely used in the treatment of hepatobiliary and ophthalmic diseases. Due to its similar morphological characterizations and chemical composition compared with other bile powders, it is difficult to accurately identify its authenticity. In addition, there are very few methods that could analyze the geographical origins of BBP. In this study, elemental analysis isotope ratio mass spectrometry (EA-IRMS) and inductively coupled plasma mass spectrometry (ICP-MS) were used to determine stable isotope ratios and elemental contents, respectively. Combined these variables with chemometrics, the discrimination models were established successfully for identifying the authenticity and geographical origins of BBP. Meanwhile, the discrimination markers were identified by calculating the variable importance for the projection (VIP) value of each variable. A total of 13 discrimination markers (δ13C, δ15N, C, Li, Mg, K, Ca, Cr, Ni, Zn, As, Se, and Sr) were used to further establish the fingerprint of BBP. According to similarity analysis, the authenticity and geographical origins of BBP could be identified without chemometrics. In conclusion, the present study established a reliable method for authenticity identification and origin traceability of BBP, which will provide references for the quality control of bile medicines.

Identification of the Authenticity and Geographical Origin of Bear Bile Powder by Using High Performance Liquid Chromatography - Charged Aerosol Detector Fingerprints Combined with Chemometrics.

Bear bile powder (BBP) is a rare animal-derived traditional Chinese medicine, and it has been widely used to treat visual disorders and hepatobiliary diseases in East Asia. However, there is still a lack of reliable quality control methods for BBP. This study was designed to establish a comprehensive quality map of BBP based on bile acids. High-performance liquid chromatography coupled with charged aerosol detector (HPLC-CAD) was used for fingerprint establishment and quantitative analysis of BBP. The similarities of HPLC-CAD chromatograms for 50 batches of BBP were more than 0.95, while the similarities of reference chromatograms between 6 other animal bile and BBP were low than 0.7. Additionally, five bile acids in BBP, including tauroursodeoxycholic acid, taurocholic acid, taurochenodeoxycholic acid, ursodesoxycholic acid, and chenodeoxycholic acid, were simultaneously quantified. This method has been validated with good regression as well as satisfactory precision, sensitivity, stability, repeatability, and accuracy. Using this method, the contents of five bile acids in BBP samples from five producing areas were determined and compared. Furthermore, Fisher linear discriminant analysis was performed to discriminate the geographic origins of BBP. The result demonstrated that HPLC-CAD fingerprint combined with multi-components quantification is an effective and reliable method for quality control of BBP, it could be a meaningful reference for the quality evaluation of medicinal bile.

Chlorphoxim induces neurotoxicity in zebrafish embryo through activation of oxidative stress.

It is known that chlorphoxim is a broad-spectrum and high-effective pesticide. With the wide use in agricultural practice, chlorphoxim residue is also frequently detected in water, but its potential toxicity to aquatic life is still unclear. In this study, zebrafish is used as a model to detect the toxicity of chlorphoxim. Our results showed that exposure of high concentration of chlorphoxim at 96 h post-fertilization (hpf) resulted in a high mortality and pericardium edema rate, a low hatchability rate and heart rate. The nervous system damage, swimming behavior alteration and acetylcholinesterase (AChE) inhibition were measured in zebrafish embryos after a 6 days post-fertilization (dpf) of chlorphoxim exposure. The expression of neural-related genes is abnormal in zebrafish embryos. Chlorphoxim exposure significantly increases oxidative stress in zebrafish embryos by inhibiting antioxidant enzyme (SOD and CAT) and activating reactive oxygen species (ROS). As expected, chlorphoxim exposure induces apoptosis by enhancing the expression of apoptotic genes (Bax, Bcl2, and p53). Astaxanthin (ATX), an effective antioxidant, was found to be able to rescue the neurotoxicity of chlorphoxim through relieving oxidative stress and apoptosis. Altogether, the results showed that chlorphoxim exposure led to severe neurotoxicity to zebrafish embryos, which was contributed to a more comprehensive understanding of the safety use of the organophosphorus pesticide.

Biosynthesis and the Transcriptional Regulation of Terpenoids in Tea Plants (Camellia sinensis).

Terpenes, especially volatile terpenes, are important components of tea aroma due to their unique scents. They are also widely used in the cosmetic and medical industries. In addition, terpene emission can be induced by herbivory, wounding, light, low temperature, and other stress conditions, leading to plant defense responses and plant-plant interactions. The transcriptional levels of important core genes (including HMGR, DXS, and TPS) involved in terpenoid biosynthesis are up- or downregulated by the MYB, MYC, NAC, ERF, WRKY, and bHLH transcription factors. These regulators can bind to corresponding cis-elements in the promoter regions of the corresponding genes, and some of them interact with other transcription factors to form a complex. Recently, several key terpene synthesis genes and important transcription factors involved in terpene biosynthesis have been isolated and functionally identified from tea plants. In this work, we focus on the research progress on the transcriptional regulation of terpenes in tea plants (Camellia sinensis) and thoroughly detail the biosynthesis of terpene compounds, the terpene biosynthesis-related genes, the transcription factors involved in terpene biosynthesis, and their importance. Furthermore, we review the potential strategies used in studying the specific transcriptional regulation functions of candidate transcription factors that have been discriminated to date.

Grass carp (Ctenopharyngodon idella) DYRK2 modulates cell apoptosis through phosphorylating p53.

In mammals, DYRK2 increases p53 phosphorylation level by interacting with it and then promotes cell apoptosis. However, the function of fish DYRK2 has not yet been elucidated. In this paper, we cloned and identified the coding sequence (CDS) of a grass carp DYRK2 (CiDYRK2) which is 1773 bp in length and encodes 590 amino acids. SMART predictive analysis showed that CiDYRK2 possesses a serine/threonine kinase domain. Subsequently, we used the dsRNA analog polyinosinic-polycytidylic acid (poly (I:C) and Grass carp reovirus (GCRV) to stimulate grass carp and CIK cells for different times and found that CiDYRK2 mRNA was significantly up-regulated both in fish tissues and cells. To explore the function of CiDYRK2, we carried out overexpression and knockdown experiments of CiDYRK2 in CIK cells. Real-time quantitative PCR (Q-PCR), TdT-mediated dUTP nick end labeling (TUNEL) assay and flow cytometry were used to detect the ratio of BAX/BCL-2 mRNA, the number of TUNEL positive cells, the proportion of Annexin V-positive cells respectively. The results showed that CiDYRK2 significantly up-regulated BAX/Bcl-2 mRNA ratio and increased the number of TUNEL-positive cells, as well as the proportion of Annexin V-positive cells. On the contrary, knock-down of CiDYRK2 significantly down-regulated BAX/Bcl-2 mRNA ratio in the cells. Therefore, CiDYRK2 promoted cell apoptosis. To study the molecular mechanism by which CiDYRK2 promoting cell apoptosis, subcellular localization and immunoprecipitation experiments were used to study the relationship between grass carp DYRK2 and the pro-apoptotic protein p53. The results showed that CiDYRK2 and Cip53 were located and co-localized in the nucleus. Co-immunoprecipitation experiment also showed that CiDYRK2 and Cip53 can bind with each other. We further found that DYRK2 can increase the phosphorylation level of p53. In a word, our results showed that grass carp DYRK2 induces cell apoptosis by increasing the phosphorylation level of p53.

Polyethyleneimine/hydrated titanium oxide-functionalized fibrous adsorbent for removing cobalt: Adsorption performance and irradiation stability.

Radionuclides of 60Co often encountered in the fields of radiation therapy, medical preparation, and equipment sterilization, which have been considered fatal. Therefore, developing efficient and irradiation-stable adsorbents for the removal of 60Co in wastewater is urgently needed. An irradiation-stable fibrous adsorbent was fabricated through the surface functionalization of collagen fibers (CFs) by polyethyleneimine (PEI) and hydrated titanium oxide (TiO) (PEI-TiO-CFs). PEI-TiO-CFs, including their adsorption performance and irradiation stability, were systematically investigated. Results showed that PEI-TiO-CFs exhibit a maximum adsorption capacity of 0.5575 mmol g-1. In addition, the adsorption capacity of PEI-TiO-CFs only demonstrated a slight decrease in the selectivity investigation of Co2+ mixed with another coexisting ion, such as Na+, K+, and NO3-, Cl-. Furthermore, breakthrough point of PEI-TiO-CFs in column is high at 80 BV (bed volume) and the PEI-TiO-CF column can be mostly regenerated using 12 BV of Na2EDTA solution. Excellent irradiation stability of PEI-TiO-CFs was confirmed by the maintained morphology and adsorption capacity after irradiation at 350 kGy of 60Co γ-ray. Results indicated that PEI-TiO-CFs are an effective adsorbent for radioactive cobalt removal from aqueous solutions.

Diflubenzuron Induces Cardiotoxicity in Zebrafish Embryos.

Diflubenzuron is an insecticide that serves as a chitin inhibitor to restrict the growth of many harmful larvae, including mosquito larvae, cotton bollworm and flies. The residue of diflubenzuron is often detected in aquaculture, but its potential toxicity to aquatic organisms is still obscure. In this study, zebrafish embryos (from 6 h to 96 h post-fertilization, hpf) were exposed to different concentrations of diflubenzuron (0, 0.5, 1.5, 2.5, 3.5 and 4.5 mg/L), and the morphologic changes, mortality rate, hatchability rate and average heart rate were calculated. Diflubenzuron exposure increased the distance between the venous sinus and bulbar artery (SV-BA), inhibited proliferation of myocardial cells and damaged vascular development. In addition, diflubenzuron exposure also induced contents of reactive oxygen species (ROS) and malondialdehyde (MDA) and inhibited the activity of antioxidants, including SOD (superoxide dismutase) and CAT (catalase). Moreover, acridine orange (AO) staining showed that diflubenzuron exposure increased the apoptotic cells in the heart. Q-PCR also indicated that diflubenzuron exposure promoted the expression of apoptosis-related genes (bax, bcl2, p53, caspase3 and caspase9). However, the expression of some heart-related genes were inhibited. The oxidative stress-induced apoptosis damaged the cardiac development of zebrafish embryos. Therefore, diflubenzuron exposure induced severe cardiotoxicity in zebrafish embryos. The results contribute to a more comprehensive understanding of the safety use of diflubenzuron.

Identification of population-level differentially expressed genes in one-phenotype data.

MOTIVATION: For some specific tissues, such as the heart and brain, normal controls are difficult to obtain. Thus, studies with only a particular type of disease samples (one phenotype) cannot be analyzed using common methods, such as significance analysis of microarrays, edgeR and limma. The RankComp algorithm, which was mainly developed to identify individual-level differentially expressed genes (DEGs), can be applied to identify population-level DEGs for the one-phenotype data but cannot identify the dysregulation directions of DEGs. RESULTS: Here, we optimized the RankComp algorithm, termed PhenoComp. Compared with RankComp, PhenoComp provided the dysregulation directions of DEGs and had more robust detection power in both simulated and real one-phenotype data. Moreover, using the DEGs detected by common methods as the 'gold standard', the results showed that the DEGs detected by PhenoComp using only one-phenotype data were comparable to those identified by common methods using case-control samples, independent of the measurement platform. PhenoComp also exhibited good performance for weakly differential expression signal data. AVAILABILITY AND IMPLEMENTATION: The PhenoComp algorithm is available on the web at https://github.com/XJJ-student/PhenoComp. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Qualitative diagnostic signature for pancreatic ductal adenocarcinoma based on the within-sample relative expression orderings.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) accounts for about 90% of pancreatic cancer, which is one of the most aggressive malignant neoplasms with a 9.3% five-year survival rate. The pathological biopsy is the current golden standard for confirming suspicious lesions of PDAC, but it is not entirely reliable because of the insufficient sampling amount and inaccurate sampling location. Therefore, developing a robust signature to aid the accurate diagnosis of PDAC is critical. METHODS: Based on the within-sample relative expression orderings of gene pairs, we identified a qualitative signature to discriminate both PDAC and adjacent samples from both chronic pancreatitis and normal samples in the training datasets and validated it in other independent datasets produced by different laboratories with different measuring platforms. RESULTS: A six-gene-pair signature was identified in the training data and validated in eight independent datasets. For surgical samples, 96.63% of 356 PDAC tissues, 100% of 11 pancreatitis tissues of non-cancer patients, and 23 of 24 normal pancreatic tissues were correctly classified. Especially, 59 of 60 cancer-adjacent normal tissues of PDAC patients were correctly identified as PDAC. For biopsy samples, all of 11 PDAC biopsy tissues were correctly classified as PDAC. CONCLUSION: The signature can distinguish both PDAC and PDAC-adjacent normal tissues from both chronic pancreatitis and normal tissues of non-cancer patients even when the sampling locations are inaccurate, which can aid the diagnosis of PDAC.

A qualitative transcriptional signature of recurrence risk for stages II-III gastric cancer patients after surgical resection.

BACKGROUND AND AIM: Metastasis is the leading cause of recurrence in gastric cancer. However, the imaging techniques and pathological examinations for tumor metastasis have a high false-positive rate or a high false-negative rate, and many proposed that metastasis-related molecular biomarkers can hardly be validated in independent datasets. METHODS: We propose to use significantly stable gene pairs with reversal relative expression orderings (REOs) between non-metastasis and metastasis gastric cancer samples as the metastasis-related gene pairs. Based on the REOs of these gene pairs, we developed a qualitative transcriptional signature for predicting the recurrence risk of stages II-III gastric cancer patients after surgical resection. RESULTS: A REOs-based signature, consisting of 19 gene pairs (19-GPS), was selected from 77 stages II-III gastric cancer patients and validated in two independent datasets. Samples in the high-risk group had shorter disease-free survival time and overall survival time than those in the low-risk group. Differentially expressed genes (DEGs) between the high- and low-risk groups classified by 19-GPS were highly reproducible comparing with those between lymph node metastasis and lymph node non-metastasis groups. Functional enrichment analysis showed that these DEGs were significantly enriched in metastasis-related pathways, such as PI3K-Akt and Rap1 signaling pathways. The multi-omics analyses suggested that the epigenetic and genomic features might cause transcriptional differences between two subgroups, which help to characterize the mechanism of gastric cancer metastasis. CONCLUSIONS: The signature could robustly identify patients at high recurrence risk after resection surgery, and the multi-omics analyses might aid in revealing the metastasis-related characteristics of gastric cancer.

Ethoprophos induces cardiac toxicity in zebrafish embryos.

Ethoprophos is an effective and widely pesticide that used in controlling nemathelminth and soil insect. However, ethoprophos has been frequently detected in environment and freshwater. The potential toxicity to aquatic organisms is still not be explored. In this study, zebrafish embryo model was used to evaluated the toxicity of ethoprophos during cardiovascular developmental process of zebrafish. Zebrafish embryos were separately exposed to 10 mg/L, 20 mg/L, 30 mg/L, 40 mg/L and 50 mg/L of ethoprophos exposure at 96 h post-fertilization (hpf), which induced cardiac defects, such as low heart rate, pericardium edema and long SV-BA distance, but had no influence to vascular development. Mechanistically, the expression of cardiac-related genes were abnormal. Moreover, ethoprophos exposure significantly increased oxidative stress in zebrafish embryos by inhibiting the production of antioxidant enzyme (SOD) and activating reactive oxygen species. Expectedly, some apoptosis genes were induced and the apoptotic cardiomyocytes were detected by acridine orange staining. In addition, ethoprophos exposure also inhibited the expression of genes in wnt signaling pathway, such as ß-catenin, Axin2, GSK3ß and Sox9b. BML284, an activator of wnt signaling pathway, can rescue the cardiotoxic effect of embryos. These results indicated that oxidative stress and blocking wnt signaling pathway were molecular basis of ethoprophos-induced injure in zebrafish. Generally, our study showed that ethoprophos exposure led to severe cardiotoxicity to zebrafish embryo.

IRF9 promotes apoptosis and innate immunity by inhibiting SIRT1-p53 axis in fish.

As a NAD+-dependent deacetylase, SIRT1 is widely involved in apoptosis and cellular inflammation via multiple pathways such as p53, NF-кB and STAT. More and more studies have shown that p53 is the first non-histone deacetylation target of SIRT1. SIRT1-p53 axis thus plays an important role in mammalian cells. IRF9 is an important member of interferon regulator factor family and performs an important role in innate immunity against foreign virus invasion. More importantly, human IRF9 can suppress the SIRT1-p53 axis. However, the functions and relationship between IRF9 and SIRT1-p53 axis are rarely studied in fish. To this end, we made a preliminary research on the functions of grass carp (Ctenopharyngodon idella) IRF9, SIRT1 and p53 in apoptosis and innate immunity. Firstly, we cloned and identified the ORF of SIRT1 (named CiSIRT1, MN125614) from C. idella and demonstrated that CiIRF9 promoted apoptosis, while CiSIRT1 inhibited apoptosis by flow cytometry and TUNEL experiments. Next, we found the interaction between CiSIRT1 and Cip53 in vivo by co-immunoprecipitation experiments. Moreover, the colocalization analysis also showed CiSIRT1 and Cip53 were mainly distributed in nucleus. Thirdly, we got a conclusion that CiIRF9 can repress the expression of CiSIRT1, implying that CiIRF9 regulates CiSIRT1-p53 axis. Finally, CiSIRT1 mRNA level was significantly up-regulated and the expression reached the highest level at 24 h post poly (I:C) stimulation in CIK cells. So, CiSIRT1 may exert an important function in innate immunity. Furthermore, we found CiSIRT1 down-regulated the expression of CiIFN1. In summary, CiIRF9 promotes apoptosis and innate immunity by inhibiting SIRT1-p53 axis. These findings will provide a new theoretical basis for the research on teleost innate immunity.

Combination of c oxidase subunit I based deoxyribonucleic acid barcoding and HPLC techniques for the identification and quality evaluation of Pheretima aspergillum.

This study aimed to identify Pheretima aspergillum (Guang-Pheretima) and its adulterants using the cytochrome c oxidase subunit I based deoxyribonucleic acid barcoding technology, and further to evaluate their quality using an optimized high-performance liquid chromatography method. For deoxyribonucleic acid barcoding identification, the Kimura-2-Parameter model was used to analyze genetic distance, and phylogenetic neighbor-joining tree was constructed for species identification of 20 labeled Guang-Pheretima samples. A high-performance liquid chromatography method was developed for the simultaneous determination of seven nucleoside components for quality evaluation. Compared with the GenBank database, 10 samples were identified as real Guang-Pheretima (P. aspergillum), and the others as the adulterants-Metaphire magna. The maximum intraspecific genetic distances of c oxidase subunit I sequence for P. aspergillum were smaller than the minimum interspecific genetic distances between P. aspergillum and M. magna. Ten P. aspergillum and 10 M. magna samples were clearly clustered in the neighbor-joining tree. The contents of seven nucleosides components in P. aspergillum were significantly higher than that in its adulterant-M. magna. The incidence of adulterants for Guang-Pheretima was high (up to 50%) with an alarming quality. This study provided a powerful idea for the quality evaluation of other highly valuable plant- or animal-derived products for safety concerns to avoid misidentification.

One functional variant in the 3'-untranslated region of TLR4 is associated with the elevated risk of ventilator-associated pneumonia in the patients with chronic obstructive pulmonary disease.

The aim of this study was to identify the association polymorphism (rs11536889) in the 3'-untranslated region (3'-UTR) of Toll-like receptors 4 (TLR4) and the risk for ventilator-associated pneumonia (VAP). miRNA database online and luciferase assays were used to validate TLR4 as the target gene of miR-1236. Enzyme-linked immunosorbent assay analysis and western blot were used to analyze the level of TLR4 in different genotype groups. In the present study, miR-1236 was predicted to bind to the rs11536889 G allele rather than the rs11536889 C allele, which was further confirmed by the luciferase activity suppressed by a fragment of 3'-UTR containing the rs11536889 G allele induced by lipopolysaccharide (LPS) and interleukin-6 (IL-6). Bronchial epithelial cells isolated from participants genotyped as GG, GC, and CC, with no remarkable difference in TLR4 messenger RNA (mRNA) levels were observed among these genotype groups. After stimulating by LPS, a TLR4 ligand, the CC-genotyped cells expressed higher levels of IL-8, IL-6, and tumor necrosis factor alpha (TNF-α) on their surfaces than cells with the other genotypes. Finally, the western blot analysis results showed that the expression level of IL-8, IL-6, and TNF-α protein was much higher in the CC group than the GC and GG groups subsequent to stimulation by LPS, and the IL-8, IL-6, and TNF-α protein levels in the GC were grouped much lower compared with the GG group. These findings indicated the regulatory association of miR-1236 with TLR4 and the abnormal expression of TLR4 caused by the presence of rs11536889 in the 3'-UTR of mRNA, which interfere with its interaction with the miR-1236, contributing to the risk of VAP.

A robust qualitative transcriptional signature for the correct pathological diagnosis of gastric cancer.

BACKGROUND: Currently, pathological examination of gastroscopy biopsy specimens is the gold standard for gastric cancer (GC) diagnosis. However, it has a false-negative rate of 10-20% due to inaccurate sampling locations and/or insufficient sampling amount. A signature should be developed to aid the early diagnosis of GC using biopsy specimens even when they are sampled from inaccurate locations. METHODS: We extracted a robust qualitative transcriptional signature, based on the within-sample relative expression orderings (REOs) of gene pairs, to discriminate both GC tissues and adjacent-normal tissues from non-GC gastritis, intestinal metaplasia and normal gastric tissues. RESULTS: A signature consisting of two gene pairs for GC diagnosis was identified and validated in data of both biopsy specimens and surgical resection specimens pooled from publicly available datasets measured by different laboratories with different platforms. For gastroscopy biopsy specimens, 96.20% of 79 non-GC tissues were correctly identified as non-GC, and 96.84% of 158 GC tissues and six of seven adjacent-normal tissues were correctly identified as GC. For surgical resection specimens, 98.37% of 2560 GC tissues and 97.28% of 221 adjacent-normal tissues were correctly identified as GC. Especially, 97.67% of the 257 GC patients at stage I were exactly diagnosed as GC. We additionally measured 21 GC tissues from seven different GC patients, each with three specimens sampled from three tumor locations with different proportions of the tumor epithelial cell. All these GC tissues were correctly identified as GC, even when the proportion of the tumor epithelial cell was as low as 14%. CONCLUSIONS: The qualitative transcriptional signature can distinguish both GC and adjacent-normal tissues from normal, gastritis and intestinal metaplasia tissues of non-GC patients even using inaccurately sampled biopsy specimens, which can be applied robustly at the individual level to aid the early GC diagnosis.

Identification of the SAMHD1 gene in grass carp and its roles in inducing apoptosis and inhibiting GCRV proliferation.

SAMHD1 is an innate immunity restriction factor that inhibits virus infection through IRF3-mediated antiviral and apoptotic responses. Fish SAMHD1 shares some similar properties with those in mammals. In this study, a SAMHD1 orthologue from grass carp (Ctenopharyngodon idellus) was cloned and characterized. The full-length cDNA of CiSAMHD1 is 2792 bp with an ORF of 1884 bp encoding a polypeptide of 627 amino acids. Multiple alignments showed that SAMHD1 is highly conserved among different species. Phylogenetic tree analysis revealed that CiSAMHD1 shared a high degree of homology with Sinocyclocheilus rhinocerous SAMHD1. Expression analysis indicated that CiSAMHD1 was widely expressed in all tissues tested including the brain, eyes, spleen, gill, intestine, liver, heart and kidney. It was significantly up-regulated in spleen, liver and intestines after treatment with poly I:C. Also, CiSAMHD1 can be induced following stimulation with recombinant IFN in CIK cells. The promoter sequence of CiSAMHD1 was identified to explore the mechanism underlying the transcriptional regulation of CiSAMHD1. The promoter sequence of CiSAMHD1 (1370 bp) consists of IRF1, IRF3, IRF9 and p65 binding elements. Gel mobility shift assay also showed that IRF1, IRF3, IRF9 and p65 prokaryotic proteins can separately interact with CiSAMHD1 promoter. Dual luciferase assay and q-PCR suggested that the promoter of CiSAMHD1 can be activated by the overexpression of CiIRF3 and CiIRF9, but cannot be triggered by CiIRF1 and Cip65. In contrast, knockdown of CiIRF3 or CiIRF9 inhibits the transcription of CiSAMHD1. Intriguingly, CCK assay suggested that CiSAMHD1 decreased cell viability. TUNEL apoptosis assay and Hoechst 33258 staining assay indicated that apoptosis is induced by the overexpression of CiSAMHD1. Crystal violet staining, detection of two GCRV genes (vp3 and vp5) and viral titration showed that CiSAMHD1 can suppress the proliferation of grass carp reovirus (GCRV) in CIK cells.

Endoplasmic Reticulum Transmembrane Proteins ZDHHC1 and STING Both Act as Direct Adaptors for IRF3 Activation in Teleost.

IFN regulatory factor (IRF)3 is a central regulator for IFN-ß expression in different types of pathogenic infections. Mammals have various pathogenic sensors that are involved in monitoring pathogen intrusions. These sensors can trigger IRF3-mediated antiviral responses through different pathways. Endoplasmic reticulum-associated proteins stimulator of IFN gene (STING) and zinc finger DHHC-type containing 1 (ZDHHC1) are critical mediators of IRF3 activation in response to viral DNA infections. In this study, grass carp STING and ZDHHC1 were found to have some similar molecular features and subcellular localization, and both were upregulated upon stimulation with polyinosinic:polycytidylic acid, B-DNA, or Z-DNA. Based on these results, we suggest that grass carp STING and ZDHHC1 might possess some properties similar to their mammalian counterparts. Overexpression of ZDHHC1 and STING in Ctenopharyngodon idella kidney cells upregulated IFN expression, whereas knockdown of IRF3 inhibited IFN activation. In addition, coimmunoprecipitation and GST pull-down assays demonstrated that STING and ZDHHC1 can interact separately with IRF3 and promote the dimerization and nuclear translocation of IRF3. Furthermore, we also found that small interfering RNA-mediated knockdown of STING could inhibit the expression of IFN and ZDHHC1 in fish cells. Similarly, knockdown of STING resulted in inhibition of the IFN promoter. In contrast, ZDHHC1 knockdown also inhibited IFN expression but had no apparent effect on STING, which indicates that STING is necessary for IFN activation through ZDHHC1. In conclusion, STING and ZDHHC1 in fish can respond to viral DNA or RNA molecules in cytoplasm, as well as activate IRF3 and, eventually, trigger IFN expression.

Identification of key aromatic compounds in Congou black tea by partial least-square regression with variable importance of projection scores and gas chromatography-mass spectrometry/gas chromatography-olfactometry.

BACKGROUND: Gas chromatography-olfactometry (GC-O) is the most frequently used method to estimate the sensory contribution of single odorant, but disregards the interactions between volatiles. In order to select the key volatiles responsible for the aroma attributes of Congou black tea (Camellia sinensis), instrumental, sensory and multivariate statistical approaches were applied. RESULTS: Using sensory analysis, nine panellists developed eight descriptors: floral, sweet, fruity, green, roasted, oil, spicy, and off-odour. Linalool, (E)-furan linalool oxide, (Z)-pyran linalool oxide, methyl salicylate, ß-myrcene, and phenylethyl alcohol, which were identified from the most representative samples by the GC-O procedure, were the essential aroma-active compounds in the formation of basic Congou black tea aroma. In addition, 136 volatiles were identified by gas chromatography-mass spectrometry (GC-MS), among which 55 compounds were determined as the key factors for six sensory attributes by partial least-square regression (PLSR) with variable importance of projection scores. CONCLUSION: Our results demonstrated that headspace solid-phase microextraction/GC-MS/GC-O was a fast approach for isolation and quantification aroma-active compounds. The PLSR method was also considered to be a useful tool in selecting important variables for sensory attributes. These two strategies, which allowed us to comprehensively evaluate the sensorial contribution of a single volatile from different perspectives, can be applied to related products for comprehensive quality control. © 2018 Society of Chemical Industry.