Sertraline-Induced Acute Pancreatitis: A Case Report and Literature Review.

Objective: Acute pancreatitis (AP) is a process of acute inflammation and cell damage of the pancreas. Gallstones and alcohol abuse are the most common cause for AP. Drug-induced pancreatitis (DIP), accounting for less than 3% of the AP, has become increasingly recognized as an additional and vitally important etiology of acute pancreatitis. Sertraline is an antidepressant of the selective serotonin reuptake inhibitor (SSRI)class that has a range of side effects even when used at the recommended dose. A recognized but rare association in teenagers is acute pancreatitis. The report is of a 15-year-old male teenager with a history of depression who developed acute pancreatitis following self-overdose of his sertraline prescription. Case Report: A 15-year-old teenager with an overdose of sertraline, which was the only medication he took, presented abdominal pain, nausea, and vomiting. The common causes of alcohol consumption, gallstones, biliary duct obstruction, malignancy, trauma, hypertriglyceridemia, and hypercalcemia were eliminated. The increased level of amylase and parenchymal edema of the pancreas revealed in computed tomography supported the diagnosis of acute pancreatitis. After discontinuation of the drug and conventional acute pancreatitis treatment, he recovered evenly. Conclusion: With the increasing use of antidepressant medications in patients of teenagers, this report is a reminder that clinicians should be aware of the association between SSRIs such as sertraline, particularly in cases of overdose, and the development of acute pancreatitis.

Preparation of novel fluorescent probe based on carbon dots for sensing and imaging Fe(III) and pyrophosphate in cells and zebrafish.

Ferric ions (Fe3+) and pyrophosphate anions (PPi) are involved in many physiological processes and play important roles in biological systems. The abnormal level of Fe3+ and PPi will cause serious damage to the environment and life. At present, the application of such probes in life, especially in vivo, is still very scarce. So, the development of a fluorescent probe to simultaneously detect Fe3+ and PPi has great significance to the health of the environment and organisms. Herein, nitrogen-doped carbon quantum dots (N-CDs) were synthesized via solvothermal treatment, using biuret and citric acid as precursors. The synthesized N-CDs showed highly selective and sensitive detection of Fe3+ through a photoluminescence quenching effect. The fluorescence of N-CDs quenched by Fe3+ could be restored with PPi, rendering the N-CDs/Fe3+ sensor promising for PPi detection ('OFF-ON'). The linear ranges of detection for Fe3+ and PPi were 3-30 and 2-12 µM, and the limits of detection were 2.71 and 1.12 µM, respectively. The practical applications of N-CDs were tested using tap water samples. Furthermore, N-CDs can be used for the detection and imaging of Fe3+ and PPi in HeLa cells and zebrafish owing to their excellent optical properties.

Pingyangmycin inhibits glycosaminoglycan sulphation in both cancer cells and tumour tissues.

Pingyangmycin is a clinically used anticancer drug and induces lung fibrosis in certain cancer patients. We previously reported that the negatively charged cell surface glycosaminoglycans are involved in the cellular uptake of the positively charged pingyangmycin. However, it is unknown if pingyangmycin affects glycosaminoglycan structures. Seven cell lines and a Lewis lung carcinoma-injected C57BL/6 mouse model were used to understand the cytotoxicity of pingyangmycin and its effect on glycosaminoglycan biosynthesis. Stable isotope labelling coupled with LC/MS method was used to quantify glycosaminoglycan disaccharide compositions from pingyangmycin-treated and untreated cell and tumour samples. Pingyangmycin reduced both chondroitin sulphate and heparan sulphate sulphation in cancer cells and in tumours. The effect was persistent at different pingyangmycin concentrations and at different exposure times. Moreover, the cytotoxicity of pingyangmycin was decreased in the presence of soluble glycosaminoglycans, in the glycosaminoglycan-deficient cell line CHO745, and in the presence of chlorate. A flow cytometry-based cell surface FGF/FGFR/glycosaminoglycan binding assay also showed that pingyangmycin changed cell surface glycosaminoglycan structures. Changes in the structures of glycosaminoglycans may be related to fibrosis induced by pingyangmycin in certain cancer patients.

miR-135b promotes proliferation and metastasis by targeting APC in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype. The aim of our study was to investigate the functional role of microRNA-135b (miR-135b) in TNBC. A real-time polymerase chain reaction assay was used to quantify miR-135b expression levels in 90 paired TNBC tissue and adjacent normal tissue samples. Wound-healing and transwell assays were performed to evaluate the effects of miR-135b expression on the migration and invasion of TNBC cells. Luciferase reporter and western blot analyses were used to verify whether the mRNA encoding APC is a major target of miR-135b. In the current study, we found that miR-135b was highly expressed in TNBC tissue and cells, and the expression levels were correlated with lymph node status and TNM stage. In TNBC cells, the ectopic expression of miR-135b promoted cell proliferation and invasion in vitro. In addition, our study proved that the overexpression of miR-135b significantly suppressed APC expression by targeting the 3'-untranslated region of APC, whereas enhanced APC expression could partially abrogate the miR-135b-mediated promotion of carcinogenic traits in TNBC cells. Taken together, our study demonstrated that miR-135b expression promoted the proliferation and invasion of TNBC by downregulating APC expression, indicating that miR-135b may serve as a promising target for the treatment of TNBC patients.

Fibroblast growth factor 21 protects against pathological cardiac remodeling by modulating galectin-3 expression.

BACKGROUND/AIMS: Fibroblast growth factor 21 (FGF21) plays a protective role in ischemia/reperfusion induced cardiac injury. However, the exact molecular mechanism of FGF21 action remains unclear. This study was designed the protective effect of FGF21 on the heart and its mechanism. METHOD: Adenovirus vector expressing FGF21 or control ß-galactosidase was injected into the myocardium of mice. Myocardial injury was observed by tissue staining and immunohistochemical staining. The expression level of caspases-3 and galectin-3 in myocardial cells were observed by immunoblotting. Then, hypoxia-induced cell model was established. Small interfering RNA (SiRNA) and plasmid were transfected into H9c2 using Lipofectamine 2000 reagent (Invitrogen). The expression levels of galectin-3, ECM and cystatin-3 in cells were observed by immunoblotting, and the relationship between fibroblast growth factor 21 and galectin-3 was analyzed. RESULT: Cell test in vitro showed that FGF21 could inhibit apoptosis and decrease the expression of ECM (ColIaI, fibronectin, and alpha-SMA) under hypoxia. Western blot data showed that hypoxia-induced cell damage increased galectin-3 levels, while FGF21 decreased galactose lectin-3 levels. In addition, inhibition of galactose agglutinin-3 expression by siRNA enhanced the cardioprotective effect of FGF21, while overexpression of galectin-3 reduced the cardioprotective effect of fibroblast growth factor 21. CONCLUSION: FGF21 may be a novel therapy for hypoxia-induced cardiac injury by regulating the expression of galectin-3.

Streptomyces inhibens sp. nov., a novel actinomycete isolated from rhizosphere soil of wheat (Triticum aestivum L.).

A novel actinomycete, designated strain NEAU-D10T, was isolated from rhizosphere soil of wheat (Triticum aestivum L.) collected from Northeast Agricultural University in Harbin, Heilongjiang Province, north-east China. A polyphasic approach was employed to determine the status of strain NEAU-D10T. Morphological and chemotaxonomic characteristics were consistent with those of members of the genus Streptomyces. The menaquinones detected were MK-9 (H6), MK-9 (H8) and MK-9 (H4). The phospholipid profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, hydroxy-phosphatidylethanolamine, phosphatidylinositol and three unidentified lipids. The major fatty acids were identified as iso-C16 : 0, C16 : 0, anteiso-C15 : 0 and iso-C14 : 0. Analysis of the 16S rRNA gene sequence showed that strain NEAU-D10T belongs to the genus Streptomyces with high sequence similarity to Streptomyces sioyaensis DSM 40032T (99.0 %) and Streptomyces auratus DSM 41897T (98.8 %). Moreover, multilocus sequence analysis based on five other housekeeping genes (atpD, gyrB, rpoB, recAand trpB) and the low level of DNA-DNA relatedness and phenotypic differences allowed the novel isolate to be differentiated from its most closely related strains, S. sioyaensis DSM 40032T and S. auratus DSM 41897T. It is concluded that the organism can be classified as representing a novel species of the genus Streptomyces, for which the name Streptomycesinhibens sp. nov. is proposed. The type strain is NEAU-D10T (=CGMCC 4.7469T=DSM 106197T).

Streptacidiphilus monticola sp. nov., a novel actinomycete isolated from soil.

A novel actinobacterium, designated strain NEAU-SW11T, was isolated from soil collected from Binxian, Heilongjiang province, north China. The isolate was found to have chemical and morphological properties of the genus Streptacidiphilus, with the highest sequence similarities to Streptacidiphilus anmyonensis JCM 16223T (98.1 %), Streptacidiphilus jiangxiensis JCM 12277T (97.8 %), Streptacidiphilus melanogenes JCM 16224T (97.6 %) and Streptacidiphilus rugosus JCM 16225T (97.4 %) and it phylogenetically clustered with these four strains. The cell wall contained ll-diaminopimelic acid as the major diamino acid and the whole-cell hydrolysates were rhamnose, ribose, glucose and galactose. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside and two unidentified phospholipids. The predominant menaquinones were MK-9(H8) and MK-9(H6). The major fatty acids were C16 : 0, anteiso-C17 : 0, C14 : 0 and C15 : 0. The DNA G+C content was 71.0 mol%. However, DNA-DNA hybridization, physiological and biochemical data showed that strain NEAU-SW11T could be distinguished from its closest relatives. Therefore, strain NEAU-SW11T represents a novel species of the genus Streptacidiphilus, for which the name Streptacidiphilus monticola sp. nov. is proposed. The type strain is NEAU-SW11T (=CGMCC 4.7427T=DSM 105744T).

Streptomyces flavalbus sp. nov., an actinobacterium isolated from rhizosphere of maize (Zea mays L.).

A novel actinobacterium, designated strain NEAU-QY24T, was isolated from the rhizosphere of corn (Zea mays L.). A polyphasic approach was employed to determine the taxonomic status of strain NEAU-QY24T. The isolate was found to have chemical and morphological properties of the genus Streptomyces, with high 16S rRNA gene sequence similarity to Streptomyces lanatus JCM 4332T (98.3%) and clustered phylogenetically with Streptomyces lannensis JCM 16578T (98.2%). The cell wall was found to contain meso-diaminopimelic acid and the whole cell sugars were identified as glucose and ribose. The predominant menaquinones were identified as MK-9(H6), MK-9(H4) and MK-9(H8). The phospholipid profile was found to consisted of diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol. The major fatty acids were identified as iso-C16:0, C16:0, anteiso-C17:0 and C15:0. A combination of DNA-DNA hybridization experiments and phenotypic tests were carried out between strain NEAU-QY24T and its closely related strains, which clarified their relatedness and demonstrated that strain NEAU-QY24T could be distinguished from these strains. These data indicate that the isolate should be recognised as a new species of the genus Streptomyces, for which the name Streptomyces flavalbus sp. nov. is proposed. The type strain is NEAU-QY24T (= CGMCC 4.7400T = DSM 104539T).

Streptomyces urticae sp. nov., isolated from rhizosphere soil of Urtica urens L.

Two novel Gram-stain positive, spore-forming, aerobic actinomycetes, designated NEAU-PCY-1T and NEAU-PCY-2, were isolated from rhizosphere soil of Urtica urens L. collected from Anshan, Liaoning Province, northeast China. The 16S rRNA gene sequence analysis showed that strains NEAU-PCY-1T and NEAU-PCY-2 exhibited 99.8% similarity with each other and are closely related to Streptomyces abietis DSM 42080T (98.2, 98.3%) and Streptomyces fildesensis DSM 41987T (98.0, 98.1%). Phylogenetic analysis based on the 16S rRNA gene sequences indicated that the two strains formed a cluster with these two closely related species. Moreover, DNA-DNA hybridization results and some phenotypic, physiological and biochemical properties differentiated the two strains from their close relatives in the genus Streptomyces. Based on a polyphasic taxonomy study, strains NEAU-PCY-1T and NEAU-PCY-2 are considered to represent a novel species of the genus Streptomyces, for which the name Streptomyces urticae sp. nov. is proposed, with NEAU-PCY-1T (= DSM 105115T = CCTCC AA 2017015T) as the type strain.

Promicromonospora viridis sp. nov., a novel actinomycete isolated from soil.

Two Gram-stain positive, aerobic actinomycete strains, designated NEAU-JGR1T and NEAU-JGC41, were isolated from soil collected from Fairy Lake Botanical Garden in Shenzhen, Guangdong Province, south of China. The 16S rRNA gene sequences analysis showed that the two strains exhibited 99.5% 16S rRNA gene sequence similarity with each other and were closely related to Promicromonospora thailandica JCM 17130T (99.4, 99.3%) and Promicromonospora citrea DSM 43110T (99.2, 99.2%). Phylogenetic analysis based on the 16S rRNA gene sequences indicated that the two strains clustered together and formed a cluster with P. thailandica JCM 17130T and P. citrea DSM 43110T. Both strains were observed to contain MK-9(H4) and MK-9(H2) as predominant menaquinones. Their whole cell sugar profiles were found to main contained rhamnose, ribose, glucose and galactose. The phospholipid profile contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, glycophosphatidylinositol, phosphatidylinositol mannoside, an unidentified glycolipid and an unidentified phospholipid. The predominant cellular fatty acids for the two strains were identified as anteiso-C15:0, iso-C15:0 and anteiso-C17:0. The DNA-DNA hybridization value between strains NEAU-JGR1T and NEAU-JGC41 was 85.1 ± 0.3%, and the values between the two strains and their close phylogenetic relatives were well below 70%, supporting the conclusion that they represent a distinct genomic species. An array of phenotypic characteristics also differentiated the isolates from closely related species. On the basis of the genetic and phenotypic properties, strains NEAU-JGR1T and NEAU-JGC41 can be classified as representatives of a novel species of the genus Promicromonospora, for which the name Promicromonospora viridis sp. nov., is proposed. The type strain is NEAU-JGR1T (= DSM 105536T = CGMCC 4.7473T).

Silencing of Prrx2 Inhibits the Invasion and Metastasis of Breast Cancer both In Vitro and In Vivo by Reversing Epithelial-Mesenchymal Transition.

BACKGROUND/AIMS: Epithelial-mesenchymal transition (EMT) is recognized as a crucial mechanism in breast cancer progression and metastasis. Paired-related homeobox 2 (Prrx2) has been identified as a new EMT inducer in cancer, but the underlying mechanisms are still poorly understood. METHODS: The expression of Prrx2 was assessed by immunohistochemistry in breast cancer tissues to evaluate the clinicopathological significance of Prrx2, as well as the correlation between Prrx2 and EMT. Short hairpin RNA knockdown of Prrx2 was used to examine cellular effects of Prrx2, detecte the expression of Wnt/ß-catenin signaling and EMT-associated proteins, and observe cell proliferation, invasion and migration abilities in vitro and in vivo. RESULTS: Clinical association studies showed that Prrx2 expression was related to tumor size, lymph node metastasis, tumor node metastasis stages, EMT and poor survival. Results also showed that knockdown of Prrx2 could alter cell morphology, suppressed the abilities of cell proliferation, invasion and migration in breast cancer. Moreover, silencing of Prrx2 induced the mesenchymal-epithelial transition and prevented nuclear translocation of ß-catenin, inhibited wnt/ß-catenin signaling pathway. CONCLUSION: Our study indicated that Prrx2 may be an important activator of EMT in human breast cancer and it can serve as a molecular target of therapeutic interventions for breast cancer.

MiR-212-5p Suppresses the Epithelial-Mesenchymal Transition in Triple-Negative Breast Cancer by Targeting Prrx2.

BACKGROUND/AIMS: Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype. Our study investigated the functional role of miR-212-5p in TNBC. METHODS: Realtime PCR was used to quantify miR-212-5p expression levels in 30 paired TNBC samples and adjacent normal tissues. Wound healing and Transwell assays were used to evaluate the effects of miR-212-5p expression on the invasiveness of TNBC cells. Luciferase reporter and Western blot assays were used to verify whether the mRNA encoding Prrx2 is a major target of miR-212-5p. RESULTS: MiR-212-5p was downregulated in TNBC, and its expression levels were related to tumor size, lymph node status and vascular invasion in breast cancer. We also observed that the miR-212-5p expression level was significantly correlated with a better prognosis in TNBC. Ectopic expression of miR-212-5p induced upregulation of E-cadherin expression and downregulation of vimentin expression. The expression of miR212-5p also suppressed the migration and invasion capacity of mesenchymal-like cancer cells accompanied by a morphological shift towards the epithelial phenotype. Moreover, our study observed that miR-212-5p overexpression significantly suppressed Prrx2 by targeting its 3'-untranslated region (3'-UTR) region, and Prrx2 overexpression partially abrogated miR-212-5p-mediated suppression. CONCLUSIONS: Our study demonstrated that miR-212-5p inhibits TNBC from acquiring the EMT phenotype by downregulating Prrx2, thereby inhibiting cell migration and invasion during cancer progression.

Two new species of the genus Streptomyces: Streptomyces camponoti sp. nov. and Streptomyces cuticulae sp. nov. isolated from the cuticle of Camponotus japonicus Mayr.

Two novel actinomycetes, designated strains 2C-SSA16(2)T and 1C-GS8T, were isolated from the cuticle of Camponotus japonicus Mayr, collected from Northeast Agricultural University, Heilongjiang Province, north China. Both of them contained genes (involved in antibiotics biosynthesis) of the ketosynthase (KS) and methyl malonyl transferase domains (PKS-I) and the adenylation domain (NRPS). A polyphasic study was carried out to establish the taxonomic positions of these strains. The 16S rRNA gene sequence analysis showed that the two novel isolates 2C-SSA16(2)T and 1C-GS8T exhibited 98.8% similarity with each other and that they are most closely related to Streptomyces umbrinus JCM 4521T (99.0, 98.6%), Streptomyces ederensis JCM 4958T (98.9, 98.7%), Streptomyces aurantiacus JCM 4453T (98.6, 98.2%), Streptomyces glomeroaurantiacus JCM 4677T (98.6, 98.1%), Streptomyces tauricus JCM4837T (98.2, 98.0%) and Streptomyces phaeochromogenes JCM 4070T (98.2, 99.2%). The corresponding phylogenetic analysis based on partial gyrB gene sequences showed that strains 2C-SSA16(2)T and 1C-GS8T formed a cluster with the above-mentioned strains. The DNA-DNA hybridization data and phenotypic characteristics indicated that strains 2C-SSA16(2)T and 1C-GS8T could be readily distinguished from each other and their closest phylogenetic relatives. Therefore, these two strains are suggested to represent two novel species of the genus Streptomyces, for which the names Streptomyces camponoti sp. nov. and Streptomyces cuticulae sp. nov. are proposed. The type strains are 2C-SSA16(2)T (=CGMCC 4.7276T = DSM 100522T) and 1C-GS8T (=CGMCC 4.7348 = DSM 103127T), respectively.

Longispora urticae sp. nov., isolated from rhizosphere soil of Urtica urens L., and emended descriptions of the species Longisporaalbida and Longisporafulva.

Two Gram-stain-positive, aerobic actinomycete strains, designated NEAU-PCY-3T and NEAU-PCY-4, were isolated from rhizosphere soil of Urtica urens L. collected from Anshan, Liaoning Province, northeast PR China. The 16S rRNA gene sequence analysis showed that the two strains exhibited 99.9 % 16S rRNA gene sequence similarity with each other and that they were most closely to Longispora fulva DSM 45356T (98.7, 98.9 %) and Longispora albida JCM 11711T (97.1, 97.2 %). Phylogenetic analysis based on the 16S rRNA gene sequences indicated that the two strains were located in the same lineage and formed a cluster with the genus Longispora. Both strains were observed to contain MK-10(H4) and MK-10(H6) as the predominant menaquinones. The cell wall peptidoglycan was found to contain meso-diaminopimelic acid, d-glutamic acid, glycine and l-alanine. Whole-cell hydrolysates mainly contained galactose, ribose and xylose. The phospholipid profile contained diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, several glycolipids and several unknown lipids. The major cellular fatty acids for strain NEAU-PCY-3T were iso-C16 : 0, iso-C17 : 0, anteiso-C17 : 0 and C18 : 1ω5c. The DNA-DNA hybridization value between strains NEAU-PCY-3T and NEAU-PCY-4 was 83.6±0.4 %, and the values between the two strains and their closest phylogenetic relatives, belonging to the genus Longispora, were well below 70 %, supporting that they represented a distinct genomic species. An array of phenotypic characteristics also differentiated the strains from their closely related species, the only two validly published Longispora species. On the basis of the genetic, chemotaxonomic and phenotypic properties, strains NEAU-PCY-3T and NEAU-PCY-4 were classified as representatives of a novel species of the genus Longispora, for which the name Longispora urticae sp. nov. is proposed. The type strain is NEAU-PCY-3T (=DSM 105119T=CCTCC AA 2017017T).

miR-655 suppresses epithelial-to-mesenchymal transition by targeting Prrx1 in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer subtype that lacks effective targeted therapies. The epithelial-to-mesenchymal transition (EMT) is a key contributor in the metastatic process. In this study, we found that miR-655 was down-regulated in TNBC, and its expression levels were associated with molecular-based classification and lymph node metastasis in breast cancer. These findings led us to hypothesize that miR-655 overexpression may inhibit EMT and its associated traits of TNBC. Ectopic expression of miR-655 not only induced the up-regulation of cytokeratin and decreased vimentin expression but also suppressed migration and invasion of mesenchymal-like cancer cells accompanied by a morphological shift towards the epithelial phenotype. In addition, we found that miR-655 was negatively correlated with Prrx1 in cell lines and clinical samples. Overexpression of miR-655 significantly suppressed Prrx1, as demonstrated by Prrx1 3'-untranslated region luciferase report assay. Our study demonstrated that miR-655 inhibits the acquisition of the EMT phenotype in TNBC by down-regulating Prrx1, thereby inhibiting cell migration and invasion during cancer progression.

Silencing of Prrx1b suppresses cellular proliferation, migration, invasion and epithelial-mesenchymal transition in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a highly aggressive tumour subtype associated with poor prognosis. The mechanisms involved in TNBC progression remains largely unknown. To date, there are no effective therapeutic targets for this tumour subtype. Paired-related homeobox 1b (Prrx1b), one of major isoforms of Prrx1, has been identified as a new epithelial-mesenchymal transition (EMT) inducer. However, the function of Prrx1b in TNBC has not been elucidated. In this study, we found that Prrx1b was significantly up-regulated in TNBC and associated with tumour size and vascular invasion of breast cancer. Silencing of Prrx1b suppressed the proliferation, migration and invasion of basal-like cancer cells. Moreover, silencing of Prrx1b prevented Wnt/ß-catenin signaling pathway and induced the mesenchymal-epithelial transition (MET). Taken together, our data indicated that Prrx1b may be an important regulator of EMT in TNBC cells and a new therapeutic target for interventions against TNBC invasion and metastasis.

[Construction of recombinant baculovirus vectors started by EF1alpha].

OBJECTIVE: To improve the transduction efficiency of baculovirus and exogenous gene expression level, we chose a mammalian cell-specific promoter-human extension factor 1alpha promoter (EF1-alpha), used virus pseudotyped tools--truncated vessicular stomatitis virus protein G (VSV-GED), added woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and adenovirus inverted terminal repeats (ITRs). METHOD: We constructed two improved recombinant baculoviruses transfer vectors named pWK and pWK-ITR with the pFB-VSV-GED-WPRE. The recombinant transfer vectors pWK-eGFP, pWK-ITR-eGFP and pWK (-)-eGFP were constructed by inserting the Enhanced Green Fluorescent Protein (eGFP) reporter gene into the downstream of EF1alpha promoter. Constructed recombinant plasmid transfected Sf9 insect cells, and observed the expression of green fluorescent protein by using the inverted fluorescence microscope. RESULTS: The fluorescence expression rate of BV-WK-eGFP, BV-WK-ITR-eGFP containing WPRE and ITRs was significantly higher than the negative control, ITRs can effectively extend the expression time of eGFP, the expression time of eGFP in BV-WK-eGFP and BV-WK-ITR-eGFP increased 72 hours compared to the negative control BV-WK (-) -eGFP. The transduction time of VSV-GED pseudotyped baculovirus BV-WK-eGFP, BV-WK-ITR-eGFP was obviously shorten in OL cells, and reduced 24 hours compared to the negative control BV-WK (-) -eGFP, transduction efficiency were higher 25.7% and 36.5% than the negative control BV-WK (-) -eGFP, respectively. CONCLUSION: The experiments proved that the VSV-GED could effectively improve the transduction efficiency of baculovirus, WPRE could enhance the expression efficiency of the exogenous gene significantly, and ITRs extend the expression time. The research will lay a foundation to explore improved recombinant baculovirus express exogenous genes in vertebrate cells and research the new recombinant live vector vaccine.

[Construction of baculovirus vector with WPRE regulatory element to express Newcastle disease virus F gene in primary chicken embryo cells].

OBJECTIVE: To construct the recombinant baculovirus with mammaliancell-specific promoter and woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), to highly express Newcastle disease virus (NDV) F gene in the primary chicken embryo cells. METHOD: We extracted total RNAs from NDV La Sota strain. Then the F gene was amplified by reverse transcription polymerase chain reaction. We constructed the baculoviral vector (pCMV-WPRE-F) with F gene fused with the WPRE near its 3'end, which expressed under the control of the CMV promoter. The F gene recombinant bacmid was obtained by Bac-to-Bac system and transfected into sf9 insect cells to acquire F gene recombinant baculovirus. After amplification of recombinant baculovirus, the recombinant virus was transfected into chicken primary cells with 50 multiplicity of infection, and the proteins were harvested at 72 h after infection. The F protein expression levels mediated by WPRE regulatory element were analyzed. RESULTS: Western blot results show that the F gene was successfully expressed in chicken primary cells. The product was a 56kDa protein and could be recognized by anti-NDV serum. The WPRE fusion significantly improved the F gene expression as 10 mmol/L butyrate did, but different to butyrate, the WPRE regulatory element was nontoxic to cells. CONCLUSION: The optimized recombinant baculovirus could efficiently deliver NDV F gene into chicken primary cells and express the F antigen protein. In addition, the WPRE regulatory element could increase the expression levels of exogenous gene mediated by baculovirus in chicken primary cells. The research provides us a potential basis for the gene engineered vaccines of NDV and other avian infectious disease based on baculovirus vector.

[Construction of baculovirus vector with Cytomegaloviruse promoter to express eGFP in primary chicken embryo cells].

OBJECTIVE: Baculovirus is known as a safe vector candidate due to its non-replication in mammalian cells. The tropism to different cells and transduction efficiency can be improved by introducing cell-specific promoter, VSV-GED and different functional regulatory elements. The optimized pseudotyped recombinant baculovirus can express eGFP gene in primary chicken cells, which provides us a new approach to develop engineered poultry vaccines. METHOD: The pseudotyped recombinant baculoviruses were constructed with cytomegaoviyns (CMV) promotor, VSV-GED, woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and inverted terminal repeats (ITRs). The recombinant baculoviruses contained eGFP reporter gene were transfected chicken primary cells, and the eGFP protein expression levels mediated by different baculoviruses were analyzed. RESULTS: The expression of eGFP was detected at 12 hours after infection. The transduction efficiency of the pseudotyped recombinant baculoviruses increased from 36% to 48.2% by inserting VSV-GED. The expression effect of eGFP in recombinant baculovirus carrying WPRE element was similar to that by adding 10 mmol/L butyrate. However, the WPRE elements are nontoxic to cells. Within 72 hours, the expression intensity of eGFP in the recombinant baculovirus with ITRs increased gradually. CONCLUSION: The VSV-GED element can improve the transduction efficiency and WPRE can increase the reporter gene eGFP expression levels mediated by baculovirus in chicken primary cells. The recombinant baculovirus with the ITRs elements can extend the expression time of eGFP.

Metal-free auxiliary pyrophosphate detection based on near-infrared carbon dots.

The excessive use of pyrophosphate (PPi) anions as additives poses a serious threat to human health and the environment. Considering the current status of PPi probes, the development of metal-free auxiliary PPi probes has important applications. In this study, a novel near-infrared nitrogen and sulfur co-doped carbon dots (N,S-CDs) were prepared. The average particle size of N,S-CDs was 2.25 ± 0.32 nm with average height was 3.05 nm. The probe N,S-CDs showed a special response to PPi, and a good linear relationship was obtained with PPi concentrations ranging from 0 to 1 µM, with the limit of detection being 0.22 nM. Tap water and milk were used for practical inspection, and ideal experimental results were acquired. In addition, the probe N,S-CDs also showed good results in biological systems, such as cell and zebrafish experiments.