Comparative transcriptomics provides insights into the pathogenic immune response of brown leaf spots in weeping forsythia.

Weeping forsythia is an important ornamental, ecological and medicinal plant. Brown leaf spots limit the large-scale production of weeping forsythia as a medicinal crop. Alternaria alternata is a pathogen causing brown leaf spots in weeping forsythia; however, its pathogenesis and the immune response mechanisms of weeping forsythia remain unclear. In this study, we identified two mechanisms based on morphological anatomy, physiological indexes and gene expression analyses. Our results showed that A. alternata induced leaf stomata to open, invaded the mesophyll, dissolved the cell wall, destroyed the cell membrane and decreased the number of chloroplasts by up-regulating the expression of auxin-activated signaling pathway genes. Alternaria alternata also down-regulated iron-ion homeostasis and binding-related genes, which caused an increase in the levels of iron ions and reactive oxygen species in leaves. These processes eventually led to programmed cell death, destroying palisade and spongy tissues and causing the formation of iron rust spots. Alternaria alternata also caused defense and hypersensitive responses in weeping forsythia through signaling pathways mediated by flg22-like and elf18-like polypeptides, ethylene, H2O2 and bacterial secretion systems. Our study provides a theoretical basis for the control of brown leaf spots in weeping forsythia.

[Determination of seven ingredients of different grades Alismatis Rhizoma by QAMS method].

The present study is to establish a quantitative analysis of multi-components by single marker(QAMS) for determining contents of seven compositions in Alismatis Rhizoma, alismoxide, alisol C 23-acetate, alisol A, alismol, alisol B, alisol B 23-acetate and 11-deoxy-alisol B. Six relative correction factors(RCFs) of alismoxide, alisol C 23-acetate, alisol A, alismol, alisol B and 11-deoxy-alisol B were established in the UPLC method with alisol B 23-acetate as the internal standard, which was to calculate the mass fraction of each. The mass fraction of seven effective constituents in Alismatis Rhizoma was calculated by the external standard method(ESM) at the same time. Compared with the content results determined by the ESM and QAMS, the feasibility and accuracy of QAMS method were verified. Within the linear range, the RCFs of alismoxide, alisol C 23-acetate, alisol A, alismol, alisol B, 11-deoxy-alisol B were 0.946, 4.183, 0.915, 1.039, 0.923 and 1.244, respectively, with good repeatability in different experimental conditions. There was no significant difference between the QAMS method and ESM method. Then, QAMS method was applied to determination of the different degree Alismatis Rhizoma from different areas. As a result, the concentrations of 7 components have differences in different areas, but no significant differences in different grades. The QAMS method is feasible and accurate for the determination of the seven chemical compositions, and which can be used for quality control of Alismatis Rhizoma.

Pharmacokinetics and disposition of anlotinib, an oral tyrosine kinase inhibitor, in experimental animal species.

Anlotinib is a new oral tyrosine kinase inhibitor; this study was designed to characterize its pharmacokinetics and disposition. Anlotinib was evaluated in rats, tumor-bearing mice, and dogs and also assessed in vitro to characterize its pharmacokinetics and disposition and drug interaction potential. Samples were analyzed by liquid chromatography/mass spectrometry. Anlotinib, having good membrane permeability, was rapidly absorbed with oral bioavailability of 28%-58% in rats and 41%-77% in dogs. Terminal half-life of anlotinib in dogs (22.8±11.0 h) was longer than that in rats (5.1±1.6 h). This difference appeared to be mainly associated with an interspecies difference in total plasma clearance (rats, 5.35±1.31 L·h-1·kg-1; dogs, 0.40±0.06 L·h-1/kg-1). Cytochrome P450-mediated metabolism was probably the major elimination pathway. Human CYP3A had the greatest metabolic capability with other human P450s playing minor roles. Anlotinib exhibited large apparent volumes of distribution in rats (27.6±3.1 L/kg) and dogs (6.6±2.5 L/kg) and was highly bound in rat (97%), dog (96%), and human plasma (93%). In human plasma, anlotinib was predominantly bound to albumin and lipoproteins, rather than to α1-acid glycoprotein or γ-globulins. Concentrations of anlotinib in various tissue homogenates of rat and in those of tumor-bearing mouse were significantly higher than the associated plasma concentrations. Anlotinib exhibited limited in vitro potency to inhibit many human P450s, UDP-glucuronosyltransferases, and transporters, except for CYP3A4 and CYP2C9 (in vitro half maximum inhibitory concentrations, <1 µmol/L). Based on early reported human pharmacokinetics, drug interaction indices were 0.16 for CYP3A4 and 0.02 for CYP2C9, suggesting that anlotinib had a low propensity to precipitate drug interactions on these enzymes. Anlotinib exhibits many pharmacokinetic characteristics similar to other tyrosine kinase inhibitors, except for terminal half-life, interactions with drug metabolizing enzymes and transporters, and plasma protein binding.

[Clinical Study of Cytomegalovirus Infection and Preemptive Therapy after Allogenic Hematopoietic Stem Cell Transplantation].

OBJECTIVE: To analyze the clinical characteristics of cytomegalovirus(CMV) infection after allogenic hematopoietic stem cell transplantation(allo-HSCT) and the effect of preemptive therapy. METHODS: A total of 134 patients who underwent allo-HSCT from March 2010 to March 2015 in the Department of Hematology of our hospital were enrolled in this study. The CMV infection rate, the median time of CMV infection occurence, and the risk factors for CMV infection after allo-HSCT, the response rate of preemptive treatment and the median time of CMV-DNA turning negative were analyzed. Five-year overall survival rate was compared between the patients with or without CMV infection. RESULTS: The incidence of CMV viremia was 55.2％(74／134), and the median time for the CMV with CMV-DNA positive for the first time was 34 days(14-283) after allo-HSCT．Both univariate and multivariate analysis showed that the thymoglobulin(ATG) used in conditioning regimen and Ⅱ-Ⅳ grade of aGVHD were the risk factors for CMV viremia. After preemptive treatment the 85.1% of patient with CMV viremia turned negative, and the median time of CMV-DNA turning negative were 15 days(5-82), only 2 patients died of CMV pneumonia. Five-year overall survival rate of the patients with or wihout CMV viremia was 49% and 66.3% respectively, and the difference between the 2 groups was significant(P=0.041）. CONCLUSION: The ATG used in conditioning regimen and Ⅱ-Ⅳ grade of aGVHD may increase the incidence of CMV infection after allo-HSCT, and the preemptive thrapy can effectively prevent the CMV viremia turning to CMV disease.

A Newly Isolated Penicillium oxalicum 16 Cellulase with High Efficient Synergism and High Tolerance of Monosaccharide.

Compared to Trichoderma reesei RUT-C30 cellulase (Trcel), Penicillium oxalicum 16 cellulase (P16cel) from the fermentation supernatant produced a 2-fold higher glucose yield when degrading microcrystalline cellulose (MCC), possessed a 10-fold higher ß-glucosidase (BGL) activity, but obtained somewhat lower other cellulase component activities. The optimal temperature and pH of ß-1,4-endoglucanase, cellobiohydrolase, and filter paperase from P16cel were 50-60 °C and 4-5, respectively, but those of BGL reached 70 °C and 5. The cellulase cocktail of P16cel and Trcel had a high synergism when solubilizing MCC and generated 1.7-fold and 6.2-fold higher glucose yields than P16cel and Trcel at the same filter paperase loading, respectively. Additional low concentration of fructose enhanced the glucose yield during enzymatic hydrolysis of MCC; however, additional high concentration of monosaccharide (especially glucose) reduced cellulase activities and gave a stronger monosaccharide inhibition on Trcel. These results indicate that P16cel is a more excellent cellulase than Trcel.

Alternative messenger RNA splicing of autophagic gene Beclin 1 in human B-cell acute lymphoblastic leukemia cells.

Beclin 1 is a key factor for initiation and regulation of autophagy, which is a cellular catabolic process involved in tumorigenesis. To investigate the role of alternative splicing of Beclin1 in the regulation of autophagy in leukemia cells, Beclin1 mRNA from 6 different types of cell lines and peripheral blood mononuclear cells from 2 healthy volunteers was reversely transcribed, subcloned, and screened for alternative splicing. New transcript variants were analyzed by DNA sequencing. A transcript variant of Beclin 1 gene carrying a deletion of exon 11, which encoded a C-terminal truncation of Beclin 1 isoform, was found. The alternative isoform was assessed by bioinformatics, immunoblotting and subcellular localization. The results showed that this variable transcript is generated by alternative 3' splicing, and its translational product displayed a reduced activity in induction of autophagy by starvation, indicating that the spliced isoform might function as a dominant negative modulator of autophagy. Our findings suggest that the alternative splicing of Beclin 1 might play important roles in leukemogenesis regulated by autophagy.

Inordinate glucose variation poststroke is associated with poor neurological improvement in patients without history of diabetes.

INTRODUCTION: Glucose variation is an important risk factor for the complications of diabetes mellitus. The plasma glucose level poststroke is in continuous fluctuation. However, whether the variation influences neurological improvement remains unknown. AIMS: This observational study aimed to investigate the association of glucose variation with neurological improvement poststroke. METHODS: We consecutively enrolled 216 ischemic stroke patients with no history of diabetes mellitus within 72 h of onset, with instant blood glucose <11.1 mmol/L at admission. The glucometabolic status was evaluated by an oral glucose tolerance test 1 day after admission and 14 days after stroke, respectively. The severity of neurological deficit was assessed with the National Institute of Health Stroke Scale (NIHSS). RESULTS: Fourteen days after stroke, 31% patients were found to have impaired glucose tolerance and 30.6% were newly diagnosed diabetes mellitus by oral glucose tolerance test. A higher level of instant blood glucose at admission or fasting plasma glucose (FPG) at 1 day correlated with a less neurological improvement. The number of patients with no <20% decrease in NIHSS was significantly decreased in patient group with higher than 30% variation of either FPG or 2-h postprandial glucose. Similar correlation between glucose variation and neurological improvement was also found in 117 patients with 2-h postprandial glucose ≥7.8 mmol/L at 1 day. CONCLUSIONS: Inordinate glucose variation correlated with less neurological improvement poststroke, giving the evidence that the fluctuation of glucose levels in stroke patients should be taken into consideration during glucose modulation.

Role of autophagy in acute myeloid leukemia therapy.

Despite its dual role in determining cell fate in a wide array of solid cancer cell lines, autophagy has been robustly shown to suppress or kill acute myeloid leukemia cells via degradation of the oncogenic fusion protein that drives leukemogenesis. However, autophagy also induces the demise of acute leukemia cells that do not express the known fusion protein, though the molecular mechanism remains elusive. Nevertheless, since it can induce cooperation with apoptosis and differentiation in response to autophagic signals, autophagy can be manipulated for a better therapy on acute myeloid leukemia.

Astragaloside IV attenuates inflammatory cytokines by inhibiting TLR4/NF-кB signaling pathway in isoproterenol-induced myocardial hypertrophy.

ETHNOPHARMACOLOGICAL RELEVANCE: Astragaloside IV (As IV) is one of the main effective components isolated from the traditional Chinese medical herb Astragalus membranaceus. The protective effect of Astragalus membranaceus on myocardial hypertrophy has been extensively proved. To test the hypothesis that Astragaloside IV can ameliorate the myocardial hypertrophy and inflammatory effect induced by ß-adrenergic hyperactivity, we carried out in vivo and in vitro experiments. MATERIAL AND METHODS: In in vivo study, the isoproterenol (Iso) (5 mg kg(-1) d(-1)) was used as a model of myocardial hypertrophy by intraperitoneal injection. SD rats were randomly assigned to following six groups: A: the control; B: Iso group; C: Iso plus As IV 20 mg kg(-1) d(-1); D: Iso plus As IV 40 mg kg(-1) d(-1); E: Iso plus As IV 80 mg kg(-1) d(-1); F: Iso plus Propranolol 40 mg kg(-1) d(-1). In in vitro study, cultured neonatal rat cardiomyocytes were pretreated with As IV (3, 10, 30 µ mol L(-1)), Propranolol (2 µ mol L(-1)) and BAY11-7082 (5 µ mol L(-1)) for 30 min, and then incubated with Iso (10 µ mol L(-1)) for 48 h. For the rats in each group, the heart mass index (HMI) and the left ventricular mass index (LVMI) were measured. To measure the transverse diameter of left ventricular myocardial cells (TDM), the hematoxylin-eosin (HE) staining method was applied. In addition, the volume and the total protein content of cardiomyocytes were measured, the mRNA expression of ANP and TLR4 were quantified by RT-PCR, the protein expression of TLR4, IκBα and p65 were quantified by Western blot, and the level of TNF-α and IL-6 were measured by ELISA. RESULTS: In vivo: Comparing the Iso group to the control, the HMI, LVMI, TDM were significantly increased; the protein expression of TLR4 and p65 were increased, while the IκBα were decreased; the expression of ANP, TLR4 mRNA, and TNF-α, IL-6 in serum were significantly increased. These changes could be partly prevented by As IV and Pro. In vitro: the over-expression of the cell size, total protein content could remarkably down-regulated by As IV and Pro, and the results of RT-PCR, Western blot and ELISA were similar to those of in vivo. CONCLUSIONS: The results of these studies indicate that Astragaloside IV has good protective effect on myocardial hypertrophy induced by isoproterenol. More specifically, the cardioprotection is related to inhibiting the TLR4/NF-кB signaling pathway and the attenuating inflammatory effect.

Cloning, expression and characterization of a lipase gene from the Candida antarctica ZJB09193 and its application in biosynthesis of vitamin A esters.

Lipase is one of the most important industrial enzymes, which has been widely used in the preparation of food additives, cosmetics and pharmaceuticals industries. In order to obtain a large amount of lipase, the lipase gene from Candida antarctica ZJB09193 was cloned, and expressed in Pichia pastoris with the vector pPICZαA. Under the optimal conditions, the yield of recombinant lipase in the culture broth reached 3.0 g/L. After purification, the properties of recombinant lipase were studied: the optimum pH and temperature were pH 8.0 and 52°C, Ca(2+) activated the activity of lipase, and the apparent K(m) and V(max) values for p-nitrophenyl acetate were 0.34 mM and 7.36 µmol min(-1) mg(-1), respectively. Furthermore, the recombinant lipase was immobilized on pretreated textile for biosynthesis of vitamin A esters. In a system of n-hexane, 0.3 g immobilized recombinant lipase was used in the presence of 0.06 g vitamin A acetate and 0.55 mmol fatty acid (nine different fatty acids were tested). The yield of all vitamin A esters exceeded 78% in 7h at 30°C except using lactic acid and hexanoic acid as substrates. After optimization, the yield of vitamin A palmitate reached 87%. This study has the potential to be developed into industrial application.

[Mechanism study of adaptive response in high background radiation area of Yangjiang in China].

OBJECTIVE: To study the adaptive response mechanisms in high background radiation area (HBRA) among Yangjiang local people through gene and protein expression of receptor for advanced glycation end products (RAGE) and S100A6 in peripheral blood and sputum in inhabitants of HBRA. METHODS: A total of 53 male inhabitants were selected from HBRA in Yangjiang as the exposure group, while 53 male inhabitants were selected from Enping (control area, CA)as the control group. The content of RAGE and S100A6 gene and protein were detected by RT-PCR and Western blotting assay. Thermo luminescent dosemeter(TLD) assay was used to measure the outside dose and estimate the effective dose. RESULTS: The effective dose in CA and HBRA was respectively 1.95 mSv and 6.24 mSv, which was 3 fold difference. Compared with CA, RAGE and S100A6 expression were significantly reduced in both gene and protein level in HBRA. The relative median mRNA expression of RAGE and S100A6 in peripheral blood were respectively 0.28, 1.06 and 0.16, 0.79 in CA and HBRA group, there was significance (with analysis Z values of -2.587 and -2.328 respectively, P < 0.05) with Wilcoxon rank test. For the protein of sputum, the relative median expression were respectively 2.98, 2.25 and 0.53, 0.47 with significant difference (with analysis Z values of -2.201 and -2.366 respectively, P < 0.05) by Wilcoxon rank test. CONCLUSION: The low expression of RAGE and S100A6 in HBRA group might be correlated with the adaptive response and the low mortality of cancer in HBRA.

Proteomic alterations in progeny of irradiated human liver cells.

This study was designed to characterize the differential protein expression in the progeny of human liver cells surviving exposure to ionizing radiation. The progeny of irradiated cells were derived from a human liver cell line exposed to 0, 2, 4, or 6 Gy of (60)Co gamma-irradiation. Total protein of the cells was extracted by two-dimensional electrophoresis (2-DE) and analyzed with ImageMaster 2D Platinum software. In total, 42 differentially expressed proteins from the progeny of irradiated cells were screened, of which 17 were identified by matrix assistant laser desorption ion-top flight-mass spectrometry (MALDI-TOF-MS) analysis. There were 4 upregulated and 13 downregulated proteins detected. The upregulated expression of two proteins, mitochondrial heat-shock 60-kD protein (HSP60) and globin transcription factor 1 (GATA-1), was further confirmed by immunoblotting. Database search revealed that these differentially expressed proteins may function in cell cycle regulation, cytoskeleton maintenance, stress response, and tumor metastasis, indicating an effect of radiation-induced genomic instability (RIGI) in the progeny of irradiated cells. Analysis on functional roles of the screened proteins may provide insight into further mechanistic investigations underlying molecular events induced by RIGI.

Proteomic analysis of lung tissue of rats exposed to cigarette smoke and radon.

This study examined the protein expression in lung tissues of rats exposed to radon and cigarette smoke using a proteomic approach. Male Wistar rats were exposed daily to radon at a concentration of 100,000 Bq/m(3) for 16 h, and then exposed to 20% cigarette smoke for 1 h for a period of 75 d, with the radon cumulative dose reaching 200 WLM (working level months). Proteins from rat lung tissue were separated by two-dimensional gel electrophoresis (2-DE), stained with Coomassie blue, and analyzed with ImageMaster two-dimensional (2D) platinum software. Differentially expressed proteins were analyzed by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MALDI-time of flight [TOF] MS or MALDI TOF/TOF-MS). Twenty prominent proteins that were correlated with signal transduction, metabolism, heat shock and stress, and cytoskeleton construction were identified. Some of the differential expression proteins were verified by Western blot analysis and immunohistochemical staining, and the results were consistent with 2-DE analysis. The identified proteins and peptides might be potential diagnostic markers of lung impairment induced by radon and cigarette smoke exposure.

Up-regulation of RAGE and S100A6 in rats exposed to cigarette smoke.

Cigarette smoke has been widely investigated in terms of epidemiology and pathological endpoints in relation to human lung diseases and animal study. In this study we exposed Wistar rats to cigarette smoke at concentrations of 20% and 60% to explore potential molecular mechanisms at the protein level. Exposures were conducted twice a day, 5 days a week for 43 weeks. As a major metabolite of nicotine in cigarette, cotinine level in rat urine was determined by HPLC-MS. A dose-dependent analysis indicated that cotinine may be used as an exposure marker of cigarette smoke. Expression of receptor for advanced glycation endproducts (RAGE), an immunoglobulin super family that triggers the intracellular signal cascade reaction leading to inflammation and its ligand S100A6 (calgranulin) in bronchial epithelial cells and lung tissues of rats, were found to be positive correlated with cotinine levels, indicating that RAGE and S100A6 may be attributable to inflammation and oxidative damage caused by cigarette smoke.

Radon-induced proteomic profile of lung tissue in rats.

The aim of this study was to investigate the differential expression of proteins in lung of rats following long-term exposure to radon. The total proteins of lung tissue from Wistar rats exposed to radon for cumulative doses up to 100, 200, or 400 WLM (working level months) were isolated by two-dimensional electrophoresis (2-DE) and analyzed with ImageMaster 2D Platinum software. Comparison of the 2-DE images between the control and radon-exposed groups resulted in 14 upregulated and 9 downregulated protein spots, of which 15 were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) or matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). The simultaneous up-expressions of RAGE and S100A6 indicated that both proteins might be applied as biomarkers for lung injury induced by long-term radon exposure.