The antiplasmodial activity of the carboxylic acid metabolite of piperaquine and its pharmacokinetic profiles in healthy volunteers.

BACKGROUND: The long-acting antimalarial drug piperaquine can be metabolized into the carboxylic acid metabolite (PQM). However, the clinical relevance of PQM remains unclear. OBJECTIVES: The pharmacodynamics/pharmacokinetics of PQM were studied. METHODS: The antimalarial activity of PQM was studied in vitro (Plasmodium strains Pf3D7 and PfDd2) and in vivo (murine Plasmodium yoelii). The toxicity of PQM was evaluated in mice, in terms of the general measures of animal well-being, serum biochemical examination and ECG monitoring. The pharmacokinetic profiles of piperaquine and its metabolite PQM were investigated in healthy subjects after recommended oral doses of piperaquine. RESULTS: PQM showed no relevant in vitro antimalarial activity (IC50 > 1.0 µM) with no significant toxicity. After recommended oral administration of piperaquine to healthy subjects, the maximum concentration of PQM was less than 30.0 nM, and it did not accumulate after repeated dosing. CONCLUSIONS: With a low antimalarial potency, PQM should not contribute to the efficacy of piperaquine with clinically acceptable doses.

VmSom1 is essential for growth, development, maintenance of cell wall integrity and virulence in Valsa mali.

Apple Valsa canker disease, caused by Valsa mali Miyabe et Yamada, severely endangers the healthy growth of apple trees. The Som1, located downstream of the cyclic AMP-dependent protein kinase A (cAMP-PKA) pathway, plays crucial roles in the growth, development, morphological differentiation, and virulence of filamentous fungi. In this study, we identify and functionally characterize VmSom1, a homolog of Som1, in Valsa mali. The VmSom1 gene is located on chromosome 12, encoding an 824 amino acid protein. Phylogenetic analysis reveals VmSom1 as a fungal Som1 homolog. The VmSom1 deletion mutants exhibit slower growth rates and fail to produce pycnidia. Additionally, their hyphal growth is significantly inhibited on media containing Calcofluor White, Congo Red, NaCl, and sorbitol. The growth rate of VmSom1 deletion mutants is reduced on maltose, lactose, sucrose and fructose media but increases on glucose medium. Moreover, the mycelial growth rate of the VmSom1 deletion mutant is significantly lower than that of the wild-type strain in peptone, NH4SO4, NaNO3, and no nitrogen. Notably, the distances between the septa increase, and chitin concentration shifts to the hyphal tip in the VmSom1 deletion mutant. Furthermore, compared with the wild-type strain, the VmSom1 deletion mutant exhibits fewer diseased spots on apple fruit and branches. Overall, our findings demonstrate that VmSom1 is involved in regulating the growth and development, colony surface hydrophobicity, osmotic stress, cell wall integrity maintenance, carbon and nitrogen source utilization, septa formation, and virulence of V. mali.

The Regulation of the Growth and Pathogenicity of Valsa mali by the Carbon Metabolism Repressor CreA.

Carbon catabolite repression (CCR) is a very important mechanism for efficient use of carbon sources in the environment and is necessary for the regulation of fungal growth, development, and pathogenesis. Although there have been extensive studies conducted regarding this mechanism in fungi, little is yet known about the effects of CreA genes on Valsa mali. However, based on the results obtained in this study for the identification of the VmCreA gene in V. mali, it was determined that the gene was expressed at all stages of fungal growth, with self-repression observed at the transcriptional level. Furthermore, the functional analysis results of the gene deletion mutants (ΔVmCreA) and complements (CTΔVmCreA) showed that the VmCreA gene played an important role in the growth, development, pathogenicity, and carbon source utilization of V. mali.

Molecular characterization of a novel endornavirus isolated from Ophiostoma bicolor associated with bark beetles.

Ophiostoma bicolor is a pathogenic fungus associated with bark beetles that can cause serious damage to host plants. In this study, a novel fungal virus, "Ophiostoma bicolor endornavirus 1" (ObEV1), was obtained from O. bicolor, and its complete genome sequence was determined. ObEV1 has a single-stranded positive-sense (+ ss) RNA genome of 10,119 nucleotides. Sequence annotation and comparison showed that the viral genome has a single large open reading frame (ORF) encoding a polyprotein of 362.48 kDa. The polyprotein contains seven conserved domains: RNA-dependent RNA polymerase (RdRp), viral RNA helicase 1 (VHel1), viral methyltransferase (VMet), DEAD-like helicase (DEXDc), gliding-GltJ (G1), large tegument protein UL36 (PHA), and YlqF-related-GTPase (Y). Sequence comparisons and phylogenetic analysis showed that ObEV1 is a novel mycovirus belonging to the genus Betaendornavirus of the family Endornaviridae. This is the first report of a mycovirus in the ophiostomatoid fungus O. bicolor.

Sustainable nano-pesticide platform based on Pyrethrins II for prevention and control Monochamus alternatus.

BACKGROUND: Pine wilt disease as a devastating forest disaster result from Bursaphelenchus xylophilus that spread by stem-borers Monochamus alternatus feeding on pine leaves, which has brought inestimable economic losses to the world's forestry due to lack of effective prevention and control measures. In this paper, we put forward a proposal for utilizing nanoHKUST-1 to encapusulate the Pyrethrins II that a nerve agent extracted from plant to control M. alternatus, including toxicity mechanism research, traceable biopesticide monitoring, and environment assessment for the first time. The highly biocompatible nanoHKUST-1 can solve the problems of poor water solubility, easy degradation and low control efficiency of Pyrethrins II. RESULTS: The results illustrated the biopesticide loading efficiency of PthII@HKUST-1 reached 85% and the cumulative release of pH-dependent PthII@HKUST-1 was up to 15 days (90%), and also effectively avoid photodegradation (pH 7.0, retention 60.9%). 50 nm PthII@HKUST-1 made it easily penetrate the body wall of MA larvae and transmit to tissue cells through contact and diffusion. Moreover, PthII@HKUST-1 can effectively enhance the cytotoxicity and utilization of Pyrethrins II, which will provide valuable research value for the application of typical plant-derived nerve agents in the prevention and control of forestry pests. PthII@HKUST-1 as an environmentally friendly nano-pesticide can efficiently deliver Pyrethrins II to the larval intestines and absorbed by the larvae. PthII@HKUST-1 could also be transmitted to the epidemic wood and dead wood at a low concentration (10 mg/L). CONCLUSION: Here we speculate that nanoHKUST-1 will bring new opportunity to research biopesticide inhibition mechanism of different agricultural and forestry pests, which will break through the existing research limitations on development, utilization and traceable monitoring of biopesticide, especially for the study of targeting specific proteins.

FgPsd2, a phosphatidylserine decarboxylase of Fusarium graminearum, regulates development and virulence.

Phosphatidylserine decarboxylases (Psds) are enzymes regulating phosphatidylethanolamine biosynthesis in prokaryotes and eukaryotes, and have the central role in lipid metabolism. To date, the functions of Psds in plant pathogenic fungi are not fully understood. In this study, we have characterized two yeast Psd orthologues: FgPsd1 and FgPsd2, in Fusarium graminearum. Our results indicate that FgPsd1 and FgPsd2 are localized in mitochondria and Golgi, respectively. In addition, we have determined that FgPsd1 is a lethal gene and deletion of FgPsd2 resulted in a significant reduction of mycelial growth and conidiation. Futhermore, the FgPsd2 deletion mutant (ΔFgPsd2) is defective in ascospore production and virulence in wheat. Our study has also found that the ΔFgPsd2 mutant is more sensitive to osmotic and oxygen stresses. Moreover, deletion of FgPsd2 reduced the formation of lipid droplets and aggravated autophagy in F. graminearum. In summary, our findings indicate that FgPsd2 is important for mycelial growth, sexual and asexual reproduction, virulence, lipid droplet formation and autophagy in F. graminearum.

Metabolic Retroversion of Piperaquine (PQ) via Hepatic Cytochrome P450-Mediated N-Oxidation and Reduction: Not an Important Contributor to the Prolonged Elimination of PQ.

As a partner antimalarial with an extremely long elimination half-life (∼30 days), piperaquine (PQ) is mainly metabolized into a pharmacologically active N-oxide metabolite [piperaquine N-oxide (PN1)] in humans. In the present work, the metabolic retroversion of PQ and PN1, potentially associated with decreased clearance of PQ, was studied. The results showed that interconversion existed for PQ and its metabolite PN1. The N-oxidation of PQ to PN1 was mainly mediated by CYP3A4, and PN1 can rapidly reduce back to PQ via cytochrome P450 (P450)/flavin-containing monooxygenase enzymes. In accordance with these findings, the P450 nonselective inhibitor (1-ABT) or CYP3A4 inhibitor (ketoconazole) inhibited the N-oxidation pathway in liver microsomes (>90%), and the reduction metabolism was inhibited by 1-ABT (>90%) or methimazole (∼50%). Based on in vitro physiologic and enzyme kinetic studies, quantitative prediction of hepatic clearance (CLH) of PQ was performed, which indicated its negligible decreased elimination in humans in the presence of futile cycling, with the unbound CLH decreasing by 2.5% (0.069 l/h per kilogram); however, a minor decrease in unbound CLH (by 12.8%) was found in mice (0.024 l/h per kilogram). After an oral dose of PQ (or PN1) to mice, the parent form predominated in the blood circulation, and PN1 (or PQ) was detected as a major metabolite. Other factors probably associated with delayed elimination of PQ (intestinal metabolism and enterohepatic circulation) did not play a key role in PQ elimination. These data suggested that the metabolic interconversion of PQ and its N-oxide metabolite contributes to but may not significantly prolong its duration in humans. SIGNIFICANCE STATEMENT: This paper investigated the interconversion metabolism of piperaquine (PQ) and its N-oxide metabolite in vitro as well as in mice. The metabolic profiles of PQ were reestablished by this futile cycling, which contributes to but may not significantly prolong its elimination in humans. Enzyme phenotyping indicated a low possibility of interaction of PQ during artemisinin drug-based combination therapy treatment.

Genome Assembly and Annotation of Botryosphaeria dothidea sdau11-99, a Latent Pathogen of Apple Fruit Ring Rot in China.

Botryosphaeria dothidea is a latent and important fungal pathogen on a wide range of woody plants. Fruit ring rot caused by B. dothidea is a major disease in China on apple. This study establishes a high-quality, nearly complete, and well-annotated genome sequence of B. dothidea strain sdau11-99. The findings of this research provide a reference genome resource for further research on the apple fruit ring rot pathogen on apple and other hosts.

The gender-related variability in the pharmacokinetics and antiplasmodial activity of naphthoquine in rodents.

BACKGROUND: Naphthoquine (NQ) is a suitable partner anti-malarial for the artemisinin-based combination therapy (ACT), which is recommended to be taken orally as a single-dose regimen. The metabolism of NQ was mainly mediated by CYP2D6, which is well-known to show gender-specific differences in its expression. In spite of its clinical use, there is limited information on the pharmacokinetics of NQ, and no data are available for females. In this study, the effect of gender on the pharmacokinetics and antiplasmodial efficacy of NQ in rodents was evaluated. The underlying factors leading to the potential gender difference, i.e., plasma protein binding and metabolic clearance, were also evaluated. METHODS: The pharmacokinetic profiles of NQ were investigated in healthy male or female rats after a single oral administration of NQ. The antiplasmodial efficacy of NQ was studied in male or female mice infected with Plasmodium yoelii. The recrudescence and survival time of infected mice were also recorded after drug treatment. Plasma protein binding of NQ was determined in pooled plasma collected from male or female mice, rat or human. In vitro metabolism experiments were performed in the liver microsomes of male or female mice, rat or human. RESULTS: The results showed that the gender of rats did not affect NQ exposure (AUC0-t and Cmax) significantly (P > 0.05). However, a significant (P < 0.05) longer t1/2 was found for NQ in male rats (192.1 ± 47.7), compared with female rats (143.9 ± 27.1). Slightly higher but not significant (P > 0.05) antiplasmodial activity was found for NQ in male mice (ED90, 1.10 mg/kg) infected with P. yoelii, compared with female mice (ED90, 1.67 mg/kg). The binding rates of NQ to plasma protein were similar in males and females. There was no metabolic difference for NQ in male and female mice, rat or human liver microsomes. CONCLUSIONS: These results indicated that the pharmacokinetic profiles of NQ were similar between male and female rats, except for a longer t1/2 in male rats. The difference was not associated with plasma protein binding or hepatic metabolic clearance. Equivalent antiplasmodial activity was found for NQ in male and female mice infected with P. yoelii. This study will be helpful for the rational design of clinical trials for NQ.

Molecular characterization of a novel fusarivirus infecting the plant-pathogenic fungus Botryosphaeria dothidea.

A novel virus, Botryosphaeria dothidea fusarivirus 1 (BdFV1), was isolated from a fungal strain, SDAU11-86 of Botryosphaeria dothidea, and its complete genome sequence was determined. BdFV1 has a single-stranded positive-sense (+ssRNA) genome with 6,179 nucleotides, excluding the poly(A) tail. The genome of BdFV1 contains two putative open reading frames (ORFs). The first ORF encodes a large polyprotein of 1,544 amino acids (aa) with conserved RNA-dependent RNA polymerase and viral helicase domains. The second ORF encodes a putative 481-aa protein with unknown function. Sequence comparisons and phylogenetic analysis suggested that BdFV1 is a novel mycovirus belonging to the newly proposed family "Fusariviridae". This is the first report of a +ssRNA mycovirus in B. dothidea.

A Suppression Method of Concentration Background Noise by Transductive Transfer Learning for a Metal Oxide Semiconductor-Based Electronic Nose.

Signal drift caused by sensors or environmental changes, which can be regarded as data distribution changes over time, is related to transductive transfer learning, and the data in the target domain is not labeled. We propose a method that learns a subspace with maximum independence of the concentration features (MICF) according to the Hilbert-Schmidt Independence Criterion (HSIC), which reduces the inter-concentration discrepancy of distributions. Then, we use Iterative Fisher Linear Discriminant (IFLD) to extract the signal features by reducing the divergence within classes and increasing the divergence among classes, which helps to prevent inconsistent ratios of different types of samples among the domains. The effectiveness of MICF and IFLD was verified by three sets of experiments using sensors in real world conditions, along with experiments conducted in the authors' laboratory. The proposed method achieved an accuracy of 76.17%, which was better than any of the existing methods that publish their data on a publicly available dataset (the Gas Sensor Drift Dataset). It was found that the MICF-IFLD was simple and effective, reduced interferences, and deftly managed tasks of transfer classification.

Liquid chromatography-tandem mass spectrometry assay for the simultaneous determination of three major flavonoids and their glucuronidated metabolites in rats after oral administration of Artemisia annua L. extract at a therapeutic ultra-low dose.

The traditional antimalarial herb Artemisia annua L., from which artemisinin is isolated, is widely used in endemic regions. It has been suggested that artemisinin activity can be enhanced by flavonoids in A. annua; however, how fast and how long the flavonoids are present in the body remains unknown. In the present study, a rapid and sensitive liquid chromatography with tandem mass spectrometry method was developed and validated for the simultaneous determination of three major flavonoids components, i.e. chrysosplenol D, chrysoplenetin, and artemetin and their glucuronidated metabolites in rats after oral administrations of A. annua extracts at a therapeutic ultra-low dose. The concentration of the intact form was determined directly, and the concentration of the glucuronidated form was assayed in the form of flavonoids aglycones, after treatment with ß-glucuronidase/sulfatase. The method was linear in the range of 0.5-300.0 ng/mL for chrysoplenetin and artemetin, and 2-600 ng/mL for chrysosplenol D. All the validation data conformed to the acceptance requirements. The study revealed a significantly higher exposure of the flavonoid constituents in conjugated forms in rats, with only trace intact from. Multiple oral doses of A. annua extracts led to a decreased plasma concentration levels for three flavonoids.

Metabolism of Piperaquine to Its Antiplasmodial Metabolites and Their Pharmacokinetic Profiles in Healthy Volunteers.

As a partner antimalarial for artemisinin drug-based combination therapy (ACT), piperaquine (PQ) can be metabolized into two major metabolites, including piperaquine N-oxide (M1) and piperaquine N,N-dioxide (M2). To better understand the antimalarial potency of PQ, the antimalarial activity of the PQ metabolites (M1 and M2) was studied in vitro (in Plasmodium falciparum strains Pf3D7 and PfDd2) and in vivo (in the murine species Plasmodium yoelii) in this study. The recrudescence and survival time of infected mice were also recorded after drug treatment. The pharmacokinetic profiles of PQ and its two metabolites (M1 and M2) were investigated in healthy subjects after oral doses of two widely used ACT regimens, i.e., dihydroartemisinin plus piperaquine phosphate (Duo-Cotecxin) and artemisinin plus piperaquine (Artequick). Remarkable antiplasmodial activity was found for PQ (50% growth-inhibitory concentration [IC50], 4.5 nM against Pf3D7 and 6.9 nM against PfDd2; 90% effective dose [ED90], 1.3 mg/kg of body weight), M1 (IC50, 25.5 nM against Pf3D7 and 38.7 nM against PfDd2; ED90, 1.3 mg/kg), and M2 (IC50, 31.2 nM against Pf3D7 and 33.8 nM against PfDd2; ED90, 2.9 mg/kg). Compared with PQ, M1 showed comparable efficacy in terms of recrudescence and survival time and M2 had relatively weaker antimalarial potency. PQ and its two metabolites displayed a long elimination half-life (∼11 days for PQ, ∼9 days for M1, and ∼4 days for M2), and they accumulated after repeated administrations. The contribution of the two PQ metabolites to the efficacy of piperaquine as a partner drug of ACT for the treatment of malaria should be considered for PQ dose optimization.

Bionic Electronic Nose Based on MOS Sensors Array and Machine Learning Algorithms Used for Wine Properties Detection.

In this study, a portable electronic nose (E-nose) prototype is developed using metal oxide semiconductor (MOS) sensors to detect odors of different wines. Odor detection facilitates the distinction of wines with different properties, including areas of production, vintage years, fermentation processes, and varietals. Four popular machine learning algorithms-extreme gradient boosting (XGBoost), random forest (RF), support vector machine (SVM), and backpropagation neural network (BPNN)-were used to build identification models for different classification tasks. Experimental results show that BPNN achieved the best performance, with accuracies of 94% and 92.5% in identifying production areas and varietals, respectively; and SVM achieved the best performance in identifying vintages and fermentation processes, with accuracies of 67.3% and 60.5%, respectively. Results demonstrate the effectiveness of the developed E-nose, which could be used to distinguish different wines based on their properties following selection of an optimal algorithm.

Simultaneous determination of piperaquine and its N-oxidated metabolite in rat plasma using LC-MS/MS.

A sensitive and efficient liquid chromatography tandem mass spectrometry method was developed and validated for the simultaneous determination of piperaquine (PQ) and its N-oxidated metabolite (PQ-M) in plasma. A simple protein precipitation procedure was used for sample preparation. Adequate chromatographic retention was achieved on a C18 column under gradient elution with acetonitrile and 2 mm aqueous ammonium acetate containing 0.15% formic acid and 0.05% trifluoroacetic acid. A triple-quadrupole mass spectrometer equipped with an electrospray source was set up in the positive ion mode and multiple reaction monitoring mode. The method was linear in the range of 2.0-400.0 ng/mL for PQ and 1.0-50.0 ng/mL for PQ-M with suitable accuracy, precision and extraction recovery. The lower limits of detection (LLOD) were established at 0.4 and 0.2 ng/mL for PQ and PQ-M, respectively, using 40 µL of plasma sample. The matrix effect was negligible under the current conditions. No effect was found for co-administrated artemisinin drugs or hemolysis on the quantification of PQ and PQ-M. Stability testing showed that two analytes remained stable under all relevant analytical conditions. The validated method was successfully applied to a pharmacokinetic study performed in rats after a single oral administration of PQ (60 mg/kg).

Unraveling the specific regulation of the central pathway for anaerobic degradation of 3-methylbenzoate.

The mbd cluster encodes the anaerobic degradation of 3-methylbenzoate in the ß-proteobacterium Azoarcus sp. CIB. The specific transcriptional regulation circuit that controls the expression of the mbd genes was investigated. The PO, PB 1, and P3 R promoters responsible for the expression of the mbd genes, their cognate MbdR transcriptional repressor, as well as the MbdR operator regions (ATACN10GTAT) have been characterized. The three-dimensional structure of MbdR has been solved revealing a conformation similar to that of other TetR family transcriptional regulators. The first intermediate of the catabolic pathway, i.e. 3-methylbenzoyl-CoA, was shown to act as the inducer molecule. An additional MbdR-dependent promoter, PA, which contributes to the expression of the CoA ligase that activates 3-methylbenzoate to 3-methylbenzoyl-CoA, was shown to be necessary for an efficient induction of the mbd genes. Our results suggest that the mbd cluster recruited a regulatory system based on the MbdR regulator and its target promoters to evolve a distinct central catabolic pathway that is only expressed for the anaerobic degradation of aromatic compounds that generate 3-methylbenzoyl-CoA as the central metabolite. All these results highlight the importance of the regulatory systems in the evolution and adaptation of bacteria to the anaerobic degradation of aromatic compounds.

Metabolite identification of the antimalarial piperaquine in vivo using liquid chromatography-high-resolution mass spectrometry in combination with multiple data-mining tools in tandem.

Artemisinin-based combination therapy is widely used for the treatment of uncomplicated Plasmodium falciparum malaria, and piperaquine (PQ) is one of important partner drugs. The pharmacokinetics of PQ is characterized by a low clearance and a large volume of distribution; however, metabolism of PQ has not been thoroughly investigated. In this work, the metabolite profiling of PQ in human and rat was studied using liquid chromatography tandem high-resolution LTQ-Orbitrap mass spectrometry (HRMS). The biological samples were pretreated by solid-phase extraction. Data processes were carried out using multiple data-mining techniques in tandem, i.e., isotope pattern filter followed by mass defect filter. A total of six metabolites (M1-M6) were identified for PQ in human (plasma and urine) and rat (plasma, urine and bile). Three reported metabolites were also found in this study, which included N-oxidation (M1, M2) and carboxylic products (M3). The subsequent N-oxidation of M3 resulted in a new metabolite M4 detected in urine and bile samples. A new metabolic pathway N-dealkylation was found for PQ in human and rat, leading to two new metabolites (M5 and M6). This study demonstrated that LC-HRMS(n) in combination with multiple data-mining techniques in tandem can be a valuable analytical strategy for rapid metabolite profiling of drugs. Copyright © 2016 John Wiley & Sons, Ltd.

Agrobacterium tumefaciens-mediated transformation of Botryosphaeria dothidea.

Botryosphaeria dothidea is a severe causal agent of die-back and cankers of many woody plants and causes great losses in many regions. The pathogenic mechanism of this pathogen has not been well explored due to lack of mutants and genetic information. In this study, we developed an Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for B. dothidea protoplasts using vector pBHt2 containing the hph gene as a selection marker under the control of trp C promoter. Using this protocol we successfully generated the B. dothidea transformants with efficiency about 23 transformants per 10(5) protoplasts. This is the first report of genetic transformation of B. dothidea via ATMT and this protocol provides an effective tool for B. dothidea genome manipulation, gene identification and functional analysis.

Magnesium Lithospermate B Protects Neurons Against Amyloid ß (1-42)-Induced Neurotoxicity Through the NF-κB Pathway.

Magnesium lithospermate B (MLB) is one of the major bioactive components of Radix Salviae miltiorrhizae, a Chinese traditional herbal medicine colloquially known as Dan Shen. In this study, we investigated the neuroprotective effect of MLB against oligomeric amyloid ß (Aß) (1-42)-induced neurotoxicity in cultured FVB mouse hippocampal neurons. We found that pretreatment with MLB not only prevents a loss in neuronal cell viability following exposure to Aß (1-42), but also attenuates Aß (1-42)-induced release of pro-inflammatory cytokines and neuronal apoptosis in a dose-dependent manner. Mechanistic studies show that MLB counteracts Aß (1-42)-induced activation of the nuclear factor kappa B (NF-κB) pathway, evidenced by the suppression of NF-κB luciferase reporters, decreased expression of phosphorylated Inhibitor κB α and IκB kinase α, and reduced nuclear translocation of p65 in response to pre-treatment with 50 µg/ml MLB prior to Aß (1-42) exposure. MLB was able to reverse the increase in phosphorylated c-Jun N-terminal kinase (JNK) levels as well as the decrease in phosphorylated Akt levels that are induced by Aß (1-42), although this finding did not extend to extracellular signal-regulated kinase or p38 kinases. Furthermore, combining MLB with the JNK inhibitor SP600125 synergistically counteracts the Aß (1-42)-induced reduction in cell viability and neurite growth, and the neuroprotective effects of MLB could be attenuated by the Akt inhibitor triciribine. In conclusion, these results suggest that MLB can protect against Aß (1-42)-induced neuronal damage, which is most likely to be mediated by the NK-κB pathway.

Efficient transformation and expression of gfp gene in Valsa mali var. mali.

Valsa mali var. mali, the causal agent of valsa canker of apple, causes great loss of apple production in apple producing regions. The pathogenic mechanism of the pathogen has not been studied extensively, thus a suitable gene marker for pathogenic invasion analysis and a random insertion of T-DNA for mutants are desirable. In this paper, we reported the construction of a binary vector pKO1-HPH containing a positive selective gene hygromycin phosphotransferase (hph), a reporter gene gfp conferring green fluorescent protein, and an efficient protocol for V. mali var. mali transformation mediated by Agrobacterium tumefaciens. A transformation efficiency up to about 75 transformants per 10(5) conidia was achieved when co-cultivation of V. mali var. mali and A. tumefaciens for 48 h in A. tumefaciens inductive medium agar plates. The insertions of hph gene and gfp gene into V. mali var. mali genome verified by polymerase chain reaction and southern blot analysis showed that 10 randomly-selected transformants exhibited a single, unique hybridization pattern. This is the first report of A. tumefaciens-mediated transformation of V. mali var mali carrying a 'reporter' gfp gene that stably and efficiently expressed in the transformed V. mali var. mali species.