Insight into the evolutionary and domesticated history of the most widely cultivated mushroom Agaricus bisporus via mitogenome sequences of 361 global strains.

Agaricus bisporus is the most widely cultivated edible mushroom in the world with a only around three hundred years known history of cultivation. Therefore, it represents an ideal organism not only to investigate the natural evolutionary history but also the understanding on the evolution going back to the early era of domestication. In this study, we generated the mitochondrial genome sequences of 352 A. bisporus strains and 9 strains from 4 closely related species around the world. The population mitogenomic study revealed all A. bisporus strains can be divided into seven clades, and all domesticated cultivars present only in two of those clades. The molecular dating analysis showed this species origin in Europe on 4.6 Ma and we proposed the main dispersal routes. The detailed mitogenome structure studies showed that the insertion of the plasmid-derived dpo gene caused a long fragment (MIR) inversion, and the distributions of the fragments of dpo gene were strictly in correspondence with these seven clades. Our studies also showed A. bisporus population contains 30 intron distribution patterns (IDPs), while all cultivars contain only two IDPs, which clearly exhibit intron loss compared to the others. Either the loss occurred before or after domestication, that could suggest that the change facilitates their adaptation to the cultivated environment.

Postsynaptic glutamate response downregulates within presynaptic exaggerated glutamate release by activating TRPV1 in the spinal dorsal horn.

Activating primary afferent TRPV1-positive (TRPV1+) fibers in the spinal dorsal horn triggers exaggerated glutamate release and induces acute pain. However, whether the glutamate postsynaptic responses on dorsal horn neurons are regulated by excessive glutamate is unknown, largely due to intrinsic technical difficulties. In the present study, capsaicin, a specific TRPV1 agonist, was used to activate TRPV1+ fibers in the spinal dorsal horn. Combining three-dimensional (3-D) holographic photostimulation and whole-cell recordings on acute spinal cord slices from adult rodents, we found that postsynaptic glutamate responses were attenuated when activating TRPV1+ fibers with capsaicin. Electron microscopy and Western blot studies found that postsynaptic GluA1 (a subtype of ionotropic glutamate receptors) on the postsynaptic membrane was decreased by acute capsaicin treatment. Therefore, postsynaptic glutamate receptor occupancy and/or downmodulation may underlie this postsynaptic attenuation. Our data thus clarify a scenario in which postsynaptic glutamate responses are largely downregulated upon TRPV1+ activation, and this change may contribute to homeostasis in the dorsal horn circuit when "acute pain" occurs.

Cardiotoxicity induced by Cochinchina momordica seed extract in zebrafish.

Momordica cochinchinensis (Lour.) Spreng is an indigenous South Asian edible fruit, and seeds of Momordica cochinchinensis have been used therapeutically in traditional Chinese medicine. Previous studies have shown that M. cochinchinensis seed (Momordicae Semen) has various pharmaceutical properties such as antioxidant and anti-ulcer effects as well as contains secondary metabolites with potential anticancer activities such as triterpenoids and saponins. Recent studies reported that water extract and ethanol extract of M. cochinchinensi seed were tested on mammals using an acute toxic classic method as OECD guidelines 420. No matter injected intravenously or intramuscularly, animals died within several days. In this study, zebrafish embryos were exposed to various doses of Cochinchina momordica seed extract (CMSE) from 2 dpf (days post fertilization, dpf) to 3 dpf. CMSE-induced cardiotoxicity such as pericardial edema, cardiac apoptosis, increased ROS production, cardiac neutrophil infiltration, decreased blood flow velocity, and reduced expression of three marker genes of cardiac functions were found in zebrafish roughly in a dose-dependent manner. These results suggest that CMSE may induce cardiotoxicity through pathways involved in inflammation, oxidative stress, and apoptosis.

Cyclophilin BcCyp2 Regulates Infection-Related Development to Facilitate Virulence of the Gray Mold Fungus Botrytis cinerea.

Cyclophilin (Cyp) and Ca2+/calcineurin proteins are cellular components related to fungal morphogenesis and virulence; however, their roles in mediating the pathogenesis of Botrytis cinerea, the causative agent of gray mold on over 1000 plant species, remain largely unexplored. Here, we show that disruption of cyclophilin gene BcCYP2 did not impair the pathogen mycelial growth, osmotic and oxidative stress adaptation as well as cell wall integrity, but delayed conidial germination and germling development, altered conidial and sclerotial morphology, reduced infection cushion (IC) formation, sclerotial production and virulence. Exogenous cyclic adenosine monophosphate (cAMP) rescued the deficiency of IC formation of the ∆Bccyp2 mutants, and exogenous cyclosporine A (CsA), an inhibitor targeting cyclophilins, altered hyphal morphology and prevented host-cell penetration in the BcCYP2 harboring strains. Moreover, calcineurin-dependent (CND) genes are differentially expressed in strains losing BcCYP2 in the presence of CsA, suggesting that BcCyp2 functions in the upstream of cAMP- and Ca2+/calcineurin-dependent signaling pathways. Interestingly, during IC formation, expression of BcCYP2 is downregulated in a mutant losing BcJAR1, a gene encoding histone 3 lysine 4 (H3K4) demethylase that regulates fungal development and pathogenesis, in B. cinerea, implying that BcCyp2 functions under the control of BcJar1. Collectively, our findings provide new insights into cyclophilins mediating the pathogenesis of B. cinerea and potential targets for drug intervention for fungal diseases.

The H3K4 demethylase Jar1 orchestrates ROS production and expression of pathogenesis-related genes to facilitate Botrytis cinerea virulence.

Histone 3 Lysine 4 (H3K4) demethylation is ubiquitous in organisms, however the roles of H3K4 demethylase JARID1(Jar1)/KDM5 in fungal development and pathogenesis remain largely unexplored. Here, we demonstrate that Jar1/KDM5 in Botrytis cinerea, the grey mould fungus, plays a crucial role in these processes. The BcJAR1 gene was deleted and its roles in fungal development and pathogenesis were investigated using approaches including genetics, molecular/cell biology, pathogenicity and transcriptomic profiling. BcJar1 regulates H3K4me3 and both H3K4me2 and H3K4me3 methylation levels during vegetative and pathogenic development, respectively. Loss of BcJAR1 impairs conidiation, appressorium formation and stress adaptation; abolishes infection cushion (IC) formation and virulence, but promotes sclerotium production in the ΔBcjar1 mutants. BcJar1 controls reactive oxygen species (ROS) production and proper assembly of Sep4, a core septin protein and virulence determinant, to initiate infection structure (IFS) formation and host penetration. Exogenous cAMP partially restored the mutant appressorium, but not IC, formation. BcJar1 orchestrates global expression of genes for ROS production, stress response, carbohydrate transmembrane transport, secondary metabolites, etc., which may be required for conidiation, IFS formation, host penetration and virulence of the pathogen. Our work systematically elucidates BcJar1 functions and provides novel insights into Jar1/KDM5-mediated H3K4 demethylation in regulating fungal development and pathogenesis.

Cytotoxic Activity of Inositol Angelates and Tirucallane-Type Alkaloids from Amoora Dasyclada.

Three new inositol angelate compounds (1-3) and two new tirucallane-type alkaloids (4 and 5) were isolated from the Amoora dasyclada, and their structures were established mainly by means of combination of 1D and 2D nuclear magnetic resonance and HR-ESI-MS. Based on cytotoxicity testing, compounds 4 and 5 exhibited significant cytotoxic activity against human cancer cell line HepG2 with IC50 value at 8.4 and 13.2 µM. In addition, compounds 4 and 5 also showed remarkable growth inhibitory activity to Artemia salina larvae.

The pre-rRNA processing factor Nop53 regulates fungal development and pathogenesis via mediating production of reactive oxygen species.

Botrytis cinerea is a necrotrophic plant fungal pathogen that annually causes enormous economic losses worldwide. The ribosome is an organelle for cellular protein biosynthesis. However, little is known about how the ribosome operates as a machine to mediate microbial pathogenesis. Here, we demonstrate that Nop53, a late-acting factor for 60S ribosomal subunit maturation, is crucial for the pathogen's development and virulence. BcNop53 is functionally equivalent to yeast nop53p. Complementation of BcNOP53 completely restored the growth defect of the yeast Δnop53 mutant. BcNop53 is located in nuclei and disruption of BcNOP53 also dramatically impaired pathogen growth. Deletion of BcNOP53 blocked infection structure formation and abolished virulence of the pathogen, possibly due to reduced production of reactive oxygen species. Moreover, loss of BcNOP53 impaired pathogen conidiation and stress adaptation, altered conidial and sclerotial morphology, retarded conidium and sclerotium germination as well as reduced the activities of cell-wall degradation-associated enzymes. Sclerotium production was, however, increased. Complementation with the wild-type BcNOP53 allele rescued defects found in the ΔBcnop53 mutant. Our work establishes a systematic elucidation of Nop53 in regulating microbial development and pathogenesis, provides novel insights into ribosomal processes that regulate fungal pathogenesis, and may open up new targets for addressing fungal diseases.

The key gluconeogenic gene PCK1 is crucial for virulence of Botrytis cinerea via initiating its conidial germination and host penetration.

The process of initiation of host invasion and survival of some foliar phytopathogenic fungi in the absence of external nutrients on host leaf surfaces remains obscure. Here, we demonstrate that gluconeogenesis plays an important role in the process and nutrient-starvation adaptation before the pathogen host invasion. Deletion of phosphoenolpyruvate carboxykinase gene BcPCK1 in gluconeogenesis in Botrytis cinerea, the causative agent of grey mould, resulted in the failure of the ΔBcpck1 mutant conidia to germinate on hard and hydrophobic surface and penetrate host cells in the absence of glucose, reduction in conidiation and slow conidium germination in a nutrient-rich medium. The wild-type and ΔBcpck1 conidia germinate similarly in the presence of glucose (higher concentration) as the sole carbon source. Conidial glucose-content should reach a threshold level to initiate germination and host penetration. Infection structure formation by the mutants displayed a glucose-dependent fashion, which corresponded to the mutant virulence reduction. Exogenous glucose or complementation of BcPCK1 completely rescued all the developmental and virulence defects of the mutants. Our findings demonstrate that BcPCK1 plays a crucial role in B. cinerea pathogenic growth and virulence, and provide new insights into gluconeogenesis mediating pathogenesis of plant fungal pathogens via initiation of conidial germination and host penetration.

Theoretical Study of the C2H5 + HO2 Reaction: Mechanism and Kinetics.

The mechanism and kinetics for the reaction of the HO2 radical with the ethyl (C2H5) radical have been investigated theoretically. The electronic structure information of the potential energy surface (PES) is obtained at the MP2/6-311++G(d,p) level of theory, and the single-point energies are refined by the CCSD(T)/6-311+G(3df,2p) level of theory. The kinetics of the reaction with multiple channels have been studied by applying variational transition-state theory (VTST) and Rice⁻Ramsperger⁻Kassel⁻Marcus (RRKM) theory over wide temperature and pressure ranges (T = 220⁻3000 K; P = 1 × 10-4⁻100 bar). The calculated results show that the HO2 radical can attack C2H5 via a barrierless addition mechanism to form the energy-rich intermediate IM1 C2H5OOH (68.7 kcal/mol) on the singlet PES. The collisional stabilization intermediate IM1 is the predominant product of the reaction at high pressures and low temperatures, while the bimolecular product P1 C2H5O + OH becomes the primary product at lower pressures or higher temperatures. At the experimentally measured temperature 293 K and in the whole pressure range, the reaction yields P1 as major product, which is in good agreement with experiment results, and the branching ratios are predicted to change from 0.96 at 1 × 10-4 bar to 0.66 at 100 bar. Moreover, the direct H-abstraction product P16 C2H6 + ³O2 on the triplet PES is the secondary feasible product with a yield of 0.04 at the collisional limit of 293 K. The present results will be useful to gain deeper insight into the understanding of the kinetics of the C2H5 + HO2 reaction under atmospheric and practical combustion conditions.

Four new species of Russula from the Xizang Autonomous Region and other provinces of China.

Russula is the largest genus in the Russulales and is widespread throughout the world. Almost all Russula species are known to be ectomycorrhizal with high ecological and edible values, and some are lethal poisonous. In this study, four new species belonging to the subgenus Russula crown clade are identified based on morphological and phylogenetic evidence from the Xizang Autonomous Region and other provinces of China. Morphologically, Russula paragraveolens (sect. Polychromae, subsect. Xerampelinae) is mainly characterised by a cherry red to blood red pileus centre, a reddish orange pileus margin; R. pseudograveolens (sect. Polychromae, subsect. Xerampelinae) is characterised by a violet brown to brownish red pileus centre, a pale red to pastel red pileus margin and short basidia; R. shigatseensis (sect. Flavisiccantes, subsect. Lepidinae) is characterised by a brownish orange to madder red pileus centre, pinkish red pileus margin, and having lateral branches or branches of hyphal terminations in pileipellis; R. yadongensis (sect. Tenellae, subsect. Laricinae) is characterised by a dark purplish red pileus centre with brownish purple tints and having isolated to clustered spines of spore ornamentations. Their distinct taxonomic status is confirmed by the positions of the four new species in both the ITS and 4-locus (nucLSU, mtSSU, rpb2, tef1) phylogenetic trees.

Protective effect of sevoflurane on lung function of elderly chronic obstructive pulmonary disease patients undergoing total hip arthroplasty.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a common respiratory disorder that affects the elderly population and increases the risk of postoperative pulmonary complications (PPCs) after major surgeries. Sevoflurane is a volatile anesthetic that has been shown to have anti-inflammatory and antioxidant properties and attenuate lung injury in animal models. AIM: To evaluate the protective effect of sevoflurane on the lung function of elderly COPD patients undergoing total hip arthroplasty (THA). METHODS: In this randomized controlled trial, we randomly assigned 120 elderly patients with COPD, who were scheduled for THA, to receive either sevoflurane (sevoflurane group) or propofol (propofol group) as the maintenance anesthetic. The primary outcome was the incidence of PPCs within seven days after surgery. The secondary outcomes were changes in the lung function parameters, inflammatory markers, oxidative stress markers, and postoperative pain scores. RESULTS: The results showed that the incidence of PPCs was significantly lower in the sevoflurane group than in the propofol group (10% vs 25%, P = 0.02). Furthermore, the decline in the forced expiratory volume in 1 s, forced vital capacity, and peak expiratory flow was significantly lesser in the sevoflurane group than in the propofol group at 24 h and 48 h after surgery (P < 0.05). The interleukin-6, tumor necrosis factor-alpha, malondialdehyde, and 8-hydroxy-2 α-deoxyguanosine levels were significantly lower in the sevoflurane group than in the propofol group at 24 h after surgery (P < 0.05). The sevoflurane group showed significantly lower postoperative pain scores than the propofol group at 6 h, 12 h, and 24 h after surgery (P < 0.05). CONCLUSION: Sevoflurane protects the lung function of elderly COPD patients undergoing THA under general anesthesia by reducing the incidence of PPCs, attenuating inflammatory and oxidative stress responses, and alleviating postoperative pain.

A Lesion Microenvironment-Responsive Fungicide Nanoplatform for Crop Disease Prevention and Control.

Controlled fungicide delivery in response to the specific microenvironment produced by fungal pathogens is an advisable strategy to improve the efficacy of fungicides. Herein, the authors construct a smart fungicide nanoplatform, using mesoporous silica nanoparticles (MSNs) as nanocarriers loaded with eugenol (EU) and Ag+ coordinated polydopamine (Ag+ -PDA) as a coating to form Ag+ -PDA@MSNs-EU NPs for Botrytis cinerea (B. cinerea) control. As a botanical fungicide, EU offers an eco-friendly alternative to synthetic fungicides and can upregulate several defense-related genes in the tomato plant. The Ag+ -PDA coating can lock the EU inside the nanocarriers and respond to the oxalic acid produced by B. cinerea to corelease the loaded EU and Ag+ . The results demonstrate that Ag+ -PDA@MSNs-EU NPs can effectively inhibit the mycelial growth of B. cinerea on detached and potted tomato leaves. The construction of such a smart fungicide nanoplatform provides new guidance to design controlled fungicides release systems, which can respond to the microenvironment associated with plant pathogen to realize fungus control.

The increased in vivo firing of pyramidal cells but not interneurons in the anterior cingulate cortex after neuropathic pain.

Chronic pain damages the balance between excitation and inhibition in the sensory cortex. It has been confirmed that the activity of cortical glutamatergic pyramidal cells increases after chronic pain. However, whether the activity of inhibitory interneurons synchronized changed remains obscure, especially in in vivo conditions. In the present study, we checked the firing rate of pyramidal cells and interneurons in the anterior cingulate cortex, a main cortical area for the regulation of nociceptive information in mice with spared nerve injury by using in vivo multi-channel recording system. We found that the firing rate of pyramidal cells but not interneurons increased in the ACC, which was further confirmed by the increased FOS expression in pyramidal cells but not interneurons, in mice with neuropathic pain. Selectively high frequency stimulation of the ACC nociceptive afferent fibers only potentiated the activity of pyramidal cells either. Our results thus suggest that the increased activity of pyramidal cells contributes to the damaged E/I balance in the ACC and is important for the pain hypersensitivity in mice with neuropathic pain.

Rare Cases of Bronchial Aneurysm and Comparison of Interventional Embolization in the Treatment of True Bronchial Aneurysm and Pseudobronchial Aneurysm.

Background: Bronchial artery aneurysm (BAA) is a rare disease. Rupture of BAA can lead to life-threatening hemoptysis, and once diagnosed, treatment is needed regardless of symptoms. Transcatheter artery embolization is the first choice of treatment because it is minimally invasive and effective. This study aimed to retrospectively compare the embolization treatment of a case of true BAA and that of a pseudobranchial aneurysm and explore the choice of embolization method for BAA with short neck or no neck. Materials and Methods: Embolization treatment and imaging characteristics of one case of true BAA and one case of pseudobronchial aneurysm admitted to our hospital were analyzed retrospectively. Embolization methods and therapeutic effects of two cases of BAAs were compared. Results: Case 1 was that of an intact true BAA inside the mediastinum located at the opening of the bronchial artery. The distal end of the aneurysm was embolized, and tumor cavity was occluded. No recurrence of BAA was found after the operation. Case 2 was that of a ruptured and hemorrhagic pseudobronchial aneurysm of the mediastinum. Coil embolization combined with covered stent graft exclusion of the thoracic aorta were performed, and the left bronchial artery and BAA were almost occluded. Nine months postoperatively, the mediastinal hematoma was almost completely absorbed. Conclusion: Endovascular embolization has become the most commonly used for the treatment of BAA. Different methods should be selected according to the location and nature of the aneurysm.

A revision of Agaricus section Arvenses with nine new species from China.

Agaricus sect. Arvenses includes numerous species that are potential candidates for cultivation, and some have high nutritional and medicinal interests. Between 2012 and 2017, 147 specimens of A. sect. Arvenses were collected in China. For this study, nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS), nuc 28S rDNA (28S), and translation elongation factor 1-alpha (tef1) sequences were used to assess species boundaries of these samples from China. Combined with morphological examination, we recognize 22 species of A. sect. Arvenses from China, of which 12 are known species, one is new record for China, and nine are proposed as new.

Supramolecular Nanoplatform Based on Mesoporous Silica Nanocarriers and Pillararene Nanogates for Fungus Control.

Synthetic fungicides have been widely used to protect crops from fungal diseases. However, excessive use of synthetic fungicides leads to the generation of fungicide resistance in fungal pathogens. Recently, smart cargo delivery systems have been introduced for the construction of a pesticide delivery nanoplatform, benefiting from their controlled release performance. Herein, a fungal pathogen microenvironment-responsive supramolecular fungicide nanoplatform has been designed and constructed, using quaternary ammonium salt (Q)-modified mesoporous silica nanoparticles (MSN-Q NPs) as nanocarriers loaded with berberine hydrochloride (BH) and carboxylatopillar[5]arene (CP[5]A) as nanogates to form BH-loaded CP[5]A@MSN-Q NPs for effective inhibition of Botrytis cinerea. CP[5]A as nanogates can endow the fungicide nanoplatform with pH stimuli-responsive release features for the control of fungicide release. The loaded BH, as a natural plant fungicide, provides an ecofriendly alternative to synthetic fungicides for controlling B. cinerea. Interestingly, we use oxalic acid (OA) secreted by B. cinerea as a trigger so that BH can be released from the fungicide nanoplatform on demand under pathogen microenvironments for controlling B. cinerea. The experimental results indicate that the fabricated fungicide nanoplatform could effectively inhibit the mycelial growth and spore germination, providing a new way for the management of B. cinerea in actual application.

Sub-lethal Camphor Exposure Triggers Oxidative Stress, Cardiotoxicity, and Cardiac Physiology Alterations in Zebrafish Embryos.

Camphor is a terpene ketone with aromatic and volatile properties in nature derived from the bark of Cinnamomum camphora or synthesized from turpentine. Camphor exhibits various biological properties such as anti-microbial, anti-viral, anti-coccidial, and anti-cancer. It is also used as a form of topical medication for skin irritation, joint pain, and as a relief for itching from insect bites. However, even though the high dose of camphor has been documented to be toxic/lethal in humans in different studies, camphor's developmental toxicity has not yet been explored, and its extensive mechanism of action is still unclear. In the present study, we aimed to assess the toxic effects of camphor in zebrafish embryos in the initial developmental stages. The obtained results demonstrated that a sub-lethal dose of camphor caused a decrease in hatching rate, body length, and substantial elevation in malformation rate on zebrafish embryos. On further observation, in the following time frame, curved body and pericardial edema of zebrafish were also observed. Furthermore, exposure to a sub-lethal dose of camphor was also able to trigger cardiotoxicity in zebrafish larvae. Later, on subsequent biochemical analysis, it was found that the antioxidant capacity inhibition and oxidative stress elevation that occurred after camphor exposure might be associated with the inhibition of total superoxide dismutase (SOD) activity and an increase in reactive oxygen species (ROS) and malondialdehyde (MDA) concentration. In addition, compared to the control group, several apoptotic cells in treated zebrafish were also found to be elevated. Finally, after further investigation on marker gene expressions, we conclude that the developmental toxicity of camphor exposure might be associated with apoptosis elevation and oxidative stress. Taken together, the current study provides a better understanding of the developmental toxicity of camphor on zebrafish, a promising alternative animal model to assess the developmental toxicity of chemical compounds.

Neurotensin Attenuates Nociception by Facilitating Inhibitory Synaptic Transmission in the Mouse Spinal Cord.

Neurotensin (NT) is an endogenous tridecapeptide in the central nervous system. NT-containing neurons and NT receptors are widely distributed in the spinal dorsal horn (SDH), indicating their possible modulatory roles in nociception processing. However, the exact distribution and function of NT, as well as NT receptors (NTRs) expression in the SDH, have not been well documented. Among the four NTR subtypes, NTR2 is predominantly involved in central analgesia according to previous reports. However, the expression and function of NTR2 in the SDH has not yet been directly elucidated. Specifically, it remains unclear how NT-NTR2 interactions contribute to NT-mediated analgesia. In the present study, by using immunofluorescent histochemical staining and immunohistochemical staining with in situ hybridization histochemical staining, we found that dense NT- immunoreactivity (NT-ir) and moderate NTR2-ir neuronal cell bodies and fibers were localized throughout the superficial laminae (laminae I-II) of the SDH at the light microscopic level. In addition, γ-aminobutyric acid (GABA) and NTR2 mRNA were colocalized in some neuronal cell bodies, predominantly in lamina II. Using confocal and electron microscopy, we also observed that NT-ir terminals made both close contacts and asymmetrical synapses with the local GABA-ir neurons. Second, electrophysiological recordings showed that NT facilitated inhibitory synaptic transmission but not glutamatergic excitatory synaptic transmission. Inactivation of NTR2 abolished the NT actions on both GABAergic and glycinergic synaptic release. Moreover, a behavioral study revealed that intrathecal injection of NT attenuated thermal pain, mechanical pain, and formalin induced acute inflammatory pain primarily by activating NTR2. Taken together, the present results provide direct evidence that NT-containing terminals and fibers, as well as NTR2-expressing neurons are widely distributed in the spinal dorsal horn, GABA-containing neurons express NTR2 mainly in lamina II, GABA coexists with NTR2 mainly in lamina II, and NT may directly increase the activity of local inhibitory neurons through NTR2 and induce analgesic effects.

Transcriptome analysis and functional validation reveal a novel gene, BcCGF1, that enhances fungal virulence by promoting infection-related development and host penetration.

Simultaneous transcriptome analyses of both host plants and pathogens, and functional validation of the identified differentially expressed genes (DEGs) allow us to better understand the mechanisms underlying their interactions. Here, we analyse the mixed transcriptome derived from Botrytis cinerea (the causal agent of grey mould) infected tomato leaves at 24 hr after inoculation, a critical time point at which the pathogen has penetrated and developed in the leaf epidermis, whereas necrotic symptoms have not yet appeared. Our analyses identified a complex network of genes involved in the tomato-B. cinerea interaction. The expression of fungal transcripts encoding candidate effectors, enzymes for secondary metabolite biosynthesis, hormone and reactive oxygen species (ROS) production, and autophagy-related proteins was up-regulated, suggesting that these genes may be involved in the initial infection processes. Specifically, tomato genes involved in phytoalexin production, stress responses, ATP-binding cassette transporters, pathogenesis-related proteins, and WRKY DNA-binding transcription factors were up-regulated. We functionally investigated several B. cinerea DEGs via gene replacement and pathogenicity assays, and demonstrated that BcCGF1 was a novel virulence-associated factor that mediates fungal development and virulence via regulation of conidial germination, conidiation, infection structure formation, host penetration, and stress adaptation. The fungal infection-related development was controlled by BcCGF-mediated ROS production and exogenous cAMP restored the mutant infection-related development. Our findings provide new insights into the elucidation of the simultaneous tactics of pathogen attack and host defence. Our systematic elucidation of BcCGF1 in mediating fungal pathogenesis may open up new targets for fungal disease control.

Online preconcentration and two-dimensional separation of cationic compounds via hyphenation of capillary zone electrophoresis with cyclodextrin-modified micellar electrokinetic capillary chromatography.

A novel preconcentration/separation approach, which online combined CZE with CD-modified MEKC, was developed for simultaneous enhancing resolving power and detection sensitivity. CZE with cation-selective exhaustive injection and transient ITP preconcentration was used as the first dimension, from which the effluent fractions were further analyzed by CD-modified MEKC acting as the second dimension. As the key to successful integration of CZE with MEKC, a new interface was designed and electroaccumulation focusing strategy was employed to avoid analyte band diffusion at the interface. The comprehensive 2-D system was successfully established with only one high voltage and four electrodes. The grouping of two orthogonal separation techniques, together with analytes preconcentration techniques, significantly enhanced resolution and sensitivity for 2-D separation of cationic compounds. The resulting electrophoregram was quite different from that of either single CZE or MEKC. Up to 14,000- to 35,000-fold improvement in sensitivity was obtained relative to conventional electrokinetic injection method. The limits of detection (S/N=3) were in the range of 0.03-0.1 microg/L. The number of theoretical plates was in the range of 103,000-184,000. This method was successfully applied to the analysis of trace cationic cardiovascular drugs in wastewater.