The mitogen-activated protein kinase module CcSte11-CcSte7-CcPmk1 regulates pathogenicity via the transcription factor CcSte12 in Cytospora chrysosperma.

The pathogen Cytospora chrysosperma is the causal agent of poplar canker disease and causes considerable economic losses in China. Mitogen-activated protein kinase (MAPK) cascades play a crucial role in mediating cellular responses and Pmk1-MAPKs are indispensable for pathogenic related processes in plant pathogenic fungi. In previous studies, we demonstrated that the CcPmk1 acts as a core regulator of fungal pathogenicity by modulating a small number of master downstream targets, such as CcSte12. In this study, we identified and characterized two upstream components of CcPmk1: MAPKKK CcSte11 and MAPKK CcSte7. Deletion of CcSte11 and CcSte7, resulted in slowed growth, loss of sporulation and virulence, similar to the defects observed in the CcPmk1 deletion mutant. In addition, CcSte11, CcSte7 and CcPmk1 interact with each other, and the upstream adaptor protein CcSte50 interact with CcSte11 and CcSte7. Moreover, we explored the global regulation network of CcSte12 by transcriptional analysis between CcSte12 deletion mutants and wild-type during the simulated infection process. Two hydrolase activity GO terms (GO:0004553 and GO:0016798) and starch and sucrose metabolism (mgr00500) KEGG pathway were significantly enriched in the down-regulated genes of CcSte12 deletion mutants. In addition, a subset of glycosyl hydrolase genes and putative effector genes were significantly down-regulated in the CcSte12 deletion mutant, which might be important for fungal pathogenicity. Especially, CcSte12 bound to the CcSp84 promoter region containing the TGAAACA motif. Moreover, comparison of CcSte12-regulated genes with CcPmk1-regulated genes revealed 116 overlapping regulated genes in both CcSte12 and CcPmk1, including some virulence-associated genes. Taken together, the protein complexes CcSte11-CcSte7-CcPmk1 receive signals transmitted by upstream CcSte50 and transmit signals to downstream CcSte12, which regulates hydrolase, effectors and other genes to promote virulence. Overall, these results indicate that the CcPmk1-MAPK signaling pathway of C. chrysosperma plays a key role in the pathogenicity.

A putative terpene cyclase gene (CcPtc1) is required for fungal development and virulence in Cytospora chrysosperma.

Cytospora chrysosperma is a destructive plant pathogenic fungus, which causes canker disease on numerous woody plants. However, knowledge concerning the interaction between C. chrysosperma and its host remains limited. Secondary metabolites produced by phytopathogens often play important roles in their virulence. Terpene cyclases (TC), polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS) are the key components for the synthesis of secondary metabolites. Here, we characterized the functions of a putative terpene type secondary metabolite biosynthetic core gene CcPtc1 in C. chrysosperma, which was significantly up-regulated in the early stages of infection. Importantly, deletion of CcPtc1 greatly reduced fungal virulence to the poplar twigs and they also showed significantly reduced fungal growth and conidiation compared with the wild-type (WT) strain. Furthermore, toxicity test of the crude extraction from each strain showed that the toxicity of crude extraction secreted by ΔCcPtc1 were strongly compromised in comparison with the WT strain. Subsequently, the untargeted metabolomics analyses between ΔCcPtc1 mutant and WT strain were conducted, which revealed 193 significantly different abundant metabolites (DAMs) inΔCcPtc1 mutant compared to the WT strain, including 90 significantly downregulated metabolites and 103 significantly up-regulated metabolites, respectively. Among them, four key metabolic pathways that reported to be important for fungal virulence were enriched, including pantothenate and coenzyme A (CoA) biosynthesis. Moreover, we also detected significant alterations in a series of terpenoids, among which (+)-ar-turmerone, pulegone, ethyl chrysanthemumate, and genipin were significantly down-regulated, while cuminaldehyde and (±)-abscisic acid were significantly up-regulated. In conclusion, our results demonstrated that CcPtc1 acts as a virulence-related secondary metabolism factor and provides new insights into the pathogenesis of C. chrysosperma.

Comparative Transcriptomic Analysis of MAPK-Mediated Regulation of Pathogenicity, Stress Responses, and Development in Cytospora chrysosperma.

Mitogen-activated protein kinase (MAPK) cascades are highly conserved signal transduction pathways that mediate cellular responses to various biotic and abiotic signals in plant-pathogenic fungi. Generally, there are three MAPKs in filamentous pathogenic fungi: Pmk1/Fus3/Kss1, Hog1, and Stl2. Our previous studies have shown that CcPmk1 is a core regulator of fungal pathogenicity in Cytospora chrysosperma, the causal agent of canker disease in a wide range of woody plants. Here, we identified and functionally characterized the other two MAPK genes (CcHog1 and CcSlt2) and then compared the transcriptional differences among these three MAPKs in C. chrysosperma. We found that the MAPKs shared convergent and distinct roles in fungal development, stress responses, and virulence. For example, CcHog1, CcSlt2, and CcPmk1 were all involved in conidiation and response to stresses, including hyperosmotic pressure, cell wall inhibition agents, and H2O2, but only CcPmk1 and CcSlt2 were required for hyphal growth and fungal pathogenicity. Transcriptomic analysis showed that numerous hyperosmosis- and cell wall-related genes significantly reduced their expression levels in ΔCcHog1 and ΔCcSlt2, respectively. Interestingly, RNA- and ribosome-related processes were significantly enriched in the upregulated genes of ΔCcSlt2, whereas they were significantly enriched in the downregulated genes of ΔCcPmk1. Moreover, two secondary metabolite gene clusters were significantly downregulated in ΔCcPmk1, ΔCcSlt2, and/or ΔCcHog1. Importantly, some virulence-associated genes were significantly downregulated in ΔCcPmk1 and/or ΔCcSlt2, such as candidate effector genes. Collectively, these results suggest that the similar and distinct phenotypes of each MAPK deletion mutant may result from the transcriptional regulation of a series of common or specific downstream genes, which provides a better understanding of the regulation network of MAPKs in C. chrysosperma.

Genetic Predisposition to Severe COVID-19 Might Increase the Risk of Stroke: A Two-Sample Mendelian Randomization Study.

Aims: The causal relationship between COVID-19 infection and stroke has not yet been fully established. This study aimed to explore this causality using two-sample Mendelian randomization (MR). Materials and Methods: Genetic variants associated with COVID-19 infection and stroke were both obtained from genome-wide association study (GWAS) summary data. The single nucleotide polymorphisms (SNPs) were selected as instrumental variables. The standard inverse variance weighted (IVW) was primarily used to assess this causality. Finally, sensitivity analysis was performed to evaluate the reliability and stability. Results: The results showed that being hospitalized due to COVID-19 had a positive effect on stroke [OR = 1.05; 95% CI= (1.01, 1.10); p = 2.34 × 10-5] and ischemic stroke [OR = 1.06; 95% CI= (1.02, 1.11); p = 2.28 × 10-6] analyzed by inverse variance weighted. Moreover, the results revealed that severe respiratory symptoms due to COVID-19 had a positive effect on stroke [OR = 1.04; 95% CI= (1.00, 1.06); p = 0.04] and that the causal effect of severe respiratory symptoms due to COVID-19 on ischemic stroke estimated by IVW suggested a positive effect [OR = 1.06; 95% CI= (1.02, 1.09); p = 0.0068], too. Conclusion: In summary, this study showed that severe COVID-19 might increase the risk of stroke, thus much more attention should be paid to patients with severe COVID-19.

Identification and quantification of main flavonoids in the leaves of Bambusa multiplex cv. Fernleaf.

The chemical profile of Bambusa multiplex cv. Fernleaf (B. multiplex) leaves was analysed by UPLC-DAD-Q-TOF-MS. Twelve compounds were identified and C-glycosyl flavonoids, including vitexin, isovitexin, isoorientin and its derivatives, are the main constitutes of the plant. Besides, a HPLC method for isoorientin quantification was developed. The RSD of retention time and peak area were 0.05% and 2.04% for six times analysis of isoorientin with concentration of 20 µg/mL. The recovery of isoorientin in real sample was 99.2%. The general trend of isoorientin content in B. multiplex leaves was that it steady increased from Jan. to May, and then quickly decreased. The maximum was found on May with value of 4.7 mg/g. The lowest level of isoorientin was found during Aug. to Nov. with value of about 1.66 mg/g. In different seasons, isoorientin is always the most dominant flavonoid which was accounted for about 50% of total flavonoids in the sample.

Yolk-shell structured Au@Ag@mSiO2 as a probe for sensing cysteine enantiomers and Cu2+ based on circular dichroism.

A yolk-shell structured Au@Ag@mSiO2 probe was fabricated by coating a layer of mesoporous silica on the surface of a Au@Ag core-shell nanosphere, followed by partially removing the Ag shell. Mirror symmetrical chiral signals at ∼258 nm in the UV region were observed for the probe upon coupling with cysteine enantiomers on the surface of Au@Ag. The intensity of the CD signal of the probe is enhanced by increasing l/d-Cys concentration, allowing the quantitative determination of the cysteine enantiomers. The developed method shows an excellent linear relationship between the CD signal and l-Cys concentration ranging from 10 µM to 90 µM with a limit of detection of 8.5 µM. In the presence of Cu2+, the CD signal of the probe weakened due to the oxidation of l-cysteine to l-cystine catalyzed by Cu2+. Based on this phenomenon, a new strategy for the detection of Cu2+ can be developed. Under the optimized conditions, the CD signal decreases linearly with the log of the concentration of Cu2+ in the range from 1 to 250 nM with a detection limit of 0.1 nM.

Chemical profile and pancreatic lipase inhibitory activity of Sinobambusa tootsik (Sieb.) Makino leaves.

BACKGROUND: Sinobambusa tootsik (Sieb.) Makino (S. tootsik) is one species of bamboo distributed in China, Japan and Vietnam. The chemical profile of its leaves and its potential application was unknown yet. METHODS: The chemical profile of S. tootsik was studied by HPLC and UPLC-DAD-QTOF-MS. The S. tootsik extract was prepared by extraction with 50% aqueous ethanol, followed by H103 macroporous resins adsorption and desorption processes. Pancreatic lipase inhibitory activity was determined using p-nitrophenyl palmitate as the substance, which was hydrolyzed by lipase to form coloured p-nitrophenol. RESULTS: Eighteen compounds were identified in S. tootsik. Most of them were the C-glycosylated derivatives of luteolin and apigenin, such as isoorientin, isoorientin-2″-O-rhamnoside and isovitexin. Isoorientin-2″-O-rhamnoside was the most dominant flavonoid in the sample. S. tootsik extract was prepared through resin adsorption/desorption with yield of 1.12 ± 015% and total flavonoids content of 82 ± 2 mg/g (in term of isoorientin). The extract exhibited pancreatic lipase inhibitory activity with IC50 value of 0.93 mg/mL. CONCLUSION: The chemical profile of S. tootsik leaves was uncovered for the first time. C-glycosyl flavonoids were the main constituents in the plant. The extract exhibited pancreatic lipase inhibitory activity and may have potential for use as a food supplement for controlling obesity.

Anchoring of silver nanoparticles on graphitic carbon nitride sheets for the synergistic catalytic reduction of 4-nitrophenol.

In this paper, a facile process was developed for anchoring of silver nanoparticles on graphitic carbon nitride sheets (Ag/g-C3N4) with high catalytic activity for reduction of 4-nitrophenol. The morphology and structure of the as-prepared Ag/g-C3N4 composite were investigated by FESEM, TEM, XRD and XPS. The reaction mechanism and the reduction kinetics of 4-nitrophenol under different light irradiation were systematically studied. The results showed that the obtained Ag/g-C3N4 composite exhibited a much higher electro/photo catalytic activity and stability for reduction of 4-nitrophenol. Significantly, due to the synergistic effect and interaction between highly dispersed Ag nanoparticles (Ag NPs, ∼7.2 nm) and lamellar g-C3N4, not only transfer of interfacial charge, but also the separation of photoinduced electrons occurred when the reaction was proceeded under light. In addition, the composite exhibited high stability and reusability during the cycling experiments. The results showed that the Ag/g-C3N4 composite is an effective and stable electro/photo catalyst for reduction of 4-nitrophenol.

The crystal structure of Cpf1 in complex with CRISPR RNA.

The CRISPR-Cas systems, as exemplified by CRISPR-Cas9, are RNA-guided adaptive immune systems used by bacteria and archaea to defend against viral infection. The CRISPR-Cpf1 system, a new class 2 CRISPR-Cas system, mediates robust DNA interference in human cells. Although functionally conserved, Cpf1 and Cas9 differ in many aspects including their guide RNAs and substrate specificity. Here we report the 2.38 Å crystal structure of the CRISPR RNA (crRNA)-bound Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1). LbCpf1 has a triangle-shaped architecture with a large positively charged channel at the centre. Recognized by the oligonucleotide-binding domain of LbCpf1, the crRNA adopts a highly distorted conformation stabilized by extensive intramolecular interactions and the (Mg(H2O)6)(2+) ion. The oligonucleotide-binding domain also harbours a looped-out helical domain that is important for LbCpf1 substrate binding. Binding of crRNA or crRNA lacking the guide sequence induces marked conformational changes but no oligomerization of LbCpf1. Our study reveals the crRNA recognition mechanism and provides insight into crRNA-guided substrate binding of LbCpf1, establishing a framework for engineering LbCpf1 to improve its efficiency and specificity for genome editing.

Structural basis for hijacking CBF-ß and CUL5 E3 ligase complex by HIV-1 Vif.

The human immunodeficiency virus (HIV)-1 protein Vif has a central role in the neutralization of host innate defences by hijacking cellular proteasomal degradation pathways to subvert the antiviral activity of host restriction factors; however, the underlying mechanism by which Vif achieves this remains unclear. Here we report a crystal structure of the Vif-CBF-ß-CUL5-ELOB-ELOC complex. The structure reveals that Vif, by means of two domains, organizes formation of the pentameric complex by interacting with CBF-ß, CUL5 and ELOC. The larger domain (α/ß domain) of Vif binds to the same side of CBF-ß as RUNX1, indicating that Vif and RUNX1 are exclusive for CBF-ß binding. Interactions of the smaller domain (α-domain) of Vif with ELOC and CUL5 are cooperative and mimic those of SOCS2 with the latter two proteins. A unique zinc-finger motif of Vif, which is located between the two Vif domains, makes no contacts with the other proteins but stabilizes the conformation of the α-domain, which may be important for Vif-CUL5 interaction. Together, our data reveal the structural basis for Vif hijacking of the CBF-ß and CUL5 E3 ligase complex, laying a foundation for rational design of novel anti-HIV drugs.

Influence of water on room-temperature preparation of silver nanoparticles using poly(vinyl pyrrolidone) as reducing agent.

Silver nanoparticles (Ag NPs) were prepared via a wet-chemical method in the presence of poly(vinyl pyrrolidone) (PVP) without other reducing agents at room temperature. The influence of the addition of water on the preparation of Ag NPs was investigated. It was found that water addition has a significant influence on the reduction reaction, resulting in changes of shape, size and optical properties of the particles. When large amounts of water were added, the reduction rate was very slow. However, when small amounts of water were used, the opposite effects on the reaction process were observed, initial inhibition effect and final promotion effect. Two main possible mechanisms were proposed to explain the opposite effects of two reaction stages with small amounts of water addition: (1) the initial inhibition effect was induced by free oxygen in water, which would react preferentially with the reducing species in the system; (2) the promotion effect thereafter may be due to the differences of chain extension of PVP molecules and electron transfer rate in ethanol and water.

Facile, template-free synthesis of silver nanodendrites with high catalytic activity for the reduction of p-nitrophenol.

Here we report a facile, surfactant-free and template-free synthesis process of highly uniform dendritic silver nanostructures with high catalytic activity for the reduction of p-nitrophenol. By controlling the concentration of AgNO(3) aqueous solution and the reaction time, various shapes of silver nanodendrites (SNDs) could be obtained easily. The effects of different parameters such as concentrations of the reagents and reaction time on the morphology and structure of as-prepared tree-like nanostructures have also been investigated by X-ray diffraction (XRD), field-emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM). Also, the X-ray photoelectron spectroscopy (XPS) has been used to identify the oxidation state of SNDs. In addition, the catalytic activity of the as-prepared SNDs samples at 200 mM AgNO(3) aqueous solution was evaluated by a redox reaction of p-nitrophenol in the presence of an excess amount of NaBH(4). It was found that the highly symmetrical SNDs with roughly 60-120 nm in stem and branch diameter and 3-12 µm in length obtained after 120 s reaction time do have higher catalytic activity than other SNDs prepared at different reaction time, several times stronger catalytic activity in the sodium borohydride reduction of p-nitrophenol to p-aminophenol, compared to some other silver nanoparticles reported in literature. The crystallinity provided by X-ray diffraction (XRD) analysis indicates that the improvement of the crystallinity is also very crucial for SNDs' catalytic activities. The SNDs are very promising catalytic candidates for the reduction of p-nitrophenol because of easily simple preparation route and high catalytic activity.

Convenient synthesis of silver nanowires with adjustable diameters via a solvothermal method.

Silver nanowires have been successfully synthesized via a simple solvothermal method by adding sodium sulfide (Na(2)S) into the solution. The Ag(2)S colloids produced in the initial stage help reduce the concentration of free Ag(+) ions in the initial formation of silver seeds and subsequently release Ag(+) ions to the solution. Otherwise, there is no oxidative etching owing to the absence of oxygen. In these cases, silver nanowires are grown preferentially. Furthermore, silver nanowires with adjustable diameters can be obtained by adjusting the concentration of Na(2)S. Electron microscopy, X-ray diffraction, and absorption spectra have been used to investigate the products, and a mechanism is proposed to interpret the controlled synthesis of silver nanowires. Finally, our results indicate that this approach provides a versatile route to prepare silver nanowires with controllable diameters.

Convenient, rapid synthesis of silver nanocubes and nanowires via a microwave-assisted polyol method.

Silver nanostructures have been synthesized via a microwave-assisted polyol method by adding sodium sulfide (Na(2)S) into the solution. An interesting morphology evolution can be observed by adjusting the concentration of Na(2)S and the heating power. It is found that the ideal concentration of Na(2)S is 31.25-500 microM for the fast reduction of Ag(+) at 300 W under optimal conditions for producing monodispersed silver nanocubes. When the heating power is increased to 400 W, 62.5-250 microM is the ideal concentration of Na(2)S for the synthesis of silver nanocubes. On increasing the concentration of Na(2)S (>500 microM), a mixture of silver nanowires, nanocubes, bipyramids, and irregular/quasispherical particles is synthesized at 300 and 400 W. In particular, an increase in the concentration of Na(2)S to 750 microM at 400 W leads to the production of a quantity of silver nanowires. In addition, silver nanocubes with controllable sizes can be obtained by changing the concentration of Na(2)S and the heating power. Compared to traditional wet-chemical methods, this method has the advantage of a marked decrease in reaction time to 3.5 min. Finally, our work provides a simple strategy for fabricating silver nanostructures with controllable morphologies and sizes.

Immunological effect of subunit influenza vaccine entrapped by liposomes.

OBJECTIVE: To elevate the immunological effect of subunit influenza vaccine in infants and aged people (over 60) using liposomal adjuvant in the context of its relatively low immunity and to investigate the relation between vaccine antigens and liposomal characteristics. METHODS: Several formulations of liposomal subunit influenza vaccine were prepared. Their relevant characteristics were investigated to optimize the preparation method. Antisera obtained from immunizinged mice were used to evaluate the antibody titers of various samples by HI and ELISA. RESULTS: Liposomal trivalent influenza vaccine prepared by film evaporation in combinedation with freeze-drying significantly increased its immunological effect in SPF Balb/c mice. Liposomal vaccine stimulated the antibody titer of H3N2, H1N1, and B much stronger than conventional influenza vaccine. As a result, liposomal vaccine (mean size: 4.5-5.5 microm, entrapment efficiency: 30%-40%) significantly increased the immunological effect of subunit influenza vaccine. CONCLUSION: The immune effect of liposomal vaccine depends on different antigens, and enhanced immunity is not positively correlated with the mean size of liposome or its entrapped efficiency.