The Gß-like protein Bcgbl1 regulates development and pathogenicity of the gray mold Botrytis cinerea via modulating two MAP kinase signaling pathways.

The fungal Gß-like protein has been reported to be involved in a variety of biological processes, such as mycelial growth, differentiation, conidiation, stress responses and infection. However, molecular mechanisms of the Gß-like protein in regulating fungal development and pathogenicity are largely unknown. Here, we show that the Gß-like protein gene Bcgbl1 in the gray mold fungus Botrytis cinerea plays a pivotal role in development and pathogenicity by regulating the mitogen-activated protein (MAP) kinases signaling pathways. The Bcgbl1 deletion mutants were defective in mycelial growth, sclerotial formation, conidiation, macroconidial morphogenesis, plant adhesion, and formation of infection cushions and appressorium-like structures, resulting in a complete loss of pathogenicity. Bcgbl1 interacted with BcSte50, the adapter protein of the cascade of MAP kinase (MAPK). Bcgbl1 mutants had reduced phosphorylation levels of two MAPKs, namely Bmp1 and Bmp3, thereby reducing infection. However, deletion of Bcgbl1 did not affect the intracellular cAMP level, and exogenous cAMP could not restore the defects. Moreover, Bcgbl1 mutants exhibited defects in cell wall integrity and oxidative stress tolerance. Transcriptional profiling revealed that Bcgbl1 plays a global role in regulation of gene expression upon hydrophobic surface induction. We further uncovered that three target genes encoding the hydrophobic surface binding proteins (HsbAs) contributed to the adhesion and virulence of B. cinerea. Overall, these findings suggest that Bcgbl1 had multiple functions and provided new insights for deciphering the Bcgbl1-mediated network for regulating development and pathogenicity of B. cinerea.

Recent Advances in Carbon-Nitrogen/Carbon-Oxygen Bond Formation Under Transition-Metal-Free Conditions.

Carbon-heteroatom bond formation under transition-metal free conditions provides a powerful synthetic alternative for the efficient synthesis of valuable molecules. In particular, C-N and C-O bonds are two important types of carbon-heteroatom bonds. Thus, continuous efforts have been deployed to develop novel C-N/C-O bond formation methodologies involving various catalysts or promoters under TM-free conditions, which enables the synthesis of various functional molecules comprising C-N/C-O bonds in a facile and sustainable manner. Considering the significance of C-N/C-O bond construction in organic synthesis and materials science, this review aims to comprehensively present selected examples on the construction of C-N (including amination and amidation) and C-O (including etherification and hydroxylation) bonds without transition metals. Besides, the involved promoters/catalysts, substrate scope, potential application and possible reaction mechanisms are also systematically discussed.

Stepwise Strategy for One-Pot Synthesis of Single-Stranded DNA Rings from Multiple Short Fragments.

Cyclic rings of single-stranded (ss) DNA have various unique properties, but wider applications have been hampered by their poor availability. This paper reports a convenient one-pot method in which these rings are efficiently synthesized by using T4 DNA ligase through convergent cyclization of easily available short DNA fragments. The key to the present method is to separate all the splint oligonucleotides into several sets, and add each set sequentially at an appropriate interval to the solutions containing all the short DNA fragments. Compared with simple one-pot strategies involving simultaneous addition of all the splints at the beginning of the reaction, both the selectivity and the yields of target ssDNA rings are greatly improved. This convergent method is especially useful for preparing large-sized rings that are otherwise hard to obtain. By starting from six short DNA fragments (71-82 nt), prepared by a DNA synthesizer, a ssDNA ring of 452-nt size was synthesized in 35 mol % yield and in high selectivity. Satisfactorily pure DNA rings were obtainable simply by treating the crude products with exonuclease.

Facile Characterization of Topology of DNA Catenanes.

During the preparation of single-stranded DNA catenanes, topological isomers of different linking numbers (Lk) are intrinsically produced, and they must be separated from each other to construct sophisticated nanostructures accurately. In many previous studies, however, mixtures of these isomers were directly employed to construct nanostructures without sufficient characterization. Here, we present a method that easily and clearly characterizes the isomers by polyacrylamide gel electrophoresis. To the mixtures of topological isomers of [2]catenanes, two-strut oligonucleotides, which are complementary with a part of both rings, were added to connect the rings and fix the whole conformations of isomers. As a result, the order of migration rate was always Lk3 > Lk2 > Lk1, irrespective of gel concentration. Thus, all the topological isomers were unanimously characterized by only one polyacrylamide gel electrophoresis experiment. Well-characterized DNA catenanes are obtainable by this two-strut strategy, opening the way to more advanced nanotechnology.

Ginsenoside Rg3 has effects comparable to those of ginsenoside re on diabetic kidney disease prevention in db/db mice by regulating inflammation, fibrosis and PPARγ.

Ginsenoside Rg3 (Rg3) is an adjuvant antitumor drug, while ginsenoside Re (Re) is an adjuvant antidiabetic drug. Our previous studies demonstrated that Rg3 and Re both have hepatoprotective effects in db/db mice. The present study aimed to observe the renoprotective effects of Rg3 on db/db mice, with Re as the control. The db/db mice were randomly assigned to receive daily oral treatment with Rg3, Re or vehicle for 8 weeks. Body weight and blood glucose were examined weekly. Blood lipids, creatinine, and BUN were examined by biochemical assay. Hematoxylin and eosin and Masson staining were used for pathological examination. The expression of peroxisome proliferator­activated receptor gamma (PPARγ) and inflammation and fibrosis biomarkers was examined by immunohistochemical and reverse transcription­quantitative PCR. Although neither had a significant effect on body weight, blood glucose or lipids, Rg3 and Re were both able to decrease the creatinine and blood urea nitrogen levels of db/db mice to levels similar to those of wild type mice and inhibit pathological changes. The expression of PPARγ was upregulated and biomarkers of inflammation and fibrosis were downregulated by Rg3 and Re. The results showed that the potential of Rg3 as a preventive treatment of diabetic kidney disease was similar to that of Re.

The Astaxanthin Aggregation Pattern Greatly Influences Its Antioxidant Activity: A Comparative Study in Caco-2 Cells.

Astaxanthin is an excellent antioxidant that can form unstable aggregates in biological or artificial systems. The changes of astaxanthin properties caused by molecular aggregation have gained much attention recently. Here, water-dispersible astaxanthin H- and J-aggregates were fabricated and stabilized by a natural DNA/chitosan nanocomplex (respectively noted as H-ADC and J-ADC), as evidenced by ultraviolet and visible spectrophotometry, Fourier transform infrared spectroscopy, and Raman spectroscopy. Compared with J-ADC, H-ADC with equivalent astaxanthin loading capacity and encapsulation efficiency showed smaller particle size and similar zeta potential. To explore the antioxidant differences between astaxanthin H- and J-aggregates, H-ADC and J-ADC were subjected to H2O2-pretreated Caco-2 cells. Compared with astaxanthin monomers and J-aggregates, H-aggregates showed a better cytoprotective effect by promoting scavenging of intracellular reactive oxygen species. Furthermore, in vitro 1,1-diphenyl-2-picrylhydrazyl and hydroxyl free radical scavenging studies confirmed a higher efficiency of H-aggregates than J-aggregates or astaxanthin monomers. These findings give inspiration to the precise design of carotenoid aggregates for efficient utilization.

RNA ligation of very small pseudo nick structures by T4 RNA ligase 2, leading to efficient production of versatile RNA rings.

T4 RNA ligase 2 catalyses two types of reactions: (i) sealing of a nick structure in double-stranded RNA and (ii) connection of two single-stranded RNA strands. In order to obtain comprehensive views on these two types of reactions and widen the application scope of this RNA ligase, we here systematically analysed the connection of single-stranded RNA strands having different secondary structures. It has been found that the ligation is enormously promoted when a stem of only 4-bp or longer is formed in the 3'-OH side of the joining site. Additional placement of a stem in the 5'-phosphate side further facilitates the ligation. In contrast, perturbation of the stem structures in RNA substrates suppresses the ligation. These results indicate that ligation of two single-stranded RNA strands by T4 RNA ligase 2 is greatly promoted by forming a "nick-like intermediate". Even the unstable intermediate, formed only temporarily in the solution, is sufficiently effective. By designing the synthetic systems in terms of this finding, short single-stranded RNA rings of versatile sizes, which are otherwise hard to be obtained, are efficiently prepared in high selectivity and yield.

Efficient Preparation of Large-Sized Rings of Single-Stranded DNA through One-Pot Ligation of Multiple Fragments.

Circular single-stranded DNA (c-ssDNA) has significant applications in DNA detection, the development of nucleic acid medicine, and DNA nanotechnology because it shows highly unique features in mobility, dynamics, and topology. However, in most cases, the efficiency of c-ssDNA preparation is very low because polymeric byproducts are easily formed due to intermolecular reaction. Herein, we report a one-pot ligation method to efficiently prepare large c-ssDNA. By ligating several short fragments of linear single-stranded DNA (l-ssDNA) in one-pot by using T4 DNA ligase, longer l-ssDNAs intermediates are formed and then rapidly consumed by the cyclization. Since the intramolecular cyclization reaction is much faster than intermolecular polymerization, the formation of polymeric products is suppressed and the dominance of intramolecular cyclization is promoted. With this simple approach, large-sized single-stranded c-ssDNAs (e.g., 200-nt) were successfully synthesized in high selectivity and yield.

Tandem blocking of PCR extension to form a single-stranded overhang for facile, visual, and ultrasensitive gene detection.

In order to detect a predetermined gene in a field test, a facile, visual, and ultrasensitive approach without the need of special and expensive machines is required. In this study, a gene in the Ebola virus was targeted as an example for diagnosis. The key strategy is to incorporate molecular blockers (azobenzene-bearing moieties or thymine dimers) in tandem in one of the PCR primers and stop the polymerase extension there to form a single-stranded overhang. The PCR product was added to the dispersion of gold nanoparticles which were labelled with a probe oligonucleotide. When the Ebola virus-specific gene existed in the specimen, the oligonucleotide on the gold particles formed a double-helix with the single-stranded overhang, and thus the dispersion remained red in color. In the absence of the gene, however, the dispersion rapidly turned to blue because of nanoparticle aggregation. The difference was explicit even when the initial specimen involved only 1 copy of the gene. Accordingly, "whether the patient is infected by the virus or not" can be easily and visually judged by the naked eye.