Dynamic changes and correlation analysis of microorganisms and flavonoids/ amino acids during white tea storage.

White tea stored for various times have different flavors. However, the mechanism of flavor conversion remains elusive. Flavonoids and amino acids are two typical flavor components in tea. Herein, the contents of 46 flavonoids and 40 amino acids were measured in white tea (Shoumei) stored for 1, 3, 5 and 7 years, respectively. L-tryptophan, L-ornithine and L-theanine contribute to the refreshing taste of Shoumei 1 and 3. Quercetin, rutin and hesperidin contribute to aging charm and grain aroma of Shoumei 5 and 7. 306 bacterial OTUs and 268 fungal OTUs core microbiota existed in all samples. Interestingly, white teas contained higher richness of fungi than bacteria. The correlation analysis showed that the cooperation with bacteria and fungi may result in the flavonoids and amino acids composition changes in white teas during storage. Overall, this study provides new insights into flavor conversion of white tea during storage.

Characterization of a multi-domain exo-ß-1,3-galactanase from Paenibacillus xylanexedens.

ß-1,3-Galactanases selectively degrade ß-1,3-galactan, thus it is an attractive enzyme technique to map high-galactan structure and prepare galactooligosaccharides. In this work, a gene encoding exo-ß-1,3-galactanase (PxGal43) was screened form Paenibacillus xylanexedens, consisting of a GH43 domain, a CBM32 domain and α-L-arabinofuranosidase B (AbfB) domain. Using ß-1,3-galactan (AG-II-P) as substrate, the recombined enzyme expressed in Escherichia coli BL21 (DE3) exhibited an optimal activity at pH 7.0 and 30 °C. The enzyme was thermostable, retaining >70 % activity after incubating at 50 °C for 2 h. In addition, it showed high tolerance to various metal ions, denaturants and detergents. Substrate specificity indicated that PxGal43 hydrolysis only ß-1,3-linked galactosyl oligosaccharides and polysaccharides, releasing galactose as an exo-acting manner. The function of the CBM32 and AbfB domain was revealed by their sequential deletion and suggested that their connection to the catalytic domain was crucial for the oligomerization, catalytic activity, substrate binding and thermal stability of PxGal43. The substrate docking and site-directed mutagenesis proposed that Glu191, Gln244, Asp138 and Glu81 served as the catalytic acid, catalytic base, pKa modulator, and substrate identifier in PxGal43, respectively. These results provide a better understanding and optimization of multi-domain bacterial GH43 ß-1,3-galactanase for the degradation of arabinogalactan.

Overexpression of PvBiP2 improved biomass yield and cadmium tolerance in switchgrass (Panicum virgatum L.).

Switchgrass (Panicum virgatum L.), the prime bioenergy feedstock crop, is one ideal candidate for phytoremediation of cadmium (Cd). The absorption of Cd imposes severe endoplasmic reticulum (ER)-stress in plants. ER chaperone binding proteins (BiPs) are important modulators in ER-stress responses. The objective of this study was to characterize one Cd-responsive BiP gene, PvBiP2, in switchgrass for its roles in Cd tolerance and plant growth. PvBiP2 was up-regulated by Cd and the ER-stress inducer, dithiothreitol (DTT) and could be trans-activated by one Cd-responsive heat shock transcription factor PvHsfA4. Overexpression of PvBiP2 in switchgrass significantly increased its plant growth with higher height, stem diameter, leaf width, internode length, and tiller numbers than those of the wildtype (WT) plants under non-stress conditions. After 30 days of Cd treatment, the PvBiP2 over-expression transgenic lines showed 40-45% higher dry biomass accumulation with net photosynthesis rate (Pn), but lower electrolyte leakage (EL), malondialdehyde (MDA), and glutathione (GSH) levels than WT. Moreover, over-expressing PvBiP2 led to ∼90-140% Cd accumulation in plants but 46-57% lower Cd translocation rates to shoots. Together, the PvHSFA4-PvBiP2 module acted as positive regulators in plant Cd tolerance, and over-expressing PvBiP2 promoted plant vegetative growth as well as Cd tolerance making it an ideal molecular target for genetic improvement in switchgrass in the future.

Yeast phospholipase D, Spo14, is not required for macroautophagy.

Autophagy-related gene (Atg) proteins are key players in autophagy. Some proteins that function in vesicle trafficking and lipid metabolism are also involved in autophagy. The SPO14 in yeast, which encodes phospholipase D (PLD), is involved in membrane trafficking and plays a vital role in sporulation during meiosis. Crosstalk has been identified between autophagy and sporulation. Although the PLD is required for macroautophagy in mammals, its role in yeast macroautophagy remains unclear. We observed that Spo14 is not required for macroautophagy in yeast and that it is dispensable for Atg8 lipidation, which plays an important role in phagophore extension. Our results also revealed that green fluorescent protein (GFP)-Atg8 degradation is not completely blocked in atg1Δ/atg1Δ cells under sporulation condition. Therefore, Spo14 is not required for macroautophagy in yeast.

Highly Chemoselective [2+1] Annulation of α-Alkylidene Pyrazolones with α-Bromonitroalkenes: Synthesis of Pyrazolone-Based Vinylcyclopropanes and Computational Studies.

A highly chemoselective [2+1] annulation of α-alkylidene pyrazolones with α-bromonitroalkenes has been achieved under mild conditions. α-Alkylidene pyrazolones were unprecedentedly used as a C1 synthon to participate in annulation reactions, providing access to diverse vinylcyclopropane-based pyrazolone products. In addition, a spectrum of pharmaceutically interesting pyrazole-fused pyranone oximes could be rapidly obtained through a [2+1] annulation/rearrangement sequential process. Computational studies disclosed the origin of the observed chemoselectivity of the [2+1] cycloaddition.

Enantio- and diastereoselective synthesis of spiropyrazolones via an organocatalytic [1 + 2 + 3] multicomponent reaction.

An asymmetric catalytic multicomponent reaction of malononitrile, benzaldehyde, and α-arylidene pyrazolinones to produce spiropyrazolones has been reported. The [1 + 2 + 3] multicomponent reaction was catalyzed by chiral cinchona alkaloids to provide spiropyrazolones in high yields, with excellent enantioselectivities and good diastereoselectivities. We also performed control experiments and proposed a plausible catalytic cycle based on the observed experimental results to explain the reaction process and stereoselectivity of the asymmetric multicomponent reaction.

Recent advances in the synthesis of C2-spiropseudoindoxyls.

Compared with the numerous reviews on the construction of C3 spirooxindoles, few reviews have examined the synthesis of structurally analogous C2-spiropseudoindoxyl derivatives. This scaffold has attracted substantial attention from synthetic chemists because of its relevance to medicinal chemistry. This review summarizes the recent progress in the synthesis of many heterocycle and carbocycle fused spiropseudoindoxyl compounds. The article is divided into sections according to the type of catalysis, including metal catalysis, high-iodine reagent mediation, and organic catalysis. We also discuss challenges and future directions based on the progress in the field. We believe that this review will update researchers focused on the synthesis of C2-spiropseudoindoxyl compounds and will encourage further growth in this field.

Chemo- and Stereoselective Cross Rauhut-Currier-Type Reaction of Tri-substituted Alkenes Containing Trifluoromethyl Groups.

A chemoselective cross Rauhut-Currier-type reaction has been developed involving a tri-substituted alkene (trifluoromethyl-containing acrylonitrile derivative) with a di- or tri-substituted alkene to yield tetra-substituted double bonds in RC-type products. This approach can support the synthesis of trifluoromethylated tetra-substituted olefins and synthetically important, structurally complex 3-allylic-type oxindole skeletons. The asymmetric version of this RC-type reaction can be realized by combining a Brønsted acid and Lewis base for bifunctional H-bonding-tertiary amine catalysis. Subsequent transformation of multi-functionalized RC-type product leads to pharmacologically interesting cyclohexane-based spiro-pyrazolones bearing six contiguous chiral centers and two highly congested, vicinal quaternary carbon centers.

Efficient sample clean-up and online preconcentration for sensitive determination of melamine in milk samples by capillary electrophoresis with contactless conductivity detection.

Based on an efficient sample clean-up and field-amplified sample injection online preconcentration technique in capillary electrophoresis with contactless conductivity detection, a new analytical method for the sensitive determination of melamine in milk samples was established. In order to remove the complex matrix interference, which resulted in a serious problem during field-amplified sample injection, liquid-liquid extraction was utilized. As a result, liquid-liquid extraction provides excellent sample clean-up efficiency when ethyl acetate was used as organic extraction by adjusting the pH of the sample solution to 9.5. Both inorganic salts and biological macromolecules are effectively removed by liquid-liquid extraction. The sample clean-up procedure, capillary electrophoresis separation parameters and field-amplified sample injection conditions are discussed in detail. The capillary electrophoresis separation was achieved within 5 min under the following conditions: an uncoated fused-silica capillary, 12 mM HAc + 10 mM NaAc (pH = 4.6) as running buffer, separation voltage of +13 kV, electrokinetic injection of +12 kV × 10 s. Preliminary validation of the method performance with spiked melamine provided recoveries >90%, with limits of detection and quantification of 0.015 and 0.050 mg/kg, respectively. The relative standard deviations of intra- and inter-day were below 6%. This newly developed method is sensitive and cost effective, therefore, suitable for screening of melamine contamination in milk products.

[Characteristics of three spiroplasma isolates from honeybee (Apis mellifera)].

OBJECTIVE: To investigate the kinds and characteristics of spiroplasma in honeybees, as well as to study the taxonomy and transmission of honeybee spiroplasma under natural conditions. METHODS: We examined the morphology of spiroplasma isolates by dark field and transmission electron microscopy and studied the biological characteristics by using conventional culture-dependent methods and molecular biology and serological methods. RESULTS: Three spiroplasma isolates were obtained from healthy Apis mellifera. All isolates exhibited helicity during their growth phase, with one isolate (MF0905) being shorter and having less helicity. This isolate also differed from the other two (MF0903 and MF0904) in having larger colonies with an irregular margin instead of being round. In addition, isolate MF0905 could not hydrolyze arginine whereas MF0903 and MF0904 could. All three isolates could use glucose and D-fructose as a carbon source but did not hydrolyse urea. Phylogenetic analysis based on the 16S rDNA, ITS and the rpoB gene showed that MF0903 and MF0904 had a close relationship with Spiroplasma melliferum, and MF0905 was close to S. clarkii. Serological studies including the growth inhibition test, metabolic inhibition test and deformation test gave the same result as the phylogenetic analysis. CONCLUSION: The spiroplasma isolate MF0905 might be S. clarkii and other two isolates were S. melliferum. This result indicated that Spiroplasma melliferum is not the only spiroplasma species in honeybees in China.

A new Neotyphodium species from Festuca parvigluma Steud. grown in China.

Neotyphodium species evolved from Epichloë species and are asexual, seedborne endophytes in many cool-season grasses. Here we propose a new species inhabiting Festuca parvigluma native to China. Morphology, host specificity and molecular phylogenetic evidence supported recognition of this new species. Sequences of beta-tubulin gene (tubB) introns and translation elongation factor 1-alpha gene (tefA) introns were present as two copies in all five isolates examined. In phylogenetic analyses copy 1 was closely related to E. yangzii in the EBY clade and copy 2 with E. typhina in the ETC clade, indicating this new species might have originated as a result of hybridization between members of these two clades. Referring to the distribution area of host plants, Neotyphodium sinofestucae is proposed for this new species.

A new stromata-producing Neotyphodium species symbiotic with clonal grass Calamagrostis epigeios (L.) Roth. grown in China.

We describe a new stromata-producing Neotyphodium species symbiotic with clonal Calamagrostis epigeios (L.) Roth. Stromata on the grass, 47.5-186 mm long, occurred frequently, but neither perithecium nor mature ascus was observed. Morphology of fungal isolates obtained from symptomatic and asymptomatic tillers were identical to each other and similar to those of epichloë endophytes. In phylogenetic analysis all selected five fungal isolates clustered into a significantly distinct clade based on sequences of beta-tubulin gene (tubB) introns and translation elongation factor 1-alpha gene (tefA) introns with bootstrap values of 99%, supporting erection of a new species. Concerning the production of extremely long stromata on the host plants and absence of sexual spores, we propose the name Neotyphodium stromatolongum Y. Ji, L. Zhan et Z. Wang, sp. nov.

A new species of Epichloë symbiotic with Chinese grasses.

Epichloë species are fungal symbionts (endophytes) of grasses, six European and four North American biological species in genus Epichloë have been described in previous researches. In this study we describe a new Epichloë species, Epichloë yangzii Li et Wang, found in natural symbioses with Roegneria kamoji native to China. We investigated the host specificity, morphology, interfertility tests and molecular phylogenetic evidences of this new species. The results indicated that E. yangzii is host specific and seedborne. Most morphological characteristics of this new species are typical in the genus. However differences are evident in several features including size of perithecia, asci and ascospores. In mating tests E. yangzii was not interfertile with E. elymi isolates from related hosts in genera Elymus. Phylogenetic relationships based on sequences of beta-tubulin gene (tub2) introns and translation elongation factor 1-alpha gene (tef1) introns showed that members of the new species grouped into exclusive clades with high bootstrap value.