Mechanistic study on the side arm effect in a palladium/Xu-Phos-catalyzed enantioselective alkoxyalkenylation of γ-hydroxyalkenes.

Recently, the asymmetric bifunctionalization of alkenes has received much attention. However, the development of enantioselective alkoxyalkenylation has posed a considerable challenge and has lagged largely behind. Herein, we report a new palladium-catalyzed enantioselective alkoxyalkenylation reaction, using a range of primary, secondary, and tertiary γ-hydroxy-alkenes with alkenyl halides. By employing newly identified Xu-Phos (Xu8 and Xu9) with a suitable side-arm adjacent to the PCy2 motif, a series of allyl-substituted tetrahydrofurans were obtained in good yields with up to 95% ee. Besides (E)-alkenyl halides, (Z)-alkenyl halide was also examined and provided the corresponding (Z)-product as a single diastereomer, supporting a stereospecific oxidative addition and reductive elimination step. Moreover, deuterium labeling and VCD experiments were employed to determine a cis-oxypalladation mechanism. DFT calculations helped us gain deeper insight into the side-arm effect on the chiral ligand. Finally, the practicability of this method is further demonstrated through a gram-scale synthesis and versatile transformations of the products.

Composition and contents of fatty acids and amino acids in the mycelia of Lentinula edodes.

With the global shortages of animal protein foods, mycoprotein as a low-cost alternative source of protein by its high-protein and low-fat content has become a development trend. Lentinula edodes (L. edodes) is a healthy food with high protein and low fiber. This work evaluated the nutritional value of L. edodes mycelia, and determined the composition and contents of fatty acids and amino acids. Eleven saturated fatty acids (SFAs) and 12 unsaturated fatty acids (UFAs) were detected in the mycelia of L. edodes. The UFA content accounted for 75.7% and 73.1% of the total fatty acid content in the mycelia of strains 18 and 18N44, respectively. Linoleic acid was the major polyunsaturated fatty acid (PUFA) in the mycelia, accounting for 91.0% and 86.3% of the UFAs, respectively. The mycelia of the two strains contained 17 types of amino acids, and the essential amino acids were sufficient (357.92 ± 0.42 and 398.38 ± 4.52 mg/g pro, respectively), both close to the WHO/FAO reference protein pattern value. The most abundant essential amino acid was Lys, and the limiting amino acids were Met + Cys and Ile, respectively. The SRC values in the mycelia of the two strains were 68.07 and 54.86, and the EAAI values were 67.70 and 74.42, respectively, both being close to those of ovalbumin. It is concluded that L. edodes mycelia are rich in easily absorbed high-quality proteins and PUFAs, and can be used as a source for meat analog required by vegetarians. This study provides a scientific basis for the further utilization of mycelial resources.

Ketone Body Rescued Seizure Behavior of LRP1 Deficiency in Drosophila by Modulating Glutamate Transport.

LRP1, the low-density lipoprotein receptor 1, would be a novel candidate gene of epilepsy according to our bioinformatic results and the animal study. In this study, we explored the role of LRP1 in epilepsy and whether beta-hydroxybutyrate, the principal ketone body of the ketogenic diet, can treat epilepsy caused by LRP1 deficiency in drosophila. UAS/GAL4 system was used to establish different genotype models. Flies were given standard, high-sucrose, and ketone body food randomly. The bang-sensitive test was performed on flies and seizure-like behavior was assessed. In morphologic experiments, we found that LRP1 deficiency caused partial loss of the ellipsoidal body and partial destruction of the fan-shaped body. Whole-body and glia LRP1 defect flies had a higher seizure rate compared to the control group. Ketone body decreased the seizure rate in behavior test in all LRP1 defect flies, compared to standard and high sucrose diet. Overexpression of glutamate transporter gene Eaat1 could mimic the ketone body effect on LRP1 deficiency flies. This study demonstrated that LRP1 defect globally or in glial cells or neurons could induce epilepsy in drosophila. The ketone body efficaciously rescued epilepsy caused by LRP1 knockdown. The results support screening for LRP1 mutations as discriminating conduct for individuals who require clinical attention and further clarify the mechanism of the ketogenic diet in epilepsy, which could help epilepsy patients make a precise treatment case by case.

Location affects fatty acid composition in Camellia sinensis cv Tieguanyin fresh leaves.

Tieguanyin tea is a typical representative of oolong tea in China, and is famous for its orchid-like aroma. Fatty acids are one of the important precursors for aroma production. However, fatty acid contents and compositions in Tieguanyin largely remain undefined. In this study, we quantified the fatty acid composition in Tieguanyin and its offspring by gas chromatography-flame ionization detector, and compared the effects of growth sites and harvest time on the leaf fatty acid composition. The results showed that total fatty acid contents in Tieguanyin fresh leaves were higher than its offspring. Growth sites had significant impacts on fatty acid contents. Tieguanyin grown in Anxi County showed higher total fatty acid contents, and higher ratio of total unsaturated fatty acids to total saturated fatty acids. The fresh leaves in the morning showed higher total fatty acid contents compared to samples harvested in the afternoon or evening, suggesting a dynamic fatty acid degradation during day period. During tea processing, unsaturated fatty acids including linolenic acid, linoleic acid and oleic acid (18:1Δ9c) decreased 13.1%, 13.2% and 84.2%, respectively. The ratio of unsaturated fatty acids to saturated fatty acids still was above 300%. We found that Tieguanyin was a typical 18:3 plant, and the higher ratio of unsaturated fatty acids to saturated fatty acids of Tieguanyin grown in Anxi County may contribute to its characteristics aroma.

ITS1/5.8S/ITS2, a Good Marker for Initial Classification of Shiitake Culinary-Medicinal Lentinus edodes (Agaricomycetes) Strains in China.

China is home to rich wild and cultivated strains of Lentinus edodes, an important edible and medicinal mushroom. Artificial selection of L. edodes has a long history, and the widely cultivated strains belong to populations different from those of most wild strains. Internal transcribed spacer (ITS) regions have been used as good markers to identify L. edodes populations. But because ITS regions exhibit incomplete concerted evolution, the use of an ITS to identify L. edodes populations has been questioned. The objective of this study was to determine whether the ITS region is suitable for identifying L. edodes populations and which populations the widely cultivated strains and the most wild strains belong to by investigating intraindividual and differential ITS polymorphisms between 44 cultivars and 44 wild strains of L. edodes in China. Intraindividual ITS polymorphism is common in L. edodes strains, and most strains possessed 2 different ITS sequences, which came from their heterokaryons. The genetic polymorphisms of ITS1, 5.8S, and ITS2 in L. edodes strains are distinct. All strains were divided into one 5.8S type (5.8S-A), 2 ITS1 types (ITS1-A and ITS1-B), and 2 ITS2 types (ITS2-A and ITS2-B), which were subdivided into 2 branches (ITS2-A1 and ITS2-A2; ITS2-B1 and ITS2-B2). ITS1/5.8S/ITS2 could be used as a good marker in preliminary classification of L. edodes strains in China. It not only exhibited classified information of ITS1, 5.8S, and ITS2 for each strain at the same time, it also indicated whether the strain was heterozygous. The 44 cultivated strains were mainly the A/A/A1 type, and the 44 wild strains were mainly the A/A/A2 and other mixed types.

Mechanism of Glucose Regulates the Fruiting Body Formation in the Beech Culinary-Medicinal Mushroom, Hypsizygus marmoreus (Agaricomycetes).

To understand the fruiting process of Hypsizygus marmoreus, a synthetic liquid medium (SLM) was optimized to induce fruiting body initiation. Dependent on the SLM, the effect of a monofactor (glucose) on the fruiting bodies of H. marmoreus was studied at different concentrations (10 and 40 g/L). Primordia appeared approximately 10 days earlier in low-glucose media (LGM) than in high-glucose media (HGM), whereas mature fruiting bodies formed on mushrooms approximately 7 days earlier and more primordia developed into mature fruiting bodies when cultured in HGM. In addition, the morphogenesis of the primordia was clustered in HGM, which was different than what was observed in LGM. Furthermore, differentially expressed genes (DEGs) that encoded various proteins involved in cell structure, general metabolism, signal transduction, and transcription and translation were analyzed by transcriptome sequencing. Six DEGs were detected by quantitative reverse-transcriptase polymerase chain reaction, and the results were consistent with the altered patterns of gene expression revealed by the transcriptome. This study not only identifies new candidate genes involved in the development of H. marmoreus but also provides a new research platform for studying the development of other edible mushrooms.

Paper/PMMA Hybrid 3D Cell Culture Microfluidic Platform for the Study of Cellular Crosstalk.

Studying cellular crosstalk is important for understanding tumor initiation, progression, metastasis, and therapeutic resistance. Moreover, a three-dimensional (3D) cell culture model can provide a more physiologically meaningful culture microenvironment. However, studying cellular crosstalk in a 3D cell culture model involves tedious processing. In this study, a paper/poly(methyl methacrylate) (PMMA) hybrid 3D cell culture microfluidic platform was successfully developed for the study of cellular crosstalk. The platform was a paper substrate with culture microreactors placed on a PMMA substrate with hydrogel-infused channels. Different types of cells were directly seeded and cultured in the microreactors. Aberrant cell proliferation of the affected cells was induced by secretions from transfected cells, and the proliferation ratios were investigated using a colorimetric method. The results showed that the responses of cellular crosstalk were different in different types of cells. Moreover, neutralizing and competitive assays were performed to show the functionality of the platform. Additionally, the triggered signaling pathways of the affected cells were directly analyzed by a subsequent immunoassay. The microfluidic platform provides a simple method for studying cellular crosstalk and the corresponding signaling pathways in a 3D culture model.

Anticancer effect of icaritin on human lung cancer cells through inducing S phase cell cycle arrest and apoptosis.

Icaritin, a prenylflavonoid derivative from Epimedium Genus, has been shown to exhibit many pharmacological and biological activities. However, the function and the underlying mechanisms of icaritin in human non-small cell lung cancer have not been fully elucidated. The purpose of this study was to investigate the anticancer effects of icaritin on A549 cells and explore the underlying molecular mechanism. The cell viability after icaritin treatment was tested by MTT assay. The cell cycle distribution, apoptosis and reactive oxygen species (ROS) levels were analyzed by flow cytometry. The mRNA and protein expression levels of the genes involved in proliferation and apoptosis were respectively detected by RT-PCR and Western blotting. The results demonstrated that icaritin induced cell cycle arrest at S phase, and down-regulated the expression levels of S regulatory proteins such as Cyclin A and CDK2. Icaritin also induced cell apoptosis characterized by positive Hoechst 33258 staining, accumulation of the Annexin V-positive cells, increased ROS level and alteration in Bcl-2 family proteins expression. Moreover, icaritin induced sustained phosphorylation of ERK and p38 MAPK. These findings suggested that icaritin might be a new potent inhibitor by inducing S phase arrest and apoptosis in human lung carcinoma A549 cells.

Identification and surgical repair of familial thoracic aortic aneurysm and dissection caused by TGFBR1 mutation.

BACKGROUND: This study aimed at exploring the causative gene and summarizing the clinical characteristics in a Chinese thoracic aortic aneurysm and dissection (TAAD) family. METHODS: Family members were examined for features of syndromic genetic diseases by clinician and geneticist. Genomic DNA was extracted from 2 distantly related members with definite TAAD for exome sequencing. RESULTS: A pathogenic mutation (rs111426349, c.1459C >T) of transforming growth factor ß receptor 1 (TGFBR1) was confirmed, which result in the amino acid substitution p.R487W. Fourteen TGFBR1 mutation carriers were detected among 39 tested members in this family. The average age at diagnosis of aortic root dilatation or aneurysm was 23.2 ± 12.6 years (range 3-37 years). Early onset of aortic root dilatation was significant in this family without reported phenotypes. The David procedure was performed prophylactically in 3 carriers of this family. CONCLUSIONS: Familial TAAD caused by TGFBR1 mutation (c.1459C >T) was confirmed in a large Chinese Han ethnic family using exome sequencing. Aggressively prophylactic David procedure may be not necessary at a smaller aortic size in familial TAAD patients with TGFBR1 mutation and further observation is warranted.

Fucoidan induces apoptosis of HepG2 cells by down-regulating p-Stat3.

Fucoidan is one of the main bioactive components of polysaccharides. The current study was focused on the anti-tumor effects of fucoidan on human heptoma cell line HepG2 and the possible mechanisms. Fucoidan treatment resulted in cell cycle arrest and apoptosis of HepG2 cells in a dose-dependent manner detected by MTT assay, flow cytometry and fluorescent microscopy. The results of flow cytometric analysis revealed that fucoidan induced G2/M arrest in the cell cycle progression. Hoechst 33258 and Annexin V/PI staining results showed that the apoptotic cell number was increased, which was associated with a dose-dependent up-regulation of Bax and down-regulation of Bcl-2 and p-Stat3. In parallel, the up-regulation of p53 and the increase in reactive oxygen species were also observed, which may play important roles in the inhibition of HepG2 growth by fucoidan. In the meantime, Cyclin B1 and CDK1 were down-regulated by fucoidan treatment. Down-regulation of p-Stat3 by fucoidan resulted in apoptosis and an increase in ROS in response to fucoidan exposure. We therefore concluded that fucoidan induces apoptosis through the down-regulation of p-Stat3. These results suggest that fucoidan may be used as a novel anti-cancer agent for hepatocarcinoma.

Per-O-acetylation of cellulose in dimethyl sulfoxide with catalyzed transesterification.

Cellulose acetylation was investigated in dimethyl sulfoxide (DMSO) with isopropenyl acetate (IPA) as acetylating reagent and 1,8-diazabicyclo[5,4,0]undec-7-ene (DBU) as catalyst at 70-130 °C for 3-12 h. The degree of substitution (DS) of acetylated cellulose was comparatively determined by titration and ¹H NMR and confirmed by FT-IR analysis. The results indicated that per-O-acetylation was achieved at >90 °C for a relatively long duration. The three well-resolved peaks of carbonyl carbons in ¹³C NMR spectra also provided evidence of per-O-acetylation. The solubility of cellulose acetates in common organic solvents was examined, and the result showed that chloroform can be an alternative choice as a solvent for fully acetylated cellulose formed in this study besides DMSO. The intrinsic viscosity of acetylated cellulose solution implied almost no degradation of cellulose during acetylation in DMSO except at higher temperature (130 °C) for a long time.

An efficient Agrobacterium-mediated transformation method for the edible mushroom Hypsizygus marmoreus.

Hypsizygus marmoreus is one of the major edible mushrooms in East Asia. As no efficient transformation method, the molecular and genetics studies were hindered. The glyceraldehyde-3-phosphate dehydrogenase (GPD) gene of H. marmoreus was isolated and its promoter was used to drive the hygromycin B phosphotransferase (HPH) and enhanced green fluorescent protein (EGFP) in H. marmoreus. Agrobacterium tumefaciens-mediated transformation (ATMT) was successfully applied in H. marmoreus. The transformation parameters were optimized, and it was found that co-cultivation of bacteria with protoplast at a ratio of 1000:1 at a temperature of 26 °C in medium containing 0.3 mM acetosyringone resulted in the highest transformation efficiency for Agrobacterium strain. Besides, three plasmids, each carrying a different promoter (from H. marmoreus, Ganoderma lucidum and Lentinula edodes) driving the expression of an antibiotic resistance marker, were also tested. The construct carrying the H. marmoreus gpd promoter produced more transformants than other constructs. Our analysis showed that over 85% of the transformants tested remained mitotically stable even after five successive rounds of subculturing. Putative transformants were analyzed for the presence of hph gene by PCR and Southern blot. Meanwhile, the expression of EGFP in H. marmoreus transformants was detected by fluorescence imaging. This ATMT system increases the transformation efficiency of H. marmoreus and may represent a useful tool for molecular genetic studies in this mushroom species.

Dual-component system dimethyl sulfoxide/LiCl as a solvent and catalyst for homogeneous ring-opening grafted polymerization of ε-caprolactone onto xylan.

The preparation of xylan-graft-poly(ε-caprolactone) (xylan-g-PCL) copolymers was investigated by homogeneous ring-opening polymerization (ROP) in a dual-component system containing Lewis base LiCl and strong polar aprotic solvent dimethyl sulfoxide (DMSO). DMSO/LiCl acted as solvent, base, and catalyst for the ROP reaction. The effects of the parameters, including the reaction temperature, molar ratio of ε-caprolactone (ε-CL) to anhydroxylose units (AXU) in xylan, and reaction time, on the degree of substitution (DS) and weight percent of PCL side chain (WPCL) were investigated. The results showed that xylan-g-PCL copolymers with low DS in the range of 0.03-0.39 were obtained under the given conditions. The Fourier transform infrared spectroscopy (FTIR), (1)H nuclear magnetic resonance (NMR), (13)C NMR, (1)H-(1)H correlation spectroscopy (COSY), and (1)H-(13)C correlation two-dimensional (2D) NMR [heteronuclear single-quantum coherence (HSQC)] characterization provided more evidence of the attachment of side chains onto xylan. Only one ε-CL was confirmed to be attached onto xylan with each side chain. Integration of resonances assigned to the substituted C2 and C3 in the HSQC spectrum also indicated 69.23 and 30.77% of PCL side chains attached to AXU at C3 and C2 positions, respectively. Although the attachment of PCL onto xylan led to the decreased thermal stability of xylan, the loss of unrecovered xylan fractions with low molecular weight because of the high solubility of xylan in DMSO/LiCl resulted in the increased thermal stability of the samples. This kind of xylan derivative has potential application in environmentally friendly and biodegradable materials considering the good biodegradability of xylan and PCL.

Fucoidan induces G1 phase arrest and apoptosis through caspases-dependent pathway and ROS induction in human breast cancer MCF-7 cells.

Fucoidan is an active component of seaweed, which inhibits proliferation and induces apoptosis of several tumor cells while the detailed mechanisms underlying this process are still not clear. In this study, the effect of Fucoidan on the proliferation and apoptosis of human breast cancer MCF-7 cells and the molecular mechanism of Fucoidan action were investigated. Viable cell number of MCF-7 cells was decreased by Fucoidan treatment in a dose-dependent manner as measured by MTT assay. Fucoidan treatment resulted in G1 phase arrest of MCF-7 cells as revealed by flow cytometry, which was associated with the decrease in the gene expression of cyclin D1 and CDK-4. Annexin V/PI staining results showed that the number of apoptotic cells was associated with regulation of cytochrome C, caspase-8, Bax and Bcl-2 at transcriptional and translational levels. Both morphologic observation and Hoechst 33258 assay results confirmed the pro-apoptotic effect of Fucoidan. Meanwhile, the ROS production was also increased by Fucoidan treatment, which suggested that Fucoidan induced oxidative damage in MCF-7 cells. The results of present study demonstrated that Fucoidan could induce G1 phase arrest and apoptosis in MCF-7 cells through regulating the cell cycle and apoptosis-related genes or proteins expression, and ROS generation is also involved in these processes.

Germacrone induces apoptosis in human hepatoma HepG2 cells through inhibition of the JAK2/STAT3 signalling pathway.

Previous studies have shown that STAT3 plays a vital role in the genesis and progression of cancer. In this study, we investigated the relationship between the JAK2/STAT3 signalling pathway and germacrone-induced apoptosis in HepG2 cells. HepG2 cells were incubated with germacrone for 24 h, the protein expression of p-STAT3, STAT3, p-JAK2 and JAK2 was detected by Western Blotting, and RT-PCR was used to determine the expression of STAT3, p53, Bcl-2 and Bax at transcriptional levels. Besides that, HepG2 cells were pre-treated with AG490 or IL-6 for 2 h, and then incubated with germacrone for 24 h. The expression of p-JAK2, JAK2, p-STAT3, STAT3, p53, Bax and Bcl-2 was detected by Western blotting. The activity of HepG2 cells was tested by MTT assay. The apoptosis of HepG2 cells and levels of reactive oxygen species (ROS) were flow cytometrically measured. The results showed that germacrone exposure decreased p-STAT3 and p-JAK2 and regulated expression of p53 and Bcl-2 family members at the same time. Moreover, IL-6 enhanced the activation of the JAK2/STAT3 signalling pathway and therefore attenuated the germacrone-induced apoptosis. Suppression of JAK2/STAT3 signalling pathway by AG490, an inhibitor of JAK2, resulted in apoptosis and an increase in ROS in response to germacrone exposure. We therefore conclude that germacrone induces apoptosis through the JAK2/STAT3 signalling pathway.

Isolation and characterization of an endosperm-specific promoter from wheat (Triticum aestivum L.).

Genes coding for avenin-like proteins (ALP) represent a new family of wheat storage protein genes. To find a wheat endosperm-specific promoter, a 1644-bp fragment upstream of the ALP type-B gene (GenBank accession number JN622144) was isolated. The important promoter elements of the ALP type-B gene were ascertained through sequence analysis which revealed that this fragment contains the TATA and CAAT boxes, which are important elements in gene expression. A prolamin box containing an endosperm motif and a GCN4-like motif (GLM) is present at about 300 bp upstream of the translation start site. The promoter sequence has two ESP-like elements and one of them is followed by an RY motif with the nucleotides CATG overlapping. The RY motif is considered the core functional sequence in a promoter. In an attempt to confirm the promoter activity, a series of 5'-deletions of the promoter were fused with the beta-glucuronidase (GUS) gene, and the constructs were stably introduced into tobacco plants. GUS staining confirmed that the AVL type-B promoter is an endosperm-specific promoter in tobacco seeds. Quantitative analysis of GUS expression in transgenic plants showed that even the shortest 5'-deletion, i.e. a 290-bp promoter sequence within the prolamin box, was sufficient to drive GUS expression in the endosperm. The highest expression level was found in transgenic plants containing the 5'-deletion vector construct pALP-8. This suggests that the ESP-like element overlapping with the RY motif may play a crucial role in the regulatory function of the promoter.

An axenic culture system for fruiting body formation by an edible bolete phylogenetically related to culinary-medicinal penny bun mushroom, Boletus edulis Bull.:Fr. strains from China.

The ability of two freshly isolated Boletus stains to fruit under axenic conditions was tested using different solid and liquid nutrient media. One strain (YNCX04) produced numerous primordia from which fruiting bodies, 12 mm and 10 mm in length, with grey, convex pilei, and yellow-white, clavate stipes developed between 15 and 30 d after inoculation of fungal mycelium onto a solid medium consisting of mineral salts, thiamine, glucose, potato, an extract of Cunninghamia lanceolata root, and agar. The other strain (YNB200) produced numerous primordia but no sporophores. Strain YNCX04 lost the ability to form fruiting bodies in axenic culture 6 mo after initial isolation but retained the ability to form primordia for up to 18 mo. Based on internal transcribed spacer sequencing data, strains YNB200 and YNCX04 formed a sub-cluster together with four previously designated Boletus edulis strains from China. Phylogenetic analysis placed the Chinese strains closer to B. aestivalis than to European and North American strains of B. edulis, although a 29-bp fragment specific to all the B. aestivalis strains was absent from all the Chinese strains. Furthermore, partial 18S rDNA sequences from strains YNB200 and YNCX04 exhibited 98% similarity with an 18S rDNA sequence from B. edulis strain Be3. Further molecular studies are indicated to more accurately establish the taxonomic positions ofF3 and F4-3, as well as the Chinese strains designated as B. edulis.

[The phylogenetic relationship of cultivated isolates of Ganoderma in China inferred from nuclear ribosomal DNA ITS sequences].

Nuclear ribosomal DNA ITS sequences have been used to investigate phylogenetic relationships between 34 Ganoderma isolates cultivated in China. Five distinct groups were identified: the subgenus Elfvingia, the sect. Phaeonema, and three groups within the sect. Ganoderma. Most of the Ganoderma isolates (85.7%) formed a single group within the sect. Gantoderma. The result indicated clear genetic diversity between the subgenus Elfvingia and the sects Phaeonema and Ganoderma, but a smaller degree of genetic diversity between the three groups placed within the sect. Ganoderma. Analysis of molecular data is a more effective and useful approach for studying the taxonomy of Ganoderma, and for establishing phylogenetic relationships within the genus, compared to methods based on fruiting body morphology.

[Inheritance of the foreign gene 1Ax1 in transgenic wheat (Triticum aestivum L.) with gene cassettes lacking vector backbone sequences].

Transformation with gene minimal expression cassettes without vector backbone sequence is a new transformation technique. In the current research, the inheritance of the foreign 1Ax1 gene cassettes were analyzed in transgenic plants recovered with gene cassettes lacking vector backbone sequences. The results showed that linear 1Ax1 gene cassettes were stably expressed and separated at the rate of 3 to 1 in the seeds of T1 generation. It suggested the functional copies of 1Ax1 gene cassettes were integrated in one sites, abiding by Mendelian genetic law. It revealed firstly the feasibility of the transformation technique with gene cassettes, and proved the inheritance of transgene cassettes in subsequent generations.