Phylogeny and molecular dating of the cerato-platanin-encoding genes.

The cerato-platanin family consists of proteins that can induce immune responses, cause necrosis, change chemotaxis and locomotion and may be related to the growth and development of various fungi. In this work, we analyzed the phylogenetic relationships among genes encoding members of the cerato-platanin family and computed the divergence times of the genes and corresponding fungi. The results showed that cerato-platanin-encoding genes could be classified into 10 groups but did not cluster according to fungal classes or their functions. The genes transferred horizontally and showed duplication. Molecular dating and adaptive evolution analyses indicated that the cerato-platanin gene originated with the appearance of saprophytes and that the gene was under positive selection. This finding suggests that cerato-platanin-encoding genes evolved with the development of fungal parasitic characteristics.

The function of snodprot in the cerato-platanin family from Dactylellina cionopaga in nematophagous fungi.

Dactylellina cionopaga is a potential biocontrol agent of phytoparasitic nematodes. Here the functions of snodprot of D. cionopaga were analysed. The gene was transcribed with a higher level under inducing conditions with nematodes. The recombinant protein expressed in Pichia pastoris had a molecular weight of 14 kDa and might form polymers in its native state. In a concentration-dependent manner, snodprot changed the chemotaxis and increased the body-bend frequency of Caenorhabditis elegans, but did not induce immunity in the indicated plants significantly. The results of an immunofluorescence assay proved that snodprot was expressed during the development of traps and conidia. According to the parasitism mechanisms of nematophagous fungi and the chemotaxis and locomotion mechanisms of C. elegans, the possible active sites of snodprot were speculated to be ASE or ASI. The gene identification indicated that snodprot is a novel parasitism-related protein of nematophagous fungi, and possesses novel activity, different from other members of the cerato-platanin family.

Implantable Bioresponsive Hydrogel Prevents Local Recurrence of Breast Cancer by Enhancing Radiosensitivity.

Breast cancer is one of the most common types of cancer. Patients are often concerned about regional recurrence after breast cancer surgery. Radiotherapy plays a vital role in reducing recurrence and prolonging the survival of patients undergoing breast-conserving surgery and high-risk mastectomy. However, 8-15% of patients still have disease progression due to radiation resistance. Therefore, new strategies for combination radiotherapy sensitization must be investigated. In this study, an implantable drug loading system, sunitinib nanoparticles @ matrix metalloproteinases -response hydrogel (NSMRH), uses enzyme-sensitive hydrogel as a carrier to load sunitinib nanoparticles, was identified. The releasing profile demonstrated that sunitinib nanoparticles may be continuously released from the hydrogels. Functional experiments revealed that, when paired with NSMRH, radiation may significantly inhibit tumor cell proliferation, migration, and invasion in vitro. Further animal experiments showed that NSMRH combined with radiotherapy could more effectively control the recurrence of subcutaneous xenograft tumors, prolong the survival time, and have no obvious toxicity in nude mice. Finally, by studying the molecular mechanism of NSMRH, it was hypothesized that in breast cancer cells, NSMRH cooperated with sensitized radiotherapy, mainly due to significantly blocking the G2/M phase, reducing the DNA repair efficiency, inhibiting tumor angiogenesis, promoting apoptosis, and reversing the abnormal expression of platelet-derived growth factor receptor alpha (PDGFRA) after radiotherapy. These findings suggest that NSMRH's radiation sensitization and anti-tumor activity may aid in the development of a novel method in future clinical applications.

Neuregulin 4 alleviates hepatic steatosis via activating AMPK/mTOR-mediated autophagy in aged mice fed a high fat diet.

Neuregulin 4 (Nrg4) is a brown fat-enriched endocrine factor that exerts beneficial metabolic effects on insulin resistance and hepatic steatosis. Autophagy is a mechanism that is essential for preventing hepatic steatosis. The aim of this study was to explore whether Nrg4 ameliorates hepatic steatosis by inducing autophagy. Aged C57BL/6 mice were maintained on a high fat diet with or without Nrg4 intervention for 3 months. Lipid accumulation in the liver was investigated. Autophagy related protein levels along with related signaling pathways that regulate autophagy were evaluated. In addition, the effects of Nrg4 on autophagy were also determined in cultured L-02 cells. Nrg4 decreased high-fat induced intrahepatic lipid content both in vivo and in vitro. Autophagy level in the liver also decreased in obese mice and Nrg4 intervention reactivated autophagy. Further, Nrg4 intervention was found to have activated autophagy via the adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway. Moreover, when the AMPK/mTOR pathway was suppressed or autophagy was inhibited, the beneficial effects of Nrg4 intervention on hepatic steatosis were diminished. These results indicated that Nrg4 intervention attenuated hepatic steatosis by promoting autophagy in the liver of aged obese mice. Additionally, Nrg4 induced autophagy via the AMPK/mTOR signaling pathway.

[Cloning and expression of arom gene of Sclerotinia sclerotiorum].

Arom gene, encoding a single polypeptide that catalyses steps two to six of the aromatic amino acid (phenylalanine, tyrosine and tryptophan) biosynthetic pathway, has been amplified from Scleortinia sclerotiorum genomic DNA by PCR and sequenced. In order to identify the fragment encoding AROM protein experimentally and search a method of obtaining the enzyme in a large amount, the open reading frame of arom gene of S. sclerotiorum was amplified by Pyrobest DNA Polymerase and inserted between Kpn I and Not I sites of the vector pYES2 to construct the expression vector pYES2-arom. The construct was transformed into Saccharomyces cerevisiae H158 by the method of LiAc/ SSDNA/PEG. The rate of transformation was 2 x 10(2)/microg DNA, which was enough for the selection of the positive transformants. PCR using the extracted plasmids as the templates and restriction enzyme analysis of the plasmids extracted from E. coli cells transformed by the above plasmids were performed respectively to screen the positive S. cerevisiae transformants since the copy number of the plasmid in S. cerevisiae was low. Subsequently, the transformant activated by the SC-U medium containing 2% raffinose was inoculated into the SC-U medium containing 2% galactose and the SC-U medium containing 2% glucose respectively to induce and depress the expression of the foreign arom gene. The results of RT-PCR analysis showed: there was not any DNA band in the negative control without the anti-transcriptase, which indicated there was no DNA contamination in the extracted total RNA; there was an expected DNA band in the positive control using the expression vector pYES2-arom as the template, which indicated the used amplification condition was proper; there was not any DNA band in the negative control using total RNA from the depressed transformant as the template, which indicated the DNA bands amplified from total RNA of the induced transformant were not false; there were the expected DNA bands in the samples using total RNA of the transformant induced for 48h, 60h, 72h or 84h as the templates, which indicated the heterogeneous arom gene was transcribed in S. cerevisiae H158 cells. The result of Northern hybridization was consistent with that of RT-PCR, and showed that arom gene of S. sclerotiorum had been transcribed in S. cerevisiae H158 cells when the cells were induced for 48h in the SC-U medium containing 2% galactose at 30 degrees C at 180r/min. 5-enolpyruvylshikimate-3-phosphate synthase activity of the transformant, which was one of AROM protein activities, was measured by estimating the rate of Pi release to check the expressed AROM protein was active or not. The results of enzyme assay in the different culture period indicated that the transformant had 5-enolpyruvylshikimate-3-phosphate synthase activity and the activity reached the peak when the transformant was induced for 72h in SC-U medium at 30 degrees C at 180r/min. The molecular weight of AROM protein is high, it exists in cytoplasm as a dimmer and its expression is controlled by the amounts of amino acids. Therefore, it is very difficult to purify the enzyme. A great lot protein can be obtained by heterogeneous expression. S. cerevisiae expression system has the merits of safe status, authentic posttranslational modification, fast cultivation etc. and usually is the first choice eukaryotic expression system. S. cerevisiae expression system of AROM protein from S. sclerotiorum was successfully constructed for the first time, which provided the basis for the research on the catalysis mechanism of the enzyme and an economical means of simultaneously synthesizing five aromatic amino acid biosynthetic pathway enzymes.