Biocontrol and Action Mechanism of Bacillus subtilis Lipopeptides' Fengycins Against Alternaria solani in Potato as Assessed by a Transcriptome Analysis.

Alternaria solani is an airborne fungus and the primary causal agent of potato early blight worldwide. No available fungicides that are both effective and environmentally friendly are usable to control this fungus. Therefore, biological control is a potential approach for its suppression. In this study, Bacillus subtilis strain ZD01's fermentation broth strongly reduced A. solani pathogenicity under greenhouse conditions. The effects of strain ZD01's secondary metabolites on A. solani were investigated. The exposure of A. solani hyphae to the supernatant resulted in swelling and swollen sacs, and the ZD01 supernatant reduced A. solani conidial germination significantly. Matrix-assisted laser desorption/ionization time of flight mass spectrometry and pure product tests revealed that fengycins were the main antifungal lipopeptide substances. To elucidate the molecular mechanism of the fengycins' biological control, RNA sequencing analyses were performed. A transcriptome analysis revealed that 304 and 522 genes in A. solani were differentially expressed after 2-h and 6-h fengycin treatments, respectively. These genes were respectively mapped to 53 and 57 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. In addition, the most enriched KEGG pathway analysis indicated that the inhibitory mechanisms of fengycins against A. solani regulated the expression of genes related to cell wall, cell membrane, transport, energy process, protein synthesis and genetic information. In particular, cell wall and cell membrane metabolism were the main processes affected by fengycin stress. Scanning and transmission electron microscope results revealed hyphal enlargement and a wide range of abnormalities in A. solani cells after exposure to fengycins. Furthermore, fengycins induced chitin synthesis in treated cells, and also caused the capture of cellular fluorescent green labeling and the release of adenosine triphosphate (ATP) from outer membranes of A. solani cells, which may enhance the fengycins ability to alter cell membrane permeability. Thus, this study increases the transcriptome data resources available and supplies a molecular framework for B. subtilis ZD01 inhibition of A. solani HWC-168 through various mechanisms, especially damaging A. solani cell walls and membranes. The transcriptomic insights may lead to an effective control strategy for potato early blight.

Toxicity mechanism of Rhododendri Mollis Flos: based on serum metabolomics and network toxicology / 中国中药杂志

This study aims to explore the toxicity mechanism of Rhododendri Mollis Flos(RMF) based on serum metabolomics and network toxicology. The toxic effect of RMF on normal rats was evaluated according to the symptoms, serum biochemical indexes, and histopathology. Serum metabolomics was combined with multivariate statistical analysis to search endogenous differential metabolites and related metabolic pathways. The toxic components, targets, and signaling pathways of RMF were screened by network toxicology technique, and the component-target-metabolite-metabolic pathway network was established with the help of serum metabolomics. The result suggested the neurotoxicity, hepatotoxicity, and cardiotoxicity of RMF. A total of 31 differential metabolites and 10 main metabolic pathways were identified by serum metabolomics, and 11 toxic components, 332 related target genes and 141 main signaling pathways were screened out by network toxicology. Further analysis yielded 7 key toxic components: grayanotoxin Ⅲ,grayanotoxinⅠ, rhodojaponin Ⅱ, rhodojaponin Ⅴ, rhodojaponin Ⅵ, rhodojaponin Ⅶ, and kalmanol, which acted on the following 12 key targets: androgen receptor(AR), albumin(ALB), estrogen receptor β(ESR2), sex-hormone binding globulin(SHBG), type 11 hydroxysteroid(17-beta) dehydrogenase(HSD17 B11), estrogen receptor α(ESR1), retinoic X receptor-gamma(RXRG), lactate dehydrogenase type C(LDHC), Aldo-keto reductase(AKR) 1 C family member 3(AKR1 C3), ATP binding cassette subfamily B member 1(ABCB1), UDP-glucuronosyltransferase 2 B7(UGT2 B7), and glutamate-ammonia ligase(GLUL). These targets interfered with the metabolism of gamma-aminobutyric acid, estriol, testosterone, retinoic acid, 2-oxobutyric acid, and affected 4 key metabolic pathways of alanine, aspartate and glutamate metabolism, cysteine and methionine metabolism, steroid hormone biosynthesis, and retinol metabolism. RMF exerts toxic effect on multiple systems through multiple components, targets, and pathways. Through the analysis of key toxic components, target genes, metabolites, and metabolic pathways, this study unveiled the mechanism of potential neurotoxicity, cardiotoxicity, and hepatotoxicity of RMF, which is expected to provide a clue for the basic research on toxic Chinese medicinals.

Mechanism of a Volatile Organic Compound (6-Methyl-2-Heptanone) Emitted From Bacillus subtilis ZD01 Against Alternaria solani in Potato.

The antagonistic mechanisms of soluble non-volatile bioactive compounds, such as proteins and lipopeptides emitted from Bacillus have been widely studied. However, there are limited studies on the antifungal mechanisms of volatile organic compounds (VOCs) produced by Bacillus against plant fungal diseases. In this study, the antagonistic mechanisms of one specific VOC, 6-methyl-2-heptanone, against Alternaria solani were investigated. To optimize the extraction conditions of headspace solid-phase microextraction, a 50/30-µm divinylbenzene/carboxen/polydimethylsiloxane fiber at 50°C for 40 min was used. For gas chromatography-mass spectrometry using a free fatty acid phase capillary column, 6-methyl-2-heptanone accounted for the highest content, at 22.27%, of the total VOCs from Bacillus subtilis ZD01, which inhibited A. solani mycelial growth strongly in vitro. Therefore, 6-methyl-2-heptanone was selected as the main active chemical to elucidate the action mechanisms against A. solani. Scanning and transmission electron microscopy analyses revealed that after exposure to an EC50 dose of 6-methyl-2-heptanone, A. solani hyphal cells had a wide range of abnormalities. 6-Methyl-2-heptanone also caused the capture of cellular fluorescent green label and the release of adenosine triphosphate (ATP) from outer membranes A. solani cells, which may enhance 6-methyl-2-heptanone ability to reach the cytoplasmic membrane. In addition, 6-methyl-2-heptanone showed strong inhibitory effect on A. solani conidial germination. It also damaged conidial internal structures, with the treated group having collapsed shrunken small vesicles as observed by transmission electron microscopy. Because 6-methyl-2-heptanone showed strong effects on mycelial integrity and conidial structure, the expression levels of related pathogenic genes in A. solani treated with 6-methyl-2-heptanone were investigated. The qRT-PCR results showed that transcriptional expression levels of slt2 and wetA genes were strongly down-regulated after exposure to 6-methyl-2-heptanone. Finally, because identifying the functions of pathogenic genes will be important for the biological control of A. solani, the wetA gene was identified as a conidia-associated gene that plays roles in regulating sporulation yield and conidial maturation. These findings provide further insights into the mechanisms of VOCs secreted by Bacillus against A. solani.

Optimization of One-step Pelletization Technology of Rouganbao Granules with Orthogonal Test Methodology / 中国中医药信息杂志

Objective To optimize the process conditions of one-step pelletization of Rouganbao Granules. Methods The factors influencing the pelletization of Rouganbao Granules were investigated by L9(34)orthogonal test, with the indexes of forming rate and fluidity. Results The spraying speed had the greatest effect on one-step pelletization, followed by atomization pressure and material temperature, and wind temperature had the smallest effect. At last, the best process parameters were relative density 1.15 (60 ℃), spray speed 55 mL/min, atomization pressure 0.25 MPa, wind temperature 75 ℃, material temperature 55 ℃, and critical relative humidity was 63%. Conclusion One-step pelletization technology can improve the preparation level and product quality, which can be used for the industrial production of Rouganbao Granules.

Clinical analysis of retroperitoneal laparoscopic renal adipose capsule outer pathway in the treatment of chyluria / 重庆医学

Objective To evaluate the surgical techniques and clinical efficacy of retroperitoneoscopic ligation of renal lymphatic vessels outside adipose capsule and inside adipose capsule for comparison in the treatment of chyluria.Methods Retrospective anal-ysis of the retroperitoneal laparoscopic adipose capsule of kidney pedicle lymphatic duct ligation(group Ⅰ)and adipose capsule pathways in the kidney totally or subtotally free renal pedicle lymphatic ligation(group Ⅱ)and 1 1 1 patients with chyluria clinical data,compared two groups of operation time,bleeding volume,the rate of analgesia,postoperative gastrointestinal function recovery time,drainage time,postoperative recovery time,postoperative hospital stay and postoperative complications and other index differ-ence.Results Retroperitoneal laparoscopic operation group of adipose capsule in operation time,bleeding volume,postoperative an-algesia,postoperative gastrointestinal function recovery time,drainage time,postoperative recovery time,postoperative hospital stay and postoperative complications were better than the adipose capsule of kidney totally or subtotally free operation group,the differ-ence was statistically significant(P<0.05).Conclusion Retroperitoneal laparoscopic renal adipose capsule of kidney pedicle lym-phatic disconnection for the treatment of chyluria effect,and adipose capsule operation ways,the method less trauma,quicker recov-ery,can completely replace the renal adipose capsule total or Sub-total of free kidney pedicle lymphatic disconnection operation,wor-thy of clinical application.

Amelogenin exons 8 and 9 encoded peptide enhances leucine rich amelogenin peptide mediated dental pulp repair.

Amelogenins containing exons 8 and 9 are alternatively spliced variants of amelogenin. Some amelogenin spliced variants have been found to promote pulp regeneration following pulp exposure. The function of the amelogenin spliced variants with the exons 8 and 9 remains unknown. In this study, we synthesized recombinant leucine rich amelogenin peptide (LRAP, A-4), LRAP plus exons 8 and 9 peptide (LRAP 8, 9) or exons 8 and 9 peptide (P89), to determine their effects on odontoblasts. In vivo analyses were completed following the insertion of agarose beads containing LRAP or LRAP 8, 9 into exposed cavity preparations of rat molars. After 8, 15 or 30 days' exposure, the pulp tissues were analyzed for changes in histomorphometry and cell proliferation by PCNA stainings. In vitro analyses included the effects of the addition of the recombinant proteins or peptide on cell proliferation, differentiation and adhesion of postnatal human dental pulp cells (DPCs). These studies showed that in vivo LRAP 8, 9 enhanced the reparative dentin formation as compared to LRAP. In vitro LRAP 8, 9 promoted DPC proliferation and differentiation to a greater extent than LRAP. These data suggest that amelogenin exons 8 and 9 may be useful in amelogenin-mediated pulp repair.

Plexin-B1 is a target of miR-214 in cervical cancer and promotes the growth and invasion of HeLa cells.

Plexin-B1, the receptor for Sema4D, has been reported to trigger multiple and sometimes opposing cellular responses in various types of tumor cells. It has been implicated in the regulation of tumor-cell survival, proliferation, angiogenesis, invasion and metastasis. However, the plexin-B1 gene expression and its regulatory mechanism in cervical cancer remain unclear. The present study shows that plexin-B1 is over-expressed in cervical tumor tissues compared to normal cervical tissues by immunohistochemistry, Western blotting and quantitative RT-PCR. The expression of plexin-B1 is significantly associated with cervical tumor metastasis and invasion according to the analysis of the clinicopathologic data. Plexin-B1 also promotes proliferation, migration and invasion in human cervical cancer HeLa cells. We also found that the plexin-B1 levels are inversely correlated with miR-214 amounts in both cervical cancer tissues and HeLa cells. And miR-214 expression level is also associated with metastasis and invasion of cervical tumor. Furthermore, we demonstrate that plexin-B1 is inhibited by miR-214 through a miR-214 binding site within the 3'UTR of plexin-B1 in HeLa cells. Ectopic expression of miR-214 could inhibit the proliferation capacity, migration and invasion ability of HeLa cells. Our findings suggest that plexin-B1, a target of miR-214, may function as an oncogene in human cervical cancer HeLa cells.