Analysis of the main active ingredients and bioactivities of essential oil from <Emphasis Type="Italic">Osmanthus fragrans</Emphasis> Var. <Emphasis Type="Italic">thunbergii</Emphasis> using a complex network approach

Background

Osmanthus fragrans has been used as folk medicine for thousands of years. The extracts of Osmanthus fragrans flowers were reported to have various bioactivities including free radical scavenging, anti-inflammation, neuroprotection and antitumor effects. However, there is still lack of knowledge about its essential oil.

Methods

In this work, we analyzed the chemical composition of the essential oil from Osmanthus fragrans var. thunbergii by GC-MS. A complex network approach was applied to investigate the interrelationships between the ingredients, target proteins, and related pathways for the essential oil. Statistical characteristics of the networks were further studied to explore the main active ingredients and potential bioactivities of O. fragrans var. thunbergii essential oil.

Results

A total of 44 ingredients were selected from the chemical composition of O. fragrans var. thunbergii essential oil, and that 191 potential target proteins together with 70 pathways were collected for these compounds. An ingredient-target-pathway network was constructed based on these data and showed scale-free property as well as power-law degree distribution. Eugenol and geraniol were screened as main active ingredients with much higher degree values. Potential neuroprotective and anti-tumor effect of the essential oil were also found. A core subnetwork was extracted from the ingredient-target-pathway network, and indicated that eugenol and geraniol contributed most to the neuroprotection of this essential oil. Furthermore, a pathway-based protein association network was built and exhibited small-world property. MAPK1 and MAPK3 were considered as key proteins with highest scores of centrality indices, which might play an important role in the anti-tumor effect of the essential oil.

Conclusions

This work predicted the main active ingredients and bioactivities of O. fragrans var. thunbergii essential oil, which would benefit the development and utilization of Osmanthus fragrans flowers. The application of complex network theory was proved to be effective in bioactivities studies of essential oil. Moreover, it provides a novel strategy for exploring the molecular mechanisms of traditional medicines.

     

Long non-coding RNA H19 regulates E2F1 expression by competitively sponging endogenous miR-29a-3p in clear cell renal cell carcinoma

Background

Numerous recent studies indicate that the long non-coding RNAs (lncRNAs) are frequently abnormal expressed and take critical roles in many cancers. Renal cell carcinoma is the secondary malignant tumors in the urinary system and has high mortality and morbidity. Around 80% of RCCs is clear cell renal cell carcinoma (ccRCC) and is characterized by high metastasis and relapse rate. However, the clinical significances of lncRNAs in ccRCC are still unknown.

Methods

The human cancer lncRNA PCR array (Yingbio) was performed to detect the differentially expressed lncRNAs in human ccRCC samples. Real-time PCR (RT-PCR), dual-luciferase assay, RNA binding protein immunoprecipitation (RIP) assay, transwell assay, CCK-8 assay, and western blot were performed to explore the molecular mechanism of lncRNAs in ccRCC cell migration and invasion.

Results

In this study, lncRNA-H19 was high expressed and negatively correlated with miR-29a-3p in ccRCC. By bioinformatics software, dual-luciferase reporter and RIP assays, we verified that miR-29a-3p was identified as a direct target of lncRNA-H19. RT-PCR and western blot demonstrated that down-regulated lncRNA-H19 could affect the expression of miR-29a-3p targeting E2F1 with competitively binding miR-29a-3p. Furthermore, transwell assays indicated that lncRNA-H19 knockdown inhibited cells migration and invasion, but this effect was attenuated by co-transfection of lncRNA-H19 siRNA and miR-29a-3p inhibitor. Over expression of E2F1 could rescue lncRNA-H19 siRNA induced suppression on cell migration and invasion in ccRCC cells.

Conclusions

These results show a possible competing endogenous RNAs regulatory network involving lncRNA-H19 regulates E2F1 expression by competitively sponging endogenous miR-29a-3p in ccRCC. This mechanism may contribute to a better understanding of ccRCC pathogenesis, and lncRNA-H19 may be further considered as a potential therapeutic target for ccRCC intervention.

     

Loss of nucleoplasmic LAP2alpha-lamin A complexes causes erythroid and epidermal progenitor hyperproliferation

Lamina-associated polypeptide (LAP) 2alpha is a chromatin-associated protein that binds A-type lamins. Mutations in both LAP2alpha and A-type lamins are linked to human diseases called laminopathies, but the molecular mechanisms are poorly understood. The A-type lamin-LAP2alpha complex interacts with and regulates retinoblastoma protein (pRb), but the significance of this interaction in vivo is unknown. Here we address the function of the A-type lamin-LAP2alpha complex with the use of LAP2alpha-deficient mice. We show that LAP2alpha loss causes relocalization of nucleoplasmic A-type lamins to the nuclear envelope and impairs pRb function. This causes inefficient cell-cycle arrest in dense fibroblast cultures and hyperproliferation of epidermal and erythroid progenitor cells in vivo, leading to tissue hyperplasia. Our results support a disease-relevant model in which LAP2alpha defines A-type lamin localization in the nucleoplasm, which in turn affects pRb-mediated regulation of progenitor cell proliferation and differentiation in highly regenerative tissues.     

Protein tyrosine phosphatase SHP2 is involved in Semaphorin 4D-induced axon repulsion

Semaphorin-4D (Sema4D), a member of class 4 membrane-bound Semaphorins, acts as a chemorepellant to the axons of retinal ganglion cells and hippocampal neurons. Plexin-B1, a neuronal Sema4D receptor, associates with either one of receptor tyrosine kinases, c-Met or ErbB2, to mediate Sema4D-signaling. In contrast to this significance, the involvement of protein tyrosine phosphatases in Semaphorin-signaling remains unknown. We here show that Src homology 2-containing protein-tyrosine phosphatase 2 (SHP2) participates in Sema4D-signaling. SHP2 was localized in the growth cones of chick embryonic retinal ganglion neurons. Phenylarsine oxide, a protein tyrosine phosphatase inhibitor, suppressed Sema4D-induced contractile response in COS-7 cells expressing Plexin-B1. Ectopic expression of a phosphatase-inactive mutant of SHP2 in the retinal ganglion cells attenuated Sema4D-induced growth cone collapse response. A SHP1/2 specific inhibitor, 8-hydroxy-7-(6-sulfonaphthalen-2-yl)diazenyl-quinoline-5-sulfonic acid (NSC-87877), also suppressed this collapse response. These results suggest that SHP2-mediated tyrosine dephosphorylation is an important step in Sema4D-induced axon repulsion.     

珍珠黄杨ITS-PCR体系的建立与优化

本研究利用改良CTAB法从珍珠黄杨叶片中提取基因组DNA作为模板,在TaqDNA聚合酶量不变的基础上,利用正交设计L_9(3~4)对4个因素(模板DNA、Mg~(2+)、dNTP和引物)在3个水平上对珍珠黄杨ITS-PCR反应体系进行优化.实验结果表明,在总体积50μL的反应体系中,建立了最佳ITS-PCR扩增条件:Mg~(2+)浓度2.0 mmol/L、引物浓度0.3 μmol/L、dNTP浓度0.3 mmol/L、DNA模板浓度240 ng/50μL、ToqDNA聚合酶的用量1.75 U/50μL和退火温度56℃,该优化体系保证了珍珠黄杨ITS-PCR产物的纯度和质量要求.珍珠黄杨ITS片段克隆测序后获得的序列长度为642 bp,其系统学信息将为珍珠黄杨的起源进化提供有力的分子水平证据.     

Developmental constraints and convergent evolution in Drosophila sex comb formation

SUMMARY The most complex and diverse secondary sexual character in Drosophila is the sex comb (SC), an arrangement of modified bristles on the forelegs of a subclade of male fruit flies. We examined SC formation in six representative nonmodel fruit fly species, in an effort to understand how the variation in comb patterning arises. We first compared SC development in two species with relatively small combs, Drosophila takahashii , where the SCs remain approximately transverse, and Drosophila biarmipes , where two rows of SC teeth rotate and move in an anterior direction relative to other bristle landmarks. We then analyzed comb ontogeny in species with prominent extended SCs parallel to the proximodistal axis, including Drosophila ficusphila and species of the montium subgroup. Our study allowed us to identify two general methods of generating longitudinal combs on the tarsus, and we showed that a montium subgroup species ( Drosophila nikananu ) with a comb convergently similar in size, orientation and position to the model organism Drosophila melanogaster , forms its SC through a different developmental mechanism. We also found that the protein product of the leg patterning gene, dachshund (dac) , is strongly reduced in the SC in all species, but not in other bristles. Our results suggest that an apparent constraint on SC position in the adult may be attributable to at least two different lineage-specific developmental processes, although external forces could also play a role.     

Cell dynamics and developmental bias in the ontogeny of a complex sexually dimorphic trait in Drosophila melanogaster

SUMMARY The Drosophila sex comb (SC) has been hailed as a powerful tool for integrative studies in development, evolution, and behavior, but its ontogeny is poorly understood, even in the model organism Drosophila melanogaster . Using 4D live imaging and other techniques, we carried out a detailed analysis of the cellular events that take place during the development of the SC. We showed that the comb and other contiguous bristle formations assemble from noncontiguous precursor cells, which join together through intercalation. Most of the rotation of the SC (which has a longitudinal orientation in D. melanogaster but is initially transverse) occurs after this stage, when the structure is a single unit. We have provided evidence that male-specific convergent extension through cell rearrangement is responsible for both this rotation and another sexually dimorphic bristle trait. Contiguous bristle formations act as barriers to cell movement within the epithelium, and we demonstrated that a particularly rapid rotation of the proximal region of the comb is associated with the presence of a constricted area between a portion of the SC and a transverse row of contiguous bristle precursors. Our results suggest that the cell dynamics in the neighborhood of the SC may have biased its evolution.     

Genetic Mechanisms in Apc-Mediated Mammary Tumorigenesis

Many components of Wnt/β-catenin signaling pathway also play critical roles in mammary tumor development, yet the role of the tumor suppressor gene APC (adenomatous polyposis coli) in breast oncongenesis is unclear. To better understand the role of Apc in mammary tumorigenesis, we introduced conditional Apc mutations specifically into two different mammary epithelial populations using K14-cre and WAP-cre transgenic mice that express Cre-recombinase in mammary progenitor cells and lactating luminal cells, respectively. Only the K14-cre–mediated Apc heterozygosity developed mammary adenocarcinomas demonstrating histological heterogeneity, suggesting the multilineage progenitor cell origin of these tumors. These tumors harbored truncation mutation in a defined region in the remaining wild-type allele of Apc that would retain some down-regulating activity of β-catenin signaling. Activating mutations at codons 12 and 61 of either H-Ras or K-Ras were also found in a subset of these tumors. Expression profiles of acinar-type mammary tumors from K14-cre; Apc^CKO/+ mice showed luminal epithelial gene expression pattern, and clustering analysis demonstrated more correlation to MMTV-neu model than to MMTV-Wnt1. In contrast, neither WAP-cre–induced Apc heterozygous nor homozygous mutations resulted in predisposition to mammary tumorigenesis, although WAP-cre–mediated Apc deficiency resulted in severe squamous metaplasia of mammary glands. Collectively, our results suggest that not only the epithelial origin but also a certain Apc mutations are selected to achieve a specific level of β-catenin signaling optimal for mammary tumor development and explain partially the colon- but not mammary-specific tumor development in patients that carry germline mutations in APC.     

Immunomodulatory strategies prevent the development of autoimmune emphysema

Background

The presence of anti-endothelial cell antibodies and pathogenic T cells may reflect an autoimmune component in the pathogenesis of emphysema. Whether immune modulatory strategies can protect against the development of emphysema is not known.

Methods

Sprague Dawley rats were immunized with human umbilical vein endothelial cells (HUVEC) to induce autoimmune emphysema and treated with intrathymic HUVEC-injection and pristane. Measurements of alveolar airspace enlargement, cytokine levels, immuno histochemical, western blot analysis, and T cell repertoire of the lung tissue were performed.

Results

The immunomodulatory strategies protected lungs against cell death as demonstrated by reduced numbers of TUNEL and active caspase-3 positive cells and reduced levels of active caspase-3, when compared with lungs from HUVEC-immunized rats. Immunomodulatory strategies also suppressed anti-endothelial antibody production and preserved CNTF, IL-1alpha and VEGF levels. The immune deviation effects of the intrathymic HUVEC-injection were associated with an expansion of CD4+CD25+Foxp3+ regulatory T cells. Pristane treatment decreased the proportion of T cells expressing receptor beta-chain, Vβ16.1 in the lung tissue.

Conclusions

Our data demonstrate that interventions classically employed to induce central T cell tolerance (thymic inoculation of antigen) or to activate innate immune responses (pristane treatment) can prevent the development of autoimmune emphysema.