Microbiome assembly of avian eggshells and their potential as transgenerational carriers of maternal microbiota

The microbiome is essential for development, health and homeostasis throughout an animal's life. Yet, the origins and transmission processes governing animal microbiomes remain elusive for non-human vertebrates, oviparous vertebrates in particular. Eggs may function as transgenerational carriers of the maternal microbiome, warranting characterisation of egg microbiome assembly. Here, we investigated maternal and environmental contributions to avian eggshell microbiota in wild passerine birds: woodlark Lullula arborea and skylark Alauda arvensis. Using 16S rRNA gene sequencing, we demonstrated in both lark species, at the population and within-nest levels, that bacterial communities of freshly laid eggs were distinct from the female cloacal microbiome. Instead, soil-borne bacteria appeared to thrive on freshly laid eggs, and eggshell microbiota composition strongly resembled maternal skin, body feather and nest material communities, sources in direct contact with laid eggs. Finally, phylogenetic structure analysis and microbial source tracking underscored species sorting from directly contacting sources rather than in vivo-transferred symbionts. The female-egg-nest system allowed an integrative assessment of avian egg microbiome assembly, revealing mixed modes of symbiont acquisition not previously documented for vertebrate eggs. Our findings illuminated egg microbiome origins, which suggested a limited potential of eggshells for transgenerational transmission, encouraging further investigation of eggshell microbiome functions in vertebrates

Clitocine reversal of P-glycoprotein associated multi-drug resistance through down-regulation of transcription factor NF-κB in R-HepG2 cell line.

Multidrug resistance (MDR) is one of the major reasons for failure in cancer chemotherapy and its suppression may increase the efficacy of therapy. The human multidrug resistance 1 (MDR1) gene encodes the plasma membrane P-glycoprotein (P-gp) that pumps various anti-cancer agents out of the cancer cell. R-HepG2 and MES-SA/Dx5 cells are doxorubicin induced P-gp over-expressed MDR sublines of human hepatocellular carcinoma HepG2 cells and human uterine carcinoma MES-SA cells respectively. Herein, we observed that clitocine, a natural compound extracted from Leucopaxillus giganteus, presented similar cytotoxicity in multidrug resistant cell lines compared with their parental cell lines and significantly suppressed the expression of P-gp in R-HepG2 and MES-SA/Dx5 cells. Further study showed that the clitocine increased the sensitivity and intracellular accumulation of doxorubicin in R-HepG2 cells accompanying down-regulated MDR1 mRNA level and promoter activity, indicating the reversal effect of MDR by clitocine. A 5'-serial truncation analysis of the MDR1 promoter defined a region from position -450 to -193 to be critical for clitocine suppression of MDR1. Mutation of a consensus NF-κB binding site in the defined region and overexpression of NF-κB p65 could offset the suppression effect of clitocine on MDR1 promoter. By immunohistochemistry, clitocine was confirmed to suppress the protein levels of both P-gp and NF-κB p65 in R-HepG2 cells and tumors. Clitocine also inhibited the expression of NF-κB p65 in MES-SA/Dx5. More importantly, clitocine could suppress the NF-κB activation even in presence of doxorubicin. Taken together; our results suggested that clitocine could reverse P-gp associated MDR via down-regulation of NF-κB

Clitocine inhibits the expression of XIAP and activation of NF-κB in R-HepG2 cells.

<p>(A) After treatment with 0.2 µM clitocine, 1.2 µM doxorubicin or combined agents for 48 h, Western blot was performed to assess the expression of NF-κB p65 and its' target mediator XIAP. (B) After treatment with 0.2 µM clitocine, 1.2 µM doxorubicin or combined for 60 min, nuclear protein was extract and analyzed using ESMA with a double-stranded oligo nucleotide probe containing NF-kB consensus sequence. Excess molar ratio of cold probe (lane 6), anti-p65 subunit of NF-kB antibody (lane 7) and IgG (lane 8) were used for the specificity assay.</p

Reversal effect of clitocine in drug resistant cancer cells.

<p>(A) P-gp was detected in HepG2, R-HepG2, MES-SA and MES-SA/Dx5 cells by Western blot analysis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040720#s2" target="_blank">Materials and Methods</a>. (B) After treatment with different concentrations of clitocine in R-HepG2 cells for 48 h, cell viability was determined by MTT assay. Data are mean ± SD, <i>N</i> = 6. (C) R-HepG2 cells were treated with different concentrations of doxorubicin together with 0, 0.05, 0.1, 0.2 or 0.4 µM clitocine respectively for 48 h. Cell viability was determined by MTT assay. Data are mean ± SD, <i>N</i> = 3. (D) After treatment with different concentrations of clitocine in MES-SA/Dx5 cells for 48 h, cell viability was determined by MTT assay. Data are mean ± SD, <i>N</i> = 3. (E) MES-SA/Dx5 cells were treated with different concentrations of doxorubicin together with 0, 0.05, 0.1, 0.2 or 0.4 µM clitocine respectively for 48 h. Cell viability was determined by MTT assay. Data are mean ± SD, <i>N</i> = 3. (F) Cellular doxorubicin accumulation level in R-HepG2 cells was measured by flow cytometry. The cells were incubated with 2 µM doxorubicin (Dox) alone or together with 0.2 µM clitocine for 24 h. The amount of doxorubicin accumulated in treated cells was quantified by flow cytometric method. (G) MDR1 mRNA level in R-HepG2 cells after clitocine treatment. After treatment with 0.2 µM clitocine for 48 h, total RNA in R-HepG2 cells was extracted. The MDR1 mRNA level in R-HepG2 cells was then measured by qRT-PCR analysis. Data are mean ± SD, <i>N</i> = 6. (H) The relative luciferase activity of full length MDR1 promoter reporter as determined by Dual-Luciferase Reporter Assay System. Cells were transiently transfected with full length MDR1 promoter reporter followed by treatment with 0.2 µM clitocine for 24 h. After that, the cells were lysed and the luciferase activity was measure. Data are mean ± SD, <i>N</i> = 6. ^*<i>P</i><0.05 vs. control, ^**<i>P</i><0.01 vs. control.</p

Clitocine inhibits the expressions of NF-κB p65 and P-gp in R-HepG2 cells <i>in vitro</i> and <i>in vivo</i> tumor tissue from nude mice.

<p>(A) After treatment with 0.2 µM clitocine for 48 h, immunocytochemistry assay was perpormed in R-HepG2 cells using antibodies: NF-κB p65 and P-gp. (B) After intravenous injection of clitocine (10 mg/kg, once a day for one week), tumors were excised from tumor bearing animals. Tumors were fixed with 4% paraformaldehyde in PBS and cut into 6 µm frozen sections and immunocytochemistry assay was performed. The expression levels of these two proteins were observed with a LSM 510 fluorescence microscope fitted with appropriate filters and the images captured with an Orca II CCD camera (Hamamatsu, Bridgewater, NJ). The pixel intensity was analyzed with Image J. A representative experiment was shown.</p

NF-κB mediated the clitocine suppression on MDR1.

<p>(A) P-gp in R-HepG2 cells with BAY-11-7085 (10 µM) treatment was assessed by Western blot assay. After treatment with 10 µM BAY-11-7085 for 48 h, P-gp protein level in R-HepG2 cells was measured by Western blot analysis. (B) The relative luciferase activity of reporter carrying the full length MDR1 promoter was determined by Dual-Luciferase Reporter Assay System. R-HepG2 cells were transiently transfected with full length MDR1 promoter reporter followed by treatment with 10 µM BAY-11-7085 for 24 h. After that the cells were lysed and the luciferase activity measured. Data are mean ± SD, <i>N</i> = 3. ^**<i>P</i><0.01 vs. control. (C) After treatment with 0.2 µM clitocine for 48 h, Western blot assay was performed in R-HepG2 cells. (D) R-HepG2 cells were transiently transfected with pcDNA3-RELA and empty pcDNA3 (negative control) followed by treatment with 0.2 µM clitocine for 48 h. P-gp protein level was measured by western blot analysis. Data are mean ± SD, <i>N</i> = 3. (E) After treatment with 0.2 µM clitocine for 48 h, Western blot assay was performed in R-HepG2, HepG2, MES-SA and MES-SA/Dx5 cells to measure the NF-κB p65 level.</p

Truncation analysis of the MDR1 promoter with clitocine treatment.

<p>(A) The relative luciferase activity of 5′-end truncated MDR1 promoters was detected by Dual-Luciferase Reporter Assay System. R-HepG2 cells were transiently transfected with the truncated MDR1 promoter reporters followed by treatment with 0.2 µM clitocine for 24 h. After that, the cells were lysed and the luciferase activity was measured. Data are mean ± SD, <i>N</i> = 8. (B) Computational analysis of the putative transcription factor binding sites in the full length MDR1 promoter was performed. A single consensus binding site for transcription factor NF-κB was identified in the region −450 to −193. (C) R-HepG2 cells were transiently transfected with wild type (WT) and mutated type (MT) full length (−988∼+525) MDR1 promoter reporter followed by treatment with 0.2 µM clitocine in R-HepG2 cells for 24 h. After that, the cells were lysed and the luciferase activity was measured. Data are mean ± SD, <i>N</i> = 4. ^*<i>P</i><0.05 vs. control. (D) Immunoprecipitaition (CHIP) assay was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040720#s2" target="_blank">Materials and Methods</a>. Chromatin from R-HepG2 cells was cross-linked, sheard and immunoprecipitated with anti-NF-κB p65 antibody. Normal rabbit IgG was included as the negative control (IgG) and the input DNA from fragmented chromatin before immunoprecipitation was used as internal control. The recovered chromatin was subjected to PCR analysis using primers covering the putative NF-κB binding motif of the MDR1 promoter and the PCR products were resolved in 1.5% agarose gel. A representative experiment is shown, and similar results were obtained from three independent experiments.</p

The structure of clitocine.

<p>The structure of clitocine.</p