Protective Effect of a Fucose-Rich Fucoidan Isolated from Saccharina japonica against Ultraviolet B-Induced Photodamage In Vitro in Human Keratinocytes and In Vivo in Zebrafish

A fucose-rich fucoidan was purified from brown seaweed Saccharina japonica, of which the UVB protective effect was investigated in vitro in keratinocytes of HaCaT cells and in vivo in zebrafish. The intracellular reactive oxygen species levels and the viability of UVB-irradiated HaCaT cells were determined. The results indicate that the purified fucoidan significantly reduced the intracellular reactive oxygen species levels and improved the viability of UVB-irradiated HaCaT cells. Furthermore, the purified fucoidan remarkably decreased the apoptosis by regulating the expressions of Bax/Bcl-xL and cleaved caspase-3 in UVB-irradiated HaCaT cells in a dose-dependent manner. In addition, the in vivo UV protective effect of the purified fucoidan was investigated using a zebrafish model. It significantly reduced the intracellular reactive oxygen species level, the cell death, the NO production, and the lipid peroxidation in UVB-irradiated zebrafish in a dose-dependent manner. These results suggest that purified fucoidan has a great potential to be developed as a natural anti-UVB agent applied in the cosmetic industry

Targeted sequencing of circulating cell-free DNA in stage II-III resectable oesophageal squamous cell carcinoma patients

Background The aim of this study was to investigate the potential of cell-free DNA (cfDNA) as a disease biomarker in oesophageal squamous cell carcinoma (ESCC) that can be used for treatment response evaluation and early detection of tumour recurrence. Methods Matched tumour tissue, pre- and post-surgery plasma and WBCs obtained from 17 ESCC patients were sequenced using a panel of 483 cancer-related genes. Results Somatic mutations were detected in 14 of 17 tumour tissues. Putative harmful mutations were observed in genes involved in well-known cancer-related pathways, including PI3K-Akt/mTOR signalling, Proteoglycans in cancer, FoxO signalling, Jak-STAT signalling, Chemokine signalling and Focal adhesion. Forty-six somatic mutations were found in pre-surgery cfDNA in 8 of 12 patients, with mutant allele frequencies (MAF) ranging from 0.24 to 4.91%. Three of the 8 patients with detectable circulating tumour DNA (ctDNA) had stage IIA disease, whereas the others had stage IIB-IIIB disease. Post-surgery cfDNA somatic mutations were detected in only 2 of 14 patients, with mutant allele frequencies of 0.28 and 0.36%. All other somatic mutations were undetectable in post-surgery cfDNA, even in samples collected within 3-4 h after surgery. Conclusion Our study shows that somatic mutations can be detected in pre-surgery cfDNA in stage IIA to IIIB patients, and at a lower frequency in post-surgery cfDNA. This indicates that cfDNA could potentially be used to monitor disease load, even in low disease-stage patients

Vitamin D deficiency causes defective resistance to Aspergillus fumigatus in mice via aggravated and sustained inflammation.

BACKGROUND: Vitamin D plays an important role in pulmonary resistance and immunity, and its deficiency has been linked to various respiratory infections. Little is known about the effect of vitamin D deficiency on host pulmonary defense to Aspergillus fumigatus (A. fumigatus). METHODS: Mice raised on vitamin D sufficient or deficient diets were infected intratracheally with A. fumigatus conidia. Mortality, fungal growth, weight loss and lung histology were monitored. Alveolar macrophages (AMs) were stimulated with A. fumigatus conidia in vitro. The kinetics of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6), chemokines (CXCL1, CCL3), and pattern recognition receptors (Toll-like receptor [TLR] 2, TLR 4 and dectin-1) expression in the lungs and AMs were measured. RESULTS: Upon A. fumigatus infection, vitamin D deficient mice showed higher mortality, greater fungal load, and more weight loss than its sufficient counterparts. Vitamin D deficient mice demonstrated aggravated and prolonged histological evidence of lung inflammation as well as enhanced BAL cell counts, dominated by neutrophils after A. fumigatus inoculation. Increased basal levels of pro-inflammatory cytokines in the lungs and AMs from naïve vitamin D deficient mice were observed. Upon A. fumigatus exposure, vitamin D deficiency led to enhanced and sustained expression of TNF-α, IL-1β, IL-6, CXCL1 and CCL3 both in vivo and in vitro. Up-regulation of TLR2, TLR4 and dectin-1was observed in the lungs and AMs from vitamin D deficient mice both at baseline and after A. fumigatus exposure. CONCLUSIONS: Vitamin D deficiency causes defective pulmonary resistance to A. fumigatus in mice, possibly by the enhanced basal expression of pattern recognition receptors and pro-inflammatory cytokines, which induced excessive inflammatory response in response to A. fumigatus challenge

Additional file 5: of Targeted sequencing of circulating cell-free DNA in stage II-III resectable oesophageal squamous cell carcinoma patients

Overview of the pathway analysis by DAVID. All genes with predictive pathogenic mutation were analysed through DAVID. The table shows the KEGG pathways, genes in each pathway and p-values. (XLSX 14 kb

Enhanced and prolonged up-regulation of PRRs in the lungs of <i>A. fumigatus</i> challenged VitD- mice.

<p>Mice were infected intratracheally with 2×10⁷<i>A. fumigatus</i> conidia. At specific time p.i., mice were sacrificed and lungs were excised, and total RNA and protein was extracted. Expression of dectin-1, TLR2 and TLR4 at the mRNA level was quantified by real-time RT-PCR, and protein levels were quantified by Western blotting (shown in middle panel). Representative data from three independent experiments are shown with n = 3 per group (mean±SE). p<0.001(###), p<0.01(##), p<0.05(#), for comparison of <i>A. fumigatus</i>-challenged VitD+/VitD- versus baseline levels; p<0.001(***), p<0.01(**), p<0.05(*), for comparison of VitD+ and VitD- mice using paired Student's T-test.</p

Enhanced pro-inflammatory cytokine and chemokine production in the lungs of <i>A. fumigatus</i>-challenged VitD- mice.

<p>A. Mice were infected intratracheally with 2×10⁷<i>A. fumigatus</i> conidia. At specific times p.i. BALF collected from mice was analyzed for cytokine and chemokine content by ELISA. Data are representative of three independent experiments with n = 5 per group (mean±SE). p<0.001(###), p<0.01(##), p<0.05(#), for comparison of <i>A. fumigatus</i>-challenged VitD+/VitD- mice versus baseline levels; p<0.001(***), p<0.01(**), p<0.05(*), for comparison of VitD+ with VitD- mice using paired Student's T-test. B. AMs isolated from naïve mouse lungs by BAL, were incubated in medium alone or in the presence of viable <i>A. fumigatus</i> conidia (MOI = 0.1) <i>in vitro</i>. Total RNA from AMs was extracted at indicated times. Transcript abundance was measured by real-time RT-PCR. Representative data from three independent experiments with n = 3 per group are shown. P<0.001(###), P<0.01(##), P<0.05(#), for comparison of <i>A. fumigatus</i>-challenged VitD+/VitD- AMs versus basal levels; P<0.001(***), P<0.01(**), P<0.05(*), for comparison of VitD+ and VitD- AMs using paired Student's T-test.</p

Additional file 1: of Targeted sequencing of circulating cell-free DNA in stage II-III resectable oesophageal squamous cell carcinoma patients

Overview of the 483 genes present in the Illumina cancer gene panel. (XLSX 14 kb

Histological evidence of aggravated and sustained inflammation in VitD- mice challenged with <i>A. fumigatus</i> conidia.

<p>A. Representative lung sections from VitD+ and VitD- mice three days post intratracheal challenge with 5×10⁷<i>A. fumigatus</i> conidia. Sections were stained with HE (upper, magnification 100x) for analysis of inflammation, and with GMS (lower, magnification 400x) for the detection of conidia and hyphae. B. Representative HE-stained lung sections (magnification 100x) from VitD- and VitD+ mice are shown at the indicated times after intratracheal challenge with 2×10⁷<i>A. fumigatus</i> conidia. C. HPS of serial HE-stained lung sections between 6 h and 7 d after intratracheal challenge with 2×10⁷<i>A. fumigatus</i> conidia. HPS is presented as mean±SE. Data are representative of three independent experiments (n = 3/group). Statistical analysis was performed by a Mann-Whitney rank sum test. p<0.001(***), p<0.01(**), p<0.05(*).</p

Enhanced and prolonged up-regulation of PRRs on <i>A. fumigatus</i>-stimulated VitD- AMs.

<p>A. Expression of surface TLR2, TLR4 and dectin-1 on AMs isolated from VitD- or VitD+ mice. AMs were isolated from mouse lungs by BAL, and stained for CD11c, dectin-1, TLR2 or TLR4 and then analyzed using flow cytometry. The data show the expression of dectin-1, TLR2 and TLR4 on CD11c-positive cells (red line, isotype control; blue line, VitD+ AMs; green line, VitD- AMs). The values shown in the flow cytometry profiles are the mean fluorescence intensity (MFI) indices. Data are representative of three independent experiments with n = 3/group. p<0.01(**), p<0.05(*) using paired Student's T-test. B. mRNA expression of TLR2, TLR4 and dectin-1 in AMs after co-cultured with live <i>A. fumigatus</i> conidia <i>in vitro</i>. AMs were isolated from naïve mouse lungs by BAL, incubated in medium alone or in the presence of viable <i>A. fumigatus</i> conidia (MOI = 0.1). Total RNA from AMs was extracted at indicated times. Transcript abundance was measured by real-time RT-PCR. Representative data from three independent experiments with n = 3 per group are shown. P<0.001(###), P<0.01(##), P<0.05(#), for comparison of A. f.-challenged VitD+/VitD- versus baseline levels; P<0.001(***), P<0.01(**), P<0.05(*), for comparison of VitD+ and VitD- using paired Student's T-test.</p