Table_1_Blood Gene Expression and Vascular Function Biomarkers in Professional Saturation Diving.XLSX

<p>Saturation diving is an established way to conduct subsea operations with human intervention. While working, the divers must acclimatize to the hyperbaric environments. In this study, genome-wide gene expression and selected plasma biomarkers for vascular function were investigated. We also examined whether antioxidant vitamin supplements affected the outcome. The study included 20 male professional divers, 13 of whom took vitamin C and E supplements in doses of 1,000 and 30 mg daily during saturation periods that lasted 7–14 days. The dives were done in a heliox atmosphere with 40 kPa oxygen partial pressure (ppO₂) to a depth of 100–115 m of sea-water (msw), from which the divers performed in-water work excursions to a maximum depth of 125 msw with 60 kPa ppO₂. Venous blood was collected immediately before and after saturation. Following gene expression profiling, post-saturation gene activity changes were analyzed. Protein biomarkers for inflammation, endothelial function, and fibrinolysis: Il-6, CRP, ICAM-1, fibrinogen, and PAI-1, were measured in plasma. Post-saturation gene expression changes indicated acclimatization to elevated ppO₂ by extensive downregulation of factors involved in oxygen transport, including heme, hemoglobin, and erythrocytes. Primary endogenous antioxidants; superoxide dismutase 1, catalase, and glutathione synthetase, were upregulated, and there was increased expression of genes involved in immune activity and inflammatory signaling pathways. The antioxidant vitamin supplements had no effect on post-saturation gene expression profiles or vascular function biomarkers, implying that the divers preserved their homeostasis through endogenous antioxidant defenses.</p

Image_1_Physiological hypoxia improves growth and functional differentiation of human intestinal epithelial organoids.tiff

BackgroundThe epithelium in the colonic mucosa is implicated in the pathophysiology of various diseases, including inflammatory bowel diseases and colorectal cancer. Intestinal epithelial organoids from the colon (colonoids) can be used for disease modeling and personalized drug screening. Colonoids are usually cultured at 18-21% oxygen without accounting for the physiological hypoxia in the colonic epithelium (3% to MethodsGrowth from single cells to differentiated colonoids was monitored by brightfield images and evaluated with a linear mixed model. Cell composition was identified by immunofluorescence staining of cell markers and single-cell RNA-sequencing (scRNA-seq). Enrichment analysis was used to identify transcriptomic differences within cell populations. Pro-inflammatory stimuli induced chemokines and Neutrophil gelatinase-associated lipocalin (NGAL) release were analyzed by Multiplex profiling and ELISA. Direct response to a lower oxygen level was analyzed by enrichment analysis of bulk RNA sequencing data.ResultsColonoids established in a 2% oxygen environment acquired a significantly larger cell mass compared to a 20% oxygen environment. No differences in expression of cell markers for cells with proliferation potential (KI67 positive), goblet cells (MUC2 positive), absorptive cells (MUC2 negative, CK20 positive) and enteroendocrine cells (CGA positive) were found between colonoids cultured in 2% and 20% oxygen. However, the scRNA-seq analysis identified differences in the transcriptome within stem-, progenitor- and differentiated cell clusters. Both colonoids grown at 2% and 20% oxygen secreted CXCL2, CXCL5, CXCL10, CXCL12, CX3CL1 and CCL25, and NGAL upon TNF + poly(I:C) treatment, but there appeared to be a tendency towards lower pro-inflammatory response in 2% oxygen. Reducing the oxygen environment from 20% to 2% in differentiated colonoids altered the expression of genes related to differentiation, metabolism, mucus lining, and immune networks.ConclusionsOur results suggest that colonoids studies can and should be performed in physioxia when the resemblance to in vivo conditions is important.</p

Table_1_Physiological hypoxia improves growth and functional differentiation of human intestinal epithelial organoids.xlsx

BackgroundThe epithelium in the colonic mucosa is implicated in the pathophysiology of various diseases, including inflammatory bowel diseases and colorectal cancer. Intestinal epithelial organoids from the colon (colonoids) can be used for disease modeling and personalized drug screening. Colonoids are usually cultured at 18-21% oxygen without accounting for the physiological hypoxia in the colonic epithelium (3% to MethodsGrowth from single cells to differentiated colonoids was monitored by brightfield images and evaluated with a linear mixed model. Cell composition was identified by immunofluorescence staining of cell markers and single-cell RNA-sequencing (scRNA-seq). Enrichment analysis was used to identify transcriptomic differences within cell populations. Pro-inflammatory stimuli induced chemokines and Neutrophil gelatinase-associated lipocalin (NGAL) release were analyzed by Multiplex profiling and ELISA. Direct response to a lower oxygen level was analyzed by enrichment analysis of bulk RNA sequencing data.ResultsColonoids established in a 2% oxygen environment acquired a significantly larger cell mass compared to a 20% oxygen environment. No differences in expression of cell markers for cells with proliferation potential (KI67 positive), goblet cells (MUC2 positive), absorptive cells (MUC2 negative, CK20 positive) and enteroendocrine cells (CGA positive) were found between colonoids cultured in 2% and 20% oxygen. However, the scRNA-seq analysis identified differences in the transcriptome within stem-, progenitor- and differentiated cell clusters. Both colonoids grown at 2% and 20% oxygen secreted CXCL2, CXCL5, CXCL10, CXCL12, CX3CL1 and CCL25, and NGAL upon TNF + poly(I:C) treatment, but there appeared to be a tendency towards lower pro-inflammatory response in 2% oxygen. Reducing the oxygen environment from 20% to 2% in differentiated colonoids altered the expression of genes related to differentiation, metabolism, mucus lining, and immune networks.ConclusionsOur results suggest that colonoids studies can and should be performed in physioxia when the resemblance to in vivo conditions is important.</p

Image_2_Physiological hypoxia improves growth and functional differentiation of human intestinal epithelial organoids.tiff

BackgroundThe epithelium in the colonic mucosa is implicated in the pathophysiology of various diseases, including inflammatory bowel diseases and colorectal cancer. Intestinal epithelial organoids from the colon (colonoids) can be used for disease modeling and personalized drug screening. Colonoids are usually cultured at 18-21% oxygen without accounting for the physiological hypoxia in the colonic epithelium (3% to MethodsGrowth from single cells to differentiated colonoids was monitored by brightfield images and evaluated with a linear mixed model. Cell composition was identified by immunofluorescence staining of cell markers and single-cell RNA-sequencing (scRNA-seq). Enrichment analysis was used to identify transcriptomic differences within cell populations. Pro-inflammatory stimuli induced chemokines and Neutrophil gelatinase-associated lipocalin (NGAL) release were analyzed by Multiplex profiling and ELISA. Direct response to a lower oxygen level was analyzed by enrichment analysis of bulk RNA sequencing data.ResultsColonoids established in a 2% oxygen environment acquired a significantly larger cell mass compared to a 20% oxygen environment. No differences in expression of cell markers for cells with proliferation potential (KI67 positive), goblet cells (MUC2 positive), absorptive cells (MUC2 negative, CK20 positive) and enteroendocrine cells (CGA positive) were found between colonoids cultured in 2% and 20% oxygen. However, the scRNA-seq analysis identified differences in the transcriptome within stem-, progenitor- and differentiated cell clusters. Both colonoids grown at 2% and 20% oxygen secreted CXCL2, CXCL5, CXCL10, CXCL12, CX3CL1 and CCL25, and NGAL upon TNF + poly(I:C) treatment, but there appeared to be a tendency towards lower pro-inflammatory response in 2% oxygen. Reducing the oxygen environment from 20% to 2% in differentiated colonoids altered the expression of genes related to differentiation, metabolism, mucus lining, and immune networks.ConclusionsOur results suggest that colonoids studies can and should be performed in physioxia when the resemblance to in vivo conditions is important.</p

Top 10 overexpressed and down-regulated pathways in post- versus pre-resistance tumour in KEGG pathways map.

<p>Several interesting and important systems were deregulated after resistance. Note that overexpressed genes in GO metabolic process was 89 and in KEGG only 18. The reason is that KEGG includes new genes in a pathway only when several publications have confirmed it, and thus is more conservative but with a high level of evidence. Most pronounced were the metabolism and modification of DNA and RNA through pyrimidine and purine metabolism and aminoacyl-t-RNA biosynthesis. Interestingly, down-regulation of cell adhesion molecules and leukocyte transendothelial migration as well as cytokine-cytokine receptor interaction was pronounced, also an expression characteristic of mesothelioma versus normal pleura.</p

Serum biomarker changes over time.

<p>Ca125 and Mesothelin (SMRP) biomarkers in serum were elevated at diagnosis and decreased and increased according to the clinical and radiological regression and progression respectively. The straight horizontal dashed lines depict the maximum normal values of the two markers. CYFRA 21-1 is not shown.</p

Genes changed more than 2-fold after resistance.

<p>Expression of genes that changed more then 2-fold in the post-resistance tumour, compared to initial tumour. Down-regulated genes are shown to the left (30 in total, two not shown) and the overexpressed to the right (15, three not shown). Green bars represent the relative gene expression before and red bars after acquired resistance.</p

Pathway signature of VEGF and NOTCH mediated EMT in ccRCC.

<p>Comparison of gene expression data from the FFPE and from the RNAlater^® dataset with published results [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149743#pone.0149743.ref020" target="_blank">20</a>] and between themselves. <i>F = FFPE samples</i>, <i>R = RNAlater</i>^<i>®</i> <i>samples</i>, <i>Numbers = fold change of up-regulation (red) or down-regulation (blue)</i>.</p

Characteristic patient features at the time of surgery.

<p>eGFR was calculated with the MDRD formula. The staging was performed based on the EAU Guidelines on renal cell carcinoma: 2014 update [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149743#pone.0149743.ref043" target="_blank">43</a>].</p

Correlation of gene expression data.

<p>The correlation of commonly differentially expressed genes is given with respect to (A) average expression and (B) log2 fold changes.</p