Pia Kiilerich

Metagenomic analysis of faecal microbiome as a tool towards targeted non-invasive biomarkers for colorectal cancer

Objective To evaluate the potential for diagnosing colorectal cancer (CRC) from faecal metagenomes. Design We performed metagenome-wide association studies on faecal samples from 74 patients with CRC and 54 controls from China, and validated the results in 16 patients and 24 controls from Denmark. We further validated the biomarkers in two published cohorts from France and Austria. Finally, we employed targeted quantitative PCR (qPCR) assays to evaluate diagnostic potential of selected biomarkers in an independent Chinese cohort of 47 patients and 109 controls. Results Besides confirming known associations of Fusobacterium nucleatum and Peptostreptococcus stomatis with CRC, we found significant associations with several species, including Parvimonas micra and Solobacterium moorei. We identified 20 microbial gene markers that differentiated CRC and control microbiomes, and validated 4 markers in the Danish cohort. In the French and Austrian cohorts, these four genes distinguished CRC metagenomes from controls with areas under the receiver-operating curve (AUC) of 0.72 and 0.77, respectively. qPCR measurements of two of these genes accurately classified patients with CRC in the independent Chinese cohort with AUC=0.84 and OR of 23. These genes were enriched in early-stage (I–II) patient microbiomes, highlighting the potential for using faecal metagenomic biomarkers for early diagnosis of CRC. Conclusions We present the first metagenomic profiling study of CRC faecal microbiomes to discover and validate microbial biomarkers in ethnically different cohorts, and to independently validate selected biomarkers using an affordable clinically relevant technology. Our study thus takes a step further towards affordable non-invasive early diagnostic biomarkers for CRC from faecal samples.

Multiplicity of expression of Na+,K+–ATPaseα-subunit isoforms in the gill of Atlantic salmon (Salmosalar): cellular localisation and absolute quantification in response tosalinity change

SUMMARY The ability to reverse the net direction of gill ion transport in response to a salinity change is critical for euryhaline teleosts and involves a complex cellular and molecular remodelling of the gill epithelium. The present study aimed to clarify the cellular localisation and exact quantitative inter-relationship of Na+,K+–ATPase α- andβ -subunit transcripts in Atlantic salmon gill during salinity change. The combined expression level of all α-isoforms in the gill increased by 100% after freshwater (FW) to seawater (SW) transfer. The α1a and α1b isoforms were both in the range 1–6 amol 20 ng–1 total RNA; α1a decreased andα 1b increased after SW-transfer, their ratio changing from 5:1 in FW to 0.26:1 in SW. The α1c and α3 levels were 10- and 100-fold lower, respectively. Theβ 1-subunit mRNA level was 0.1–0.3 amol 20 ng–1 total RNA, thus much lower than the sum ofα -subunits. Even though increasing 3-fold after SW-transfer,β -subunit availability may still limit functional pump synthesis. The mRNAs of the predominant α1a and α1b isoforms were localised by in situ hybridisation in specific gill cells of both FW and SW salmon. Labelling occurred mainly in presumed chloride cells and cells deep in the filament but occasionally also on lamellae. Overall, the salinity-induced variation in labelling pattern and intensity matched the quantification data. In conclusion, the predominant switching of Na+,K+–ATPase α-subunit isoform mRNA during salinity acclimation reflects a marked remodelling of mitochondrion-rich cells (MRCs) in the gill and probably tuning of the pump performance to accomplish a net reversal of gill ion transport in hypo- and hypertonic environments.

Chronic Trichuris muris Infection Decreases Diversity of the Intestinal Microbiota and Concomitantly Increases the Abundance of Lactobacilli.

The intestinal microbiota is vital for shaping the local intestinal environment as well as host immunity and metabolism. At the same time, epidemiological and experimental evidence suggest an important role for parasitic worm infections in maintaining the inflammatory and regulatory balance of the immune system. In line with this, the prevalence of persistent worm infections is inversely correlated with the incidence of immune-associated diseases, prompting the use of controlled parasite infections for therapeutic purposes. Despite this, the impact of parasite infection on the intestinal microbiota, as well as potential downstream effects on the immune system, remain largely unknown. We have assessed the influence of chronic infection with the large-intestinal nematode Trichuris muris, a close relative of the human pathogen Trichuris trichiura, on the composition of the murine intestinal microbiota by 16S ribosomal-RNA gene-based sequencing. Our results demonstrate that persistent T. muris infection dramatically affects the large-intestinal microbiota, most notably with a drop in the diversity of bacterial communities, as well as a marked increase in the relative abundance of the Lactobacillus genus. In parallel, chronic T. muris infection resulted in a significant shift in the balance between regulatory and inflammatory T cells in the intestinal adaptive immune system, in favour of inflammatory cells. Together, these data demonstrate that chronic parasite infection strongly influences the intestinal microbiota and the adaptive immune system. Our results illustrate the complex interactions between these factors in the intestinal tract, and contribute to furthering the understanding of this interplay, which is of crucial importance considering that 500 million people globally are suffering from these infections and their potential use for therapeutic purposes.

Cortisol regulation of ion transporter mRNA in Atlantic salmon gill and the effect of salinity on the signaling pathway

Based on real-time RT-PCR, analysis of transcripts of selected ion-regulatory proteins (Na(+), K(+)-ATPase alpha1a and alpha1b subunit, Na(+), K(+), 2Cl(-) cotransporter, cystic fibrosis transmembrane conductance regulator (CFTR), and H(+)-ATPase B-subunit), the regulatory role of cortisol and the associated receptor signaling pathway (glucocorticoid (GR) versus mineralocorticoid (MR)) of cortisol was investigated in the salmon gill. Using a gill organ culture technique, the effect of cortisol with and without addition of specific hormone receptor antagonists (RU486 and spironolactone) was analyzed in gills from freshwater (FW) and seawater (SW) acclimated fish. The effect of cortisol was highly dependent on acclimation to salinity. In FW, cortisol stimulated the transcript levels of CFTR-I and Na(+), K(+)-ATPase alpha1a and alpha1b. Addition of RU486 totally abolished the cortisol effects on CFTR-I and Na(+), K(+)-ATPase alpha1b, suggesting that signaling was mediated via GR. Interestingly, both spironolactone and RU486 were able to inhibit the cortisol effect on Na(+), K(+)-ATPase alpha1a indicating a role for both MR and GR in regulation of this target gene. In SW, cortisol increased the transcript levels of CFTR-I, CFTR-II, Na(+), K(+)-ATPase alpha1a and alpha1b, and NKCC. Interestingly, the effect of cortisol on CFTR-I and Na(+), K(+)-ATPase alpha1a was mediated through GR and MR respectively, while both GR and MR signaling were required in the regulation of CFTR-II and Na(+), K(+)-ATPase alpha1b. In FW gills, GR1 and MR transcript levels were not significantly affected by cortisol. In SW gills, GR1 and MR transcripts were downregulated by cortisol; GR1 was regulated via the MR and MR regulation was mediated via GR.

Branchial Expression Patterns of Claudin Isoforms in Atlantic Salmon During Seawater Acclimation and Smoltification

In euryhaline teleosts, permeability changes in gill epithelia are essential during acclimation to changed salinity. This study examined expression patterns of branchial tight junction proteins called claudins, which are important determinants of ion selectivity and general permeability in epithelia. We identified Atlantic salmon genes belonging to the claudin family by screening expressed sequence tag libraries available at NCBI, and classification was performed with the aid of maximum likelihood analysis. In gill libraries, five isoforms (10e, 27a, 28a, 28b, and 30) were present, and quantitative PCR analysis confirmed tissue-specific expression in gill when compared with kidney, intestine, heart, muscle, brain, and liver. Expression patterns during acclimation of freshwater salmon to seawater (SW) and during the smoltification process were examined. Acclimation to SW reduced the expression of claudin 27a and claudin 30 but had no overall effect on claudin 28a and claudin 28b. In contrast, SW induced a fourfold increase in expression of claudin 10e. In accord, a peak in branchial claudin 10e was observed during smoltification in May, coinciding with optimal SW tolerance. Smoltification induced no significant changes in expression of the other isoforms. This study demonstrates the expression of an array of salmon claudin isoforms and shows that SW acclimation involves inverse regulation, in the gill, of claudin 10e vs. claudin 27a and 30. It is possible that claudin 10e is an important component of cation selective channels, whereas reduction in claudin 27a and 30 may change permeability conditions in favor of the ion secretory mode of the SW gill.

Osmoregulation and expression of ion transport proteins and putative claudins in the gill of Southern Flounder (Paralichthys lethostigma)

The southern flounder is a euryhaline teleost that inhabits ocean, estuarine, and riverine environments. We investigated the osmoregulatory strategy of juvenile flounder by examining the time-course of homeostatic responses, hormone levels, and gill Na(+),K(+)-ATPase and Na(+),K(+),2Cl(-) cotransporter protein expression after salinity challenge. Transfer of freshwater (FW)-acclimated flounder to sea water (SW) induced an increase in plasma osmolality and cortisol and a decrease in muscle water content, plasma insulin-like growth factor I (IGF-I) and hepatic IGF-I mRNA, all returning to control levels after 4 days. Gill Na(+),K(+)-ATPase and Na(+),K(+),2Cl(-) cotransporter protein levels were elevated in response to SW after 4 days. Transfer of SW-acclimated flounder to FW reduced gill Na(+),K(+)-ATPase and Na(+),K(+),2Cl(-) cotransporter protein, increased plasma IGF-I, but did not alter hepatic IGF-I mRNA or plasma cortisol levels. Gill claudin-3 and claudin-4 immunoreactive proteins were elevated in FW versus SW acclimated flounder. The study demonstrates that successful acclimation of southern flounder to SW or FW occurs after an initial crisis period and that the salinity adaptation process is associated with changes in branchial expression of ion transport and putative tight junction claudin proteins known to regulate epithelial permeability in mammalian vertebrates.

A reference gene catalogue of the pig gut microbiome

The pig is a major species for livestock production and is also extensively used as the preferred model species for analyses of a wide range of human physiological functions and diseases1. The importance of the gut microbiota in complementing the physiology and genome of the host is now well recognized2. Knowledge of the functional interplay between the gut microbiota and host physiology in humans has been advanced by the human gut reference catalogue3,4. Thus, establishment of a comprehensive pig gut microbiome gene reference catalogue constitutes a logical continuation of the recently published pig genome5. By deep metagenome sequencing of faecal DNA from 287 pigs, we identified 7.7 million non-redundant genes representing 719 metagenomic species. Of the functional pathways found in the human catalogue, 96% are present in the pig catalogue, supporting the potential use of pigs for biomedical research. We show that sex, age and host genetics are likely to influence the pig gut microbiome. Analysis of the prevalence of antibiotic resistance genes demonstrated the effect of eliminating antibiotics from animal diets and thereby reducing the risk of spreading antibiotic resistance associated with farming systems.

ACBP--a PPAR and SREBP modulated housekeeping gene.

The acyl-CoA binding protein (ACBP) is a 10 kD intracellular lipid binding protein that binds and transports acyl-CoA esters. The protein is expressed in most cell types at low levels; however, expression differs markedly between different cell types with expression being particularly high in e.g. cells with a high turnover of fatty acids. We show here that the relatively high basal promoter activity of the rat ACBP gene in fibroblasts and hepatoma cells relies on sequences between −331 to −182 and on the Sp1 and NF-Y sites at −172 and −143, respectively. The basal transcription is modulated by members of the PPAR and SREBP families. In adipocytes, PPARγ is in part responsible for the induction during adipocyte differentiation, but other transcription factors appear to play a role as well. In hepatocytes, SREBP-1c is the main regulator of ACBP in response to changes in insulin levels during fasting/refeeding. PPARα counteracts this effect by stimulating ACBP expression during fasting. In addition, PPARα mediates the induction of ACBP expression in response to peroxisome proliferators. PPARα and PPARγ do not require sequences upstream of −182 for transactivation; however, SREBP-1c requires the synergistic action of sequences in intron 1 for transactivation of the ACBP promoter.

Implication of the mineralocorticoid axis in rainbow trout osmoregulation during salinity acclimation

Cortisol and glucocorticoid receptors (GRs) play an important role in fish osmoregulation, whereas the involvement of the mineralocorticoid receptor (MR) and its putative ligand 11-deoxycorticosterone (DOC) is poorly investigated. In this study, we assessed the implication of DOC and MR in rainbow trout (Oncorhynchus mykiss) osmoregulation during hypo- and hypersaline acclimation in parallel with the cortisol-GR system. A RIA for DOC was developed to measure plasma DOC levels, and a MR-specific antibody was developed to localize MR protein in the gill, intestine, and kidney. This is the first study to report DOC plasma levels during salinity change and MR localization in fish osmoregulatory tissue. Corticosteroid receptor mRNA abundance was investigated in osmoregulatory tissue during salinity acclimation, and the effect of cortisol and DOC on ionic transporters gene expression was assayed using an in vitro gill incubation method. Differential tissue-, salinity-, and time-dependent changes in MR mRNA levels during both hyper- and hyposaline acclimations and the ubiquitous localization of MR in osmoregulatory tissue suggest a role for the MR in osmoregulation. Presumably, DOC does not act as ligand for MR in osmoregulation because there were no changes in plasma DOC levels during either freshwater-seawater (FW-SW) or SW-FW acclimation or any effect of DOC on gill ionic transporter mRNA levels in the gill. Taken together, these results suggest a role for MR, but not for DOC, in osmoregulation and confirm the importance of cortisol as a major endocrine regulator of trout osmoregulation.

Differential effects of cortisol and 11-deoxycorticosterone on ion transport protein mRNA levels in gills of two euryhaline teleosts, Mozambique tilapia (Oreochromis mossambicus) and striped bass (Morone saxatilis).

The role of cortisol as the only corticosteroid in fish osmoregulation has recently been challenged with the discovery of a mineralocorticoid-like hormone, 11-deoxycorticosterone (DOC), and necessitates new studies of the endocrinology of osmoregulation in fish. Using an in vitro gill explant incubation approach, DOC-mediated regulation of selected osmoregulatory target genes in the gill was investigated and compared with that of cortisol in two euryhaline teleosts, Mozambique tilapia (Oreochromis mossambicus) and striped bass (Morone saxatilis). The effects were tested in gills from both fresh water (FW)- and seawater (SW)-acclimated fish. Both cortisol and DOC caused an up-regulation of the Na(+),K(+)-ATPase α1 subunit in SW-acclimated tilapia but had no effect in FW-acclimated fish. Cortisol conferred an increase in Na(+),K(+),2Cl(-) cotransporter (NKCC) isoform 1a transcript levels in FW- and SW-acclimated tilapia, whereas DOC had a stimulatory effect only in SW-acclimated fish. Cortisol had no effect on NKCC isoform 1b mRNA levels at both salinities, while DOC stimulated this isoform in SW-acclimated fish. In striped bass, cortisol conferred an up-regulation of Na(+),K(+)-ATPase α1 and NKCC transcript levels in FW- and SW-acclimated fish, whereas DOC resulted in down-regulation of these transcripts in FW-acclimated fish. It was also found that both corticosteroids may rapidly (30 min) alter the mitogen-activated protein kinase signalling pathway in gill, inducing phosphorylation of extracellular signal-regulated kinase 1 (ERK1) and ERK2 in a salinity-dependent manner. The study shows a disparate organisation of corticosteroid signalling mechanisms involved in ion regulation in the two species and adds new evidence to a role of DOC as a mineralocorticoid hormone in teleosts.