Molecular responses to toxicological stressors: profiling microRNAs in wild Atlantic salmon (Salmo salar) exposed to acidic aluminum-rich water.

Atlantic salmon (Salmo salar) is among the most sensitive organisms toward acidic, aluminum exposure. Main documented responses to this type of stress are a combination of hypoxia and loss of blood plasma ions. Physiological responses to stress in fish are often grouped into primary, secondary and tertiary responses, where the above mentioned effects are secondary responses, while primary responses include endocrine changes as measurable levels of catecholamines and corticosteroids. In this study we have exposed young (14 months) Atlantic salmon to acid/Al water (pH ≈ 5.6, Al(i) ≈ 80 µg L⁻¹) for 3 days, and obtained clear and consistent decrease of Na⁺ and Cl⁻ ions, and increases of glucose in blood plasma, hematocrit and P(CO2) in blood. We did not measure plasma cortisol (primary response compound), but analyzed effects on microRNA level (miRNA) in muscle tissue, as this may represent initial markers of primary stress responses. miRNAs regulate diverse biological processes, many are evolutionarily conserved, and hundreds have been identified in various animals, although only in a few fish species. We used a novel high-throughput sequencing (RNA-Seq) method to identify miRNAs in Atlantic salmon and specific miRNAs as potential early markers for stress. A total of 18 miRNAs were significantly differentially expressed (FDR<0.1) in exposed compared to control fish, four down-regulated and 14 up-regulated. An unsupervised hierarchical clustering of significant miRNAs revealed two clusters representing exposed and non-exposed individuals. Utilizing the genome of the zebrafish and bioinformatic tools, we identified 224 unique miRNAs in the Atlantic salmon samples sequenced. Additional laboratory studies focusing on function, stress dose-responses and temporal expression of the identified miRNAs will facilitate their use as initial markers for stress responses.

Differential expression of miRNAs in colorectal cancer: comparison of paired tumor tissue and adjacent normal mucosa using high-throughput sequencing.

We present the results of a global study of dysregulated miRNAs in paired samples of normal mucosa and tumor from eight patients with colorectal cancer. Although there is existing data of miRNA contribution to colorectal tumorigenesis, these studies are typically small to medium scale studies of cell lines or non-paired tumor samples. The present study is to our knowledge unique in two respects. Firstly, the normal and adjacent tumor tissue samples are paired, thus taking into account the baseline differences between individuals when testing for differential expression. Secondly, we use high-throughput sequencing, thus enabling a comprehensive survey of all miRNAs expressed in the tissues. We use Illumina sequencing technology to perform sequencing and two different tools to statistically test for differences in read counts per gene between samples: edgeR when using the pair information and DESeq when ignoring this information, i.e., treating tumor and normal samples as independent groups. We identify 37 miRNAs that are significantly dysregulated in both statistical approaches, 19 down-regulated and 18 up-regulated. Some of these miRNAs are previously published as potential regulators in colorectal adenocarcinomas such as miR-1, miR-96 and miR-145. Our comprehensive survey of differentially expressed miRNAs thus confirms some existing findings. We have also discovered 16 dysregulated miRNAs, which to our knowledge have not previously been associated with colorectal carcinogenesis: the following significantly down-regulated miR-490-3p, -628-3p/-5p, -1297, -3151, -3163, -3622a-5p, -3656 and the up-regulated miR-105, -549, -1269, -1827, -3144-3p, -3177, -3180-3p, -4326. Although the study is preliminary with only eight patients included, we believe the results add to the present knowledge on miRNA dysregulation in colorectal carcinogenesis. As such the results would serve as a robust training set for validation of potential biomarkers in a larger cohort study. Finally, we also present data supporting the hypothesis that there are differences in miRNA expression between adenocarcinomas and neuroendocrine tumors of the colon.

Wobble-enhanced ARMS method for detection of KRAS and BRAF mutations.

Monoclonal antibodies targeting the epidermal growth factor receptor have expanded the range of treatment options for patients with metastatic colorectal cancer. However, somatic mutations in the KRAS and BRAF genes have proven to be molecular predictors of resistance to treatment with anti-epidermal growth factor receptor therapy in these patients. Thus, we have developed a sensitive mutation assay, wobble-enhanced amplification refractory mutation system, for detecting the 8 most commonly reported mutations of clinical importance in the KRAS and BRAF genes; KRAS g.34G>C (p.G12R), g.34G>A (p.G12S), g.34G>T (p.G12C), g.35G>A (p.G12D), g.35G>C (p.G12A), g.35G>T (p.G12V), g.38G>A (p.G13D), and BRAF g.1799T>A (p.V600E). A total of 28 candidate setups were designed based on bioinformatics and primer/probe design. Eight candidate setups were thus selected using a synthetic oligonucleotide model. The setups were further validated through several experiments using formalin-fixed paraffin-embedded tissue and cell lines. The results confirm that the wobble-enhanced amplification refractory mutation system method is quick, cost effective, and sensitive. The method is optimized to handle a typical template input of 1 to 20 ng DNA per polymerase chain reaction and can be implemented in any laboratory with ease with a real-time polymerase chain reaction instrument capable of handling TaqMan techonology. The steps used to develop this method can be implemented to design assays for other mutations located in KRAS, BRAF, or other candidate genes.

Airway inflammation in paper mill workers.

Paper mill workers are exposed to culturable microorganisms (MOs). We hypothesized that inflammatory airway response could be detected in sputum of nonsymptomatic workers. From four paper mills, we included 29 healthy nonsmoking men. Workers exposed to high levels of MOs (HMOE, n = 17) were compared with workers exposed to low levels of MO (LMOE, n = 12). A reference group of 22 healthy, nonsmoking, nonexposed (NE) men were also included. We performed differential cell counts of induced sputum, studied gene expressions of isolated sputum macrophages and analyzed inflammatory parameters, including matrix metalloproteinases. Sputum from HMOE workers had a significantly higher percentage of neutrophils than that from LMOE workers (P < 0.05) and NE controls (P < 0.001). There was also an increased gene expression of different pro-inflammatory cytokines, interleukin-6, tumor necrosis factor-alpha, and macrophage inflammatory protein-1beta in isolated airway macrophages and increased levels of total matrix metalloprotease-9 activity in induced sputum from the HMOE group. Our findings indicate that paper industry workers exposed to MOs develop subclinical airway inflammation.