Multi-tissue proteogenomic analysis for mechanistic toxicology studies in non-model species.

New approach methodologies (NAM), including omics and in vitro approaches, are contributing to the implementation of 3R (reduction, refinement and replacement) strategies in regulatory science and risk assessment. In this study, we present an integrative transcriptomics and proteomics analysis workflow for the validation and revision of complex fish genomes and demonstrate how proteogenomics expression matrices can be used to support multi-level omics data integration in non-model species in vivo and in vitro. Using Atlantic salmon as an example, we constructed proteogenomic databases from publicly available transcriptomic data and in-house generated RNA-Seq and LC-MS/MS data. Our analysis identified ∼80,000 peptides, providing direct evidence of translation for over 40,000 RefSeq structures. The data also highlighted 183 co-located peptide groups that supported a single transcript each, and in each case, either corrected a previous annotation, supported Ensembl annotations not present in RefSeq, or identified novel previously unannotated genes. Proteogenomics data-derived expression matrices revealed distinct profiles for the different tissue types analyzed. Focusing on proteins involved in defense against xenobiotics, we detected distinct expression patterns across different salmon tissues and observed homology in the expression of chemical defense proteins between in vivo and in vitro liver systems. Our study demonstrates the potential of proteogenomic analyses in extending our understanding of complex fish genomes and provides an advanced bioinformatic toolkit to support the further development of NAMs and their application in regulatory science and (eco)toxicological studies of non-model species.

Dietary beauvericin and enniatin B exposure cause different adverse health effects in farmed Atlantic salmon.

The extensive use of plant ingredients in novel aquafeeds have introduced mycotoxins to the farming of seafood. The emerging enniatin B (ENNB) and beauvericin (BEA) mycotoxins have been found in the novel aquafeeds and farmed fish. Little is known about the potential toxicity of ENNs and BEA in farmed fish and their feed-to-organ transfer. Atlantic salmon (Salmo salar) pre-smolt (75.3 ± 8.10 g) were fed four graded levels of spiked chemical pure ENNB or BEA feeds for three months, in triplicate tanks. Organismal adverse health end-point assessment included intestinal function (protein digestibility), disturbed hematology (red blood cell formation), bone formation (spinal deformity), overall energy use (feed utilization), and lipid oxidative status (vitamin E). Both dietary BEA and ENNB had a low (<∼0.01%) transfer to organs (kidney > liver > brain > muscle), with a higher transfer for ENNB compared to BEA. BEA caused a growth reduction combined with a decreased protein digestion and feed conversion rate- ENNB caused a stunted growth, unrelated to feed utilization capacity. In addition, ENNB caused anemia while BEA gave an oxidative stress response. Lower bench-mark dose regression assessment showed that high background levels of ENNB in commercial salmon feed could pose a risk for animal health, but not in the case of BEA.

Prenatal fever and autism risk.

Some studies suggest that prenatal infection increases risk of autism spectrum disorders (ASDs). This study was undertaken in a prospective cohort in Norway to examine whether we could find evidence to support an association of the prenatal occurrence of fever, a common manifestation of infection, with ASD risk. Prospective questionnaires provided maternal exposure data; case status was established from clinical assessments and registry linkages. In a large, prospectively ascertained cohort of pregnant mothers and their offspring, we examined infants born ⩾32 weeks for associations between fever exposure in each trimester and ASD risk using logistic regression. Maternal exposure to second-trimester fever was associated with increased ASD risk, adjusting for presence of fever in other trimesters and confounders (adjusted odds ratio (aOR), 1.40; 95% confidence interval, 1.09-1.79), with a similar, but nonsignificant, point estimate in the first trimester. Risk increased markedly with exposure to three or more fever episodes after 12 weeks' gestation (aOR, 3.12; 1.28-7.63). ASD risk appears to increase with maternal fever, particularly in the second trimester. Risk magnified dose dependently with exposure to multiple fevers after 12 weeks' gestation. Our findings support a role for gestational maternal infection and innate immune responses to infection in the pathogenesis of at least some cases of ASD.

The Autism Birth Cohort: a paradigm for gene-environment-timing research.

The reported prevalence of autism spectrum disorders (ASDs) has increased by 5- to 10-fold over the past 20 years. Whether ASDs are truly more frequent is controversial; nonetheless, the burden is profound in human and economic terms. Although autism is among the most heritable of mental disorders, its pathogenesis remains obscure. Environmental factors are proposed; however, none is implicated. Furthermore, there are no biomarkers to screen for ASD or risk of ASD. The Autism Birth Cohort (ABC) was initiated to analyze gene x environment x timing interactions and enable early diagnosis. It uses a large, unselected birth cohort in which cases are prospectively ascertained through population screening. Samples collected serially through pregnancy and childhood include parental blood, maternal urine, cord blood, milk teeth and rectal swabs. More than 107,000 children are continuously screened through questionnaires, referral, and a national registry. Cases are compared with a control group from the same cohort in a 'nested case-control' design. Early screening and diagnostic assessments and re-assessments are designed to provide a rich view of longitudinal trajectory. Genetic, proteomic, immunologic, metagenomic and microbiological tools will be used to exploit unique biological samples. The ABC is a paradigm for analyzing the role of genetic and environmental factors in complex disorders.

Transcriptional effects on glutathione S-transferases in first feeding Atlantic cod (Gadus morhua) larvae exposed to crude oil.

Polycyclic aromatic hydrocarbons (PAHs) and other oil compounds are known to induce stress and impact health of marine organisms. Water-soluble fractions of oil contain components known to induce glutathione S-transferases (GSTs), one of the major classes of phase II detoxifying enzymes present in essentially all eukaryotic organisms. In this study, the transcriptional responses of six GSTs (GST pi, GST mu, GST omega, GST theta, GSY zeta and GST kappa) were examined in early larvae of Atlantic cod Gadus morhua exposed to five concentrations of dispersed oil (containing oil droplets and water-soluble fraction) and water-soluble fractions (WSF) of oil. When Atlantic cod larvae were exposed to WSF (containing 1.31+/-0.31microg summation PAH/L for 4 days), expression of GSTM3 and GSTO1 was significantly increased, whereas no differences in GST expression were observed in larvae exposed to a corresponding 50% lower amount of dispersed oil (containing 0.36+/-0.10 microg summation PAH/L for 4 days). The study suggest that although the oil clearly had severe negative effects on the larvae (i.e. concentration-dependent lethality and growth reduction), only minor effects on GST transcription could be observed using RNA obtained from pooled whole-larvae homogenates. This result indicates that the expression of these important detoxification enzymes is only moderately inducible at such an early developmental stage either reflecting low tolerance of cod larvae to dispersed oil or alternatively that using whole-larvae homogenates may have masked tissue-specific mRNA induction.

Characterisation of salmon and trout CD8alpha and CD8beta.

The genes and corresponding cDNAs of both alpha and beta chains of the Atlantic salmon (Salmo salar) CD8 molecule have been sequenced and characterized. In addition, the cDNAs for alpha and beta chains of brown trout (Salmo trutta) and for the beta chain in rainbow trout (Oncorhynchus mykiss) have been sequenced. The cDNAs code for signal sequences which are preceded by short 5' UTRs. These are followed by typical immunoglobulin superfamily variable sequences all of which contain two conserved cysteines for the intra-chain disulphide bond. The hinge regions display conserved cysteines for dimerisation and several O-glycosylation motifs for each predicted protein. The domain sharing the highest sequence identity with mammals is the single pass transmembrane domain for all sequences. In salmon, each domain is predominantly coded for by a single exon except the cytoplasmic/3' UTR domains, which are coded for by 3 and 2 exons for the alpha and beta genes, respectively. In the alpha gene, the second cytoplasmic exon may be spliced out to form an alternative shorter transcript which if expressed would exhibit a truncated cytoplasmic tail. A splice variant found for the salmon beta gene introduces a stop codon after only 40 amino acids. Overall amino acid identities between salmonid sequences were higher than 90%, whereas they shared only 15-20% identity with species such as, chicken and human. Analysis of the expression patterns of the two salmon genes using quantitative RT-PCR shows a very high expression in the thymus. This is mirrored by the expression of the TCRalpha gene, which is known to be co-expressed with CD8 on mammalian T cells. This is the first report of a sequence for CD8beta in a teleost and together with the CD8alpha sequence, it encodes the ortholog of the CD8 co-receptor molecule on mammalian T cells.

[Nasal congestion and earache--upper respiratory tract infections in 4-year-old children]. / Tett i nesen og vondt i øret--øvre luftveisinfeksjoner hos fireåringer.

BACKGROUND: Few population-based studies in Norway have addressed upper respiratory infections in children. MATERIAL AND METHODS: A questionnaire concerning health, diseases and living condition was administered to parents of all four-year-old children in Vestfold county (n = 1912). Participation rate was 79%. RESULTS: Close to one out of three of the children suffered from recurrent common cold (four episodes or more) during the last year. One third experienced otitis at least once, and one out of twenty experienced four episodes of otitis or more. Day-care centre attendance was the only significant risk factor for recurrent common cold (odds ratio 1.50; 95% CI 1.18-1.92) or otitis (odds ratio 1.42; 95% CI 1.12-1.77). The odds ratios were essentially unchanged after logistic regression analysis, when mother's education, housing, other children in the family, passive smoking, and history of breastfeeding were included in the analysis. Children with otitis or recurrent common cold consulted a doctor almost ten times as often as other children. INTERPRETATION: Day-care centre attendance has significant influence on the occurrence of upper respiratory infections in four-year-olds, but less influence than reported for younger children. Other environmental differences seem to be of little importance and do not represent any potential for prophylaxis.

[Drug prescription to 4-year-old children in the county of Vestfold]. / Legemiddelforskrivning til fireåringer i Vestfold.

We carried out a study of four-year-old children's general health, diseases and use of medicines over a 12-month period in 1995-96. The study was based on a questionnaire administered in connection with the regular health controls. Questionnaires from 1,912 children were returned, representing 79% of children born during one year in the county. More than half the children had received medicine prescribed by a physician, corresponding to 557 preparations per 1,000 children. Some children had used several types of drugs, with a mean of 1.4 preparations per child. Antibiotics were prescribed to 63% of the children. Compared to what has been found in other studies, the use of prescribed medicine was moderate. More pharmacoepidemiological studies are needed to assess whether the use of medicine in children is at an acceptable level.

[Health status survey in a district with many immigrant children]. / En helsestasjonsundersøkelse i et distrikt med mange innvandrerbarn.

In Norway, prophylactic child health care is organized through local mother and child clinics, each of which serves a given population. This study summarizes the findings from the routine check-up of four-year-olds in a district with a high proportion of immigrant families. The sample consisted of 70 children, 33 of them from immigrant families. The ordinary check-up was supplemented by information obtained by means of a questionnaire, which was filled in by the public health nurse and the doctor in consultation with the parents. A validated scale, or checklist (BCL), for ordinary behavioural problems among pre-school children was also included. The findings must be interpreted with caution, one reason being the large socio-economic differences between the immigrant and the Norwegian families included in the study. Nevertheless, the difference between the eating habits of the two groups of children was striking, in spite of the emphasis placed on diet at the clinic. Another important finding was the poor knowledge of Norwegian among the immigrant children, which was obviously associated with the poor language skills of the mothers. Therefore, an important element of the preventive health work among immigrant children should be to encourage the mothers to attend courses in Norwegian.

[Infection problems connected to child day care centers]. / Infeksjonsproblemer knyttet til barnehager.

International studies show that children in day care centres have approximately twice as many episodes of infectious diseases as children cared for at home, while, according to most studies, children in family day care experience an intermediate number of infections. After some months in day care the risk of infection decreases. The diseases in question are usually transferred from person to person through close physical contact. Children's general mode of behaviour tends to favour such transmission. The risk of infection in pregnant women on the day care staff and among parents is considered. Finally, the author discusses the potential to control infection in a day care setting. Strict rules for isolation of sick children probably have little effect on the spread of the infections. Good routines for washing hands and for changing nappies are considered to be the most important ways of controlling infection in day care centres.

Clinical and pharmacologic studies with adriamycin-DNA complex in children with malignant disease.

During the last 4 years, we have studied the adriamycin-DNA complex originally developed by Trouet and co-workers (1972). This paper summarizes the results of our pharmacologic and clinical studies. The complex is taken up by cells through an adsorptive pinocytosis, with DNA as the binding molecule. Excess DNA prevents uptake of the drug. Administration of the drug as the complex results in much higher serum concentration and a reduced urinary excretion. The complex is well tolerated, but side effects are probably of the same order as those seen with the free drug. An exception may be the heart. The acute toxicity is not seen when infusing the complex. Our experience with 20 children who have received more than 500 mg/m2 indicates that the chronic cardiac toxicity may be reduced, too. Spectacular, but anecdotal, results have been observed in a variety of solid tumors. Of 16 children with acute myelogenous leukemia, 14 went into a complete remission on a protocol of cytosine arabinoside in combination with the complex. Three of these children are now off therapy, with the longest observation period being 4 years and 4 months.

A pharmacokinetic evaluation of free and DNA-complexed adriamycin: a preliminary study in children with malignant disease.

The pharmacokinetics of adriamycin given as free and in DNA-complexed form was compared in six children with malignant disease. The two types of adriamycin were given to the same child at 3--4-week intervals, thereby excluding genetic variations when comparing the results. Plasma and urine were collected during and after the drug infusion, and the drug concentrations were measured by means of a sensitive fluorimetric procedure. The study shows that one obtains: 1. a much higher plasma concentration of adriamycin when it is given in the complexed form. 2. a lower urine excretion of adriamycin and fluorescent metabolites when adriamycin is administered as the DNA-complex.