Deficiency of spns1 exacerbates per- and polyfluoroalkyl substances mediated hepatic toxicity and steatosis in zebrafish (Danio rerio).

Per- and polyfluoroalkyl substances (PFAS) are man-made long-lasting chemical compounds that are found in everyday household items. Today they occur in the environment as a major group of pollutants. These compounds are broadly used in commercial product preparation such as, for food packaging, nonstick coatings, and firefighting foam. In humans, PFAS can cause immune disorders, impaired fetal development, abnormal skeletal tissue development, osteoarthritis, thyroid dysfunctions, cholesterol changes, affect insulin regulation and lipid metabolism, and are also involved in the development of fatty liver disease. In the current study, we investigated the effect of low, but physiologically relevant, concentrations of perfluorooctanoic acid (PFOA), heptafluorobutyric acid (HFBA), and perfluorotetradecanoic acid (PFTA) on gene expression markers of an inflammatory response (tnfa, il-1b, il-6, rplp0, edem1, and dnajc3a), unfolded protein response (UPR) (bip, atf4a, atf6, xbp1, and ddit3), senescence (p21, pai1, smp30, mdm2, and baxa), lipogenesis (scd1, acc, srebp1, pparγ, and fasn) and autophagy (p62, atg3, atg7, rab7, lc3b, and becn1) in AB wild-type (+/+), spns1-wt sibling (+/+), (+/-) and spns1 homozygous mutant (-/-) zebrafish embryos. Exposure to PFOA and HFBA (50 and 100 nM) specifically modulated inflammatory, UPR, senescence, lipogenic, and autophagy signaling in spns1-wt (+/+), (+/-), and spns1-mutant (-/-) zebrafish embryos. Furthermore, PFOA, but not HFBA, upregulated lipogenic-related gene expression and enhanced hepatic steatosis in spns1-wt (+/+), (+/-) zebrafish embryos. Combined exposure to PFOA, HFBA, and PFTA differentially expressed inflammatory, senescence, lipogenic, and autophagy-associated gene expression in spns1-mutant (-/-) zebrafish embryos compared with spns1-wt (+/+), (+/-) and AB-wt (+/+) zebrafish embryos. In addition, chronic exposure (∼2 months) to PFOA (120-600 nM) upregulated the expression of hepatic lipogenic and steatosis biomarkers in AB-wt (+/+) zebrafish. Collectively, our data suggest that acute/chronic physiologically relevant concentrations of PFOA upregulate inflammatory, UPR, senescence, and lipogenic signaling in spns1-wt (+/+), (+/-) and spns1-mutant (-/-) zebrafish embryos as well as in two-month-old AB-wt zebrafish, by targeting autophagy and hence induces toxicity that could promote nonalcoholic fatty liver disease.

Identifying Distal cis-acting Gene-Regulatory Sequences by Expressing BACs Functionalized with loxP-Tn10 Transposons in Zebrafish.

Bacterial Artificial Chromosomes (BACs) are large pieces of DNA from the chromosomes of organisms propagated faithfully in bacteria as large extra-chromosomal plasmids. Expression of genes contained in BACs can be monitored after functionalizing the BAC DNA with reporter genes and other sequences that allow stable maintenance and propagation of the DNA in the new host organism. The DNA in BACs can be altered within its bacterial host in several ways. Here we discuss one such approach, using Tn10 mini-transposons, to introduce exogenous sequences into BACs for a variety of purposes. The largely random insertions of Tn10 transposons carrying lox sites have been used to position mammalian cell-selectable antibiotic resistance genes, enhancer-traps and inverted repeat ends of the vertebrate transposon Tol2 precisely at the ends of the genomic DNA insert in BACs. These modified BACs are suitable for expression in zebrafish or mouse, and have been used to functionally identify important long-range gene regulatory sequences in both species. Enhancer-trapping using BACs should prove uniquely useful in analyzing multiple discontinuous DNA domains that act in concert to regulate expression of a gene, and is not limited by genome accessibility issues of traditional enhancer-trapping methods.

Using BAC transgenesis in zebrafish to identify regulatory sequences of the amyloid precursor protein gene in humans.

BACKGROUND: Non-coding DNA in and around the human Amyloid Precursor Protein (APP) gene that is central to Alzheimer's disease (AD) shares little sequence similarity with that of appb in zebrafish. Identifying DNA domains regulating expression of the gene in such situations becomes a challenge. Taking advantage of the zebrafish system that allows rapid functional analyses of gene regulatory sequences, we previously showed that two discontinuous DNA domains in zebrafish appb are important for expression of the gene in neurons: an enhancer in intron 1 and sequences 28-31 kb upstream of the gene. Here we identify the putative transcription factor binding sites responsible for this distal cis-acting regulation, and use that information to identify a regulatory region of the human APP gene. RESULTS: Functional analyses of intron 1 enhancer mutations in enhancer-trap BACs expressed as transgenes in zebrafish identified putative binding sites of two known transcription factor proteins, E4BP4/ NFIL3 and Forkhead, to be required for expression of appb. A cluster of three E4BP4 sites at -31 kb is also shown to be essential for neuron-specific expression, suggesting that the dependence of expression on upstream sequences is mediated by these E4BP4 sites. E4BP4/ NFIL3 and XFD1 sites in the intron enhancer and E4BP4/ NFIL3 sites at -31 kb specifically and efficiently bind the corresponding zebrafish proteins in vitro. These sites are statistically over-represented in both the zebrafish appb and the human APP genes, although their locations are different. Remarkably, a cluster of four E4BP4 sites in intron 4 of human APP exists in actively transcribing chromatin in a human neuroblastoma cell-line, SHSY5Y, expressing APP as shown using chromatin immunoprecipitation (ChIP) experiments. Thus although the two genes share little sequence conservation, they appear to share the same regulatory logic and are regulated by a similar set of transcription factors. CONCLUSION: The results suggest that the clock-regulated and immune system modulator transcription factor E4BP4/ NFIL3 likely regulates the expression of both appb in zebrafish and APP in humans. It suggests potential human APP gene regulatory pathways, not on the basis of comparing DNA primary sequences with zebrafish appb but on the model of conservation of transcription factors.

Enhanced cortisol response to stress in children in autism.

Children with Autism often show difficulties in adapting to change. Previous studies of cortisol, a neurobiologic stress hormone reflecting hypothalamic-pituitary-adrenal (HPA) axis activity, in children with autism have demonstrated variable results. This study measured cortisol levels in children with and without Autism: (1) at rest; (2) in a novel environment; and (3) in response to a blood draw stressor. A significantly higher serum cortisol response was found in the group of children with autism. Analysis showed significantly higher peak cortisol levels and prolonged duration and recovery of cortisol elevation following the blood-stick stressor in children with autism. This study suggests increased reactivity of the HPA axis to stress and novel stimuli in children with autism.

Twenty-year survival after coronary artery surgery: an institutional perspective from Emory University.

BACKGROUND: Coronary artery bypass graft (CABG) surgery has been performed frequently for symptomatic coronary atherosclerotic heart disease for more than 30 years. However, uncertainty exists regarding the relationship between long-term survival after CABG and readily available clinical correlates of mortality. METHODS AND RESULTS: We studied outcome at 20 years by age, sex, and other variables in 3939 patients who had CABG surgery from 1973 to 1979 in the Emory University System of Healthcare. Twenty-year survival, freedom from myocardial infarction, and freedom from repeat CABG were 35.6% (95% confidence interval [CI], 33.9% to 37.3%), 66.6% (95% CI, 64.6% to 68.6%), and 59.1% (95% CI, 56.9% to 61.5%). Multivariate correlates of late mortality were age (hazard ratio [HR], 1.46 per 10 years), female sex (HR, 1.21), hypertension (HR, 1.44), angina class (HR, 1.07 per class increase of 1), prior CABG (HR, 1.72), ejection fraction (HR, 1.07 per 10-point decrease), number of vessels diseased (HR, 1.11 per 1-vessel increase), and weight (HR, 1.04 per 10 kg). Twenty-year survival by age was 55%, 38%, 22%, and 11% for age <50, 50 to 59, 60 to 69, and >70 years at the time of initial surgery. Survival at 20 years after surgery with and without hypertension was 27% and 41%, respectively. Similarly, 20-year survival was 37% and 29% for men and women. CONCLUSIONS: Symptomatic coronary atherosclerotic heart disease requiring surgical revascularization is progressive with continuing events and mortality. Clinical correlates of mortality significantly impact survival over time and may help identify long-term benefits after CABG.