Short Copy Number Variations Potentially Associated with Tonic Immobility Responses in Newly Hatched Chicks

<div><p>Introduction</p><p>Tonic immobility (TI) is fear-induced freezing that animals may undergo when confronted by a threat. It is principally observed in prey species as defence mechanisms. In our preliminary research, we detected large inter-individual variations in the frequency and duration of freezing behavior among newly hatched domestic chicks (<i>Gallus gallus</i>). In this study we aim to identify the copy number variations (CNVs) in the genome of chicks as genetic candidates that underlie the behavioral plasticity to fearful stimuli.</p><p>Methods</p><p>A total of 110 domestic chicks were used for an association study between TI responses and copy number polymorphisms. Array comparative genomic hybridization (aCGH) was conducted between chicks with high and low TI scores using an Agilent 4×180 custom microarray. We specifically focused on 3 genomic regions (>60 Mb) of chromosome 1 where previous quantitative trait loci (QTL) analysis showed significant F-values for fearful responses.</p><p>Results</p><p>ACGH successfully detected short CNVs within the regions overlapping 3 QTL peaks. Eleven of these identified loci were validated by real-time quantitative polymerase chain reaction (qPCR) as copy number polymorphisms. Although there wkas no significant <i>p</i> value in the correlation analysis between TI scores and the relative copy number within each breed, several CNV loci showed significant differences in the relative copy number between 2 breeds of chicken (White Leghorn and Nagoya) which had different quantitative characteristics of fear-induced responses.</p><p>Conclusion</p><p>Our data shows the potential CNVs that may be responsible for innate fear response in domestic chicks.</p></div

Candidate short Copy Number Variations identified by array Comparative Genomic Hybridization and subsequent qPCR validation.

<p><b>Note</b>: Only loci displaying quantitative difference in qPCR validation are shown here.</p

The induction and duration of TI response in each chicken breed/strain.

<p><b>Note</b>: Standard error of the mean (SEM) are shown with the time until righting (sec).</p

Probe coverage on chicken chromosome 1 for array comparative genomic hybridization.

<p>Probes are designed in 3 regions (>60 Mb) where significant <i>F</i>-values have been identified by previous quantitative trait loci analysis. Genome-wide <i>F</i>-values for tonic immobility duration (thick line) and induction attempts (thin line) are quoted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080205#pone.0080205-Schtz1" target="_blank">[7]</a>.</p

Comparison of relative copy number between chicken breeds with different sensitivity to fear.

<p>Relative copy number is calculated as log2^ΔCt, where ΔCt = Ct_β-actin – Ct_target. NG and WL indicate Nagoya and White Leghorn, respectively. The number of samples in each group was; <i>n</i> = 13 (NG; TI induction 1), <i>n</i> = 58 (NG; TI induction 2∼7), <i>n</i> = 13 (WL; TI induction 1), and <i>n</i> = 26 (WL; TI induction 2∼7).</p

Urine concentrations (mean ± SEM µmol/mg creatinine) of amino acids in bottlenose dolphins and mice.

<p>†Levels of carnosine were described as mean ± SEM × 10⁻² µmol/mg creatinine.</p><p>‡Carnosine was only detected in four samples of mouse urine.</p><p>*Significantly different between the bottlenose dolphins and mice (<i>P</i><0.001, LME-associated ANOVA).</p

Hierarchical cluster analysis reveals different plasma aminogram patterns in cetaceans and mice.

<p>Dendrograms obtained from a hierarchical cluster analysis of the plasma aminograms are shown. Each amino acid concentration was normalized before the analysis, and the red and green squares represent high and low relative concentrations, respectively. Ward's linkage method was performed on the Euclidean distances obtained from the standardized aminogram. Briefly, this method seeks to choose successive clustering steps so as to minimize the increase in the error sum of squares found at each level. Bottlenose dolphins (n = 51), pacific white-sided dolphins (n = 16), Risso's dolphins (n = 19), false-killer whales (n = 4), C57BL/6J mice (n = 8) and ICR mice (n = 5) were used for this analysis. Orn, ornithine; Arg, Arginine; Cit, Citrulline, Thr, threonine; Lys, lysine; Ser, serine; Asp, Aspartate; Glu, Glutamate; Tau, Taurine; Gln, Glutamine; Gly, Glycine; Val, Valine; Ile, Isoleucine; Leu, Leucine; Asn, Asparagine; Cys, Cystine; Ala, Alanine; Pro, Proline; Met, Methionine; Tyr, Tyrosine; Trp, Tryptophan; Phe, Phenylalanine; His, Histidine; Cysthi, Cystathionine; 3-MH. 3-methylhistidine; Car, Carnosine.</p

Plasma levels of 3-MH and carnosine are higher in cetaceans.

<p>The plasma levels of 3-MH (A) and carnosine (B) are shown. Individual data points are shown as open circles, and bars indicate the average values. The different letters indicate significant differences (<i>P</i><0.05) among groups as determined by Tukey's test. ND  =  not detected. Bottlenose dolphins (n = 51), pacific white-sided dolphins (n = 16), Risso's dolphins (n = 19), false-killer whales (n = 4), C57BL/6J mice (n = 8) and ICR mice (n = 5) were used for this analysis.</p

Data_Sheet_1_Decreased circulating branched-chain amino acids are associated with development of Alzheimer’s disease in elderly individuals with mild cognitive impairment.docx

BackgroundNutritional epidemiology has shown that inadequate dietary protein intake is associated with poor brain function in the elderly population. The plasma free amino acid (PFAA) profile reflects nutritional status and may have the potential to predict future changes in cognitive function. Here, we report the results of a 2-year interim analysis of a 3-year longitudinal study following mild cognitive impairment (MCI) participants.MethodIn a multicenter prospective cohort design, MCI participants were recruited, and fasting plasma samples were collected. Based on clinical assessment of cognitive function up to 2 years after blood collection, MCI participants were divided into two groups: remained with MCI or reverted to cognitively normal (“MCI-stable,” N = 87) and converted to Alzheimer’s disease (AD) (“AD-convert,” N = 68). The baseline PFAA profile was compared between the two groups. Stratified analysis based on apolipoprotein E ε4 (APOE ε4) allele possession was also conducted.ResultsPlasma concentrations of all nine essential amino acids (EAAs) were lower in the AD-convert group. Among EAAs, three branched-chain amino acids (BCAAs), valine, leucine and isoleucine, and histidine (His) exhibited significant differences even in the logistic regression model adjusted for potential confounding factors such as age, sex, body mass index (BMI), and APOE ε4 possession (p ConclusionThe PFAA profile, especially decreases in BCAAs and His, is associated with development of AD in MCI participants, and the difference was larger in the APOE ε4-negative population, suggesting that the PFAA profile is an independent risk indicator for AD development. Measuring the PFAA profile may have importance in assessing the risk of AD conversion in the MCI population, possibly reflecting nutritional status.Clinical trial registration[https://center6.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000025322], identifier [UMIN000021965].</p