Promoter activity analysis of the bovine <i>PDHB</i> gene.

<p>a. We transferred six serial deletion constructs in pGL3-basic into C2C12 cells. After 5 h we replaced the transfection mixture with DMEM with 5% FBS (myoblasts) or 2% HS (myotubes). b. We transferred the same constructs into 3T3-L1 cells. We normalized relative luciferase activities to Renilla luciferase activity. The transcription factor binding sites of MYOG and C/EBPß are indicated with closed circles and ellipses, respectively. *, P<0.05. Error bars represent the SD (n = 3).</p

Structure characteristics of the bovine <i>PDHB</i> gene.

<p>a. Here we show the genomic, mRNA and protein components in detail. 5’- UTR:5’- untranslated region, 3’- UTR:3’- untranslated region, ORF: open reading frame, Transketpyr: transketolase, pyrimidine binding domain. b. 5’-RACE. Lane 1 and 2 are products of the first and second PCR, respectively. Lane M represents the marker of DL2000. c. 5’-regulatory region sequence of bovine <i>PDHB</i> gene. Arrows mark the transcription initiation sites. The cytosine residue is designated as +1. The transcription factor binding sites are boxed. The primers are underlined with the respective names below the line. The CpG island is indicated with red color.</p

Functional analysis of the mutated MYOG and C/EBPß sites.

<p>We transferred the mutated sites MYOG and C/EBPß into C2C12 myotubes (a) and 3T3-L1 cells (b). **, P<0.01. Error bars represent the SD (n = 3).</p

ChIP assay of MYOG and C/EBPß binding to PDHB promoter in vivo.

<p>We analyzed immunoprecipitated products for MYOG (a) and C/EBPß (b) antibodies via RT-PCR. We analyzed immunoprecipitated products for MYOG (c) and C/EBPß (d) antibodies via ChIP-QPCR. We used total chromatin from muscle (a and c) and fat (b and d) as the input. We used normal mouse IgG as the negative control antibodies. **, P<0.01. Error bars represent the SD (n = 3).</p

Spatial expression analysis of bovine <i>PDHB</i> mRNA.

<p>We normalized the mRNA expression levels of <i>PDHB</i> to those of <i>GAPDH</i>. Error bars represent the standard deviation (SD) (n = 3).</p

Primers utilized in this study.

<p>Primers utilized in this study.</p

Multi-alignments sequence analysis of the core functional promoter of bovine <i>PDHB</i> in relation toother mammals.

<p>The transcription factor binding sites are marked with boxes. The nucleotide sequence is numbered in 5'-regulatory sequence of the bovine <i>PDHB</i> gene (GenBank No. KJ649747).</p

EMSA involving 5’-biotin labeled MYOG and C/EBPß probes.

<p>a. 5’-biotin labeled MYOG probes and nuclear extracts of C2C12 myotubes. Lane 1: MYOG probes; lane 2: MYOG probes with nuclear extracts of C2C12 myotubes; lane 3: MYOG probes and nuclear extracts with a 125-fold unlabeled MYOG oligonucleotides; lane 4: MYOG probes and nuclear extracts with a 125-fold unlabeled mMYOG oligonucleotides. lane 5: MYOG probes and nuclear extracts with myogenin antibodies. b. 5’-biotin labeled C/EBPß probes and nuclear extracts of 3T3-L1 cells. Lane 1: C/EBPß probes; lane 2: C/EBPß probes with nuclear extracts of 3T3-L1 cells; lane 3: C/EBPß probes and nuclear extracts with 125-fold unlabeled C/EBPß oligonucleotides; lane 4: C/EBPß probes and nuclear extracts with 125-fold unlabeled mC/EBPß oligonucleotides. lane 5: C/EBPß probes and nuclear extracts with C/EBPß antibodies.</p

Study of golden pompano (<i>Trachinotus ovatus</i>) freshness forecasting method by utilising Vis/NIR spectroscopy combined with electronic nose

<p>Golden pompano (<i>Trachinotus ovatus</i>) quality forecasting method utilising Vis/NIR spectroscopy combined with electronic nose (EN) was investigated in this article. Responses of Vis/NIR spectroscopy and EN to pompanos stored at 4°C were measured for 6 days. Physical/chemical indexes including texture, total volatile basic nitrogen, pH, total viable counts, and human sensory evaluation were synchronously examined as quality references. Chemometric methods including principal component analysis (PCA) and stochastic resonance (SR) were employed for spectroscopic and EN data analysis. Physicochemical examination demonstrated that fish quality decreased rapidly during storage. PCA qualitatively classified freshness degree of pompano samples, while SR signal-to-noise ratio (SNR) spectrum using SNR maximum quantitatively characterised quality for all samples. Golden pompano quality predictive models were developed based on spectroscopy, EN, and spectroscopy combined with EN, respectively. Results demonstrated that the model developed based on spectroscopy combined with EN presented a forecasting accuracy of 93.3%.</p