MOESM1 of Metabolic engineering of Saccharomyces cerevisiae for efficient production of glucaric acid at high titer

Additional file 1: Table S1. Primers used in this study. Figure S1. MIOX4 activity of the episomal expression plasmid in the wild-type strain and opi1 mutant strain with myo-inositol. All experiments were performed in triplicates and the error bar represented mean Âą standard deviation. Figure S2. myo-inositol residue in shake flask (A) and fed-batch (B) cultures when fed 60 mM (10.8 g/L) myo-inositol to the culture. All experiments were performed in triplicates and the error bar represented mean Âą standard deviation

Distinct and common expression of receptors for inflammatory mediators in vagal nodose versus jugular capsaicin-sensitive/TRPV1-positive neurons detected by low input RNA sequencing

<div><p>Capsaicin-sensitive sensory C-fibers derived from vagal ganglia innervate the visceral organs, and respond to inflammatory mediators and noxious stimuli. These neurons play an important role in maintenance of visceral homeostasis, and contribute to the symptoms of visceral inflammatory diseases. Vagal sensory neurons are located in two ganglia, the jugular ganglia (derived from the neural crest), and the nodose ganglia (from the epibranchial placodes). The functional difference, especially in response to immune mediators, between jugular and nodose neurons is not fully understood. In this study, we microscopically isolated murine nodose and jugular capsaicin-sensitive / <i>Trpv1</i>-expressing C-fiber neurons and performed transcriptome profiling using ultra-low input RNA sequencing. RNAseq detected genes with significantly differential expression in jugular and nodose neurons, which were mostly involved in neural functions. Transcriptional regulators, including <i>Cited1</i>, <i>Hoxb5</i> and <i>Prdm12</i> showed distinct expression patterns in the two C-fiber neuronal populations. Common and specific expression of immune receptor proteins was characterized in each neuronal type. The expression of immune receptors that have received little or no attention from vagal sensory biologists is highlighted including receptors for certain chemokines (CXCLs), interleukins (IL-4) and interferons (IFNα, IFNγ). Stimulation of immune receptors with their cognate ligands led to activation of the C-fibers in isolated functional assays.</p></div

Response of isolated vagal sensory neurons to interferon-α (1000iu/ml) interferon- γ (200 ng/ml) CXCL8 (100ng/ml), and S-1P (10 μM) in a calcium assay.

<p>The vagal neurons were isolated from <i>Pirt-GCaMP3</i> heterozygote mice in which the genetically encoded Ca²⁺ indicator GCaMP3 is expressed under the sensory neuron-specific <i>Pirt</i> promoter. Capsaicin sensitivity was determined at the end of the experiment by adding 1 μM capsaicin. The capsaicin response is shown in the experiments with interferon-α to provide some insight into efficacy of the mediator responses relative to a maximal capsaicin response. Different colors indicate response in different representative neurons (Total of N>100 neurons were tested in each condition). F/F0: The intensity of whole-cell fluorescence for each cell under study as a function of time, normalized to the resting fluorescence.</p

Jugular and nodose C-fiber neurons were isolated based on capsaicin sensitivity and <i>Trpv1</i> expression.

<p>(A) Dissection of right jugular/nodose complex in the mouse. SCG denotes superior cervical ganglion (modified from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185985#pone.0185985.ref012" target="_blank">12</a>]. (B) The rostral part of the jugular/nodose ganglion (JNG) complex is formed by jugular (J) neurons while the caudal part is formed by nodose (N) neurons. The jugular (neural crest-derived) neurons are visualized by X-Gal staining while nodose (placodes-derived) neurons remain unstained in Wnt1Cre/R26R mouse [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185985#pone.0185985.ref012" target="_blank">12</a>]. Neurons isolated from the rostral section of the ganglion (J) were considered jugular neurons, whereas those from the more caudal aspect (N) were considered nodose neurons. The upper-central part of the ganglion (X) often comprise a mixture of nodose and jugular neurons and was avoided. (C-D) The collection of capsaicin-responsive / Trpv1-positive C-fiber neurons. The jugular or nodose portions of JNG were enzymatically dissociated, capsaicin-sensitive C-fiber neurons were identified by one of two methods and collected. (C) Identification of C-fiber neurons by capsaicin-responsiveness by intracellular calcium assay in wild-type mice (capsaicin selection). (D) Identification of C-fiber neurons by tdTomato immunofluorescence in the Trpv1-tdTomato mice (Trpv1-tdTomato selection).</p

RNA sequencing revealed distinct gene expression between Trpv1-tdTomato positive and negative nodose C-fiber neurons.

<p>(A) Principle component analysis showing the overall expression patterns of jugular, Trpv1-tdTomato positive and negative nodose C-fiber neurons. Log2 transformed CPM from Trpv1-tdTomato selection dataset was used. (B) Heatmap showing expression of genes with significant expression difference between Trpv1-tdTomato positive and negative nodose C-fiber neurons. Log2 transformed CPM was used. Red indicates high expression, green indicates low expression. Color bar indicates z score. (C) Selected genes that are differentially expressed in Trpv1-tdTomato positive and negative nodose C-fiber neurons. Y-axis indicates average CPM from Trpv1-tdTomato selection dataset. Blue indicates Trpv1-tdTomato positive nodose neurons, red indicates Trpv1-tdTomato negative nodose neurons. P values were calculated based on group comparison. * p value ≤ 0.05; ** p value ≤ 0.01; *** p value ≤ 0.001. Data from TRPV1 selection dataset.</p

Concordance between RNAseq analysis^a and functional, histological, and single cell RT-PCR analysis^b.

<p>Concordance between RNAseq analysis<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185985#t001fn001" target="_blank">^a</a> and functional, histological, and single cell RT-PCR analysis<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185985#t001fn002" target="_blank">^b</a>.</p

RNA sequencing revealed distinct gene expression between nodose and jugular C-fiber neurons.

<p>(A) Heatmap showing expression of genes with significant expression difference between nodose and jugular C-fiber neurons. Log2 transformed CPM was used. Red indicates high expression, green indicates low expression. Color bar indicates z score. (B) Pie plot showing functional annotation of genes with significant expression difference between nodose and jugular C-fiber neurons. The number of genes in each category is indicated. (C) Top 20 Gene Ontology (molecular function and biological process) pathways enriched for genes with significant expression difference between nodose and jugular C-fiber neurons. Pathways are ranked by fold of enrichment. Blue bar and upper horizontal axis indicate fold of enrichment; red line and lower horizontal axis indicate–log10 (FDR). Data from TRPV1 selection dataset.</p

RNA sequencing revealed distinct expression of transcription factors between nodose and jugular C-fiber neurons.

<p>(A) Transcription factors differentially expressed in nodose and jugular C-fiber neurons from the Trpv1 selection. Y-axis indicates average of normalized counts per million (CPM) from Trpv1-tdTomato selection. Blue indicates nodose, red indicates jugular C-fiber neurons. P values were calculated based on group comparison. * p value ≤ 0.05; ** p value ≤ 0.01; *** p value ≤ 0.001. (B) Transcription factors differentially expressed in nodose and jugular C-fiber neurons from the capsaicin selection. Y-axis indicates average CPM from capsaicin selection. Blue indicates nodose, red indicates jugular C-fiber neurons. P values were calculated based on group comparison. * p value ≤ 0.05; ** p value ≤ 0.01; *** p value ≤ 0.001.</p

Action potential discharge of a C-fibers terminating in the mouse lungs.

<p>(A) Left, representative tracing showing action potential discharge evoked by S-1P in nodose C-fiber terminating in the mouse lung. The peak frequency of action potential discharge was 6±2Hz (N = 10). Right: S-1P evoked action potential discharge in 3 of 5 jugular C-fibers. (B), The PAR1 receptor selective agonist TFLLR stimulated nodose C-fibers (19 ± 3 Hz), but not jugular C-fibers (n = 4).</p

Expression of receptors for selected immune mediators in nodose and jugular afferent neurons^a.

<p>Expression of receptors for selected immune mediators in nodose and jugular afferent neurons<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185985#t002fn001" target="_blank">^a</a>.</p