Angular-Resolved Polarized Surface Enhanced Raman Spectroscopy

Surface immobilized gold nanospheres (SIGNs) on a metallic substrate are widely used for surface enhanced Raman scattering (SERS) spectroscopy. Since SIGNs significantly enhance the local electric fields of light, due to localized surface plasmon resonance, it is a highly promising SERS platform. To understand the mechanism of enhancement of Raman scattering in the presence of colloidal metals, it is necessary to know how the molecules are adsorbed on the SIGNs. We performed angular-resolved polarized SERS (AP-SERS) spectroscopy to probe the adsorption structure of the molecules on the SIGN. A theoretical analysis is given for the AP-SERS from the molecules on the SIGNs, based on the quasi-static approximation and the local field approximation. The molecules are adsorbed only in the upper hemisphere region of the SIGN in the high coverage SIGN substrate, whereas at low coverage, the molecules are adsorbed in both upper and lower regions. This is because the solution does not intrude into the space between the nanospheres, as a result of the surface tension of ethanol. This result explains the unstable and irreproducible SERS signal from the aggregated nanospheres

Effect of <i>Skn-1a</i> deficiency on the functional differentiation of <i>Trpm5</i>-expressing brush cells in trachea.

<p>A: Skn-1a-expressing cells were characterized using immunohistochemistry with anti-Skn-1a and anti-villin antibodies. Villin-positive brush cells were divided into two types, Skn-1a-positive (arrowhead) and Skn-1a-negative brush cells (arrow). B: <i>Skn-1a</i>-expressing cells were characterized by two-color <i>in situ</i> hybridization with RNA probes for <i>Skn-1a</i> and <i>Trpm5</i>. <i>Skn-1a-</i>positive brush cells were co-labeled with <i>Trpm5</i> riboprobe (arrowheads). Scale bars, 20 μm. C: The impact of <i>Skn-1a</i> deficiency on the functional differentiation of Trpm5/Skn1a-positive brush cells in the tracheal epithelium was examined by <i>in situ</i> hybridization using probes for taste signaling molecules of <i>Tas1r3</i>, <i>Tas2rs</i> (<i>Tas2r105</i>, <i>Tas2r108</i>, <i>Tas2r131</i>), <i>Gnat3</i>, <i>Plcb2</i> and <i>Trpm5</i>. The mRNA signals of taste signaling molecules observed in wild-type mice were completely absent in the <i>Skn-1a-</i>/- mice, indicating that <i>Skn-1a</i> is required for the functional differentiation of Trpm5-positive brush cells. Scale bar, 100 μm. D: The expression of taste signaling molecules (<i>Tas1r3</i>, <i>Tas2r105</i>, <i>Tas2r108</i>, <i>Tas2r131</i>, <i>Gnat3</i>, <i>Plcb2</i>, and <i>Trpm5</i>) in wild-type (WT) and <i>Skn-1a</i>-/- (KO) trachea was examined by RT-PCR. The expression of taste signaling molecule genes was not detected in <i>Skn-1a-/-</i> trachea. A housekeeping gene, <i>GAPDH</i> was used as a positive control.</p

Skn-1a/Pou2f3 functions as a master regulator to generate Trpm5-expressing chemosensory cells in mice

<div><p>Transient receptor potential channel M5 (Trpm5)-expressing cells, such as sweet, umami, and bitter taste cells in the oropharyngeal epithelium, solitary chemosensory cells in the nasal respiratory epithelium, and tuft cells in the small intestine, that express taste-related genes function as chemosensory cells. Previous studies demonstrated that Skn-1a/Pou2f3, a POU homeodomain transcription factor is expressed in these Trpm5-expressing chemosensory cells, and is necessary for their generation. Trpm5-expressing cells have recently been found in trachea, auditory tube, urethra, thymus, pancreatic duct, stomach, and large intestine. They are considered to be involved in protective responses to potential hazardous compounds as Skn-1a-dependent bitter taste cells, respiratory solitary chemosensory cells, and intestinal tuft cells are. In this study, we examined the expression and function of Skn-1a/Pou2f3 in Trpm5-expressing cells in trachea, auditory tube, urethra, thymus, pancreatic duct, stomach, and large intestine. Skn-1a/Pou2f3 is expressed in a majority of Trpm5-expressing cells in all tissues examined. In <i>Skn-1a/Pou2f3-</i>deficient mice, the expression of Trpm5 as well as marker genes for Trpm5-expressing cells were absent in all tested tissues. Immunohistochemical analyses demonstrated that two types of microvillous cells exist in trachea, urethra, and thymus, Trpm5-positive and Trpm5-negative cells. In <i>Skn-1a/Pou2f3-</i>deficient mice, a considerable proportion of Trpm5-negative and villin-positive microvillous cells remained present in these tissues. Thus, we propose that Skn-1a/Pou2f3 is the master regulator for the generation of the Trpm5-expressing microvillous cells in multiple tissues.</p></div

Skn-1a is required for the functional differentiation of Trpm5-positive tracheal brush cells.

<p>A: Immunostaining of Trpm5 and ChAT on coronal sections of the trachea of wild-type and <i>Skn-1a</i>-/- mice. Trpm5-positive brush cells were ChAT positive in the wild-type trachea (arrows), whereas no immunoreactive signals for Trpm5 and ChAT was observed in the <i>Skn-1a</i>-/- trachea. B: Immunostaining of Trpm5 and villin on coronal sections of the trachea of wild-type and <i>Skn-1a</i>-/- mice. In wild-type mice, both Trpm5 and villin-double positive (arrowhead) and villin-single positive (arrow) brush cells were observed. In <i>Skn-1a</i>-/- mice, Trpm5-positive brush cells were absent and only villin-single positive brush cells (arrows) were observed. Scale bars, 20 μm. C: Quantification of the number of immunosignals for Trpm5 and villin in the wild-type and <i>Skn-1a</i>-/- tracheal epithelium. The signals of Trpm5 were completely absent in the <i>Skn-1a</i>-/- tracheal epithelium, and the number of villin-single positive cells was significantly decreased in <i>Skn-1a</i>-/- mice. Each symbol represents an individual mouse. The error bars represent the mean ± SEM (n = 3, *P < 0.05, Student’s <i>t</i>-test).</p

Loss of the Trpm5-positive chemosensory cells in multiple tissues in <i>Skn-1a</i>-/- mice.

<p>A: Two-color <i>in situ</i> hybridization of <i>Skn-1a</i> (green) and <i>Trpm5</i> (magenta) in various tissues of auditory tube, urethra, thymus, and pancreatic duct of wild-type adult mice. The mRNA signals of <i>Skn-1a</i> were co-labeled with <i>Trpm5</i> signals (arrowheads) in all tissues examined. Scale bar, 20 μm. B: Co-immunostaining using antibodies against Skn-1a (green) and villin (magenta) on cryosections of auditory tube, urethra, thymus, and pancreatic duct of wild-type adult mice. Skn-1a-positive cells were overlapped with villin-positive cells (arrowheads). The arrows indicate Skn-1a negative and villin-single positive cells. Scale bar, 20 μm. C: Double-label immunohistochemistry of Trpm5 and villin on sections of auditory tube, urethra, thymus, and pancreatic duct of wild-type (top) and <i>Skn-1a</i>-/- mice (bottom) was carried out to examine the impact of <i>Skn-1a</i> deficiency on Trpm5-positive chemosensory cells. Trpm5-positive cells were co-labeled with anti-villin antibody (arrowheads) in wild-type mice, whereas the expression of Trpm5 was abolished in all tested tissues in <i>Skn-1a</i>-/- mice. The immunoreactive signals for villin were detected in <i>Skn-1a</i>-/- urethral epithelium and thymic medulla (arrows), but not in auditory tube and pancreatic duct. Scale bar, 20 μm. D: The expression of taste signaling molecules (<i>Tas1r3</i>, <i>Tas2r105</i>, <i>Tas2r108</i>, <i>Tas2r131</i>, <i>Gnat3</i>, <i>Plcb2</i>, and <i>Trpm5</i>) in auditory tube, urethra, thymus, and pancreatic duct was examined by RT-PCR in wild-type (WT) and <i>Skn-1a</i>-/- (KO) mice. The expression of taste signaling molecules observed in wilt-type mice was not detected in <i>Skn-1a</i>-/- mice. A housekeeping gene, <i>GAPDH</i> was used as a positive control.</p

Impact of <i>Skn-1a</i> deficiency on Trpm5-positive tuft cells in digestive tracts.

<p>A: Two-color <i>in situ</i> hybridization of <i>Skn-1a</i> (green) and <i>Trpm5</i> (magenta) on sections of digestive tracts of stomach, small intestine, and large intestine of wild-type adult mice. The mRNA signals of <i>Skn-1a</i> were co-labeled with <i>Trpm5</i> signals (arrowheads) in all tissues examined. Scale bar, 20 μm. B: Co-immunostaining using antibodies against Skn-1a (green) and villin (magenta) on sections of stomach, small intestine, and large intestine of wild-type adult mice. Skn-1a-positive cells were overlapped with villin-positive cells (arrowheads). Scale bar, 20 μm. C: The impact of <i>Skn-1a</i> deficiency on Trpm5-positive tuft cells was examined by double-label immunohistochemistry of Trpm5 and villin using sections of stomach, small intestine, and large intestine of wild-type (top) and <i>Skn-1a</i>-/- mice. Trpm5-positive cells were co-labeled with anti-villin antibody (arrowheads) in wild-type mice, whereas the expression of Trpm5 was abolished in all tested tissues in <i>Skn-1a</i>-/- mice. Scale bars, 20 μm. The immunoreactive signals for villin detected in wild-type mice (arrows) were not observed in <i>Skn-1a</i>-/- mice. D: The signals of intestinal tuft cells marker gene, Dclk1 mRNA were observed in wild-type digestive tracts, whereas no signals of Dclk1 mRNA were observed in <i>Skn-1a</i>-/-. Scale bars, 100 μm.</p

QC data from NGS measurement for comparison of variances among samples subjected to different conditions.

(XLSX)</p

Stability of plasma in storage periods up to 30 days at 4°C.

The library concentrations (A), identification rate (B) and UMI ratios (C) were compared between samples stored for 1 hour and 1 day, 3 days, 5 days, 7 days and 30 days after blood collection. MiRNA expression profiles at 1 hour vs. 1 day (D), 1 hour vs. 3 days (E), 1 hour vs. 5 days (F), 1 hour vs. 7 days (G), and 1 hour vs. 30 days (H) were compared. MiRNAs with a fold change greater than 2-fold or less than half-fold are indicated outside of the dashed lines. The X-axis or Y-axis of the scatter plot indicates log2(miRNA expression counts). Significant differences were identified using one-way ANOVA with a post hoc Dunnett’s test.</p

RNA concentrations in the purified RNA samples.

Concentration of RNAs obtained from samples isolated using different blood collection tube conditions (A). The crossbar indicates the mean, and the error bar indicates the standard deviation (SD). Significant differences were identified using one-way ANOVA with a post hoc Tukey test and are indicated as * p (TIFF)</p

MiRNAs with higher expression at 2 days and 3 days than at 1 hour.

MiRNAs with higher expression at 2 days and 3 days than at 1 hour.</p