Increasing the Modulation Depth of Gd^III-Based Pulsed Dipolar EPR Spectroscopy (PDS) with Porphyrin–Gd^III Laser-Induced Magnetic Dipole Spectroscopy

Distance determination with pulsed EPR has become an important technique for the structural investigation of biomacromolecules, with double electron–electron resonance spectroscopy (DEER) as the most important method. GdIII-based spin labels are one of the most frequently used spin labels for DEER owing to their stability against reduction, high magnetic moment, and absence of orientation selection. A disadvantage of GdIII–GdIII DEER is the low modulation depth due to the broad EPR spectrum of GdIII. Here, we introduce laser-induced magnetic dipole spectroscopy (LaserIMD) with a spin pair consisting of GdIII(PymiMTA) and a photoexcited porphyrin as an alternative technique. We show that the excited state of the porphyrin is not disturbed by the presence of the GdIII complex and that herewith modulation depths of almost 40% are possible. This is significantly higher than the value of 7.2% that was achieved with GdIII–GdIII DEER

Gene Set Enrichment Analysis with microarray expression data from the renal cortex and medulla of rats fed lithium for 6 months.

<p>The mean signal intensity of a probe set (a surrogate value of expression level) across all lithium-fed samples (y-axis) is plotted against the mean signal intensity across all control samples (x-axis). Each point represents a single probe. <b>(A)</b> Gene sets with functional gene annotation enrichment for MARGS, fibrosis, TGFβ2 and ECM genes in the renal cortex; <b>(B)</b> Gene sets with functional gene annotation enrichment for MARGS, immune response, TGFβ2 and ECM genes in the medulla. With the exception of the MARGS gene set, all gene sets in this figure were extracted from <a href="http://www.SABiosciences.com" target="_blank">www.SABiosciences.com</a>.</p

Sections of rat kidney stained with Masson’s Trichrome.

<p>Low power sections of normal rat kidney (A) and rat fed a lithium diet for six months (B), in which collecting duct tubules show cyst-like dilatations. The lithium treated rats show glomerulosclerosis (C, arrow), collecting duct dilations # (C, D), and epithelial linings either greatly attenuated (D, arrow) or with enlarged cells, often appearing multinucleate (E, arrow). The cortex has areas of focal fibrosis(F). Scale bar, A, B, 200 microns; C-F, 50 microns.</p

Immunohistochemical location of TGFβ2 in the kidneys of control rats and rats fed a lithium-containing diet for 6 months.

<p>(A) Cortical region of a control rat, showing weak cytoplasmic staining in the proximal tubules but an absence of stain in the distal tubule. (B) In the outer medulla, the collecting duct is unstained but there is some staining in the thick ascending limbs. (C) In the lithium-treated animals there is strong staining in the basolateral regions of the distal tubules, but no staining in the cortical collecting ducts. (D) This pattern is repeated in the outer medulla, where the thick ascending limbs show strong basolateral staining but the collecting duct is devoid of stain. Scale bar, 50 microns.</p

Sections of kidneys of control and lithium-treated rats probed for NOV/CCN3.

<p>In control rats the protein is apparently absent from the cortex (A), and barely detectable in the outer medulla (B), in basolateral margins of collecting ducts, but absent from thick ascending limbs. In contrast NOV occurs basolaterally in epithelial cells of the distal tubules and cortical collecting ducts of lithium-treated rats, (C, arrows) and weakly in collecting ducts of the outer medulla (D). Scale bar, 50 microns.</p

Scatter plots of signal intensities of NOV, TGFβ2, MMP2 and CD44 in the renal cortex (A) and medulla (B) from control rats and rats fed lithium for 6 months.

<p>Each point represents the signal intensity (a surrogate value of expression level) of mRNA in a sample. The expression differences between control and lithium-treated samples are clear (p values included).</p

Fibrosis classifier showing 100% correct classification rate of Lithium versus Control (for explanation see text).

<p>Fibrosis classifier showing 100% correct classification rate of Lithium versus Control (for explanation see text).</p

MARGS expression changes in the renal cortex between control and lithium-treated rats.

<p>MARGS expression changes in the renal cortex between control and lithium-treated rats.</p

Principal component analysis (PCA) and hierarchical cluster analyses of MARGS in the renal cortex (A) and medulla (B) of kidneys from control and lithium-fed rats (6 months).

<p><b>A.</b> PCA (left) and hierarchical cluster analysis (right) using 5 MARGS genes from cortex (expression filtered, p-value < 0.05, |FC| > 1.5); <b>B.</b> PCA (left) and hierarchical cluster analysis (right) using 11 MARGS genes from medulla (expression filtered, p-value < 0.05, |FC| > 1.5). MARGS are sufficient to separate the two groups. In PCA, each sphere represents one sample. The ellipsoids represent 95% of the data of a group. In both PCA plots in A and B, red indicates expression above median, blue indicates expression below median. The depth of the colour indicates extent of expression above or below median.</p

Sections of kidneys of control and lithium-treated rats probed for CD44.

<p>In control kidneys (A, B) there is strong labelling basolaterally in the distal tubule (A), and weak labelling in the glomeruli (B). The collecting ducts (A) are unstained. In the lithium-treated rats there is strong staining in both collecting ducts (C) and thick ascending limbs (C), while the staining of the glomeruli is unchanged (D). Scale bar, 50 microns.). Scale bar, 50 microns.</p