<i>CSH</i> is a powerful tool for a variety of stains.

<p>Common histological stains, displaying the fidelity of <i>CSH</i>. (Top panels): A mouse aorta stained with alkaline phosphatase (ALP, red) for detection of early calcification, with Gill's hematoxylin as counterstaining (purple), which depicts advanced calcification. ALP stain is scarlet red (denoted “A” in the top left panel), while hematoxylin is a shade of purple (denoted “H” in the top left panel). Visually, the hematoxylin interferes with the ALP, making it difficult to see where the ALP stain begins and ends. We analyzed the section for ALP-positive area using both CSH and an RGB-based method. (Bottom panels): A mouse liver stained with picrosirius red staining visualized using polarized light microscopy for detection of fibrosis. We analyzed the section using both CSH and an RGB-based method. The RGB method was unable to register the brightest parts of the stain as positive (gray), and falsely interpreted stain artifacts as positive areas (green in both “Merge” images).</p

<i>CSH</i> processing on an infarcted mouse heart.

<p>A) An infarcted mouse heart section cut 8 µm thick. Muscle tissue is scarlet red, while collagen fibers appear blue, and necrotic regions are purple-black. Insets show enlarged areas of muscle, collagen and necrotic region. B) The same mouse heart, post-processing by <i>CSH</i>. The areas that <i>CSH</i> determined as collagen are blue, and the areas that <i>CSH</i> determined as muscle are red. The background is yellow. C) A plot of the pixels from the original heart image mapped to HSV space. The gray arrows indicate the direction from which this 3-D graph will be displayed in the following 2-D images. D) A plot of the pixels from the original image in the Hue-Saturation plane. The borders collagen and the muscle rectangular thresholds are visible at Hue = {200, 300, 385}. E) A plot of the pixels from the original image in the Hue-Value plane. F) A plot of the pixels from the original image in the Value-Saturation plane. This graph most clearly shows the different shapes of the collagen peak (blue) and the muscle peak (red).</p

Analytic performance across diverse section thicknesses.

<p>A) A section of infarcted mouse heart cut to 4 µm and stained with Masson's trichrome. Descending from the original image, we see the RGB binary image, the HSV binary image, a density map of the pixels mapped to the RGB color space, and a density map of the pixels mapped to the HSV color space. B) A section of infarcted mouse heart cut to 6 µm and stained with Masson's trichrome. C) A section of infarcted mouse heart cut to 8 µm and stained with Masson's trichrome. D) For each of four experimental hearts and each of the three section thicknesses, the area identified as muscle is plotted next to the area identified as collagen using the RGB method. Because each heart has a different size infarction, these results for each heart are normalized as a percentage of the measured area in the 6 µm sample. As the section thickness increases, RGB analysis decreases the perceived collagen area, despite analyzing adjacent sections of heart. E) For each of four experimental hearts and each of the three section thicknesses, the area identified as muscle is plotted next to the area identified as collagen using the HSV method. Because each heart has a different size infarction, these results for each heart are normalized as a percentage of the measured area in the 6 µm sample. There is no discernible change in perceived muscle or collagen area as the section thickness increases when using the HSV method.</p

<i>CSH</i> is consistent between individuals.

<p>Apoe−/− mouse innominate arteries stained with MAC3 antibody for detection of macrophages. RGB1(threshold1) was optimized for Cross Section 1, and overestimates the positive area when applied to Cross Section 2. RGB2(threshold2) was optimized for Cross Section 2, and underestimates the positive area when applied to Cross Section 1. <i>CSH</i> was able to effectively use a single HSV threshold on both cross sections. In the overlays between the HSV and RGB1 and RGB2, yellow area shows where there is agreement between the HSV method and the RGB method. Green area in the overlays may indicate false positive area reported by the RGB method, while red area in the overlays may represent false negative area reported by the RGB method.</p

A Combined Gas-Phase Separation Strategy for ADP-ribosylated Peptides

ADP-ribosylation (ADPr) is a post-translational modification that is best studied using mass spectrometry. Method developments that are permissive with low inputs or baseline levels of protein ribosylation represent the next frontier in the field. High-field asymmetric waveform ion mobility spectrometry (FAIMS) reduces peptide complexity in the gas phase, providing a means to achieve maximal ADPr peptide sequencing depth. We therefore investigated the extent to which FAIMS with or without traditional gas-phase fractionation–separation (GPS) can increase the number of ADPr peptides. We examined ADPr peptides enriched from mouse spleens. We gleaned additional insight by also reporting findings from the corresponding non-ADPr peptide contaminants and the peptide inputs for ADPr peptide enrichment. At increasingly higher negative compensation voltages, ADPr peptides were more stable, whereas the non-ADPr peptides were filtered out. A combination of 3 GPS survey scans, each with 8 compensation voltages, resulted in 790 high-confidence ADPr peptides, compared to 90 with GPS alone. A simplified acquisition strategy requiring only two injections corresponding to two MS1 scan ranges coupled to optimized compensation voltage settings provided 402 ADPr peptides corresponding to 234 ADPr proteins. We conclude that our combined GPS strategy is a valuable addition to any ADP-ribosylome workflow. The data are available via ProteomeXchange with identifier PXD040898

A Combined Gas-Phase Separation Strategy for ADP-ribosylated Peptides

ADP-ribosylation (ADPr) is a post-translational modification that is best studied using mass spectrometry. Method developments that are permissive with low inputs or baseline levels of protein ribosylation represent the next frontier in the field. High-field asymmetric waveform ion mobility spectrometry (FAIMS) reduces peptide complexity in the gas phase, providing a means to achieve maximal ADPr peptide sequencing depth. We therefore investigated the extent to which FAIMS with or without traditional gas-phase fractionation–separation (GPS) can increase the number of ADPr peptides. We examined ADPr peptides enriched from mouse spleens. We gleaned additional insight by also reporting findings from the corresponding non-ADPr peptide contaminants and the peptide inputs for ADPr peptide enrichment. At increasingly higher negative compensation voltages, ADPr peptides were more stable, whereas the non-ADPr peptides were filtered out. A combination of 3 GPS survey scans, each with 8 compensation voltages, resulted in 790 high-confidence ADPr peptides, compared to 90 with GPS alone. A simplified acquisition strategy requiring only two injections corresponding to two MS1 scan ranges coupled to optimized compensation voltage settings provided 402 ADPr peptides corresponding to 234 ADPr proteins. We conclude that our combined GPS strategy is a valuable addition to any ADP-ribosylome workflow. The data are available via ProteomeXchange with identifier PXD040898

PTX3 associates negatively with obesity and metabolic syndrome in non-diabetic subject.

<p>PTX3 associates negatively with obesity but positively with diabetes mellitus (A). Mean plasma PTX3 levels among 779 individuals, comparing non-obese (BMI <30) and obese individuals (BMI ≥30) (left), or non-diabetic and diabetic control individuals (right). Mean values were estimated from linear regression models, adjusted for age, sex, smoking status, triglycerides, HDL cholesterol, and hypertension. Models for diabetes also adjusted for BMI. PTX3 negatively associates with obesity among non-diabetic individuals (B). Mean values of plasma PTX3 levels by BMI category in non-diabetic (left) and diabetic individuals (right) estimated from linear regression models. Mean values adjusted for age, sex, smoking status, triglycerides, HDL cholesterol, and hypertension. PTX3 associates negatively with the number of metabolic syndrome components (C). Mean values of plasma PTX3 levels according to presence of 0, 1, 2, 3, and 4 or more components of metabolic syndrome in all individuals (left) and non-diabetic individuals (right). Mean values estimated from linear regression models, adjusted for age, sex, and smoking status.</p

Baseline characteristics of the 366 controls in the CARE populations by quartiles of PTX3.

<p>IQR  =  interquartile range. MDRD-GFR  =  Modification of Diet in Renal Disease-Glomerular Filtration Rate.</p

PTX3 levels associate adversely with metabolic syndrome components.

<p>Mean values of selected characteristics (BMI, waist circumference, triglyceride, and HDL cholesterol) by quartile of PTX3. Mean values were estimated from linear regression models, adjusted for age.</p

Lack of association between PTX3 and short pentraxin.

<p>Spearman correlations revealed no association between plasma levels of the long pentraxin PTX3 and the short pentraxin CRP (A). Plasma PTX3 levels positively correlated with plasma SAA levels (B), and plasma CRP and SAA levels correlated strongly with each other (C). After exclusion of participants with high CRP levels (>10 mg/L), PTX3 showed no significant correlations with CRP and SAA (D and E), whereas CRP and SAA correlated positively with each other (F).</p