Macrophages have key functional role in the pathogenesis are various cardiovascular diseases, such as atherosclerosis and aortic aneurysm. Their accumulation within the vessel wall leads to sustained local inflammatory responses characterized by secretion of chemokines, cytokines, and matrix protein degrading enzymes. Here, we summarize recent findings on macrophage contribution to cardiovascular disease published in ATVB . In this issue, we focus on the origin, survival/death, and phenotypic switching of macrophages within vessel walls.
We found that certain mid-range consumer level digital SLR cameras using full-frame CMOS sensors outperform x-ray film in acquiring signals from immunoblots that use enhanced chemiluminescence for detection. These cameras exhibit a sensitivity comparable to x-ray film, yet provide a 3-fold increase in linear dynamic range and substantial cost saving over time, are more convenient to use, and eliminate the chemical waste associated with processing film. Keywords camera; chemiluminescence; CMOS; dot blot; ECL; imaging; immunoblot; SLR; Western blot; xray film Due to its high sensitivity, ECL (enhanced chemiluminescence) is currently the dominant signal-generating method for immuno-detection of proteins via Western blots and dot blots [1]. Signal acquisition is typically accomplished either with x-ray film or specialized cameras that employ cooled CCD (charge-coupled device) sensors. X-ray film is more popular because of its greater sensitivity and/or resolution for a given exposure time, and because it requires only a modest expenditure for equipment. The main drawbacks of film are its limited and nonlinear dynamic range, and the fact that automated film processors are expensive to maintain and generate chemical waste. Dedicated CCD-based imagers exhibit a significantly better dynamic range and linearity than film and do not generate chemical waste, but their combination of sensitivity and pixel resolution are generally inferior to film. Moreover, the cost of these imagers ($30-60k) is out of the range of many academic research labs.Several new single lens reflex (SLR) digital cameras in the mid-range consumer market have been shown to achieve remarkable low-light sensitivity using high resolution full-frame 35 mm sensors based on CMOS (complementary metal oxide semiconductor) technology in combination with advanced noise-reduction firmware. Here we report a comparison of one such camera vs x-ray film for acquiring ECL signals from dot blots and Western blots. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptIn our first test we spotted serial dilutions (in triplicate) of horseradish peroxidase-coupled antibody (HRP-Ab) directly onto two separate, but identically treated, nitrocellulose membranes. The membranes were developed with a commercial ECL reagent kit. One was imaged by a 4.0 min exposure to a Nikon D700 SLR camera fitted with a 50 mm f/1.4 lens and 12 mm extension tube, and the other by a 4.0 min exposure to x-ray film. To process the SLR image, the resulting 14-bit R...
Extracellular vesicle (EV) secretion is an important mechanism used by cells to release biomolecules. A common necroptosis effector—mixed lineage kinase domain like (MLKL)—was recently found to participate in the biogenesis of small and large EVs independent of its function in necroptosis. The objective of the current study is to gain mechanistic insights into EV biogenesis during necroptosis. Assessing EV number by nanoparticle tracking analysis revealed an increased number of EVs released during necroptosis. To evaluate the nature of such vesicles, we performed a newly adapted, highly sensitive mass spectrometry‐based proteomics on EVs released by healthy or necroptotic cells. Compared to EVs released by healthy cells, EVs released during necroptosis contained a markedly higher number of unique proteins. Receptor interacting protein kinase‐3 (RIPK3) and MLKL were among the proteins enriched in EVs released during necroptosis. Further, mouse embryonic fibroblasts (MEFs) derived from mice deficient of Rab27a and Rab27b showed diminished basal EV release but responded to necroptosis with enhanced EV biogenesis as the wildtype MEFs. In contrast, necroptosis‐associated EVs were sensitive to Ca2+ depletion or lysosomal disruption. Neither treatment affected the RIPK3‐mediated MLKL phosphorylation. An unbiased screen using RIPK3 immunoprecipitation‐mass spectrometry on necroptotic EVs led to the identification of Rab11b in RIPK3 immune‐complexes. Our data suggests that necroptosis switches EV biogenesis from a Rab27a/b dependent mechanism to a lysosomal mediated mechanism.
Isoaspartate (isoAsp) formation is a major source of protein damage that is kept in check by the repair function of protein L-isoaspartyl methyltransferase (PIMT). Mice deficient in PIMT accumulate isoAsp-containing proteins, resulting in cognitive deficits, abnormal neuronal physiology and cytoarchitecture, and fatal epileptic seizures 30–60 days after birth. Synapsins I and II, dynamin-1, collapsin response mediator protein 2 (CRMP2), and α/β-tubulin are major targets of PIMT in brain. To investigate links between isoAsp accumulation and the neurological phenotype of the KO mice, we used Western blotting to compare patterns of in vivo phosphorylation or acetylation of the major PIMT targets listed above. Phosphorylations of synapsins I and II at Ser-9 were increased in female KO vs. WT mice, and acetylation of tubulin at Lys-40 was decreased in male KO vs. WT mice. Average levels of dynamin-1 phosphorylation at Ser-778 and Ser-795 were higher in male KO vs. WT mice, but the statistical significance (P>0.1) was low. No changes in phosphorylation were found in synapsins I and II at Ser-603, in CRMP2 at Ser-522 or Thr-514, in DARPP-32 at Thr-34, or in PDK1 at Ser-241. General levels of phosphorylation assessed with Pro-Q Diamond stain, or an anti-phosphotyrosine antibody, appeared similar in the WT and KO mice. We conclude that isoAsp accumulation is associated with altered functional status of several neuronal proteins that are highly susceptible to this type of damage. We also uncovered unexpected differences in how male and female mice respond to isoAsp accumulation in the brain.
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