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FLEXIQinase, a mass spectrometry-based assay, to unveil multi-kinase mechanisms
We introduce a mass spectrometry-based method that provides residue-resolved quantitative information about protein phosphorylation. In this FLEXIQinase assay we combined our Full-Length Expressed Stable Isotope-labeled Protein for Quantification strategy (FLEXIQuant) with a traditional kinase assay to determine the mechanisms of multi-kinase substrate phosphorylation such as priming-dependent kinase activities. The assay monitors the decrease in signal intensity of the substrate peptides and the concomitant increase in the (n×80 Da)-shifted phosphorylated peptide. We analyzed the c-Jun N-terminal Kinase (JNK)-dependent glycogen synthase kinase 3β (GSK3β) activity on doublecortin (DCX) revealing mechanistic details about the role of phosphorylation cross-talk in GSK3β activity and permitting an advanced model for GSK3β-mediated signaling
The impact of PARPs and ADP-ribosylation on inflammation and host–pathogen interactions
This work is licensed under a Creative Commons Attribution 4.0 International License.Poly-adenosine diphosphate-ribose polymerases (PARPs) promote ADP-ribosylation, a highly conserved, fundamental posttranslational modification (PTM). PARP catalytic domains transfer the ADP-ribose moiety from NAD+ to amino acid residues of target proteins, leading to mono- or poly-ADP-ribosylation (MARylation or PARylation). This PTM regulates various key biological and pathological processes. In this review, we focus on the roles of the PARP family members in inflammation and host–pathogen interactions. Here we give an overview the current understanding of the mechanisms by which PARPs promote or suppress proinflammatory activation of macrophages, and various roles PARPs play in virus infections. We also demonstrate how innovative technologies, such as proteomics and systems biology, help to advance this research field and describe unanswered questions
Enrichment of calcifying extracellular vesicles using density-based ultracentrifugation protocol
Calcifying extracellular vesicles (EVs) released from cells within atherosclerotic plaques have received increased attention for their role in mediating vascular calcification, a major predictor of cardiovascular morbidity and mortality. However, little is known about the difference between this pathologic vesicle population and other EVs that contribute to physiological cellular processes. One major challenge that hinders research into these differences is the inability to selectively isolate calcifying EVs from other vesicle populations. In this study, we hypothesized that the formation of mineral within calcifying EVs would increase the density of the vesicles such that they would pellet at a faster rate during ultracentrifugation. We show that after 10 min of ultracentrifugation at 100,000×g, calcifying EVs are depleted from the conditioned media of calcifying coronary artery smooth muscle cells and are enriched in the pelleted portion. We utilized mass spectrometry to establish functional proteomic differences between the calcifying EVs enriched in the 10 min ultracentrifugation compared to other vesicle populations preferentially pelleted by longer ultracentrifugation times. The procedures established in this study will allow us to enrich the vesicle population of interest and perform advanced proteomic analyses to find subtle differences between calcifying EVs and other vesicle populations that may be translated into therapeutic targets for vascular calcification. Finally, we will show that the differences in ultracentrifugation times required to pellet the vesicle populations can also be used to estimate physical differences between the vesicles
Elastogenesis Correlates With Pigment Production in Murine Aortic Valve Leaflets
Objective: Aortic valve (AV) leaflets rely on a precise extracellular matrix (ECM)
microarchitecture for appropriate biomechanical performance. The ECM structure is
maintained by valvular interstitial cells (VICs), which reside within the leaflets. The
presence of pigment produced by a melanocytic population of VICs in mice with dark
coats has been generally regarded as a nuisance, as it interferes with histological analysis
of the AV leaflets. However, our previous studies have shown that the presence of
pigment correlates with increased mechanical stiffness within the leaflets as measured
by nanoindentation analyses. In the current study, we seek to better characterize the
phenotype of understudied melanocytic VICs, explore the role of these VICs in ECM
patterning, and assess the presence of these VICs in human aortic valve tissues.
Approach and Results: Immunofluorescence and immunohistochemistry revealed that
melanocytes within murine AV leaflets express phenotypic markers of either neuronal or
glial cells. These VIC subpopulations exhibited regional patterns that corresponded to
the distribution of elastin and glycosaminoglycan ECM proteins, respectively. VICs with
neuronal and glial phenotypes were also found in human AV leaflets and showed ECM
associations similar to those observed in murine leaflets. A subset of VICs within human
AV leaflets also expressed dopachrome tautomerase, a common melanocyte marker. A
spontaneous mouse mutant with no aortic valve pigmentation lacked elastic fibers and
had reduced elastin gene expression within AV leaflets. A hyperpigmented transgenic
mouse exhibited increased AV leaflet elastic fibers and elastin gene expression.
Conclusions: Melanocytic VIC subpopulations appear critical for appropriate
elastogenesis in mouse AVs, providing new insight into the regulation of AV ECM
homeostasis. The identification of a similar VIC population in human AVs suggests
conservation across species
Melting Pot 2.0
Multi-agent artificial intelligence research promises a path to develop
intelligent technologies that are more human-like and more human-compatible
than those produced by "solipsistic" approaches, which do not consider
interactions between agents. Melting Pot is a research tool developed to
facilitate work on multi-agent artificial intelligence, and provides an
evaluation protocol that measures generalization to novel social partners in a
set of canonical test scenarios. Each scenario pairs a physical environment (a
"substrate") with a reference set of co-players (a "background population"), to
create a social situation with substantial interdependence between the
individuals involved. For instance, some scenarios were inspired by
institutional-economics-based accounts of natural resource management and
public-good-provision dilemmas. Others were inspired by considerations from
evolutionary biology, game theory, and artificial life. Melting Pot aims to
cover a maximally diverse set of interdependencies and incentives. It includes
the commonly-studied extreme cases of perfectly-competitive (zero-sum)
motivations and perfectly-cooperative (shared-reward) motivations, but does not
stop with them. As in real-life, a clear majority of scenarios in Melting Pot
have mixed incentives. They are neither purely competitive nor purely
cooperative and thus demand successful agents be able to navigate the resulting
ambiguity. Here we describe Melting Pot 2.0, which revises and expands on
Melting Pot. We also introduce support for scenarios with asymmetric roles, and
explain how to integrate them into the evaluation protocol. This report also
contains: (1) details of all substrates and scenarios; (2) a complete
description of all baseline algorithms and results. Our intention is for it to
serve as a reference for researchers using Melting Pot 2.0.Comment: 59 pages, 54 figures. arXiv admin note: text overlap with
arXiv:2107.0685
Second Heart Field–Derived Cells Contribute to Angiotensin II–Mediated Ascending Aortopathies
BACKGROUND: The ascending aorta is a common location for aneurysm and dissection. This aortic region is populated by a mosaic of medial and adventitial cells that are embryonically derived from either the second heart field (SHF) or the cardiac neural crest. SHF-derived cells populate areas that coincide with the spatial specificity of thoracic aortopathies. The purpose of this study was to determine whether and how SHF-derived cells contribute to ascending aortopathies.
METHODS: Ascending aortic pathologies were examined in patients with sporadic thoracic aortopathies and angiotensin II (AngII)–infused mice. Ascending aortas without overt pathology from AngII-infused mice were subjected to mass spectrometry– assisted proteomics and molecular features of SHF-derived cells were determined by single-cell transcriptomic analyses. Genetic deletion of either Lrp1 (low-density lipoprotein receptor–related protein 1) or Tgfbr2 (transforming growth factor–β receptor type 2) in SHF-derived cells was conducted to examine the effect of SHF-derived cells on vascular integrity.
RESULTS: Pathologies in human ascending aortic aneurysmal tissues were predominant in outer medial layers and adventitia. This gradient was mimicked in mouse aortas after AngII infusion that was coincident with the distribution of SHF-derived cells. Proteomics indicated that brief AngII infusion before overt pathology occurred evoked downregulation of smooth muscle cell proteins and differential expression of extracellular matrix proteins, including several LRP1 ligands. LRP1 deletion in SHFderived cells augmented AngII-induced ascending aortic aneurysm and rupture. Single-cell transcriptomic analysis revealed that brief AngII infusion decreased Lrp1 and Tgfbr2 mRNA abundance in SHF-derived cells and induced a unique fibroblast population with low abundance of Tgfbr2 mRNA. SHF-specific Tgfbr2 deletion led to embryonic lethality at E12.5 with dilatation of the outflow tract and retroperitoneal hemorrhage. Integration of proteomic and single-cell transcriptomics results identified PAI1 (plasminogen activator inhibitor 1) as the most increased protein in SHF-derived smooth muscle cells and fibroblasts during AngII infusion. Immunostaining revealed a transmural gradient of PAI1 in both ascending aortas of AngIIinfused mice and human ascending aneurysmal aortas that mimicked the gradient of medial and adventitial pathologies.
CONCLUSIONS: SHF-derived cells exert a critical role in maintaining vascular integrity through LRP1 and transforming growth factor–β signaling associated with increases of aortic PAI1
A prospective study of differences in duodenum compared to remaining small bowel motion between radiation treatments: Implications for radiation dose escalation in carcinoma of the pancreas
PURPOSE: As a foundation for a dose escalation trial, we sought to characterize duodenal and non-duodenal small bowel organ motion between fractions of pancreatic radiation therapy. PATIENTS AND METHODS: Nine patients (4 women, 5 men) undergoing radiation therapy were enrolled in this prospective study. The patients had up to four weekly CT scans performed during their course of radiation therapy. Pancreas, duodenum and non-duodenal small bowel were then contoured for each CT scan. On the initial scan, a four-field plan was generated to fully cover the pancreas. This plan was registered to each subsequent CT scan. Dose-volume histogram (DVH) analyses were performed for the duodenum, non-duodenal small bowel, large bowel, and pancreas. RESULTS: With significant individual variation, the volume of duodenum receiving at least 80% of the prescribed dose was consistently greater than the remaining small bowel. In the patient with the largest inter-fraction variation, the fractional volume of non-duodenal small bowel irradiated to at least the 80% isodose line ranged from 1% to 20%. In the patient with the largest inter-fraction variation, the fractional volume of duodenum irradiated to at least the 80% isodose line ranged from 30% to 100%. CONCLUSION: The volume of small bowel irradiated during four-field pancreatic radiation therapy changes substantially between fractions. This suggests dose escalation may be possible. However, dose limits to the duodenum should be stricter than for other segments of small bowel
<sup>18</sup>F-Fluoride Signal Amplification Identifies Microcalcifications Associated With Atherosclerotic Plaque Instability in Positron Emission Tomography/Computed Tomography Images
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