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Patent Title: Carrier. Patent Number: 2,, Class: Assignee: assignor of one-half to Clarence C. Nall Chickasaw, AL. Information here. Name: Powers, Sam R. City: Clanton County: Chilton. Date: Oct 7, Patent Title: Level wind attachment for winch. Name: Powers, Samuel R.
Date: Oct 24, Patent Title: Safety trip device. Notes: Speed, James O. Birmingham, AL - joint inventors. Name: Powers, William T. City: Huntsville County: Madison. Date: Sep 21, Patent Title: Cryogenic high pressure sensor module. Patent Number: 5,, Class:.
Notes: Chapman, John J. Yorktown, VA - joint inventors. Date: Jun 5, Patent Title: Cryogenic, absolute, high pressure sensor. Patent Number: 6,, Class: Name: Prabhakarpandian, Balabhaskar. City: Madison County: Madison. Date: Sep 19, Patent Title: Spacer for delivery of medications from an inhaler to children and breathing impaired patients.
Patent Number: 7,, Class: Date: May 25, Patent Title: Synthetic microfluidic microvasculature network. Date: May 8, Patent Number: 8,, Class: Date: Jan 15, Patent Title: Microfluidic assay for selection and optimization of drug delivery vehicles to tumors.
Date: Apr 9, Patent Title: Synthetic microfluidic blood-brain barrier. Date: May 7, Patent Title: Microfluidic biological extraction chip. Date: Nov 19, Patent Title: Synthetic microfluidic microvasculature networks. Date: Sep 9, Patent Title: Particle adhesion assay for microfluidic bifurcations.
Madison, AL - joint inventors. Date: Jan 27, Patent Title: Microfluidic assay in idealized microvascular network for characterization of leukocyte adhesion cascade. Date: Mar 22, Patent Title: Synthetic microfluidic systems for wound healing and hemostasis.
Patent Number: 9,, Class: 1. Date: Sep 27, Patent Title: Microfluidic assay in idealized microvascular network for selection and optimization of drug delivery vehicles to simulated tumors. Date: Oct 10, For the next three years, Daly fought between Cage Rage and regional bouts, earning herself a , with 8 finishes.
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The purpose of the Conference is to share the experience of leading experts in the application of innovative technologies, mathematical methods and information management systems in industrial production, in the fields of aerospace, energy and chemical engineering, materials science and design of new materials, as well as engineering and automation of technological processes. Improving the distribution uniformity of surface hardness of the cured CFRP molniezaschita coating during processing in a microwave field elektromagnitnom.
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Karshi Engineering and Economics Institute Uzbekistan. On the features of production, design and reliability assessment of enclosing composite structures with effective thermal insulation. Victor M Bobryashov and Nikolay Bushuev. CdAs2 — MnAs system investigation.
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Models for dispatching machine-building divisions. Approaches to standardization in the automatic control of water quality for water supply and wastewater systems. G A Sambursky. Development of a methodology for evaluating the operational properties of elastic elements for various purposes by acoustic emission. Kirov", St. Analysis of the uniform distribution of herbicides in the interstitial zone with a rod with a deviating section. Determination of the energy efficiency of drying hawthorn fruit in a drum dryer with a paddle mixing device.
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Refer to Figure 9—source data 1. Polyubiquitination of signaling proteins by the Cbl family can lead to their degradation via the endolysosome or proteasome and to non-degradation-mediated inhibition Duan et al. These E3 ligases must in turn be tightly regulated to prevent hyperresponsive or aberrant immune signaling while maintaining proper pathogen clearance Zhu et al. It is also an SFK substrate, requiring phosphorylation to become fully activated. These binding interfaces promote general c-Cbl interaction with all the SFKs.
In this paper we report that c-Cbl targets LynA for rapid and specific degradation, a process that occurs with a half-life of a minute in BMDMs. Exploiting the similarity between LynA and LynB to make a limited set of point mutations, we have discovered that, while activation-loop phosphorylation and SH3-domain interactions mediate slower-phase targeting by c-Cbl and other E3 ligases, rapid degradation of LynA is triggered by an interaction peculiar to its unique-domain insert region.
We have discovered that phosphorylation of Y32 in the unique-region insert of LynA is required for its rapid degradation. This phosphorylation occurs within seconds of LynA activation in macrophages, and flags LynA for rapid, c-Cbl-mediated polyubiquitination. Phosphorylation of LynA Y32 had been observed in neuroblastoma cell lines upon receptor-tyrosine-kinase activation Palacios-Moreno et al.
We now present evidence that LynA Y32 is phosphorylated in primary macrophages, where this potentially positive-regulatory function paradoxically induces LynA to trigger its own c-Cbl-mediated polyubiquitination and degradation, a process underlying its function as a rheostat controlling the LynA checkpoint.
Interestingly, the shorter isoform of Hck can also induce degradation of LynA, although less efficiently than Lyn itself. Degradation of LynA depends on a minimum of two phosphorylation events: the phosphorylation of LynA Y32 and activating phosphorylation of c-Cbl Feshchenko et al.
The ability of individual Src family members to induce LynA degradation necessarily relies on the efficiency of these two processes. This suggests, perhaps surprisingly Lock et al. On the other hand, Syk and FynT have both been reported to phosphorylate and activate c-Cbl Feshchenko et al. Again, we conclude that the substrate preferences of the Src family members are tightly regulated, contradicting the assumption that downstream players in the ITAM signaling pathway are activated en bloc by semi-promiscuous kinases.
In this ultrafast feedback process, activation-loop- and Yautophosphorylated LynA could efficiently phosphorylate and activate c-Cbl and trigger its own degradation, potentially then releasing c-Cbl for other positive- and negative-regulatory signaling functions. It is also possible that, by analogy to a similarly situated but non-homologous phosphorylation site in Hck pY29 Johnson et al. Hyperactivated LynA could then efficiently phosphorylate and activate nearby or pre-complexed c-Cbl, triggering its own degradation more aggressively than do the other SFKs.
LynA-upregulated tumors have the more invasive phenotype Tornillo et al. Aberrant activation driven by ITAM signaling pathways in macrophages is a known driver of autoimmune and inflammatory disease, with activated macrophages accumulating in chronically inflamed tissues in the absence of an active infection Wynn et al.
Regulation of LynA protein expression and deactivation kinetics by c-Cbl could, via the LynA checkpoint, prevent the initiation of pathological signaling. The threshold for macrophage activation is modulated by changes to their local environment. Dynamic changes in SFK and c-Cbl levels could modulate the macrophage activation threshold, ensuring that macrophages respond appropriately during times of infection low threshold and limiting aberrant activation in response to cellular debris and small-scale antibody complexes during inflammatory resolution high threshold.
Like macrophages, mast cells reside in nearly every tissue and perform environment-specific functions in addition to sensing non-self Galli et al. While macrophages have distinct anti-inflammatory roles as professional phagocytes in the silent clearance of apoptotic cells and agents of wound healing, mast cells are constitutively primed for ITAM-induced triggering, releasing preformed granules that contain inflammatory cytokines, chemokines, prostaglandins, and proteases.
Pursuant to these differing functions, macrophages continuously gauge ITAM ligand valency i. One striking difference between the macrophage and mast-cell ITAM regulatory machinery is that mast cells express almost no c-Cbl. Regulated degradation tunes down the LynA rheostat, blocking cell signaling via the LynA checkpoint at low cellular doses of LynA and overriding the LynA checkpoint at higher doses.
Mast cells express almost no c-Cbl and maintain high levels of LynA at steady state and over time, consistent with permissive signaling in the absence of a large-scale ITAM receptor clustering event nucleated by multivalent receptor ligation. Appropriate regulation of immune receptor thresholds is critical for maintaining the function of innate immune cells; threshold dysregulation can lead to chronic feedback loops that drive inflammatory signaling in autoimmune disease and conversely tumor-supporting immunosuppressive signaling Wynn et al.
Elucidating the mechanisms by which the LynA checkpoint is regulated may allow us to tune immune-cell sensitivity and reprogram pathological cells. Farrar University of Minnesota Chiang et al. Animals are kept under supervision of a licensed doctor of veterinary medicine and supporting veterinary staff under strict NIH guidelines.
Shimizu University of Minnesota , respectively. Louis, MO as described previously Phee et al. Jurkat and JCaM1. A construct containing eGFP alone was used as a control. Cells were resuspended in ice-cold PBS and coverslipped on glass slides directly before imaging. Exposure times for GFP were set to normalize the brightness of the GFP across the different transfection conditions, as cell-to-cell expression was variable. Weiss University of California, San Francisco.
Lowell University of California, San Francisco. BMDMs were prepared using standard methods Zhu et al. Briefly, bone marrow was extracted from femura and tibiae of mice aged 6—8 weeks. All myeloid cells were rested or primed overnight before stimulation. Weiss, University of California, San Francisco. Control double-stranded RNA from IDT was not predicted to be complementary to any sequence in the human or mouse transcriptome. Transfections were pooled from several cuvettes to obtain enough cells for several stimulation conditions.
Cells were used 48 hr after transfection. An saRNA sequence designed to upregulate transcription of Cbl was designed using an algorithm published previously Voutila et al. On day three the cells were transferred into fresh mast-cell medium and rested overnight. BMDM stimulations have been described previously Freedman et al.
Shokat University of California, San Francisco. Whole-cell lysates were prepared for immunoblotting by sonication with a Diagenode Bioruptor Diagenode, Inc, Denville, NJ for 3 min and boiling for 15 min. For immunoblotting 0. Images were background-subtracted, and bands of the appropriate molecular weight were demarcated and analyzed for each gel lane.
Unless specified otherwise, error bars represent standard error of the mean from at least three independent experiments. Asterisks reflect specified P-values. No gamma or other nonlinear correction was applied. Images were rotated for figure preparation only after densitometry analysis.
Louis, MO. LynA-specific antibody Freedman et al. A bicinchoninic acid BCA protein assay was performed on the whole-cell lysates Thermo Fisher to ensure that equal amounts of protein from untreated and 3-IB-PP1-treated cells would be subjected to immunoprecipitation. A bovine serum albumin BSA standard curve was used to quantifty the total protein in each IP sample and ensure that equal mass quantities were subjected to trypsin digest.
For this calibration, we ran 0. Total protein was visualized without fixation using SimplyBlue Safestain Thermo Fisher according to manufacturer's instructions. Staining was quantified by densitometry, and BSA signals vs. This function was then used to quantify the total protein in each immunoprecipitate.
Digested samples were submitted for identification at the University of Minnesota Center for Mass Spectrometry and Proteomics. Peptide elution was performed using a tripartite gradient, decreasing the fraction of Buffer A 0. The column was mounted in a nanospray source in line with an Orbitrap Fusion mass spectrometer Thermo Scientific. Spray voltage was 2. The acquisition method combined two scan events corresponding to a full scan event and a parallel reaction monitoring PRM event targeting the singly-, doubly-, and triply-charged precursor ions of the three phosphorylated peptides without scheduling.
The precursor ion of each targeted peptide was isolated using an isolation window of 1. PRM data were collected in centroid mode. Experiments were run in triplicate, and the entire experiment was performed independently twice. All data generated or analysed during this study are included in the manuscript and supporting files.
Source data files have been provided for graphs in Figure 1, Figure 1-figure supplement 1, Figure 2, Figure 3, Figure 3-figure supplement 2, Figure 4, Figure 4-figure supplement 1, Figure 4-figure supplement 5, Figure 5, Figure 6, Figure 6-figure supplement 1, Figure 7, Figure 8, and Figure 9.
In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included. Thank you for submitting your article "An orthogonal c-Cbl recognition mode targets LynA for rapid degradation and builds specificity into the LynA checkpoint" for consideration by eLife.
Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Tadatsugu Taniguchi as the Senior Editor. The reviewers have opted to remain anonymous. The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. This is an interesting study that builds upon previous work by these authors that demonstrates a possible check-point role for LynA in macrophage activation caused by activation-induced degradation.
This study demonstrates that this degradation is mediated by the E3 Ub ligase cCbl that targets the insert region on LynA and requires Tyr The data supporting the authors' conclusions are strong. The major weakness of the study is that cCbl has already been reported to cause Lyn degradation.
Nevertheless, the current study has merit because it demonstrates that this role is specific for LynA and because the study presents these observations within an important biological context. Freedman and co-workers demonstrate a rapid negative feedback loop in a specific spliced form of the Src family tyrosine kinase LynA that is not seen with the alternatively spliced form, LynB, or with other members of the SFK family.
Why does its phosphorylation enhance ubiquitination and degradation? It is suggested that this could be a binding site for cCBL as well as an activating phosphorylation? Was the binding of cCBL tested? This would be a logical mechanism of action. Does Y32 phosphorylation induce relocalization of Lyn into the cytoplasm from the membrane?
That could also explain the mechanism. If so, is this phosphorylation in cis or trans? For example, is autophosphorylation concentration-dependent in vitro. Can the Y32 phosphorylation be reconstituted using kinase-inactive and phosphorylation site mutated proteins? Would it result in loss of resting LynA? Would that attenuate ERK activation? In the original manuscript we reported that unique-region Y32 is the key residue marking LynA for rapid polyubiquitination and degradation.
We speculated that this might be a site of phosphorylation, based on a prediction algorithm NetPhos 2. However, we had no evidence that this site was actually phosphorylated in non-cancer cells or in hematopoietic cells, and thus the mechanism of Y32 in targeting LynA for rapid polyubiquitination was unclear. The results of these experiments are reported in new Figure 4 and Figure 4—figure supplements We can now unequivocally state that in primary macrophages LynA Y32 is a site of phosphorylation, induced upon SFK activation.
Moreover, pY32 is massively enriched in polyubiquitinated LynA. We therefore conclude that activation of the SFKs leads to phosphorylation of LynA at position Y32, and that this is the mechanism by which LynA is targeted specifically for polyubiquitination and degradation. These mass spectrometry experiments were quite labor intensive and occupied the entire revision time frame. We did perform co-immunoprecipitation experiments and found inducible association of c-Cbl with LynA, but we were unable to demonstrate that this association was due to Y32 phosphorylation.
Like all SFKs, LynA interacts with c-Cbl via several binding interfaces, including the SH3 domain, the activation-loop peptide binding site, and the activation-loop phosphotyrosine. Additional cloning to isolate the contributions of each of these interfaces was not possible before resubmission.
We were unable to get good staining of total LynA, but we were able to visualize eGFP-tagged unique-region constructs. As shown in new Figure 3—figure supplement 1. All three constructs were membrane localized in the presence and absence of 3-IB-PP1, even in the presence of a kinase-active LynB construct. While the LynA kinase domain may add some localization functionality, we can say that the Y32 mutation does not itself change the localization of the LynA unique region.
We used targeted mass spectrometry to detect phosphorylation of LynA Y32 with increasing doses of recombinant LynA to probe cis vs. Unfortunately, the data were uninterpretable because recombinant LynA purified from insect cells is already phosphorylated at position Y We are continuing to troubleshoot these experiments, but they are likely to take several more months and so unfortunately were not possible for this revision. These data, presented in new Figure 7 and Figure 7—figure supplement , reveal SFK-specific regulation of LynA degradation, with LynA itself likely inducing its own degradation via Y32 autophosphorylation.
One of these constructs was found to increase c-Cbl expression an average of 2-fold, and we were able to detect an increase in LynA degradation and corresponding decrease in Syk and Erk phosphorylation in response to 3-IB-PP1 in c-Cbl overexpressing mast cells relative to mock-transfected cells new panels, Figure 9G-J. With this modest increase in c-Cbl expression, steady-state expression of LynA was not significantly affected.
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. This article is distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use and redistribution provided that the original author and source are credited. Article citation count generated by polling the highest count across the following sources: Crossref , PubMed Central , Scopus.
Clustering of receptors associated with immunoreceptor tyrosine-based activation motifs ITAMs initiates the macrophage antimicrobial response. Macrophages also encounter nonpathogenic molecules that cluster receptors weakly and must tune their sensitivity to avoid inappropriate responses. To investigate this response threshold, we compared signaling in the presence and absence of receptor clustering using a small-molecule inhibitor of Csk, which increased SFK activation and produced robust membrane-proximal signaling.
Inflammatory priming of macrophages upregulated LynA and promoted receptor-independent signaling. Together, the basal-state signaling checkpoint regulated by LynA expression and degradation and the signaling reorganization initiated by receptor clustering allow cells to discriminate optimally between pathogens and nonpathogens.
Ageing is associated with increased vulnerability to environmental cold exposure. Previously, we identified the role of the cold-sensitive transient receptor potential TRP A1, M8 receptors as vascular cold sensors in mouse skin. We hypothesised that this dynamic cold-sensor system may become dysfunctional in ageing.
This implies the reliance of the already reduced cold-induced vascular response in ageing mice on remaining TRP receptor activity. The cold-induced vascular response is important for sensing cold and retaining body heat and health. These findings reveal that cold sensors, essential for this neurovascular pathway, decline as ageing onsets.
Experiments with hippocampal neurons indicate that this mechanism contributes toward LTD. This non-canonical mode of PKA regulation may underlie many other cellular processes. Cited 5 Views 1, Annotations Open annotations. The current annotation count on this page is being calculated.
Cite this article as: eLife ;8:e doi: Figure 1 with 2 supplements see all. Download asset Open asset. Figure 2. Figure 3 with 2 supplements see all. Figure 3—source data 1 Quantification of c-Cbl and Lyn proteins coexpressed in Jurkat cells. Figure 4 with 5 supplements see all. Figure 5. Figure 6 with 1 supplement see all. Figure 6—source data 1 Quantification of kinase-impaired LynA proteins expressed in Jurkat cells. Birmingham, AL - joint inventors.
Name: Powers, William T. City: Huntsville County: Madison. Date: Sep 21, Patent Title: Cryogenic high pressure sensor module. Patent Number: 5,, Class:. Notes: Chapman, John J. Yorktown, VA - joint inventors. Date: Jun 5, Patent Title: Cryogenic, absolute, high pressure sensor. Patent Number: 6,, Class: Name: Prabhakarpandian, Balabhaskar. City: Madison County: Madison. Date: Sep 19, Patent Title: Spacer for delivery of medications from an inhaler to children and breathing impaired patients.
Patent Number: 7,, Class: Date: May 25, Patent Title: Synthetic microfluidic microvasculature network. Date: May 8, Patent Number: 8,, Class: Date: Jan 15, Patent Title: Microfluidic assay for selection and optimization of drug delivery vehicles to tumors. Date: Apr 9, Patent Title: Synthetic microfluidic blood-brain barrier. Date: May 7, Patent Title: Microfluidic biological extraction chip.
Date: Nov 19, Patent Title: Synthetic microfluidic microvasculature networks. Date: Sep 9, Patent Title: Particle adhesion assay for microfluidic bifurcations. Madison, AL - joint inventors. Date: Jan 27, Patent Title: Microfluidic assay in idealized microvascular network for characterization of leukocyte adhesion cascade. Date: Mar 22, Patent Title: Synthetic microfluidic systems for wound healing and hemostasis. Patent Number: 9,, Class: 1. Date: Sep 27, Patent Title: Microfluidic assay in idealized microvascular network for selection and optimization of drug delivery vehicles to simulated tumors.
Date: Oct 10, Patent Title: Synthetic microfluidic systems for wound healing. Date: Apr 3, Patent Title: Synthetic microfluidic systems for tumor metastasis. Patent Title: Multi-chambered cell culture device to model organ microphysiology. Date: Jul 3, Patent Title: Cell culture device with an array of microfluidic networks. Patent Number: 10,, Class: 1. Date: Feb 25, Date: May 5, Date: Sep 15, Patent Title: Synthetic microfluidic systems for hypoxia.
Name: Prabhakarpandian, Balabhasker. Date: Jul 15,
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