Signaling Entities Research Articles

PLxIS-containing domains are biochemically flexible regulators of interferons and metabolism

Signal transduction proteins containing a pLxIS motif induce interferon (IFN) responses central to antiviral immunity. Apart from their established roles in activating the IFN regulator factor (IRF) transcription factors, the existence of additional pathways and functions associated with the pLxIS motif is unknown. Using a synthetic biology-based platform, we identified two orphan pLxIS-containing proteins that stimulate IFN responses independent of all known pattern-recognition receptor pathways. We further uncovered a diversity of pLxIS signaling mechanisms, where the pLxIS motif represents one component of a multi-motif signaling entity, which has variable functions in activating IRF3, the TRAF6 ubiquitin ligase, IκB kinases, mitogen-activated protein kinases, and metabolic activities. The most diverse pLxIS signaling mechanisms were associated with the highest antiviral activities in human cells. The flexibility of domains that regulate IFN signaling may explain their prevalence in nature.

Click chemical ligation-enabled digital particle counting for multiplexed microRNA analysis

Digital counting assays, that quantify targets by counting individual signal entities, provide a promising way for the sensitive analysis of biomarkers even at the single-molecule level. Considering the requirements of complex enzyme-catalyzed amplification techniques and specialized instruments in traditional digital counting biosensors, herein, a simple digital counting platform for microRNA (miRNA) analysis is developed by employing the miRNA-templated click chemical ligation to hinge ultrabright quantum dot-doped nanoparticles (QDNPs) on the bottom of microplate well. Compared with the traditional short miRNA-mediated sandwich hybridization mechanism, the click chemistry-mediated ligation featured enhanced stability, achieving higher sensitivity by directly counting the number of QDNPs with a common wide-field fluorescence microscope. Furthermore, enzyme-free cycling click ligation strategy is adopted to push the detection limit of miRNA down to a low level of 8 fM. What is more, taking advantages of the tunable emission wavelength and narrow emission spectra of fluorescent nanoparticles, the platform enables simultaneous detection of multiplex miRNA targets without cross interference. Benefiting from the simple operation, high sensitivity, and good generality, miRNA analysis in complex samples is successfully achieved. This method not only pioneers a new route for digital counting assays but also holds great potential in miRNA-related biological researches.

Platelet Spreading and Clot Retraction are Regulated by two Distinct αIIbβ3 Outside-in Signaling Pathways.

Bi-directional signaling through platelet integrin αIIbβ3 is essential in hemostasis and thrombosis. In quiescent platelets αIIbβ3 is in a low-affinity ligand binding state. However, upon platelet activation by agonists through inside-out signaling, a rapid switch in the conformation of the integrin results in a high affinity ligand binding state capable of binding soluble fibrinogen. Ligand binding to the αIIbβ3 induces a signaling termed outside-in signaling that regulate platelet spreading and clot retraction. These events are often interchangeably used to represent outside-in signaling pathway. Using pharmacological inhibitors of known signaling molecules that have been implicated to regulate outside-in signaling, we assessed human platelet spreading and clot retraction. We found that inhibition of PI3K, PLC, PKC, and FAK strongly attenuated both platelet spreading and clot retraction suggesting that they are essential for both clot retraction and platelet spreading. Whereas inhibition of Rac1, ROCK, p38, and MEK did not affect platelet spreading but significantly delayed clot retraction suggesting that these signaling molecules do not participate in platelet spreading. Interestingly, Src family kinases (SFKs) are required for platelet spreading and FAK activation but suppresses clot retraction since their inhibition causes faster clot retraction. Thus, it becomes evident that platelet spreading, and clot retraction are differently regulated through αIIbβ3 outside-in signaling and should not be used interchangeably as readout for αIIbβ3 outside-in signaling assessment. Significance Statement Current anti-platelet drugs have increased risk of bleeding and low efficacy. There is an increased effort to identify novel anti-platelet agents that have improved efficacy with reduced risk of bleeding. It is increasingly felt that inhibition of αIIbβ3-induced outside-in signaling may inhibit thrombosis without compromising hemostasis. However, the signaling entities regulating outside-in signaling is poorly understood. Our work included in this manuscript delineates the distinct signaling pathways involved in outside-in signaling and identify potential novel targets for intervention of thrombosis.

Unraveling the Connection: Cholesterol, Calcium Signaling, and Neurodegeneration.

Cholesterol and calcium play crucial roles as integral structural components and functional signaling entities within the central nervous system. Disruption in cholesterol homeostasis has been linked to Alzheimer's, Parkinson's, and Huntington's Disease while alterations in calcium signaling is hypothesized to be a key substrate for neurodegeneration across many disorders. Despite the importance of regulated cholesterol and calcium homeostasis for brain health there has been an absence of research investigating the interdependence of these signaling molecules and how they can tune each other's abundance at membranes to influence membrane identity. Here, we discuss the role of cholesterol in shaping calcium dynamics in a neurodegenerative disorder that arises due to mutations in the lysosomal cholesterol transporter, Niemann Pick Type C1 (NPC1). We discuss the molecular mechanisms through which altered lysosomal cholesterol transport influences calcium signaling pathways through remodeling of ion channel distribution at organelle-organelle membrane contacts leading to neurodegeneration. This scientific inquiry not only sheds light on NPC disease but also holds implications for comprehending other cholesterol-associated neurodegenerative disorders.

Modulating the dynamics of NFκB and PI3K enhances the ensemble-level TNFR1 signaling mediated apoptotic response

Cell-to-cell variability during TNFα stimulated Tumor Necrosis Factor Receptor 1 (TNFR1) signaling can lead to single-cell level pro-survival and apoptotic responses. This variability stems from the heterogeneity in signal flow through intracellular signaling entities that regulate the balance between these two phenotypes. Using systematic Boolean dynamic modeling of a TNFR1 signaling network, we demonstrate that the signal flow path variability can be modulated to enable cells favour apoptosis. We developed a computationally efficient approach “Boolean Modeling based Prediction of Steady-state probability of Phenotype Reachability (BM-ProSPR)” to accurately predict the network’s ability to settle into different phenotypes. Model analysis juxtaposed with the experimental observations revealed that NFκB and PI3K transient responses guide the XIAP behaviour to coordinate the crucial dynamic cross-talk between the pro-survival and apoptotic arms at the single-cell level. Model predicted the experimental observations that ~31% apoptosis increase can be achieved by arresting Comp1 – IKK* activity which regulates the NFκB and PI3K dynamics. Arresting Comp1 – IKK* activity causes signal flow path re-wiring towards apoptosis without significantly compromising NFκB levels, which govern adequate cell survival. Priming an ensemble of cancerous cells with inhibitors targeting the specific interaction involving Comp1 and IKK* prior to TNFα exposure could enable driving them towards apoptosis.

NO-ferroheme is a signaling entity in the vasculature

Despite wide appreciation of the biological role of nitric oxide (NO) synthase (NOS) signaling, questions remain about the chemical nature of NOS-derived bioactivity. Here we show that NO-like bioactivity can be efficiently transduced by mobile NO-ferroheme species, which can transfer between proteins, partition into a hydrophobic phase and directly activate the sGC–cGMP–PKG pathway without intermediacy of free NO. The NO-ferroheme species (with or without a protein carrier) efficiently relax isolated blood vessels and induce hypotension in rodents, which is greatly potentiated after the blockade of NOS activity. While free NO-induced relaxations are abolished by an NO scavenger and in the presence of red blood cells or blood plasma, a model compound, NO-ferroheme-myoglobin preserves its vasoactivity suggesting the physiological relevance of NO-ferroheme species. We conclude that NO-ferroheme behaves as a signaling entity in the vasculature.

Ultrasound-Guided Posterior Iliac Crest Aspiration

Background: Bone Marrow Aspirate (BMA) contains growth factors and signaling entities essential for local cellular repair. BMA and concentrated BMA injections (BMC) are used for orthopedic procedures, such as in treating cartilaginous and bony defects. Several techniques exist for harvesting BMA. A technique termed ultrasound-guided posterior iliac crest aspiration (UPICA) is of special interest. This case series wasundertaken to determine if practitioners can effectively perform the UPICA technique in a clinical setting. Methods: In accordance with the UPICA technique, ultrasound guidance was used to localize the posterior iliac crest. Following local anesthesia, aspiration was performed using 10 mL syringes at multiple depths. The periosteum was entered once. A needle was advanced 0.5 cm with each new depth. At each penetration level, 10 mL was drawn before rotating 90 degrees and aspirating an additional 10 mL. Following aspiration at various depths, approximately 60 mL of BMA was obtained. The inclusion criteria for this study were patients with diagnosed glenohumeral osteoarthritis, knee osteoarthritis, hip osteoarthritis, or rotator cuff tears whose condition had failed to improve with conservative care. The UPICA technique was performed in each case by the senior author (BG). Results: For the 10 cases, the average patient age was 72 (SD 7.6; range 58–81) years old. The sample included 8 males and 2 females. The mean total nucleated cell count (TNCC)/mL BMA was 9.7E+06 (SD 2.9E+06), while the TNCC/mL bone marrow concentrate (BMC) was 43.9E+06 (SD 29.5E+06), a 4.3 (SD 2.3)-fold increase in concentration. The mean cell colony forming units-fibroblasts (CFU-f)/mL BMA was 520 (SD 155) with a mean CFU-f/mL BMC of 4899 (SD 2887) resulting in a 9.6 (SD 4.6)-fold increase in concentration. The viability for the BMA was 97.5 (SD 2.3) %, and the BMC was 94.9 (SD 4.55) %. Conclusions: Utilizing the UPICA technique, similar yields for the BMC CFU-f/mL counts were obtained to those reported previously by other clinicians. Additionally, the results demonstrated the ability of the bone marrow concentration system to successfully concentrate the BMA. Based on these findings, the research team endorses the UPICA technique as a viable method for bone marrow aspiration.

DNA repair byproduct 8-oxoguanine base promotes myoblast differentiation

Muscle contraction increases the level of reactive oxygen species (ROS), which has been acknowledged as key signaling entities in muscle remodeling and to underlie the healthy adaptation of skeletal muscle. ROS inevitably endows damage to various cellular molecules including DNA. DNA damage ought to be repaired to ensure genome integrity; yet, how DNA repair byproducts affect muscle adaptation remains elusive. Here, we showed that exercise elicited the generation of 8-oxo-7,8-dihydroguanine (8-oxoG), that was primarily found in mitochondrial genome of myofibers. Upon exercise, TA muscle's 8-oxoG excision capacity markedly enhanced, and in the interstitial fluid of TA muscle from the post-exercise mice, the level of free 8-oxoG base was significantly increased. Addition of 8-oxoG to myoblasts triggered myogenic differentiation via activating Ras-MEK-MyoD signal axis. 8-Oxoguanine DNA glycosylase1 (OGG1) silencing from cells or Ogg1 KO from mice decreased Ras activation, ERK phosphorylation, MyoD transcriptional activation, myogenic regulatory factors gene (MRFs) expression. In reconstruction experiments, exogenously added 8-oxoG base enhanced the expression of MRFs and accelerated the recovery of the injured skeletal muscle. Collectively, these data not only suggest that DNA repair metabolite 8-oxoG function as a signal entity for muscle remodeling and contribute to exercise-induced adaptation of skeletal muscle, but also raised the potential for utilizing 8-oxoG in clinical treatment to skeletal muscle damage-related disorders.

Extracellular vesicles - on the cusp of a new language in the biological sciences.

Extracellular vesicles (EVs) play a key role both in physiological balance and homeostasis and in disease processes through their ability to participate in intercellular signaling and communication. An ever-expanding knowledge pool and a myriad of functional properties ascribed to EVs point to a new language of communication in biological systems that has opened a path for the discovery and implementation of novel diagnostic applications. EVs originate in the endosomal network and via non-random shedding from the plasma membrane by mechanisms that allow the packaging of functional cargoes, including proteins, lipids, and genetic materials. Deciphering the molecular mechanisms that govern packaging, secretion and targeted delivery of extracellular vesicle-borne cargo will be required to establish EVs as important signaling entities, especially when ascribing functional properties to a heterogeneous population of vesicles. Several molecular cascades operate within the endosomal network and at the plasma membrane that recognize and segregate cargos as a prelude to vesicle budding and release. EVs are transferred between cells and operate as vehicles in biological fluids within tissues and within the microenvironment where they are responsible for short- and long-range targeted information. In this review, we focus on the remarkable capacity of EVs to establish a dialogue between cells and within tissues, often operating in parallel to the endocrine system, we highlight selected examples of past and recent studies on the functions of EVs in health and disease.

The carboxyl-terminal TSP1-homology domain is the biologically active effector peptide of matricellular protein CCN5 that counteracts profibrotic CCN2

Cellular Communication Network (CCN) proteins have multi-modular structures important for their roles in cellular responses associated with organ development and tissue homeostasis. CCN2 has previously been reported to be secreted as a preproprotein that requires proteolytic activation to release its bioactive carboxyl-terminal fragment. Here, our goal was to resolve whether CCN5, a divergent member of the CCN family with converse functions relative to CCN2, releases the TSP1 homology domain as its bioactive signaling entity. The recombinant CCN5 or CCN3 TSP1 homology domains were produced in ExpiCHO-S or DG44 CHO cells as secretory fusion proteins appended to the carboxyl-terminal end of His-Halo-Sumo or amino-terminal end of human albumin, and purified from the cell culture medium. We tested these fusion proteins in various phosphokinase signaling pathways or cell physiologic assays. Fusion proteins with the CCN5 TSP1 domain inhibited key signaling pathways previously reported to be stimulated by CCN2, irrespective of fusion partner. The fusion proteins also efficiently inhibited CCN1/2-stimulated cell migration and gap closure following scratch wound of fibroblasts. Fusion protein with the CCN3 TSP1 domain inhibited these functions with similar efficacy and potency as that of the CCN5 TSP1 domain. The CCN5 TSP1 domain also recapitulated a positive regulatory function previously assigned to full-length CCN5, i.e. induction of estrogen receptor-α mRNA expression in triple negative MDA-MB-231 mammary adenocarcinoma cells and inhibited epithelial-to-mesenchymal transition and CCN2-induced mammosphere formation of MCF7 adenocarcinoma cells. In conclusion, the CCN5 TSP1 domain is the bioactive entity that confers the biologic functions of unprocessed CCN5.

Lipolysis-derived linoleic acid drives beige fat progenitor cell proliferation.

De novo beige adipocyte biogenesis involves the proliferation of progenitor cells in white adipose tissue (WAT); however, what regulates this process remains unclear. Here, we report that in mouse models but also in human tissues, WAT lipolysis-derived linoleic acid triggers beige progenitor cell proliferation following cold acclimation, β3-adrenoceptor activation, and burn injury. A subset of adipocyte progenitors, as marked by cell surface markers PDGFRα or Sca1 and CD81, harbored cristae-rich mitochondria and actively imported linoleic acid via a fatty acid transporter CD36. Linoleic acid not only was oxidized as fuel in the mitochondria but also was utilized for the synthesis of arachidonic acid-derived signaling entities such as prostaglandin D2. Oral supplementation of linoleic acid was sufficient to stimulate beige progenitor cell proliferation, even under thermoneutral conditions, in a CD36-dependent manner. Together, this study provides mechanistic insights into how diverse pathophysiological stimuli, such as cold and burn injury, promote de novo beige fat biogenesis.

Extracellular membrane vesicles from Limosilactobacillus reuteri strengthen the intestinal epithelial integrity, modulate cytokine responses and antagonize activation of TRPV1.

Bacterial extracellular membrane vesicles (MV) are potent mediators of microbe-host signals, and they are not only important in host-pathogen interactions but also for the interactions between mutualistic bacteria and their hosts. Studies of MV derived from probiotics could enhance the understanding of these universal signal entities, and here we have studied MV derived from Limosilactobacillus reuteri DSM 17938 and BG-R46. The production of MV increased with cultivation time and after oxygen stress. Mass spectrometry-based proteomics analyses revealed that the MV carried a large number of bacterial cell surface proteins, several predicted to be involved in host-bacteria interactions. A 5'-nucleotidase, which catalyze the conversion of AMP into the signal molecule adenosine, was one of these and analysis of enzymatic activity showed that L. reuteri BG-R46 derived MV exhibited the highest activity. We also detected the TLR2 activator lipoteichoic acid on the MV. In models for host interactions, we first observed that L. reuteri MV were internalized by Caco-2/HT29-MTX epithelial cells, and in a dose-dependent manner decreased the leakage caused by enterotoxigenic Escherichia coli by up to 65%. Furthermore, the MV upregulated IL-1β and IL-6 from peripheral blood mononuclear cells (PBMC), but also dampened IFN-γ and TNF-α responses in PBMC challenged with Staphylococcus aureus. Finally, we showed that MV from the L. reuteri strains have an antagonistic effect on the pain receptor transient receptor potential vanilloid 1 in a model with primary dorsal root ganglion cells from rats. In summary, we have shown that these mobile nanometer scale MV reproduce several biological effects of L. reuteri cells and that the production parameters and selection of strain have an impact on the activity of the MV. This could potentially provide key information for development of innovative and more efficient probiotic products.

Structural principles of B cell antigen receptor assembly.

The B cell antigen receptor (BCR) is composed of a membrane-bound class M, D, G, E or A immunoglobulin for antigen recognition1-3 and a disulfide-linked Igα (also known as CD79A) and Igβ (also known as CD79B) heterodimer (Igα/β) that functions as the signalling entity through intracellular immunoreceptor tyrosine-based activation motifs (ITAMs)4,5. The organizing principle of the BCR remains unknown. Here we report cryo-electron microscopy structures of mouse full-length IgM BCR and its Fab-deleted form. At the ectodomain (ECD), the Igα/β heterodimer mainly uses Igα to associate with Cµ3 and Cµ4 domains of one heavy chain (µHC) while leaving the other heavy chain (µHC') unbound. The transmembrane domain (TMD) helices of µHC and µHC' interact with those of the Igα/β heterodimer to form a tight four-helix bundle. The asymmetry at the TMD prevents the recruitment of two Igα/β heterodimers. Notably, the connecting peptide between the ECD and TMD of µHC intervenes in between those of Igα and Igβ to guide TMD assembly through charge complementarity. Weaker but distinct density for the Igβ ITAM nestles next to the TMD, suggesting potential autoinhibition of ITAM phosphorylation. Interfacial analyses suggest that all BCR classes utilize a general organizational architecture. Our studies provide a structural platform for understanding B cell signalling and designing rational therapies against BCR-mediated diseases.

Co-culture platform for neuron-astrocyte interaction using optogenetic modulation.

For decades, the role of glial cells has attracted attention in the neuroscience field. Particularly, although the astrocyte is the most abundant glial cell type, it was believed to function as a passive support cell. However, recent evidence suggests that astrocytes actively release various gliotransmitters and signaling entities that regulate the excitability of pre-and post-synaptic neurons in the brain. In this study, we optimized the ratio of astrocytes and neurons to investigate the interaction between astrocytes and neurons. To this end, postnatal day 0-1 rodent hippocampi were dissociated and cultured. The neuron-astrocyte ratio was monitored for up to 3weeks after treating the cultures with 0, 1, and 5µM of cytosine arabinoside (Ara-C) at DIV 2. Subsequently, from postnatal transgenic (TG) mouse expressing ChR2 on astrocytes, hippocampi were cultured on the microelectrode array (MEA) with the desired neuron-astrocyte ratio. The astrocyte was irradiated using a 473nm blue laser for 30s in a cycle of 10Hz and electrophysiological recording was performed to verify the activities of neurons induced by the stimulated astrocytes. Astrocytes and neurons in both co-cultures increased at an identical ratio when treated with 1µM Ara-C, whereas they decreased significantly when treated with 5µM Ara-C. Particularly, the laser-stimulated astrocytes induced an increase in the frequency of neuronal activities and lasted after illumination. The proposed co-culture platform is expected to be used in experiments to investigate the network between astrocytes and neurons in vitro.

T-cell trans-synaptic vesicles are distinct and carry greater effector content than constitutive extracellular vesicles

The immunological synapse is a molecular hub that facilitates the delivery of three activation signals, namely antigen, costimulation/corepression and cytokines, from antigen-presenting cells (APC) to T cells. T cells release a fourth class of signaling entities, trans-synaptic vesicles (tSV), to mediate bidirectional communication. Here we present bead-supported lipid bilayers (BSLB) as versatile synthetic APCs to capture, characterize and advance the understanding of tSV biogenesis. Specifically, the integration of juxtacrine signals, such as CD40 and antigen, results in the adaptive tailoring and release of tSV, which differ in size, yields and immune receptor cargo compared with steadily released extracellular vesicles (EVs). Focusing on CD40L+ tSV as model effectors, we show that PD-L1 trans-presentation together with TSG101, ADAM10 and CD81 are key in determining CD40L vesicular release. Lastly, we find greater RNA-binding protein and microRNA content in tSV compared with EVs, supporting the specialized role of tSV as intercellular messengers.

Mechanisms and mathematical modeling of ROS production by the mitochondrial electron transport chain.

Reactive oxygen species (ROS) are recognized both as damaging molecules and intracellular signaling entities. In addition to its role in ATP generation, the mitochondrial electron transport chain (ETC) constitutes a relevant source of mitochondrial ROS, in particular during pathological conditions. Mitochondrial ROS homeostasis depends on species- and site-dependent ROS production, their bioreactivity, diffusion, and scavenging. However, our quantitative understanding of mitochondrial ROS homeostasis has thus far been hampered by technical limitations, including a lack of truly site- and/or ROS-specific reporter molecules. In this context, the use of computational models is of great value to complement and interpret empirical data, as well as to predict variables that are difficult to assess experimentally. During the past decades, various mechanistic models of ETC-mediated ROS production have been developed. Although these often-complex models have generated novel insights, their parameterization, analysis, and integration with other computational models are not straightforward. In contrast, phenomenological (sometimes termed "minimal") models use a relatively small set of equations to describe empirical relationship(s) between ROS-related and other parameters and generally aim to explore system behavior and generate hypotheses for experimental validation. In this review, we first discuss ETC-linked ROS homeostasis and introduce various detailed mechanistic models. Next, we present how bioenergetic parameters (e.g., NADH/NAD+ ratio and mitochondrial membrane potential) relate to site-specific ROS production within the ETC and how these relationships can be used to design minimal models of ROS homeostasis. Finally, we illustrate how minimal models have been applied to explore pathophysiological aspects of ROS.

Analysis of Plasma Using Flow Cytometry Reveals Increased Immune Cell-Derived Extracellular Vesicles in Untreated Relapsing-Remitting Multiple Sclerosis.

Extracellular vesicles (EVs) are secreted from cells under physiological and pathological conditions, and are found in biological fluids while displaying specific surface markers that are indicative of their cell of origin. EVs have emerged as important signaling entities that may serve as putative biomarkers for various neurological conditions, including multiple sclerosis (MS). The objective of this study was to measure and compare immune cell-derived EVs within human plasma between untreated RRMS patients and healthy controls. Using blood plasma and peripheral blood mononuclear cells (PBMCs) collected from RRMS patients and controls, PBMCs and EVs were stained and quantified by flow cytometry using antibodies against CD9, CD61, CD45, CD3, CD4, CD8, CD14, and CD19. While several immune cell-derived EVs, including CD3+, CD4+, CD8+, CD14+, and CD19+ were significantly increased in RRMS vs. controls, no differences in immune cell subsets were observed with the exception of increased circulating CD19+ cells in RRMS patients. Our study demonstrated that plasma-derived EVs secreted from T cells, B cells, and monocytes were elevated in untreated RRMS cases with low disability, despite very limited changes in circulating immune cells, and suggest the utility of circulating EVs as biomarkers in MS.

Endothelin-1 mediated glycosaminoglycan synthesizing gene expression involves NOX-dependent transactivation of the transforming growth factor-β receptor.

G protein-coupled receptor (GPCR) agonist endothelin-1 (ET-1) through transactivation of the transforming growth factor(TGF) β receptor (TGFBR1) stimulates glycosaminoglycan (GAG) elongation on proteoglycans. GPCR agonists thrombin and lysophosphatidic acid (LPA) via respective receptors transactivate the TGFBR1 via Rho/ROCK dependent pathways however mechanistic insight for ET-1 transactivation of the TGFBR1 remains unknown. NADPH oxidase (NOX) generates reactive oxygen species (ROS) and is a signalling entity implicated in the pathogenesis of many diseases including atherosclerosis. If implicated in this pathway, NOX/ROS would be a potential therapeutic target. In this study, we investigated the involvement of NOX in ET-1/ET receptor-mediated transactivation of TGFBR1 to stimulate mRNA expression of GAG chain synthesizing enzymes chondroitin 4-O-sulfotransferase 1 (C4ST-1) and chondroitin sulfate synthase 1 (ChSy-1). The invitro model used vascular smooth muscle cells that were treated with pharmacological antagonists in the presence and absence of ET-1 or TGF-β. Proteins and phosphoproteins isolated from treated cells were quantified by western blotting and quantitative real-time PCR was used to assess mRNA expression of GAG synthesizing enzymes. In the presence of diphenyliodonium (DPI) (NOX inhibitor), ET-1 stimulated phospho-Smad2C levels were inhibited. ET-1 mediated mRNA expression of GAG synthesizing enzymes C4ST-1 and ChSy-1 was also blocked by TGBFR1 antagonists, SB431542, broad spectrum ET receptor antagonist bosentan, DPI and ROS scavenger N-acetyl-L-cysteine. This work shows that NOX and ROS play an important role in ET-1mediated transactivation of the TGFBR1 and downstream gene targets associated with GAG chainelongation. As ROS is involved in GPCR to protein tyrosine kinase receptor transactivation, the NOX/ROS axis presents as the first common biochemical target in all GPCR to kinase receptor transactivation signalling.

CRISPR deletions in cell lines for reconstitution studies of pseudokinase function.

The non-catalytic cousins of protein kinases, the pseudokinases, have grown to prominence as indispensable signaling entities over the past decade, despite their lack of catalytic activity. Because their importance has only been fully embraced recently, many of the 10% of the human kinome categorized as pseudokinases are yet to be attributed biological functions. The advent of CRISPR-Cas9 editing to genetically delete pseudokinases in a cell line of interest has proven invaluable to dissecting many functions and remains the method of choice for gene knockout. Here, using the terminal effector pseudokinase in the necroptosis cell death pathway, MLKL, as an exemplar, we describe a method for genetic knockout of pseudokinases in cultured cells. This method does not retain the CRISPR guide sequence in the edited cells, which eliminates possible interference in subsequent reconstitution studies where mutant forms of the pseudokinase can be reintroduced into cells exogenously for detailed mechanistic characterization.

New Insights Into the Regulatory Roles of Extracellular Vesicles in Tumor Angiogenesis and Their Clinical Implications.

Angiogenesis is required for tumor growth and development. Extracellular vesicles (EVs) are important signaling entities that mediate communication between diverse types of cells and regulate various cell biological processes, including angiogenesis. Recently, emerging evidence has suggested that tumor-derived EVs play essential roles in tumor progression by regulating angiogenesis. Thousands of molecules are carried by EVs, and the two major types of biomolecules, noncoding RNAs (ncRNAs) and proteins, are transported between cells and regulate physiological and pathological functions in recipient cells. Understanding the regulation of EVs and their cargoes in tumor angiogenesis has become increasingly important. In this review, we summarize the effects of tumor-derived EVs and their cargoes, especially ncRNAs and proteins, on tumor angiogenesis and their mechanisms, and we highlight the clinical implications of EVs in bodily fluids as biomarkers and as diagnostic, prognostic, and therapeutic targets in cancer patients.