Integrated Stress Response Potentiates Ponatinib-Induced Cardiotoxicity.

BACKGROUND: Mitochondrial dysfunction is a primary driver of cardiac contractile failure; yet, the cross talk between mitochondrial energetics and signaling regulation remains obscure. Ponatinib, a tyrosine kinase inhibitor used to treat chronic myeloid leukemia, is among the most cardiotoxic tyrosine kinase inhibitors and causes mitochondrial dysfunction. Whether ponatinib-induced mitochondrial dysfunction triggers the integrated stress response (ISR) to induce ponatinib-induced cardiotoxicity remains to be determined. METHODS: Using human induced pluripotent stem cells-derived cardiomyocytes and a recently developed mouse model of ponatinib-induced cardiotoxicity, we performed proteomic analysis, molecular and biochemical assays to investigate the relationship between ponatinib-induced mitochondrial stress and ISR and their role in promoting ponatinib-induced cardiotoxicity. RESULTS: Proteomic analysis revealed that ponatinib activated the ISR in cardiac cells. We identified GCN2 (general control nonderepressible 2) as the eIF2α (eukaryotic translation initiation factor 2α) kinase responsible for relaying mitochondrial stress signals to trigger the primary ISR effector-ATF4 (activating transcription factor 4), upon ponatinib exposure. Mechanistically, ponatinib treatment exerted inhibitory effects on ATP synthase activity and reduced its expression levels resulting in ATP deficits. Perturbed mitochondrial function resulting in ATP deficits then acts as a trigger of GCN2-mediated ISR activation, effects that were negated by nicotinamide mononucleotide, an NAD+ precursor, supplementation. Genetic inhibition of ATP synthase also activated GCN2. Interestingly, we showed that the decreased abundance of ATP also facilitated direct binding of ponatinib to GCN2, unexpectedly causing its activation most likely because of a conformational change in its structure. Importantly, administering an ISR inhibitor protected human induced pluripotent stem cell-derived cardiomyocytes against ponatinib. Ponatinib-treated mice also exhibited reduced cardiac function, effects that were attenuated upon systemic ISRIB administration. Importantly, ISRIB does not affect the antitumor effects of ponatinib in vitro. CONCLUSIONS: Neutralizing ISR hyperactivation could prevent or reverse ponatinib-induced cardiotoxicity. The findings that compromised ATP production potentiates GCN2-mediated ISR activation have broad implications across various cardiac diseases. Our results also highlight an unanticipated role of ponatinib in causing direct activation of a kinase target despite its role as an ATP-competitive kinase inhibitor.

A Critical Role for ERO1α in Arterial Thrombosis and Ischemic Stroke.

BACKGROUND: Platelet adhesion and aggregation play a crucial role in arterial thrombosis and ischemic stroke. Here, we identify platelet ERO1α (endoplasmic reticulum oxidoreductase 1α) as a novel regulator of Ca2+ signaling and a potential pharmacological target for treating thrombotic diseases. METHODS: Intravital microscopy, animal disease models, and a wide range of cell biological studies were utilized to demonstrate the pathophysiological role of ERO1α in arteriolar and arterial thrombosis and to prove the importance of platelet ERO1α in platelet activation and aggregation. Mass spectrometry, electron microscopy, and biochemical studies were used to investigate the molecular mechanism. We used novel blocking antibodies and small-molecule inhibitors to study whether ERO1α can be targeted to attenuate thrombotic conditions. RESULTS: Megakaryocyte-specific or global deletion of Ero1α in mice similarly reduced platelet thrombus formation in arteriolar and arterial thrombosis without affecting tail bleeding times and blood loss following vascular injury. We observed that platelet ERO1α localized exclusively in the dense tubular system and promoted Ca2+ mobilization, platelet activation, and aggregation. Platelet ERO1α directly interacted with STIM1 (stromal interaction molecule 1) and SERCA2 (sarco/endoplasmic reticulum Ca2+-ATPase 2) and regulated their functions. Such interactions were impaired in mutant STIM1-Cys49/56Ser and mutant SERCA2-Cys875/887Ser. We found that ERO1α modified an allosteric Cys49-Cys56 disulfide bond in STIM1 and a Cys875-Cys887 disulfide bond in SERCA2, contributing to Ca2+ store content and increasing cytosolic Ca2+ levels during platelet activation. Inhibition of Ero1α with small-molecule inhibitors but not blocking antibodies attenuated arteriolar and arterial thrombosis and reduced infarct volume following focal brain ischemia in mice. CONCLUSIONS: Our results suggest that ERO1α acts as a thiol oxidase for Ca2+ signaling molecules, STIM1 and SERCA2, and enhances cytosolic Ca2+ levels, promoting platelet activation and aggregation. Our study provides evidence that ERO1α may be a potential target to reduce thrombotic events.

Soil selenium enhanced the resistance of oilseed rape to Sclerotinia sclerotiorum by optimizing the plant microbiome.

Plant can recruit beneficial microbes to enhance their ability to resist disease. Selenium is well established as a beneficial element in plant growth, but its role in mediating microbial disease resistance remained poorly understood. Here, we investigated the correlation between selenium, oilseed rape rhizosphere microbes and Sclerotinia sclerotiorum. Soil application of 0.5 and 1.0 mg/kg selenium significantly increased the resistance of oilseed rape to Sclerotinia sclerotiorum compared with no selenium application, and the disease inhibition rate was higher than 20%. The disease resistance of oilseed rape was related to rhizosphere microorganisms, and beneficial bacteria isolated from the rhizosphere inhibited Sclerotinia stem rot. Burkholderia cepacia, and synthetic community enhanced plant disease resistance through transcriptional regulation and activated plant-induced systemic resistance to protect plants. Besides, inoculation of isolated bacteria optimized the bacterial community structure of leaves and enriched beneficial microorganisms such as Bacillus, Pseudomonas and Sphingomonas. Bacillus isolated from the leaves were sprayed on the detached leaves, and it also performed a significant inhibition effect on Sclerotinia sclerotiorum. Overall, our results suggested that selenium drive plant rhizosphere microorganisms to increase resistance to Sclerotinia sclerotiorum in oilseed rape.

Gα13 Mediates Transendothelial Migration of Neutrophils by Promoting Integrin-Dependent Motility without Affecting Directionality.

Neutrophil migration requires ß2 integrins and chemoattractant receptor signaling for motility and directionality. G protein subunit Gα13 can facilitate cell migration by mediating RhoA activation induced by G protein-coupled receptors. However, the possible role of Gα13-integrin interaction in migration is unclear. In this study, we show that Gα13 -/- neutrophils are deficient in transendothelial migration and migration on ß2 integrin ligand ICAM-1. However, unlike G protein-coupled receptors and integrin inside-out signaling pathways, Gα13 is important in migration velocity and neutrophil spreading but not in directionality nor cell adhesion. Importantly, neutrophil recruitment in vivo was also inhibited in Gα13 -/- mice, suggesting the importance of Gα13 in transendothelial migration of neutrophils in vitro and in vivo. Furthermore, a synthetic peptide (MB2mP6) derived from the Gα13 binding site of ß2 inhibited Gα13-ß2 interaction and Gα13-mediated transient RhoA inhibition in neutrophils, suggesting that this peptide inhibited integrin outside-in signaling. MB2mP6 inhibited migration of control neutrophils through endothelial cell monolayers or ICAM-1-coated filters, but was without further effect on Gα13 -/- neutrophils. It also inhibited integrin-dependent neutrophil migration velocity without affecting directionality. In vivo, MB2mP6 markedly inhibited neutrophil infiltration into the cardiac tissues induced by ischemia/reperfusion injury. Thus, Gα13-dependent outside-in signaling enables integrin-dependent neutrophil motility without affecting directionality and may be a new therapeutic target for inhibiting neutrophil trafficking but not adhesion.

Shear and Integrin Outside-In Signaling Activate NADPH-Oxidase 2 to Promote Platelet Activation.

[Figure: see text].

Signaling mechanisms of the platelet glycoprotein Ib-IX complex.

The glycoprotein Ib-IX (GPIb-IX) complex mediates initial platelet adhesion to von Willebrand factor (VWF) immobilized on subendothelial matrix and endothelial surfaces, and transmits VWF binding-induced signals to stimulate platelet activation. GPIb-IX also functions as part of a mechanosensor to convert mechanical force received via VWF binding into intracellular signals, thereby greatly enhancing platelet activation. Thrombin binding to GPIb-IX initiates GPIb-IX signaling cooperatively with protease-activated receptors to synergistically stimulate the platelet response to low-dose thrombin. GPIb-IX signaling may also occur following the binding of other GPIb-IX ligands such as leukocyte integrin αMß2 and red cell-derived semaphorin 7A, contributing to thrombo-inflammation. GPIb-IX signaling requires the interaction between the cytoplasmic domains of GPIb-IX and 14-3-3 protein and is mediated through Src family kinases, the Rho family of small GTPases, phosphoinositide 3-kinase-Akt-cGMP-mitogen-activated protein kinase, and LIM kinase 1 signaling pathways, leading to calcium mobilization, integrin activation, and granule secretion. This review summarizes the current understanding of GPIb-IX signaling.

[Expression of Glutathione Peroxidases and Its Effect on Clinical Prognosis in Glioma Patients].

Objective To investigate the relationship between the expression of glutathione peroxidase(GPX)genes and the clinical prognosis in glioma patients,and to construct and evaluate the model for predicting the prognosis of glioma. Methods The clinical information and GPX expression of 663 patients,including 153 patients of glioblastoma(GBM)and 510 patients of low-grade glioma(LGG),were obtained from The Cancer Genome Atlas(TCGA)database.The relationship between GPX expression and patient survival was analyzed.The key GPX affecting the prognosis of glioma was screened out by single- and multi-factor Cox's proportional-hazards regression models and validated by least absolute shrinkage and selection operator(Lasso)regression.Finally,we constructed the model for predicting the prognosis of glioma with the screening results and then used concordance index and calibration curve respectively to evaluate the discrimination and calibration of model. Results Compared with those in the control group,the expression levels of GPX1,GPX3,GPX4,GPX7,and GPX8 were up-regulated in glioma patients(all P<0.001).Moreover,the expression levels of other GPX except GPX3 were higher in GBM patients than in LGG patients(all P<0.001).The Kaplan-Meier curves showed that the progression-free survival of GBM with high expression of GPX1(P=0.013)and GPX4(P=0.040),as well as the overall survival,disease-specific survival,and progression-free survival of LGG with high expression of GPX1,GPX7,and GPX8,was shortened(all P<0.001).GPX7 and GPX8 were screened out as the key factors affecting the prognosis of LGG.The results were further used to construct a nomogram model,which suggested GPX7 was the most important variable.The concordance index of the model was 0.843(95%CI=0.809-0.853),and the calibration curve showed that the predicted and actual results had good consistency. Conclusion GPX7 is an independent risk factor affecting the prognosis of LGG,and the nomogram model constructed with it can be used to predict the survival rate of LGG.

Super electro-optic modulation in bulk KTN:Cu based on electric-field-enhanced permittivity.

The electric-field-enhanced effect of permittivity can improve the performance of electro-optic modulators and deflectors. A theoretical model of super electro-optic modulation based on the field-enhanced effect of the permittivity was proposed. Results showed that a strong field-enhanced effect can greatly reduce the half-wave voltage and increase the modulation depth as a result of increased relative dielectric permittivity and permittivity gradient to the electric field. For bulk paraelectric KTN:Cu near the Curie temperature, we found a novel phenomenon that the response of relative dielectric permittivity to the bias electric field was closely related to the frequency, including attenuation, invariance, and enhancement. We effectively selected the frequencies corresponding to the strong field-enhanced effect by measuring the dielectric-frequency spectrum under the bias voltage. At these frequencies, a phase retardation of π was achieved through 2Vpp AC modulation voltage, indicating that the half-wave voltage was reduced by one order of magnitude.

Knockdown of circ_0006528 Suppresses Cell Proliferation, Migration, Invasion, and Adriamycin Chemoresistance via Regulating the miR-1236-3p/CHD4 Axis in Breast Cancer.

BACKGROUND: Adriamycin (ADM) is one of the postoperative chemotherapy drugs for breast cancer (BCa) patients. Circular RNAs have been shown to modulate ADM resistance in many cancers. However, it is unclear whether circ_0006528 can modulate the ADM chemoresistance in BCa. METHODS: Levels of circ_0006528, microRNA-1236-3p (miR-1236-3p), and chromodomain helicase DNA-binding protein 4 (CHD4) were detected by quantitative real-time polymerase chain reaction or western blot. Cell proliferation, the half maximal inhibitory concentration (IC50) value of ADM, and cell migration and invasion were evaluated by cell counting kit-8 and transwell assays, respectively. The interaction among circ_0006528, miR-1236-3p, and CHD4 was confirmed using dual-luciferase reporter assays. Tumor formation in nude mice was performed to explore the effect of circ_0006528 in vivo. RESULTS: Higher levels of circ_0006528 and CHD4 and lower level of miR-1236-3p were found in ADM-resistant BCa tissues and cells, and patients with high circ_0006528 had a shorter overall survival. Circ_0006528 could directly bind to miR-1236-3p, and circ_0006528 knockdown or miR-1236-3p overexpression could suppress cell proliferation, migration, invasion, and ADM resistance in ADM-resistant BCa cells. Moreover, circ_0006528-regulated CHD4 expression by sponging miR-1236-3p, and CHD4 elevation reversed the inhibitory effect of circ_0006528 knockdown on ADM-resistant BCa cells. Consistently, circ_0006528 inhibition retarded ADM-resistant BCa tumor growth in vivo by decreasing CHD4 and increasing miR-1236-3p. CONCLUSIONS: Downregulation of circ_0006528 restrained cell proliferation, migration, invasion, and drug resistance of ADM-resistant BCa cells through inhibiting CHD4 and inducing miR-1236-3p.

A Variant of sNASP Exacerbates Lymphocyte Subset Disorder and Nephritis in a Spontaneous Lupus Model Sle1.Yaa Mouse.

A variant of somatic nuclear autoantigenic sperm protein (sNASP) was identified from the murine lupus susceptibility locus Sle2c1 by whole exome sequencing (WES). Previous studies have shown that mutant sNASP could synergize with the Faslpr mutation in exacerbating autoimmunity and aggravating end-organ inflammation. In the current study, the sNASP mutation was introduced into Sle1.Yaa mice to detect whether it has a synergistic effect with Sle1 or Yaa loci. As expected, compared with Sle1.Yaa mice, Sle1.Yaa.ΔsNASP mice showed enlarged lymph nodes, aggravated renal inflammation, and shortened survival time. The proportions of CD3+ T cells, activated CD19+CD86+ B cells, Th1 cells in the spleen and lymph nodes, and Th17 cells in lymph nodes in Sle1.Yaa.ΔsNASP mice were increased compared to those in Sle1.Yaa mice. The levels of IFN-γ and TNF-α in the serum of Sle1.Yaa.ΔsNASP mice were higher than those of Sle1.Yaa mice. The above results show that mutant sNASP can interact with different lupus susceptibility genes and promote the disease process of systemic lupus erythematosus.

Platelet Protein Disulfide Isomerase Promotes Glycoprotein Ibα-Mediated Platelet-Neutrophil Interactions Under Thromboinflammatory Conditions.

BACKGROUND: Platelet-neutrophil interactions contribute to vascular occlusion and tissue damage in thromboinflammatory disease. Platelet glycoprotein Ibα (GPIbα), a key receptor for the cell-cell interaction, is believed to be constitutively active for ligand binding. Here, we established the role of platelet-derived protein disulfide isomerase (PDI) in reducing the allosteric disulfide bonds in GPIbα and enhancing the ligand-binding activity under thromboinflammatory conditions. METHODS: Bioinformatic analysis identified 2 potential allosteric disulfide bonds in GPIbα. Agglutination assays, flow cytometry, surface plasmon resonance analysis, a protein-protein docking model, proximity ligation assays, and mass spectrometry were used to demonstrate a direct interaction between PDI and GPIbα and to determine a role for PDI in regulating GPIbα function and platelet-neutrophil interactions. Also, real-time microscopy and animal disease models were used to study the pathophysiological role of PDI-GPIbα signaling under thromboinflammatory conditions. RESULTS: Deletion or inhibition of platelet PDI significantly reduced GPIbα-mediated platelet agglutination. Studies using PDI-null platelets and recombinant PDI or Anfibatide, a clinical-stage GPIbα inhibitor, revealed that the oxidoreductase activity of platelet surface-bound PDI was required for the ligand-binding function of GPIbα. PDI directly bound to the extracellular domain of GPIbα on the platelet surface and reduced the Cys4-Cys17 and Cys209-Cys248 disulfide bonds. Real-time microscopy with platelet-specific PDI conditional knockout and sickle cell disease mice demonstrated that PDI-regulated GPIbα function was essential for platelet-neutrophil interactions and vascular occlusion under thromboinflammatory conditions. Studies using a mouse model of ischemia/reperfusion-induced stroke indicated that PDI-GPIbα signaling played a crucial role in tissue damage. CONCLUSIONS: Our results demonstrate that PDI-facilitated cleavage of the allosteric disulfide bonds tightly regulates GPIbα function, promoting platelet-neutrophil interactions, vascular occlusion, and tissue damage under thromboinflammatory conditions.

14-3-3 proteins in platelet biology and glycoprotein Ib-IX signaling.

Members of the 14-3-3 family of proteins function as adapters/modulators that recognize phosphoserine/phosphothreonine-based binding motifs in many intracellular proteins and play fundamental roles in signal transduction pathways of eukaryotic cells. In platelets, 14-3-3 plays a wide range of regulatory roles in phosphorylation-dependent signaling pathways, including G-protein signaling, cAMP signaling, agonist-induced phosphatidylserine exposure, and regulation of mitochondrial function. In particular, 14-3-3 interacts with several phosphoserine-dependent binding sites in the major platelet adhesion receptor, the glycoprotein Ib-IX complex (GPIb-IX), regulating its interaction with von Willebrand factor (VWF) and mediating VWF/GPIb-IX-dependent mechanosignal transduction, leading to platelet activation. The interaction of 14-3-3 with GPIb-IX also plays a critical role in enabling the platelet response to low concentrations of thrombin through cooperative signaling mediated by protease-activated receptors and GPIb-IX. The various functions of 14-3-3 in platelets suggest that it is a possible target for the treatment of thrombosis and inflammation.

Shear-induced integrin signaling in platelet phosphatidylserine exposure, microvesicle release, and coagulation.

It is currently unclear why agonist-stimulated platelets require shear force to efficiently externalize the procoagulant phospholipid phosphatidylserine (PS) and release PS-exposed microvesicles (MVs). We reveal that integrin outside-in signaling is an important mechanism for this requirement. PS exposure and MV release were inhibited in ß3-/- platelets or by integrin antagonists. The impaired MV release and PS exposure in ß3-/- platelets were rescued by expression of wild-type ß3 but not a Gα13 binding-deficient ß3 mutant (E733EE to AAA), which blocks outside-in signaling but not ligand binding. Inhibition of Gα13 or Src also diminished agonist/shear-dependent PS exposure and MV release, further indicating a role for integrin outside-in signaling. PS exposure in activated platelets was induced by application of pulling force via an integrin ligand, which was abolished by inhibiting Gα13-integrin interaction, suggesting that Gα13-dependent transmission of mechanical signals by integrins induces PS exposure. Inhibition of Gα13 delayed coagulation in vitro. Furthermore, inhibition or platelet-specific knockout of Gα13 diminished laser-induced intravascular fibrin formation in arterioles in vivo. Thus, ß3 integrins serve as a shear sensor activating the Gα13-dependent outside-in signaling pathway to facilitate platelet procoagulant function. Pharmacological targeting of Gα13-integrin interaction prevents occlusive thrombosis in vivo by inhibiting both coagulation and platelet thrombus formation.

Serum amyloid A promotes LPS clearance and suppresses LPS-induced inflammation and tissue injury.

Lipopolysaccharide (LPS) is a major microbial mediator for tissue injury and sepsis resulting from Gram-negative bacterial infection. LPS is an external factor that induces robust expression of serum amyloid A (SAA), a major constituent of the acute-phase proteins, but the relationship between SAA expression and LPS-induced tissue injury remains unclear. Here, we report that mice with inducible transgenic expression of human SAA1 are partially protected against inflammatory response and lung injury caused by LPS and cecal ligation and puncture (CLP). In comparison, transgenic SAA1 does not attenuate TNFα-induced lung inflammation and injury. The SAA1 expression level correlates inversely with the endotoxin concentrations in serum and lung tissues since SAA1 binds directly to LPS to form a complex that promotes LPS uptake by macrophages. Disruption of the SAA1-LPS interaction with a SAA1-derived peptide partially reduces the protective effect and exacerbates inflammation. These findings demonstrate that acute-phase SAA provides innate feedback protection against LPS-induced inflammation and tissue injury.

Oxidant Signaling Mediated by Nox2 in Neutrophils Promotes Regenerative Myelopoiesis and Tissue Recovery following Ischemic Damage.

Ischemic tissue damage activates hematopoietic stem and progenitor cells (HSPCs) in the bone marrow (BM)-generating myeloid cells, and persistent HSPC activity may drive chronic inflammation and impair tissue recovery. Although increased reactive oxygen species in the BM regulate HSPC functions, their roles in myelopoiesis of activated HSPCs and subsequent tissue recovery during ischemic damage are not well understood. In this paper, we report that deletion of Nox2 NADPH oxidase in mice results in persistent elevations in BM HSPC activity and levels of inflammatory monocytes/macrophages in BM and ischemic tissue in a model of hindlimb ischemia. Ischemic tissue damage induces oxidants in BM such as elevations of hydrogen peroxide and oxidized phospholipids, which activate redox-sensitive Lyn kinase in a Nox2-dependent manner. Moreover, during tissue recovery after ischemic injury, this Nox2-ROS-Lyn kinase axis is induced by Nox2 in neutrophils that home to the BM, which inhibits HSPC activity and inflammatory monocyte generation and promotes tissue regeneration after ischemic damage. Thus, oxidant signaling in the BM mediated by Nox2 in neutrophils regulates myelopoiesis of HSPCs to promote regeneration of damaged tissue.

Differential Levels of Endoplasmic Reticulum Stress in Peripheral Blood Mononuclear Cells from Patients with Sudden Sensorineural Hearing Loss.

BACKGROUND Sudden sensorineural hearing loss (SSNHL) is currently treated with a combination of drugs, predominantly with glucocorticoids (GCs). However, the mechanisms of action of GCs in SSNHL are unknown. This study aimed to analyze the role of endoplasmic reticulum stress (ERS) in SSNHL pathogenesis and prognosis. MATERIAL AND METHODS In this study, we evaluated the expression and activation status of the protein kinase RNA-like endoplasmic reticulum kinase (PERK)-C/EBP homologous protein (CHOP) pathway in peripheral blood mononuclear cells (PBMCs) from patients with SSNHL and compared them with those in healthy controls. We also compared differences in expression of activating transcription factor 4 (ATF4) and CHOP before and after glucocorticoid treatment in patients with improved and unimproved SSNHL. RESULTS Treatment with GCs significantly improved hearing in 55% of patients with SSNHL. Levels of phosphorylated PERK (p-PERK) and phosphorylated eukaryotic initiation factor 2alpha were increased in PBMCs from patients with SSNHL compared with healthy controls. ATF4 and CHOP expression were also significantly elevated. After treatment, the amount of ATF4 and CHOP proteins in PBMCs in the patients whose SSNHL improved was significantly reduced compared with the levels measured before treatment in all patients with SSNHL. The expression of the ATF4 and CHOP proteins in PBMCs in the unimproved group, however, was not significantly changed relative to pretreatment levels. CONCLUSIONS ERS may play a significant role in the pathogenesis of SSNHL, and the responsiveness of the condition to GC-mediated mitigation of ERS may be one of the key factors that affect patient prognosis.

Twinfilin 2a regulates platelet reactivity and turnover in mice.

Regulated reorganization of the actin cytoskeleton is a prerequisite for proper platelet production and function. Consequently, defects in proteins controlling actin dynamics have been associated with platelet disorders in humans and mice. Twinfilin 2a (Twf2a) is a small actin-binding protein that inhibits actin filament assembly by sequestering actin monomers and capping filament barbed ends. Moreover, Twf2a binds heterodimeric capping proteins, but the role of this interaction in cytoskeletal dynamics has remained elusive. Even though Twf2a has pronounced effects on actin dynamics in vitro, only little is known about its function in vivo. Here, we report that constitutive Twf2a-deficient mice (Twf2a-/-) display mild macrothrombocytopenia due to a markedly accelerated platelet clearance in the spleen. Twf2a-/- platelets showed enhanced integrin activation and α-granule release in response to stimulation of (hem) immunoreceptor tyrosine-based activation motif (ITAM) and G-protein-coupled receptors, increased adhesion and aggregate formation on collagen I under flow, and accelerated clot retraction and spreading on fibrinogen. In vivo, Twf2a deficiency resulted in shortened tail bleeding times and faster occlusive arterial thrombus formation. The hyperreactivity of Twf2a-/- platelets was attributed to enhanced actin dynamics, characterized by an increased activity of n-cofilin and profilin 1, leading to a thickened cortical cytoskeleton and hence sustained integrin activation by limiting calpain-mediated integrin inactivation. In summary, our results reveal the first in vivo functions of mammalian Twf2a and demonstrate that Twf2a-controlled actin rearrangements dampen platelet activation responses in a n-cofilin- and profilin 1-dependent manner, thereby indirectly regulating platelet reactivity and half-life in mice.

Intronic (TTTGA)n insertion in SAMD12 also causes familial cortical myoclonic tremor with epilepsy.

BACKGROUND: Intronic (TTTCA)n insertions in the SAMD12, TNRC6A, and RAPGEF2 genes have been identified as causes of familial cortical myoclonic tremor with epilepsy. OBJECTIVE: To identify the cause of familial cortical myoclonic tremor with epilepsy pedigrees without (TTTCA)n insertions in SAMD12, TNRC6A, and RAPGEF2. METHODS: Repeat-primed polymerase chain reaction, long-range polymerase chain reaction, and Sanger sequencing were performed to identify the existence of a novel (TTTGA)n insertion. Targeted long-read sequencing was performed to confirm the accurate structure of the (TTTGA)n insertion. RESULTS: We identified a novel expanded intronic (TTTGA)n insertion at the same site as the previously reported (TTTCA)n insertion in SAMD12. This insertion cosegregated with familial cortical myoclonic tremor with epilepsy in 1 Chinese pedigree with no (TTTCA)n insertion. In the targeted long-read sequencing of 2 patients and 1 asymptomatic carrier in this pedigree, with 1 previously reported (TTTCA)n -insertion-carrying patient as a positive control, a respective total of 302, 159, 207, and 50 on-target subreads (predicated accuracy: ≥90%) spanning the target repeat expansion region were generated. These sequencing data revealed the accurate repeat expansion structures as (TTTTA)114-123 (TTTGA)108-116 in the pedigree and (TTTTA)38 (TTTCA)479 in (TTTCA)n -insertion-carrying patient. CONCLUSION: The targeted long-read sequencing helped us to elucidate the accurate structures of the (TTTGA)n and (TTTCA)n insertions. Our finding offers a novel possible cause for familial cortical myoclonic tremor with epilepsy and might shed light on the identification of genetic causes of this disease in pedigrees with no detected (TTTCA)n insertion in the reported causative genes. © 2019 International Parkinson and Movement Disorder Society.

A directional switch of integrin signalling and a new anti-thrombotic strategy.

Integrins have a critical role in thrombosis and haemostasis. Antagonists of the platelet integrin αIIbß3 are potent anti-thrombotic drugs, but also have the life-threatening adverse effect of causing bleeding. It is therefore desirable to develop new antagonists that do not cause bleeding. Integrins transmit signals bidirectionally. Inside-out signalling activates integrins through a talin-dependent mechanism. Integrin ligation mediates thrombus formation and outside-in signalling, which requires Gα13 and greatly expands thrombi. Here we show that Gα13 and talin bind to mutually exclusive but distinct sites within the integrin ß3 cytoplasmic domain in opposing waves. The first talin-binding wave mediates inside-out signalling and also ligand-induced integrin activation, but is not required for outside-in signalling. Integrin ligation induces transient talin dissociation and Gα13 binding to an EXE motif (in which X denotes any residue), which selectively mediates outside-in signalling and platelet spreading. The second talin-binding wave is associated with clot retraction. An EXE-motif-based inhibitor of Gα13-integrin interaction selectively abolishes outside-in signalling without affecting integrin ligation, and suppresses occlusive arterial thrombosis without affecting bleeding time. Thus, we have discovered a new mechanism for the directional switch of integrin signalling and, on the basis of this mechanism, designed a potent new anti-thrombotic drug that does not cause bleeding.