GSK-3β Is Required for Memory Reconsolidation in Adult Brain

Activation of GSK-3β is presumed to be involved in various neurodegenerative diseases, including Alzheimer's disease (AD), which is characterized by memory disturbances during early stages of the disease. The normal function of GSK-3β in adult brain is not well understood. Here, we analyzed the ability of heterozygote GSK-3β knockout (GSK+/−) mice to form memories. In the Morris water maze (MWM), learning and memory performance of GSK+/− mice was no different from that of wild-type (WT) mice for the first 3 days of training. With continued learning on subsequent days, however, retrograde amnesia was induced in GSK+/− mice, suggesting that GSK+/− mice might be impaired in their ability to form long-term memories. In contextual fear conditioning (CFC), context memory was normally consolidated in GSK+/− mice, but once the original memory was reactivated, they showed reduced freezing, suggesting that GSK+/− mice had impaired memory reconsolidation. Biochemical analysis showed that GSK-3β was activated after memory reactivation in WT mice. Intraperitoneal injection of a GSK-3 inhibitor before memory reactivation impaired memory reconsolidation in WT mice. These results suggest that memory reconsolidation requires activation of GSK-3β in the adult brain.     

Peptide Bβ15-42 Preserves Endothelial Barrier Function in Shock

Loss of vascular barrier function causes leak of fluid and proteins into tissues, extensive leak leads to shock and death. Barriers are largely formed by endothelial cell-cell contacts built up by VE-cadherin and are under the control of RhoGTPases. Here we show that a natural plasmin digest product of fibrin, peptide Bß15-42 (also called FX06), significantly reduces vascular leak and mortality in animal models for Dengue shock syndrome. The ability of Bß15-42 to preserve endothelial barriers is confirmed in rats i.v.-injected with LPS. In endothelial cells, Bß15-42 prevents thrombin-induced stress fiber formation, myosin light chain phosphorylation and RhoA activation. The molecular key for the protective effect of Bß15-42 is the src kinase Fyn, which associates with VE-cadherin-containing junctions. Following exposure to Bß15-42 Fyn dissociates from VE-cadherin and associates with p190RhoGAP, a known antagonists of RhoA activation. The role of Fyn in transducing effects of Bß15-42 is confirmed in Fyn^−/− mice, where the peptide is unable to reduce LPS-induced lung edema, whereas in wild type littermates the peptide significantly reduces leak. Our results demonstrate a novel function for Bß15-42. Formerly mainly considered as a degradation product occurring after fibrin inactivation, it has now to be considered as a signaling molecule. It stabilizes endothelial barriers and thus could be an attractive adjuvant in the treatment of shock.     

Targeting TGF-β for treatment of osteogenesis imperfecta

BACKGROUNDCurrently, there is no disease-specific therapy for osteogenesis imperfecta (OI). Preclinical studies demonstrate that excessive TGF-β signaling is a pathogenic mechanism in OI. Here, we evaluated TGF-β signaling in children with OI and conducted a phase I clinical trial of TGF-β inhibition in adults with OI.METHODSHistology and RNA-Seq were performed on bones obtained from children. Gene Ontology (GO) enrichment assay, gene set enrichment analysis (GSEA), and Ingenuity Pathway Analysis (IPA) were used to identify dysregulated pathways. Reverse-phase protein array, Western blot, and IHC were performed to evaluate protein expression. A phase I study of fresolimumab, a TGF-β neutralizing antibody, was conducted in 8 adults with OI. Safety and effects on bone remodeling markers and lumbar spine areal bone mineral density (LS aBMD) were assessed.RESULTSOI bone demonstrated woven structure, increased osteocytes, high turnover, and reduced maturation. SMAD phosphorylation was the most significantly upregulated GO molecular event. GSEA identified the TGF-β pathway as the top activated signaling pathway, and IPA showed that TGF-β1 was the most significant activated upstream regulator mediating the global changes identified in OI bone. Treatment with fresolimumab was well-tolerated and associated with increases in LS aBMD in participants with OI type IV, whereas participants with OI type III and VIII had unchanged or decreased LS aBMD.CONCLUSIONIncreased TGF-β signaling is a driver pathogenic mechanism in OI. Anti–TGF-β therapy could be a potential disease-specific therapy, with dose-dependent effects on bone mass and turnover.TRIAL REGISTRATIONClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT03064074","term_id":"NCT03064074"}}NCT03064074.FUNDINGBrittle Bone Disorders Consortium (U54AR068069), Clinical Translational Core of Baylor College of Medicine Intellectual and Developmental Disabilities Research Center (P50HD103555) from National Institute of Child Health and Human Development, USDA/ARS (cooperative agreement 58-6250-6-001), and Sanofi Genzyme.     

TGF-β is a critical mediator of acute lung injury

We have shown that the integrin alphavbeta6 activates latent TGF-beta in the lungs and skin. We show here that mice lacking this integrin are completely protected from pulmonary edema in a model of bleomycin-induced acute lung injury (ALI). Pharmacologic inhibition of TGF-beta also protected wild-type mice from pulmonary edema induced by bleomycin or Escherichia coli endotoxin. TGF-beta directly increased alveolar epithelial permeability in vitro by a mechanism that involved depletion of intracellular glutathione. These data suggest that integrin-mediated local activation of TGF-beta is critical to the development of pulmonary edema in ALI and that blocking TGF-beta or its activation could be effective treatments for this currently untreatable disorder.     

Targeting the αv integrin/TGF-β axis improves natural killer cell function against glioblastoma stem cells

Glioblastoma multiforme (GBM), the most aggressive brain cancer, recurs because glioblastoma stem cells (GSCs) are resistant to all standard therapies. We showed that GSCs, but not normal astrocytes, are sensitive to lysis by healthy allogeneic natural killer (NK) cells in vitro. Mass cytometry and single-cell RNA sequencing of primary tumor samples revealed that GBM tumor–infiltrating NK cells acquired an altered phenotype associated with impaired lytic function relative to matched peripheral blood NK cells from patients with GBM or healthy donors. We attributed this immune evasion tactic to direct cell-to-cell contact between GSCs and NK cells via αv integrin–mediated TGF-β activation. Treatment of GSC-engrafted mice with allogeneic NK cells in combination with inhibitors of integrin or TGF-β signaling or with TGFBR2 gene–edited allogeneic NK cells prevented GSC-induced NK cell dysfunction and tumor growth. These findings reveal an important mechanism of NK cell immune evasion by GSCs and suggest the αv integrin/TGF-β axis as a potentially useful therapeutic target in GBM.     

Phosphatidylserine-dependent ingestion of apoptotic cells promotes TGF-β1 secretion and the resolution of inflammation

Ingestion of apoptotic cells in vitro by macrophages induces TGF-beta1 secretion, resulting in an anti-inflammatory effect and suppression of proinflammatory mediators. Here, we show in vivo that direct instillation of apoptotic cells enhanced the resolution of acute inflammation. This enhancement appeared to require phosphatidylserine (PS) on the apoptotic cells and local induction of TGF-beta1. Working with thioglycollate-stimulated peritonea or LPS-stimulated lungs, we examined the effect of apoptotic cell uptake on TGF-beta1 induction. Viable or opsonized apoptotic human Jurkat T cells, or apoptotic PLB-985 cells, human monomyelocytes that do not express PS during apoptosis, failed to induce TGF-beta1. PS liposomes, or PS directly transferred onto the PLB-985 surface membranes, restored the TGF-beta1 induction. Apoptotic cell instillation into LPS-stimulated lungs reduced proinflammatory chemokine levels in the bronchoalveolar lavage fluid (BALF). Additionally, total inflammatory cell counts in the BALF were markedly reduced 1-5 days after apoptotic cell instillation, an effect that could be reversed by opsonization or coinstillation of TGF-beta1 neutralizing antibody. This reduction resulted from early decrease in neutrophils and later decreases in lymphocytes and macrophages. In conclusion, apoptotic cell recognition and clearance, via exposure of PS and ligation of its receptor, induce TGF-beta1 secretion, resulting in accelerated resolution of inflammation.     

TGF-β signaling in myeloproliferative neoplasms contributes to myelofibrosis without disrupting the hematopoietic niche

Myeloproliferative neoplasms (MPNs) are associated with significant alterations in the bone marrow microenvironment that include decreased expression of key niche factors and myelofibrosis. Here, we explored the contribution of TGF-β to these alterations by abrogating TGF-β signaling in bone marrow mesenchymal stromal cells. Loss of TGF-β signaling in Osx-Cre–targeted MSCs prevented the development of myelofibrosis in both MPL^W515L and Jak2^V617F models of MPNs. In contrast, despite the absence of myelofibrosis, loss of TGF-β signaling in mesenchymal stromal cells did not rescue the defective hematopoietic niche induced by MPL^W515L, as evidenced by decreased bone marrow cellularity, hematopoietic stem/progenitor cell number, and Cxcl12 and Kitlg expression, and the presence of splenic extramedullary hematopoiesis. Induction of myelofibrosis by MPL^W515L was intact in Osx-Cre Smad4^fl/fl recipients, demonstrating that SMAD4-independent TGF-β signaling mediates the myelofibrosis phenotype. Indeed, treatment with a c-Jun N-terminal kinase (JNK) inhibitor prevented the development of myelofibrosis induced by MPL^W515L. Together, these data show that JNK-dependent TGF-β signaling in mesenchymal stromal cells is responsible for the development of myelofibrosis but not hematopoietic niche disruption in MPNs, suggesting that the signals that regulate niche gene expression in bone marrow mesenchymal stromal cells are distinct from those that induce a fibrogenic program.     

Adhesion of Activated Platelets to Endothelial Cells: Evidence for a GPIIbIIIa-dependent Bridging Mechanism and Novel Roles for Endothelial Intercellular Adhesion Molecule 1 (ICAM-1), αvβ3 Integrin, and GPIbα

Although it has been reported that activated platelets can adhere to intact endothelium, the receptors involved have not been fully characterized. Also, it is not clear whether activated platelets bind primarily to matrix proteins at sites of endothelial cell denudation or directly to endothelial cells. Thus, this study was designed to further clarify the mechanisms of activated platelet adhesion to endothelium. Unstimulated human umbilical vein endothelial cell (HUVEC) monolayers were incubated with washed, stained, and thrombin-activated human platelets. To exclude matrix involvement, HUVEC were harvested mechanically and platelet binding was measured by flow cytometry. Before the adhesion assay, platelets or HUVEC were treated with different receptor antagonists. Whereas blockade of platelet β₁ integrins, GPIbα, GPIV, P-selectin, and platelet-endothelial cell adhesion molecule (PECAM)-1 did not reduce platelet adhesion to HUVEC, blockade of platelet GPIIbIIIa by antibodies or Arg-Gly-Asp (RGD) peptides markedly decreased adhesion. Moreover, when platelets were treated with blocking antibodies to GPIIbIIIa-binding adhesive proteins, including fibrinogen and fibronectin, and von Willebrand factor (vWF), platelet binding was also reduced markedly. Addition of fibrinogen, fibronectin, or vWF further increased platelet adhesion, indicating that both endogenous platelet-exposed and exogenous adhesive proteins can participate in the binding process. Evaluation of the HUVEC receptors revealed predominant involvement of intercellular adhesion molecule (ICAM)-1 and α_vβ₃ integrin. Blockade of these two receptors by antibodies decreased platelet binding significantly. Also, there was evidence that a component of platelet adhesion was mediated by endothelial GPIbα. Blockade of β₁ integrins, E-selectin, P-selectin, PECAM-1, vascular cell adhesion molecule (VCAM)-1 and different matrix proteins on HUVEC did not affect platelet adhesion. In conclusion, we show that activated platelet binding to HUVEC monolayers is mediated by a GPIIbIIIa-dependent bridging mechanism involving platelet-bound adhesive proteins and the endothelial cell receptors ICAM-1, α_vβ₃ integrin, and, to a lesser extent, GPIbα.     

Development of γG, γA, γM, β1C/β1A, C′1 esterase inhibitor, ceruloplasmin, transferrin, hemopexin, haptoglobin, fibrinogen, plasminogen, α1-antitrypsin, orosomucoid, β-lipoprotein, α2-macroglobulin, and prealbumin in the human conceptus

The synthesis of γG, γA, γM, β_1C/β_1A, C′1 esterase inhibitor, ceruloplasmin, transferrin, hemopexin, haptoglobin, fibrinogen, α₁-antitrypsin, orosomucoid, β-lipoprotein, α₂-macroglobulin, and prealbumin was studied in 15 normal human embryos and fetuses of 29 days to 18 wk gestation and in the yolk sacs of four embryos from 5.5 to 11.5 wk gestation using tissue culture in ¹⁴C-labeled amino acids followed by radioimmunoelectrophoresis. The human embryo as early as 29 day gestation synthesized β_1C/β_1A, C′1 esterase inhibitor, transferrin, hemopexin, α₁-antitrypsin, β-lipoprotein, α₂-macroglobulin, and prealbumin in culture. At 32 days gestation ceruloplasmin and orosomucoid were also synthesized, but synthesis of fibrinogen was not observed before 5.5 wk. Synthesis of γM occurred as early as 10.5 wk gestation, and γG synthesis was found in cultures as early as 12 wk gestation; γA synthesis was not detected in any of the tissue cultures. With the exception of the γ-globulins, each of the proteins studied was synthesized by the liver, but additional sites of synthesis for some of these proteins were also found. Synthesis of γG and γM occurred primarily in the spleen, but other sites of synthesis were noted as well.     

Thermal stability and oxidation characteristics of α-pinene, β-pinene and α-pinene/β-pinene mixture

Turpentine is a renewable resource, has good combustion performance, and is considered to be a fuel or promising additive to diesel fuel. This is very important for the investigation of thermal stability and energy oxidation characteristics, because evaluation of energy or fuel quality assurance and use safety are necessary. The main components of turpentine are α-pinene and β-pinene, which have unsaturated double bonds and high chemical activity. By investigating their thermal stability and oxidation reaction characteristics, we know the chemical thermal properties and thermal explosion hazard of turpentine. In this present study, the thermal stability and oxidation characteristics of α-pinene, β-pinene and α-pinene/β-pinene mixture were investigated using a high sensitivity accelerating rate calorimeter (ARC) and C80 calorimeter. The important parameters of oxidation reaction and thermal stability were obtained from the temperature, pressure and exothermic behavior in chemical reaction. The results show that α-pinene and β-pinene are thermally stable without chemical reaction under a nitrogen atmosphere even when the temperature reaches 473 K. The initial exothermic temperature of the two pinenes and their mixture is 333–338 K, and the heat release (−ΔH) of their oxidation is 2745–2973 J g⁻¹. The oxidation activation energy (E_a) of α-pinene, β-pinene and α-pinene/β-pinene mixture is 116.25 kJ mol⁻¹, 121.85 kJ mol⁻¹, and 115.95 kJ mol⁻¹, respectively. There are three steps in the oxidation of pinenes: the first is the induction period of the oxidation reaction; the second is the main oxidation stage, and the pressure is reduced; the third is thermal decomposition to produce gas.

Turpentine is a renewable resource, has good combustion performance, and is considered to be a fuel or promising additive to diesel fuel.     

Electrophilic halogenations of propargyl alcohols: paths to α-haloenones, β-haloenones and mixed β,β-dihaloenones

The Meyer–Schuster rearrangement of propargyl alcohols or alkynols leading to α,β-unsaturated carbonyl compounds is well known. Yet, electrophilic halogenations of the same alkynols and their alkoxy, ester and halo derivatives are inconspicuous. This review on the halogenation reactions of propargyl alcohols and derivatives intends to give a perspective from its humble direct halogenation beginning to the present involving metal catalysis. The halogenation products of propargyl alcohols include α-fluoroenones, α-chloroenones, α-bromoenones and α-iodoenones, as well as β-haloenones and symmetrical and mixed β,β-dihaloenones. They are, in essence, tri and tetrasubstituted alkenes carrying halo-functionalization at the α- or β-carbon. This is a potential stepping stone for further construction towards challenging substituted alkenones via Pd-catalysed coupling reactions.

This review highlights the development of α-haloenone, β-haloenone and mixed β,β-dihaloenone formations from propargyl alcohols via direct electrophilic halogenations and metal catalysed-halonium interception rearrangements.     

Oxygen Equilibrium Characteristics of Abnormal Hemoglobins: Hirose (α2β237Ser), L Ferrara (α247Glyβ2), Broussais (α290Asnβ2), and Dhofar (α2β258Arg)

The oxygen equilibrium characteristics of four structural variants of hemoglobin A were correlated with their amino acid substitutions.Hemoglobin Dhofar, in which the proline at E2(58)beta is replaced by arginine, had normal oxygen equilibrium characteristics.Hemoglobin L Ferrara. in which the aspartic acid at CD5(47)alpha is replaced by glycine, and hemoglobin Broussais, in which the lysine at FG2(90)alpha is replaced by asparagine, both showed a slightly elevated oxygen affinity; nevertheless both demonstrated a normal heme-heme interaction and a normal Bohr effect.Hemoglobin Hirose, in which the tryptophan at C3 (37)beta is replaced by serine, showed abnormalities of all oxygen equilibrium characteristics; i.e., increased oxygen affinity, diminished heme-heme interaction, and reduced Bohr effect.These results suggest that aspartic acid at CD5(47)alpha and lysine at FG2(90)alpha are involved in the function of the hemoglobin molecule, despite the fact that these positions are not located directly in the heme or the alpha-beta-contact regions.Tryptophan at C3(37)beta is located at contact between alpha(1)- and beta(2)-subunits. It is suggested that the substitution by serine might disturb the quarternary structure of the mutant hemoglobin molecule during transition from oxy-form to deoxy-form resulting in an alteration of the heme function.     

Pharmacologic Inhibition of the TGF-β Type I Receptor Kinase Has Anabolic and Anti-Catabolic Effects on Bone

During development, growth factors and hormones cooperate to establish the unique sizes, shapes and material properties of individual bones. Among these, TGF-β has been shown to developmentally regulate bone mass and bone matrix properties. However, the mechanisms that control postnatal skeletal integrity in a dynamic biological and mechanical environment are distinct from those that regulate bone development. In addition, despite advances in understanding the roles of TGF-β signaling in osteoblasts and osteoclasts, the net effects of altered postnatal TGF-β signaling on bone remain unclear. To examine the role of TGF-β in the maintenance of the postnatal skeleton, we evaluated the effects of pharmacological inhibition of the TGF-β type I receptor (TβRI) kinase on bone mass, architecture and material properties. Inhibition of TβRI function increased bone mass and multiple aspects of bone quality, including trabecular bone architecture and macro-mechanical behavior of vertebral bone. TβRI inhibitors achieved these effects by increasing osteoblast differentiation and bone formation, while reducing osteoclast differentiation and bone resorption. Furthermore, they induced the expression of Runx2 and EphB4, which promote osteoblast differentiation, and ephrinB2, which antagonizes osteoclast differentiation. Through these anabolic and anti-catabolic effects, TβRI inhibitors coordinate changes in multiple bone parameters, including bone mass, architecture, matrix mineral concentration and material properties, that collectively increase bone fracture resistance. Therefore, TβRI inhibitors may be effective in treating conditions of skeletal fragility.     

Abrogation of TGF-β signaling enhances chemokine production and correlates with prognosis in human breast cancer

In human breast cancer, loss of carcinoma cell–specific response to TGF-β signaling has been linked to poor patient prognosis. However, the mechanisms through which TGF-β regulates these processes remain largely unknown. In an effort to address this issue, we have now identified gene expression signatures associated with the TGF-β signaling pathway in human mammary carcinoma cells. The results strongly suggest that TGF-β signaling mediates intrinsic, stromal-epithelial, and host-tumor interactions during breast cancer progression, at least in part, by regulating basal and oncostatin M–induced CXCL1, CXCL5, and CCL20 chemokine expression. To determine the clinical relevance of our results, we queried our TGF-β–associated gene expression signatures in 4 human breast cancer data sets containing a total of 1,319 gene expression profiles and associated clinical outcome data. The signature representing complete abrogation of TGF-β signaling correlated with reduced relapse-free survival in all patients; however, the strongest association was observed in patients with estrogen receptor–positive (ER-positive) tumors, specifically within the luminal A subtype. Together, the results suggest that assessment of TGF-β signaling pathway status may further stratify the prognosis of ER-positive patients and provide novel therapeutic approaches in the management of breast cancer.     

Tumor Necrosis Factor α and Lymphotoxin α Are Not Required for Induction of Acute Experimental Autoimmune Encephalomyelitis

Immunization of mice with myelin components results in experimental autoimmune encephalomyelitis (EAE), which is mediated by myelin-specific CD4⁺ T cells and anti-myelin antibodies. Tumor necrosis factor α (TNF-α) and lymphotoxin α (LT-α) are thought to be involved in the events leading to inflammatory demyelination in the central nervous system. To ascertain this hypothesis 129 × C57BL/6 mice with an inactivation of the tnf and lta genes (129 × C57BL/6^−/−) and SJL/J mice derived from backcrosses of the above mentioned mutant mice (SJL^−/−) were immunized with mouse spinal cord homogenate (MSCH) or proteolipid protein. Both 129 × C57BL/6^−/− mice and SJL^−/− mice developed EAE. In SJL^−/− mice immunized with MSCH, a very severe form of EAE with weight loss, paralysis of all four limbs, and lethal outcome was observed. The histologic hallmark was an intense perivascular and parenchymal infiltration with predominantly CD4⁺ T cells and some CD8⁺ T cells associated with demyelination in both brain and spinal cord. These results indicate that TNF-α and LT-α are not essential for the development of EAE.