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Objectives: Recently, necroptosis has attracted increasing attention in arthritis research; however, it remains unclear whether its regulation is involved in osteoarthritis (OA) pathogenesis. Since receptor-interacting protein kinase-3 (RIP3) plays a pivotal role in necroptosis and its dysregulation is involved in various pathological processes, we investigated the role of the RIP3 axis in OA pathogenesis.Methods: Experimental OA was induced in wild-type or Rip3 knockout mice by surgery to destabilise the medial meniscus (DMM) or the intra-articular injection of adenovirus carrying a target gene (Ad-Rip3 and Ad-Trim24 shRNA). RIP3 expression was examined in OA cartilage from human patients; Trim24, a negative regulator of RIP3, was identified by microarray and in silico analysis. Connectivity map (CMap) and in silico binding approaches were used to identify RIP3 inhibitors and to examine their direct regulation of RIP3 activation in OA pathogenesis.Results: RIP3 expression was markedly higher in damaged cartilage from patients with OA than in undamaged cartilage. In the mouse model, adenoviral RIP3 overexpression accelerated cartilage disruption, whereas Rip3 depletion reduced DMM-induced OA pathogenesis. Additionally, TRIM24 knockdown upregulated RIP3 expression; its downregulation promoted OA pathogenesis in knee joint tissues. The CMap approach and in silico binding assay identified AZ-628 as a potent RIP3 inhibitor and demonstrated that it abolished RIP3-mediated OA pathogenesis by inhibiting RIP3 kinase activity.Conclusions: TRIM24-RIP3 axis perturbation promotes OA chronicity by activating RIP3 kinase, suggesting that the therapeutic manipulation of this pathway could provide new avenues for treating OA. [ABSTRACT FROM AUTHOR]

Background and Purpose: Severe acute pancreatitis (SAP) is characterized by trypsinogen activation, infiltration of leucocytes and tissue necrosis but the intracellular signalling mechanisms regulating organ injury in the pancreas remain elusive. Rho-kinase is a potent regulator of specific cellular processes effecting several pro-inflammatory activities. Herein, we examined the role of Rho-kinase signalling in acute pancreatitis.Experimental Approach: Pancreatitis was induced by infusion of taurocholate into the pancreatic duct in C57BL/6 mice. Animals were treated with a Rho-kinase inhibitor Y-27632 (0.5-5 mg·kg⁻¹) before induction of pancreatitis.Key Results: Taurocholate infusion caused a clear-cut increase in blood amylase, pancreatic neutrophil infiltration, acinar cell necrosis and oedema formation in the pancreas. Levels of pancreatic myeloperoxidase (MPO), macrophage inflammatory protein-2 (MIP-2), trypsinogen activation peptide (TAP) and lung MPO were significantly increased, indicating local and systemic disease. Inhibition of Rho-kinase activity dose-dependently protected against pancreatitis. For example, 5 mg·kg⁻¹ Y-27632 reduced acinar cell necrosis, leucocyte infiltration and pancreatic oedema by 90%, 89% and 58%, respectively, as well as tissue levels of MPO by 75% and MIP-2 by 84%. Moreover, Rho-kinase inhibition decreased lung MPO by 75% and blood amylase by 83%. Pancreatitis-induced TAP levels were reduced by 61% in Y-27632-treated mice. Inhibition of Rho-kinase abolished secretagogue-induced activation of trypsinogen in pancreatic acinar cells in vitro.Conclusions and Implications: Our novel data suggest that Rho-kinase signalling plays an important role in acute pancreatitis by regulating trypsinogen activation and subsequent CXC chemokine formation, neutrophil infiltration and tissue injury. Thus, these results indicate that Rho-kinase may constitute a novel target in the management of SAP. [ABSTRACT FROM AUTHOR]

Background and Purpose: The δ-subunit-containing GABAA receptors, α4 β1 δ and α4 β3 δ, in dentate gyrus granule cells (DGGCs) are known to exhibit both spontaneous channel openings (i.e. constitutive activity) and agonist-induced current. The functional implications of spontaneous gating are unclear. In this study, we tested the hypothesis that constitutively active α4 β1/3 δ receptors limit agonist efficacy.Experimental Approach: Whole-cell electrophysiological recordings of adult male rat and mouse hippocampal DGGCs were used to characterize known agonists and antagonists at δ-subunit-containing GABAA receptors. To separate constitutive and agonist-induced currents, different recording conditions were employed.Key Results: Recordings at either 24°C or 34°C, including the PKC autoinhibitory peptide (19-36) intracellularly, removed spontaneous gating by GABAA receptors. In the absence of spontaneous gating, DGGCs responded to the α4 β1/3 δ orthosteric agonist Thio-THIP with a four-fold increased efficacy relative to recording conditions favouring constitutive activity. Surprisingly, the neutral antagonist gabazine was unable to antagonize the current by Thio-THIP. Furthermore, a current was elicited by gabazine alone only when the constitutive current was silenced (EC50 2.1 μM). The gabazine-induced current was inhibited by picrotoxin, potentiated by DS2, completely absent in δ-/- mice and reduced in β1-/- mice, but could not be replicated in human α4 β1/3 δ receptors expressed heterologously in HEK cells.Conclusion and Implications: Kinase activity infers spontaneous gating in α4 β1/3 δ receptors in DGGCs. This significantly limits the efficacy of GABAA agonists and has implications in pathologies involving aberrant excitability caused by phosphorylation (e.g. addiction and epilepsy). In such cases, the efficacy of δ-preferring GABAA ligands may be reduced. [ABSTRACT FROM AUTHOR]

Background: Autosomal recessive mutations in the glucocerebrosidase gene, Beta-glucocerebrosidase 1 (GBA1), cause the lysosomal storage disorder Gaucher's disease. Heterozygous carriers of most GBA1 mutations have dramatically increased Parkinson's disease (PD) risk, but the mechanisms and cells affected remain unknown. Glucocerebrosidase expression is relatively enriched in astrocytes, yet the impact of its mutation in these cells has not yet been addressed.Objectives: Emerging data supporting non-cell-autonomous mechanisms driving PD pathogenesis inspired the first characterization of GBA1-mutant astrocytes. In addition, we asked whether LRRK2, likewise linked to PD and enriched in astrocytes, intersected with GBA1 phenotypes.Methods: Using heterozygous and homozygous GBA1 D409V knockin mouse astrocytes, we conducted rigorous biochemical and image-based analyses of lysosomal function and morphology. We also examined basal and evoked cytokine response at the transcriptional and secretory levels.Results: The D409V knockin astrocytes manifested broad deficits in lysosomal morphology and function, as expected. This, however, is the first study to show dramatic defects in basal and TLR4-dependent cytokine production. Albeit to different extents, both the lysosomal dysfunction and inflammatory responses were normalized by inhibition of LRRK2 kinase activity, suggesting functional intracellular crosstalk between glucocerebrosidase and LRRK2 activities in astrocytes.Conclusions: These data demonstrate novel pathologic effects of a GBA1 mutation on inflammatory responses in astrocytes, indicating the likelihood of broader immunologic changes in GBA-PD patients. Our findings support the involvement of non-cell-autonomous mechanisms contributing to the pathogenesis of GBA1-linked PD and identify new opportunities to correct these changes with pharmacological intervention. © 2020 International Parkinson and Movement Disorder Society. [ABSTRACT FROM AUTHOR]

Background and Purpose: Immunosuppressive drugs have shown great promise in treating autoimmune diseases in recent years. A series of novel oxazole derivatives were screened for their immunosuppressive activity. PO-322 [1H-indole-2,3-dione 3-(1,3-benzoxazol-2-ylhydrazone)] was identified as the most effective of these compounds. Here, we have investigated the mechanism(s) underlying the inhibition of T-cell proliferation in vitro by PO-322, as well as its effects on the delayed-type hypersensitivity (DTH) response and imiquimod-induced dermatitis in vivo.Experimental Approach: T-cell proliferation and apoptosis were analysed with flow cytometry. Cell viability was assessed with a CCK-8 assay. Protein kinase activity was assessed by SelectScreen Kinase Profiling Services. The phosphorylation of signal-regulated molecules was measured by Western blot. Cytokine levels were determined by elisa. The effect of PO-322 on DTH and imiquimod-induced dermatitis was evaluated in BALB/c mice.Key Results: PO-322 inhibited human T-cell proliferation with anti-CD3/anti-CD28 mAbs or alloantigen without significant cytotoxicity. Importantly, PO-322 was a selective inhibitor of the serum- and glucocorticoid-regulated kinase 1 (SGK1) and decreased NDRG1 phosphorylation but not p70S6K, STAT5, Akt, or ERK1/2 phosphorylation. Furthermore, PO-322 inhibited IFN-γ, IL-6, and IL-17 expression but not IL-10 expression. Finally, treatment with PO-322 was safe and effective for ameliorating the DTH response and imiquimod-induced dermatitis in mice.Conclusions and Implications: PO-322 exerted immunosuppressive activity in vitro and in vivo by selectively inhibiting SGK1 activity. PO-322 represents a potential lead compound for the design and development of new drugs for the treatment of autoimmune diseases. [ABSTRACT FROM AUTHOR]

Introduction: The impact of eccentric exercise on mitochondrial function has only been poorly investigated and remains unclear. This study aimed to identify the changes in skeletal muscle mitochondrial respiration, specifically triggered by a single bout of eccentric treadmill exercise. Methods: Male adult mice were randomly divided into eccentric (ECC; downhill running), concentric (CON; uphill running), and unexercised control groups (n = 5/group). Running groups performed 18 bouts of 5 min at 20 cm·s−1 on an inclined treadmill (±15° to 20°). Mice were sacrificed 48 h after exercise for blood and quadriceps muscles collection. Deep proximal (red) and superficial distal (white) muscle portions were used for high-resolution respirometric measurements. Results: Plasma creatine kinase activity was significantly higher in the ECC compared with CON group, reflecting exercise-induced muscle damage (P < 0.01). The ECC exercise induced a significant decrease in oxidative phosphorylation capacity in both quadriceps femoris parts (P = 0.032 in proximal portion, P = 0.010 in distal portion) in comparison with the CON group. This observation was only made for the nicotinamide adenine dinucleotide (NADH) pathway using pyruvate + malate as substrates. When expressed as a flux control ratio, indicating a change related to mitochondrial quality rather than quantity, this change seemed more prominent in distal compared with proximal portion of quadriceps muscle. No significant difference between groups was found for the NADH pathway with glutamate or glutamate + malate as substrates, for the succinate pathway or for fatty acid oxidation. Conclusions: Our data suggest that ECC exercise specifically affects pyruvate mitochondrial transport and/or oxidation 48 h after exercise, and this alteration mainly concerns the distal white muscle portion. This study provides new perspectives to improve our understanding of the mitochondrial adaptation associated with ECC exercise. [ABSTRACT FROM AUTHOR]

Background: Myocarditis refers to an autoimmune inflammatory response of the myocardium with characterization of self-reactive CD4+ T cell activation, which lacks effective treatment and has a poor prognosis. Acacetin is a natural flavonoid product that has been reported to have anti-inflammatory effects. However, acacetin has not been investigated in myocarditis. Methods: Oral acacetin treatment was administered in an experimental autoimmune myocarditis model established with myosin heavy chain-alpha peptide. Echocardiography, pathological staining, and RT-qPCR were used to detect cardiac function, myocardial injury, and inflammation levels. Flow cytometry was utilized to detect the effect of acacetin on CD4+ T cell function. RNA-seq, molecular docking, and microscale thermophoresis (MST) were employed to investigate potential mechanisms. Seahorse analysis, mitoSOX, JC-1, and mitotracker were utilized to detect the effect of acacetin on mitochondrial function. Results: Acacetin attenuated cardiac injury and fibrosis as well as heart dysfunction, and reduced cardiac inflammatory cytokines and ratio of effector CD4+ T and Th17 cells. Acacetin inhibited CD4+ T cell activation, proliferation, and Th17 cell differentiation. Mechanistically, the effects of acacetin were related to reducing mitochondrial complex II activity thereby inhibiting mitochondrial respiration and mitochondrial reactive oxygen species in CD4+ T cells. Conclusion: Acacetin may be a valuable therapeutic drug in treating CD4+ T cell-mediated myocarditis. [ABSTRACT FROM AUTHOR]

Simple Summary: Neuroblastoma is the most common cancer in infants and the most common solid tumor outside the brain in children, and responds poorly to current therapies. The sphingolipid-modifying drug opaganib, which has anticancer and anti-inflammatory activity in many preclinical models, was tested for inhibition of neuroblastoma cell proliferation in cell culture and in tumors growing in mice. Opaganib inhibited cell proliferation regardless of the MYCN gene status of the neuroblastoma cells. Treatment of tumor-bearing mice with opaganib in combination with the established drugs irinotecan and temozolomide reduced tumor growth and increased survival compared with irinotecan plus temozolomide alone. Because opaganib has already demonstrated safety in patients with cancer, this new drug may provide improved therapy for neuroblastoma patients. Neuroblastoma (NB), the most common cancer in infants and the most common solid tumor outside the brain in children, grows aggressively and responds poorly to current therapies. We have identified a new drug (opaganib, also known as ABC294640) that modulates sphingolipid metabolism by inhibiting the synthesis of sphingosine 1-phosphate (S1P) by sphingosine kinase-2 and elevating dihydroceramides by inhibition of dihydroceramide desaturase. The present studies sought to determine the potential therapeutic activity of opaganib in cell culture and xenograft models of NB. Cytotoxicity assays demonstrated that NB cells, including cells with amplified MYCN, are effectively killed by opaganib concentrations well below those that accumulate in tumors in vivo. Opaganib was shown to cause dose-dependent decreases in S1P and hexosylceramide levels in Neuro-2a cells, while concurrently elevating levels of dihydroceramides. As with other tumor cells, opaganib reduced c-Myc and Mcl-1 protein levels in Neuro-2a cells, and also reduced the expression of the N-Myc protein. The in vivo growth of xenografts of human SK-N-(BE)2 cells with amplified MYCN was suppressed by oral administration of opaganib at doses that are well tolerated in mice. Combining opaganib with temozolomide plus irinotecan, considered the backbone for therapy of relapsed or refractory NB, resulted in increased antitumor activity in vivo compared with temozolomide plus irinotecan or opaganib alone. Mice did not lose additional weight when opaganib was combined with temozolomide plus irinotecan, indicating that the combination is well tolerated. Opaganib has additive antitumor activity toward Neuro-2a tumors when combined with the checkpoint inhibitor anti-CTLA-4 antibody; however, the combination of opaganib with anti-PD-1 or anti-PD-L1 antibodies did not provide increased antitumor activity over that seen with opaganib alone. Overall, the data demonstrate that opaganib modulates sphingolipid metabolism and intracellular signaling in NB cells and inhibits NB tumor growth alone and in combination with other anticancer drugs. Amplified MYCN does not confer resistance to opaganib, and, in fact, the drug attenuates the expression of both c-Myc and N-Myc. The safety of opaganib has been established in clinical trials with adults with advanced cancer or severe COVID-19, and so opaganib has excellent potential for treating patients with NB, particularly in combination with temozolomide and irinotecan or anti-CTLA-4 antibody. [ABSTRACT FROM AUTHOR]

Background and purpose:Mushrooms are popular both as food and as a source of natural compounds of biopharmaceutical interest. Some mushroom-derived compounds such as β-glucan have been shown to be immunostimulatory; this study explores the anti-inflammatory properties of hispidin analogues derived from the mushroom, Inonotus xeranticus. We sought to identify the molecular mechanism of action of these hispidin analogues by determining their effects on lipopolysaccharide (LPS)-mediated inflammatory responses in a macrophage cell line.Experimental approach:The production of inflammatory mediators was determined by Griess assay, reverse transcription-PCR and ELISA. The inhibitory effect of davalliactone on LPS-induced activation of signalling cascades was assessed by western blotting, immunoprecipitation and direct kinase assay.Key results:In activated RAW264.7 cells, davallialactone strongly downregulated LPS-mediated inflammatory responses, including NO production, prostaglandin E2 release, expression of proinflammatory cytokine genes and cell surface expression of co-stimulatory molecules. Davallialactone treatment did not alter cell viability or morphology. Davallialactone was found to exert its anti-inflammatory effects by inhibiting a signalling cascade that activates nuclear factor kappa B via PI3K, Akt and IKK, but not mitogen-activated protein kinases. Treatment with davallialactone affected the phosphorylation of these signalling proteins, but not their level of expression. These inhibitory effects were not due to the interruption of toll-like receptor 4 binding to CD14. In particular, davallialactone strongly inhibited the LPS-induced phosphorylation and kinase activity of Src, implying that Src may be a potential pharmacological target of davallialactone.Conclusions and implications:Our data suggest that davallialactone, a small molecule found in edible mushrooms, has anti-inflammatory activity. Davallialactone can be developed as a pharmaceutically valuable anti-Src kinase agent.British Journal of Pharmacology (2008) 154, 852–863; doi:10.1038/bjp.2008.136; published online 5 May 2008 [ABSTRACT FROM AUTHOR]

Insulin-like growth factor binding protein (IGFBP)-1 has been shown to alter cellular responses to insulin-like growth factor 1 (IGF-1). Human IGFBP-1 undergoes serine phosphorylation, and this enhances both its affinity for IGF-1 by six- to eightfold and its capacity to inhibit IGF-1 actions. To investigate the physiological role of IGFBP-1 in vivo, transgenic mice have been generated using either the human IGFBP-1 or rat IGFBP-1 transgene. Both lines of mice expressed high concentrations of IGFBP-1 in serum and tissues; however, human IGFBP-1 transgenic mice did not show glucose intolerance and exhibited no significant intrauterine growth retardation, whereas rat IGFBP-1 transgenic mice showed fasting hyperglycemia and intrauterine growth restriction. The aim of this study was to investigate the physiological differences in the phosphorylation state of human IGFBP-1 and rat IGFBP-1 in these transgenic mice. The phosphorylation status of IGFBP-1 in transgenic mouse serum was analyzed by nondenaturing PAGE. Almost all of the IGFBP-1 in serum from the human IGFBP-1 transgenic mice was present as a nonphosphorylated form. Most of the rat IGFBP-1 in the serum of the mice expressing the rat IGFBP-1 was phosphorylated. Immunoprecipitation showed that mouse hepatoma (Hepa 1-6) cells (exposed to [32P]H3PO4) secrete 32P-labeled IGFBP-1. When the human IGFBP-1 transgene was transfected into Hepa 1-6 cells, all of the IGFBP-1 was secreted in the nonphosphorylated form. However, when the rat IGFBP-1 transgene was transfected into these cells, phosphorylated forms of IGFBP-1 were secreted. To confirm this result, the mouse hepatoma cell protein kinase was partially purified. This kinase activity phosphorylated mouse and rat IGFBP-1 in vitro, but it did not phosphorylate human IGFBP-1. Scatchard analysis showed that the affinity of phosphorylated rat IGFBP-1 for IGF-1 was 3.9-fold higher than that of nonphosphorylated human IGFBP-1. We conclude that the mouse IGFBP-1 kinase activity cannot phosphorylate human IGFBP-1, whereas it can phosphorylate rat IGFBP-1. The phosphorylation state of human IGFBP-1 may account for part of the phenotypic differences noted in the two studies of transgenic mice, and it is an important determinant of the capacity of human IGFBP-1 to inhibit IGF-1 actions in vivo. [ABSTRACT FROM AUTHOR]

Anemia in β-thalassemia is related to ineffective erythropoiesis and reduced red cell survival. Excess free heme and accumulation of unpaired α-globin chains impose substantial oxidative stress on β-thalassemic erythroblasts and erythrocytes, impacting cell metabolism. We hypothesized that increased pyruvate kinase activity induced by mitapivat (AG-348) in the Hbbth3/+ mouse model for β-thalassemia would reduce chronic hemolysis and ineffective erythropoiesis through stimulation of red cell glycolytic metabolism. Oral mitapivat administration ameliorated ineffective erythropoiesis and anemia in Hbbth3/+ mice. Increased ATP, reduced reactive oxygen species production, and reduced markers of mitochondrial dysfunction associated with improved mitochondrial clearance suggested enhanced metabolism following mitapivat administration in β-thalassemia. The amelioration of responsiveness to erythropoietin resulted in reduced soluble erythroferrone, increased liver Hamp expression, and diminished liver iron overload. Mitapivat reduced duodenal Dmt1 expression potentially by activating the pyruvate kinase M2-HIF2α axis, representing a mechanism additional to Hamp in controlling iron absorption and preventing β-thalassemia-related liver iron overload. In ex vivo studies on erythroid precursors from patients with β-thalassemia, mitapivat enhanced erythropoiesis, promoted erythroid maturation, and decreased apoptosis. Overall, pyruvate kinase activation as a treatment modality for β-thalassemia in preclinical model systems had multiple beneficial effects in the erythropoietic compartment and beyond, providing a strong scientific basis for further clinical trials. [ABSTRACT FROM AUTHOR]

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Background: The cyclic AMP (adenosine monophosphate; cAMP)-hydrolyzing protein PDE4B (phosphodiesterase 4B) is a key negative regulator of cardiac β-adrenergic receptor stimulation. PDE4B deficiency leads to abnormal Ca2+ handling and PDE4B is decreased in pressure overload hypertrophy, suggesting that increasing PDE4B in the heart is beneficial in heart failure.Methods: We measured PDE4B expression in human cardiac tissues and developed 2 transgenic mouse lines with cardiomyocyte-specific overexpression of PDE4B and an adeno-associated virus serotype 9 encoding PDE4B. Myocardial structure and function were evaluated by echocardiography, ECG, and in Langendorff-perfused hearts. Also, cAMP and PKA (cAMP dependent protein kinase) activity were monitored by Förster resonance energy transfer, L-type Ca2+ current by whole-cell patch-clamp, and cardiomyocyte shortening and Ca2+ transients with an Ionoptix system. Heart failure was induced by 2 weeks infusion of isoproterenol or transverse aortic constriction. Cardiac remodeling was evaluated by serial echocardiography, morphometric analysis, and histology.Results: PDE4B protein was decreased in human failing hearts. The first PDE4B-transgenic mouse line (TG15) had a ≈15-fold increase in cardiac cAMP-PDE activity and a ≈30% decrease in cAMP content and fractional shortening associated with a mild cardiac hypertrophy that resorbed with age. Basal ex vivo myocardial function was unchanged, but β-adrenergic receptor stimulation of cardiac inotropy, cAMP, PKA, L-type Ca2+ current, Ca2+ transients, and cell contraction were blunted. Endurance capacity and life expectancy were normal. Moreover, these mice were protected from systolic dysfunction, hypertrophy, lung congestion, and fibrosis induced by chronic isoproterenol treatment. In the second PDE4B-transgenic mouse line (TG50), markedly higher PDE4B overexpression, resulting in a ≈50-fold increase in cardiac cAMP-PDE activity caused a ≈50% decrease in fractional shortening, hypertrophy, dilatation, and premature death. In contrast, mice injected with adeno-associated virus serotype 9 encoding PDE4B (1012 viral particles/mouse) had a ≈50% increase in cardiac cAMP-PDE activity, which did not modify basal cardiac function but efficiently prevented systolic dysfunction, apoptosis, and fibrosis, while attenuating hypertrophy induced by chronic isoproterenol infusion. Similarly, adeno-associated virus serotype 9 encoding PDE4B slowed contractile deterioration, attenuated hypertrophy and lung congestion, and prevented apoptosis and fibrotic remodeling in transverse aortic constriction.Conclusions: Our results indicate that a moderate increase in PDE4B is cardioprotective and suggest that cardiac gene therapy with PDE4B might constitute a new promising approach to treat heart failure. [ABSTRACT FROM AUTHOR]

Salt-inducible kinases (SIKs) are key regulators of cellular metabolism and growth, but their role in cardiomyocyte plasticity and heart failure pathogenesis remains unknown. Here, we showed that loss of SIK1 kinase activity protected against adverse cardiac remodeling and heart failure pathogenesis in rodent models and cardiomyocytes derived from human induced pluripotent stem cells. We found that SIK1 phosphorylated and stabilized histone deacetylase 7 (HDAC7) protein during cardiac stress, an event that is required for pathologic cardiomyocyte remodeling. Gain- and loss-of-function studies of HDAC7 in cultured cardiomyocytes implicated HDAC7 as a prohypertrophic signaling effector that can induce c-Myc expression, indicating a functional departure from the canonical MEF2 corepressor function of class IIa HDACs. Taken together, our findings reveal what we believe to be a previously unrecognized role for a SIK1/HDAC7 axis in regulating cardiac stress responses and implicate this pathway as a potential target in human heart failure. [ABSTRACT FROM AUTHOR]

Exosomes, as functional paracrine units of therapeutic cells, can partially reproduce the reparative properties of their parental cells. The constitution of exosomes, as well as their biological activity, largely depends on the cells that secrete them. We isolated exosomes from explant-derived cardiac stromal cells from patients with heart failure (FEXO) or from normal donor hearts (NEXO) and compared their regenerative activities in vitro and in vivo. Patients in the FEXO group exhibited an impaired ability to promote endothelial tube formation and cardiomyocyte proliferation in vitro. Intramyocardial injection of NEXO resulted in structural and functional improvements in a murine model of acute myocardial infarction. In contrast, FEXO therapy exacerbated cardiac function and left ventricular remodeling. microRNA array and PCR analysis revealed dysregulation of miR-21-5p in FEXO. Restoring miR-21-5p expression rescued FEXO's reparative function, whereas blunting miR-21-5p expression in NEXO diminished its therapeutic benefits. Further mechanistic studies revealed that miR-21-5p augmented Akt kinase activity through the inhibition of phosphatase and tensin homolog. Taken together, the heart failure pathological condition altered the miR cargos of cardiac-derived exosomes and impaired their regenerative activities. miR-21-5p contributes to exosome-mediated heart repair by enhancing angiogenesis and cardiomyocyte survival through the phosphatase and tensin homolog/Akt pathway. [ABSTRACT FROM AUTHOR]

Background: Fundc1 (FUN14 domain containing 1), an outer mitochondrial membrane protein, is important for mitophagy and mitochondria-associated endoplasmic reticulum membranes (MAMs). The roles of Fundc1 and MAMs in diabetic hearts remain unknown. The aims of this study, therefore, were to determine whether the diabetes mellitus-induced Fundc1 expression could increase MAM formation, and whether disruption of MAM formation improves diabetic cardiac function.Methods: Levels of FUNDC1 were examined in the hearts from diabetic patients and nondiabetic donors. Levels of Fundc1-induced MAMs and mitochondrial and heart function were examined in mouse neonatal cardiomyocytes exposed to high glucose (HG, 30 mmol/L d-glucose for 48 hours), and in streptozotocin-treated cardiac-specific Fundc1 knockout mice and cardiac-specific Fundc1 knockout diabetic Akita mice, as well.Results: FUNDC1 levels were significantly elevated in cardiac tissues from diabetic patients in comparison with those from nondiabetic donors. In cultured mouse neonatal cardiomyocytes, HG conditions increased levels of Fundc1, the inositol 1,4,5-trisphosphate type 2 receptor (Ip3r2), and MAMs. Genetic downregulation of either Fundc1 or Ip3r2 inhibited MAM formation, reduced endoplasmic reticulum-mitochondrial Ca2+ flux, and improved mitochondrial function in HG-treated cardiomyocytes. Consistently, adenoviral overexpression of Fundc1 promoted MAM formation, mitochondrial Ca2+ increase, and mitochondrial dysfunction in cardiomyocytes exposed to normal glucose (5.5 mmol/L d-glucose). In comparison with nondiabetic controls, levels of Fundc1, Ip3r2, and MAMs were significantly increased in hearts from streptozotocin-treated mice and Akita mice. Furthermore, in comparison with control hearts, diabetes mellitus markedly increased coimmunoprecipitation of Fundc1 and Ip3r2. The binding of Fundc1 to Ip3r2 inhibits Ip3r2 ubiquitination and proteasome-mediated degradation. Cardiomyocyte-specific Fundc1 deletion ablated diabetes mellitus-induced MAM formation, prevented mitochondrial Ca2+ increase, mitochondrial fragmentation, and apoptosis with improved mitochondrial functional capacity and cardiac function. In mouse neonatal cardiomyocytes, HG suppressed AMP-activated protein kinase activity. Furthermore, in cardiomyocytes of Prkaa2 knockout mice, expression of Fundc1, MAM formation, and mitochondrial Ca2+ levels were significantly increased. Finally, adenoviral overexpression of a constitutively active mutant AMP-activated protein kinase ablated HG-induced MAM formation and mitochondrial dysfunction.Conclusions: We conclude that diabetes mellitus suppresses AMP-activated protein kinase, initiating Fundc1-mediated MAM formation, mitochondrial dysfunction, and cardiomyopathy, suggesting that AMP-activated protein kinase-induced Fundc1 suppression is a valid target to treat diabetic cardiomyopathy. [ABSTRACT FROM AUTHOR]

Background: The ataxia telangiectasia mutated and Rad3-related kinase (ATR) has a key role in the signalling of stalled replication forks and DNA damage to cell cycle checkpoints and DNA repair. It has long been recognised as an important target for cancer therapy but inhibitors have proved elusive. As NU6027, originally developed as a CDK2 inhibitor, potentiated cisplatin in a CDK2-independent manner we postulated that it may inhibit ATR.Methods: Cellular ATR kinase activity was determined by CHK1 phosphorylation in human fibroblasts with inducible dominant-negative ATR-kinase dead expression and human breast cancer MCF7 cells. Cell cycle effects and chemo- and radiopotentiation by NU6027 were determined in MCF7 cells and the role of mismatch repair and p53 was determined in isogenically matched ovarian cancer A2780 cells.Results: NU6027 is a potent inhibitor of cellular ATR activity (IC(50)=6.7 μM) and enhanced hydroxyurea and cisplatin cytotoxicity in an ATR-dependent manner. NU6027 attenuated G2/M arrest following DNA damage, inhibited RAD51 focus formation and increased the cytotoxicity of the major classes of DNA-damaging anticancer cytotoxic therapy but not the antimitotic, paclitaxel. In A2780 cells sensitisation to cisplatin was greatest in cells with functional p53 and mismatch repair (MMR) and sensitisation to temozolomide was greatest in p53 mutant cells with functional MMR. Importantly, NU6027 was synthetically lethal when DNA single-strand break repair is impaired either through poly(ADP-ribose) polymerase (PARP) inhibition or defects in XRCC1.Conclusion: NU6027 inhibits ATR, impairing G2/M arrest and homologous recombination thus increasing sensitivity to DNA-damaging agents and PARP inhibitors. It provides proof of concept data for clinical development of ATR inhibitors. [ABSTRACT FROM AUTHOR]

Abstract: Over-expression of EGFR, as in most cases of ovarian cancer, is associated with advanced-stage disease and poor prognosis. Activation of EGFR signaling pathway is involved in increased cell proliferation, angiogenesis, metastasis and decreased apoptosis. Tyrosine kinase activity is essential for signal transduction and receptor down-regulation. However, we found in this study that tyrosine kinase activity is not necessary in ligand-induced EGFR down-regulation in ovarian cancer cell line CaOV3 cells. EGFR tyrosine kinase inhibitors, such as PD153035, AG1478, as well as non-specific tyrosine kinase inhibitor PP2 cannot reverse EGF-induced down-regulation of EGFR. These findings thus permit us to develop the following exciting but unconventional strategy to sensitize cancer cells, namely, by priming ovarian cancer cells with EGF and EGFR inhibitor PD153035, before chemotherapy. This priming procedure down-regulates EGFR without induction of mitogenic signals such as ERK and PI3K/AKT. EGF plus EGFR inhibitor-primed ovarian cancer cells display increased sensitivity to taxol-induced cell death, resistant to EGF-induced cell migration and cell proliferation as well as ERK and PI3K/AKT activation. Further studies showed that PD153035, which does not reverse ligand-induced EGFR down-regulation, blocks EGF-induced EGFR activation as well as EGFR’s binding to c-cbl and Grb2. Taken together, we contend that priming with EGFR inhibitors plus EGF inhibits cell signaling pathways leading to cell proliferation and survival, while down-regulating EGFR. This priming approach sensitizes ovarian cancer cells and would ultimately result in better chemotherapeutical outcome. [Copyright &y& Elsevier]

Background: Two mechanisms exist for inducing renal proximal tubule hypertrophy. One is characterized by regulation of the G1 cell cycle kinase (cell cycle-dependent mechanism), while the other mechanism involves an imbalance between rates of protein synthesis and degradation, and occurs independently of cell cycle kinase regulation (cell cycle-independent mechanism). The present studies examined whether the compensatory proximal tubule growth following uninephrectomy is mediated by the cell cycle-dependent or -independent mechanism.Methods: Studies were done in both rats and C57Bl6 mice on tissue harvested from sham-operated or uninephrectomized animals. The magnitude of BrdU incorporation was used as the hyperplasia marker, while the proximal tubule protein: DNA ratio was used as the hypertrophy marker. Cdk4/cyclin D and cdk2/cyclin E kinase activities were assayed on renal cortex (rat studies) or isolated proximal tubules (mouse studies) using an in vitro kinase assay.Results: In both rats and mice, compensatory proximal tubule growth was hypertrophic, not hyperplastic, evidenced by an increase in the protein:DNA ratio without a change in BrdU incorporation. In mice, cdk4/cyclin D kinase activity progressively increased between days 4 and 7, while cdk2/cyclin E kinase activity was decreased at both 4 and 7 days. In rats the development of hypertrophy was associated with an increase in cdk4/cyclin D kinase at days 4, 7, and 10, and an increase in cdk2/cyclin E kinase activity at days 2, 4, and 7. Roscovitine, a cdk2/cyclin E kinase inhibitor, inhibited cdk2/cyclin E kinase activity in both sham and nephrectomized rats; however, it did not prevent the development of proximal tubule hypertrophy.Conclusions: Uninephrectomy-induced compensatory proximal tubule growth is a hypertrophic form of growth that is mediated by a cell cycle-dependent mechanism. [ABSTRACT FROM AUTHOR]

1 The role of substance P and its high affinity neurokinin-1 receptor in colitis has not been fully elucidated. We assessed the participation of neurokinin-1 receptor in colitis using the 2,4,6,-trinitrobenzensulphonic add and dextran sulphate-induced animal models of colitis and genetically-engineered, neurokinin-1 receptor-deficient mice. 2 Clinical signs, macroscopic and histologic damage associated with 2,4,6,-trinitrobenzensulphonic acid (12 days) and dextran sulphate (5 days) colitis were more severe in neurokinin-1 deficient than in wild-type mice, while immuno reactivities for epidermal growth factor and its receptor were similar in the colon of both mice strains before and after colitis. 3 Substance P. dose-dependently induced intestinal fibroblast proliferation and enhanced epidermal growth factor-induced proliferation in intestinal fibroblasts isolated from wild-type, but not from neurokinin-1 receptor deficient mice. 4 Substance P-induced intestinal fibroblast proliferation required the presence of epidermal growth factor receptor with kinase activity. Furthermore, substance P induced epidermal growth factor tyrosine phosphorylation and activation in normal intestinal fibroblasts. 5 Our results indicate that in mice lacking the neurokinin-I receptor, substance P plays a protective role in prolonged experimental colitis. [ABSTRACT FROM AUTHOR]

Aberrant expression of FLT3 has been found in most cases of B-lineage ALL and AML, and subsets of T cell ALL, CML in blast crisis and CLL. In 20% of patients with AML the receptor has small internal tandem duplications of the juxtamembrane region which appear to contitutively activate the receptor. To investigate whether FLT3 activation could play a role in leukemia, we generated a constitutively activated FLT3 by fusing its cytoplasmic domain to the helix-loop-helix domain of TEL in analogy to the fusion that occurs with TEL-PDGFR in CMML. In vitro translation assays demonstrated oligomerization and intrinsic tyrosine kinase activity of the TEL-FLT3 chimeric receptor. Constitutively activated TEL-FLT3 conferred IL-3 independence and long-term proliferation to transfected Ba/F3 cells. Immunoblot analyses showed that JAK 2, STAT 3, STAT 5a, STAT 5b and CBL were tyrosine-phosphorylated in TEL-FLT3 expressing Ba/F3 cells in the absence of IL-3. These data suggest a possible role for the JAK/STAT pathway in FLT3 signaling. Transplantation of TEL-FLT3 expressing Ba/F3 cells into syngeneic mice caused mortality in all mice by 3 weeks after injection. Histopathologic analysis demonstrated a massive infiltration of mononuclear cells in the liver, spleen and bone marrow. The mimicking of naturally occurring TEL fusions provides an approach to assess aspects of the biology of activated FLT3, or other receptor-type tyrosine kinases (RTKs) in leukemic transformation. [ABSTRACT FROM AUTHOR]

A decline in plasma concentration of insulin-like growth factor-1 (IGF-1) has been hypothesized to contribute to a decrease in tissue protein synthesis and function in aging animals and man. In this study, the effects of aging and long-term caloric restriction on growth hormone receptor signal transduction were assessed in hepatic tissue to determine whether alterations in tissue responsiveness to growth hormone contribute to the decline in IGF-1 gene expression. Liver slices from female C57/BL mice (10, 17, and 31 months) were prepared in media and stimulated with growth hormone (2 nM). An increase in growth hormone receptor binding was observed in 31-month ad libitum-fed animals (p < .01) compared to 10- or 17-month-old animals), and this effect was partially attenuated by moderate caloric restriction. However, growth hormone (2 nM)-induced IGF-1 gene expression was significantly lower in old ad libitum-fed animals (p < .05 compared to 10-month-old ad libitum and 31-month-old caloric-restricted animals). Further analysis revealed that growth hormone receptor and JAK2 kinase phosphorylation as well as mitogen-activated protein (MAP) kinase activity were significantly lower in old animals compared to the adult or middle-age groups (p < .05). Old caloric-restricted animals demonstrated a significant increase in growth hormone receptor and JAK2 kinase phosphorylation and MAP kinase activity in response to growth hormone. The results demonstrate that growth hormone increases growth hormone receptor and JAK2 kinase phosphorylation as well as MAP kinase activity in liver. These responses decrease with age and are attenuated by moderate, long-term caloric restriction. [ABSTRACT FROM AUTHOR]

Primary palatogenesis in mice is similar to that in humans, and spontaneous cleft lip appears to be multifactorially determined in both. Binding of a ligand to erbB4 has been shown to stimulate the receptor's protein kinase activity, which subsequently stimulates a signal-transduction cascade leading to cell growth and differentiation, and to morphogenesis during development. In this study, an immunohistochemical technique was used to investigate the temporal and spatial expression of erbB4 in the primary palate of cleft (A/WySn) and noncleft strains of mice (BALB/cBy). Positive staining of erbB4 was found in ectodermal and mesenchymal cells of facial prominences before the primary palate formation stage (day 10, hour 20) in both strains. During the primary palate formation stage (day 11, hour 20), positive staining of erbB4 was found in the ectodermal and mesenchymal cells of the facial prominences of the noncleft strain, but not in those of the cleft strain. These results suggest erbB4 expression may be associated with normal primary palatogenesis of mice and, conversely, cleft lip may be associated with a deficiency of erbB4 expression during primary palate formation in mice. [ABSTRACT FROM AUTHOR]

Objective: Osteoarthritis (OA) is one of the most common degenerative joint diseases and is associated with autophagy suppression. However, the molecular mechanism of autophagy regulation in the context of OA is not fully understood. In this study, we sought to determine the role that HECTD1 plays in the pathogenesis of OA. Methods: We used RNA sequencing analysis to explore the differential expression of E3 ubiquitin ligase genes in healthy human cartilage and human cartilage affected by OA. Using surgery‐ and aging‐induced OA mouse models, we comprehensively analyzed the function of the screened gene Hectd1 in the development of OA; furthermore, we dissected the mechanism by which HECTD1 regulates autophagy and OA progression using a combination of molecular biologic, cell biologic, and biochemical approaches. Results: HECTD1 was significantly down‐regulated in human OA cartilage samples compared to healthy cartilage samples. Overexpression of HECTD1 in mouse joints alleviated OA pathogenesis, whereas conditional depletion of Hectd1 in cartilage samples aggravated surgery‐ and aging‐induced OA pathogenesis. Mechanistically, HECTD1 bound to Rubicon and ubiquitinated Rubicon at lysine residue 534, which targets Rubicon for proteasomal degradation. More importantly, HECTD1‐mediated Rubicon degradation regulated chondrocyte autophagy, leading to mitigation of stress‐induced chondrocyte death and the subsequent progression of OA. Conclusion: HECTD1 plays a crucial role in the pathogenesis of OA, in that HECTD1 regulates chondrocyte autophagy by ubiquitinating and targeting Rubicon for proteasomal degradation. [ABSTRACT FROM AUTHOR]

Mutant Kras and chronic pancreatitis are the most common pathological events involved in human pancreatic cancer. It has been demonstrated that c-Raf is responsible for transmitting signals from mutant Ras to its downstream signals including MEK-ERK and for initiating carcinogenesis. The soluble epoxide hydrolase (sEH), a pro-inflammatory enzyme, generally inactivates anti-inflammatory and anti-pain epoxyeicosatrienoic acids (EETs). Herein, we have synthesized a novel compound of trans-4-{4-[3-(4-chloro-3-trifluoromethyl-phenyl)-ureido]-cyclohexyloxy}-pyridine-2-carboxylic acid methylamide (t-CUPM) via modifying the central phenyl ring of sorafenib and confirmed its dual inhibition of sEH and c-Raf by recombinant kinase activity assay. Pharmacokinetic analysis revealed that oral dosing of t-CUPM resulted in higher blood levels than that of sorafenib throughout the complete time course (48 h). The effect of t-CUPM on the inhibition of mutant Kras(G12D)-initiated murine pancreatic cancer cell growth was determined using the mouse pancreatic carcinoma cell model obtained from LSL-Kras(G12D)/Pdx1-Cre mice and showed that t-CUPM significantly inhibited this murine pancreatic carcinoma cell growth both in vitro and in mice in vivo. Inhibition of mutant Kras-transmitted phosphorylations of cRAF/MEK/ERK was demonstrated in these pancreatic cancer cells using Western blot assay and immunohistochemical approach. Modulation of oxylipin profile, particularly increased EETs/DHET ratio by sEH inhibition, was observed in mice treated with t-CUPM. These results indicate that t-CUPM is a highly potential agent to treat pancreatic cancer via simultaneously targeting c-Raf and sEH. [ABSTRACT FROM AUTHOR]

Activated macrophages have been classified into classical (M1) and alternative (M2) macrophages. We aimed to establish a method to yield enough number of macrophages to analyze their molecular, biological and immunological functions. We used drugs; adjuvant albumin from chicken egg whites--Imject Alum (OVA-Alum) and OVA Complete Freund Adjuvant (OVA-CFA), to induce macrophages to M2 and M1 respectively. We analyzed the phenotype of purified macrophages induced under these immune conditions, using flow cytometry (FACS) to detect cell-surface molecules and the enzyme-linked immunosorbent assay (ELISA) was used to detect cytokines. The cDNA microarray was employed to measure changes in expression level of cell surface protein between M1 and M2 macrophages. Phenotype analysis of purified macrophages, induced under these immune conditions, showed macrophages induced by OVA-Alum was almost M2 while the proportion of M1 macrophages induced by OVA-CFA was significantly higher. The results also showed higher expression level of macrophage galactose N- acetyl-galactosamine specific lectin-2 protein (MGL1/2-PE), a known M2 macrophage marker, on the surface of Alum-induced macrophages. On the basis of these preliminary data, ELISA results revealed that after macrophage stimulation with lipopolysaccharides (LPS), the level of interleukin (IL)-10 produced by Alum- induced macrophages was higher than the level of IL-10 produced by CFA-induced macrophages. In contrast, the level of tumor necrosis factor-alpha (TNF-α) produced by CFA-induced macrophages was higher than Alum-induced macrophages. The cDNA microarray confirmed previous results and suggest immunoglobulin-like type 2 receptor alpha (Pilra) as a new marker for M1, macrophage galactose N-acetylgalactosamine-specific lectin 2 (Mgl2) as M2 macrophages marker. [ABSTRACT FROM AUTHOR]

Study Question: What biological processes are linked to the signaling of the energy sensor 5'-AMP-activated protein kinase (AMPK) in mouse and human granulosa cells (GCs)?Summary Answer: The lack of α1AMPK in GCs impacted cell cycle, adhesion, lipid metabolism and induced a hyperandrogenic response.What Is Known Already: AMPK is expressed in the ovarian follicle, and its activation by pharmacological medications, such as metformin, inhibits the production of steroids. Polycystic ovary syndrome (PCOS) is responsible for infertility in approximately 5-20% of women of childbearing age and possible treatments include reducing body weight, improving lifestyle and the administration of a combination of drugs to improve insulin resistance, such as metformin.Study Design, Size, Duration: AMPK signaling was evaluated by analyzing differential gene expression in immortalized human granulosa cells (KGNs) with and without silencing α1AMPK using CRISPR/Cas9. In vivo studies included the use of a α1AMPK knock-out mouse model to evaluate the role of α1AMPK in folliculogenesis and fertility. Expression of α1AMPK was evaluated in primary human granulosa-luteal cells retrieved from women undergoing IVF with and without a lean PCOS phenotype (i.e. BMI: 18-25 kg/m2).Participants/materials, Setting, Methods: α1AMPK was disrupted in KGN cells and a transgenic mouse model. Cell viability, proliferation and metabolism were evaluated. Androgen production was evaluated by analyzing protein levels of relevant enzymes in the steroid pathway by western blots, and steroid levels obtained from in vitro and in vivo models by mass spectrometry. Differential gene expression in human GC was obtained by RNA sequencing. Analysis of in vivo murine folliculogenesis was performed by histology and immunochemistry, including evaluation of the anti-Müllerian hormone (AMH) marker. The α1AMPK gene expression was evaluated by quantitative RT-PCR in primary GCs obtained from women with the lean PCOS phenotype (n = 8) and without PCOS (n = 9).Main Results and the Role Of Chance: Silencing of α1AMPK in KGN increased cell proliferation (P < 0.05 versus control, n = 4), promoted the use of fatty acids over glucose, and induced a hyperandrogenic response resulting from upregulation of two of the enzymes involved in steroid production, namely 3β-hydroxysteroid dehydrogenase (3βHSD) and P450 side-chain cleavage enzyme (P450scc) (P < 0.05, n = 3). Female mice deficient in α1AMPK had a 30% decrease in their ovulation rate (P < 0.05, n = 7) and litter size, a hyperandrogenic response (P < 0.05, n = 7) with higher levels of 3βHSD and p450scc levels in the ovaries, and an increase in the population of antral follicles (P < 0.01, n = 10) compared to controls. Primary GCs from lean women with PCOS had lower α1AMPK mRNA expression levels than the control group (P < 0.05, n = 8-9).Large Scale Data: The FastQ files and metadata were submitted to the European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB46048.Limitations, Reasons For Caution: The human KGN is a not fully differentiated, transformed cell line. As such, to confirm the role of AMPK in GC and the PCOS phenotype, this model was compared to two others: an α1AMPK transgenic mouse model and primary differentiated granulosa-lutein cells from non-obese women undergoing IVF (with and without PCOS). A clear limitation is the small number of patients with PCOS utilized in this study and that the collection of human GCs was performed after hormonal stimulation.Wider Implications Of the Findings: Our results reveal that AMPK is directly involved in steroid production in human GCs. In addition, AMPK signaling was associated with other processes frequently reported as dysfunctional in PCOS models, such as cell adhesion, lipid metabolism and inflammation. Silencing of α1AMPK in KGN promoted folliculogenesis, with increases in AMH. Evaluating the expression of the α1AMPK subunit could be considered as a marker of interest in infertility cases related to hormonal imbalances and metabolic disorders, including PCOS.Study Funding/competing Interest(s): This study was financially supported by the Institut National de la Recherche Agronomique (INRA) and the national programme « FERTiNERGY » funded by the French National Research Agency (ANR). The authors report no intellectual or financial conflicts of interest related to this work. R.K. is identified as personnel of the International Agency for Research on Cancer/World Health Organization. R.K. alone is responsible for the views expressed in this article and she does not necessarily represent the decisions, policy or views of the International Agency for Research on Cancer/World Health Organization.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Study Question: Are mutations in MOS (MOS proto-oncogene, serine/threonine kinase) involved in early embryonic arrest in infertile women?Summary Answer: We identified mutations in MOS that may cause human female infertility characterized by preimplantation embryonic arrest (PREMBA), and the effects of the mutations in human embryonic kidney 293T (HEK293T cells) and mouse oocytes provided evidence for a causal relation between MOS and female infertility.What Is Known Already: MOS, an activator of mitogen-activated protein kinase, mediates germinal vesicle breakdown and metaphase II arrest. Female MOS knockout mice are viable but sterile. Thus, MOS seems to be an important part of the mammalian cell cycle mechanism that regulates female meiosis.Study Design, Size, Duration: Whole-exome sequencing, bioinformatics filtering analysis and genetic analysis were performed to identify two different biallelic mutations in MOS in two independent families. The infertile patients presenting with early embryonic arrest were recruited from October 2018 to June 2020.Participants/materials, Setting, Methods: The female patients diagnosed with primary infertility were recruited from the reproduction centres of local hospitals. Genomic DNA from the affected individuals, their family members and healthy controls was extracted from peripheral blood. We performed whole-exome sequencing in patients diagnosed with PREMBA. Functional effects of the mutations were investigated in HEK293T cells by western blotting and in mouse oocytes by microinjection and immunofluorescence.Main Results and the Role Of Chance: We identified the homozygous missense mutation c.285C>A (p.(Asn95Lys)) and the compound heterozygous mutations c.467delG (p.(Gly156Alafs*18)) and c.956G>A (p.(Arg319His)) in MOS in two independent patients. The mutations c.285C>A (p.(Asn95Lys)) and c.467delG (p.(Gly156Alafs*18)) reduced the protein level of MOS, and all mutations reduced the ability of MOS to phosphorylate its downstream target, extracellular signal-regulated kinase1/2. In addition, the identified mutations reduced the capacity of exogenous human MOS to rescue the metaphase II exit phenotype, and the F-actin cytoskeleton of mouse oocytes was affected by the patient-derived mutations.Limitations, Reasons For Caution: Owing to the lack of in vivo data from patient oocytes, the exact molecular mechanism affected by MOS mutations and leading to PREMBA is still unknown and should be further investigated using knock-out or knock-in mice.Wider Implications Of the Findings: We identified recessive mutations in MOS in two independent patients with the PREMBA phenotype. Our findings reveal the important role of MOS during human oocyte meiosis and embryonic development and suggest that mutations in MOS may be precise diagnostic markers for clinical genetic counselling.Study Funding/competing Interest(s): This work was supported by the National Natural Science Foundation of China (81725006, 81822019, 81771581, 81971450, 81971382,82001538 and 82071642), the project supported by the Shanghai Municipal Science and Technology Major Project (2017SHZDZX01), the Project of the Shanghai Municipal Science and Technology Commission (19JC1411001), the Natural Science Foundation of Shanghai (19ZR1444500 and 21ZR1404800), the Shuguang Program of the Shanghai Education Development Foundation and the Shanghai Municipal Education Commission (18SG03), the Foundation of the Shanghai Health and Family Planning Commission (20154Y0162), the Capacity Building Planning Program for Shanghai Women and Children's Health Service and the collaborative innovation centre project construction for Shanghai Women and Children's Health. The authors have no conflicts of interest to declare.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Background: Long noncoding RNA NEAT1 has been implicated in glioma progression. However, the effect of NEAT1 on glycolysis of glioma cell and the potential mechanism remain unclear.Methods: In vitro experiments, including CCK-8, colony formation, ECAR, and lactate detection assays were performed to evaluate the effect of NEAT1 on proliferation and glycolysis of glioma cell. RNA pulldown and RIP assays were performed to identify the interaction between NEAT1 and PGK1. Truncated mutation of NEAT1 and PGK1 was used to confirm the specific interactive domains between NEAT1 and PGK1. Animal studies were performed to analyze the effect of NEAT1/PGK1 on glioma progression.Results: NEAT1 knockdown significantly suppressed the proliferation and glycolysis of glioma cells. NEAT1 could specifically interact with PGK1, which promotes PGK1 stability. Hairpin A of NEAT1 is essential for interaction with M1 domain of PGK1. Depletion of NEAT1 markedly inhibited tumor growth in mice, while PGK1 could reverse this effect. Higher expression of NEAT1 was associated with poor overall survival of GBM patients.Conclusions: NEAT1 over expression promotes glioma progression through stabilizing PGK1. NEAT1/PGK1 axis is a candidate therapeutic target for glioma treatment. [ABSTRACT FROM AUTHOR]

Background: Cancer screening provides the opportunity to detect cancer early, ideally before symptom onset and metastasis, and offers an increased opportunity for a better prognosis. The ideal biomarkers for cancer screening should discriminate individuals who have not developed invasive cancer yet but are destined to do so from healthy subjects. However, most cancers lack effective screening recommendations. Therefore, further studies on novel screening strategies are urgently required.Methods: We used a simple suboptimal inoculation melanoma mouse model to obtain 'pre-diagnostic samples' of mice with macroscopic melanomas. High-throughput sequencing and bioinformatic analysis were employed to identify differentially expressed RNAs in platelet signatures of mice injected with a suboptimal number of melanoma cells (eDEGs) compared with mice with macroscopic melanomas and negative controls. Moreover, 36 genes selected from the eDEGs via bioinformatics analysis were verified in a mouse validation cohort via quantitative real-time PCR. LASSO regression was utilized to generate the prediction models with gene expression signatures as the best predictors for occult tumor progression in mice.Results: These RNAs identified from eDEGs of mice injected with a suboptimal number of cancer cells were strongly enriched in pathways related to immune response and regulation. The prediction models generated by 36 gene qPCR verification data showed great diagnostic efficacy and predictive value in our murine validation cohort, and could discriminate mice with occult tumors from control group (area under curve (AUC) of 0.935 (training data) and 0.912 (testing data)) (gene signature including Cd19, Cdkn1a, S100a9, Tap1, and Tnfrsf1b) and also from macroscopic tumor group (AUC of 0.920 (training data) and 0.936 (testing data)) (gene signature including Ccr7, Cd4, Kmt2d, and Ly6e).Conclusions: Our proof-of-concept study provides evidence for potential clinical relevance of blood platelets as a platform for liquid biopsy-based early detection of cancer. [ABSTRACT FROM AUTHOR]

DNA damage is a well-known initiator of tumorigenesis. Studies have shown that most cancer cells rely on aerobic glycolysis for their bioenergetics. We sought to identify a molecular link between genomic mutations and metabolic alterations in neoplastic transformation. We took advantage of the intrinsic genomic instability arising in xeroderma pigmentosum C (XPC). The XPC protein plays a key role in recognizing DNA damage in nucleotide excision repair, and patients with XPC deficiency have increased incidence of skin cancer and other malignancies. In cultured human keratinocytes, we showed that lentivirus-mediated knockdown of XPC reduced mitochondrial oxidative phosphorylation and increased glycolysis, recapitulating cancer cell metabolism. Accumulation of unrepaired DNA following XPC silencing increased DNA-dependent protein kinase activity, which subsequently activated AKT1 and NADPH oxidase-1 (NOX1), resulting in ROS production and accumulation of specific deletions in mitochondrial DNA (mtDNA) over time. Subcutaneous injection of XPC-deficient keratinocytes into immunodeficient mice led to squamous cell carcinoma formation, demonstrating the tumorigenic potential of transduced cells. Conversely, simultaneous knockdown of either NOX1 or AKT1 blocked the neoplastic transformation induced by XPC silencing. Our results demonstrate that genomic instability resulting from XPC silencing results in activation of AKT1 and subsequently NOX1 to induce ROS generation, mtDNA deletions, and neoplastic transformation in human keratinocytes. [ABSTRACT FROM AUTHOR]

Purpose: Protein phosphorylation mediated by protein kinases controls numerous cellular processes. A genetically encoded, generalizable split firefly luciferase (FL)-assisted complementation system was developed for noninvasive monitoring phosphorylation events and efficacies of kinase inhibitors in cell culture and in small living subjects by optical bioluminescence imaging.Procedures: An Akt sensor (AST) was constructed to monitor Akt phosphorylation and the effect of different PI-3K and Akt inhibitors. Specificity of AST was determined using a non-phosphorylable mutant sensor containing an alanine substitution (ASA).Results: The PI-3K inhibitor LY294002 and Akt kinase inhibitor perifosine led to temporal- and dose-dependent increases in complemented FL activities in 293T human kidney cancer cells stably expressing AST (293T/AST) but not in 293T/ASA cells. Inhibition of endogenous Akt phosphorylation and kinase activities by perifosine also correlated with increase in complemented FL activities in 293T/AST cells but not in 293T/ASA cells. Treatment of nude mice bearing 293T/AST xenografts with perifosine led to a 2-fold increase in complemented FL activities compared to that of 293T/ASA xenografts. Our system was used to screen a small chemical library for novel modulators of Akt kinase activity.Conclusion: This generalizable approach for noninvasive monitoring of phosphorylation events will accelerate the discovery and validation of novel kinase inhibitors and modulators of phosphorylation events. [ABSTRACT FROM AUTHOR]

Genes crucial for cancer development can be mutated via various mechanisms, which may reflect the nature of the mutagen. In thyroid papillary carcinomas, mutations of genes coding for effectors along the MAPK pathway are central for transformation. BRAF point mutation is most common in sporadic tumors. By contrast, radiation-induced tumors are associated with paracentric inversions activating the receptor tyrosine kinases RET and NTRK1. We report here a rearrangement of BRAF via paracentric inversion of chromosome 7q resulting in an in-frame fusion between exons 1-8 of the AKAP9 gene and exons 9-18 of BRAF. The fusion protein contains the protein kinase domain and lacks the autoinhibitory N-terminal portion of BRAF. It has elevated kinase activity and transforms NIH3T3 cells, which provides evidence, for the first time to our knowledge, of in vivo activation of an intracellular effector along the MAPK pathway by recombination. The AKAP9-BRAF fusion was preferentially found in radiation-induced papillary carcinomas developing after a short latency, whereas BRAF point mutations were absent in this group. These data indicate that in thyroid cancer, radiation activates components of the MAPK pathway primarily through chromosomal paracentric inversions, whereas in sporadic forms of the disease, effectors along the same pathway are activated predominantly by point mutations. [ABSTRACT FROM AUTHOR]

In collapsing focal segmental glomerulosclerosis (FSGS) of HIV-associated nephropathy (HIVAN), podocytes exhibit a high proliferation rate and loss of differentiation markers. We have found previously that the nef gene of HIV-1 is responsible for these changes. Here, we investigated the signaling pathways induced by Nef and its role in the pathogenesis of HIVAN. Using conditionally immortalized podocytes after differentiation, we found that infection of podocytes with nef increased Src kinase activity and signal transducer and activator of transcription 3 (Stat3) phosphorylation and activated the Ras-c-Raf-MAPK1,2 pathway. A dominant negative mutant of Src abolished the Nef effect, whereas inhibition of MAPK1,2 or dominant negative Stat3 reduced Nef effects partially. Reducing the expression of Nef with small interference RNA reversed the Nef effect. Mutation of Nef in the PxxP or R105R106 motifs diminished Nef signaling and the phenotypic changes in podocytes. Both phospho-MAPK1,2 and phospho-Stat3 staining increased in podocytes of kidneys from HIV-1 transgenic mice compared with their littermates and in podocytes of kidneys from HIVAN patients compared with HIV patients with non-HIVAN kidney diseases or non-HIV patients with idiopathic FSGS, classic FSGS, or minimal-change disease. These data suggest that Nef-induced activation of Stat3 and Ras-MAPK1,2 via Src-dependent pathways is responsible for podocyte proliferation and dedifferentiation, a characteristic finding in collapsing FSGS of HIVAN. [ABSTRACT FROM AUTHOR]

Introduction: Autoreactivity against pulmonary vascular structures is thought to be involved in idiopathic pulmonary arterial hypertension (IPAH), but the underlying mechanisms remain poorly understood. We hypothesised that aberrant B-cell activation contributes to IPAH aetiology.Methods: Mice with enhanced B-cell activation due to B-cell-specific overexpression of the B-cell receptor (BCR) signalling molecule Bruton's tyrosine kinase (BTK) were subjected to lung injury and examined for several pulmonary hypertension (PH) indices. Peripheral blood lymphocytes from patients with IPAH (n=13), connective tissue disease-associated PAH (CTD-PAH, n=9), congenital heart disease PAH (n=7), interstitial lung disease associated PH (n=17) and healthy controls (n=19) were characterised by 14-colour flow cytometry.Results: Following pulmonary injury, BTK-overexpressing mice showed prolonged activation of B cells and CXCR5+ follicular T-helper (Tfh) cells, as well as features of PH development. Patients with CTD-PAH and CHD-PAH displayed reduced proportions of circulating non-switched-memory B cells (p=0.03, p=0.02, respectively). Interestingly, we observed increased BTK protein expression in naive (p=0.007) and memory B-cell subsets of patients with IPAH and CTD-PAH. BTK was particularly high in patients with IPAH with circulating autoantibodies (p=0.045). IPAH patients had low frequencies of circulating CXCR5+ Tfh cells (p=0.005). Hereby, the increased BTK protein expression in B cells was associated with high proportions of Tfh17 (p=0.018) and Tfh17.1 (p=0.007) cells within the circulating Tfh population.Conclusions: Our study shows that pulmonary injury in combination with enhanced B-cell activation is sufficient to induce PH symptoms in mice. In parallel, immune homeostasis in patients with IPAH is compromised, as evidenced by increased BCR signalling and cTfh17 polarisation, indicating that adaptive immune activation contributes to IPAH disease induction or progression. [ABSTRACT FROM AUTHOR]

Combination treatment of PKD utilizing dual inhibition of EGF-receptor activity and ligand bioavailability. Background. We have previously demonstrated an essential role for increased epidermal growth factor receptor (EGFR) activity in mediating renal cyst formation and biliary ductal ectasia (BDE) in murine models of autosomal-recessive polycystic kidney disease (ARPKD) such as the BPK mouse. The current study was designed to determine (1 ) if treatment with a second-generation inhibitor of EGFR tyrosine kinase activity, EKB-569, was effective in treatment of ARPKD; (2 ) if tyrosine kinase inhibitor therapy used in combination with pharmacologic reduction of the availability of transforming growth factor-α (TGF-α), using WTACE2, could provide improved therapeutic efficacy and/or decrease potential toxicity; and (3 ) if effectiveness of treatment could be monitored noninvasively in murine ARPKD models by use of serial ultrasonography. Methods. BPK litters were treated with EKB-569 by intraperitoneal injection from postnatal day 7 to postnatal day 21. EKB-569's effectiveness alone or in combination with WTACE2 was measured by reduction in kidney weight/body weight ratios, morphometric renal cystic index, and evaluation of renal function. Renal ultrasound was performed on normal and cystic animals, under different therapeutic regimens, utilizing a 15 mHz linear array transducer, and ultrasound data were compared with histology and renal functional data. Results. Treatment of BPK mice with EKB-569 alone resulted in a marked reduction of kidney weight/body weight ratios, dramatically reduced collecting tubule cystic index, as well as BDE, and improved renal function. The combined treatment with EKB-569 and WTACE2 permitted a 67% reduction in EKB-569 dosage necessary to achieve results equivalent to those produced with EKB-569 alone. Untreated cystic animals died of renal failure, on average, at postnatal day 24 with a collecting tubule cystic index of 4.8, significant BDE, and maximal urine osmolarity of 361 mOsm. Cystic animals treated with EKB-569 and WTACE2 to postnatal day 21 were alive and well with normal renal function, a reduced collecting tubule cystic index of 1.7 (P < 0.02), improvement in BDE, and a threefold increase in maximum urinary concentrating ability (P < 0.01). Renal ultrasound could reliably detect cystic kidneys as early as postnatal day 7 and the natural history as well as effects of therapeutic intervention were clearly delineated by ultrasound evaluation. Conclusion. This study demonstrates that in murine ARPKD (1 ) EKB-569 is as effective as first-generation EGFR tyrosine kinase inhibitors in reducing cyst formation and preserving renal function; (2 ) combination therapy with EKB-569 and WTACE2 provides maximum efficacy in improving renal and biliary abnormalities, at lower doses, thereby minimizing potential toxicity; and (3 ) renal ultrasound provides a simple, reliable, noninvasive method of following natural history and effect of treatment regimens. [ABSTRACT FROM AUTHOR]

Activation of mammalian sterile 20-like kinase 1 (Mst1) by genotoxic compounds is known to stimulate apoptosis in some cell types. The importance of Mst1 in cell death caused by clinically relevant pathologic stimuli is unknown, however. In this study, we show that Mst1 is a prominent myelin basic protein kinase activated by proapoptotic stimuli in cardiac myocytes and that Mst1 causes cardiac myocyte apoptosis in vitro in a kinase activity-dependent manner. In vivo, cardiac-specific overexpression of Mst1 in transgenic mice results in activation of caspases, increased apoptosis, and dilated cardiomyopathy. Surprisingly, however, Mst1 prevents compensatory cardiac myocyte elongation or hypertrophy despite increased wall stress, thereby obscuring the use of the Frank-Starling mechanism, a fundamental mechanism by which the heart maintains cardiac output in response to increased mechanical load at the single myocyte level. Furthermore, Mst1 is activated by ischemia/reperfusion in the mouse heart in vivo. Suppression of endogenous Mst1 by cardiac-specific overexpression of dominant-negative Mst1 in transgenic mice prevents myocyte death by pathologic insults. These results show that Mst1 works as both an essential initiator of apoptosis and an inhibitor of hypertrophy in cardiac myocytes, resulting in a previously unrecognized form of cardiomyopathy. [ABSTRACT FROM AUTHOR]

Aberrant expression and activating mutations of the class III receptor tyrosine kinase Flt3 (Flk-2, STK-1) have been linked to poor prognosis in acute myeloid leukemia (AML). Inhibitors of Flt3 tyrosine kinase activity are, therefore, of interest as potential therapeutic compounds. We previously described bis(1H-2-indolyl)-1-methanones as a novel class of selective inhibitors for platelet-derived growth factor receptors (PDGFR). Several bis(1H-2-indolyl)-1-methanone derivatives, represented by the compounds D-64406 and D-65476, are also potent inhibitors of Flt3. They inhibit proliferation of TEL-Flt3-transfected BA/F3 cells with IC(50) values of 0.2-0.3 microM in the absence of IL-3 but >10 microM in the presence of IL-3. Ligand-stimulated autophosphorylation of Flt3 in EOL-1 cells and corresponding downstream activation of Akt/PKB are effectively inhibited by bis(1H-2-indolyl)-1-methanones whereas autophosphorylation of c-Kit/SCF receptor or c-Fms/CSF-1 receptor is less sensitive or insensitive, respectively. Flt3 kinase purified by different methods is potently inhibited in vitro, demonstrating a direct mechanism of inhibition. 32D cells, expressing a constitutively active Flt3 variant with internal tandem duplication are greatly sensitized to radiation-induced apoptosis in the presence of D-64406 or D-65476 in the absence but not in the presence of IL-3. Thus, bis(1H-2-indolyl)-1-methanones are potential candidates for the treatment of Flt3-driven leukemias. [ABSTRACT FROM AUTHOR]

Background: Periodontal disease(s) and metabolic illnesses negatively impact the quality of life and, eventually mental health.Objective: This study investigated the effect of Porphyromonas gingivalis (W83) oral infection on the development of Alzheimer's disease (AD) pathophysiology in a wild-type obese, diabetic (db/db) mouse model.Methods: The db/db mice were either orally infected with P. gingivalis and Fusobacterium nucleatum or sham infected for 16 weeks. The presence of amyloid-β (Aβ) and neurofibrillary tangles (NFTs) were assessed using a silver impregnation technique and subsequently by immunohistochemistry for tau and neuroinflammation. The mRNA abundance of a panel of 184 genes was performed using quantitative real-time PCR, and the differentially expressed genes were analyzed by Ingenuity Pathway Analysis.Results: While no Aβ plaques and NFTs were evident by silver impregnation, immunohistochemistry (glial cell markers) of the P. gingivalis-infected mice tissue sections exhibited neuroinflammation in the form of reactive microglia and astrocytes. Anti-tau immunopositivity, in addition to cells, was prominent in thickened axons of hippocampal CA neurons. The mRNA abundance of crucial genes in the insulin signaling pathway (INSR, IGF1, IRS, IDE, PIK3R, SGK1, GYS, GSK3B, AKT1) were upregulated, potentially exacerbating insulin resistance in the brain by P. gingivalis oral infection. Increased mRNA abundance of several kinases, membrane receptors, transcription factors, and pro-inflammatory mediators indicated hyperactivation of intracellular cascades with potential for tau phosphorylation and Aβ release in the same infection group.Conclusion: P. gingivalis W83 infection of db/db mice provides a disease co-morbidity model with the potential to reproduce AD pathophysiology with induced periodontal disease. [ABSTRACT FROM AUTHOR]

Autoantibodies are a hallmark of numerous neurological disorders, including multiple sclerosis, autoimmune encephalitides and neuromyelitis optica. Whilst well understood in peripheral myeloid cells, the pathophysiological significance of autoantibody-induced Fc receptor signalling in microglia remains unknown, in part due to the lack of a robust in vivo model. Moreover, the application of therapeutic antibodies for neurodegenerative disease also highlights the importance of understanding Fc receptor signalling in microglia. Here, we describe a novel in vivo experimental paradigm that allows for selective engagement of Fc receptors within the CNS by peripherally injecting anti-myelin oligodendrocyte glycoprotein (MOG) monoclonal antibodies into normal wild-type mice. MOG antigen-bound immunoglobulins were detected throughout the CNS and triggered a rapid and tightly regulated proliferative response in both brain and spinal cord microglia. This microglial response was abrogated when anti-MOG antibodies were deprived of Fc receptor effector function or injected into Fcγ receptor knockout mice and was associated with the downregulation of Fc receptors in microglia, but not peripheral myeloid cells, establishing that this response was dependent on central Fc receptor engagement. Downstream of the Fc receptors, BTK was a required signalling node for this response, as microglia proliferation was amplified in BtkE41K knock-in mice expressing a constitutively active form of the enzyme and blunted in mice treated with a CNS-penetrant small molecule inhibitor of BTK. Finally, this response was associated with transient and stringently regulated changes in gene expression predominantly related to cellular proliferation, which markedly differed from transcriptional programs typically associated with Fc receptor engagement in peripheral myeloid cells. Together, these results establish a physiologically-meaningful functional response to Fc receptor and BTK signalling in microglia, while providing a novel in vivo tool to further dissect the roles of microglia-specific Fc receptor and BTK-driven responses to both pathogenic and therapeutic antibodies in CNS homeostasis and disease. [ABSTRACT FROM AUTHOR]

Objective: The MAST family of microtubule-associated serine-threonine kinases (STKs) have distinct expression patterns in the developing and mature human and mouse brain. To date, only MAST1 has been conclusively associated with neurological disease, with de novo variants in individuals with a neurodevelopmental disorder, including a mega corpus callosum.Methods: Using exome sequencing, we identify MAST3 missense variants in individuals with epilepsy. We also assess the effect of these variants on the ability of MAST3 to phosphorylate the target gene product ARPP-16 in HEK293T cells.Results: We identify de novo missense variants in the STK domain in 11 individuals, including 2 recurrent variants p.G510S (n = 5) and p.G515S (n = 3). All 11 individuals had developmental and epileptic encephalopathy, with 8 having normal development prior to seizure onset at <2 years of age. All patients developed multiple seizure types, 9 of 11 patients had seizures triggered by fever and 9 of 11 patients had drug-resistant seizures. In vitro analysis of HEK293T cells transfected with MAST3 cDNA carrying a subset of these patient-specific missense variants demonstrated variable but generally lower expression, with concomitant increased phosphorylation of the MAST3 target, ARPP-16, compared to wild-type. These findings suggest the patient-specific variants may confer MAST3 gain-of-function. Moreover, single-nuclei RNA sequencing and immunohistochemistry shows that MAST3 expression is restricted to excitatory neurons in the cortex late in prenatal development and postnatally.Interpretation: In summary, we describe MAST3 as a novel epilepsy-associated gene with a potential gain-of-function pathogenic mechanism that may be primarily restricted to excitatory neurons in the cortex. ANN NEUROL 2021;90:274-284. [ABSTRACT FROM AUTHOR]

The defining feature of pancreatic islet β-cell function is the precise coordination of changes in blood glucose levels with insulin secretion to regulate systemic glucose homeostasis. While ATP has long been heralded as a critical metabolic coupling factor to trigger insulin release, glucose-derived metabolites have been suggested to further amplify fuel-stimulated insulin secretion. The mitochondrial export of citrate and isocitrate through the citrate-isocitrate carrier (CIC) has been suggested to initiate a key pathway that amplifies glucose-stimulated insulin secretion, though the physiological significance of β-cell CIC-to-glucose homeostasis has not been established. Here, we generated constitutive and adult CIC β-cell knockout (KO) mice and demonstrate that these animals have normal glucose tolerance, similar responses to diet-induced obesity, and identical insulin secretion responses to various fuel secretagogues. Glucose-stimulated NADPH production was impaired in β-cell CIC KO islets, whereas glutathione reduction was retained. Furthermore, suppression of the downstream enzyme cytosolic isocitrate dehydrogenase (Idh1) inhibited insulin secretion in wild-type islets but failed to impact β-cell function in β-cell CIC KO islets. Our data demonstrate that the mitochondrial CIC is not required for glucose-stimulated insulin secretion and that additional complexities exist for the role of Idh1 and NADPH in the regulation of β-cell function. [ABSTRACT FROM AUTHOR]

The expression of the BCR-ABL fusion oncoprotein in primitive hematopoietic cells results in chronic myeloid leukemia. Over the past decade studies of several in vitro and in vivo cell systems revealed multiple signal transduction pathways activated by BCR-ABL. However, the precise function of BCR-ABL in the pathogenesis of CML is still unclear. The goal of this review is to synthesize data on intracellular signaling in the context of the diverse murine assay systems employed. We emphasize the importance of in vivo assays and assays using primary cells in understanding the biology of CML and the molecular mechanisms by which BCR-ABL exerts its effects. [ABSTRACT FROM AUTHOR]

Mitochondrial glycerol phosphate dehydrogenase (mGPD) is the rate-limiting enzyme of the glycerol phosphate redox shuttle. It was recently claimed that metformin, a first-line drug used for the treatment of type 2 diabetes, inhibits liver mGPD 30-50%, suppressing gluconeogenesis through a redox mechanism. Various factors cast doubt on this idea. Total-body knockout of mGPD in mice has adverse effects in several tissues where the mGPD level is high but has little or no effect in liver, where the mGPD level is the lowest of 10 tissues. Metformin has beneficial effects in humans in tissues with high levels of mGPD, such as pancreatic β-cells, where the mGPD level is much higher than that in liver. Insulin secretion in mGPD knockout mouse β-cells is normal because, like liver, β-cells possess the malate aspartate redox shuttle whose redox action is redundant to the glycerol phosphate shuttle. For these and other reasons, we used four different enzyme assays to reassess whether metformin inhibited mGPD. Metformin did not inhibit mGPD in homogenates or mitochondria from insulin cells or liver cells. If metformin actually inhibited mGPD, adverse effects in tissues where the level of mGPD is much higher than that in the liver could prevent the use of metformin as a diabetes medicine. [ABSTRACT FROM AUTHOR]

Bioactive peptides attract growing concerns for their participation in multiple biological processes. Their roles in the pathogenesis of type 1 diabetes mellitus remain poorly understood. In this study, we used LC-MS/MS technology to compare the peptide profiling between pancreatic tissue of T1DM mice and pancreatic tissue of matched control groups. A total of 106 peptides were differentially expressed in T1DM pancreatic tissue, including 43 upregulated and 63 downregulated peptides. Most of the precursor proteins are insulin. Further bioinformatics analysis (GO and pathway analysis) indicated that the potential functions of these differential peptides were tightly related to regulation of endoplasmic reticulum stress. In conclusion, this study highlights new candidate peptides and provides a new perspective for exploring T1DM pathogenesis and clinical treatments. [ABSTRACT FROM AUTHOR]

Background: Curculigoside is a natural phenolic glycoside compound produced by Curculigo orchioides Gaertn. This study aimed to explore the effects of curculigoside in promoting the osteogenic differentiation of adipose-derived stem cells (ADSCs) as well as the underlying mechanism. Methods: ADSCs were treated with curculigoside at different concentrations (0 μmol/L, 1 μmol/L, 2.5 μmol/L, 5 μmol/L, 10 μmol/L, and 20 μmol/L), and cell viability was assessed by CCK-8 assay. Then, the alkaline phosphatase (ALP) activity was determined, and alizarin red S (ARS) staining was performed to measure the extracellular mineralization of curculigoside. Information about protein-chemical interactions is provided by the search tool for interactions of chemicals (STITCH) database. Then, LY294002 was administered to explore the mechanism by which curculigoside promotes the osteogenic differentiation of ADSCs. Western blot assays were performed to assess changes in the expression of osteogenic-related markers and the phosphorylation of PI3K and AKT. Finally, we established an ovariectomized (OVX)-induced osteoporosis mouse model and administered curculigoside to explore the effects of curculigoside in preventing bone loss in vivo. Results: The CCK-8 assay indicated that curculigoside did not induce cytotoxicity at a concentration of 5 μmol/L after 48 h. The ALP and ARS results revealed that the induced group had higher ALP activity and calcium deposition than the control group. Moreover, the curculigoside group exhibited increased biomineralization, ALP activity, and ARS staining compared to the induced and control groups, and these effects were partially inhibited by LY294002. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that the target genes of curculigoside were mainly involved in the PI3K-Akt signaling pathway. PCR and western blot analysis showed that the expression of RUNX2, ALP, and Osterix was upregulated in curculigoside-treated ADSCs, but this effect was partially reversed by the PI3K inhibitor LY294002. Moreover, the curculigoside-treated group exhibited significantly increased phosphorylation of AKT to P-AKT compared with the osteogenic induction group. After treatment with curculigoside, the mice had a higher bone volume than the OVX mice, suggesting partial protection from cancellous bone loss. In addition, when LY294002 was added, the protective effects of curculigoside could be neutralized. Conclusions: Curculigoside could induce the osteogenic differentiation of ADSCs and prevent bone loss in an OVX model through the PI3K/Akt signaling pathway. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are noncoding small RNAs that regulate various pathophysiological cellular processes. Here, we report that expression of the miR-378 family was significantly induced by metabolic inflammatory inducers, a high-fructose diet, and inflammatory cytokine tumor necrosis factor-α. Hepatic miRNA profiling revealed that expression of miR-378a was highly upregulated, which, in turn, targeted the 3'-untranslated region of PPARα mRNA, impaired mitochondrial fatty acid β-oxidation, and induced mitochondrial and endoplasmic reticulum stress. More importantly, the upregulated miR-378a can directly bind to and activate the double-strand RNA (dsRNA)-dependent protein kinase R (PKR) to sustain the metabolic stress. In vivo, genetic depletion of miR-378a prevented PKR activation and ameliorated inflammatory stress and insulin resistance. Counterbalancing the upregulated miR-378a using nanoparticles encapsulated with an anti-miR-378a oligonucleotide restored PPARα activity, inhibited PKR activation and ER stress, and improved insulin sensitivity in fructose-fed mice. Our study delineated a novel mechanism of miR-378a in the pathogenesis of metabolic inflammation and insulin resistance through targeting metabolic signaling at both mRNA (e.g., PPARα) and protein (e.g., PKR) molecules. This novel finding of functional interaction between miRNAs (e.g., miR-378a) and cellular RNA binding proteins (e.g., PKR) is biologically significant because it greatly broadens the potential targets of miRNAs in cellular pathophysiological processes. [ABSTRACT FROM AUTHOR]

Renal epidermal growth factor receptor (EGFR) signaling is activated in models of diabetic nephropathy (DN), and inhibition of the EGFR signaling pathway protects against the development of DN. We have now determined that in cultured podocytes, high glucose led to increases in activation of EGFR signaling but decreases in autophagy activity as indicated by decreased beclin-1 and inhibition of LC3B autophagosome formation as well as increased rubicon (an autophagy inhibitor) and SQSTM1 (autophagy substrate). Either genetic (small interfering [si]EGFR) or pharmacologic (AG1478) inhibition of EGFR signaling attenuated the decreased autophagy activity. In addition, rubicon siRNA knockdown prevented high glucose-induced inhibition of autophagy in podocytes. We further examined whether selective EGFR deletion in podocytes affected the progression of DN in type 2 diabetes. Selective podocyte EGFR deletion had no effect on body weight or fasting blood sugars in either db/db mice or nos3-/-; db/db mice, a model of accelerated type 2 DN. However selective podocyte EGFR deletion led to relative podocyte preservation and marked reduction in albuminuria and glomerulosclerosis, renal proinflammatory cytokine/chemokine expression, and decreased profibrotic and fibrotic components in nos3-/-; db/db mice. Podocyte EGFR deletion led to decreased podocyte expression of rubicon, in association with increased podocyte autophagy activity. Therefore, activation of EGFR signaling in podocytes contributes to progression of DN at least in part by increasing rubicon expression, leading to subsequent autophagy inhibition and podocyte injury. [ABSTRACT FROM AUTHOR]

Murine protein serine-threonine kinase 38 (MPK38)/maternal embryonic leucine zipper kinase (MELK) is implicated in diverse biological processes, including the cell cycle, apoptosis, and tumorigenesis; however, its physiological role is unknown. Using mice lacking MPK38 (MPK38-/-), we found that MPK38-/- male, but not female, mice (7 months of age) became obese while consuming a standard diet, displayed impairments in metabolism and inflammation, became more obese than wild-type mice while consuming a high-fat diet, and exhibited no castration/testosterone replacement-induced metabolic changes. The adenoviral restoration of MPK38 ameliorated the obesity-induced adverse metabolic profile of the obese male, but not female, mice. Seven-month-old MPK38-/- males displayed typical postcastration concentrations of serum testosterone with an accompanying decrease in serum luteinizing hormone (LH) levels, suggesting a role for MPK38 in the age-related changes in serum testosterone in aged mature adult male mice. The stability and activity of MPK38 were increased by dihydrotestosterone but reduced by estradiol (E2). These findings suggest MPK38 as a therapeutic target for obesity-related metabolic disorders in males. [ABSTRACT FROM AUTHOR]

Aberrant activation of eIF4E signalling pathway is common in breast cancer and holds potential therapeutic options. In our work, galeterone as a chemical compound under clinical trials for the treatment of prostate cancer, was identified to be effective in targeting breast cancer cells via suppressing MNK-eIF4E and β-catenin. In despite of varying IC50, galeterone at nanomolar concentrations significantly decreased viability, proliferation and migration of a panel of breast cancer cell lines regardless of clinical subtypes and genetic mutations, and to a higher extent than in normal breast cells. Galeterone significantly enhanced the effects of chemotherapeutic drugs in reducing proliferation and viability but not migration. The in vivo efficacy of galeterone as single drug alone and its ability in augmenting chemotherapy's efficacy were also shown in breast cancer xenograft mouse model. Mechanism analysis demonstrated that galeterone decreased MNK1/2 level and phosphorylation of eIF4E. In addition, galeterone decreased β-catenin level via promoting GSK-3β-mediated β-catenin degradation, and furthermore that Akt but not CK1 was involved in β-catenin degradation by galeterone. Rescue studies demonstrated that both MNK/eIF4E and β-catenin were responsible for anti-breast cancer activity of galeterone. Our study provides pre-clinical evidence to initialize clinical trials for breast cancer using galeterone in combination with chemotherapy. [ABSTRACT FROM AUTHOR]