NF2 is an autosomal dominant disease characterized by development of bilateral vestibular schwannomas and other benign tumors in central nervous system. Loss of the NF2 gene product, Merlin, leads to aberrant Schwann cell proliferation, motility, and survival, but the mechanisms by which this tumor suppressor functions remain unclear. One well-defined target of Merlin is the group I family of p21-activated kinases, which are allosterically inhibited by Merlin and which, when activated, stimulate cell cycle progression, motility, and increased survival. Here, we examine the effect of Pak inhibition on cells with diminished Merlin function.

Here we provide evidence in support of an inherent role for Arpc1b, a component of the Arp2/3 complex, in regulation of mitosis and demonstrate that its depletion inhibits Aurora A activation at the centrosome and impairs the ability of mammalian cells to enter mitosis. We discovered that Arpc1b colocalizes with gamma-tubulin at centrosomes and stimulates Aurora A activity. Aurora A phosphorylates Arpc1b on threonine 21, and expression of Arpc1b but not a nonphosphorylatable Arpc1b mutant in mammalian cells leads to Aurora A kinase activation and abnormal centrosome amplification in a Pak1-independent manner. Together, these findings reveal a new function for Arpc1b in centrosomal homeostasis. Arpc1b is both a physiological activator and substrate of Aurora A kinase and these interactions help to maintain mitotic integrity in mammalian cells.

Pak1 plays an important role in various cellular processes, including cell motility, polarity, survival and proliferation. To date, its role in atherogenesis has not been explored. Here we report the effect of Pak1 on atherogenesis using atherosclerosis-prone apolipoprotein E-deficient (ApoE(-/-)) mice as a model. Disruption of Pak1 in ApoE(-/-) mice results in reduced plaque burden, significantly attenuates circulating IL-6 and MCP-1 levels, limits the expression of adhesion molecules and diminishes the macrophage content in the aortic root of ApoE(-/-) mice. We also observed reduced oxidized LDL uptake and increased cholesterol efflux by macrophages and smooth muscle cells of ApoE(-/-):Pak1(-/-) mice as compared with ApoE(-/-) mice. In addition, we detect increased Pak1 phosphorylation in human atherosclerotic arteries, suggesting its role in human atherogenesis. Altogether, these results identify Pak1 as an important factor in the initiation and progression of atherogenesis.

Tumour necrosis factor-α (TNF-α) plays a key role in the regulation of cardiac contractility. Although cardiomyocytes are known to express the TNF-α receptors (TNFRs), the mechanism of TNF-α signal transmission is incompletely understood. The aim of this study was to investigate whether the tumour suppressor Ras-association domain family protein 1 isoform A (RASSF1A) modulates TNF-α signalling in cardiomyocytes.

Mutations in the inositol 5-phosphatase OCRL cause Lowe syndrome and Dent's disease. Although OCRL, a direct clathrin interactor, is recruited to late-stage clathrin-coated pits, clinical manifestations have been primarily attributed to intracellular sorting defects. Here we show that OCRL loss in Lowe syndrome patient fibroblasts impacts clathrin-mediated endocytosis and results in an endocytic defect. These cells exhibit an accumulation of clathrin-coated vesicles and an increase in U-shaped clathrin-coated pits, which may result from sequestration of coat components on uncoated vesicles. Endocytic vesicles that fail to lose their coat nucleate the majority of the numerous actin comets present in patient cells. SNX9, an adaptor that couples late-stage endocytic coated pits to actin polymerization and which we found to bind OCRL directly, remains associated with such vesicles. These results indicate that OCRL acts as an uncoating factor and that defects in clathrin-mediated endocytosis likely contribute to pathology in patients with OCRL mutations.

Calcium (Ca2+) is vital for multiple processes in the body, and maintenance of the electrolyte concentration is required for everyday physiological function. In the kidney, and more specifically, in the late distal convoluted tubule and connecting tubule, the fine-tuning of Ca2+ reabsorption from the pro-urine takes place. Here, Ca2+ enters the epithelial cell via the transient receptor potential vanilloid receptor type 5 (TRPV5) channel, diffuses to the basolateral side bound to calbindin-D28k and is extruded to the blood compartment via the Na+/Ca2+ exchanger 1 (NCX1) and the plasma membrane Ca2+ ATPase (PMCA). Traditionally, PMCA1 was considered to be the primary Ca2+ pump in this process. However, in recent studies TRPV5-expressing tubules were shown to highly express PMCA4. Therefore, PMCA4 may have a predominant role in renal Ca2+ handling. This study aimed to elucidate the role of PMCA4 in Ca2+ homeostasis by characterizing the Ca2+ balance, and renal and duodenal Ca2+-related gene expression in PMCA4 knockout mice. The daily water intake of PMCA4 knockout mice was significantly lower compared to wild type littermates. There was no significant difference in serum Ca2+ level or urinary Ca2+ excretion between groups. In addition, renal and duodenal mRNA expression levels of Ca2+-related genes, including TRPV5, TRPV6, calbindin-D28k, calbindin-D9k, NCX1 and PMCA1 were similar in wild type and knockout mice. Serum FGF23 levels were significantly increased in PMCA4 knockout mice. In conclusion, PMCA4 has no discernible role in normal renal Ca2+ handling as no urinary Ca2+ wasting was observed. Further investigation of the exact role of PMCA4 in the distal convoluted tubule and connecting tubule is required.

The p21-activated protein kinases (Paks) have been implicated in the regulation of smooth muscle contractility, but the physiologic effects of Pak activation on airway reactivity in vivo are unknown. A mouse model with a genetic deletion of Pak1 (Pak1(-/-)) was used to determine the role of Pak in the response of the airways in vivo to challenge with inhaled or intravenous acetylcholine (ACh). Pulmonary resistance was measured in anesthetized mechanically ventilated Pak1(-/-) and wild type mice. Pak1(-/-) mice exhibited lower airway reactivity to ACh compared with wild type mice. Tracheal segments dissected from Pak1(-/-) mice and studied in vitro also exhibited reduced responsiveness to ACh compared with tracheas from wild type mice. Morphometric assessment and pulmonary function analysis revealed no differences in the structure of the airways or lung parenchyma, suggesting that that the reduced airway responsiveness did not result from structural abnormalities in the lungs or airways due to Pak1 deletion. Inhalation of the small molecule synthetic Pak1 inhibitor, IPA3, also significantly reduced in vivo airway responsiveness to ACh and 5-hydroxytryptamine (5-Ht) in wild type mice. IPA3 inhibited the contractility of isolated human bronchial tissues to ACh, confirming that this inhibitor is also effective in human airway smooth muscle tissue. The results demonstrate that Pak is a critical component of the contractile activation process in airway smooth muscle, and suggest that Pak inhibition could provide a novel strategy for reducing airway hyperresponsiveness.

Hypertension is a well-established risk factor for adverse cardiovascular events, and older age is a risk factor for the development of hypertension. Genomewide association studies have linked ATP2B1, the gene for the plasma membrane calcium ATPase 1 (PMCA1), to blood pressure (BP) and hypertension. Here, we present the effects of reduction in the expression of PMCA1 on BP and small artery structure and function when combined with advancing age. Heterozygous PMCA1 null mice (PMCA1Ht ) were generated and conscious BP was measured at 6 to 18 months of age. Passive and active properties of isolated small mesenteric arteries were examined by pressure myography. PMCA1Ht mice exhibited normal BP at 6 and 9 months of age but developed significantly elevated BP when compared to age-matched wild-type controls at ≥12 months of age. Decreased lumen diameter, increased wall thickness and increased wall:lumen ratio were observed in small mesenteric arteries from animals 9 months of age and older, indicative of eutrophic remodelling. Increases in mesenteric artery intrinsic tone and global intracellular calcium were evident in animals at both 6 and 18 months of age. Thus, decreased expression of PMCA1 is associated with increased BP when combined with advancing age. Changes in arterial structure precede the elevation of BP. Pathways involving PMCA1 may be a novel target for BP regulation in the elderly.

Adult fibroblasts can be reprogrammed into induced pluripotent stem cells (iPSC) for use in various applications. However, there are challenges in iPSC generation including low reprogramming efficiency, yield, cell survival and viability. Since the Hippo signalling pathway is a key pathway involved in regulating cell proliferation and survival, we here test whether modification of the Hippo pathway will enhance the efficiency of iPSC generation and improve their survival. The Hippo pathway was modified by genetic ablation of the mammalian sterile-20 like kinase 1 (Mst1), a major component of the pathway. Using adult skin fibroblasts isolated from Mst1 knockout mice (Mst1-/-) as a source of iPSC we found that genetic ablation of Mst1 leads to significantly increased reprogramming efficiency by 43.8%. Moreover, Mst1-/- iPSC displayed increase proliferation by 12% as well as an increase in cell viability by 20% when treated with a chemical hypoxic inducer. Mechanistically, we found higher activity of YAP, the main downstream effector of the Hippo pathway, in iPSC lacking Mst1. In conclusion, our data suggests that Mst1 can be targeted to improve the efficiency of adult somatic cell reprogramming as well as to enhance iPSC proliferation and survival.

Targeted BRAF inhibition (BRAFi) and combined BRAF and MEK inhibition (BRAFi and MEKi) therapies have markedly improved the clinical outcomes of patients with metastatic melanoma. Unfortunately, the efficacy of these treatments is often countered by the acquisition of drug resistance. Here we investigated the molecular mechanisms that underlie acquired resistance to BRAFi and to the combined therapy. Consistent with previous studies, we show that resistance to BRAFi is mediated by ERK pathway reactivation. Resistance to the combined therapy, however, is mediated by mechanisms independent of reactivation of ERK in many resistant cell lines and clinical samples. p21-activated kinases (PAKs) become activated in cells with acquired drug resistance and have a pivotal role in mediating resistance. Our screening, using a reverse-phase protein array, revealed distinct mechanisms by which PAKs mediate resistance to BRAFi and the combined therapy. In BRAFi-resistant cells, PAKs phosphorylate CRAF and MEK to reactivate ERK. In cells that are resistant to the combined therapy, PAKs regulate JNK and β-catenin phosphorylation and mTOR pathway activation, and inhibit apoptosis, thereby bypassing ERK. Together, our results provide insights into the molecular mechanisms underlying acquired drug resistance to current targeted therapies, and may help to direct novel drug development efforts to overcome acquired drug resistance.

To establish magnetic resonance (MR)-based molecular imaging paradigms for the noninvasive monitoring of extracellular pH (pHe) as a functional surrogate biomarker for metabolic changes induced by locoregional therapy of liver cancer.

Volcanoes commonly inflate or deflate during episodes of unrest or eruption. Continuum mechanics models that assume linear elastic deformation of the Earth's crust are routinely used to invert the observed ground motions. The source(s) of deformation in such models are generally interpreted in terms of magma bodies or pathways, and thus form a basis for hazard assessment and mitigation. Using discontinuum mechanics models, we show how host-rock fracturing (i.e. non-elastic deformation) during drainage of a magma body can progressively change the shape and depth of an elastic-deformation source. We argue that this effect explains the marked spatio-temporal changes in source model attributes inferred for the March-April 2007 eruption of Piton de la Fournaise volcano, La Reunion. We find that pronounced deflation-related host-rock fracturing can: (1) yield inclined source model geometries for a horizontal magma body; (2) cause significant upward migration of an elastic-deformation source, leading to underestimation of the true magma body depth and potentially to a misinterpretation of ascending magma; and (3) at least partly explain underestimation by elastic-deformation sources of changes in sub-surface magma volume.

Advanced glycation end products (AGE) are central to the development of cardiovascular complications associated with diabetes mellitus. AGE may alter cellular function through cross-linking of cellular proteins or by activating the AGE receptor (RAGE). However, the signalling molecules involved during AGE stimulation in cardiomyocytes remain unclear. Here, we investigated the effects of AGE treatment on intracellular calcium homeostasis of isolated cardiomyocytes and studied the activation of signalling molecules involved in this process. Treatment of cardiomyocytes with AGE for 24 h resulted in a dose-dependent reduction in calcium transient amplitude, reaching a maximum 50% reduction at a dose of 1 mg·mL-1. This was accompanied with a 32% reduction in sarcoplasmic reticulum calcium content but without any detectable changes in the expression of major calcium channels. Mechanistically, we observed a significant increase in the production of reactive oxygen species (ROS) in AGE-treated cardiomyocytes and enhancement of NADPH oxidase activity. This was accompanied with activation of p38 kinase and nuclear translocation of NF-κB, and subsequently induction of inducible NO synthase (iNOS) expression, leading to excessive nitric oxide production. Overall, our data reveal the molecular signalling that may underlie the alteration of intracellular calcium homeostasis in cardiac myocytes due to AGE stimulation. This may provide new insights into the pathophysiological mechanisms of the development of diabetic cardiomyopathy.

Inflammation plays a key role during cardiac hypertrophy and the development of heart failure. Interleukin-10 (IL-10) is a major anti-inflammatory cytokine that is expressed in the heart and may play a crucial role in cardiac remodeling. Based on the evidence that IL-10 potentially reduces pathological hypertrophy, it was hypothesized that signaling via the IL-10 receptor (IL10R) in the heart produces a protective role in reducing cardiac hypertrophy. The aim of this study was to investigate the effects of the ablation of Il-10-r1 gene during pathological cardiac hypertrophy in mice. We found that IL-10R1 gene silencing in cultured cardiomyocytes diminished the anti-hypertrophic effect of Il-10 in TNF-α induced hypertrophy model. We then analyzed mice deficient in the Il-10-r1 gene (IL-10R1-/- mice) and subjected them to transverse aortic constriction or isoproterenol infusion to induce pathological hypertrophy. In response to transverse aortic constriction for 2 weeks, IL-10R1-/- mice displayed a significant increase in the hypertrophic response as indicated by heart weight/body weight ratio, which was accompanied by significant increases in cardiomyocyte surface area and interstitial fibrosis. In contrast, there was no difference in hypertrophic response to isoproterenol infusion (10 days) between the knockout and control groups. Analysis of cardiac function using echocardiography and invasive hemodynamic studies did not show any difference between the WT and IL-10R1-/- groups, most likely due to the short term nature of the models. In conclusion, our data shows that signaling via the IL-10 receptor may produce protective effects against pressure overload-induced hypertrophy but not against β-adrenergic stimuli in the heart. Our data supports previous evidence that signaling modulated by IL-10 and its receptor may become a potential target to control pathological cardiac hypertrophy.

Myocardial insulin resistance contributes to heart failure in response to pathological stresses, therefore, a therapeutic strategy to maintain cardiac insulin pathways requires further investigation. We demonstrated that insulin receptor substrate 1 (IRS1) was reduced in failing mouse hearts post-myocardial infarction (MI) and failing human hearts. The mice manifesting severe cardiac dysfunction post-MI displayed elevated mir128-3p in the myocardium. Ischemia-upregulated mir128-3p promoted Irs1 degradation. Using rat cardiomyocytes and human-induced pluripotent stem cell-derived cardiomyocytes, we elucidated that mitogen-activated protein kinase 7 (MAPK7, also known as ERK5)-mediated CCAAT/enhancer-binding protein beta (CEBPβ) transcriptionally represses mir128-3p under hypoxia. Therapeutically, functional studies demonstrated gene therapy-delivered cardiac-specific MAPK7 restoration or overexpression of CEBPβ impeded cardiac injury after MI, at least partly due to normalization of mir128-3p. Furthermore, inhibition of mir128-3p preserved Irs1 and ameliorated cardiac dysfunction post-MI. In conclusion, we reveal that targeting mir128-3p mitigates myocardial insulin resistance, thereafter slowing down the progression of heart failure post-ischemia.

Limited view tomographic reconstruction aims to reconstruct a tomographic image from a limited number of projection views arising from sparse view or limited angle acquisitions that reduce radiation dose or shorten scanning time. However, such a reconstruction suffers from severe artifacts due to the incompleteness of sinogram. To derive quality reconstruction, previous methods use UNet-like neural architectures to directly predict the full view reconstruction from limited view data; but these methods leave the deep network architecture issue largely intact and cannot guarantee the consistency between the sinogram of the reconstructed image and the acquired sinogram, leading to a non-ideal reconstruction. In this work, we propose a cascaded residual dense spatial-channel attention network consisting of residual dense spatial-channel attention networks and projection data fidelity layers. We evaluate our methods on two datasets. Our experimental results on AAPM Low Dose CT Grand Challenge datasets demonstrate that our algorithm achieves a consistent and substantial improvement over the existing neural network methods on both limited angle reconstruction and sparse view reconstruction. In addition, our experimental results on Deep Lesion datasets demonstrate that our method is able to generate high-quality reconstruction for 8 major lesion types.

Methylthioadenosine phosphorylase (MTAP) is a key enzyme in the methionine salvage pathway that converts the polyamine synthesis byproduct 5'-deoxy-5'-methylthioadenosine (MTA) into methionine. Inactivation of MTAP, often by homozygous deletion, is found in both solid and hematologic malignancies and is one of the most frequently observed genetic alterations in human cancer. Previous work established that MTAP-deleted cells accumulate MTA and contain decreased amounts of proteins with symmetric dimethylarginine (sDMA). These findings led to the hypothesis that accumulation of intracellular MTA inhibits the protein arginine methylase (PRMT5) responsible for bulk protein sDMAylation. Here, we confirm that MTAP-deleted cells have increased MTA accumulation and reduced protein sDMAylation. However, we also show that addition of extracellular MTA can cause a dramatic reduction of the steady-state levels of sDMA-containing proteins in MTAP+ cells, even though no sustained increase in intracellular MTA is found because of catabolism of MTA by MTAP. We determined that inhibition of protein sDMAylation by MTA occurs within 48 h, is reversible, and is specific. In addition, we have identified two enhancer-binding proteins, FUBP1 and FUBP3, that are differentially sDMAylated in response to MTAP and MTA. These proteins work via the far upstream element site located upstream of Myc and other promoters. Using a transcription reporter construct containing the far upstream element site, we demonstrate that MTA addition can reduce transcription, suggesting that the reduction in FUBP1 and FUBP3 sDMAylation has functional consequences. Overall, our findings show that extracellular MTA can inhibit protein sDMAylation and that this inhibition can affect FUBP function.

Reduction in cardiac contractility is common in severe sepsis. However, the pathological mechanism is still not fully understood. Recently it has been found that circulating histones released after extensive immune cell death play important roles in multiple organ injury and disfunction, particularly in cardiomyocyte injury and contractility reduction. How extracellular histones cause cardiac contractility depression is still not fully clear. In this work, using cultured cardiomyocytes and a histone infusion mouse model, we demonstrate that clinically relevant histone concentrations cause significant increases in intracellular calcium concentrations with subsequent activation and enriched localization of calcium-dependent protein kinase C (PKC) α and βII into the myofilament fraction of cardiomyocytes in vitro and in vivo. Furthermore, histones induced dose-dependent phosphorylation of cardiac troponin I (cTnI) at the PKC-regulated phosphorylation residues (S43 and T144) in cultured cardiomyocytes, which was also confirmed in murine cardiomyocytes following intravenous histone injection. Specific inhibitors against PKCα and PKCβII revealed that histone-induced cTnI phosphorylation was mainly mediated by PKCα activation, but not PKCβII. Blocking PKCα also significantly abrogated histone-induced deterioration in peak shortening, duration and the velocity of shortening, and re-lengthening of cardiomyocyte contractility. These in vitro and in vivo findings collectively indicate a potential mechanism of histone-induced cardiomyocyte dysfunction driven by PKCα activation with subsequent enhanced phosphorylation of cTnI. These findings also indicate a potential mechanism of clinical cardiac dysfunction in sepsis and other critical illnesses with high levels of circulating histones, which holds the potential translational benefit to these patients by targeting circulating histones and downstream pathways.

Mitochondrial dysfunction is a feature of type I and type II diabetes, but there is a lack of consistency between reports and links to disease development. We aimed to investigate if mitochondrial structure-function remodelling occurs in the early stages of diabetes by employing a mouse model (GENA348) of Maturity Onset Diabetes in the Young, exhibiting hyperglycemia, but not hyperinsulinemia, with mild left ventricular dysfunction. Employing 3-D electron microscopy (SBF-SEM) we determined that compared to wild-type, WT, the GENA348 subsarcolemma mitochondria (SSM) are ~ 2-fold larger, consistent with up-regulation of fusion proteins Mfn1, Mfn2 and Opa1. Further, in comparison, GENA348 mitochondria are more irregular in shape, have more tubular projections with SSM projections being longer and wider. Mitochondrial density is also increased in the GENA348 myocardium consistent with up-regulation of PGC1-α and stalled mitophagy (down-regulation of PINK1, Parkin and Miro1). GENA348 mitochondria have more irregular cristae arrangements but cristae dimensions and density are similar to WT. GENA348 Complex activity (I, II, IV, V) activity is decreased but the OCR is increased, potentially linked to a shift towards fatty acid oxidation due to impaired glycolysis. These novel data reveal that dysregulated mitochondrial morphology, dynamics and function develop in the early stages of diabetes.

Endoplasmic Reticulum (ER) stress and oxidative stress have been highly implicated in the pathogenesis of cardiac hypertrophy and heart failure (HF). However, the mechanisms involved in the interplay between these processes in the heart are not fully understood. The present study sought to determine a causative link between Pak2-dependent UPR activation and oxidative stress via Nrf2 regulation under pathological ER stress. We report that sustained ER stress and Pak2 deletion in cardiomyocytes enhance Nrf2 expression. Conversely, AAV9 mediated Pak2 delivery in the heart leads to a significant decrease in Nrf2 levels. Pak2 overexpression enhances the XBP1-Hrd1 UPR axis and ameliorates tunicamycin induced cardiac apoptosis and dysfunction in mice. We found that Pak2 deletion and altered proteostasis render Nrf2 detrimental by switching from its antioxidant role to renin-angiotensin aldosterone system (RAAS) gene regulator. Mechanistically, Pak2 mediated Hrd1 expression targets Nrf2 for ubiquitination and degradation thus preventing its aberrant activation. Moreover, we find a significant increase in Nrf2 with a decrease in Pak2 in human myocardium of dilated heart disease. Using human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), we find that Pak2 is able to ameliorate Nrf2 induced RAAS activation under ER stress. These findings demonstrate that Pak2 is a novel Nrf2 regulator in the stressed heart. Activation of XBP1-Hrd1 is attributed to prevent ER stress-induced Nrf2 RAAS component upregulation. This mechanism explains the functional dichotomy of Nrf2 in the stressed heart. Thus, Pak2 regulation of Nrf2 homeostasis may present as a potential therapeutic route to alleviate detrimental ER stress and heart failure.