Senescence of lung mesenchymal stem cells of preterm infants by cyclic stretch and hyperoxia via p21.

Phenotype distortion of lung resident mesenchymal stem cells (MSC) in preterm infants is a hallmark event in the pathogenesis of bronchopulmonary dysplasia (BPD). Here, we evaluated the impact of cyclic mechanical stretch (CMS) and hyperoxia (HOX). The negative action of HOX on proliferation and cell death was more pronounced at 80% than at 40%. Although the impact of CMS alone was modest, CMS plus HOX displayed the strongest effect sizes. Exposure to CMS and/or HOX induced the downregulation of PDGFRα, and cellular senescence preceded by p21 accumulation. p21 interference interfered with cellular senescence and resulted in aggravated cell death, arguing for a prosurvival mechanism. HOX 40% and limited exposure to HOX 80% prevailed in a reversible phenotype with reuptake of proliferation, while prolonged exposure to HOX 80% resulted in definite MSC growth arrest. Our mechanistic data explain how HOX and CMS induce the effects on MSC phenotype disruption. The results are congruent with the clinical observation that preterm infants requiring supplemental oxygen plus mechanical ventilation are at particular risk for BPD. Although inhibiting p21 is not a feasible approach, limiting the duration and magnitude of the exposures is promising.NEW & NOTEWORTHY Rarefication of lung mesenchymal stem cells (MSC) due to exposure to cyclic mechanical stretch (CMS) during mechanical ventilation with oxygen-rich gas is a hallmark of bronchopulmonary dysplasia in preterm infants, but the pathomechanistic understanding is incomplete. Our studies identify a common signaling mechanism mediated by p21 accumulation, leading to cellular senescence and cell death, most pronounced during the combined exposure with in principle reversible phenotype change depending on strength and duration of exposures.

Does Cell-Type-Specific Silencing of Monoamine Oxidase B Interfere with the Development of Right Ventricle (RV) Hypertrophy or Right Ventricle Failure in Pulmonary Hypertension?

Increased mitochondrial reactive oxygen species (ROS) formation is important for the development of right ventricular (RV) hypertrophy (RVH) and failure (RVF) during pulmonary hypertension (PH). ROS molecules are produced in different compartments within the cell, with mitochondria known to produce the strongest ROS signal. Among ROS-forming mitochondrial proteins, outer-mitochondrial-membrane-located monoamine oxidases (MAOs, type A or B) are capable of degrading neurotransmitters, thereby producing large amounts of ROS. In mice, MAO-B is the dominant isoform, which is present in almost all cell types within the heart. We analyzed the effect of an inducible cardiomyocyte-specific knockout of MAO-B (cmMAO-B KO) for the development of RVH and RVF in mice. Right ventricular hypertrophy was induced by pulmonary artery banding (PAB). RV dimensions and function were measured through echocardiography. ROS production (dihydroethidium staining), protein kinase activity (PamStation device), and systemic hemodynamics (in vivo catheterization) were assessed. A significant decrease in ROS formation was measured in cmMAO-B KO mice during PAB compared to Cre-negative littermates, which was associated with reduced activity of protein kinases involved in hypertrophic growth. In contrast to littermates in which the RV was dilated and hypertrophied following PAB, RV dimensions were unaffected in response to PAB in cmMAO-B KO mice, and no decline in RV systolic function otherwise seen in littermates during PAB was measured in cmMAO-B KO mice. In conclusion, cmMAO-B KO mice are protected against RV dilatation, hypertrophy, and dysfunction following RV pressure overload compared to littermates. These results support the hypothesis that cmMAO-B is a key player in causing RV hypertrophy and failure during PH.

Pneumococcal hydrogen peroxide regulates host cell kinase activity.

Introduction: Protein kinases are indispensable reversible molecular switches that adapt and control protein functions during cellular processes requiring rapid responses to internal and external events. Bacterial infections can affect kinase-mediated phosphorylation events, with consequences for both innate and adaptive immunity, through regulation of antigen presentation, pathogen recognition, cell invasiveness and phagocytosis. Streptococcus pneumoniae (Spn), a human respiratory tract pathogen and a major cause of community-acquired pneumoniae, affects phosphorylation-based signalling of several kinases, but the pneumococcal mediator(s) involved in this process remain elusive. In this study, we investigated the influence of pneumococcal H2O2 on the protein kinase activity of the human lung epithelial H441 cell line, a generally accepted model of alveolar epithelial cells. Methods: We performed kinome analysis using PamGene microarray chips and protein analysis in Western blotting in H441 lung cells infected with Spn wild type (SpnWT) or with SpnΔlctOΔspxB -a deletion mutant strongly attenuated in H2O2 production- to assess the impact of pneumococcal hydrogen peroxide (H2O2) on global protein kinase activity profiles. Results: Our kinome analysis provides direct evidence that kinase activity profiles in infected H441 cells significantly vary according to the levels of pneumococcal H2O2. A large number of kinases in H441 cells infected with SpnWT are significantly downregulated, whereas this no longer occurs in cells infected with the mutant SpnΔlctOΔspxB strain, which lacks H2O2. In particular, we describe for the first time H2O2-mediated downregulation of Protein kinase B (Akt1) and activation of lymphocyte-specific tyrosine protein kinase (Lck) via H2O2-mediated phosphorylation.

The diverging roles of insulin-like growth factor binding proteins in pulmonary arterial hypertension.

Pulmonary hypertension (PH) is a progressive, severe and to date not curable disease of the pulmonary vasculature. Alterations of the insulin-like growth factor 1 (IGF-1) system are known to play a role in vascular pathologies and IGF-binding proteins (IGFBPs) are important regulators of the bioavailability and function of IGFs. In this study, we show that circulating plasma levels of IGFBP-1, IGFBP-2 and IGFBP-3 are increased in idiopathic pulmonary arterial hypertension (IPAH) patients compared to healthy individuals. These binding proteins inhibit the IGF-1 induced IGF-1 receptor (IGF1R) phosphorylation and exhibit diverging effects on the IGF-1 induced signaling pathways in human pulmonary arterial cells (i.e. healthy as well as IPAH-hPASMCs, and healthy hPAECs). Furthermore, IGFBPs are differentially expressed in an experimental mouse model of PH. In hypoxic mouse lungs, IGFBP-1 mRNA expression is decreased whereas the mRNA for IGFBP-2 is increased. In contrast to IGFBP-1, IGFBP-2 shows vaso-constrictive properties in the murine pulmonary vasculature. Our analyses show that IGFBP-1 and IGFBP-2 exhibit diverging effects on IGF-1 signaling and display a unique IGF1R-independent kinase activation pattern in human pulmonary arterial smooth muscle cells (hPASMCs), which represent a major contributor of PAH pathobiology. Furthermore, we could show that IGFBP-2, in contrast to IGFBP-1, induces epidermal growth factor receptor (EGFR) signaling, Stat-3 activation and expression of Stat-3 target genes. Based on our results, we conclude that the IGFBP family, especially IGFBP-1, IGFBP-2 and IGFBP-3, are deregulated in PAH, that they affect IGF signaling and thereby regulate the cellular phenotype in PH.

Alveolar macrophage-expressed Plet1 is a driver of lung epithelial repair after viral pneumonia.

Influenza A virus (IAV) infection mobilizes bone marrow-derived macrophages (BMDM) that gradually undergo transition to tissue-resident alveolar macrophages (TR-AM) in the inflamed lung. Combining high-dimensional single-cell transcriptomics with complex lung organoid modeling, in vivo adoptive cell transfer, and BMDM-specific gene targeting, we found that transitioning ("regenerative") BMDM and TR-AM highly express Placenta-expressed transcript 1 (Plet1). We reveal that Plet1 is released from alveolar macrophages, and acts as important mediator of macrophage-epithelial cross-talk during lung repair by inducing proliferation of alveolar epithelial cells and re-sealing of the epithelial barrier. Intratracheal administration of recombinant Plet1 early in the disease course attenuated viral lung injury and rescued mice from otherwise fatal disease, highlighting its therapeutic potential.

Cyclin D-CDK4 Disulfide Bond Attenuates Pulmonary Vascular Cell Proliferation.

BACKGROUND: Pulmonary hypertension (PH) is a chronic vascular disease characterized, among other abnormalities, by hyperproliferative smooth muscle cells and a perturbed cellular redox and metabolic balance. Oxidants induce cell cycle arrest to halt proliferation; however, little is known about the redox-regulated effector proteins that mediate these processes. Here, we report a novel kinase-inhibitory disulfide bond in cyclin D-CDK4 (cyclin-dependent kinase 4) and investigate its role in cell proliferation and PH. METHODS: Oxidative modifications of cyclin D-CDK4 were detected in human pulmonary arterial smooth muscle cells and human pulmonary arterial endothelial cells. Site-directed mutagenesis, tandem mass-spectrometry, cell-based experiments, in vitro kinase activity assays, in silico structural modeling, and a novel redox-dead constitutive knock-in mouse were utilized to investigate the nature and definitively establish the importance of CDK4 cysteine modification in pulmonary vascular cell proliferation. Furthermore, the cyclin D-CDK4 oxidation was assessed in vivo in the pulmonary arteries and isolated human pulmonary arterial smooth muscle cells of patients with pulmonary arterial hypertension and in 3 preclinical models of PH. RESULTS: Cyclin D-CDK4 forms a reversible oxidant-induced heterodimeric disulfide dimer between C7/8 and C135, respectively, in cells in vitro and in pulmonary arteries in vivo to inhibit cyclin D-CDK4 kinase activity, decrease Rb (retinoblastoma) protein phosphorylation, and induce cell cycle arrest. Mutation of CDK4 C135 causes a kinase-impaired phenotype, which decreases cell proliferation rate and alleviates disease phenotype in an experimental mouse PH model, suggesting this cysteine is indispensable for cyclin D-CDK4 kinase activity. Pulmonary arteries and human pulmonary arterial smooth muscle cells from patients with pulmonary arterial hypertension display a decreased level of CDK4 disulfide, consistent with CDK4 being hyperactive in human pulmonary arterial hypertension. Furthermore, auranofin treatment, which induces the cyclin D-CDK4 disulfide, attenuates disease severity in experimental PH models by mitigating pulmonary vascular remodeling. CONCLUSIONS: A novel disulfide bond in cyclin D-CDK4 acts as a rapid switch to inhibit kinase activity and halt cell proliferation. This oxidative modification forms at a critical cysteine residue, which is unique to CDK4, offering the potential for the design of a selective covalent inhibitor predicted to be beneficial in PH.

Pharmacology and Rationale for Seralutinib in the Treatment of Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a complex disorder characterized by vascular remodeling and a consequent increase in pulmonary vascular resistance. The histologic hallmarks of PAH include plexiform and neointimal lesions of the pulmonary arterioles, which are composed of dysregulated, apoptosis-resistant endothelial cells and myofibroblasts. Platelet-derived growth factor receptors (PDGFR) α and ß, colony stimulating factor 1 receptor (CSF1R), and mast/stem cell growth factor receptor kit (c-KIT) are closely related kinases that have been implicated in PAH progression. In addition, emerging data indicate significant crosstalk between PDGF signaling and the bone morphogenetic protein receptor type 2 (BMPR2)/transforming growth factor ß (TGFß) receptor axis. This review will discuss the importance of the PDGFR-CSF1R-c-KIT signaling network in PAH pathogenesis, present evidence that the inhibition of all three nodes in this kinase network is a potential therapeutic approach for PAH, and highlight the therapeutic potential of seralutinib, currently in development for PAH, which targets these pathways.

Right ventricular failure in pulmonary hypertension: recent insights from experimental models.

Right ventricular (RV) function is a critical determinant of the prognosis of patients with pulmonary hypertension (PH). Upon establishment of PH, RV dysfunction develops, leading to a gradual worsening of the condition over time, culminating in RV failure and premature mortality. Despite this understanding, the underlying mechanisms of RV failure remain obscure. As a result, there are currently no approved therapies specifically targeting the right ventricle. One contributing factor to the lack of RV-directed therapies is the complexity of the pathogenesis of RV failure as observed in animal models and clinical studies. In recent years, various research groups have begun utilizing multiple models, including both afterload-dependent and afterload-independent models, to investigate specific targets and pharmacological agents in RV failure. In this review, we examine various animal models of RV failure and the recent advancements made utilizing these models to study the mechanisms of RV failure and the potential efficacy of therapeutic interventions, with the ultimate goal of translating these findings into clinical practice to enhance the management of individuals with PH.

Targeting Wnt-ß-Catenin-FOSL Signaling Ameliorates Right Ventricular Remodeling.

BACKGROUND: The ability of the right ventricle (RV) to adapt to an increased pressure afterload determines survival in patients with pulmonary arterial hypertension. At present, there are no specific treatments available to prevent RV failure, except for heart/lung transplantation. The wingless/int-1 (Wnt) signaling pathway plays an important role in the development of the RV and may also be implicated in adult cardiac remodeling. METHODS: Molecular, biochemical, and pharmacological approaches were used both in vitro and in vivo to investigate the role of Wnt signaling in RV remodeling. RESULTS: Wnt/ß-catenin signaling molecules are upregulated in RV of patients with pulmonary arterial hypertension and animal models of RV overload (pulmonary artery banding-induced and monocrotaline rat models). Activation of Wnt/ß-catenin signaling leads to RV remodeling via transcriptional activation of FOSL1 and FOSL2 (FOS proto-oncogene [FOS] like 1/2, AP-1 [activator protein 1] transcription factor subunit). Immunohistochemical analysis of pulmonary artery banding -exposed BAT-Gal (ß-catenin-activated transgene driving expression of nuclear ß-galactosidase) reporter mice RVs exhibited an increase in ß-catenin expression compared with their respective controls. Genetic inhibition of ß-catenin, FOSL1/2, or WNT3A stimulation of RV fibroblasts significantly reduced collagen synthesis and other remodeling genes. Importantly, pharmacological inhibition of Wnt signaling using inhibitor of PORCN (porcupine O-acyltransferase), LGKK-974 attenuated fibrosis and cardiac hypertrophy leading to improvement in RV function in both, pulmonary artery banding - and monocrotaline-induced RV overload. CONCLUSIONS: Wnt- ß-Catenin-FOSL signaling is centrally involved in the hypertrophic RV response to increased afterload, offering novel targets for therapeutic interference with RV failure in pulmonary hypertension.

Robotic gaze and human views: A systematic exploration of robotic gaze aversion and its effects on human behaviors and attitudes.

Similar to human-human interaction (HHI), gaze is an important modality in conversational human-robot interaction (HRI) settings. Previously, human-inspired gaze parameters have been used to implement gaze behavior for humanoid robots in conversational settings and improve user experience (UX). Other robotic gaze implementations disregard social aspects of gaze behavior and pursue a technical goal (e.g., face tracking). However, it is unclear how deviating from human-inspired gaze parameters affects the UX. In this study, we use eye-tracking, interaction duration, and self-reported attitudinal measures to study the impact of non-human inspired gaze timings on the UX of the participants in a conversational setting. We show the results for systematically varying the gaze aversion ratio (GAR) of a humanoid robot over a broad parameter range from almost always gazing at the human conversation partner to almost always averting the gaze. The main results reveal that on a behavioral level, a low GAR leads to shorter interaction durations and that human participants change their GAR to mimic the robot. However, they do not copy the robotic gaze behavior strictly. Additionally, in the lowest gaze aversion setting, participants do not gaze back as much as expected, which indicates a user aversion to the robot gaze behavior. However, participants do not report different attitudes toward the robot for different GARs during the interaction. In summary, the urge of humans in conversational settings with a humanoid robot to adapt to the perceived GAR is stronger than the urge of intimacy regulation through gaze aversion, and a high mutual gaze is not always a sign of high comfort, as suggested earlier. This result can be used as a justification to deviate from human-inspired gaze parameters when necessary for specific robot behavior implementations.

Notch1 Induces Defective Epithelial Surfactant Processing and Pulmonary Fibrosis.

Rationale: Although type II alveolar epithelial cells (AEC2s) are chronically injured in idiopathic pulmonary fibrosis (IPF), they contribute to epithelial regeneration in IPF. Objectives: We hypothesized that Notch signaling may contribute to AEC2 proliferation, dedifferentiation characterized by loss of surfactant processing machinery, and lung fibrosis in IPF. Methods: We applied microarray analysis, kinome profiling, flow cytometry, immunofluorescence analysis, western blotting, quantitative PCR, and proliferation and surface activity analysis to study epithelial differentiation, proliferation, and matrix deposition in vitro (AEC2 lines, primary murine/human AEC2s), ex vivo (human IPF-derived precision-cut lung slices), and in vivo (bleomycin and pepstatin application, Notch1 [Notch receptor 1] intracellular domain overexpression). Measurements and Main Results: We document here extensive SP-B and -C (surfactant protein-B and -C) processing defects in IPF AEC2s, due to loss of Napsin A, resulting in increased intra-alveolar surface tension and alveolar collapse and induction of endoplasmic reticulum stress in AEC2s. In vivo pharmacological inhibition of Napsin A results in the development of AEC2 injury and overt lung fibrosis. We also demonstrate that Notch1 signaling is already activated early in IPF and determines AEC2 fate by inhibiting differentiation (reduced lamellar body compartment, reduced capacity to process hydrophobic SP) and by causing increased epithelial proliferation and development of lung fibrosis, putatively via altered JAK (Janus kinase)/Stat (signal transducer and activator of transcription) signaling in AEC2s. Conversely, inhibition of Notch signaling in IPF-derived precision-cut lung slices improved the surfactant processing capacity of AEC2s and reversed fibrosis. Conclusions: Notch1 is a central regulator of AEC2 fate in IPF. It induces alveolar epithelial proliferation and loss of Napsin A and of surfactant proprotein processing, and it contributes to fibroproliferation.

Video connecting families and social robots: from ideas to practices putting technology to work.

Technology use is a socially embedded process, especially when it comes to older adults and care. However, the restrictions associated with the COVID-19 pandemic have limited social contact to protect vulnerable groups in care homes, and even if technology use has increased in other areas, there is little known about the potential uptake of communication technology and changes in social interaction in the care context during a lasting crisis. This paper explores changes in communication technology use triggered by the pandemic at two care homes, using a qualitative diary study, online interviews and observations, and in-situ interviews within the care home with residents and workers. Our findings point to increasing use of tablets and video conference software triggered by COVID-related experiences, with implications for living and working in care homes. We also characterise the isolation experience of the residents, the workers' concerns about the residents and changes in social interaction. We observed new areas of technology usage, associated changing work practices, technical affinity issues and context-specific attitudes towards future technologies. While the pandemic has triggered the use of communication technology in care homes on a small scale, this has also caused increasing workload and in particular articulation work, which requires support structures and the re-definition of work roles.

Epigenetically silenced apoptosis-associated tyrosine kinase (AATK) facilitates a decreased expression of Cyclin D1 and WEE1, phosphorylates TP53 and reduces cell proliferation in a kinase-dependent manner.

Silencing of the Apoptosis associated Tyrosine Kinase gene (AATK) has been described in cancer. In our study, we specifically investigated the epigenetic inactivation of AATK in pancreatic adenocarcinoma, lower grade glioma, lung, breast, head, and neck cancer. The resulting loss of AATK correlates with impaired patient survival. Inhibition of DNA methyltransferases (DNMTs) reactivated AATK in glioblastoma and pancreatic cancer. In contrast, epigenetic targeting via the CRISPR/dCas9 system with either EZH2 or DNMT3A inhibited the expression of AATK. Via large-scale kinomic profiling and kinase assays, we demonstrate that AATK acts a Ser/Thr kinase that phosphorylates TP53 at Ser366. Furthermore, whole transcriptome analyses and mass spectrometry associate AATK expression with the GO term 'regulation of cell proliferation'. The kinase activity of AATK in comparison to the kinase-dead mutant mediates a decreased expression of the key cell cycle regulators Cyclin D1 and WEE1. Moreover, growth suppression through AATK relies on its kinase activity. In conclusion, the Ser/Thr kinase AATK represses growth and phosphorylates TP53. Furthermore, expression of AATK was correlated with a better patient survival for different cancer entities. This data suggests that AATK acts as an epigenetically inactivated tumor suppressor gene.

Sustained Endurance Training Leads to Metabolomic Adaptation.

Endurance training induces several adaptations in substrate metabolism, especially in relation to glycogen conservation. The study aimed to investigate differences in the metabolism of lipids, lipid-like substances, and amino acids between highly trained and untrained subjects using targeted metabolomics. Depending on their maximum relative oxygen uptake (VO2max), subjects were categorized as either endurance-trained (ET) or untrained (UT). Resting blood was taken and plasma isolated. It was screened for changes of 345 metabolites, including amino acids and biogenic amines, acylcarnitines, glycerophosphocholines (GPCs), sphingolipids, hexoses, bile acids, and polyunsaturated fatty acids (PUFAs) by using liquid chromatography coupled to tandem mass spectrometry. Acylcarnitine (C14:1, down in ET) and five GPCs (lysoPC a C18:2, up in ET; PC aa C42:0, up in ET; PC ae C38:2, up in ET; PC aa C38:5, down in ET; lysoPC a C26:0, down in ET) were differently regulated in ET compared to UT. TCDCA was down-regulated in athletes, while for three ratios of bile acids CA/CDCA, CA/(GCA+TCA), and DCA/(GDCA+TDCA) an up-regulation was found. TXB2 and 5,6-EET were down-regulated in the ET group and 18S-HEPE, a PUFA, showed higher levels in 18S-HEPE in endurance-trained subjects. For PC ae C38:2, TCDCA, and the ratio of cholic acid to chenodeoxycholic acid, an association with VO2max was found. Numerous phospholipids, acylcarnitines, glycerophosphocholines, bile acids, and PUFAs are present in varying concentrations at rest in ET. These results might represent an adaptation of lipid metabolism and account for the lowered cardiovascular risk profile of endurance athletes.

Comparative kinase activity profiling of pathogenic influenza A viruses reveals new anti- and pro-viral protein kinases.

Constant evolution of influenza A viruses (IAVs) leads to the occurrence of new virus strains, which can cause epidemics and occasional pandemics. Here we compared two medically relevant IAVs, namely A/Hamburg/4/09 (H1N1pdm09) of the 2009 pandemic and the highly pathogenic avian IAV human isolate A/Thailand/1(KAN-1)/2004 (H5N1), for their ability to trigger intracellular phosphorylation patterns using a highly sensitive peptide-based kinase activity profiling approach. Virus-dependent tyrosine phosphorylations of substrate peptides largely overlap between the two viruses and are also strongly overrepresented in comparison to serine/threonine peptide phosphorylations. Both viruses trigger phosphorylations with distinct kinetics by overlapping and different kinases from which many form highly interconnected networks. As approximately half of the kinases forming a signalling hub have no known function for the IAV life cycle, we interrogated selected members of this group for their ability to interfere with IAV replication. These experiments revealed negative regulation of H1N1pdm09 and H5N1 replication by NUAK [novel (nua) kinase] kinases and by redundant ephrin A (EphA) receptor tyrosine kinases.

Myeloid-cell-specific deletion of inducible nitric oxide synthase protects against smoke-induced pulmonary hypertension in mice.

BACKGROUND: Pulmonary hypertension (PH) is a common complication of COPD, associated with increased mortality and morbidity. Intriguingly, pulmonary vascular alterations have been suggested to drive emphysema development. Previously, we identified inducible nitric oxide synthase (iNOS) as an essential enzyme for development and reversal of smoke-induced PH and emphysema, and showed that iNOS expression in bone-marrow-derived cells drives pulmonary vascular remodelling, but not parenchymal destruction. In this study, we aimed to identify the iNOS-expressing cell type driving smoke-induced PH and to decipher pro-proliferative pathways involved. METHODS: To address this question we used 1) myeloid-cell-specific iNOS knockout mice in chronic smoke exposure and 2) co-cultures of macrophages and pulmonary artery smooth muscle cells (PASMCs) to decipher underlying signalling pathways. RESULTS: Myeloid-cell-specific iNOS knockout prevented smoke-induced PH but not emphysema in mice. Moreover, iNOS deletion in myeloid cells ameliorated the increase in expression of CD206, a marker of M2 polarisation, on interstitial macrophages. Importantly, the observed effects on lung macrophages were hypoxia-independent, as these mice developed hypoxia-induced PH. In vitro, smoke-induced PASMC proliferation in co-cultures with M2-polarised macrophages could be abolished by iNOS deletion in phagocytic cells, as well as by extracellular signal-regulated kinase inhibition in PASMCs. Crucially, CD206-positive and iNOS-positive macrophages accumulated in proximity of remodelled vessels in the lungs of COPD patients, as shown by immunohistochemistry. CONCLUSION: In summary, our results demonstrate that iNOS deletion in myeloid cells confers protection against PH in smoke-exposed mice and provide evidence for an iNOS-dependent communication between M2-like macrophages and PASMCs in underlying pulmonary vascular remodelling.

The Pheno- and Genotypic Characterization of Porcine Escherichia coli Isolates.

Escherichia (E.) coli is the main causative pathogen of neonatal and post-weaning diarrhea and edema disease in swine production. There is a significant health concern due to an increasing number of human infections associated with food and/or environmental-borne pathogenic and multidrug-resistant E. coli worldwide. Monitoring the presence of pathogenic and antimicrobial-resistant E. coli isolates is essential for sustainable disease management in livestock and human medicine. A total of 102 E. coli isolates of diseased pigs were characterized by antimicrobial and biocide susceptibility testing. Antimicrobial resistance genes, including mobile colistin resistance genes, were analyzed by PCR and DNA sequencing. The quinolone resistance-determining regions of gyrA and parC in ciprofloxacin-resistant isolates were analyzed. Clonal relatedness was investigated by two-locus sequence typing (CH clonotyping). Phylotyping was performed by the Clermont multiplex PCR method. Virulence determinants were analyzed by customized DNA-based microarray technology developed in this study for fast and economic molecular multiplex typing. Thirty-five isolates were selected for whole-genome sequence-based analysis. Most isolates were resistant to ampicillin and tetracycline. Twenty-one isolates displayed an ESBL phenotype and one isolate an AmpC ß-lactamase-producing phenotype. Three isolates had elevated colistin minimal inhibitory concentrations and carried the mcr-1 gene. Thirty-seven isolates displayed a multi-drug resistance phenotype. The most predominant ß-lactamase gene classes were blaTEM-1 (56%) and blaCTX-M-1 (13.71%). Mutations in QRDR were observed in 14 ciprofloxacin-resistant isolates. CH clonotyping divided all isolates into 51 CH clonotypes. The majority of isolates belonged to phylogroup A. Sixty-four isolates could be assigned to defined pathotypes wherefrom UPEC was predominant. WGS revealed that the most predominant sequence type was ST100, followed by ST10. ST131 was detected twice in our analysis. This study highlights the importance of monitoring antimicrobial resistance and virulence properties of porcine E. coli isolates. This can be achieved by applying reliable, fast, economic and easy to perform technologies such as DNA-based microarray typing. The presence of high-risk pathogenic multi-drug resistant zoonotic clones, as well as those that are resistant to critically important antibiotics for humans, can pose a risk to public health. Improved protocols may be developed in swine farms for preventing infections, as well as the maintenance and distribution of the causative isolates.

Therapeutic Potential of Regorafenib-A Multikinase Inhibitor in Pulmonary Hypertension.

Pulmonary hypertension (PH) is characterized by a progressive elevation of mean arterial pressure followed by right ventricular failure and death. Previous studies have indicated that numerous inhibitors of receptor tyrosine kinase signaling could be either beneficial or detrimental for the treatment of PH. Here we investigated the therapeutic potential of the multi-kinase inhibitor regorafenib (BAY 73-4506) for the treatment of PH. A peptide-based kinase activity assay was performed using the PamStation®12 platform. The 5-bromo-2'-deoxyuridine proliferation and transwell migration assays were utilized in pulmonary arterial smooth muscle cells (PASMCs). Regorafenib was administered to monocrotaline- and hypoxia-induced PH in rats and mice, respectively. Functional parameters were analyzed by hemodynamic and echocardiographic measurements. The kinase activity assay revealed upregulation of twenty-nine kinases in PASMCs from patients with idiopathic PAH (IPAH), of which fifteen were established as potential targets of regorafenib. Regorafenib showed strong anti-proliferative and anti-migratory effects in IPAH-PASMCs compared to the control PASMCs. Both experimental models indicated improved cardiac function and reduced pulmonary vascular remodeling upon regorafenib treatment. In lungs from monocrotaline (MCT) rats, regorafenib reduced the phosphorylation of c-Jun N-terminal kinase and extracellular signal-regulated kinase 1/2. Overall, our data indicated that regorafenib plays a beneficial role in experimental PH.

Kinases as potential targets for treatment of pulmonary hypertension and right ventricular dysfunction.

Pulmonary hypertension (PH) is a progressive pulmonary vasculopathy that causes chronic right ventricular pressure overload and often leads to right ventricular failure. Various kinase inhibitors have been studied in the setting of PH and either improved or worsened the disease, highlighting the importance of understanding the specific role of the respective kinases in a spatiotemporal cellular context. In this review, we will summarize the knowledge on the role of kinases in PH and focus on druggable targets for which certain criteria are met: (a) deregulation of the kinase in PH; (b) small-molecule inhibitors are available (e.g. from the oncology field); (c) preclinical studies have shown their efficacy in PH models; and (d) when available, therapeutic exploitation in human PH has been initiated. Along this line, clinical considerations such as personalized medicine approaches to predict therapy response and adverse side events such as cardiotoxicity together with their clinical management are discussed. LINKED ARTICLES: This article is part of a themed issue on Risk factors, comorbidities, and comedications in cardioprotection. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.1/issuetoc.