Cross-sectional survey and Bayesian network model analysis of traditional Chinese medicine in Austria: investigating public awareness, usage determinants and perception of scientific support.

OBJECTIVES: Despite the paucity of evidence verifying its efficacy and safety, traditional Chinese medicine (TCM) is expanding in popularity and political support. Decisions to include TCM diagnoses in the International Classification of Diseases 11th Revision and campaigns to integrate TCM into national healthcare systems have occurred while public perception and usage of TCM, especially in Europe, remains undetermined. Accordingly, this study investigates TCM's popularity, usage and perceived scientific support, as well as its relationship to homeopathy and vaccinations. DESIGN/SETTING: We performed a cross-sectional survey of the Austrian population. Participants were either recruited on the street (in-person) or online (web-link) via a popular Austrian newspaper. PARTICIPANTS: 1382 individuals completed our survey. The sample was poststratified according to data derived from Austria's Federal Statistical Office. OUTCOME MEASURES: Associations between sociodemographic factors, opinion towards TCM and usage of complementary medicine (CAM) were investigated using a Bayesian graphical model. RESULTS: Within our poststratified sample, TCM was broadly known (89.9% of women, 90.6% of men), with 58.9% of women and 39.5% of men using TCM between 2016 and 2019. Moreover, 66.4% of women and 49.7% of men agreed with TCM being supported by science. We found a positive relationship between perceived scientific support for TCM and trust in TCM-certified medical doctors (ρ=0.59, 95% CI 0.46 to 0.73). Moreover, perceived scientific support for TCM was negatively correlated with proclivity to get vaccinated (ρ=-0.26, 95% CI -0.43 to -0.08). Additionally, our network model yielded associations between TCM-related, homeopathy-related and vaccination-related variables. CONCLUSIONS: TCM is widely known within the Austrian general population and used by a substantial proportion. However, a disparity exists between the commonly held public perception that TCM is scientific and findings from evidence-based studies. Emphasis should be placed on supporting the distribution of unbiased, science-driven information.

Upregulation of Nox4 induces a pro-survival Nrf2 response in cancer-associated fibroblasts that promotes tumorigenesis and metastasis, in part via Birc5 induction.

BACKGROUND: A pro-oxidant enzyme, NADPH oxidase 4 (Nox4) has been reported to be a critical downstream effector of TGFß-induced myofibroblast transformation during fibrosis. While there are a small number of studies suggesting an oncogenic role of Nox4 derived from activated fibroblasts, direct evidence linking this pro-oxidant to the tumor-supporting CAF phenotype and the mechanisms involved are lacking, particularly in breast cancer. METHODS: We targeted Nox4 in breast patient-derived CAFs via siRNA-mediated knockdown or administration of a pharmaceutical inhibitor (GKT137831). We also determine primary tumor growth and metastasis of implanted tumor cells using a stable Nox4-/- syngeneic mouse model. Autophagic flux of CAFs was assessed using a tandem fluorescent-tagged ptfl-LC3 plasmid via confocal microscopy analysis and determination of the expression level of autophagy markers (beclin-1 and LC3B). Nox4 overexpressing CAFs depend on the Nrf2 (nuclear factor-erythroid factor 2-related factor 2) pathway for survival. We then determined the dependency of Nox4-overexpressing CAFs on the Nrf2-mediated adaptive stress response pathway for survival. Furthermore, we investigated the involvement of Birc5 on CAF phenotype (viability and collagen contraction activity) as well as the expression level of CAF markers, FAP and αSMA. CONCLUSIONS: We found that deletion of stroma Nox4 and pharmaceutically targeting its activity with GKT137831 significantly inhibited orthotopic tumor growth and metastasis of implanted E0771 and 4T1 murine mammary carcinoma cell lines in mice. More importantly, we found a significant upregulation of Nox4 expression in CAFs isolated from human breast tumors versus normal mammary fibroblasts (RMFs). Our in situ RNA hybridization analysis for Nox4 transcription on a human breast tumor microarray further support a role of this pro-oxidant in the stroma of breast carcinomas. In addition, we found that Nox4 promotes autophagy in CAFs. Moreover, we found that Nox4 promoted survival of CAFs via activation of Nrf2, a master regulator of oxidative stress response. We have further shown Birc5 is involved as a downstream modulator of Nrf2-mediated pro-survival phenotype. Together these studies indicate a role of redox signaling via the Nox4-Nrf2 pathway in tumorigenesis and metastasis of breast cancer cells by promoting autophagy and survival of CAFs.

Clonidine for the Treatment of Agitation After Dexmedetomidine Discontinuation in Pediatric Patients: A Retrospective Cohort Study.

OBJECTIVE: Dexmedetomidine has become a widely used drug in PICUs for sedation. We aim to determine the effects of clonidine on pediatric patients after dexmedetomidine use. METHODS: This was a retrospective cohort study that evaluated all pediatric patients admitted to a tertiary PICU who received dexmedetomidine infusion for >48 hours. Outcomes in patients exposed to clonidine (CLON) were compared with those of patients who were not exposed (NoCLON). RESULTS: A total of 216 patients were included in this study (43 CLON and 173 NoCLON). The primary outcome, agitation, was less in the CLON cohort (9.3%) than in the NoCLON cohort (9.3% versus 29.5%, respectively; p < 0.01). Hospital LOS was longer in the CLON group (59 versus 20 days, p < 0.01), as was PICU LOS (37.4 versus 11.1 days, p < 0.01). There was no significant difference in the occurrence of increased heart rate or blood pressure between the 2 cohorts. Patients exposed to concurrent midazolam and opioid infusions had higher incidence of agitation when they did not receive clonidine (CLON 8% versus NoCLON 37%, OR 0.15; 95% CI, 0.05-0.51; p < 0.01). In contrast, there was no difference in the incidence of agitation for the CLON group versus the NoCLON group when dexmedetomidine was administered alone (25% versus 19%, OR 1.4; p = 0.99). CONCLUSIONS: Our study confirms the importance and effectiveness of clonidine to treat agitation after dexmedetomidine discontinuation. A validated withdrawal scoring tool can help better define dexmedetomidine withdrawal in pediatric patients.

A Novel Method for Improving Weld Seam Properties through Inline Coupling of Welding and Forming.

Keywords: WeldForming; post weld treatment; recrystallization; thermomechanical simulation; numerical simulation; FEM.

EHD1 and RUSC2 Control Basal Epidermal Growth Factor Receptor Cell Surface Expression and Recycling.

Epidermal growth factor receptor (EGFR) is a prototype receptor tyrosine kinase and an oncoprotein in many solid tumors. Cell surface display of EGFR is essential for cellular responses to its ligands. While postactivation endocytic trafficking of EGFR has been well elucidated, little is known about mechanisms of basal/preactivation surface display of EGFR. Here, we identify a novel role of the endocytic regulator EHD1 and a potential EHD1 partner, RUSC2, in cell surface display of EGFR. EHD1 and RUSC2 colocalize with EGFR in vesicular/tubular structures and at the Golgi compartment. Inducible EHD1 knockdown reduced the cell surface EGFR expression with accumulation at the Golgi compartment, a phenotype rescued by exogenous EHD1. RUSC2 knockdown phenocopied the EHD1 depletion effects. EHD1 or RUSC2 depletion impaired the EGF-induced cell proliferation, demonstrating that the novel, EHD1- and RUSC2-dependent transport of unstimulated EGFR from the Golgi compartment to the cell surface that we describe is functionally important, with implications for physiologic and oncogenic roles of EGFR and targeted cancer therapies.

Cocaine Mediated Neuroinflammation: Role of Dysregulated Autophagy in Pericytes.

Cocaine, a known psychostimulant, results in oxidative stress and inflammation. Recent studies from our group have shown that cocaine induces inflammation in glial cells. Our current study was aimed at investigating whether cocaine exposure could also induce inflammation in non-glial cells such as the pericytes with a focus on the endoplasmic reticulum (ER) stress/autophagy axis. Our in vitro findings demonstrated that exposure of pericytes to cocaine resulted in upregulation of the pro-inflammatory cytokines (TNF-α, IL-1ß, and IL-6) in both the intracellular as well as extracellular compartments, thus underpinning pericytes as yet another source of neuroinflammation. Cocaine exposure of pericytes resulted in increased formation of autophagosomes as demonstrated by a time-dependent increase of autophagy markers, with a concomitant defect in the fusion of the autophagosome with the lysosomes. Pharmacological blocking of the sigma 1 receptor underscored its role in cocaine-mediated activation of pericytes. Furthermore, it was also demonstrated that cocaine-mediated dysregulation of autophagy involved upstream activation of the ER stress pathways, with a subsequent downstream production of pro-inflammatory cytokines in pericytes. These findings were also validated in an in vivo model wherein pericytes in the isolated brain microvessels of cocaine injected mice (7 days) exhibited increased expression of both the autophagy marker-LC3 as well as the pro-inflammatory cytokine, IL-6. This is the first report describing the role of pericytes in cocaine-mediated neuroinflammation. Interventions aimed at blocking either the sigma-1 receptor or the upstream ER stress mediators could likely be envisioned as promising therapeutic targets for abrogating cocaine-mediated inflammation in pericytes.

Experimental evolution of UV resistance in a phage.

The dsDNA bacteriophage T7 was subjected to 30 cycles of lethal ultraviolet light (UV) exposure to select increased resistance to UV. The exposure effected a 0.9999 kill of the ancestral population, and survival of the ending population was nearly 50-fold improved. At the end point, a 2.1 kb deletion of early genes and three substitutions in structural-genes were the only changes observed at high frequency throughout the 40 kb genome; no changes were observed in genes affecting DNA metabolism. The deletion accounted for only a two-fold improvement in survival. One possible explanation of its benefit is that it represents an error catastrophe, whereby the genome experiences a reduced mutation rate. The mechanism of benefit provided by the three structural-gene mutations remains unknown. The results offer some hope of artificially evolving greater protection against sunlight damage in applications of phage therapy to plants, but the response of T7 is weak compared to that observed in bacteria selected to resist ionizing radiation. Because of the weak response, mathematical analysis of the selection process was performed to determine how the protocol might have been modified to achieve a greater response, but the greatest protection may well come from evolving phages to bind materials that block the UV.

Role of the EHD Family of Endocytic Recycling Regulators for TCR Recycling and T Cell Function.

T cells use the endocytic pathway for key cell biological functions, including receptor turnover and maintenance of the immunological synapse. Some of the established players include the Rab GTPases, the SNARE complex proteins, and others, which function together with EPS-15 homology domain-containing (EHD) proteins in non-T cell systems. To date, the role of the EHD protein family in T cell function remains unexplored. We generated conditional EHD1/3/4 knockout mice using CD4-Cre and crossed these with mice bearing a myelin oligodendrocyte glycoprotein-specific TCR transgene. We found that CD4+ T cells from these mice exhibited reduced Ag-driven proliferation and IL-2 secretion in vitro. In vivo, these mice exhibited reduced severity of experimental autoimmune encephalomyelitis. Further analyses showed that recycling of the TCR-CD3 complex was impaired, leading to increased lysosomal targeting and reduced surface levels on CD4+ T cells of EHD1/3/4 knockout mice. Our studies reveal a novel role of the EHD family of endocytic recycling regulatory proteins in TCR-mediated T cell functions.

Extracellular superoxide dismutase and its role in cancer.

Reactive oxygen species (ROS) are increasingly recognized as critical determinants of cellular signaling and a strict balance of ROS levels must be maintained to ensure proper cellular function and survival. Notably, ROS is increased in cancer cells. The superoxide dismutase family plays an essential physiological role in mitigating deleterious effects of ROS. Due to the compartmentalization of ROS signaling, EcSOD, the only superoxide dismutase in the extracellular space, has unique characteristics and functions in cellular signal transduction. In comparison to the other two intracellular SODs, EcSOD is a relatively new comer in terms of its tumor suppressive role in cancer and the mechanisms involved are less well understood. Nevertheless, the degree of differential expression of this extracellular antioxidant in cancer versus normal cells/tissues is more pronounced and prevalent than the other SODs. A significant association of low EcSOD expression with reduced cancer patient survival further suggests that loss of extracellular redox regulation promotes a conducive microenvironment that favors cancer progression. The vast array of mechanisms reported in mediating deregulation of EcSOD expression, function, and cellular distribution also supports that loss of this extracellular antioxidant provides a selective advantage to cancer cells. Moreover, overexpression of EcSOD inhibits tumor growth and metastasis, indicating a role as a tumor suppressor. This review focuses on the current understanding of the mechanisms of deregulation and tumor suppressive function of EcSOD in cancer.

CSF-1 receptor signalling is governed by pre-requisite EHD1 mediated receptor display on the macrophage cell surface.

Colony stimulating factor-1 receptor (CSF-1R), a receptor tyrosine kinase (RTK), is the master regulator of macrophage biology. CSF-1 can bind CSF-1R resulting in receptor activation and signalling essential for macrophage functions such as proliferation, differentiation, survival, polarization, phagocytosis, cytokine secretion, and motility. CSF-1R activation can only occur after the receptor is presented on the macrophage cell surface. This process is reliant upon the underlying macrophage receptor trafficking machinery. However, the mechanistic details governing this process are incompletely understood. C-terminal Eps15 Homology Domain-containing (EHD) proteins have recently emerged as key regulators of receptor trafficking but have not yet been studied in the context of macrophage CSF-1R signalling. In this manuscript, we utilize primary bone-marrow derived macrophages (BMDMs) to reveal a novel function of EHD1 as a regulator of CSF-1R abundance on the cell surface. We report that EHD1-knockout (EHD1-KO) macrophages cell surface and total CSF-1R levels are significantly decreased. The decline in CSF-1R levels corresponds with reduced downstream macrophage functions such as cell proliferation, migration, and spreading. In EHD1-KO macrophages, transport of newly synthesized CSF-1R to the macrophage cell surface was reduced and was associated with the shunting of the receptor to the lysosome, which resulted in receptor degradation. These findings reveal a novel and functionally important role for EHD1 in governing CSF-1R signalling via regulation of anterograde transport of CSF-1R to the macrophage cell surface.

Cbl-family ubiquitin ligases and their recruitment of CIN85 are largely dispensable for epidermal growth factor receptor endocytosis.

Members of the casitas B-lineage lymphoma (Cbl) family (Cbl, Cbl-b and Cbl-c) of ubiquitin ligases serve as negative regulators of receptor tyrosine kinases (RTKs). An essential role of Cbl-family protein-dependent ubiquitination for efficient ligand-induced lysosomal targeting and degradation is now well-accepted. However, a more proximal role of Cbl and Cbl-b as adapters for CIN85-endophilin recruitment to mediate ligand-induced initial internalization of RTKs is supported by some studies but refuted by others. Overexpression and/or incomplete depletion of Cbl proteins in these studies is likely to have contributed to this dichotomy. To address the role of endogenous Cbl and Cbl-b in the internalization step of RTK endocytic traffic, we established Cbl/Cbl-b double-knockout (DKO) mouse embryonic fibroblasts (MEFs) and demonstrated that these cells lack the expression of both Cbl-family members as well as endophilin A, while they express CIN85. We show that ligand-induced ubiquitination of EGFR, as a prototype RTK, was abolished in DKO MEFs, and EGFR degradation was delayed. These traits were reversed by ectopic human Cbl expression. EGFR endocytosis, assessed using the internalization of (125)I-labeled or fluorescent EGF, or of EGFR itself, was largely retained in Cbl/Cbl-b DKO compared to wild type MEFs. EGFR internalization was also largely intact in Cbl/Cbl-b depleted MCF-10A human mammary epithelial cell line. Inducible shRNA-mediated knockdown of CIN85 in wild type or Cbl/Cbl-b DKO MEFs had no impact on EGFR internalization. Our findings, establish that, at physiological expression levels, Cbl, Cbl-b and CIN85 are largely dispensable for EGFR internalization. Our results support the model that Cbl-CIN85-endophilin complex is not required for efficient internalization of EGFR, a prototype RTK.

Nexus of signaling and endocytosis in oncogenesis driven by non-small cell lung cancer-associated epidermal growth factor receptor mutants.

Epidermal growth factor receptor (EGFR) controls a wide range of cellular processes, and aberrant EGFR signaling as a result of receptor overexpression and/or mutation occurs in many types of cancer. Tumor cells in non-small cell lung cancer (NSCLC) patients that harbor EGFR kinase domain mutations exhibit oncogene addiction to mutant EGFR, which confers high sensitivity to tyrosine kinase inhibitors (TKIs). As patients invariably develop resistance to TKIs, it is important to delineate the cell biological basis of mutant EGFR-induced cellular transformation since components of these pathways can serve as alternate therapeutic targets to preempt or overcome resistance. NSCLC-associated EGFR mutants are constitutively-active and induce ligand-independent transformation in nonmalignant cell lines. Emerging data suggest that a number of factors are critical for the mutant EGFR-dependent tumorigenicity, and bypassing the effects of TKIs on these pathways promotes drug resistance. For example, activation of downstream pathways such as Akt, Erk, STAT3 and Src is critical for mutant EGFR-mediated biological processes. It is now well-established that the potency and spatiotemporal features of cellular signaling by receptor tyrosine kinases such as EGFR, as well as the specific pathways activated, is determined by the nature of endocytic traffic pathways through which the active receptors traverse. Recent evidence indicates that NSCLC-associated mutant EGFRs exhibit altered endocytic trafficking and they exhibit reduced Cbl ubiquitin ligase-mediated lysosomal downregulation. More recent work has shown that mutant EGFRs undergo ligand-independent traffic into the endocytic recycling compartment, a behavior that plays a key role in Src pathway activation and oncogenesis. These studies are beginning to delineate the close nexus between signaling and endocytic traffic of EGFR mutants as a key driver of oncogenic processes. Therefore, in this review, we will discuss the links between mutant EGFR signaling and endocytic properties, and introduce potential mechanisms by which altered endocytic properties of mutant EGFRs may alter signaling and vice versa as well as their implications for NSCLC therapy.

A kinase inhibitor screen reveals protein kinase C-dependent endocytic recycling of ErbB2 in breast cancer cells.

ErbB2 overexpression drives oncogenesis in 20-30% cases of breast cancer. Oncogenic potential of ErbB2 is linked to inefficient endocytic traffic into lysosomes and preferential recycling. However, regulation of ErbB2 recycling is incompletely understood. We used a high-content immunofluorescence imaging-based kinase inhibitor screen on SKBR-3 breast cancer cells to identify kinases whose inhibition alters the clearance of cell surface ErbB2 induced by Hsp90 inhibitor 17-AAG. Less ErbB2 clearance was observed with broad-spectrum PKC inhibitor Ro 31-8220. A similar effect was observed with Go 6976, a selective inhibitor of classical Ca(2+)-dependent PKCs (α, ß1, ßII, and γ). PKC activation by PMA promoted surface ErbB2 clearance but without degradation, and ErbB2 was observed to move into a juxtanuclear compartment where it colocalized with PKC-α and PKC-δ together with the endocytic recycling regulator Arf6. PKC-α knockdown impaired the juxtanuclear localization of ErbB2. ErbB2 transit to the recycling compartment was also impaired upon PKC-δ knockdown. PMA-induced Erk phosphorylation was reduced by ErbB2 inhibitor lapatinib, as well as by knockdown of PKC-δ but not that of PKC-α. Our results suggest that activation of PKC-α and -δ mediates a novel positive feedback loop by promoting ErbB2 entry into the endocytic recycling compartment, consistent with reported positive roles for these PKCs in ErbB2-mediated tumorigenesis. As the endocytic recycling compartment/pericentrion has emerged as a PKC-dependent signaling hub for G-protein-coupled receptors, our findings raise the possibility that oncogenesis by ErbB2 involves previously unexplored PKC-dependent endosomal signaling.

Myocardial ischemic memory imaging with molecular echocardiography.

BACKGROUND: Diagnosing acute coronary syndrome in patients presenting with chest discomfort is a challenge. Because acute myocardial ischemia/reperfusion is associated with endothelial upregulation of leukocyte adhesion molecules, which persist even after ischemia has resolved, we hypothesized that microbubbles designed to adhere to endothelial selectins would permit echocardiographic identification of recently ischemic myocardium. METHODS AND RESULTS: Lipid microbubbles (diameter, 3.3+/-1.7 microm) were synthesized. The selectin ligand sialyl Lewis(x) was conjugated to the microbubble surface (MB(sLex)). Control bubbles (MB(CTL)) bore surface Lewis(x) or sialyl Lewis(c). Intravital microscopy of mouse cremaster muscle was performed after intravenous injection of MB(sLex) (n=11) or MB(CTL) (n=9) with or without prior intrascrotal tumor necrosis factor-alpha. There was greater adhesion of MB(sLex) to inflamed versus noninflamed endothelium (P = 0.0081). Rats (n=12) underwent 15 minutes of anterior descending coronary artery occlusion. After 30 minutes and 1 hour of reperfusion, high-mechanical-index nonlinear echocardiographic imaging was performed in which single frames were acquired at 3.5 and 4 minutes after intravenous injection of MB(sLex) or MB(CTL). Video intensity at 4 minutes was subtracted from that at 3.5 minutes to derive target-specific acoustic signal. MB(sLex) caused greater opacification in postischemic versus nonischemic myocardium at both time points (P < or = 0.002). Immunostaining confirmed endothelial P-selectin expression in the ischemic bed. CONCLUSIONS: Echocardiographic identification of recently ischemic myocardium is possible using ultrasound contrast agents targeted to selectins. This may offer a new approach to the more timely and precise diagnosis of acute coronary syndrome in patients presenting with chest pain of uncertain cardiac origin.

Targeted ultrasound contrast agents: in vitro assessment of endothelial dysfunction and multi-targeting to ICAM-1 and sialyl Lewisx.

An ultrasound-based molecular imaging technique capable of detecting endothelial cell markers of inflammation may allow early, non-invasive assessment of vascular disease. Clinical application of targeted, acoustically-active microbubbles requires optimization of microbubble-endothelial adhesion strength to maximize image signal-to-noise ratio, as well as the ability to discern the degree of inflammation along a continuum of dysfunction. Accordingly, we hypothesized that adhesion of intercellular adhesion molecule-1 (ICAM-1)-targeted microbubbles is dependent on the degree of endothelial inflammation, and that microbubbles multi-targeted to both ICAM-1 (via anti-ICAM-1 antibodies) and selectins (via sialyl Lewisx) demonstrate greater adhesion strength than microbubbles targeted to either inflammatory marker alone. In a radial flow chamber, microbubbles were perfused across endothelial cells activated with interleukin-1beta to four different levels of inflammation, as assessed by quantitative ICAM-1 expression. ICAM-1-targeted microbubble adhesion strength increased with increasing degree of inflammation, with a relationship that was both positive and linear (r > 0.99). Microbubble adhesion strength was significantly higher for the multi-targeted microbubbles than either of the single-targeted microbubbles. These data thus demonstrate that multi-targeting of contrast microbubbles may offer improved adhesion characteristics, allowing for greater sensitivity to inflammation. Furthermore, the adhesion strength of targeted microbubbles is linearly dependent on the degree of inflammation, suggesting that targeted ultrasound imaging may offer differentiation between various degrees of endothelial dysfunction, and thus detect not only the presence, but also the severity of inflammatory disease processes.