PRUNE1 and NME/NDPK family proteins influence energy metabolism and signaling in cancer metastases.

We describe here the molecular basis of the complex formation of PRUNE1 with the tumor metastasis suppressors NME1 and NME2, two isoforms appertaining to the nucleoside diphosphate kinase (NDPK) enzyme family, and how this complex regulates signaling the immune system and energy metabolism, thereby shaping the tumor microenvironment (TME). Disrupting the interaction between NME1/2 and PRUNE1, as suggested, holds the potential to be an excellent therapeutic target for the treatment of cancer and the inhibition of metastasis dissemination. Furthermore, we postulate an interaction and regulation of the other Class I NME proteins, NME3 and NME4 proteins, with PRUNE1 and discuss potential functions. Class I NME1-4 proteins are NTP/NDP transphosphorylases required for balancing the intracellular pools of nucleotide diphosphates and triphosphates. They regulate different cellular functions by interacting with a large variety of other proteins, and in cancer and metastasis processes, they can exert pro- and anti-oncogenic properties depending on the cellular context. In this review, we therefore additionally discuss general aspects of class1 NME and PRUNE1 molecular structures as well as their posttranslational modifications and subcellular localization. The current knowledge on the contributions of PRUNE1 as well as NME proteins to signaling cascades is summarized with a special regard to cancer and metastasis.

Tankyrase inhibition interferes with junction remodeling, induces leakiness, and disturbs YAP1/TAZ signaling in the endothelium.

Tankyrase inhibitors are increasingly considered for therapeutic use in malignancies that are characterized by high intrinsic ß-catenin activity. However, how tankyrase inhibition affects the endothelium after systemic application remains poorly understood. In this study, we aimed to investigate how the tankyrase inhibitor XAV939 affects endothelial cell function and the underlying mechanism involved. Endothelial cell function was analyzed using sprouting angiogenesis, endothelial cell migration, junctional dynamics, and permeability using human umbilical vein endothelial cells (HUVEC) and explanted mouse retina. Underlying signaling was studied using western blot, immunofluorescence, and qPCR in HUVEC in addition to luciferase reporter gene assays in human embryonic kidney cells. XAV939 treatment leads to altered junctional dynamics and permeability as well as impaired endothelial migration. Mechanistically, XAV939 increased stability of the angiomotin-like proteins 1 and 2, which impedes the nuclear translocation of YAP1/TAZ and consequently suppresses TEAD-mediated transcription. Intriguingly, XAV939 disrupts adherens junctions by inducing RhoA-Rho dependent kinase (ROCK)-mediated F-actin bundling, whereas disruption of F-actin bundling through the ROCK inhibitor H1152 restores endothelial cell function. Unexpectedly, this was accompanied by an increase in nuclear TAZ and TEAD-mediated transcription, suggesting differential regulation of YAP1 and TAZ by the actin cytoskeleton in endothelial cells. In conclusion, our findings elucidate the complex relationship between the actin cytoskeleton, YAP1/TAZ signaling, and endothelial cell function and how tankyrase inhibition disturbs this well-balanced signaling.

Sufficient Cav-1 levels in the endothelium are critical for the maintenance of the neurovascular unit in the retina.

BACKGROUND: Caveolin-1 (Cav-1) is a pivotal protein in the plasma membrane. Studies on homozygous Cav-1 deficient mice revealed that Cav-1 is essential for endothelial function and angiogenesis in the retina. However, whether a reduction in Cav-1 content hampers the neurovascular unit (NVU) in the retina is unclear. Thus, this study examines the NVU in the retinas of heterozygous Cav-1 deficient (Cav-1+/-) mice and analyzes possible underlying mechanisms. METHODS: The vascular, glial and neuronal components in the retina were evaluated using retinal morphometry, whole mount retinal immunofluorescence staining, histological analysis and optical coherence tomography. In addition, immunoblotting and immunofluorescence staining, subcellular fractionation, biotin labeling of cell surface proteins, and proximity ligation assay were employed to detect expression and localization of proteins in the retina or endothelial cells (ECs) upon knockdown of Cav-1 with Cav-1 siRNA. RESULTS: Cav-1+/- retinas showed a significant reduction in pericyte coverage along with an increase in acellular capillaries compared to controls at 8 months of age, but not at 1 month. A significant loss and obvious morphological abnormalities of smooth muscle cells were observed in 8-month-old Cav-1+/- retinal arterioles. Macroglial and microglial cells were activated in the Cav-1+/- retinas. A transient significant delay in retinal angiogenesis was detected in Cav-1+/- retinas at p5, which was however no longer detectable at p10. The Cav-1+/- retinas displayed increased vascular permeability and a notable reduction in VEGFR2 content at 8 months. In vitro, siRNA-mediated knockdown experiments in ECs revealed that the loss of Cav-1 in ECs resulted in decreased levels of VEGFR2, VE-Cadherin and their interaction at the plasma membrane as well. CONCLUSION: Our results indicate that a sufficient Cav-1 level over 50% of its normal abundance is vital for the proper localization of VEGFR2 and VE-cadherin, likely in a complex, at the plasma membrane, which is essential for the maintenance of normal NVU in the retina.

The Muscarinic Acetylcholine M2 Receptor-Induced Nitration of p190A by eNOS Increases RhoA Activity in Cardiac Myocytes.

p190RhoGAP, which exists in two paralogs, p190RhoGAP-A (p190A) and p190RhoGAP-B (p190B), is a GTPase activating protein (GAP) contributing to the regulation of the cellular activity of RhoGTPases. Recent data showed that M2 muscarinic acetylcholine receptor (M2R) stimulation in neonatal rat cardiac myocytes (NRCM) induces the binding of p190RhoGAP to the long isoform of the regulator of G protein signaling 3 (RGS3L). This complex formation alters the substrate preference of p190RhoGAP from RhoA to Rac1. By analyzing carbachol-stimulated GAP activity, we show herein that p190A, but not p190B, alters its substrate preference in NRCM. Based on data that the RhoGAP activity of p190A in endothelial cells is diminished upon nitration by endothelial nitric oxide synthase (eNOS)-derived peroxynitrite, we studied whether carbachol-induced NO/peroxynitrite formation contributes to the carbachol-induced RhoA activation in NRCM. Interestingly, the carbachol-induced RhoA activation in NRCM was suppressed by the eNOS-preferring inhibitor L-NIO as well as the non-selective NOS inhibitor L-NAME. Using L-NIO, we firstly verified the carbachol-induced NO production concurrent with eNOS activation and, secondly, the carbachol-induced nitration of p190A in NRCM. By co-immunoprecipitation, the carbachol-induced complex formation of eNOS, p190A, RGS3L and caveolin-3 was detected. We thus conclude that the NO production by M2R-induced eNOS activation in caveolae in NRCM is required for the nitration of p190A, leading to the binding to RGS3L and the change in substrate preference from RhoA to Rac1. In line with this interpretation, the disruption of caveolae in NRCM by methyl-ß-cyclodextrin suppressed carbachol-induced RhoA activation in NRCM to a similar extent as the inhibition of NO production.

Liquid biopsy comprehensive genomic profiling of lung cancer in the Italian population: A real-world experience.

OBJECTIVES: Liquid biopsy with next-generation sequencing (NGS) has emerged as a promising tool for tumor mutation profiling. In this study, we describe the genomic profile of Italian lung cancer patients tested with blood-based comprehensive genomic profiling (CGP) to assess the genomic landscape complexity and its impact on enhancing treatment options for patients. MATERIALS AND METHODS: Between January 2021 and December 2021, a total of 229 lung cancer patients were profiled by FoundationOne®Liquid CDx (F1LCDx®) assay on circulating tumor DNA (ctDNA). F1LCDx® reports alterations across 324 cancer-related genes and genomic signatures, including tumor fraction (TF) and blood-based tumor mutational burden (bTMB). Detected variants were classified according to the ESMO Scale of Clinical Actionability for molecular Targets (ESCAT). RESULTS: 90.4% of patients had at least one detectable alteration in plasma. The most frequently mutated genes were TP53 (47.6%), DNMT3A (33.2%), EGFR (20.1%), and KRAS (15.7%). Elevated TF was detected in 18.3% of patients, suggesting high reliability of test results. According to the ESCAT classification, potentially actionable alterations (Tier I-II) were identified in 27.1% of samples. An additional 5.2% harbored an alteration for which an approved drug is available in other cancer types (Tier III). Furthermore, 13.1% of tumors exhibited high bTMB, which may predict response to immunotherapy. Overall, 156 (68.1%) patients were eligible for enrolment in clinical trials. CONCLUSION: Liquid biopsy NGS is a viable and valuable approach to guide personalized therapy. The use of blood-based CGP may help identify a larger number of actionable mutations and increase chances of enrolment in clinical trials.

Pathogen and human NDPK-proteins promote AML cell survival via monocyte NLRP3-inflammasome activation.

A history of infection has been linked with increased risk of acute myeloid leukaemia (AML) and related myelodysplastic syndromes (MDS). Furthermore, AML and MDS patients suffer frequent infections because of disease-related impaired immunity. However, the role of infections in the development and progression of AML and MDS remains poorly understood. We and others previously demonstrated that the human nucleoside diphosphate kinase (NDPK) NM23-H1 protein promotes AML blast cell survival by inducing secretion of IL-1ß from accessory cells. NDPKs are an evolutionary highly conserved protein family and pathogenic bacteria secrete NDPKs that regulate virulence and host-pathogen interactions. Here, we demonstrate the presence of IgM antibodies against a broad range of pathogen NDPKs and more selective IgG antibody activity against pathogen NDPKs in the blood of AML patients and normal donors, demonstrating that in vivo exposure to NDPKs likely occurs. We also show that pathogen derived NDPK-proteins faithfully mimic the catalytically independent pro-survival activity of NM23-H1 against primary AML cells. Flow cytometry identified that pathogen and human NDPKs selectively bind to monocytes in peripheral blood. We therefore used vitamin D3 differentiated monocytes from wild type and genetically modified THP1 cells as a model to demonstrate that NDPK-mediated IL-1ß secretion by monocytes is NLRP3-inflammasome and caspase 1 dependent, but independent of TLR4 signaling. Monocyte stimulation by NDPKs also resulted in activation of NF-κB and IRF pathways but did not include the formation of pyroptosomes or result in pyroptotic cell death which are pivotal features of canonical NLRP3 inflammasome activation. In the context of the growing importance of the NLRP3 inflammasome and IL-1ß in AML and MDS, our findings now implicate pathogen NDPKs in the pathogenesis of these diseases.

Contribution of the hexosamine biosynthetic pathway in the hyperglycemia-dependent and -independent breakdown of the retinal neurovascular unit.

BACKGROUND: Diabetic retinopathy (DR) remains one of the most common complications of diabetes despite great efforts to uncover its underlying mechanisms. The pathogenesis of DR is characterized by the deterioration of the neurovascular unit (NVU), showing damage of vascular cells, activation of glial cells and dysfunction of neurons. Activation of the hexosamine biosynthesis pathway (HBP) and increased protein O-GlcNAcylation have been evident in the initiation of DR in patients and animal models. SCOPE OF REVIEW: The impairment of the NVU, in particular, damage of vascular pericytes and endothelial cells arises in hyperglycemia-independent conditions as well. Surprisingly, despite the lack of hyperglycemia, the breakdown of the NVU is similar to the pathology in DR, showing activated HBP, altered O-GlcNAc and subsequent cellular and molecular dysregulation. MAJOR CONCLUSIONS: This review summarizes recent research evidence highlighting the significance of the HBP in the breakdown of the NVU in hyperglycemia-dependent and -independent manners, and thus identifies joint avenues leading to vascular damage as seen in DR and thus identifying novel potential targets in such retinal diseases.

Chemical Tools for Studying Phosphohistidine: Generation of Selective τ-Phosphohistidine and π-Phosphohistidine Antibodies.

Nonhydrolysable stable analogues of τ-phosphohistidine (τ-pHis) and π-pHis have been designed, aided by electrostatic surface potential calculations, and subsequently synthesized. The τ-pHis and π-pHis analogues (phosphopyrazole 8 and pyridyl amino amide 13, respectively) were used as haptens to generate pHis polyclonal antibodies. Both τ-pHis and π-pHis conjugates in the form of BSA-glutaraldehyde-τ-pHis and BSA-glutaraldehyde-π-pHis were synthesized and characterized by 31 P NMR spectroscopy. Commercially available τ-pHis (SC56-2) and π-pHis (SC1-1; SC50-3) monoclonal antibodies were used to show that the BSA-G-τ-pHis and BSA-G-π-pHis conjugates could be used to assess the selectivity of pHis antibodies in a competitive ELISA. Subsequently, the selectivity of the pHis antibodies generated by using phosphopyrazole 8 and pyridyl amino amide 13 as haptens was assessed by competitive ELISA against His, pSer, pThr, pTyr, τ-pHis and π-pHis. Antibodies generated by using phosphopyrazole 8 as a hapten were found to be selective for τ-pHis, and antibodies generated by using pyridyl amino amide 13 were found to be selective for π-pHis. Both τ- and π-pHis antibodies were shown to be effective in immunological experiments, including ELISA, western blot, and immunofluorescence. The τ-pHis antibody was also shown to be useful in the immunoprecipitation of proteins containing pHis.

Spatial shopping behavior during the Corona pandemic: insights from a micro-econometric store choice model for consumer electronics and furniture retailing in Germany.

During the COVID-19 pandemic, e-commerce's market share has increased dramatically, a phenomenon attributable to not only lockdowns but to voluntary changes in shopping behavior as well. The current study examines the main determinants driving shopping behavior in the context of both physical and online store availability, and investigates whether specific drivers have changed during the pandemic. The study aims to test whether fear of infection and mandatory wearing of face masks in shops have influenced consumer channel choice. The empirical analysis focuses on two product types (consumer electronics, furniture), with empirical data collected via a representative consumer survey in three German regions. The statistical analysis was performed using a hurdle model approach and the findings are compared to those of a study related to pre-pandemic shopping. The results show that the determinants of shopping behavior have largely not changed. Channel choice can be explained by shopping attitudes, age, and partially, by place of residence of consumers. Store choice is determined primarily by shopping transaction costs and store features. Fear of infection and the mandatory wearing of face masks exhibit minimal influence on channel choice, if any. The importance of cross-channel integration of stores/chains has decreased significantly, while average travel times for in-store purchases have declined. Supplementary Information: The online version contains supplementary material available at 10.1007/s10109-023-00408-x.

Lamin A/C-dependent chromatin architecture safeguards naïve pluripotency to prevent aberrant cardiovascular cell fate and function.

Tight control of cell fate choices is crucial for normal development. Here we show that lamin A/C plays a key role in chromatin organization in embryonic stem cells (ESCs), which safeguards naïve pluripotency and ensures proper cell fate choices during cardiogenesis. We report changes in chromatin compaction and localization of cardiac genes in Lmna-/- ESCs resulting in precocious activation of a transcriptional program promoting cardiomyocyte versus endothelial cell fate. This is accompanied by premature cardiomyocyte differentiation, cell cycle withdrawal and abnormal contractility. Gata4 is activated by lamin A/C loss and Gata4 silencing or haploinsufficiency rescues the aberrant cardiovascular cell fate choices induced by lamin A/C deficiency. We uncover divergent functions of lamin A/C in naïve pluripotent stem cells and cardiomyocytes, which have distinct contributions to the transcriptional alterations of patients with LMNA-associated cardiomyopathy. We conclude that disruption of lamin A/C-dependent chromatin architecture in ESCs is a primary event in LMNA loss-of-function cardiomyopathy.

Functional antagonism between CagA and DLC1 in gastric cancer.

Helicobacter (H.) pylori-induced gastritis is a risk factor for gastric cancer (GC). Deleted-in-liver-cancer-1 (DLC1/ARHGAP7) inhibits RHOA, a downstream mediator of virulence factor cytotoxin-A (CagA) signalling and driver of consensus-molecular-subtype-2 diffuse GC. DLC1 located to enterochromaffin-like and MIST1+ stem/chief cells in the stomach. DLC1+ cells were reduced in H. pylori gastritis and GC, and in mice infected with H. pylori. DLC1 positivity inversely correlated with tumour progression in patients. GC cells retained an N-terminal truncation variant DLC1v4 in contrast to full-length DLC1v1 in non-neoplastic tissues. H. pylori and CagA downregulated DLC1v1/4 promoter activities. DLC1v1/4 inhibited cell migration and counteracted CagA-driven stress phenotypes enforcing focal adhesion. CagA and DLC1 interacted via their N- and C-terminal domains, proposing that DLC1 protects against H. pylori by neutralising CagA. H. pylori-induced DLC1 loss is an early molecular event, which makes it a potential marker or target for subtype-aware cancer prevention or therapy.

Chronic isoprenaline/phenylephrine vs. exclusive isoprenaline stimulation in mice: critical contribution of alpha1-adrenoceptors to early cardiac stress responses.

Hyperactivity of the sympathetic nervous system is a major driver of cardiac remodeling, exerting its effects through both α-, and ß-adrenoceptors (α-, ß-ARs). As the relative contribution of subtype α1-AR to cardiac stress responses remains poorly investigated, we subjected mice to either subcutaneous perfusion with the ß-AR agonist isoprenaline (ISO, 30 mg/kg × day) or to a combination of ISO and the stable α1-AR agonist phenylephrine (ISO/PE, 30 mg/kg × day each). Telemetry analysis revealed similar hemodynamic responses under both ISO and ISO/PE treatment i.e., permanently increased heart rates and only transient decreases in mean blood pressure during the first 24 h. Echocardiography and single cell analysis after 1 week of exposure showed that ISO/PE-, but not ISO-treated animals established α1-AR-mediated inotropic responsiveness to acute adrenergic stimulation. Morphologically, additional PE perfusion limited concentric cardiomyocyte growth and enhanced cardiac collagen deposition during 7 days of treatment. Time-course analysis demonstrated a diverging development in transcriptional patterns at day 4 of treatment i.e., increased expression of selected marker genes Xirp2, Nppa, Tgfb1, Col1a1, Postn under chronic ISO/PE treatment which was either less pronounced or absent in the ISO group. Transcriptome analyses at day 4 via RNA sequencing demonstrated that additional PE treatment caused a marked upregulation of genes allocated to extracellular matrix and fiber organization along with a more pronounced downregulation of genes involved in metabolic processes, muscle adaptation and cardiac electrophysiology. Consistently, transcriptome changes under ISO/PE challenge more effectively recapitulated early transcriptional alterations in pressure overload-induced experimental heart failure and in human hypertrophic cardiomyopathy.

RGS3L allows for an M2 muscarinic receptor-mediated RhoA-dependent inotropy in cardiomyocytes.

The role and outcome of the muscarinic M2 acetylcholine receptor (M2R) signaling in healthy and diseased cardiomyocytes is still a matter of debate. Here, we report that the long isoform of the regulator of G protein signaling 3 (RGS3L) functions as a switch in the muscarinic signaling, most likely of the M2R, in primary cardiomyocytes. High levels of RGS3L, as found in heart failure, redirect the Gi-mediated Rac1 activation into a Gi-mediated RhoA/ROCK activation. Functionally, this switch resulted in a reduced production of reactive oxygen species (- 50%) in cardiomyocytes and an inotropic response (+ 18%) in transduced engineered heart tissues. Importantly, we could show that an adeno-associated virus 9-mediated overexpression of RGS3L in rats in vivo, increased the contractility of ventricular strips by maximally about twofold. Mechanistically, we demonstrate that this switch is mediated by a complex formation of RGS3L with the GTPase-activating protein p190RhoGAP, which balances the activity of RhoA and Rac1 by altering its substrate preference in cardiomyocytes. Enhancement of this complex formation could open new possibilities in the regulation of the contractility of the diseased heart.

The orphan receptor GPRC5B activates pro-inflammatory signaling in the vascular wall via Fyn and NFκB.

BACKGROUND AND AIMS: Atherosclerosis is driven by an inflammatory process of the vascular wall. The novel orphan G-protein coupled receptor 5B of family C (GPRC5B) is involved in drosophila sugar and lipid metabolism as well as mice adipose tissue inflammation. Here, we investigated the role of GPRC5B in the pro-atherogenic mechanisms of hyperglycemia and vascular inflammation. METHODS: Immortalized and primary endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) were used for stimulation with high glucose or different cytokines. Adenoviral- or plasmid-driven GPRC5B overexpression and siRNA-mediated knockdown were performed in these cells to analyze functional and mechanistic pathways of GPRC5B. RESULTS: In ECs and VSMCs, stimulation with high glucose, TNFα or LPS induced a significant upregulation of endogenous GPRC5B mRNA and protein levels. GPRC5B overexpression and knockdown increased and attenuated, respectively, the expression of the pro-inflammatory cytokines TNFα, IL-1ß, IL-6 as well as the pro-atherogenic vascular adhesion molecules ICAM-1 and VCAM-1. Furthermore, the expression and activity of the metalloproteinase MMP-9, a component of atherosclerotic plaque stabilization, were significantly enhanced by GPRC5B overexpression. Mechanistically, GPRC5B increased the phosphorylation of ERK1/2 and activated NFκB through a direct interaction with the tyrosine kinase Fyn. CONCLUSIONS: Our findings demonstrate that GPRC5B is upregulated in response to high glucose and pro-inflammatory signaling. GPRC5B functionally modulates the inflammatory activity in cells of the vascular wall, suggesting a pro-atherogenic GPRC5B-dependent positive feedback loop via Fyn and NFκB. Thus, GPRC5B warrants further attention as a novel pharmacological target for the treatment of vascular inflammation and possibly atherogenesis.

Glucosamine protects against neuronal but not vascular damage in experimental diabetic retinopathy.

OBJECTIVE: Glucosamine, an intermetabolite of the hexosamine biosynthesis pathway (HBP), is a widely used nutritional supplement in osteoarthritis patients, a subset of whom also suffer from diabetes. HBP is activated in diabetic retinopathy (DR). The aim of this study is to investigate the yet unclear effects of glucosamine on DR. METHODS: In this study, we tested the effect of glucosamine on vascular and neuronal pathology in a mouse model of streptozotocin-induced DR in vivo and on cultured endothelial and Müller cells to elucidate the underlying mechanisms of action in vitro. RESULTS: Glucosamine did not alter the blood glucose or HbA1c levels in the animals, but induced body weight gain in the non-diabetic animals. Interestingly, the impaired neuronal function in diabetic animals could be prevented by glucosamine treatment. Correspondingly, the activation of Müller cells was prevented in the retina as well as in cell culture. Conversely, glucosamine administration in the normal retina damaged the retinal vasculature by increasing pericyte loss and acellular capillary formation, likely by interfering with endothelial survival signals as seen in vitro in cultured endothelial cells. Nevertheless, under diabetic conditions, no further increase in the detrimental effects were observed. CONCLUSIONS: In conclusion, the effects of glucosamine supplementation in the retina appear to be a double-edged sword: neuronal protection in the diabetic retina and vascular damage in the normal retina. Thus, glucosamine supplementation in osteoarthritis patients with or without diabetes should be taken with care.

TRPV1 activation and internalization is part of the LPS-induced inflammation in human iPSC-derived cardiomyocytes.

The non-selective cation channel transient receptor potential vanilloid 1 (TRPV1) is expressed throughout the cardiovascular system. Recent evidence shows a role for TRPV1 in inflammatory processes. The role of TRPV1 for myocardial inflammation has not been established yet. Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (hiPSC-CM) from 4 healthy donors were incubated with lipopolysaccharides (LPS, 6 h), TRPV1 agonist capsaicin (CAP, 20 min) or the antagonist capsazepine (CPZ, 20 min). TRPV1 expression was studied by PCR and western blotting. TRPV1 internalization was analyzed by immunofluorescence. Interleukin-6 (IL-6) secretion and phosphorylation of JNK, p38 and ERK were determined by ELISA. TRPV1-associated ion channel current was measured by patch clamp. TRPV1-mRNA and -protein were expressed in hiPSC-CM. TRPV1 was localized in the plasma membrane. LPS significantly increased secretion of IL-6 by 2.3-fold, which was prevented by pre-incubation with CPZ. LPS induced TRPV1 internalization. Phosphorylation levels of ERK, p38 or JNK were not altered by TRPV1 stimulation or inhibition. LPS and IL-6 significantly lowered TRPV1-mediated ion channel current. TRPV1 mediates the LPS-induced inflammation in cardiomyocytes, associated with changes of cellular electrophysiology. LPS-induced inflammation results in TRPV1 internalization. Further studies have to examine the underlying pathways and the clinical relevance of these findings.

RhoGEF17-An Essential Regulator of Endothelial Cell Death and Growth.

The Rho guanine nucleotide exchange factor RhoGEF17 was described to reside in adherens junctions (AJ) in endothelial cells (EC) and to play a critical role in the regulation of cell adhesion and barrier function. The purpose of this study was to analyze signal cascades and processes occurring subsequent to AJ disruption induced by RhoGEF17 knockdown. Primary human and immortalized rat EC were used to demonstrate that an adenoviral-mediated knockdown of RhoGEF17 resulted in cell rounding and an impairment in spheroid formation due to an enhanced proteasomal degradation of AJ components. In contrast, ß-catenin degradation was impaired, which resulted in an induction of the ß-catenin-target genes cyclin D1 and survivin. RhoGEF17 depletion additionally inhibited cell adhesion and sheet migration. The RhoGEF17 knockdown prevented the cells with impeded cell-cell and cell-matrix contacts from apoptosis, which was in line with a reduction in pro-caspase 3 expression and an increase in Akt phosphorylation. Nevertheless, the cells were not able to proliferate as a cell cycle block occurred. In summary, we demonstrate that a loss of RhoGEF17 disturbs cell-cell and cell-substrate interaction in EC. Moreover, it prevents the EC from cell death and blocks cell proliferation. Non-canonical ß-catenin signaling and Akt activation could be identified as a potential mechanism.

Involvement of NDPK-B in Glucose Metabolism-Mediated Endothelial Damage via Activation of the Hexosamine Biosynthesis Pathway and Suppression of O-GlcNAcase Activity.

Our previous studies identified that retinal endothelial damage caused by hyperglycemia or nucleoside diphosphate kinase-B (NDPK-B) deficiency is linked to elevation of angiopoietin-2 (Ang-2) and the activation of the hexosamine biosynthesis pathway (HBP). Herein, we investigated how NDPK-B is involved in the HBP in endothelial cells (ECs). The activities of NDPK-B and O-GlcNAcase (OGA) were measured by in vitro assays. Nucleotide metabolism and O-GlcNAcylated proteins were assessed by UPLC-PDA (Ultra-performance liquid chromatography with Photodiode array detection) and immunoblot, respectively. Re-expression of NDPK-B was achieved with recombinant adenoviruses. Our results show that NDPK-B depletion in ECs elevated UDP-GlcNAc levels and reduced NDPK activity, similar to high glucose (HG) treatment. Moreover, the expression and phosphorylation of glutamine:fructose-6-phosphate amidotransferase (GFAT) were induced, whereas OGA activity was suppressed. Furthermore, overall protein O-GlcNAcylation, along with O-GlcNAcylated Ang-2, was increased in NDPK-B depleted ECs. Pharmacological elevation of protein O-GlcNAcylation using Thiamet G (TMG) or OGA siRNA increased Ang-2 levels. However, the nucleoside triphosphate to diphosphate (NTP/NDP) transphosphorylase and histidine kinase activity of NDPK-B were dispensable for protein O-GlcNAcylation. NDPK-B deficiency hence results in the activation of HBP and the suppression of OGA activity, leading to increased protein O-GlcNAcylation and further upregulation of Ang-2. The data indicate a critical role of NDPK-B in endothelial damage via the modulation of the HBP.

Dissecting Gq/11-Mediated Plasma Membrane Translocation of Sphingosine Kinase-1.

Diverse extracellular signals induce plasma membrane translocation of sphingosine kinase-1 (SphK1), thereby enabling inside-out signaling of sphingosine-1-phosphate. We have shown before that Gq-coupled receptors and constitutively active Gαq/11 specifically induced a rapid and long-lasting SphK1 translocation, independently of canonical Gq/phospholipase C (PLC) signaling. Here, we further characterized Gq/11 regulation of SphK1. SphK1 translocation by the M3 receptor in HEK-293 cells was delayed by expression of catalytically inactive G-protein-coupled receptor kinase-2, p63Rho guanine nucleotide exchange factor (p63RhoGEF), and catalytically inactive PLCß3, but accelerated by wild-type PLCß3 and the PLCδ PH domain. Both wild-type SphK1 and catalytically inactive SphK1-G82D reduced M3 receptor-stimulated inositol phosphate production, suggesting competition at Gαq. Embryonic fibroblasts from Gαq/11 double-deficient mice were used to show that amino acids W263 and T257 of Gαq, which interact directly with PLCß3 and p63RhoGEF, were important for bradykinin B2 receptor-induced SphK1 translocation. Finally, an AIXXPL motif was identified in vertebrate SphK1 (positions 100-105 in human SphK1a), which resembles the Gαq binding motif, ALXXPI, in PLCß and p63RhoGEF. After M3 receptor stimulation, SphK1-A100E-I101E and SphK1-P104A-L105A translocated in only 25% and 56% of cells, respectively, and translocation efficiency was significantly reduced. The data suggest that both the AIXXPL motif and currently unknown consequences of PLCß/PLCδ(PH) expression are important for regulation of SphK1 by Gq/11.

A phenomenological approach to assessing the effectiveness of COVID-19 related nonpharmaceutical interventions in Germany.

Nonpharmaceutical interventions against the spread of SARS-CoV-2 in Germany included the cancellation of mass events (from March 8), closures of schools and child day care facilities (from March 16) as well as a "lockdown" (from March 23). This study attempts to assess the effectiveness of these interventions in terms of revealing their impact on infections over time. Dates of infections were estimated from official German case data by incorporating the incubation period and an empirical reporting delay. Exponential growth models for infections and reproduction numbers were estimated and investigated with respect to change points in the time series. A significant decline of daily and cumulative infections as well as reproduction numbers is found at March 8, March 10 and March 3, respectively. Further declines and stabilizations are found in the end of March. There is also a change point in new infections at April 19, but daily infections still show a negative growth. From March 19, the reproduction numbers fluctuate on a level below one. The decline of infections in early March 2020 can be attributed to relatively small interventions and voluntary behavioural changes. Additional effects of later interventions cannot be detected clearly. Liberalizations of measures from April 20 did not induce a re-increase of infections. Thus, the effectiveness of most German interventions remains questionable. Moreover, assessing of interventions is impeded by the estimation of true infection dates and the influence of test volume.