The role of the adaptor protein lamellipodin in glioblastoma cell invasion and radiosensitivity

Background: Highly infiltrative growth and resistance to radiation as well as chemotherapy contribute to a poor prognosis of glioblastoma. To improve the survival of patients with glioblastoma, further research to uncover the complex signaling network is essential. Due to the central role of the signaling adaptor lamellipodin in nervous system development and cell migration, a function of lamellipodin in glioblastoma is conceivable. However, the specific function of lamellipodin in the invasion and radioresistance of glioblastoma is so far entirely unknown. Therefore, the present work investigates the invasion and radioresistance of glioblastoma cells and the underlying signaling mechanism under the influence of lamellipodin. Material and Methods: Expression of lamellipodin was evaluated in human astrocytes and nine glioblastoma cell lines by Western blot analysis. Localization of lamellipodin in glioblastoma cells was analyzed by applying immunofluorescence staining. The effects of lamellipodin silencing by siRNA on the glioblastoma characteristics invasion, survival, and residual DNA double strand breaks (DSB) upon X-ray irradiation, proliferation, apoptosis, and senescence were investigated. Alterations in molecular signaling were analyzed by phosphoproteome analysis upon control and lamellipodin depletion combined with/-out X-ray irradiation in A172 cells. Colony formation assays were performed after single and double knockdown of lamellipodin and the affected proteins to connect latter findings to clonogenic survival. Direct lamellipodin binding partners were explored using mass spectrometry analysis. Validation of protein-protein interaction was determined by immunoprecipitation and proximity ligation assay. Clonogenic survival assay was performed after triple knockdown of lamellipodin, previous proteins identified by phosphoproteome, and the direct interaction partner of lamellipodin. Hierarchical analysis was performed by analyzing the expression and phosphorylation of the determined proteins by immunoblot. Results: Lamellipodin was shown to be expressed and phosphorylated in varying amounts in the glioblastoma cell culture panel, with a preferential localization in membrane protrusions and the cytoplasm. The siRNA-specific depletion of lamellipodin tremendously decreased glioblastoma invasion and proliferation while exerting no impact on apoptosis or senescence. Moreover, seven of the nine studied glioblastoma cell lines were radiosensitized by lamellipodin silencing without affecting the number of residual DNA double strand breaks, while its overexpression improved radiation survival. Mechanistically, the loss of lamellipodin impaired the phosphorylation of nine proteins (EIF2A, EGFR, FOS, MKK6, NFKBIA, PRKAA2, RSK2, SRC, and TAK1), which are mostly implicated in the EGFR-MAPK signaling. The combinational silencing of lamellipodin and the relevant proteins achieved overall radiosensitization in A172 and U343MG cells. Furthermore, mass spectrometric analysis of lamellipodin immunoprecipitates demonstrated that the lamellipodin interactome alters in response to X ray irradiation conditions. RICTOR was confirmed as a direct linker of lamellipodin to the EGFR-MAPK signaling by immunoprecipitation and proximity ligation assay. In addition, triple depletion of lamellipodin, RICTOR, and EGFR resulted in similar degrees of radiosensitization as reported for lamellipodin knockdown, highlighting their superimposable role in glioblastoma radiation response. In line, lamellipodin silencing increased EGFR expression and phosphorylation, while lamellipodin phosphorylation was decreased upon EGFR deficiency. Conclusion: The results uncover the crucial function of lamellipodin for invasion, proliferation, and radiosensitivity of glioblastoma cells. Based on molecular analyses, lamellipodin was discovered as a determinant of EGFR signaling by interacting with RICTOR. Based on these data, the complexity of the signaling networks conducting radiation survival is broadened by adding the adaptor protein lamellipodin.Hintergrund: Ein stark infiltrierendes Wachstum und Resistenzen gegenüber Bestrahlung und Chemotherapie tragen zu einer schlechten Prognose des Glioblastoms bei. Um die Therapie des Glioblastoms zu verbessern, ist die weitere Erforschung des komplexen Signalnetzwerkes notwendig. Aufgrund der essentiellen Rolle des Signaladaptors Lamellipodin für die Entwicklung des Nervensystems und der Migration von Zellen ist eine Funktion von Lamellipodin im Glioblastom vorstellbar. Die spezifische Funktion von Lamellipodin in der Invasion und Strahlenresistenz des Glioblastoms ist allerdings bisher völlig unbekannt. Die vorliegende Arbeit untersucht daher die Invasion und Strahlenresistenz von Glioblastomzellen sowie den zugrundliegenden molekularen Mechanismus im Hinblick auf Lamellipodin. Material und Methoden: Die Expression von Lamellipodin wurde in humanen Astrozyten und neun Glioblastom-Zelllinien mittels Western Blot Analyse untersucht. Die Lokalisierung von Lamellipodin in Glioblastomzellen wurde mit Immunfluoreszenzfärbung analysiert. Die Auswirkungen eines Lamellipodin-Knockdown mittels siRNA auf die Glioblastom-Eigenschaften Invasion, Überleben und residuale DNA-Doppelstrangbrüche (DSB) bei Röntgenbestrahlung, Proliferation, Apoptose und Seneszenz wurden untersucht. Veränderungen in molekularen Signalwegen wurden durch Phosphoproteomanalyse nach Kontroll- und Lamellipodin-Knockdown in Kombination mit und ohne Röntgenbestrahlung in A172 Zellen analysiert. Koloniebildungsassays wurden nach einfachem und doppeltem Knockdown von Lamellipodin und den betroffenen Proteinen durchgeführt, um letztere Ergebnisse mit dem klonogenen Überleben in Verbindung zu bringen. Direkte Lamellipodin-Bindungspartner wurden mittels massenspektrometrischer Analyse untersucht. Die Validierung der Protein-Protein-Interaktion wurde durch Immunpräzipitation und Proximity Ligation Assay bestimmt. Ein klonogenes Überlebensassay wurde nach dreifachem Knockdown von Lamellipodin, durch das Phosphoproteom identifizierten Proteinen und dem direkten Interaktionspartner von Lamellipodin durchgeführt. Die hierarchische Analyse erfolgte durch Analyse der Expression und Phosphorylierung der ermittelten Proteine mittels Immunblot. Ergebnisse: Die Expression und Phosphorylierung von Lamellipodin war unterschiedlich in den Glioblastomzelllinien. Die siRNA-vermittelte Deletion von Lamellipodin verringerte die Invasion und Proliferation von Glioblastomzellen enorm, während die Reduktion von Lamellipodin keine Auswirkungen auf Apoptose oder Seneszenz hatte. Darüber hinaus wurden sieben der neun untersuchten Glioblastomzelllinien durch das Ausschalten von Lamellipodin radiosensibilisiert, ohne dass sich dies auf die Anzahl der residualen DNA-Doppelstrangbrüche auswirkte, während die Überexpression von Lamellipodin das Überleben nach Bestrahlung verbesserte. Mechanistisch beeinträchtigte der Verlust von Lamellipodin die Phosphorylierung von neun Proteinen (EIF2A, EGFR, FOS, MKK6, NFKBIA, PRKAA2, RSK2, SRC und TAK1), die hauptsächlich an der EGFR-MAPK-Signalübertragung beteiligt sind. Die kombinierte Ausschaltung von Lamellipodin und den relevanten Proteinen führte zu einer allgemeinen Radiosensibilisierung in den Zelllinien A172 und U343MG. Darüber hinaus zeigte die massenspektrometrische Analyse von Lamellipodin-Immunpräzipitaten, dass sich das Lamellipodin-Interaktom als Reaktion auf Röntgenbestrahlung verändert. RICTOR wurde durch Immunpräzipitation und Proximity Ligation Assay als direktes Bindeglied von Lamellipodin zum EGFR-MAPK-Signalweg identifiziert. Darüber hinaus führte die dreifache Deletion von Lamellipodin, RICTOR und EGFR zu einem ähnlichen Grad an Radiosensibilisierung wie der Knockdown von Lamellipodin, was ihre gemeinsame Rolle bei der Strahlenreaktion von Glioblastomen unterstreicht. Darüber hinaus erhöhte die Ausschaltung von Lamellipodin die EGFR-Expression und -Phosphorylierung, während die Lamellipodin-Phosphorylierung bei EGFR-Mangel verringert wurde. Schlussfolgerung: Die Ergebnisse decken die entscheidende Funktion von Lamellipodin für die Invasion, Proliferation und Radiosensitivität von Glioblastomzellen auf. Basierend auf molekularen Analysen wurde Lamellipodin als eine Determinante der EGFR-Signalübertragung durch Interaktion mit RICTOR identifiziert. Auf der Grundlage dieser Daten wird die Komplexität des Signalnetzwerks, welches das Überleben durch Strahlung reguliert, durch das Adaptorprotein Lamellipodin erweitert

Synthesis and Characterization of New Azido Esters Derived from Malonic Acid

Three new α,α‐diazidated azido esters derived from malonic acid were synthesized and characterized with a method of diazidation under mild reaction conditions. They are energy‐rich, small, liquid molecules which contain up to six azido moieties. The three synthesized compounds were compared to already known azidated malonic acid esters regarding their density, thermal stability, calculated heat of formation and energetic properties. Further they were tested and classified for their sensitivities according to UN guidelines

Communicative signals during joint attention promote neural processes of infants and caregivers

Communicative signals such as eye contact increase infants’ brain activation to visual stimuli and promote joint attention. Our study assessed whether communicative signals during joint attention enhance infant-caregiver dyads’ neural responses to objects, and their neural synchrony. To track mutual attention processes, we applied rhythmic visual stimulation (RVS), presenting images of objects to 12-month-old infants and their mothers (n = 37 dyads), while we recorded dyads’ brain activity (i.e., steady-state visual evoked potentials, SSVEPs) with electroencephalography (EEG) hyperscanning. Within dyads, mothers either communicatively showed the images to their infant or watched the images without communicative engagement. Communicative cues increased infants’ and mothers’ SSVEPs at central-occipital-parietal, and central electrode sites, respectively. Infants showed significantly more gaze behaviour to images during communicative engagement. Dyadic neural synchrony (SSVEP amplitude envelope correlations, AECs) was not modulated by communicative cues. Taken together, maternal communicative cues in joint attention increase infants’ neural responses to objects, and shape mothers’ own attention processes. We show that communicative cues enhance cortical visual processing, thus play an essential role in social learning. Future studies need to elucidate the effect of communicative cues on neural synchrony during joint attention. Finally, our study introduces RVS to study infant-caregiver neural dynamics in social contexts

a secondary analysis of two pharmacokinetic studies in surgical ICU patients

Background In ICU patients, glomerular filtration is often impaired, but also supraphysiological values are observed (“augmented renal clearance”, >130 mL/min/1.73 m2). Renally eliminated drugs (e.g. many antibiotics) must be adjusted accordingly, which requires a quantitative measure of renal function throughout all the range of clinically encountered values. Estimation from plasma creatinine is standard, but cystatin C may be a valuable alternative. Methods This was a secondary analysis of renal function parameters in 100 ICU patients from two pharmacokinetic studies on vancomycin and betalactam antibiotics. Estimated clearance values obtained by the Cockcroft-Gault formula (eCLCG), the CKD-EPI formula (eCLCKD-EPI) or the cystatin C based Hoek formula (eCLHoek) were compared with the measured endogenous creatinine clearance (CLCR). Agreement of values was assessed by modified Bland-Altman plots and by calculating bias (median error) and precision (median absolute error). Sensitivity and specificity of estimates to identify patients with reduced (130 mL/min/1.73 m2) CLCR were calculated. Results The CLCR was well distributed from highly compromised to supraphysiological values (median 73.2, range 16.8-234 mL/min/1.73 m2), even when plasma creatinine was not elevated (≤0.8 mg/dL for women, ≤1.1 mg/dL for men). Bias and precision were +13.5 mL/min/1.73 m2 and ±18.5 mL/min/1.73 m2 for eCLCG, +7.59 and ±16.8 mL/min/1.73 m2 for eCLCKD-EPI, and -4.15 and ±12.9 mL/min/1.73 m2 for eCLHoek, respectively, with eCLHoek being more precise than the other two (p < 0.05). The central 95% of observed errors fell between -59.8 and +250 mL/min/1.73 m2 for eCLCG, -83.9 and +79.8 mL/min/1.73 m2 for eCLCKD-EPI, and -103 and +27.9 mL/min/1.73 m2 for eCLHoek. Augmented renal clearance was underestimated by eCLCKD-EPI and eCLHoek. Patients with reduced CLCR were identified with good specificity by eCLCG, eCLCKD-EPI and eCLHoek (0.95, 0.97 and 0.91, respectively), but with less sensitivity (0.55, 0.55 and 0.83). For augmented renal clearance, specificity was 0.81, 0.96 and 0.96, but sensitivity only 0.69, 0.25 and 0.38. Conclusions Normal plasma creatinine concentrations can be highly misleading in ICU patients. Agreement of the cystatin C based eCLHoek with CLCR is better than that of the creatinine based eCLCG or eCLCKD-EPI. Detection and quantification of augmented renal clearance by estimates is problematic, and should rather rely on CLCR

Longitudinal changes in anxiety and psychological distress, and associated risk and protective factors during the first three months of the COVID‐19 pandemic in Germany

Background: The COVID-19 pandemic is related to multiple stressors and therefore may be associated with psychological distress. The aim of this study was to longitudinally assess symptoms of (un-)specific anxiety and depression along different stages of the pandemic to generate knowledge about the progress of psychological consequences of the pandemic and to test the role of potential risk and resilience factors that were derived from cross-sectional studies and official recommendations. Methods: The present study uses a longitudinal observational design with four waves of online data collection (from March 27 to June 15, 2020) in a convenience sample of the general population in Germany. A total of N = 2376 participants that completed at least two waves of the survey were included in the analyses. Findings: Specific COVID-19-related anxiety and the average daily amount of preoccupation with the pandemic decreased continuously over the four waves. Unspecific worrying and depressive symptoms decreased on average but not on median level. Self-efficacy, normalization, maintaining social contacts, and knowledge, where to get medical support, were associated with fewer symptoms relative to baseline. Suppression, unhealthy habits, and a longer average daily time of thinking about the pandemic were correlated with a relative increase of symptoms. Interpretation: Our findings provide insight into the longitudinal changes of symptoms of psychological distress along the first three months of the COVID-19 pandemic in Germany. Furthermore, we were able to reaffirm the anticipated protective and risk factors that were extracted from previous studies and recommendations

A functional yeast survival screen of tumor-derived cDNA libraries designed to identify anti-apoptotic mammalian oncogenes

Yeast cells can be killed upon expression of pro-apoptotic mammalian proteins. We have established a functional yeast survival screen that was used to isolate novel human anti-apoptotic genes overexpressed in treatment-resistant tumors. The screening of three different cDNA libraries prepared from metastatic melanoma, glioblastomas and leukemic blasts allowed for the identification of many yeast cell death-repressing cDNAs, including 28% of genes that are already known to inhibit apoptosis, 35% of genes upregulated in at least one tumor entity and 16% of genes described as both anti-apoptotic in function and upregulated in tumors. These results confirm the great potential of this screening tool to identify novel anti-apoptotic and tumor-relevant molecules. Three of the isolated candidate genes were further analyzed regarding their anti-apoptotic function in cell culture and their potential as a therapeutic target for molecular therapy. PAICS, an enzyme required for de novo purine biosynthesis, the long non-coding RNA MALAT1 and the MAST2 kinase are overexpressed in certain tumor entities and capable of suppressing apoptosis in human cells. Using a subcutaneous xenograft mouse model, we also demonstrated that glioblastoma tumor growth requires MAST2 expression. An additional advantage of the yeast survival screen is its universal applicability. By using various inducible pro-apoptotic killer proteins and screening the appropriate cDNA library prepared from normal or pathologic tissue of interest, the survival screen can be used to identify apoptosis inhibitors in many different systems

Inequality and Income Dynamics in Germany

We provide a comprehensive analysis of income inequality and income dynamics for Germany over the last two decades. Combining personal income tax and social security data allows us – for the first time – to offer a complete picture of the distribution of annual earnings in Germany. We find that cross-sectional inequality rose until 2009 for men and women. After the Great Recession inequality continued to rise at a slower rate for men and fell slightly for women due to compression at the lower tail. We further document substantial gender differences in average earnings and inequality over the life-cycle. While for men earnings rise and inequality falls as they grow older, many women reduce working hours when starting a family such that average earnings fall and inequality increases. Men’s earnings changes are on average smaller than women’s but are substantially more affected by the business cycle. During the Great Recession, men’s earnings losses become magnified and gains are attenuated. Apart from recession years, earnings changes are significantly right-skewed reflecting the good overall state of the German labor market and increasing labor supply. In the second part of the paper, we study the distribution of total income including incomes of self-employed, business owners, and landlords. We find that total inequality increased significantly more than earnings inequality. Regarding income dynamics, entrepreneurs’ income changes are more dispersed, less skewed, less leptokurtic and less dependent on average past income than workers’ income changes. Finally, we find that top income earners have become less likely to fall out of the top 1 and 0.1 percent

Triterpene Acids from Frankincense and Semi-Synthetic Derivatives That Inhibit 5-Lipoxygenase and Cathepsin G

Age-related diseases, such as osteoarthritis, Alzheimer’s disease, diabetes, and cardiovascular disease, are often associated with chronic unresolved inflammation. Neutrophils play central roles in this process by releasing tissue-degenerative proteases, such as cathepsin G, as well as pro-inflammatory leukotrienes produced by the 5-lipoxygenase (5-LO) pathway. Boswellic acids (BAs) are pentacyclic triterpene acids contained in the gum resin of the anti-inflammatory remedy frankincense that target cathepsin G and 5-LO in neutrophils, and might thus represent suitable leads for intervention with age-associated diseases that have a chronic inflammatory component. Here, we investigated whether, in addition to BAs, other triterpene acids from frankincense interfere with 5-LO and cathepsin G. We provide a comprehensive analysis of 17 natural tetra- or pentacyclic triterpene acids for suppression of 5-LO product synthesis in human neutrophils. These triterpene acids were also investigated for their direct interference with 5-LO and cathepsin G in cell-free assays. Furthermore, our studies were expanded to 10 semi-synthetic BA derivatives. Our data reveal that besides BAs, several tetra- and pentacyclic triterpene acids are effective or even superior inhibitors of 5-LO product formation in human neutrophils, and in parallel, inhibit cathepsin G. Their beneficial target profile may qualify triterpene acids as anti-inflammatory natural products and pharmacological leads for intervention with diseases related to aging

Hypoxia evokes increased PDI and PDIA6 expression in the infarcted myocardium of ex-germ-free and conventionally raised mice

The prototypic protein disulfide isomerase (PDI), encoded by the P4HB gene, has been described as a survival factor in ischemic cardiomyopathy. However, the role of protein disulfide isomerase associated 6 (PDIA6) under hypoxic conditions in the myocardium remains enigmatic, and it is unknown whether the gut microbiota influences the expression of PDI and PDIA6 under conditions of acute myocardial infarction. Here, we revealed that, in addition to the prototypic PDI, the PDI family member PDIA6, a regulator of the unfolded protein response, is upregulated in the mouse cardiomyocyte cell line HL-1 when cultured under hypoxia. In vivo, in the left anterior descending artery (LAD) ligation mouse model of acute myocardial infarction, similar to PDI, PDIA6 protein expression was enhanced in the infarcted area (LAD ) relative to uninfarcted sham tissue or the neighbouring area at risk (LAD–) of C57BL/6J mice. Interestingly, we found that ex-germ-free (ex-GF) mice subjected to the LAD ligation model for 24 h had a reduced ejection fraction compared with their conventionally raised (CONV-R) SPF controls. Furthermore, the LAD area in the infarcted heart of ex-GF mice showed reduced PDIA6 expression relative to CONV-R controls, suggesting that the presence of a gut microbiota enhanced LAD ligation-triggered PDIA6 expression. Collectively, our results demonstrate that PDIA6 is upregulated in cardiomyocytes as a consequence of hypoxia. In the LAD mouse model, PDIA6 was also increased in the infarcted area under in vivo conditions, but this increase was suppressed in ex-GF mice relative to CONV-R controls

Erythropoietin and the effect of oxygen during proliferation and differentiation of human neural progenitor cells

<p>Abstract</p> <p>Background</p> <p>Hypoxia plays a critical role in various cellular mechanisms, including proliferation and differentiation of neural stem and progenitor cells. In the present study, we explored the impact of lowered oxygen on the differentiation potential of human neural progenitor cells, and the role of erythropoietin in the differentiation process.</p> <p>Results</p> <p>In this study we demonstrate that differentiation of human fetal neural progenitor cells under hypoxic conditions results in an increased neurogenesis. In addition, expansion and proliferation under lowered oxygen conditions also increased neuronal differentiation, although proliferation rates were not altered compared to normoxic conditions. Erythropoietin partially mimicked these hypoxic effects, as shown by an increase of the metabolic activity during differentiation and protection of differentiated cells from apoptosis.</p> <p>Conclusion</p> <p>These results provide evidence that hypoxia promotes the differentiation of human fetal neural progenitor cells, and identifies the involvement of erythropoietin during differentiation as well as different cellular mechanisms underlying the induction of differentiation mediated by lowered oxygen levels.</p