European Integration Perspectives in Times of Global Crises - 13th Network Europe Conference, Athens, 19 – 22 June 2022

With the White Paper on the Future of Europe, the European Commission had launched a debate on fundamental reforms of the Union structures in 2017. A total of five reform scenarios ranged from a reduction and focusing of the Union’s competences to increased integration in the sense of a United States of Europe. However, the White Paper did not have any consequences; none of the reform scenarios presented was implemented. However, current global challenges in the areas of health, climate change and energy resources as well as the shift in the global balance of power and related security issues demonstrate the increasing importance of a strong and united Europe. The idea of an “ever closer union”, as laid down in the preamble of the 1992 EU Treaty, could experience a renaissance. Against this background, the 13th Network Europe Conference addressed the importance of the integration project in times of global crises and the challenges in various policy areas, as well as the EU’s relations with its eastern and southern neighbors and its role vis-à-vis global actors such as China and Russia. This publication contains the conference contributions

Gene expression of PMP22 is an independent prognostic factor for disease-free and overall survival in breast cancer patients

<p>Abstract</p> <p>Background</p> <p>Gene expression of peripheral myelin protein 22 (<it>PMP22</it>) and the epithelial membrane proteins (<it>EMPs</it>) was found to be differentially expressed in invasive and non-invasive breast cell lines in a previous study. We want to evaluate the prognostic impact of the expression of these genes on breast cancer.</p> <p>Methods</p> <p>In a retrospective multicenter study, gene expression of <it>PMP22 </it>and the <it>EMPs </it>was measured in 249 primary breast tumors by real-time PCR. Results were statistically analyzed together with clinical data.</p> <p>Results</p> <p>In univariable Cox regression analyses PMP22 and the EMPs were not associated with disease-free survival or tumor-related mortality. However, multivariable Cox regression revealed that patients with higher than median <it>PMP22 </it>gene expression have a 3.47 times higher risk to die of cancer compared to patients with equal values on clinical covariables but lower <it>PMP22 </it>expression. They also have a 1.77 times higher risk to relapse than those with lower <it>PMP22 </it>expression. The proportion of explained variation in overall survival due to <it>PMP22 </it>gene expression was 6.5% and thus PMP22 contributes equally to prognosis of overall survival as nodal status and estrogen receptor status. Cross validation demonstrates that 5-years survival rates can be refined by incorporating <it>PMP22 </it>into the prediction model.</p> <p>Conclusions</p> <p><it>PMP22 </it>gene expression is a novel independent prognostic factor for disease-free survival and overall survival for breast cancer patients. Including it into a model with established prognostic factors will increase the accuracy of prognosis.</p

Analysis of congenital disorder of glycosylation-Id in a yeast model system shows diverse site-specific under-glycosylation of glycoproteins

Asparagine-linked glycosylation is a common post translational modification of proteins in eukaryotes. Mutations in the human ALG3 gene cause changed levels and altered glycan structures on mature glycoproteins and are the cause of a severe congenital disorder of glycosylation (CDG-Id). Diverse glycoproteins are also under-glycosylated in Saccharomyces cerevisae alg3 mutants. Here we analyzed site-specific glycosylation occupancy in this yeast model system using peptide-N-glycosidase F to label glycosylation sites with an asparagine-aspartate conversion that creates a new endoproteinase AspN cleavage site, followed by proteolytic digestion, and detection of peptides and glycopeptides by LC-ESI-MS/MS. We used this analytical method to identify and measure site specific glycosylation occupancy in alg3 mutant and wild type yeast strains. We found decreased site specific N-glycosylation occupancy in the alg3 knockout strain preferentially at Asn-Xaa-Ser sequences located in secondary structural elements, features previously associated with poor glycosylation efficiency. Furthermore, we identified 26 previously experimentally unverified glycosylation sites. Our results provide insights into the underlying mechanisms of disease in CDG-Id, and our methodology will be useful in site specific glycosylation analysis in many model systems and clinical applications

MDL28170, a Calpain Inhibitor, Affects Trypanosoma cruzi Metacyclogenesis, Ultrastructure and Attachment to Rhodnius prolixus Midgut

BACKGROUND: Trypanosoma cruzi is the etiological agent of Chagas' disease. During the parasite life cycle, many molecules are involved in the differentiation process and infectivity. Peptidases are relevant for crucial steps of T. cruzi life cycle; as such, it is conceivable that they may participate in the metacyclogenesis and interaction with the invertebrate host. METHODOLOGY/PRINCIPAL FINDINGS: In this paper, we have investigated the effect of the calpain inhibitor MDL28170 on the attachment of T. cruzi epimastigotes to the luminal midgut surface of Rhodnius prolixus, as well as on the metacyclogenesis process and ultrastructure. MDL28170 treatment was capable of significantly reducing the number of bound epimastigotes to the luminal surface midgut of the insect. Once the cross-reactivity of the anti-Dm-calpain was assessed, it was possible to block calpain molecules by the antibody, leading to a significant reduction in the capacity of adhesion to the insect guts by T. cruzi. However, the antibodies were unable to interfere in metacyclogenesis, which was impaired by the calpain inhibitor presenting a significant reduction in the number of metacyclic trypomastigotes. The calpain inhibitor also promoted a direct effect against bloodstream trypomastigotes. Ultrastructural analysis of epimastigotes treated with the calpain inhibitor revealed disorganization in the reservosomes, Golgi and plasma membrane disruption. CONCLUSIONS/SIGNIFICANCE: The presence of calpain and calpain-like molecules in a wide range of organisms suggests that these proteins could be necessary for basic cellular functions. Herein, we demonstrated the effects of MDL28170 in crucial steps of the T. cruzi life cycle, such as attachment to the insect midgut and metacyclogenesis, as well as in parasite viability and morphology. Together with our previous findings, these results help to shed some light on the functions of T. cruzi calpains. Considering the potential roles of these molecules on the interaction with both invertebrate and vertebrate hosts, it is interesting to improve knowledge on these molecules in T. cruzi

8p22 MTUS1 Gene Product ATIP3 Is a Novel Anti-Mitotic Protein Underexpressed in Invasive Breast Carcinoma of Poor Prognosis

BACKGROUND: Breast cancer is a heterogeneous disease that is not totally eradicated by current therapies. The classification of breast tumors into distinct molecular subtypes by gene profiling and immunodetection of surrogate markers has proven useful for tumor prognosis and prediction of effective targeted treatments. The challenge now is to identify molecular biomarkers that may be of functional relevance for personalized therapy of breast tumors with poor outcome that do not respond to available treatments. The Mitochondrial Tumor Suppressor (MTUS1) gene is an interesting candidate whose expression is reduced in colon, pancreas, ovary and oral cancers. The present study investigates the expression and functional effects of MTUS1 gene products in breast cancer. METHODS AND FINDINGS: By means of gene array analysis, real-time RT-PCR and immunohistochemistry, we show here that MTUS1/ATIP3 is significantly down-regulated in a series of 151 infiltrating breast cancer carcinomas as compared to normal breast tissue. Low levels of ATIP3 correlate with high grade of the tumor and the occurrence of distant metastasis. ATIP3 levels are also significantly reduced in triple negative (ER- PR- HER2-) breast carcinomas, a subgroup of highly proliferative tumors with poor outcome and no available targeted therapy. Functional studies indicate that silencing ATIP3 expression by siRNA increases breast cancer cell proliferation. Conversely, restoring endogenous levels of ATIP3 expression leads to reduced cancer cell proliferation, clonogenicity, anchorage-independent growth, and reduces the incidence and size of xenografts grown in vivo. We provide evidence that ATIP3 associates with the microtubule cytoskeleton and localizes at the centrosomes, mitotic spindle and intercellular bridge during cell division. Accordingly, live cell imaging indicates that ATIP3 expression alters the progression of cell division by promoting prolonged metaphase, thereby leading to a reduced number of cells ungergoing active mitosis. CONCLUSIONS: Our results identify for the first time ATIP3 as a novel microtubule-associated protein whose expression is significantly reduced in highly proliferative breast carcinomas of poor clinical outcome. ATIP3 re-expression limits tumor cell proliferation in vitro and in vivo, suggesting that this protein may represent a novel useful biomarker and an interesting candidate for future targeted therapies of aggressive breast cancer

Exploiting residue-level and profile-level interface propensities for usage in binding sites prediction of proteins

<p>Abstract</p> <p>Background</p> <p>Recognition of binding sites in proteins is a direct computational approach to the characterization of proteins in terms of biological and biochemical function. Residue preferences have been widely used in many studies but the results are often not satisfactory. Although different amino acid compositions among the interaction sites of different complexes have been observed, such differences have not been integrated into the prediction process. Furthermore, the evolution information has not been exploited to achieve a more powerful propensity.</p> <p>Result</p> <p>In this study, the residue interface propensities of four kinds of complexes (homo-permanent complexes, homo-transient complexes, hetero-permanent complexes and hetero-transient complexes) are investigated. These propensities, combined with sequence profiles and accessible surface areas, are inputted to the support vector machine for the prediction of protein binding sites. Such propensities are further improved by taking evolutional information into consideration, which results in a class of novel propensities at the profile level, i.e. the binary profiles interface propensities. Experiment is performed on the 1139 non-redundant protein chains. Although different residue interface propensities among different complexes are observed, the improvement of the classifier with residue interface propensities can be negligible in comparison with that without propensities. The binary profile interface propensities can significantly improve the performance of binding sites prediction by about ten percent in term of both precision and recall.</p> <p>Conclusion</p> <p>Although there are minor differences among the four kinds of complexes, the residue interface propensities cannot provide efficient discrimination for the complicated interfaces of proteins. The binary profile interface propensities can significantly improve the performance of binding sites prediction of protein, which indicates that the propensities at the profile level are more accurate than those at the residue level.</p

CXCL12 expression by healthy and malignant ovarian epithelial cells

<p>Abstract</p> <p>Background</p> <p>CXCL12 has been widely reported to play a biologically relevant role in tumor growth and spread. In epithelial ovarian cancer (EOC), CXCL12 enhances tumor angiogenesis and contributes to the immunosuppressive network. However, its prognostic significance remains unclear. We thus compared CXCL12 status in healthy and malignant ovaries, to assess its prognostic value.</p> <p>Methods</p> <p>Immunohistochemistry was used to analyze CXCL12 expression in the reproductive tracts, including the ovaries and fallopian tubes, of healthy women, in benign and borderline epithelial tumors, and in a series of 183 tumor specimens from patients with advanced primary EOC enrolled in a multicenter prospective clinical trial of paclitaxel/carboplatin/gemcitabine-based chemotherapy (GINECO study). Univariate COX model analysis was performed to assess the prognostic value of clinical and biological variables. Kaplan-Meier methods were used to generate progression-free and overall survival curves.</p> <p>Results</p> <p>Epithelial cells from the surface of the ovary and the fallopian tubes stained positive for CXCL12, whereas the follicles within the ovary did not. Epithelial cells in benign, borderline and malignant tumors also expressed CXCL12. In EOC specimens, CXCL12 immunoreactivity was observed mostly in epithelial tumor cells. The intensity of the signal obtained ranged from strong in 86 cases (47%) to absent in 18 cases (<10%). This uneven distribution of CXCL12 did not reflect the morphological heterogeneity of EOC. CXCL12 expression levels were not correlated with any of the clinical parameters currently used to determine EOC prognosis or with HER2 status. They also had no impact on progression-free or overall survival.</p> <p>Conclusion</p> <p>Our findings highlight the previously unappreciated constitutive expression of CXCL12 on healthy epithelia of the ovary surface and fallopian tubes, indicating that EOC may originate from either of these epithelia. We reveal that CXCL12 production by malignant epithelial cells precedes tumorigenesis and we confirm in a large cohort of patients with advanced EOC that CXCL12 expression level in EOC is not a valuable prognostic factor in itself.</p> <p>Trial Registration</p> <p>ClinicalTrials.gov: <a href="http://www.clinicaltrials.gov/ct2/show/NCT00052468">NCT00052468</a></p

The Selaginella Genome Identifies Genetic Changes Associated with the Evolution of Vascular Plants

Vascular plants appeared ~410 million years ago then diverged into several lineages of which only two survive: the euphyllophytes (ferns and seed plants) and the lycophytes (1). We report here the genome sequence of the lycophyte Selaginella moellendorffii (Selaginella), the first non-seed vascular plant genome reported. By comparing gene content in evolutionary diverse taxa, we found that the transition from a gametophyte- to sporophyte- dominated life cycle required far fewer new genes than the transition from a non-seed vascular to a flowering plant, while secondary metabolic genes expanded extensively and in parallel in the lycophyte and angiosperm lineages. Selaginella differs in post- transcriptional gene regulation, including small RNA regulation of repetitive elements, an absence of the tasiRNA pathway and extensive RNA editing of organellar genes

STAT3 Regulates Monocyte TNF-Alpha Production in Systemic Inflammation Caused by Cardiac Surgery with Cardiopulmonary Bypass

BACKGROUND: Cardiopulmonary bypass (CPB) surgery initiates a controlled systemic inflammatory response characterized by a cytokine storm, monocytosis and transient monocyte activation. However, the responsiveness of monocytes to Toll-like receptor (TLR)-mediated activation decreases throughout the postoperative course. The purpose of this study was to identify the major signaling pathway involved in plasma-mediated inhibition of LPS-induced tumor necrosis factor (TNF)-α production by monocytes. METHODOLOGY/PRINCIPAL FINDINGS: Pediatric patients that underwent CPB-assisted surgical correction of simple congenital heart defects were enrolled (n = 38). Peripheral blood mononuclear cells (PBMC) and plasma samples were isolated at consecutive time points. Patient plasma samples were added back to monocytes obtained pre-operatively for ex vivo LPS stimulations and TNF-α and IL-6 production was measured by flow cytometry. LPS-induced p38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB activation by patient plasma was assessed by Western blotting. A cell-permeable peptide inhibitor was used to block STAT3 signaling. We found that plasma samples obtained 4 h after surgery, regardless of pre-operative dexamethasone treatment, potently inhibited LPS-induced TNF-α but not IL-6 synthesis by monocytes. This was not associated with attenuation of p38 MAPK activation or IκB-α degradation. However, abrogation of the IL-10/STAT3 pathway restored LPS-induced TNF-α production in the presence of suppressive patient plasma. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that STAT3 signaling plays a crucial role in the downregulation of TNF-α synthesis by human monocytes in the course of systemic inflammation in vivo. Thus, STAT3 might be a potential molecular target for pharmacological intervention in clinical syndromes characterized by systemic inflammation