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The Pauli Exclusion Principle (PEP) was introduced by the austrian physicist Wolfgang Pauli in 1925. Since then, several experiments have checked its validity. From 2006 until 2010, the VIP (VIolation of the Pauli Principle) experiment took data at the LNGS underground laboratory to test the PEP. This experiment looked for electronic 2p to 1s transitions in copper, where 2 electrons are in the 1s state before the transition happens. These transitions violate the PEP. The lack of detection of X-ray photons coming from these transitions resulted in a preliminary upper limit for the violation of the PEP of $4.7 \times 10^{-29}$. Currently, the successor experiment VIP2 is under preparation. The main improvements are, on one side, the use of Silicon Drift Detectors (SDDs) as X-ray photon detectors. On the other side an active shielding is implemented, which consists of plastic scintillator bars read by Silicon Photomultipliers (SiPMs). The employment of these detectors will improve the upper limit for the violation of the PEP by around 2 orders of magnitude. The Pauli Exclusion Principle (PEP) was introduced by the austrian physicist Wolfgang Pauli in 1925. Since then, several experiments have checked its validity. From 2006 until 2010, the VIP (Violation of the Pauli Principle) experiment took data at the LNGS underground laboratory to test the PEP. This experiment looked for electronic 2p to Is transitions in copper, where 2 electrons are in the Is state before the transition happens. These transitions violate the PEP. The lack of detection of X-ray photons coming from these transitions resulted in a preliminary upper limit for the violation of the PEP of 4.7 × 10(-29). Currently, the successor experiment VIP2 is under preparation. The main improvements are, on one side, the use of Silicon Drift Detectors (SDDs) as X-ray photon detectors. On the other side an active shielding is implemented, which consists of plastic scintillator bars read by Silicon Photomultipliers (SiPMs). The employment of these detectors will improve the upper limit for the violation of the PEP by around 2 orders of magnitude. The Pauli Exclusion Principle (PEP) was introduced by the austrian physicist Wolfgang Pauli in 1925. Since then, several experiments have checked its validity. From 2006 until 2010, the VIP (VIolation of the Pauli Principle) experiment took data at the LNGS underground laboratory to test the PEP. This experiment looked for electronic 2p to 1s transitions in copper, where 2 electrons are in the 1s state before the transition happens. These transitions violate the PEP. The lack of detection of X-ray photons coming from these transitions resulted in a preliminary upper limit for the violation of the PEP of $4.7 \times 10^{-29}$. Currently, the successor experiment VIP2 is under preparation. The main improvements are, on one side, the use of Silicon Drift Detectors (SDDs) as X-ray photon detectors. On the other side an active shielding is implemented, which consists of plastic scintillator bars read by Silicon Photomultipliers (SiPMs). The employment of these detectors will improve the upper limit for the violation of the PEP by around 2 orders of magnitude.

Research in R.B.’s laboratory was supported by the European Research Council Starting Grant AngioGenesHD (638028) and Consolidator Grant AngioUnrestUHD (101001814), the CNIC Intramural Grant Program Severo Ochoa (11-2016-IGP-SEV-2015-0505), the Ministerio de Ciencia e Innovación (MCIN) (SAF2013-44329-P, RYC-2013- 13209, and SAF2017-89299-P) and ‘La Caixa’ Banking Foundation (HR19-00120). J.V.’s laboratory was supported by MCIN (PGC2018- 097019-B-I00 and PID2021-122348NB-I00) and La Caixa (HR17-00247 and HR22-00253). K.G.’s laboratory was supported by Knut and Alice Wallenberg Foundation (2020.0057) and Vetenskapsrådet (2021-04896). The CNIC is supported by Instituto de Salud Carlos III, MCIN, and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MCIN/ AEI/10.13039/501100011033). Microscopy experiments were performed at the Microscopy and Dynamic Imaging Unit, CNIC, ICTS-ReDib, co-funded by MCIN/AEI/10.13039/501100011033 and FEDER ‘Una manera de hacer Europa’ (ICTS-2018-04-CNIC-16). M.F.-C. was supported by PhD fellowships from La Caixa (CX_E-2015-01) and Boehringer Ingelheim travel grants. S.M. was supported by the Austrian Science Fund (J4358). A.R. was supported by the Youth Employment Initiative (PEJD-2019-PRE/BMD-16990). L.G.-O. was supported by the Spanish Ministry of Economy and Competitiveness (PRE2018-085283). We thank S. Bartlett (CNIC) for English editing, as well as the members of the Transgenesis, Microscopy, Genomics, Citometry and Bioinformatic units at CNIC. We also thank F. Radtke (Swiss Institute for Experimental Cancer Research), R. H. Adams (Max Planck Institute for Molecular Biomedicine), F. Alt (Boston Children’s Hospital, Harvard Medical School), T. Honjo (Kyoto University Institute for Advanced Studies), I. Flores (CNIC), J. Lewis (Cancer Research UK London Research Institute), S. Habu (Tokai University School of Medicine), T. Gridley (Maine Health Institute for Research) and C. Brakebusch (Biotech Research and Innovation Centre) for sharing the Dll4floxed, Notch1floxed, Notch2floxed, Cdh5(PAC)-creERT2, Myc floxed, Rbpj floxed, p21−/−, Jag1floxed, Dll1floxed, Jag2floxed and Rac1floxed mice. Sí

Post-polymerisation functionalisation provides a facile and efficient way for the introduction of functional groups on the backbone of conjugated polymers. Using post-polymerisation functionalisation approaches, the polymer chain length is usually not affected, meaning that the resulting polymers only differ in their attached functional groups or side chains, which makes them particularly interesting for investigating the influence of the different groups on the polymer properties. For such functionalisations, highly efficient and selective reactions are needed to avoid the formation of complex mixtures or permanent defects in the polymer backbone. A variety of suitable synthetic approaches and reactions that fulfil these criteria have been identified and reported. In this review, a thorough overview is given of the post-polymerisation functionalisations reported to date, with the methods grouped based on the type of reaction used: cycloaddition, oxidation/reduction, nucleophilic aromatic substitution, or halogenation and subsequent cross-coupling reaction. Instead of modifications on the aliphatic side chains of the conjugated polymers, we focus on modifications directly on the conjugated backbones, as these have the most pronounced effect on the optical and electronic properties. Some of the discussed materials have been used in applications, ranging from solar cells to bioelectronics. By providing an overview of this versatile and expanding field for the first time, we showcase post-polymerisation functionalisation as an exciting pathway for the creation of new conjugated materials for a range of applications.

Invasive aspergillosis remains one of the most devastating fungal diseases and is predominantly linked to infections caused by the opportunistic human mold pathogen Aspergillus fumigatus. Major treatment regimens for the disease comprise the administration of antifungals belonging to the azole, polyene and echinocandin drug class. The prodrug 5-fluorocytosine (5FC), which is the only representative of a fourth class, the nucleobase analogs, shows unsatisfactory in vitro activities and is barely used for the treatment of aspergillosis. The main route of 5FC activation in A. fumigatus comprises its deamination into 5-fluorouracil (5FU) by FcyA, which is followed by Uprt-mediated 5FU phosphoribosylation into 5-fluorouridine monophosphate (5FUMP). In this study, we characterized and examined the role of a metabolic bypass that generates this nucleotide via 5-fluorouridine (5FUR) through uridine phosphorylase and uridine kinase activities. Resistance profiling of mutants lacking distinct pyrimidine salvage activities suggested a minor contribution of the alternative route in 5FUMP formation. We further analyzed the contribution of drug efflux in 5FC tolerance and found that A. fumigatus cells exposed to 5FC reduce intracellular fluoropyrimidine levels through their export into the environment. This release, which was particularly high in mutants lacking Uprt, generates a toxic environment for cytosine deaminase lacking mutants as well as mammalian cells. Employing the broad-spectrum fungal efflux pump inhibitor clorgyline, we demonstrate synergistic properties of this compound in combination with 5FC, 5FU as well as 5FUR. This research was funded by the Austrian Science Fund (FWF), grant P31093 to F.G. and M2867 to C.B. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Sí

Aromatic carboxylic anhydrides are ubiquitous building blocks in organic materials chemistry. In particular, dianhydrides and their conversion into imides have received considerable attention in the synthesis of organic semiconductors, pigments, and battery electrode materials. Here we extend the family of aromatic carboxylic anhydrides with a unique new member, a conjugated cyclophane with four anhydride groups. The cyclophane, named squarephaneic tetraanhydride due to its square shape, is obtained in a three-step synthesis; the anhydride groups are located at the corners and allow for efficient functionalisation, as shown by the conversion into imides and carboxylates. The crystal structures of the tetraanhydride and one of the imides demonstrate the high degree of porosity achievable with the new building block. Electrochemical measurements in solution and solid state are also presented. Considering the short synthesis and unique properties, we expect widespread use of the new building block in the development of organic materials.

π-Conjugated macrocycles are molecules with unique properties that are increasingly exploited for applications and the question of whether they can sustain global aromatic or antiaromatic ring currents is particularly intriguing. However, there are only a small number of experimental studies that investigate how the properties of π-conjugated macrocycles evolve with systematic structural changes. Here, we present such a systematic experimental study of a set of [2.2.2.2]cyclophanetetraenes, all with formally Hückel antiaromatic ground states, and combine it with an in-depth computational analysis. The study reveals the central role of local and global aromaticity for rationalizing the observed optoelectronic properties, ranging from extremely large Stokes shifts of up to 1.6 eV to reversible fourfold reduction, a highly useful feature for charge storage/accumulation applications. A recently developed method for the visualization of chemical shielding tensors (VIST) is applied to provide unique insight into local and global ring currents occurring in different planes along the macrocycle. Conformational changes as a result of the structural variations can further explain some of the observations. The study contributes to the development of structure–property relationships and molecular design guidelines and will help to understand, rationalize, and predict the properties of other π-conjugated macrocycles. It will also assist in the design of macrocycle-based supramolecular elements with defined properties., Insights into how the optoelectronic properties of π-conjugated macrocycles evolve as a result of systematic structural changes are presented, helping to develop structure–property relationships and molecular design guidelines.

The synthesis of two novel n-type molecular organic semiconductors based on a fluorinated indacenodithiophene core in combination with an electron withdrawing N-cyanoimine group is reported, and the influence of the regiochemistry of the solubilizing sidechain is investigated. The N-cyanoimine is confirmed to be a strongly electron accepting group, which in combination with the core fluorination resulted in high electron affinities for both materials. Single crystal analysis demonstrated that whilst both materials arrange in ordered slipped stacks with close π–π stacking distances (∼3.40 Å), significant differences in electron transfer integrals for the two regioisomers were observed, relating to differences in relative molecular displacement along the π-stacking direction. Organic thin-film transistors fabricated via blade-coating displayed electron mobility up to 0.13 cm2 V−1 s−1 for the isomer with the larger transfer integral.

Therapeutic modulation of vascular cell proliferation and migration is essential for the effective inhibition of angiogenesis in cancer or its induction in cardiovascular disease. The general view is that an increase in vascular growth factor levels or mitogenic stimulation is beneficial for angiogenesis, since it leads to an increase in both endothelial proliferation and sprouting. However, several recent studies showed that an increase in mitogenic stimuli can also lead to the arrest of angiogenesis. This is due to the existence of intrinsic signaling feedback loops and cell cycle checkpoints that work in synchrony to maintain a balance between endothelial proliferation and sprouting. This balance is tightly and effectively regulated during tissue growth and is often deregulated or impaired in disease. Most therapeutic strategies used so far to promote vascular growth simply increase mitogenic stimuli, without taking into account its deleterious effects on this balance and on vascular cells. Here, we review the main findings on the mechanisms controlling physiological vascular sprouting, proliferation, and senescence and how those mechanisms are often deregulated in acquired or congenital cardiovascular disease leading to a diverse range of pathologies. We also discuss alternative approaches to increase the effectiveness of pro-angiogenic therapies in cardiovascular regenerative medicine. Severin Mühleder was funded by the Austrian Science Fund (FWF) project J4358. Macarena Fernández-Chacón and Irene Garcia-Gonzalez were supported by PhD fellowships from Fundación La Caixa (CX_E-2015-01 and CX-SO-16-1, respectively). Rui Benedito was funded by the European Research Council (ERC-2014-StG—638028), the Centro Nacional de Investigaciones Cardiovasculares (CNIC), and by the Ministerio de Economia, Industria y Competitividad (MEIC: SAF2013-44329-P, SAF2017-89299-P, and RYC-2013-13209). The CNIC is supported by the Ministerio de Ciencia, Innovación y Universidades (MCNU) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505). Sí

Background: Poly(p-phenylene vinylene)s (PPVs) and [2.2.2.2]paracyclophanetetraene (PCT) are both composed of alternating π-conjugated para-phenylene and vinylene units. However, while the former constitute a class of π-conjugated polymers that has been used in organic electronics for decades, the latter is a macrocycle that only recently revealed its potential for applications such as organic battery electrodes. The cyclic structure endows PCT with unusual properties, and further tuning of these may be required for specific applications. Methods: In this article, we adopt an approach often used for tuning the properties of PPVs, the introduction of alkoxy (or alkylthio) substituents at the phenylene units, for tuning the optoelectronic properties of PCT. The resulting methoxy- and methylthio-substituted PCTs, obtained by Wittig cyclisation reactions, are studied by UV-vis absorption, photoluminescence, and cyclic voltammetry measurements, and investigated computationally using the visualisation of chemical shielding tensors (VIST) method. Results: The measurements show that substitution leads to slight changes in terms of absorption/emission energies and redox potentials while having a pronounced effect on the photoluminescence intensity. The computations show the effect of the substituents on the ring currents and chemical shielding and on the associated local and global (anti)aromaticity of the macrocycles, highlighting the interplay of local and global aromaticity in various electronic states. Conclusions: The study offers interesting insights into the tuneability of the properties of this versatile class of π-conjugated macrocycles.

Background: Poly(p-phenylene vinylene)s (PPVs) and [2.2.2.2]paracyclophanetetraene (PCT) are both composed of alternating π-conjugated para-phenylene and vinylene units. However, while the former constitute a class of π-conjugated polymers that has been used in organic electronics for decades, the latter is a macrocycle that only recently revealed its potential for applications such as organic battery electrodes. The cyclic structure endows PCT with unusual properties, and further tuning of these may be required for specific applications. Methods: In this article, we adopt an approach often used for tuning the properties of PPVs, the introduction of alkoxy (or alkylthio) substituents at the phenylene units, for tuning the optoelectronic properties of PCT. The resulting methoxy- and methylthio-substituted PCTs, obtained by Wittig cyclisation reactions, are studied by UV-vis absorption, photoluminescence, and cyclic voltammetry measurements, and investigated computationally using the visualisation of chemical shielding tensors (VIST) method. Results: The measurements show that substitution leads to slight changes in terms of absorption/emission energies and redox potentials while having a pronounced effect on the photoluminescence intensity. The computations show the effect of the substituents on the ring currents and chemical shielding and on the associated local and global (anti)aromaticity of the macrocycles, highlighting the interplay of local and global aromaticity in various electronic states. Conclusions: The study offers interesting insights into the tuneability of the properties of this versatile class of π-conjugated macrocycles.

International audience; Domestic dogs have been shown to reciprocate help received from conspecifics in food-giving tasks. However, it is not yet known whether dogs also reciprocate help received from humans. Here, we investigated whether dogs reciprocate the receipt of food from humans. In an experience phase, subjects encountered a helpful human who provided them with food by activating a food dispenser, and an unhelpful human who did not provide them with food. Subjects later had the opportunity to return food to each human type, in a test phase, via the same mechanism. In addition, a free interaction session was conducted in which the subject was free to interact with its owner and with whichever human partner it had encountered on that day. Two studies were carried out, which differed in the complexity of the experience phase and the time lag between the experience phase and test phase. Subjects did not reciprocate the receipt of food in either study. Furthermore, no difference was observed in the duration subjects spent in proximity to, or the latency to approach, the two human partners. Although our results suggest that dogs do not reciprocate help received from humans, they also suggest that the dogs did not recognize the cooperative or uncooperative act of the humans during the experience phase. It is plausible that aspects of the experimental design hindered the emergence of any potential reciprocity. However, it is also possible that dogs are simply not prosocial towards humans in food-giving contexts.

Titin, as the main protein responsible for the passive stiffness of the sarcomere, plays a key role in diastolic function and is a determinant factor in the etiology of heart disease. Titin stiffness depends on unfolding and folding transitions of immunoglobulin-like (Ig) domains of the I-band, and recent studies have shown that oxidative modifications of cryptic cysteines belonging to these Ig domains modulate their mechanical properties in vitro. However, the relevance of this mode of titin mechanical modulation in vivo remains largely unknown. Here, we describe the high evolutionary conservation of titin mechanical cysteines and show that they are remarkably oxidized in murine cardiac tissue. Mass spectrometry analyses indicate a similar landscape of basal oxidation in murine and human myocardium. Monte Carlo simulations illustrate how disulfides and S-thiolations on these cysteines increase the dynamics of the protein at physiological forces, while enabling load- and isoform-dependent regulation of titin stiffness. Our results demonstrate the role of conserved cysteines in the modulation of titin mechanical properties in vivo and point to potential redox-based pathomechanisms in heart disease. This work was supported by the Ministerio de Ciencia e Innovación (MCIN/AEI/10.13039/501100011033) grants BIO2014-54768-P, BIO2017-83640-P (AEI/FEDER, UE), PID2020-120426GB-I00, RYC2014-16604 to JAC and PGC2018-097019-B-I00 to JV, the Regional Government of Madrid grants S2018/NMT-4443 (Tec4Bio, 50% cofinanced by the European Social Fund and the European Regional Development Fund for the programming period 2014-2020) and PEJ16/MED/TL-1593 to JAC and the Instituto de Salud Carlos III (Fondo de Investigacion ´ Sanitaria grant PRB3, PT17/0019/0003- ISCIII-SGEFI /ERDF, ProteoRed), and “la Caixa” Banking Foundation (project code HR17-00247) to JV. We acknowledge funding from the European Research Area Network on Cardiovascular Diseases through grant MINOTAUR to SS (The Austrian Science Fund – FWF, I3301) and JAC (ISCIII-AC16/00045). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovacion ´ (MCIN) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MCIN/AEI/10.13039/501100011033). IMM was the recipient of a CNIC-ACCIONA Masters Fellowship and holds a fellowship from “La Caixa” Foundation (ID 100010434, fellowship code LCF/BQ/DR20/11790009). CSC is the recipient of an FPI-SO predoctoral fellowship BES-2016-076638. We thank Wolfgang A. Linke and Pablo García-Pavía for critical feedback. We are also thankful for the insights of four anonymous reviewers. Sí

Aromaticity is a central concept in chemistry, pervading areas from biochemistry to materials science. Recently, chemists also started to exploit intricate phenomena such as the interplay of local and global (anti)aromaticity or aromaticity in non‐planar systems and three dimensions. These phenomena pose new challenges in terms of our fundamental understanding and the practical visualisation of aromaticity. To overcome these challenges, a method for the visualisation of chemical shielding tensors (VIST) is developed here that allows for a 3D visualisation with quantitative information about the local variations and anisotropy of the chemical shielding. After exemplifying the method in different planar hydrocarbons, we study two non‐planar macrocycles to show the unique benefits of the VIST method for molecules with competing π‐conjugated systems and conclude with a norcorrole dimer showing clear evidence of through‐space aromaticity. We believe that the VIST method will be a highly valuable addition to the computational toolbox., A method for the visualisation of chemical shielding tensors (VIST) is developed, which provides a graphical representation of aromaticity and antiaromaticity, their local variations, and anisotropy. The method provides unique insight into fascinating phenomena such as the interplay of aromaticity and antiaromaticity in macrocycles with competing π‐conjugated systems.

A number of mono- and dioligosilanylated silocanes were prepared. Compounds included silocanes with 1-methyl-1-tris(trimethylsilyl)silyl, 1,1-bis[tris(trimethylsilyl)silyl], and 1,1-bis[tris(trimethylsilyl)germyl] substitution pattern as well as two examples where the silocane silicon atom is part of a cyclosilane or oxacyclosilane ring. The mono-tris(trimethylsilyl)silylated compound could be converted to the respective silocanylbis(trimethylsilyl)silanides by reaction with KOtBu and in similar reactions the cyclosilanes were transformed to oligosilane-1,3-diides. However, the reaction of the 1,1-bis[tris(trimethylsilyl)silylated] silocane with two equivalents of KOtBu leads to the replacement of one tris(trimethylsilyl)silyl unit with a tert-butoxy substituent followed by silanide formation via KOtBu attack at one of the SiMe3 units of remaining tris(trimethylsilyl)silyl group. For none of the silylated silocanes, signs of hypercoordinative interaction between the nitrogen and silicon silocane atoms were detected either in the solid state. by single crystal XRD analysis, nor in solution by 29Si-NMR spectroscopy. This was further confirmed by cyclic voltammetry and a DFT study, which demonstrated that the N-Si distance in silocanes is not only dependent on the energy of a potential N-Si interaction, but also on steric factors and through-space interactions of the neighboring groups at Si and N, imposing the orientation of the pz(N) orbital relative to the N-Si-X axis.

Floral scent is an important trait in plant–pollinator interactions. It not only varies among plant species but also among populations within species. Such variability might be caused by various non–selective factors, or, as has been shown in some instances, might be the result of divergent selective pressures exerted by variable pollinator climates. Cypripedium calceolus is a Eurasian deceptive orchid pollinated mainly by bees, which spans wide altitudinal and latitudinal gradients in mainly quite isolated populations. In the present study, we investigated whether pollinators and floral scents vary among different latitudes. Floral scents of three C. calceolus populations in the Southern Alps were collected by dynamic headspace and analyzed by gas chromatography coupled to mass spectrometry (GC/MS). These data were completed by previously published scent data of the Northern Alps and Scandinavia. The scent characteristics were compared with information on pollinators recorded for present study or available in the literature. More than 80 scent compounds were overall recorded from plants of the three regions, mainly aliphatics, terpenoids, and aromatics. Seven compounds were found in all samples, and most samples were dominated by linalool and octyl acetate. Although scents differed among regions and populations, the main compounds were similar among regions. Andrena and Lasioglossum species were the main pollinators in all three regions, with Andrena being relatively more abundant than Lasioglossum in Scandinavia. We discuss natural selection mediated by pollinators and negative frequency–dependent selection as possible reasons for the identified variation of floral scent within and among populations and regions.

International audience; Several recent studies have shown that SCAN, a functional belonging to the meta-generalized gradient approximation (MGGA) family, leads to significantly overestimated magnetic moments in itinerant ferromagnetic metals. However, this behavior is not inherent to the MGGA level of approximation since TPSS, for instance, does not lead to such severe overestimations. In order to provide a broader view of the accuracy of MGGA functionals for magnetism, we extend the assessment to more functionals but also to antiferromagnetic solids. The results show that to describe magnetism there is overall no real advantage in using a MGGA functional compared to GGAs. For both types of approximation, an improvement in ferromagnetic metals is necessarily accompanied by a deterioration (underestimation) in antiferromagnetic insulators, and vice versa. We also provide some analysis in order to understand in more detail the relation between the mathematical form of the functionals and the results.

Podoplanin (Pdpn), a brain-tumor-related glycoprotein identified in humans and animals, is endogenously expressed in several organs critical for life support such as kidney, lung, heart and brain. In the brain, Pdpn has been identified in proliferative nestin- positive adult neural progenitor cells and in neurons of the neurogenic hippocampal dentate gyrus (DG), a structure associated to anxiety, critical for learning and memory functions and severely damaged in people with Alzheimer’s Disease (AD). The in vivo role of Pdpn in adult neurogenesis and anxiety-like behavior remained however unexplored. Using mice with disrupted Pdpn gene as a model organism and applying combined behavioral, molecular biological and electrophysiological assays, we here show that the absence of Pdpn selectively impairs long-term synaptic depression in the neurogenic DG without affecting the CA3-Schaffer’s collateral-CA1 synapses. Pdpn deletion also enhanced the proliferative capacity of DG neural progenitor cells and diminished survival of differentiated neuronal cells in vitro. In addition, mice with podoplanin gene disruption showed increased anxiety-like behaviors in experimentally validated behavioral tests as compared to wild type littermate controls. Together, these findings broaden our knowledge on the molecular mechanisms influencing hippocampal synaptic plasticity and neurogenesis in vivo and reveal Pdpn as a novel molecular target for future studies addressing general anxiety disorder and synaptic depression-related memory dysfunctions.

Progress in biomedical science is tightly associated with the improvement of methods and genetic tools to manipulate and analyze gene function in mice, the most widely used model organism in biomedical research. The joint effort of numerous individual laboratories and consortiums has contributed to the creation of a large genetic resource that enables scientists to image cells, probe signaling pathways activities, or modify a gene function in any desired cell type or time point, à la carte. However, as these tools significantly increase in number and become more sophisticated, it is more difficult to keep track of each tool's possibilities and understand their advantages and disadvantages. Knowing the best currently available genetic technology to answer a particular biological question is key to reach a higher standard in biomedical research. In this review, we list and discuss the main advantages and disadvantages of available mammalian genetic technology to analyze cardiovascular cell biology at higher cellular and molecular resolution. We start with the most simple and classical genetic approaches and end with the most advanced technology available to fluorescently label cells, conditionally target their genes, image their clonal expansion, and decode their lineages. RB is supported by the European Research Council (ERC-2014-StG-638028), the Centro Nacional de Investigaciones Cardiovasculares (CNIC), and the Ministerio de Ciencia y Innovación (MCIN: SAF2017-89299-P). The CNIC is supported by MCIN, the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (SEV-2015-0505). IG-G and MF-C were supported by PhD fellowships from Fundación La Caixa (CX-SO- 16-1 and CX_E-2015-01, respectively). SM was funded by the Austrian Science Fund (FWF) project J4358. Sí

Coculture systems employing adipose tissue-derived mesenchymal stromal/stem cells (ASC) and endothelial cells (EC) represent a widely used technique to model vascularization. Within this system, cell-cell communication is crucial for the achievement of functional vascular network formation. Extracellular vesicles (EVs) have recently emerged as key players in cell communication by transferring bioactive molecules between cells. In this study we aimed to address the role of EVs in ASC/EC cocultures by discriminating between cells, which have received functional EV cargo from cells that have not. Therefore, we employed the Cre-loxP system, which is based on donor cells expressing the Cre recombinase, whose mRNA was previously shown to be packaged into EVs and reporter cells containing a construct of floxed dsRed upstream of the eGFP coding sequence. The evaluation of Cre induced color switch in the reporter system via EVs indicated that there is no EV-mediated RNA transmission either between EC themselves or EC and ASC. However, since Cre mRNA was not found present in EVs, it remains unclear if Cre mRNA is generally not packaged into EVs or if EVs are not taken up by the utilized cell types. Our data indicate that this technique may not be applicable to evaluate EV-mediated cell-to-cell communication in an in vitro setting using EC and ASC. Further investigations will require a functional system showing efficient and specific loading of Cre mRNA or protein into EVs. This research was partially funded by the Austrian Research promotion agency. Severin Mühleder was funded by the Austrian Science Fund (FWF). Sí

As extracellular vesicles (EVs) have become a prominent topic in life sciences, a growing number of studies are published on a regular basis addressing their biological relevance and possible applications. Nevertheless, the fundamental question of the true vesicular nature as well as possible influences on the EV secretion behavior have often been not adequately addressed. Furthermore, research regarding endothelial cell-derived EVs (EndoEVs) often focused on the large vesicular fractions comprising of microvesicles (MV) and apoptotic bodies. In this study we aimed to further extend the current knowledge of the influence of pre-isolation conditions, such as cell density and conditioning time, on EndoEV release from human umbilical vein endothelial cells (HUVECs). We combined fluorescence nanoparticle tracking analysis (NTA) and the established fluorescence-triggered flow cytometry (FT-FC) protocol to allow vesicle-specific detection and characterization of size and surface markers. We found significant effects of cell density and conditioning time on both abundance and size distribution of EndoEVs. Additionally, we present detailed information regarding the surface marker display on EVs from different fractions and size ranges. Our data provide crucial relevance for future projects aiming to elucidate EV secretion behavior of endothelial cells. Moreover, we show that the influence of different conditioning parameters on the nature of EndoEVs has to be considered. This research was funded by the Austrian Research promotion agency and Particle Metrix. Severin Mühleder was funded by the Austrian Science Fund (FWF) project J4358. Sí

Vineyard inter-rows can provide habitats for a range of plant and animal species especially when covered with vegetation. However, frequent tillage results in the degradation of habitat quality and the provision of biodiversity-based ecosystem services. Wild bees are important pollinators of crops and wild plants and depend on both, floral resources and suitable nesting sites, which are influenced by the landscape configuration. We examined effects of field and landscape parameters on wild bee species’ richness, abundance and functional traits in Austrian vineyards over two years using Generalised Linear Mixed models, Detrended Correspondence Analysis and Random Forests. Alternating tillage was compared with no tillage in two inter-rows per vineyard. Forage availability in these inter-rows was estimated by flower coverage at each sampling date, and landscape features were analysed within a radius of 750 m around the vineyards. Across all vineyards we found 84 wild bee species with a mean abundance (±SD) of 29 (±16.6). Forage availability had the strongest positive effect on wild bee diversity and abundance. In comparison to no tillage, alternating tillage slightly increased wild bee diversity and abundance. Eusocial wild bees were more abundant in untilled inter-rows, whereas solitary wild bees were more closely associated with alternating tilled vineyards. At the landscape scale, the percentage of artificial areas (mostly villages) and distance to semi-natural elements raised wild bee diversity and abundance. The proportion of woodland increased the abundance of wild bees, in particular of eusocial taxa. Solitary wild bee abundance was enhanced by the number of solitary trees. Pollination provided by wild bees in viticultural areas can be enhanced by maintaining a diversity of different soil management strategies to improve forage availability in vineyards. Furthermore, semi-natural elements such as fallows or solitary trees providing floral resources and nesting habitat should be preserved within viticultural landscapes., This research is part of the BiodivERsA project VineDivers (www.vinedivers.eu) and was funded through the BiodivERsA/FACCE JPI (2013-2014 joint call) for research proposals, with the national funders: Austrian Science Fund (grant number I 2044-B25FWF), French National Research Agency (ANR), Spanish Ministry of Economy and Competitiveness (MINECO), Romanian Executive Agency for Higher Education, Research, Development and Innovation Funding (UEFISCDI) and Federal Ministry of Education and Research (BMBF/Germany). The open access funding was provided by the Austrian Science Fund: I 2044-B25. We are grateful to Johann Grassl and to the winegrowers in the Neusiedlersee-Hügelland and Carnuntum region for allowing us to study their vineyards.

Osteosarcoma (OS) is a rare tumor of the bone occurring mainly in young adults accounting for 5% of all childhood cancers. Because of the limited therapeutic options, there has been no survival improvement for OS patients in the past 40 years. The epidermal growth factor receptor (EGFR) is highly expressed in OS; however, its clinical relevance is unclear. Here, we employed an autochthonous c-Fos-dependent OS mouse model (H2-c-fosLTR) and human OS tumor biopsies for preclinical studies aimed at identifying novel biomarkers and therapeutic benefits of anti-EGFR therapies. We show that EGFR deletion/inhibition results in reduced tumor formation in H2-c-fosLTR mice by directly inhibiting the proliferation of cancer-initiating osteoblastic cells by a mechanism involving RSK2/CREB-dependent c-Fos expression. Furthermore, OS patients with co-expression of EGFR and c-Fos exhibit reduced overall survival. Preclinical studies using human OS xenografts revealed that only tumors expressing both EGFR and c-Fos responded to anti-EGFR therapy demonstrating that c-Fos can be considered as a novel biomarker predicting response to anti-EGFR treatment in OS patients. We thank Martina Hammer for maintaining mouse colonies, Malgorzata Tryniecki for genotyping, Bilge Vasfiye Göcen for technical assistance, and Andrea Nolz for help with X‐ray imaging. We are grateful to Marlon R. Schneider for providing the ColAREG transgenic mice and to Eugenie S. Kleinerman for providing the LM7 OS cell line. This work was supported by the Austrian Science Fund (FWF) (DK W1212). M.S. is supported by an ERC‐Advanced grant (ERC‐2015‐AdG TNT‐Tumors 694883). The [11C]erlotinib PET imaging part of this work was supported by the FWF project “Transmembrane Transporters in Health and Disease” (SFB F35). Sí

Recent genome analyses have identified recurrent mutations in the cohesin complex in a wide range of human cancers. Here we demonstrate that the most frequently mutated subunit of the cohesin complex, STAG2, displays a strong synthetic lethal interaction with its paralog STAG1. Mechanistically, STAG1 loss abrogates sister chromatid cohesion in STAG2 mutated but not in wild-type cells leading to mitotic catastrophe, defective cell division and apoptosis. STAG1 inactivation inhibits the proliferation of STAG2 mutated but not wild-type bladder cancer and Ewing sarcoma cell lines. Restoration of STAG2 expression in a mutated bladder cancer model alleviates the dependency on STAG1. Thus, STAG1 and STAG2 support sister chromatid cohesion to redundantly ensure cell survival. STAG1 represents a vulnerability of cancer cells carrying mutations in the major emerging tumor suppressor STAG2 across different cancer contexts. Exploiting synthetic lethal interactions to target recurrent cohesin mutations in cancer, e.g. by inhibiting STAG1, holds the promise for the development of selective therapeutics. We would like to thank Monika Kriz and Renate Schnitzer for clonal analysis. The IMP is supported by Boehringer Ingelheim. Research in the laboratory of J-MP is funded by the Austrian Science Fund (SFB-F34 and Wittgenstein award Z196-B20) and the Austrian Research Promotion Agency (Head- quarter grants FFG-834223 and FFG-852936, Laura Bassi Centre for Optimized Structural Studies grant FFG-840283). Research in the laboratory of JZ was funded by a Starting Grant of the European Research Council (ERC no. 336860) and SFB grant F4710 of the Austrian Science Fund (FWF). Research on Ewing sarcoma in the laboratory of HK was funded by the Austrian Science Fund ERA- Net grant I 1225-B19. Work in the lab of FXR was funded by a grant from Fundacio´ n Cientı´fica de la Asociacio´ n Espan˜ ola Contra el Ca´ ncer, Madrid, Spain. Research in the laboratory of TW is supported by National Institute of Health grant R01CA169345 and an Innovation Grant from Alex’s Lemonade Stand Sí

19 pages; We show in the context of $\mathbb{Z}^k$-actions that every van der Corput set is a Heilbronn set. Furthermore we establish Diophantine inequalities of the Heilbronn type for generalized polynomials $g$ in particular for sequences $\nu(n)=\lfloor n^c\rfloor+n^k$ with $c>1$ a non-integral real number and $k\in\mathbb{N}$, as well as for $\nu(p)$ where $p$ runs through all prime numbers.

Invasive Aspergillosis (IA) is an opportunistic infection caused by Aspergillus, a ubiquitously present airborne pathogenic mold. A growing number of studies suggest a major host genetic component in disease susceptibility. Here, we evaluated whether 14 single-nucleotide polymorphisms within NF kappa B1, NF kappa B2, RelA, RelB, Rel, and IRF4 genes influence the risk of IA in a population of 834 high-risk patients (157 IA and 677 non IA) recruited through a collaborative effort involving the aspBlOmics consortium and four European clinical institutions. No significant overall associations between selected SNPs and the risk of IA were found in this large cohort. Although a hematopoietic stem cell transplantation (HSCT)-stratified analysis revealed that carriers of the IRF4(rs12203592T/T) genotype had a six-fold increased risk of developing the infection when compared with those carrying the C allele (ORREC = 6.24, 95%CI 1.25-31.2, P = 0.026), the association of this variant with IA risk did not reach significance at experiment-wide significant threshold. In addition, we found an association of the IRF4(AATC) and IRF4(GGTC) haplotypes (not including the IRF4(rs12203592T/T) risk allele) with a decreased risk of IA but the magnitude of the association was similar to the one observed in the single-SNP analysis, which indicated that the haplotypic effect on IA risk was likely due to the IRF4(rs12203592) SNP Finally, no evidence of significant interactions among the genetic markers tested and the risk of IA was found. These results suggest that the SNPs on the studied genes do not have a clinically relevant impact on the risk of developing IA., This study was supported by grants PI12/02688 from Fond de Investigaciones Sanitarias (Institute de Salud Carlos III, Madrid, Spain), the ERA-NET PathoGenoMics (03159000A; Ministerio de Ciencia e Innovation PIM2010EPA-00756, Madrid, Spain), the Collaborative Research Center / Transregio 124 FungiNet, the Austrian Science Fundation (FWF I-656-B09), the Fundacao para a Ciencia e Tecnologia (FCT), cofundcd by Programa Operacional Regional do Norte (ON.2-O Novo Norte), the Quadro de Referencia Estrategico Nacional (QREN) through the Fundo Europeu de Desenvolvimento Regional (FEDER) and the Projeto Estrategico - LA 26 - 2013-2014 (PEst-C/SAU/LA0026/2013). Agostinho Carvalho and Cristina Cunha were supported by the Fundacao para a e Tecnologia (FCT), Portugal (IF/00735/2014 and SFRH/BPD/96176/2013, respectively). The PCRAGA trial was supported by an unrestricted grant from Pfizer, which had no involvement or control over the collection, analysis, and interpretation of data; the writing of the report; or the decision to submit the paper for publication. This study was also supported by Astellas Pharma Inc. and a donation from Consuelo Gonzalez Moreno, an acute myeloid leukemia survivor., info:eu-repo/semantics/publishedVersion

International audience; Fisher's geometric model has been widely used to study the effects of pleiotropy and organismic complexity on phenotypic adaptation. Here, we study a version of Fisher's model in which a population adapts to a gradually moving optimum. Key parameters are the rate of environmental change, the dimensionality of phenotype space, and the patterns of mutational and selectional correlations. We focus on the distribution of adaptive substitutions, that is, the multivariate distribution of the phenotypic effects of fixed beneficial mutations. Our main results are based on an "adaptive-walk approximation," which is checked against individual-based simulations. We find that (1) the distribution of adaptive substitutions is strongly affected by the ecological dynamics and largely depends on a single composite parameter γ, which scales the rate of environmental change by the "adaptive potential" of the population; (2) the distribution of adaptive substitution reflects the shape of the fitness landscape if the environment changes slowly, whereas it mirrors the distribution of new mutations if the environment changes fast; (3) in contrast to classical models of adaptation assuming a constant optimum, with a moving optimum, more complex organisms evolve via larger adaptive steps.

20 p.-8 fig. Margherita Bertuzzi et alt., Destruction of the pulmonary epithelium is a major feature of lung diseases caused by the mould pathogen Aspergillus fumigatus. Although it is widely postulated that tissue invasion is governed by fungal proteases, A. fumigatus mutants lacking individual or multiple enzymes remain fully invasive, suggesting a concomitant requirement for other pathogenic activities during host invasion. In this study we discovered, and exploited, a novel, tissue non-invasive, phenotype in A.fumigatus mutants lacking the pH-responsive transcription factor PacC. Our study revealed a novel mode of epithelial entry,occurring in a cell wall-dependent manner prior to protease production, and via the Dectin-1 b-glucan receptor. DpacC mutants are defective in both contact-mediated epithelial entry and protease expression, and significantly attenuated for pathogenicity in leukopenic mice. We combined murine infection modelling, in vivo transcriptomics, and in vitro infections of human alveolar epithelia, to delineate two major, and sequentially acting, PacC-dependent processes impacting epithelial integrity in vitro and tissue invasion in the whole animal. We demonstrate that A. fumigatus spores and germlings are internalised by epithelial cells in a contact-, actin-, cell wall- and Dectin-1 dependent manner and DpacC mutants, which aberrantly remodel the cell wall during germinative growth, are unable to gain entry into epithelial cells, both in vitro and in vivo. We further show that PacC acts as a global transcriptional regulator of secreted molecules during growth in the leukopenic mammalian lung, and profile the full cohort of secreted gene products expressed during invasive infection. Our study reveals a combinatorial mode of tissue entry dependent upon sequential, and mechanistically distinct, perturbations of the pulmonary epithelium and demonstrates, for the first time a protective role for Dectin-1 blockade in epithelial defences. Infecting DpacC mutants are hypersensitive to cell wall-active antifungal agents highlighting the value of PacC signalling as a target for antifungal therapy., This work was supported in part by grants to EMB from the MRC (G0501164) and BBSRC (BB/G009619/1), to EMB and NDR from the Wellcome Trust(WT093596MA), to MB from Imperial College London (Division of Investigative Sciences PhD Studentship), to HH from the ERA-NET PathoGenoMics project TRANSPAT, Austrian Science Foundation (FWF I282-B09), to SGF from the National Institutes of Health, USA (R01AI073829). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

International audience; An increasing number of studies demonstrate phenotypic and genetic changes in natural populations that are subject to climate change, and there is hope that some of these changes will contribute to avoiding species extinctions ('evolutionary rescue'). Here, we review theoretical models of rapid evolution in quantitative traits that can shed light on the potential for adaptation to a changing climate. Our focus is on quantitative-genetic models with selection for a moving phenotypic optimum. We point out that there is no one-to-one relationship between the rate of adaptation and population survival, because the former depends on relative fitness and the latter on absolute fitness. Nevertheless, previous estimates that sustainable rates of genetically based change usually do not exceed 0.1 haldanes (i.e., phenotypic standard deviations per generation) are probably correct. Survival can be greatly facilitated by phenotypic plasticity, and heritable variation in plasticity can further speed up genetic evolution. Multivariate selection and genetic correlations are frequently assumed to constrain adaptation, but this is not necessarily the case and depends on the geometric relationship between the fitness landscape and the structure of genetic variation. Similar conclusions hold for adaptation to shifting spatial gradients. Recent models of adaptation in multispecies communities indicate that the potential for rapid evolution is strongly influenced by interspecific competition.