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Abstract In kidney transplant recipients, urine CXCL9 and CXCL10 (uCXCL9/10) chemokines have reached a sufficiently high level of evidence to be recommended by the European Society of Organ Transplantation for the monitoring of immune quiescence. To assess the risk of acute rejection (AR), the advantage of uCXCL9/10 is their cost-effectiveness and their high diagnostic performance. Here, we evaluated the feasibility of a next-generation immunoassay for quantifying uCXCL9/10 levels. It demonstrated high efficiency with minimal workflow and a 90-min time to result. Preanalytical studies indicated stability of uCXCL9/10 levels and analytical studies confirmed excellent linearity and precision. In a cohort of 1048 samples collected at biopsy, the results correlated significantly with ELISA quantification and were integrated into a previously validated 8-parameter urine chemokine model. The next generation immunoassay achieved an accuracy of 0.84 for AR diagnosis. This study validates this technology as a robust, locally available and unexpensive platform and marks a significant step towards the widespread implementation of uCXCL9/10, for immune quiescence monitoring. Therefore, we developed an open-access web application using uCXCL9/10 to calculate AR risk and improve clinical decision-making to perform biopsy, ushering in a new era in kidney transplantation, where personalized, data-driven care becomes the norm.

Abstract Oncogene-induced senescence (OIS) arrests cell proliferation in response to replication stress (RS) induced by oncogenes. OIS depends on the DNA damage response (DDR), but also on the cGAS-STING pathway, which detects cytosolic DNA and induces type I interferons (IFNs). Whether and how RS and IFN responses cooperate to promote OIS remains unknown. Here, we show that the induction of OIS by the H-RASV12 oncogene in immortalized human fibroblasts depends on the MRE11 nuclease. Indeed, treatment with the MRE11 inhibitor Mirin prevented RS, micronuclei formation and IFN response induced by RASV12. Overexpression of the cytosolic nuclease TREX1 also prevented OIS. Conversely, overexpression of a dominant negative mutant of TREX1 or treatment with IFN-β was sufficient to induce RS and DNA damage, independent of RASV12 induction. These data suggest that the IFN response acts as a positive feedback loop to amplify DDR in OIS through a process regulated by MRE11 and TREX1.

Summary: The advancement of regenerative life support systems (RLSS) is crucial to allow long-distance space travel. Within the Micro-Ecological Life Support System Alternative (MELiSSA), efficient nitrogen recovery from urine and other waste streams is vital to produce liquid fertilizer to feed food and oxygen production in subsequent photoautotrophic processes. This study explores the effects of ionizing radiation on nitrogen cycle bacteria that transform urea to nitrate. In particular, we assess the radiotolerance of Comamonas testosteroni, Nitrosomonas europaea, and Nitrobacter winogradskyi after exposure to acute γ-irradiation. Moreover, a comprehensive whole transcriptome analysis elucidates the effects of spaceflight-analogue low-dose ionizing radiation on the individual axenic strains and on their synthetic community o. This research sheds light on how the spaceflight environment could affect ureolysis and nitrification processes from a transcriptomic perspective.

Abstract Astrocytes are intimately linked with brain blood vessels, an essential relationship for neuronal function. However, astroglial factors driving these physical and functional associations during postnatal brain development have yet to be identified. By characterizing structural and transcriptional changes in mouse cortical astrocytes during the first two postnatal weeks, we find that high-mobility group box 1 (Hmgb1), normally upregulated with injury and involved in adult cerebrovascular repair, is highly expressed in astrocytes at birth and then decreases rapidly. Astrocyte-selective ablation of Hmgb1 at birth affects astrocyte morphology and endfoot placement, alters distribution of endfoot proteins connexin43 and aquaporin-4, induces transcriptional changes in astrocytes related to cytoskeleton remodeling, and profoundly disrupts endothelial ultrastructure. While lack of astroglial Hmgb1 does not affect the blood-brain barrier or angiogenesis postnatally, it impairs neurovascular coupling and behavior in adult mice. These findings identify astroglial Hmgb1 as an important player in postnatal gliovascular maturation.

Abstract Rejection remains the main cause of premature graft loss after kidney transplantation, despite the use of potent immunosuppression. This highlights the need to better understand the composition and the cell-to-cell interactions of the alloreactive inflammatory infiltrate. Here, we performed droplet-based single-cell RNA sequencing of 35,152 transcriptomes from 16 kidney transplant biopsies with varying phenotypes and severities of rejection and without rejection, and identified cell-type specific gene expression signatures for deconvolution of bulk tissue. A specific association was identified between recipient-derived FCGR3A+ monocytes, FCGR3A + NK cells and the severity of intragraft inflammation. Activated FCGR3A+ monocytes overexpressed CD47 and LILR genes and increased paracrine signaling pathways promoting T cell infiltration. FCGR3A + NK cells overexpressed FCRL3, suggesting that antibody-dependent cytotoxicity is a central mechanism of NK-cell mediated graft injury. Multiplexed immunofluorescence using 38 markers on 18 independent biopsy slides confirmed this role of FcγRIII+ NK and FcγRIII+ nonclassical monocytes in antibody-mediated rejection, with specificity to the glomerular area. These results highlight the central involvement of innate immune cells in the pathogenesis of allograft rejection and identify several potential therapeutic targets that might improve allograft longevity.

Abstract Despite significant advancements in in vitro cardiac modeling approaches, researchers still lack the capacity to obtain in vitro measurements of a key indicator of cardiac function: contractility, or stroke volume under specific loading conditions—defined as the pressures to which the heart is subjected prior to and during contraction. This work puts forward a platform that creates this capability, by providing a means of dynamically controlling loading conditions in vitro. This dynamic tissue loading platform consists of a thin magnetoresponsive hydrogel cantilever on which 2D engineered myocardial tissue is cultured. Exposing the cantilever to an external magnetic field—generated by positioning magnets at a controlled distance from the cantilever—causes the hydrogel film to stretch, creating tissue load. Next, cell contraction is induced through electrical stimulation, and the force of the contraction is recorded, by measuring the cantilever's deflection. Force–length‐based measurements of contractility are then derived, comparable to clinical measurements. In an illustrative application, the platform is used to measure contractility both in untreated myocardial tissue and in tissue exposed to an inotropic agent. Clear differences are observed between conditions, suggesting that the proposed platform has significant potential to provide clinically relevant measurements of contractility.

Ganglion cells classically respond to either light increase (ON) or decrease (OFF). Here, the authors show that during natural scene stimulation, a single ganglion cell can switch between ON and OFF depending on the visual context.

Chimeric antigen receptor engineering in T cells has been shown to be of great potential therapeutic benefit in a range of immune pathologies, although the functionality of such cell therapies can be limited due to tonic signalling and the induction of dysfunction. Here the authors show transient inhibition of mTOR can rescue their 41-BB-CAR-Tregs from tonic signalling-induced dysfunction.

Naked mole-rats are hypoxia-tolerant mammals, and during hypoxia their body temperature decreases via unknown mechanisms. Here the authors report that the hypoxia-induced body temperature decrease in naked mole rats occurs through decreased brown adipose tissue thermogenesis via decreases in a key thermogenic mitochondrial protein: UCP1.

With the growing threat of antibiotic resistance, unconventional approaches to antimicrobial discovery are needed. Here, the authors present a peptide topology that mimics virus architecture and assembles into antimicrobial capsids that disrupt bacterial membranes upon contact.

In recent years, multielectrode arrays and large silicon probes have been developed to record simultaneously between hundreds and thousands of electrodes packed with a high density. However, they require novel methods to extract the spiking activity of large ensembles of neurons. Here, we developed a new toolbox to sort spikes from these large-scale extracellular data. To validate our method, we performed simultaneous extracellular and loose patch recordings in rodents to obtain ‘ground truth’ data, where the solution to this sorting problem is known for one cell. The performance of our algorithm was always close to the best expected performance, over a broad range of signal-to-noise ratios, in vitro and in vivo. The algorithm is entirely parallelized and has been successfully tested on recordings with up to 4225 electrodes. Our toolbox thus offers a generic solution to sort accurately spikes for up to thousands of electrodes.

We study the elastic scattering time ${\tau }_{{\rm{s}}}$ of ultracold atoms propagating in optical disordered potentials in the strong scattering regime, going beyond the recent work of Richard et al (2019 Phys. Rev. Lett. 122 100403). There, we identified the crossover between the weak and the strong scattering regimes by comparing direct measurements and numerical simulations to the first order Born approximation. Here we focus specifically on the strong scattering regime, where the first order Born approximation is not valid anymore and the scattering time is strongly influenced by the nature of the disorder. To interpret our observations, we connect the scattering time ${\tau }_{{\rm{s}}}$ to the profiles of the spectral functions that we estimate using higher order Born perturbation theory or self-consistent Born approximation. The comparison reveals that self-consistent methods are well suited to describe ${\tau }_{{\rm{s}}}$ for Gaussian-distributed disorder, but fails for laser speckle disorder. For the latter, we show that the peculiar profiles of the spectral functions, as measured independently in Volchkov et al (2018 Phys. Rev. Lett. 120 060404), must be taken into account. Altogether our study characterizes the validity range of usual theoretical methods to predict the elastic scattering time of matter waves, which is essential for future close comparison between theory and experiments, for instance regarding the ongoing studies on Anderson localization.

Hundreds of double homeobox (DUX) genes map within 3.3-kb repeated elements dispersed in the human genome and encode DNA-binding proteins. Among these, we identified DUX4, a potent transcription factor that causes facioscapulohumeral muscular dystrophy (FSHD). In the present study, we performed yeast two-hybrid screens and protein co-purifications with HaloTag-DUX fusions or GST-DUX4 pull-down to identify protein partners of DUX4, DUX4c (which is identical to DUX4 except for the end of the carboxyl terminal domain) and DUX1 (which is limited to the double homeodomain). Unexpectedly, we identified and validated (by co-immunoprecipitation, GST pull-down, co-immunofluorescence and in situ Proximal Ligation Assay) the interaction of DUX4, DUX4c and DUX1 with type III intermediate filament protein desmin in the cytoplasm and at the nuclear periphery. Desmin filaments link adjacent sarcomere at the Z-discs, connect them to sarcolemma proteins and interact with mitochondria. These intermediate filament also contact the nuclear lamina and contribute to positioning of the nuclei. Another Z-disc protein, LMCD1 that contains a LIM domain was also validated as a DUX4 partner. The functionality of DUX4 or DUX4c interactions with cytoplasmic proteins is underscored by the cytoplasmic detection of DUX4/DUX4c upon myoblast fusion. In addition, we identified and validated (by co-immunoprecipitation, co-immunofluorescence and in situ Proximal Ligation Assay) as DUX4/4c partners several RNA-binding proteins such as C1QBP, SRSF9, RBM3, FUS/TLS and SFPQ that are involved in mRNA splicing and translation. FUS and SFPQ are nuclear proteins, however their cytoplasmic translocation was reported in neuronal cells where they associated with ribonucleoparticles (RNPs). Several other validated or identified DUX4/DUX4c partners are also contained in mRNP granules, and the co-localizations with cytoplasmic DAPI-positive spots is in keeping with such an association. Large muscle RNPs were recently shown to exit the nucleus via a novel mechanism of nuclear envelope budding. Following DUX4 or DUX4c overexpression in muscle cell cultures, we observed their association with similar nuclear buds. In conclusion, our study demonstrated unexpected interactions of DUX4/4c with cytoplasmic proteins playing major roles during muscle differentiation. Further investigations are on-going to evaluate whether these interactions play roles during muscle regeneration as previously suggested for DUX4c.

During development, tissue repair, and tumor growth, most blood vessel networks are generated through angiogenesis. Vascular endothelial growth factor (VEGF) is a key regulator of this process and currently both VEGF and its receptors, VEGFR1, VEGFR2, and Neuropilin1 (NRP1), are targeted in therapeutic strategies for vascular disease and cancer. NRP1 is essential for vascular morphogenesis, but how NRP1 functions to guide vascular development has not been completely elucidated. In this study, we generated a mouse line harboring a point mutation in the endogenous Nrp1 locus that selectively abolishes VEGF-NRP1 binding (Nrp1VEGF−). Nrp1VEGF− mutants survive to adulthood with normal vasculature revealing that NRP1 functions independent of VEGF-NRP1 binding during developmental angiogenesis. Moreover, we found that Nrp1-deficient vessels have reduced VEGFR2 surface expression in vivo demonstrating that NRP1 regulates its co-receptor, VEGFR2. Given the resources invested in NRP1-targeted anti-angiogenesis therapies, our results will be integral for developing strategies to re-build vasculature in disease.

Soil invertebrates are known to be much involved in soil behaviour and therefore in the provision of ecosystem services. Functional trait-based approaches are methodologies which can be used to understand soil invertebrates' responses to their environment. They (i) improve the predictions and (ii) are less dependent on space and time. The way traits have been used recently has led to misunderstandings in the integration and interpretation of data. Trait semantics are especially concerned. The aim of this paper is to propose a thesaurus for soil invertebrate trait-based approaches. T-SITA, an Internet platform, is the first initiative to deal with the semantics of traits and ecological preferences for soil invertebrates. It reflects the agreement of a scientific expert community to fix semantic properties (e.g. definition) of approximately 100 traits and ecological preferences. In addition, T-SITA has been successfully linked with a fully operational database of soil invertebrate traits. Such a link enhances data integration and improves the scientific integrity of data.

The filamentous cyanobacterium Arthrospira platensis has a long history of use as a food supply and it has been used by the European Space Agency in the MELiSSA project, an artificial microecosystem which supports life during long-term manned space missions. This study assesses progress in the field of cyanobacterial shotgun proteomics and light/dark diurnal cycles by focusing on Arthrospira platensis. Several fractionation workflows including gel-free and gel-based protein/peptide fractionation procedures were used and combined with LC-MS/MS analysis, enabling the overall identification of 1306 proteins, which represents 21% coverage of the theoretical proteome. A total of 30 proteins were found to be significantly differentially regulated under light/dark growth transition. Interestingly, most of the proteins showing differential abundance were related to photosynthesis, the Calvin cycle and translation processes. A novel aspect and major achievement of this work is the successful improvement of the cyanobacterial proteome coverage using a 3D LC-MS/MS approach, based on an immobilized metal affinity chromatography, a suitable tool that enabled us to eliminate the most abundant protein, the allophycocyanin. We also demonstrated that cell growth follows a light/dark cycle in A. platensis. This preliminary proteomic study has highlighted new characteristics of the Arthrospira platensis proteome in terms of diurnal regulation.

Gammaherpesvirinae, such as the human Epstein-Barr virus (EBV) and the Kaposi's sarcoma associated herpesvirus (KSHV) are highly prevalent pathogens that have been associated with several neoplastic diseases. As EBV and KSHV are host-range specific and replicate poorly in vitro, animal counterparts such as Murid herpesvirus-4 (MuHV-4) have been widely used as models. In this study, we used MuHV-4 in order to improve the knowledge about proteins that compose gammaherpesviruses virions. To this end, MuHV-4 extracellular virions were isolated and structural proteins were identified using liquid chromatography tandem mass spectrometry-based proteomic approaches. These analyses allowed the identification of 31 structural proteins encoded by the MuHV-4 genome which were classified as capsid (8), envelope (9), tegument (13) and unclassified (1) structural proteins. In addition, we estimated the relative abundance of the identified proteins in MuHV-4 virions by using exponentially modified protein abundance index analyses. In parallel, several host proteins were found in purified MuHV-4 virions including Annexin A2. Although Annexin A2 has previously been detected in different virions from various families, its role in the virion remains controversial. Interestingly, despite its relatively high abundance in virions, Annexin A2 was not essential for the growth of MuHV-4 in vitro. Altogether, these results extend previous work aimed at determining the composition of gammaherpesvirus virions and provide novel insights for understanding MuHV-4 biology.

UVB oxidizes proteins through the generation of reactive oxygen species. One consequence of UVB irradiation is carbonylation, the irreversible formation of a carbonyl group on proline, lysine, arginine or threonine residues. In this study, redox proteomics was performed to identify carbonylated proteins in the UVB resistant marine bacterium Photobacterium angustum. Mass-spectrometry was performed with either biotin-labeled or dinitrophenylhydrazide (DNPH) derivatized proteins. The DNPH redox proteomics method enabled the identification of 62 carbonylated proteins (5% of 1221 identified proteins) in cells exposed to UVB or darkness. Eleven carbonylated proteins were quantified and the UVB/dark abundance ratio was determined at both the protein and peptide levels. As a result we determined which functional classes of proteins were carbonylated, which residues were preferentially modified, and what the implications of the carbonylation were for protein function. As the first large scale, shotgun redox proteomics analysis examining carbonylation to be performed on bacteria, our study provides a new level of understanding about the effects of UVB on cellular proteins, and provides a methodology for advancing studies in other biological systems.

Detecting and locating prey are key to predatory success within trophic chains. Predators use various signals through specialized visual, olfactory, auditory or tactile sensory systems to pinpoint their prey. Snakes chemically sense their prey through a highly developed auxiliary olfactory sense organ, the vomeronasal organ (VNO). In natricine snakes that are able to feed on land and water, the VNO plays a critical role in predatory behavior by detecting cues, known as vomodors, which are produced by their potential prey. However, the chemical nature of these cues remains unclear. Recently, we demonstrated that specific proteins-parvalbumins-present in the cutaneous mucus of the common frog (Rana temporaria) may be natural chemoattractive proteins for these snakes. Here, we show that parvalbumins and parvalbumin-like proteins, which are mainly intracellular, are physiologically present in the epidermal mucous cells and mucus of several frog and fish genera from both fresh and salt water. These proteins are located in many tissues and function as Ca(2+) buffers. In addition, we clarified the intrinsic role of parvalbumins present in the cutaneous mucus of amphibians and fishes. We demonstrate that these Ca(2+)-binding proteins participate in innate bacterial defense mechanisms by means of calcium chelation. We show that these parvalbumins are chemoattractive for three different thamnophiine snakes, suggesting that these chemicals play a key role in their prey-recognition mechanism. Therefore, we suggest that recognition of parvalbumin-like proteins or other calcium-binding proteins by the VNO could be a generalized prey-recognition process in snakes. Detecting innate prey defense mechanism compounds may have driven the evolution of this predator-prey interaction.

Facioscapulohumeral muscular dystrophy (FSHD) is a progressive muscle disorder linked to a contraction of the D4Z4 repeat array in the 4q35 subtelomeric region. This deletion induces epigenetic modifications that affect the expression of several genes located in the vicinity. In each D4Z4 element, we identified the double homeobox 4 (DUX4) gene. DUX4 expresses a transcription factor that plays a major role in the development of FSHD through the initiation of a large gene dysregulation cascade that causes myogenic differentiation defects, atrophy and reduced response to oxidative stress. Because miRNAs variably affect mRNA expression, proteomic approaches are required to define the dysregulated pathways in FSHD. In this study, we optimized a differential isotope protein labeling (ICPL) method combined with shotgun proteomic analysis using a gel-free system (2DLC-MS/MS) to study FSHD myotubes. Primary CD56(+) FSHD myoblasts were found to fuse into myotubes presenting various proportions of an atrophic or a disorganized phenotype. To better understand the FSHD myogenic defect, our improved proteomic procedure was used to compare predominantly atrophic or disorganized myotubes to the same matching healthy control. FSHD atrophic myotubes presented decreased structural and contractile muscle components. This phenotype suggests the occurrence of atrophy-associated proteolysis that likely results from the DUX4-mediated gene dysregulation cascade. The skeletal muscle myosin isoforms were decreased while non-muscle myosin complexes were more abundant. In FSHD disorganized myotubes, myosin isoforms were not reduced, and increased proteins were mostly involved in microtubule network organization and myofibrillar remodeling. A common feature of both FSHD myotube phenotypes was the disturbance of several caveolar proteins, such as PTRF and MURC. Taken together, our data suggest changes in trafficking and in the membrane microdomains of FSHD myotubes. Finally, the adjustment of a nuclear fractionation compatible with mass spectrometry allowed us to highlight alterations of proteins involved in mRNA processing and stability.

Dystrophin is a large protein involved in the rare genetic disease Duchenne muscular dystrophy (DMD). It functions as a mechanical linker between the cytoskeleton and the sarcolemma, and is able to resist shear stresses during muscle activity. In all, 75% of the dystrophin molecule consists of a large central rod domain made up of 24 repeat units that share high structural homology with spectrin-like repeats. However, in the absence of any high-resolution structure of these repeats, the molecular basis of dystrophin central domain's functions has not yet been deciphered. In this context, we have performed a computational study of the whole dystrophin central rod domain based on the rational homology modeling of successive and overlapping tandem repeats and the analysis of their surface properties. Each tandem repeat has very specific surface properties that make it unique. However, the repeats share enough electrostatic-surface similarities to be grouped into four separate clusters. Molecular dynamics simulations of four representative tandem repeats reveal specific flexibility or bending properties depending on the repeat sequence. We thus suggest that the dystrophin central rod domain is constituted of seven biologically relevant sub-domains. Our results provide evidence for the role of the dystrophin central rod domain as a scaffold platform with a wide range of surface features and biophysical properties allowing it to interact with its various known partners such as proteins and membrane lipids. This new integrative view is strongly supported by the previous experimental works that investigated the isolated domains and the observed heterogeneity of the severity of dystrophin related pathologies, especially Becker muscular dystrophy.

If an organism's juvenile and adult life stages inhabit different environments, certain traits may need to be independently adapted to each environment. In many organisms, a move to a different environment during ontogeny is accompanied by metamorphosis. In such organisms phenotypic induction early in ontogeny can affect later phenotypes. In laboratory experiments we first investigated correlations between body morphology and the locomotor performance traits expressed in different life stages of the common frog, Rana temporaria: swimming speed and acceleration in tadpoles; and jump-distance in froglets. We then tested for correlations between these performances across life stages. We also subjected tadpoles to unchanging or decreasing water levels to explore whether decreasing water levels might induce any carry-over effects. Body morphology and performance were correlated in tadpoles; morphology and performance were correlated in froglets: hence body shape and morphology affect performance within each life stage. However, performance was decoupled across life stages, as there was no correlation between performance in tadpoles and performance in froglets. While size did not influence tadpole performance, it was correlated with performance of the metamorphosed froglets. Experiencing decreasing water levels accelerated development time, which resulted in smaller tadpoles and froglets, i.e., a carry-over effect. Interestingly, decreasing water levels positively affected the performance of tadpoles, but negatively affected froglet performance. Our results suggest that performance does not necessarily have to be correlated between life stages. However, froglet performance is size dependent and carried over from the tadpole stage, suggesting that some important size-dependent characters cannot be decoupled via metamorphosis.