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ObjectivesThe comparison of treatment efficacy for cerebral cavernous malformations (CCMs) has not yet been well researched.DesignPubMed, Cochrane Library, Science Direct, ISI Web of Science, Embase and additional sources were searched to identify cohort studies about the treatment of CCMs published between 1990 and 2020. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines were followed; the Newcastle-Ottawa Scale was used to assess the risk of bias and to evaluate limitations based on selection/outcome biases. The cumulative incidences with 95% CIs were calculated using the random effects model. The models of Poisson distribution were applied to evaluate risk factors of poorer treatment outcome by calculating rate ratios within 100 person-years with 95% CIs.ResultsA total of 100 cohorts yielding 8994 patients treated for CCMs within 41 098 person-years of follow-up were analysed. The efficacy of ensuring the prevention of haemorrhage was 97% in surgical, 86% in radiosurgical and 77% in the conservative treatment. The lowest mortality (1%) was after radiosurgery, and the highest persistent morbidity (22%) was in natural history series. Deep-seated and brainstem CCMs were associated with higher bleeding rates. Lobar localisation was a protective factor in all analyses. Patients with history of previous haemorrhage were exposed to higher risk of rebleeding. Male gender was a protective factor associated with lower risk of post-treatment haemorrhage.ConclusionsSurgical resection of CCM is effective in ensuring the prevention of haemorrhage with acceptable morbidity and mortality, but conservative and radiosurgical management is a justified treatment alternative. Brainstem and deep-seated CCMs are predominantly associated with higher haemorrhage rates.

Seroconversion after COVID-19 vaccination is impaired in kidney transplant recipients. Emerging variants of concern such as the B.1.617.2 (delta) and the B.1.1.529 (omicron) variants pose an increasing threat to these patients. In this observational cohort study, we measured anti-S1 IgG, surrogate neutralizing, and anti-receptor-binding domain antibodies three weeks after a third mRNA vaccine dose in 49 kidney transplant recipients and compared results to 25 age-matched healthy controls. In addition, vaccine-induced neutralization of SARS-CoV-2 wild-type, the B.1.617.2 (delta), and the B.1.1.529 (omicron) variants was assessed using a live-virus assay. After a third vaccine dose, anti-S1 IgG, surrogate neutralizing, and anti-receptor-binding domain antibodies were significantly lower in kidney transplant recipients compared to healthy controls. Only 29/49 (59%) sera of kidney transplant recipients contained neutralizing antibodies against the SARS-CoV-2 wild-type or the B.1.617.2 (delta) variant and neutralization titers were significantly reduced compared to healthy controls (p < 0.001). Vaccine-induced cross-neutralization of the B.1.1.529 (omicron) variants was detectable in 15/35 (43%) kidney transplant recipients with seropositivity for anti-S1 IgG, surrogate neutralizing, and/or anti-RBD antibodies. Neutralization of the B.1.1.529 (omicron) variants was significantly reduced compared to neutralization of SARS-CoV-2 wild-type or the B.1.617.2 (delta) variant for both, kidney transplant recipients and healthy controls (p < .001 for all)., Dietmar Hopp Stiftung; Heidelberg Faculty of Medicine Rahel Goitein-Straus Program; Heidelberg Faculty of Medicine Physician Scientist Program; German Federal Research Network Applied Surveillance and Testing (BFAST); Network University Medicine; Helmholtz Association Initiative and Networking Fund Project Virological and Immunological Determinants of COVID-19 Pathogenesis

Despite the importance of functional outcome, only a few scoring systems exist to predict neurologic outcome in meningioma surgery. Therefore, our study aims to identify preoperative risk factors and develop the receiver operating characteristics (ROC) models estimating the risk of a new postoperative neurologic deficit and a decrease in Karnofsky performance status (KPS). A multicentric study was conducted in a cohort of 552 consecutive patients with skull base meningiomas who underwent surgical resection from 2014 to 2019. Data were gathered from clinical, surgical, and pathology records as well as radiological diagnostics. The preoperative predictive factors of functional outcome (neurologic deficit, decrease in KPS) were analyzed in univariate and multivariate stepwise selection analyses. Permanent neurologic deficits were present in 73 (13.2%) patients and a postoperative decrease in KPS in 84 (15.2%). Surgery-related mortality was 1.3%. A ROC model was developed to estimate the probability of a new neurologic deficit (area 0.74; SE 0.0284; 95% Wald confidence limits (0.69; 0.80)) based on meningioma location and diameter. Consequently, a ROC model was developed to predict the probability of a postoperative decrease in KPS (area 0.80; SE 0.0289; 95% Wald confidence limits (0.74; 0.85)) based on the patient’s age, meningioma location, diameter, presence of hyperostosis, and dural tail. To ensure an evidence-based therapeutic approach, treatment should be founded on known risk factors, scoring systems, and predictive models. We propose ROC models predicting the functional outcome of skull base meningioma resection based on the age of the patient, meningioma size, and location and the presence of hyperostosis and dural tail.

During intestinal organogenesis, equipotent epithelial progenitors mature into phenotypically distinct stem cells that are responsible for life-long maintenance of the tissue. While the morphological changes associated with the transition are well-characterized, the molecular mechanisms underpinning the maturation process are not fully understood. Here, we leverage intestinal organoid cultures to profile transcriptional, chromatin accessibility, DNA methylation and 3D chromatin conformation landscapes defining fetal and adult epithelial cells. We observed prominent differences in gene expression and enhancer activity, accompanied by changes in 3D organization and local changes in DNA accessibility and methylation, between the two cellular states. Using integrative analyses, we identified sustained YAP transcriptional activity as a major gatekeeper of the immature fetal state. We found the YAP-associated transcriptional network to be regulated at various levels of chromatin organization, and likely to be coordinated by changes in extracellular matrix composition. Altogether, our work highlights the value of unbiased profiling of regulatory landscapes for the identification of key mechanisms underlying tissue maturation.

Expansins facilitate cell expansion by mediating pH-dependent cell wall (CW) loosening. However, the role of expansins in controlling CW biomechanical properties in specific tissues and organs remains elusive. We monitored hormonal responsiveness and spatial specificity of expression and localization of expansins predicted to be the direct targets of cytokinin signaling in Arabidopsis (Arabidopsis thaliana). We found EXPANSIN1 (EXPA1) homogenously distributed throughout the CW of columella/lateral root cap, while EXPA10 and EXPA14 localized predominantly at 3-cell boundaries in the epidermis/cortex in various root zones. EXPA15 revealed cell-type-specific combination of homogenous vs. 3-cell boundaries localization. By comparing Brillouin frequency shift and AFM-measured Young's modulus, we demonstrated Brillouin light scattering (BLS) as a tool suitable for non-invasive in vivo quantitative assessment of CW viscoelasticity. Using both BLS and AFM, we showed that EXPA1 overexpression upregulated CW stiffness in the root transition zone (TZ). The dexamethasone-controlled EXPA1 overexpression induced fast changes in the transcription of numerous CW-associated genes, including several EXPAs and XYLOGLUCAN:XYLOGLUCOSYL TRANSFERASEs (XTHs), and associated with rapid pectin methylesterification determined by in situ Fourier-transform infrared spectroscopy in the root TZ. The EXPA1-induced CW remodeling is associated with the shortening of the root apical meristem, leading to root growth arrest. Based on our results, we propose that expansins control root growth by a delicate orchestration of CW biomechanical properties, possibly regulating both CW loosening and CW remodeling.

Supplementary Methods

Acute leukemias are systemic malignancies associated with a dire outcome. Because of low immunogenicity, leukemias display a remarkable ability to evade immune control and are often resistant to checkpoint blockade. Here, we discover that leukemia cells actively establish a suppressive environment to prevent immune attacks by co-opting a signaling axis that skews macrophages toward a tumor-promoting tissue repair phenotype, namely the GAS6/AXL axis. Using aggressive leukemia models, we demonstrate that ablation of the AXL receptor specifically in macrophages, or its ligand GAS6 in the environment, stimulates antileukemic immunity and elicits effective and lasting natural killer cell– and T cell–dependent immune response against naïve and treatment-resistant leukemia. Remarkably, AXL deficiency in macrophages also enables PD-1 checkpoint blockade in PD-1–refractory leukemias. Finally, we provide proof-of-concept that a clinical-grade AXL inhibitor can be used in combination with standard-of-care therapy to cure established leukemia, regardless of AXL expression in malignant cells.Significance:Alternatively primed myeloid cells predict negative outcome in leukemia. By demonstrating that leukemia cells actively evade immune control by engaging AXL receptor tyrosine kinase in macrophages and promoting their alternative priming, we identified a target which blockade, using a clinical-grade inhibitor, is vital to unleashing the therapeutic potential of myeloid-centered immunotherapy.This article is highlighted in the In This Issue feature, p. 2659

Supplementary Figures and Figure legends

We present a rare case of cerebral venous sinus thrombosis in a COVID-19-positive, 2-month-old infant, to this day the youngest described patient with this rare combination of findings. He was hospitalized with focal seizures. The first brain imaging showed subdural hematoma and focal ischemic changes. The subdural hematoma was successfully evacuated. The control imaging, done due to lethargy, showed an extensive cerebral venous sinus thrombosis. The thrombosis was managed with low molecular weight heparin leading to clinical and radiological improvement. With this case report, we would like to add to the information pool of COVID-19 neurological manifestations in children, particularly those younger than 1 year.

Endocrine treatment regimens for breast cancer that target the estrogen receptor-α (ERα) are effective, but acquired resistance remains a limiting drawback. One mechanism of acquired resistance that has been hypothesized is functional substitution of the orphan receptor estrogen-related receptor-α (ERRα) for ERα. To examine this hypothesis, we analyzed ERRα and ERα in recurrent tamoxifen-resistant breast tumors and conducted a genome-wide target gene profiling analysis of MCF-7 breast cancer cell populations that were sensitive or resistant to tamoxifen treatment. This analysis uncovered a global redirection in the target genes controlled by ERα, ERRα, and their coactivator AIB1, defining a novel set of target genes in tamoxifen-resistant cells. Beyond differences in the ERα and ERRα target gene repertoires, both factors were engaged in similar pathobiologic processes relevant to acquired resistance. Functional analyses confirmed a requirement for ERRα in tamoxifen- and fulvestrant-resistant MCF-7 cells, with pharmacologic inhibition of ERRα sufficient to partly restore sensitivity to antiestrogens. In clinical specimens (n = 1041), increased expression of ERRα was associated with enhanced proliferation and aggressive disease parameters, including increased levels of p53 in ERα-positive cases. In addition, increased ERRα expression was linked to reduced overall survival in independent tamoxifen-treated patient cohorts. Taken together, our results suggest that ERα and ERRα cooperate to promote endocrine resistance, and they provide a rationale for the exploration of ERRα as a candidate drug target to treat endocrine-resistant breast cancer. Cancer Res; 75(4); 720–31. ©2015 AACR.

Supplementary Figure S6. Immunohistochemical stainings of ERRalpha in primary ERalpha-positive breast carcinomas on a tissue microarray (n=1041).

Supplementary Figure S3. ChIP-Seq validation by qRT-PCR.

Supplementary Figure S4. AIB1 in tamoxifen- and fulvestrant-resistant breast cancer cells.

Supplementary Tables S1-10. Supplementary Table S1: Clinico-pathological features of primary treatment-naive and relapsed tamoxifen-resistant breast carcinomas assessed by routine pathological evaluation. Supplementary Table S2: Antibodies for Western blot analyses, ChIP and immunohisto-chemistry. Supplementary Table S3: Primer sequences for qRT-PCR (5'-3'). Supplementary Table S4 : p-values. Supplementary Table S5: Top differentially expressed transcripts between MCF-7 #Wt and #TamR. Supplementary Table S6: ERalpha, ERRalpha and AIB1 target genes identified by ChIP-Seq and gene expression profiling (EP) in MCF-7 #Wt vs. # TamR cells. Supplementary Table S7: ERalpha and ERRalpha target genes identified by ChIP-Seq and gene expression profiling in MCF-7 cells. Supplemetary Table S8: Enrichment of functional categories for ERalpha and ERRalpha target genes in #TamR. Supplementary Table S9: Pairwise relationship between ERRalpha and co-variables in ERalpha-positive breast carcinomas (tumor set III, n=1041). Supplementary Table S10: Effects of co-variables on patient survival in tamoxifen-treated primary breast tumors (GSE9893) stratified to grade and PR status.

Supplementary Figure S2. Chromatin immunoprecipitation (ChIP).

Supplementary Material and Methods, Figure Legends S1-6. Supplementary Material and Methods, Figure Legends S1-6.

Supplementary Figure S5. Co-administration of anti-estrogens upon lentiviral-mediated ERRα knockdown.

N-Glycanase 1 (NGLY1) deficiency is a rare and complex genetic disorder. Although recent studies have shed light on the molecular underpinnings of NGLY1 deficiency, a systematic characterization of gene and protein expression changes in patient-derived cells has been lacking. Here, we performed RNA-sequencing and mass spectrometry to determine the transcriptomes and proteomes of 66 cell lines representing four different cell types derived from 14 NGLY1 deficient patients and 17 controls. Although NGLY1 protein levels were up to 9.5-fold downregulated in patients compared with parents, residual and likely non-functional NGLY1 protein was detectable in all patient-derived lymphoblastoid cell lines. Consistent with the role of NGLY1 as a regulator of the transcription factor Nrf1, we observed a cell type-independent downregulation of proteasomal genes in NGLY1 deficient cells. In contrast, genes involved in ribosome biogenesis and mRNA processing were upregulated in multiple cell types. In addition, we observed cell type-specific effects. For example, genes and proteins involved in glutathione synthesis, such as the glutamate-cysteine ligase subunits GCLC and GCLM, were downregulated specifically in lymphoblastoid cells. We provide a web application that enables access to all results generated in this study at https://apps.embl.de/ngly1browser. This resource will guide future studies of NGLY1 deficiency in directions that are most relevant to patients.

Control of gene expression in specific tissues and/or at certain stages of development allows the study and manipulation of gene function with high precision. Site-specific genome recombination by the flippase (FLP) and cyclization recombination (Cre) enzymes has proved particularly relevant. Joint efforts of many research groups have led to the creation of efficient FLP and Cre drivers to regulate gene expression in a variety of tissues in Caenorhabditis elegans. Here, we extend this toolkit by the addition of FLP lines that drive recombination specifically in distal tip cells, the somatic gonad, coelomocytes, and the epithelial P lineage. In some cases, recombination-mediated gene knockouts do not completely deplete protein levels due to persistence of long-lived proteins. To overcome this, we developed a spatiotemporally regulated degradation system for green fluorescent fusion proteins based on FLP-mediated recombination. Using 2 stable nuclear pore proteins, MEL-28/ELYS and NPP-2/NUP85 as examples, we report the benefit of combining tissue-specific gene knockout and protein degradation to achieve complete protein depletion. We also demonstrate that FLP-mediated recombination can be utilized to identify transcriptomes in a C. elegans tissue of interest. We have adapted RNA polymerase DamID for the FLP toolbox and by focusing on a well-characterized tissue, the hypodermis, we show that the vast majority of genes identified by RNA polymerase DamID are known to be expressed in this tissue. These tools allow combining FLP activity for simultaneous gene inactivation and transcriptomic profiling, thus enabling the inquiry of gene function in various complex biological processes., This project was funded by the Spanish State Research Agency, the European Union, and the European Regional Development Fund (BFU2016-79313-P, CEX2020-001088-M, and PID2019-105069GB-I00; doi:10.13039/501100011033).

Control of gene expression in specific tissues and/or at certain stages of development allows the study and manipulation of gene function with high precision. Site-specific genome recombination by the Flippase (FLP) and Cre enzymes has proven particularly relevant. Joint efforts of many research groups have led to the creation of efficient FLP and Cre drivers to regulate gene expression in a variety of tissues in Caenorhabditis elegans. Here, we extend this toolkit by the addition of FLP lines that drive recombination specifically in distal tip cells, the somatic gonad, coelomocytes and the epithelial P lineage. In some cases, recombination-mediated gene knockouts do not completely deplete protein levels due to persistence of long-lived proteins. To overcome this, we developed a spatiotemporally regulated degradation system for GFP fusion proteins (GFPdeg) based on FLP-mediated recombination. Using two stable nuclear pore proteins, MEL-28/ELYS and NPP-2/NUP85 as examples, we report the benefit of combining tissue-specific gene knockout and protein degradation to achieve complete protein depletion. We also demonstrate that FLP-mediated recombination can be utilized to identify transcriptomes in a C. elegans tissue of interest. We have adapted RNA polymerase DamID (RAPID) for the FLP toolbox and by focusing on a well-characterized tissue, the hypodermis, we show that the vast majority of genes identified by RAPID are known to be expressed in this tissue. These tools allow combining FLP activity for simultaneous gene inactivation and transcriptomic profiling, thus enabling the inquiry of gene function in various complex biological processes.

Gliomas are the most common central nervous tumors in children and adolescents. However, spinal cord low-grade gliomas (sLGGs) are rare, with scarce information on tumor genomics and epigenomics. To define the molecular landscape of sLGGs, we integrated clinical data, histology, and multi-level genetic and epigenetic analyses on a consecutive cohort of 26 pediatric patients. Driver molecular alteration was found in 92% of patients (24/26). A novel variant of KIAA1549:BRAF fusion (ex10:ex9) was identified using RNA-seq in four cases. Importantly, only one-third of oncogenic drivers could be revealed using standard diagnostic methods, and two-thirds of pediatric patients with sLGGs required extensive molecular examination. The majority (23/24) of detected alterations were potentially druggable targets. Four patients in our cohort received targeted therapy with MEK or NTRK inhibitors. Three of those exhibited clinical improvement (two with trametinib, one with larotrectinib), and two patients achieved partial response. Methylation profiling was implemented to further refine the diagnosis and revealed intertumoral heterogeneity in sLGGs. Although 55% of tumors clustered with pilocytic astrocytoma, other rare entities were identified in this patient population. In particular, diffuse leptomeningeal glioneuronal tumors (n = 3) and high-grade astrocytoma with piloid features (n = 1) and pleomorphic xanthoastrocytoma (n = 1) were present. A proportion of tumors (14%) had no match with the current version of the classifier. Complex molecular genetic sLGGs characterization was invaluable to refine diagnosis, which has proven to be essential in such a rare tumor entity. Moreover, identifying a high proportion of drugable targets in sLGGs opened an opportunity for new treatment modalities.

Inter-patient variability and the similarity of healthy and leukemic stem cells have impeded the characterization of leukemic stem cells (LSCs) in acute myeloid leukemia (AML), and their differentiation landscape. Here, we introduce CloneTracer, a novel method that adds clonal resolution to single-cell RNA-seq datasets. Applied to samples from 19 AML patients, CloneTracer revealed routes of leukemic differentiation. While residual healthy cells dominated the dormant stem cell compartment, active leukemic stem cells resembled their healthy counterpart and retained erythroid capacity. By contrast, downstream myeloid progenitors were highly aberrant and constituted the disease-defining compartment: Their gene expression and differentiation state determined both chemotherapy response and the leukemia’s ability to differentiate to transcriptomically normal monocytes. Finally, we demonstrated the potential of CloneTracer to identify surface markers mis-regulated specifically in leukemic cells by intra-patient comparisons. Taken together, CloneTracer revealed a differentiation landscape that mimics its healthy counterpart and determines biology and therapy response in AML.

BackgroundAxial Spondyloarthritis can lead to significant disability and impairment in quality of life. TNF inhibitors are recommended to patients enduring active disease despite conventional treatment. Nonetheless, up to 40% of patients of patients fail to respond to TNF inhibitors. In this context, it is important to identify as early as possible patients highly likely to respond. This study aims at identifying, among axial spondyloarthritis patients undergoing treatment with the TNF inhibitor adalimumab, early molecular biomarkers differentiating good responders from non-responders after 14 weeks of treatment, as measured by ASAS20.MethodsPeripheral blood RNA sequencing and serum proteins measured by mass spectrometry were evaluated in a cohort of biologic naïve axial spondyloarthritis patients (n = 35), before (baseline) and after (3-5 days, 2 weeks and 14 weeks) treatment with adalimumab. Results from differential expression analysis were used in combination with clinical data to build logistic regression models and random forest models to predict response to adalimumab at baseline.ResultsResponders to adalimumab presented higher levels of markers of innate immunity at baseline, mostly related with neutrophils, and lower levels of adaptive immunity markers, particularly B-cells. A logistic regression model incorporating ASDAS-CRP and AFF3, the top differentially expressed gene between responders and non-responders at baseline, enabled an accurate prediction of response to adalimumab in our cohort (AUC=0.96), with random forest models suggesting 80% predictive accuracy. A treatment-associated signature suggests a reduction in inflammatory activity, with C-reactive protein and Haptoglobin showing strong and early decrease in the serum of axial spondyloarthritis patients, while a cluster of apolipoproteins showed increased expression at week 14.ConclusionsDifferences in disease activity and/or blood innate/adaptive immune cell type composition at baseline may be a major contributor to response to adalimumab in axial spondyloarthritis, where a model including clinical and blood gene expression variables shows high predictive power. Our results suggest novel molecular biomarkers of response to adalimumab at baseline.Trial registrationAxial spondyloarthritis patients were selected from participants of the Bioefficacy study - Biomarkers Identification of Anti-TNFα Agent’s Efficacy in Ankylosing Spondylitis Patients Using a Transcriptome Analysis and Mass Spectrometry (clinical trials.gov identifier NCT02492217).

The utility for liquid biopsy of tumor-associated circulating single-nucleotide variants, as opposed to mutations, of the mitochondrial (mt) and nuclear genomes in neuroblastoma (NB) is unknown.Variants of the mt and nuclear genomes from tumor, blood cells, and consecutive plasma samples of five patients with metastatic NB that relapsed or progressed were analyzed. Targeted parallel sequencing results of the mt genome, and of the coding region of 139 nuclear genes and 22 miRNAs implicated in NB, were correlated with clinical imaging and laboratory data.All tumors harbored multiple somatic mt and nuclear single nucleotide variants with low allelic frequency, most of them not detected in the circulation. In one patient a tumor-associated mt somatic variant was detected in the plasma before and during progressive disease. In a second patient a circulating nuclear tumor-associated DNA variant heralded clinical relapse. In all patients somatic mt and nuclear variants not evident in the tumor biopsy at time of diagnosis were found circulating at varying timepoints. This suggests either tumor heterogeneity, evolution of tumor variants or a confounding contribution of normal tissues to somatic variants in patient plasma. The number and allelic frequency of the circulating variants did not reflect the clinical course of the tumors. Mutational signatures of mt and nuclear somatic variants differed. They varied between patients and were detected in the circulation without mirroring the patients' course.In this limited cohort of NB patients clinically informative tumor-associated mt and nuclear circulating variants were detected by targeted parallel sequencing in a minority of patients.

DSA (digital subtraction angiography) is the gold standard for measuring carotid artery stenosis (CS). Yet, the correlation between DSA and stenosis is not well documented.We compared CS as measured by DSA to carotid artery specimens obtained from carotid endarterectomy surgery. Patients were divided into three groups according to NASCET criteria (North American Symptomatic Carotid Endarterectomy Trial): stenosis of 30-49% (mild), stenosis of 50-69% (moderate), and stenosis of 70-99% (severe).This prospective cohort study involved 644 patients. The mean stenosis in the mild stenosis group (n = 128 patients) was 54% ECST (European Carotid Surgery Trial), 40% NASCET, and 72% ESs (endarterectomy specimens). The mean absolute difference between ECST and NASCET was 14%. The mean stenosis in the moderate stenosis group (n = 347 patients) was 66% ECST, 60% NASCET, and 77% ES. The mean absolute difference between ECST and NASCET was 6%. The mean stenosis in the severe group (n = 169 patients) was 80% ECST, 76% NASCET, and 79% ES. No significant correlation coefficients were found between DSA and ES methods. In the mild group, the CC was 0.16 (ESCT) and 0.13 (NASCET); in the moderate group, the CC was 0.05 (ESCT) and 0.01 (NASCET); and in the severe group, the CC was 0.23 (ESCT) and 0.10 (NASCET). For all groups combined, CC was 0.22 for the ECST and 0.20 for the NASCET method.The relationship between DSA and ES methods to measure CS is almost random. This lack of a relationship between the DSA and ES techniques questions the validity of current DSA-based guidelines.

Nanoscale zero-valent iron (nZVI) has increasingly been applied to remediate aquifers polluted by organochlorines or heavy metals. As a result, bacteria in the vicinity of remediate action can be stressed by surplus iron released from nZVI. However, the understanding of the iron stress defense pathways during this process is currently incomplete. Therefore, we aimed to elucidate the physiological and transcriptomic response of the bacterium, Pseudomonas putida NCTC 10936, to 100 mg/L of nZVI and 44.5 µg/L of dissolved iron obtained from nZVI suspension. Cell viability was neither affected by nZVI nor dissolved iron, although the dissolved iron caused stress that altered the cell physiology and caused the generation of smaller cells, whereas cells were elongated in the presence of nZVI. Transcriptomic analysis confirmed the observed stronger physiological effect caused by dissolved iron (in total 3839 differentially expressed genes [DEGs]) than by nZVI (945 DEGs). Dissolved iron (but not nZVI) activated genes involved in oxidative stress-related pathways, antioxidant activity, carbohydrate and energy metabolism, but downregulated genes associated with flagellar assembly proteins and two-component systems involved in sensing external stimuli. As a result, bacteria very effectively faced oxidative insults and cell viability was not affected.

A novel actinobacterial strain, designated 15TR583T, was isolated from a waterlogged acidic soil collected near the town of Trebon, Czech Republic, and was subjected to a polyphasic taxonomic characterization. Phylogenetic analysis based on 16S rRNA gene and whole-genome sequences revealed that the organism forms an individual line of descent related to the orderStreptosporangiales, classActinomycetia. The strain shared highest 16S rRNA gene sequence similarity, yet of only 92.8%, withActinocorallia aureaIFO 14752T. The strain grew in white colonies of aerobic, Gram-stain-positive, unbranching substrate mycelium bearing single spores at hyphae tips. The major fatty acids (>10%) were iso-C16 : 0, C16 : 0, iso-C17 : 1ω9 and 10-methyl-C17 : 0. The fatty acid pattern differed from all patterns currently described for actinobacterial genera. The organism contained as major menaquinones MK9(H6) and MK9(H8), which differentiated it from other actinobacterial families. Polar lipids were composed of six unidentified glycolipids, an unidentified phosphoglycolipid, two unidentified phospholipids and two unidentified aminolipids. Whole-cell sugars contained galactose, xylose and arabinose as major components. The peptidoglycan type was A1γmeso-diaminopimelic acid. The genomic DNA G+C content was 69.7 mol%. The distinct phylogenetic position and unusual combination of chemotaxonomic characteristics justify the proposal ofTreboniagen. nov., with the type speciesTrebonia kvetiisp. nov. (type strain 15TR583T=CCM 8942T=DSM 109105T), withinTreboniaceaefam. nov.

Dysregulation in type I IFN and IFN-stimulated genes (ISGs) induced by monocytes is one of the key features of systemic sclerosis (SSc) pathogenesis. Abnormalities in microRNA (miRNA) expression are related to excessive IFN production, however the role of miRNA remains largely elusive in SSc monocytes. This study explores global miRNA-mRNA profiling of SSc monocytes and functional attenuation of IFN and ISGs by specific miRNAs. Global sequencing of mRNA (mRNA-seq) and miRNA (miRNA-seq) samples were performed simultaneously on healthy controls and SSc monocytes. Following computational analysis, selected miRNAs-mRNA candidates were validated, correlated with clinical parameters, and tested by functional assays. Transcriptomics data and qPCR analysis confirmed IFN signature in SSc but not in rheumatoid arthritis monocytes. Based on miRNA-seq analysis, five miRNAs were selected for further validation. Only the expression patterns of miRNA-26a-2-3p and miRNA-485-3p were confirmed and negatively correlated with clinical parameters. Exogenous delivery of miRNA-26a-2-3p to TLR-stimulated monocytic THP-1 cells specifically inhibited ISGs but not inflammasome activity in functional assays. In conclusion, our miRNA-mRNA co-sequencing and functional analysis identify miRNA-26a-2-3p as a new candidate, which is predicated to negatively regulate ISGs. This implies that reduced expression of miRNA-26a-2-3 may be involved in pathogenic IFN signature in SSc monocytes.

Evidence accumulates that the functional plasticity of insulin and insulin-like growth factor signaling in insects could spring, among others, from the multiplicity of insulin receptors (InRs). Their multiple variants may be implemented in the control of insect polyphenism, such as wing or caste polyphenism. Here, we present a comprehensive phylogenetic analysis of insect InR sequences in 118 species from 23 orders and investigate the role of three InRs identified in the linden bug, Pyrrhocoris apterus, in wing polymorphism control. We identified two gene clusters (Clusters I and II) resulting from an ancestral duplication in a late ancestor of winged insects, which remained conserved in most lineages, only in some of them being subject to further duplications or losses. One remarkable yet neglected feature of InR evolution is the loss of the tyrosine kinase catalytic domain, giving rise to decoys of InR in both clusters. Within the Cluster I, we confirmed the presence of the secreted decoy of insulin receptor in all studied Muscomorpha. More importantly, we described a new tyrosine kinase-less gene (DR2) in the Cluster II, conserved in apical Holometabola for ∼300 My. We differentially silenced the three P. apterus InRs and confirmed their participation in wing polymorphism control. We observed a pattern of Cluster I and Cluster II InRs impact on wing development, which differed from that postulated in planthoppers, suggesting an independent establishment of insulin/insulin-like growth factor signaling control over wing development, leading to idiosyncrasies in the co-option of multiple InRs in polyphenism control in different taxa.

Patients with chronic obstructive pulmonary disease (COPD) are still waiting for curative treatments. Considering the environmental cause of COPD (e.g., cigarette smoke) and disease phenotypes, including stem-cell senescence and impaired differentiation, we hypothesized that COPD will be associated with altered epigenetic signaling in lung cells. We generated genome-wide DNA methylation maps at single CpG resolution of primary human lung fibroblasts (HLFs) isolated from distal parenchyma of ex-smoker controls and COPD patients, with both mild and severe disease. The epigenetic landscape is markedly changed in lung fibroblasts across COPD stages, with DNA methylation changes occurring predominantly in regulatory regions, including promoters and enhancers. RNA sequencing of matched fibroblasts demonstrated dysregulation of genes involved in proliferation, DNA repair, and extracellular matrix organization. Notably, we identified epigenetic and transcriptional dysregulation already in mild COPD patients, providing unique insights into early disease. Integration of profiling data identified 110 candidate regulators of disease phenotypes, including epigenetic factors. Using phenotypic screens, we verified the regulator capacity of multiple candidates and linked them to repair processes in the human lung.Our study provides first integrative high-resolution epigenetic and transcriptomic maps of human lung fibroblasts across stages of COPD. We reveal novel transcriptomic and epigenetic signatures associated with COPD onset and progression and identify new candidate regulators involved in the pathogenesis of chronic respiratory diseases. The presence of various epigenetic factors among the candidates demonstrates that epigenetic regulation in COPD is an exciting research field that holds promise for novel therapeutic avenues for patients.

Purpose About 15% of patients with common variable immunodeficiency (CVID) develop a small intestinal enteropathy, which resembles celiac disease with regard to histopathology but evolves from a distinct, poorly defined pathogenesis that has been linked in some cases to chronic norovirus (NV) infection. Interferon-driven inflammation is a prominent feature of CVID enteropathy, but it remains unknown how NV infection may contribute. Methods Duodenal biopsies of CVID patients, stratified according to the presence of villous atrophy (VA), IgA plasma cells (PCs), and chronic NV infection, were investigated by flow cytometry, multi-epitope-ligand cartography, bulk RNA-sequencing, and RT-qPCR of genes of interest. Results VA development was connected to the lack of intestinal (IgA+) PC, a T helper 1/T helper 17 cell imbalance, and increased recruitment of granzyme+CD8+ T cells and pro-inflammatory macrophages to the affected site. A mixed interferon type I/III and II signature occurred already in the absence of histopathological changes and increased with the severity of the disease and in the absence of (IgA+) PCs. Chronic NV infection exacerbated this signature when compared to stage-matched NV-negative samples. Conclusions Our study suggests that increased IFN signaling and T-cell cytotoxicity are present already in mild and are aggravated in severe stages (VA) of CVID enteropathy. NV infection preempts local high IFN-driven inflammation, usually only seen in VA, at milder disease stages. Thus, revealing the impact of different drivers of the pathological mixed IFN type I/III and II signature may allow for more targeted treatment strategies in CVID enteropathy and supports the goal of viral elimination. Graphical abstract

Additional file 1: Table S1. Table of primers used to validate the fusion transcripts identified by RNA sequencing.

Additional file 2: Table S2. Table showing the complete sLGGs cohort emphasizing the original histology before molecular pathology reevaluation, anatomical location, and molecular-biology data.

Control of gene expression in specific tissues and/or at certain stages of development allows the study and manipulation of gene function with high precision. Site-specific genome recombination by the Flippase (FLP) and Cre enzymes has proven particularly relevant. Joint efforts of many research groups have led to the creation of efficient FLP and Cre drivers to regulate gene expression in a variety of tissues in Caenorhabditis elegans. Here, we extend this toolkit by the addition of FLP lines that drive recombination specifically in distal tip cells, the somatic gonad, coelomocytes and the epithelial P lineage. In some cases, recombination-mediated gene knockouts do not completely deplete protein levels due to persistence of long-lived proteins. To overcome this, we developed a spatiotemporally regulated degradation system for GFP fusion proteins (GFPdeg) based on FLP-mediated recombination. Using two stable nuclear pore proteins, MEL-28/ELYS and NPP-2/NUP85 as examples, we report the benefit of combining tissue-specific gene knockout and protein degradation to achieve complete protein depletion. We also demonstrate that FLP-mediated recombination can be utilized to identify transcriptomes in a C. elegans tissue of interest. We have adapted RNA polymerase DamID (RAPID) for the FLP toolbox and by focusing on a well-characterized tissue, the hypodermis, we show that the vast majority of genes identified by RAPID are known to be expressed in this tissue. These tools allow combining FLP activity for simultaneous gene inactivation and transcriptomic profiling, thus enabling the inquiry of gene function in various complex biological processes.

The purpose of the Maleth Program, also known as Project Maleth, is Malta's first space program to evaluate human skin tissue microbiome changes in type 2 diabetes mellitus (T2DM) patients afflicted with diabetic foot ulcers (DFU). This was carried out in both ground-based models and spaceflight. The first mission (Maleth I) under this program was carried out to uncover the effects of spaceflight, microgravity and radiation on human skin tissue microbiome samples from six T2DM patients recruited into the study. Each patient human skin tissue sample was split in three, with one section processed immediately for genomic profiling by 16S typing and the rest were processed for longer term ground-control and spaceflight experiments. Ground-control and spaceflight human skin tissue samples were also processed for genomic profiling upon mission re-entry and completion. Maleth I's overall objective was achieved, as human skin tissue samples with their microbiomes travelled to space and back yielding positive results by both standard microbiology techniques and genetic typing using 16S rRNA amplicon sequencing. Preliminary findings of this mission are discussed in light of its innovative approach at DFU microbiome research, and the clinical implications that may emerge from this and other future similar studies.

BACKGROUND: Childhood thalamopeduncular gliomas arise at the interface of thalamus and cerebral peduncle. The optimal treatment is total resection but not at the cost of neurological function. We present long-term clinical and oncological outcomes of maximal safe resection. METHODS: Retrospective review of prospectively collected data: demography, symptomatology, imaging, extent of resection, surgical complications, histology, functional and oncological outcome. RESULTS: During 16-year period (2005-20) 21 patients were treated at our institution. These were 13 girls and 8 boys (mean age 7.6 years). Presentation included progressive hemiparesis in 9 patients, raised intracranial pressure in 9 patients and cerebellar symptomatology in 3 patients. The tumor was confined to the thalamus in 6 cases. Extent of resection was judged on postoperative imaging as total (6), near-total (6) and less extensive (9). Surgical complications included progression of baseline neurological status in 6 patients, 5 of these gradually improved to preoperative status. All tumors were classified as low-grade gliomas. Disease progression was observed in 9 patients (median progression free survival 7.3 years). At last follow-up (median 6.1 years) all patients were alive; median Lansky score of 90. Seven patients were without evidence of disease, 6 had stable disease, 7 stable following progression and 1 had progressive disease managed expectantly. CONCLUSION: Pediatric patients with low grade thalamopeduncular gliomas have excellent long-term functional and oncological outcomes even when gross total resection is not achievable. Surgery should aim at total resection; however neurological function should not be endangered due to excellent chance for long-term survival.

Anti-cancer therapies often exhibit only short-term effects. Tumors typically develop drug resistance causing relapses that might be tackled with drug combinations. Identification of the right combination is challenging and would benefit from high-content, high-throughput combinatorial screens directly on patient biopsies. However, such screens require a large amount of material, normally not available from patients. To address these challenges, we developed a scalable microfluidic workflow to screen hundreds of drug combinations in picoliter-size droplets using transcriptome changes as a readout for drug effects. We devised a deterministic combinatorial DNA barcoding approach to encode treatment conditions, enabling the gene expression-based readout of drug effects in a highly multiplexed fashion. We applied our method to screen the effect of 420 drug combinations on the transcriptome of K562 cells using only ∼250 single cell droplets per condition, to successfully predict synergistic and antagonistic drug pairs, as well as their pathway activities.

N-Glycanase 1 (NGLY1) deficiency is a rare and complex genetic disorder. Although recent studies have shed light on the molecular underpinnings of NGLY1 deficiency, a systematic characterization of gene and protein expression changes in patient-derived cells has been lacking. Here, we performed RNA-sequencing and mass spectrometry to determine the transcriptomes and proteomes of 66 cell lines representing 4 different cell types derived from 14 NGLY1 deficient patients and 17 controls. While gene and protein expression levels agreed well with each other, expression differences were more pronounced at the protein level. Although NGLY1 protein levels were up to 9.5-fold downregulated in patients compared to parent controls, depending on the genotype, NGLY1 protein was still detectable in all patient-derived lymphoblastoid cell lines. Consistent with the role of NGLY1 as a regulator of the transcription factor Nrf1, we observed a cell type-independent downregulation of proteasomal genes in NGLY1 deficient cells. In contrast, genes involved in ribosomal mRNA processing were upregulated in multiple cell types. In addition, we observed cell type-specific effects. For example, genes and proteins involved in glutathione synthesis, such as the glutamate-cystein ligase subunits GCLC and GCLM, were downregulated specifically in lymphoblastoid cells. We provide a web application that enables access to all results generated in this study at https://apps.embl.de/ngly1browser. This resource will guide future studies of NGLY1 deficiency in directions that are most relevant to patients.

Funder: Max-Planck-Gesellschaft (Max Planck Society); doi: https://doi.org/10.13039/501100004189, During the co-translational assembly of protein complexes, a fully synthesized subunit engages with the nascent chain of a newly synthesized interaction partner. Such events are thought to contribute to productive assembly, but their exact physiological relevance remains underexplored. Here, we examine structural motifs contained in nucleoporins for their potential to facilitate co-translational assembly. We experimentally test candidate structural motifs and identify several previously unknown co-translational interactions. We demonstrate by selective ribosome profiling that domain invasion motifs of beta-propellers, coiled-coils, and short linear motifs may act as co-translational assembly domains. Such motifs are often contained in proteins that are members of multiple complexes (moonlighters) and engage with closely related paralogs. Surprisingly, moonlighters and paralogs assemble co-translationally in only some but not all of the relevant biogenesis pathways. Our results highlight the regulatory complexity of assembly pathways.

Hybridization and polyploidization are important evolutionary processes whose impacts range from the alteration of gene expression and phenotypic variation to the triggering of asexual reproduction. We investigated fishes of the Cobitis taenia-elongatoides hybrid complex, which allowed us to disentangle the direct effects of both processes, due to the co-occurrence of parental species with their diploid and triploid hybrids. Employing morphological, ecological, and RNAseq approaches, we investigated the molecular determinants of hybrid and polyploid forms. In contrast with other studies, hybridization and polyploidy induced relatively very little transgressivity. Instead, Cobitis hybrids appeared intermediate with a clear effect of genomic dosing when triploids expressed higher similarity to the parent contributing two genome sets. This dosage effect was symmetric in the germline (oocyte gene expression), interestingly though, we observed an overall bias toward C. taenia in somatic tissues and traits. At the level of individual genes, expression-level dominance vastly prevailed over additivity or transgressivity. Also, trans-regulation of gene expression was less efficient in diploid hybrids than in triploids, where the expression modulation of homoeologs derived from the “haploid” parent was stronger than those derived from the “diploid” parent. Our findings suggest that the apparent intermediacy of hybrid phenotypes results from the combination of individual genes with dominant expression rather than from simple additivity. The efficiency of cross-talk between trans-regulatory elements further appears dosage dependent. Important effects of polyploidization may thus stem from changes in relative concentrations of trans-regulatory elements and their binding sites between hybridizing genomes. Links between gene regulation and asexuality are discussed.

The cytosolic accumulation of mitochondrial precursors is hazardous to cellular fitness and is associated with a number of diseases. However, it is not observed under physiological conditions. Individual mechanisms that allow cells to avoid cytosolic accumulation of mitochondrial precursors have recently been discovered, but their interplay and regulation remain elusive. Here, we show that cells rapidly launch a global transcriptional programme to restore cellular proteostasis after induction of a 'clogger' protein that reduces the number of available mitochondrial import sites. Cells upregulate the protein folding and proteolytic systems in the cytosol and downregulate both the cytosolic translation machinery and many mitochondrial metabolic enzymes, presumably to relieve the workload of the overstrained mitochondrial import system. We show that this transcriptional remodelling is a combination of a 'wideband' core response regulated by the transcription factors Hsf1 and Rpn4 and a unique mitoprotein-induced downregulation of the oxidative phosphorylation components, mediated by an inactivation of the HAP complex.

Natural light environments are highly variable. Flexible adjustment between light energy utilization and photoprotection is therefore of vital importance for plant performance and fitness in the field. Short-term reactions to changing light intensity are triggered inside chloroplasts and leaves within seconds to minutes, whereas long-term adjustments proceed over hours and days, integrating multiple signals. While the mechanisms of long-term acclimation to light intensity have been studied by changing constant growth light intensity during the day, responses to fluctuating growth light intensity have rarely been inspected in detail. We performed transcriptome profiling in Arabidopsis (Arabidopsis thaliana) leaves to investigate long-term gene expression responses to fluctuating light (FL). In particular, we examined whether responses differ between young and mature leaves or between morning and the end of the day. Our results highlight global reprogramming of gene expression under FL, including that of genes related to photoprotection, photosynthesis, and photorespiration and to pigment, prenylquinone, and vitamin metabolism. The FL-induced changes in gene expression varied between young and mature leaves at the same time point and between the same leaves in the morning and at the end of the day, indicating interactions of FL acclimation with leaf development stage and time of day. Only 46 genes were up- or down-regulated in both young and mature leaves at both time points. Combined analyses of gene coexpression and cis-elements pointed to a role of the circadian clock and light in coordinating the acclimatory responses of functionally related genes. Our results also suggest a possible cross talk between FL acclimation and systemic acquired resistance-like gene expression in young leaves.

Background Sepsis is associated with high platelet turnover and elevated levels of immature platelets. Changes in the platelet transcriptome and the specific impact of immature platelets on the platelet transcriptome remain unclear. Thus, this study sought to address whether and how elevated levels of immature platelets affect the platelet transcriptome in patients with sepsis. Methods Blood samples were obtained from patients with sepsis requiring vasopressor therapy (n = 8) and from a control group of patients with stable coronary artery disease and otherwise similar demographic characteristics (n = 8). Immature platelet fraction (IPF) was determined on a Sysmex XE 2100 analyser and platelet function was tested by impedance aggregometry. RNA from leukocyte-depleted platelets was used for transcriptome analysis by Next Generation Sequencing integrating the use of unique molecular identifiers. Results IPF (median [interquartile range]) was significantly elevated in sepsis patients (6.4 [5.3–8.7] % vs. 3.6 [2.6–4.6] %, p = 0.005). Platelet function testing revealed no differences in adenosine diphosphate- or thrombin receptor activating peptide-induced platelet aggregation between control and sepsis patients. Putative circular RNA transcripts were decreased in platelets from septic patients. Leukocyte contamination defined by CD45 abundance levels in RNA-sequencing was absent in both groups. Principal component analysis of transcripts showed only partial overlap of clustering with IPF levels. RNA sequencing showed up-regulation of 524 and down-regulation of 118 genes in platelets from sepsis patients compared to controls. Upregulated genes were mostly related to catabolic processes and protein translation. Comparison to published platelet transcriptomes showed a large overlap of changes observed in sepsis and COVID-19 but not with reticulated platelets from healthy donors. Conclusions Patients with sepsis appear to have a less degraded platelet transcriptome as indicated by increased levels of immature platelets and decreased levels of putative circular RNA transcripts. The present data suggests that increased protein translation is a characteristic mechanism of systemic inflammation.

SummaryVariants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are replacing the initial wild-type strain, jeopardizing current efforts to contain the pandemic. Amino acid exchanges in the spike protein are of particular concern as they can render the virus more transmissible or reduce vaccine efficacy. Here, we conducted whole genome sequencing of SARS-CoV-2 positive samples from the Rhine-Neckar district in Germany during January-March 2021. We detected a total of 166 samples positive for a variant with a distinct mutational pattern in the spike gene comprising L18F, L452R, N501Y, A653V, H655Y, D796Y and G1219V with a later gain of A222V. This variant was designated A.27.RN according to its phylogenetic clade classification. It emerged in parallel with the B.1.1.7 variant, increased to >50% of all SARS-CoV-2 variants by week five. Subsequently it decreased to

Nerve injury leads to chronic pain and exaggerated sensitivity to gentle touch (allodynia) as well as a loss of sensation in the areas in which injured and non-injured nerves come together1-3. The mechanisms that disambiguate these mixed and paradoxical symptoms are unknown. Here we longitudinally and non-invasively imaged genetically labelled populations of fibres that sense noxious stimuli (nociceptors) and gentle touch (low-threshold afferents) peripherally in the skin for longer than 10 months after nerve injury, while simultaneously tracking pain-related behaviour in the same mice. Fully denervated areas of skin initially lost sensation, gradually recovered normal sensitivity and developed marked allodynia and aversion to gentle touch several months after injury. This reinnervation-induced neuropathic pain involved nociceptors that sprouted into denervated territories precisely reproducing the initial pattern of innervation, were guided by blood vessels and showed irregular terminal connectivity in the skin and lowered activation thresholds mimicking low-threshold afferents. By contrast, low-threshold afferents-which normally mediate touch sensation as well as allodynia in intact nerve territories after injury4-7-did not reinnervate, leading to an aberrant innervation of tactile end organs such as Meissner corpuscles with nociceptors alone. Genetic ablation of nociceptors fully abrogated reinnervation allodynia. Our results thus reveal the emergence of a form of chronic neuropathic pain that is driven by structural plasticity, abnormal terminal connectivity and malfunction of nociceptors during reinnervation, and provide a mechanistic framework for the paradoxical sensory manifestations that are observed clinically and can impose a heavy burden on patients., The research leading to these results has received funding from the following sources: an ERC Advanced Investigator grant to R.K. (Pain Plasticity 294293); grants from the Deutsche Forschungsgemeinschaft to R.K. (SFB1158, projects B01, B06), to T.K. (SFB1158, project B08), to S.G.L. (SFB1158, project A01) and to V.G. (SFB1158, project A03); a grant to B.O. (project number 371923335); and grant CIDEGENT/2020/052 from Generalitat Valenciana to F.J.T. R.K. is a member of the Molecular Medicine Partnership Unit of the European Molecular Biology Laboratory and Medical Faculty Heidelberg. V.G. and T.A.N. were partially supported by a post-doctoral fellowship and physician scientist fellowship, respectively, from the Medical Faculty Heidelberg. D.M. was partially supported by a post-doctoral fellowship from Excellence Cluster CellNetworks. We acknowledge support from the Interdisciplinary Neurobehavioral Core (INBC) for the behavioural experiments, the data storage service SDS@hd and bwMLS&WISO HPC supported by the state of Baden-Württemberg and the German Research Foundation (DFG) through grants INST 35/1314-1 FUGG and INST 35/1134-1 FUGG, respectively.

Regeneration of adult mammalian central nervous system (CNS) axons is abortive, resulting in inability to recover function after CNS lesion, including spinal cord injury (SCI). Here, we show that the spiny mouse (Acomys) is an exception to other mammals, being capable of spontaneous and fast restoration of function after severe SCI, re-establishing hind limb coordination. Remarkably, Acomys assembles a scarless pro-regenerative tissue at the injury site, providing a unique structural continuity of the initial spinal cord geometry. The Acomys SCI site shows robust axon regeneration of multiple tracts, synapse formation, and electrophysiological signal propagation. Transcriptomic analysis of the spinal cord following transcriptome reconstruction revealed that Acomys rewires glycosylation biosynthetic pathways, culminating in a specific pro-regenerative proteoglycan signature at SCI site. Our work uncovers that a glycosylation switch is critical for axon regeneration after SCI and identifies beta 3gnt7, a crucial enzyme of keratan sulfate biosynthesis, as an enhancer of axon growth. Santa Casa da Misericordia de Lisboa MC-39-2019, Wings for Life WFL-PT-21/20, Portuguese Foundation for Science and Technology UID/BIM/04773/2013/CBMR info:eu-repo/semantics/publishedVersion