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Abstract N-glycosylation is one of the most common protein modifications in eukaryotes, with immense importance at the molecular, cellular, and organismal level. Accurate and reliable N-glycan analysis is essential to obtain a systems-wide understanding of fundamental biological processes. Due to the structural complexity of glycans, their analysis is still highly challenging. Here we make publicly available a consistent N-glycome dataset of 20 different mouse tissues and demonstrate a multimodal data analysis workflow that allows for unprecedented depth and coverage of N-glycome features. This highly scalable, LC-MS/MS data-driven method integrates the automated identification of N-glycan spectra, the application of non-targeted N-glycome profiling strategies and the isomer-sensitive analysis of glycan structures. Our delineation of critical sub-structural determinants and glycan isomers across the mouse N-glycome uncovered tissue-specific glycosylation patterns, the expression of non-canonical N-glycan structures and highlights multiple layers of N-glycome complexity that derive from organ-specific regulations of glycobiological pathways.

Abstract Cerebral cavernous malformations (CCMs) are anomalies of the cerebral vasculature. Loss of the CCM proteins CCM1/KRIT1, CCM2, or CCM3/PDCD10 trigger a MAPK-Krüppel-like factor 2 (KLF2) signaling cascade, which induces a pathophysiological pattern of gene expression. The downstream target genes that are activated by KLF2 are mostly unknown. Here we show that Chromobox Protein Homolog 7 (CBX7), component of the Polycomb Repressive Complex 1, contributes to pathophysiological KLF2 signaling during zebrafish cardiovascular development. CBX7/cbx7a mRNA is strongly upregulated in lesions of CCM patients, and in human, mouse, and zebrafish CCM-deficient endothelial cells. The silencing or pharmacological inhibition of CBX7/Cbx7a suppresses pathological CCM phenotypes in ccm2 zebrafish, CCM2-deficient HUVECs, and in a pre-clinical murine CCM3 disease model. Whole-transcriptome datasets from zebrafish cardiovascular tissues and human endothelial cells reveal a role of CBX7/Cbx7a in the activation of KLF2 target genes including TEK, ANGPT1, WNT9, and endoMT-associated genes. Our findings uncover an intricate interplay in the regulation of Klf2-dependent biomechanical signaling by CBX7 in CCM. This work also provides insights for therapeutic strategies in the pathogenesis of CCM.

Abstract The development of vascular networks in microfluidic chips is crucial for the long-term culture of three-dimensional cell aggregates such as spheroids, organoids, tumoroids, or tissue explants. Despite rapid advancement in microvascular network systems and organoid technologies, vascularizing organoids-on-chips remains a challenge in tissue engineering. Most existing microfluidic devices poorly reflect the complexity of in vivo flows and require complex technical set-ups. Considering these constraints, we develop a platform to establish and monitor the formation of endothelial networks around mesenchymal and pancreatic islet spheroids, as well as blood vessel organoids generated from pluripotent stem cells, cultured for up to 30 days on-chip. We show that these networks establish functional connections with the endothelium-rich spheroids and vascular organoids, as they successfully provide intravascular perfusion to these structures. We find that organoid growth, maturation, and function are enhanced when cultured on-chip using our vascularization method. This microphysiological system represents a viable organ-on-chip model to vascularize diverse biological 3D tissues and sets the stage to establish organoid perfusions using advanced microfluidics.

Abstract Marburg and Ebola filoviruses are two of the deadliest infectious agents and several outbreaks have occurred in the last decades. Although several receptors and co-receptors have been reported for Ebola virus, key host factors remain to be elucidated. In this study, using a haploid cell screening platform, we identify the guanine nucleotide exchange factor CCZ1 as a key host factor in the early stage of filovirus replication. The critical role of CCZ1 for filovirus infections is validated in 3D primary human hepatocyte cultures and human blood-vessel organoids, both critical target sites for Ebola and Marburg virus tropism. Mechanistically, CCZ1 controls early to late endosomal trafficking of these viruses. In addition, we report that CCZ1 has a role in the endosomal trafficking of endocytosis-dependent SARS-CoV-2 infections, but not in infections by Lassa virus, which enters endo-lysosomal trafficking at the late endosome stage. Thus, we have identified an essential host pathway for filovirus infections in cell lines and engineered human target tissues. Inhibition of CCZ1 nearly completely abolishes Marburg and Ebola infections. Thus, targeting CCZ1 could potentially serve as a promising drug target for controlling infections caused by various viruses, such as SARS-CoV-2, Marburg, and Ebola.

Organoid-based research has made significant discoveries and contributions to our understanding of human organ function in both health and disease. To continue making progress, it is crucial to acknowledge the crucial role of the immune system in all organs. Various immune cells, such as macrophages, T cells, and neutrophils, are resident in almost all human tissues and play essential roles in organ homeostasis, function, and disease. Using diverse methods, researchers have begun integrating immune cells into organoid models, leading to more physiologically relevant models that better represent various aspects of human disease. These methods range from immune cell injection to co-culture and tissue expansion with existing immune cells. Immune cells can be sourced from mature patients or generated from stem cells as immature immune cells. The successful incorporation of immune cells into organoids will enhance our understanding of organ function and provide a more accurate approximation of human disease.

Abstract FAM3C/ILEI is an important cytokine for tumor progression and metastasis. However, its involvement in inflammation remains elusive. Here, we show that ILEI protein is highly expressed in psoriatic lesions. Inducible keratinocyte‐specific ILEI overexpression in mice (K5‐ILEIind) recapitulates many aspects of psoriasis following TPA challenge, primarily manifested by impaired epidermal differentiation and increased neutrophil recruitment. Mechanistically, ILEI triggers Erk and Akt signaling, which then activates STAT3 via Ser727 phosphorylation. Keratinocyte‐specific ILEI deletion ameliorates TPA‐induced skin inflammation. A transcriptomic ILEI signature obtained from the K5‐ILEIind model shows enrichment in several signaling pathways also found in psoriasis and identifies urokinase as a targetable enzyme to counteract ILEI activity. Pharmacological inhibition of urokinase in TPA‐induced K5‐ILEIind mice results in significant improvement of psoriasiform symptoms by reducing ILEI secretion. The ILEI signature distinguishes psoriasis from healthy skin with uPA ranking among the top “separator” genes. Our study identifies ILEI as a key driver in psoriasis, indicates the relevance of ILEI‐regulated genes for disease manifestation, and shows the clinical impact of ILEI and urokinase as novel potential therapeutic targets in psoriasis.

Abstract The natural compound Artemisinin is the most widely used antimalarial drug worldwide. Based on its cytotoxicity, it is also used for anticancer therapy. Artemisinin and its derivates are endoperoxides that damage proteins in eukaryotic cells; their definite mechanism of action and host cell targets, however, have remained largely elusive. Using yeast and haploid stem cell screening, we demonstrate that a single cellular pathway, namely porphyrin (heme) biosynthesis, is required for the cytotoxicity of Artemisinins. Genetic or pharmacological modulation of porphyrin production is sufficient to alter its cytotoxicity in eukaryotic cells. Using multiple model systems of human brain tumor development, such as cerebral glioblastoma organoids, and patient‐derived tumor spheroids, we sensitize cancer cells to dihydroartemisinin using the clinically approved porphyrin enhancer and surgical fluorescence marker 5‐aminolevulinic acid, 5‐ALA. A combination treatment of Artemisinins and 5‐ALA markedly and specifically killed brain tumor cells in all model systems tested, including orthotopic patient‐derived xenografts in vivo. These data uncover the critical molecular pathway for Artemisinin cytotoxicity and a sensitization strategy to treat different brain tumors, including drug‐resistant human glioblastomas.

Combining high-speed AFM, single molecule recognition force spectroscopy, and molecular dynamics simulations Zhu, Canena, Sikora et al. characterize the interaction dynamics of the trimeric spike protein of SARS-CoV-2 wt, and delta and omicron variants with its entry receptor ACE2. While delta variant increases avidity by multivalent binding to ACE2, omicron variant shows an extended binding lifetime.

Type 2 diabetes often presents with reproductive disorders, including reduced male and female fertility. Here they show that diabetes disrupts the APLN signaling axis in the testes and that spermatogenesis can be restored through treatment with an APLN antagonist.

Summary: Despite similar infection rates, COVID-19 has resulted in more deaths in men than women. To understand the underlying mechanisms behind this sex-biased difference in disease severity, we infected K18-human angiotensin converting enzyme 2 (ACE2) mice of both sexes with SARS-CoV-2. Our study revealed a unique protein expression profile in the lung microenvironment of female mice. As a result, they were less vulnerable to severe infection, with higher ACE2 expression and a higher estrogen receptor α (ERα)/androgen receptor (AR) ratio that led to increased antiviral factor levels. In male mice, inhaling recombinant ACE2 neutralized the virus and maintained the ERα/AR ratio, thereby protecting the lungs. Our findings suggest that inhaling recombinant ACE2 could serve as a decoy receptor against SARS-CoV-2 and protect male mice by offsetting ERα-associated protective mechanisms. Additionally, our study supports the potential effectiveness of recombinant ACE2 therapy in human lung organoids infected with the Delta variant.

Abstract Whole‐exome sequencing of two patients with idiopathic complex neurodevelopmental disorder (NDD) identified biallelic variants of unknown significance within FIBCD1, encoding an endocytic acetyl group‐binding transmembrane receptor with no known function in the central nervous system. We found that FIBCD1 preferentially binds and endocytoses glycosaminoglycan (GAG) chondroitin sulphate‐4S (CS‐4S) and regulates GAG content of the brain extracellular matrix (ECM). In silico molecular simulation studies and GAG binding analyses of patient variants determined that such variants are loss‐of‐function by disrupting FIBCD1‐CS‐4S association. Gene knockdown in flies resulted in morphological disruption of the neuromuscular junction and motor‐related behavioural deficits. In humans and mice, FIBCD1 is expressed in discrete brain regions, including the hippocampus. Fibcd1 KO mice exhibited normal hippocampal neuronal morphology but impaired hippocampal‐dependent learning. Further, hippocampal synaptic remodelling in acute slices from Fibcd1 KO mice was deficient but restored upon enzymatically modulating the ECM. Together, we identified FIBCD1 as an endocytic receptor for GAGs in the brain ECM and a novel gene associated with an NDD, revealing a critical role in nervous system structure, function and plasticity.

Abstract The recent emergence of multiple SARS‐CoV‐2 variants has caused considerable concern due to both reduced vaccine efficacy and escape from neutralizing antibody therapeutics. It is, therefore, paramount to develop therapeutic strategies that inhibit all known and future SARS‐CoV‐2 variants. Here, we report that all SARS‐CoV‐2 variants analyzed, including variants of concern (VOC) Alpha, Beta, Gamma, Delta, and Omicron, exhibit enhanced binding affinity to clinical grade and phase 2 tested recombinant human soluble ACE2 (APN01). Importantly, soluble ACE2 neutralized infection of VeroE6 cells and human lung epithelial cells by all current VOC strains with markedly enhanced potency when compared to reference SARS‐CoV‐2 isolates. Effective inhibition of infections with SARS‐CoV‐2 variants was validated and confirmed in two independent laboratories. These data show that SARS‐CoV‐2 variants that have emerged around the world, including current VOC and several variants of interest, can be inhibited by soluble ACE2, providing proof of principle of a pan‐SARS‐CoV‐2 therapeutic.

Abstract Neuromedin B (NMB) is a member of the neuromedin family of neuropeptides with a high level of region-specific expression in the brain. Several GWAS studies on non-obese and obese patients suggested that polymorphisms in NMB predispose to obesity by affecting appetite control and feeding preference. Furthermore, several studies proposed that NMB can act as an insulin releasing peptide. Since the functional study has never been done, the in vivo role of NMB as modulator of weight gain or glucose metabolism remains unclear. Here, we generated Nmb conditional mice and nervous system deficient NmB mice. We then performed olfactory and food preference analysis, as well as metabolic analysis under standard and high fat diet. Additionally, in direct islet studies we evaluated the role of NMB on basal and glucose-stimulated insulin secretion in mouse and humans.

Cardiac regeneration is one of the grand challenges in repairing injured human hearts. Numerous studies of signaling pathways and metabolism on cardiac development and disease pave the way for endogenous cardiomyocyte regeneration. New drug delivery approaches, high-throughput screening, as well as novel therapeutic compounds combined with gene editing will facilitate the development of potential cell-free therapeutics. In parallel, progress has been made in the field of cell-based therapies. Transplantation of human pluripotent stem cell (hPSC)-derived cardiomyocytes (hPSC-CMs) can partially rescue the myocardial defects caused by cardiomyocyte loss in large animals. In this review, we summarize current cell-based and cell-free regenerative therapies, discuss the importance of cardiomyocyte maturation in cardiac regenerative medicine, and envision new ways of regeneration for the injured heart.

Endogenous ACE2 is a receptor for SARS-CoV-2 and a recombinant soluble ACE2 protein can inhibit SARS-CoV-2 infection acting as a decoy. Here the authors show that B38-CAP, an ACE2-like enzyme but not a decoy for the virus, is protective against SARS-CoV-2-induced lung injury in animal models.

Summary: This protocol presents the use of SARS-CoV-2 isolates to infect human kidney organoids, enabling exploration of the impact of SARS-CoV-2 infection in a human multicellular in vitro system. We detail steps to generate kidney organoids from human pluripotent stem cells (hPSCs) and emulate a diabetic milieu via organoids exposure to diabetogenic-like cell culture conditions. We further describe preparation and titration steps of SARS-CoV-2 virus stocks, their subsequent use to infect the kidney organoids, and assessment of the infection via immunofluorescence.For complete details on the use and execution of this protocol, please refer to Garreta et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

BackgroundThe prognosis of diffusely infiltrating glioma patients is dismal but varies greatly between individuals. While characterization of gliomas primarily relied on histopathological features, molecular markers increasingly gained importance and play a key role in the recently published 5th edition of the World Health Organization (WHO) classification. Heme biosynthesis represents a crucial pathway due to its paramount importance in oxygen transport, energy production and drug metabolism. Recently, we described a “heme biosynthesis mRNA expression signature” that correlates with histopathological glioma grade and survival. The aim of the current study was to correlate this heme biosynthesis mRNA expression signature with diagnostic molecular markers and investigate its continued prognostic relevance.Materials and methodsIn this study, patient data were derived from the “The Cancer Genome Atlas” (TCGA) lower-grade glioma and glioblastoma cohorts. We identified diffusely infiltrating gliomas correlating molecular tumor diagnosis according to the most recent WHO classification with heme biosynthesis mRNA expression. The following molecular markers were analyzed: EGFR amplification, TERT promoter mutation, CDKN2A/B homozygous loss, chromosome 7 + /10- aneuploidy, MGMT methylation, IDH mutation, ATRX loss, p53 mutation and 1p19q codeletion. Subsequently, we calculated the heme biosynthesis mRNA expression signature for correlation with distinct molecular glioma markers/molecular subgroups and performed survival analyses.ResultsA total of 649 patients with available data on up-to-date molecular markers and heme biosynthesis mRNA expression were included. According to analysis of individual molecular markers, we found a significantly higher heme biosynthesis mRNA expression signature in gliomas with IDH wildtype (p < 0.0005), without 1p19q codeletion (p < 0.0005), with homozygous CDKN2A/B loss (p < 0.0005) and with EGFR amplification (p = 0.001). Furthermore, we observed that the heme biosynthesis mRNA expression signature increased with molecular subgroup aggressiveness (p < 0.0005), being lowest in WHO grade 2 oligodendrogliomas and highest in WHO grade 4 glioblastomas. Finally, the heme biosynthesis mRNA expression signature was a statistically significant survival predictor after multivariate correction for all molecular markers (p < 0.0005).ConclusionOur data demonstrate a significant correlation between heme biosynthesis regulation and diagnostic molecular markers and a prognostic relevance independent of these established markers. Consequently, heme biosynthesis expression is a promising biomarker for glioma aggressiveness and might constitute a potential target for novel therapeutic approaches.

Abstract There is a critical need for safe and effective drugs for COVID‐19. Only remdesivir has received authorization for COVID‐19 and has been shown to improve outcomes but not decrease mortality. However, the dose of remdesivir is limited by hepatic and kidney toxicity. ACE2 is the critical cell surface receptor for SARS‐CoV‐2. Here, we investigated additive effect of combination therapy using remdesivir with recombinant soluble ACE2 (high/low dose) on Vero E6 and kidney organoids, targeting two different modalities of SARS‐CoV‐2 life cycle: cell entry via its receptor ACE2 and intracellular viral RNA replication. This combination treatment markedly improved their therapeutic windows against SARS‐CoV‐2 in both models. By using single amino‐acid resolution screening in haploid ES cells, we report a singular critical pathway required for remdesivir toxicity, namely, Adenylate Kinase 2. The data provided here demonstrate that combining two therapeutic modalities with different targets, common strategy in HIV treatment, exhibit strong additive effects at sub‐toxic concentrations. Our data lay the groundwork for the study of combinatorial regimens in future COVID‐19 clinical trials.

Abstract Muscle wasting and atrophy are regulated by multiple molecular processes, including mRNA processing. Reduced levels of the polyadenylation binding protein nucleus 1 (PABPN1), a multifactorial regulator of mRNA processing, cause muscle atrophy. A proteomic study in muscles with reduced PABPN1 levels suggested dysregulation of sarcomeric and cytoskeletal proteins. Here we investigated the hypothesis that reduced PABPN1 levels lead to an aberrant organization of the cytoskeleton. MURC, a plasma membrane-associated protein, was found to be more abundant in muscles with reduced PABPN1 levels, and it was found to be expressed at regions showing regeneration. A polarized cytoskeletal organization is typical for muscle cells, but muscle cells with reduced PABPN1 levels (named as shPAB) were characterized by a disorganized cytoskeleton that lacked polarization. Moreover, cell mechanical features and myogenic differentiation were significantly reduced in shPAB cells. Importantly, restoring cytoskeletal stability, by actin overexpression, was beneficial for myogenesis, expression of sarcomeric proteins and proper localization of MURC in shPAB cell cultures and in shPAB muscle bundle. We suggest that poor cytoskeletal mechanical features are caused by altered expression levels of cytoskeletal proteins and contribute to muscle wasting and atrophy.

As ACE2 is the critical SARS-CoV-2 receptor, we hypothesized that aerosol administration of clinical grade soluble human recombinant ACE2 (APN01) will neutralize SARS-CoV-2 in the airways, limit spread of infection in the lung, and mitigate lung damage caused by deregulated signaling in the renin-angiotensin (RAS) and Kinin pathways. Here, after demonstrating in vitro neutralization of SARS-CoV-2 by APN01, and after obtaining preliminary evidence of its tolerability and preventive efficacy in a mouse model, we pursued development of an aerosol formulation. As a prerequisite to a clinical trial, we evaluated both virus binding activity and enzymatic activity for cleavage of Ang II following aerosolization. We report successful aerosolization for APN01, retaining viral binding as well as catalytic RAS activity. Dose range-finding and IND-enabling repeat-dose aerosol toxicology testing were conducted in dogs. Twice daily aerosol administration for two weeks at the maximum feasible concentration revealed no notable toxicities. Based on these results, a Phase I clinical trial in healthy volunteers has now been initiated (NCT05065645), with subsequent Phase II testing planned for individuals with SARS-CoV-2 infection.

Angiotensin-converting enzyme 2 (ACE2) is the carboxypeptidase to degrade angiotensin II (Ang II) to angiotensin 1-7 (Ang 1-7) and improves the pathologies of cardiovascular disease and acute respiratory distress syndrome (ARDS)/acute lung injury. B38-CAP is a bacteria-derived ACE2-like carboxypeptidase as potent as human ACE2 and ameliorates hypertension, heart failure and SARS-CoV-2-induced lung injury in mice. Recombinant B38-CAP is prepared with E. coli protein expression system more efficiently than recombinant soluble human ACE2. Here we show therapeutic effects of B38-CAP on abdominal sepsis- or acid aspiration-induced acute lung injury. ACE2 expression was downregulated in the lungs of mice with cecal ligation puncture (CLP)-induced sepsis or acid-induced lung injury thereby leading to upregulation of Ang II levels. Intraperitoneal injection of B38-CAP significantly decreased Ang II levels while upregulated angiotensin 1-7 levels. B38-CAP improved survival rate of the mice under sepsis. B38-CAP suppressed the pathologies of lung inflammation, improved lung dysfunction and downregulated elevated cytokine mRNA levels in the mice with acute lung injury. Thus, systemic treatment with an ACE2-like enzyme might be a potential therapeutic strategy for the patients with severe sepsis or ARDS.

Despite tremendous progress in the understanding of COVID-19, mechanistic insight into immunological, disease-driving factors remains limited. We generated maVie16, a mouse-adapted SARS-CoV-2, by serial passaging of a human isolate. In silico modeling revealed how only three Spike mutations of maVie16 enhanced interaction with murine ACE2. maVie16 induced profound pathology in BALB/c and C57BL/6 mice, and the resulting mouse COVID-19 (mCOVID-19) replicated critical aspects of human disease, including early lymphopenia, pulmonary immune cell infiltration, pneumonia, and specific adaptive immunity. Inhibition of the proinflammatory cytokines IFNγ and TNF substantially reduced immunopathology. Importantly, genetic ACE2-deficiency completely prevented mCOVID-19 development. Finally, inhalation therapy with recombinant ACE2 fully protected mice from mCOVID-19, revealing a novel and efficient treatment. Thus, we here present maVie16 as a new tool to model COVID-19 for the discovery of new therapies and show that disease severity is determined by cytokine-driven immunopathology and critically dependent on ACE2 in vivo.

Seventeen years after the epidemic of SARS coronavirus, a novel coronavirus SARS-CoV-2-emerged resulting in an unprecedented pandemic. Angiotensin-converting enzyme 2 (ACE2) is an essential receptor for cell entry of SARS-CoV-2 as well as the SARS coronavirus. Despite many similarities to SARS coronavirus, SARS-CoV-2 exhibits a higher affinity to ACE2 and shows higher infectivity and transmissibility, resulting in explosive increase of infected people and COVID-19 patients. Emergence of the variants harboring mutations in the receptor-binding domain of the Spike protein has drawn critical attention to the interaction between ACE2 and Spike and the efficacies of vaccines and neutralizing antibodies. ACE2 is a carboxypeptidase which degrades angiotensin II, B1-bradykinin, or apelin, and thereby is a critical regulator of cardiovascular physiology and pathology. In addition, the enzymatic activity of ACE2 is protective against acute respiratory distress syndrome (ARDS) caused by viral and non-viral pneumonias, aspiration, or sepsis. Upon infection, both SARS-CoV-2 and SARS coronaviruses downregulates ACE2 expression, likely associated with the pathogenesis of ARDS. Thus, ACE2 is not only the SARS-CoV-2 receptor but might also play an important role in multiple aspects of COVID-19 pathogenesis and possibly post-COVID-19 syndromes. Soluble forms of recombinant ACE2 are currently utilized as a pan-variant decoy to neutralize SARS-CoV-2 and a supplementation of ACE2 carboxypeptidase activity. Here, we review the role of ACE2 in the pathology of ARDS in COVID-19 and the potential application of recombinant ACE2 protein for treating COVID-19.

Summary: Formalin-fixed paraffin embedding (FFPE) is the most widespread long-term tissue preservation approach. Here, we report a procedure to perform genome-wide spatial analysis of mRNA in FFPE-fixed tissue sections, using well-established, commercially available methods for imaging and spatial barcoding using slides spotted with barcoded oligo(dT) probes to capture the 3′ end of mRNA molecules in tissue sections. We applied this method for expression profiling and cell type mapping in coronal sections from the mouse brain to demonstrate the method’s capability to delineate anatomical regions from a molecular perspective. We also profiled the spatial composition of transcriptomic signatures in two ovarian carcinosarcoma samples, exemplifying the method’s potential to elucidate molecular mechanisms in heterogeneous clinical samples. Finally, we demonstrate the applicability of the assay to characterize human lung and kidney organoids and a human lung biopsy specimen infected with SARS-CoV-2. We anticipate that genome-wide spatial gene expression profiling in FFPE biospecimens will be used for retrospective analysis of biobank samples, which will facilitate longitudinal studies of biological processes and biomarker discovery.

Infection and viral entry of SARS-CoV-2 crucially depends on the binding of its Spike protein to angiotensin converting enzyme 2 (ACE2) presented on host cells. Glycosylation of both proteins is critical for this interaction. Recombinant soluble human ACE2 can neutralize SARS-CoV-2 and is currently undergoing clinical tests for the treatment of COVID-19. We used 3D structural models and molecular dynamics simulations to define the ACE2 N-glycans that critically influence Spike-ACE2 complex formation. Engineering of ACE2 N-glycosylation by site-directed mutagenesis or glycosidase treatment resulted in enhanced binding affinities and improved virus neutralization without notable deleterious effects on the structural stability and catalytic activity of the protein. Importantly, simultaneous removal of all accessible N-glycans from recombinant soluble human ACE2 yields a superior SARS-CoV-2 decoy receptor with promise as effective treatment for COVID-19 patients.

Summary: The Linear Ubiquitin Chain Assembly Complex (LUBAC), composed of HOIP, HOIL-1L, and SHARPIN, promotes tumor necrosis factor (TNF)-dependent NF-κB signaling in diverse cell types. HOIL-1L contains an Npl4 Zinc Finger (NZF) domain that specifically recognizes linear ubiquitin chains, but its physiological role in vivo has remained unclear. Here, we demonstrate that the HOIL-1L NZF domain has important regulatory functions in inflammation and immune responses in mice. We generated knockin mice (Hoil-1lT201A;R208A/T201A;R208A) expressing a HOIL-1L NZF mutant and observed attenuated responses to TNF- and LPS-induced shock, including prolonged survival, stabilized body temperature, reduced cytokine production, and liver damage markers. Cells derived from Hoil-1lT201A;R208A/T201A;R208A mice show reduced TNF-dependent NF-κB activation and incomplete recruitment of HOIL-1L into TNF Receptor (TNFR) Complex I. We further show that HOIL-1L NZF cooperates with SHARPIN to prevent TNFR-dependent skin inflammation. Collectively, our data suggest that linear ubiquitin-chain binding by HOIL-1L regulates immune responses and inflammation in vivo.

Validating crosslinking-mass spectrometry workflows is hampered by the lack of a ground truth to assess the robustness of the crosslink identifications. Here, the authors present a synthetic library of crosslinked peptides, enabling unambiguous discrimination of correct and incorrect crosslink identifications.

The enzyme ACE2 is involved in cardiac pathology and can counteract heart failure and other cardio-pulmonary diseases. Here the authors show that bacteria produce an ACE2-like enzyme that is effective in suppressing cardiac hypertrophy and fibrosis in mice.

ABSTRACTHow severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infections engage cellular host pathways and innate immunity in infected cells remains largely elusive. We performed an integrative proteo-transcriptomics analysis in SARS-CoV-2 infected Huh7 cells to map the cellular response to the invading virus over time. We identified four pathways, ErbB, HIF-1, mTOR and TNF signaling, among others that were markedly modulated during the course of the SARS-CoV-2 infection in vitro. Western blot validation of the downstream effector molecules of these pathways revealed a dose-dependent activation of Akt, mTOR, S6K1 and 4E-BP1 at 24 hours post infection (hpi). However, we found a significant inhibition of HIF-1α through 24hpi and 48hpi of the infection, suggesting a crosstalk between the SARS-CoV-2 and the Akt/mTOR/HIF-1 signaling pathways. Inhibition of the mTOR signaling pathway using Akt inhibitor MK-2206 showed a significant reduction in virus production. Further investigations are required to better understand the molecular sequelae in order to guide potential therapy in the management of severe coronavirus disease 2019 (COVID-19) patients.

Abstract Angiogenesis is a hallmark of cancer, promoting growth and metastasis. Anti‐angiogenic treatment has limited efficacy due to therapy‐induced blood vessel alterations, often followed by local hypoxia, tumor adaptation, progression, and metastasis. It is therefore paramount to overcome therapy‐induced resistance. We show that Apelin inhibition potently remodels the tumor microenvironment, reducing angiogenesis, and effectively blunting tumor growth. Functionally, targeting Apelin improves vessel function and reduces polymorphonuclear myeloid‐derived suppressor cell infiltration. Importantly, in mammary and lung cancer, Apelin prevents resistance to anti‐angiogenic receptor tyrosine kinase (RTK) inhibitor therapy, reducing growth and angiogenesis in lung and breast cancer models without increased hypoxia in the tumor microenvironment. Apelin blockage also prevents RTK inhibitor‐induced metastases, and high Apelin levels correlate with poor prognosis of anti‐angiogenic therapy patients. These data identify a druggable anti‐angiogenic drug target that reduces tumor blood vessel densities and normalizes the tumor vasculature to decrease metastases.

PR domain-containing member 12 (PRDM12) is a key developmental transcription factor in sensory neuronal specification and survival. Patients with rare deleterious variants in PRDM12 are born with congenital insensitivity to pain (CIP) due to the complete absence of a subtype of peripheral neurons that detect pain. In this paper, we report two additional CIP cases with a novel homozygous PRDM12 variant. To elucidate the function of PRDM12 during mammalian development and adulthood, we generated temporal and spatial conditional mouse models. We find that PRDM12 is expressed throughout the adult nervous system. We observed that loss of PRDM12 during mid-sensory neurogenesis but not in the adult leads to reduced survival. Comparing cellular biophysical nociceptive properties in developmental and adult-onset PRDM12 deletion mouse models, we find that PRDM12 is necessary for proper nociceptive responses throughout life. However, we find that PRDM12 regulates distinct age-dependent transcriptional programs. Together, our results implicate PRDM12 as a viable therapeutic target for specific pain therapies even in adults.

[This corrects the article DOI: 10.1371/journal.pgen.1008268.].

RANKL and RANK are expressed in different cell types and tissues throughout the body. They were originally described for their essential roles in bone remodeling and the immune system but have subsequently been shown to provide essential signals from regulating mammary gland homeostasis during pregnancy to modulating tumorigenesis. The success of RANKL/RANK research serves as a paragon for translational research from the laboratory to the bedside. The case in point has been the development of Denosumab, a RANKL-blocking monoclonal antibody which has already helped millions of patients suffering from post-menopausal osteoporosis and skeletal related events in cancer. Here we will provide an overview of the pathway from its origins to its clinical relevance in disease, with a special focus on emerging evidence demonstrating the therapeutic value of targeting the RANKL/RANK/OPG axis not only in breast cancer but also as an addition to the cancer immunotherapy arsenal.

Abstract Background Important insight into the mechanisms through which gene-environmental interactions cause schizophrenia can be achieved through preclinical studies combining prenatal immune stimuli with disease-related genetic risk modifications. Accumulating evidence associates JNK signalling molecules, including MKK7/MAP2K7, with genetic risk. We tested the hypothesis that Map2k7 gene haploinsufficiency in mice would alter the prenatal immune response to the viral mimetic polyriboinosinic-polyribocytidylic acid (polyI:C), specifically investigating the impact of maternal versus foetal genetic variants. Methods PolyI:C was administered to dams (E12.5), and cytokine/chemokine levels were measured 6 h later, in maternal plasma, placenta and embryonic brain. Results PolyI:C dramatically elevated maternal plasma levels of most cytokines/chemokines. Induction of IL-1β, IL-2, IL-10, IL-12, TNF-α and CXCL3 was enhanced, while CCL5 was suppressed, in Map2k7 hemizygous (Hz) dams relative to controls. Maternal polyI:C administration also increased embryonic brain chemokines, influenced by both maternal and embryonic genotype: CCL5 and CXCL10 levels were higher in embryonic brains from Map2k7 dams versus control dams; for CCL5, this was more pronounced in Map2k7 Hz embryos. Placental CXCL10 and CXCL12 levels were also elevated by polyI:C, the former enhanced and the latter suppressed, in placentae from maternal Map2k7 Hzs relative to control dams receiving polyI:C. Conclusions The results demonstrate JNK signalling as a mediator of MIA effects on the foetus. Since both elevated CXCL10 and supressed CXCL12 compromise developing GABAergic interneurons, the results support maternal immune challenge contributing to schizophrenia-associated neurodevelopmental abnormalities. The influence of Map2k7 on cytokine/chemokine induction converges the genetic and environmental aspects of schizophrenia, and the overt influence of maternal genotype offers an intriguing new insight into modulation of embryonic neurodevelopment by genetic risk.

Bluetongue virus (BTV), an arbovirus of ruminants, is a causative agent of numerous epidemics around the world. Due to the emergence of novel reassortant BTV strains and new outbreaks, there is an unmet need for efficacious antivirals. In this study, we used an improved haploid screening platform to identify the relevant host factors for BTV infection. Our screening tool identified and validated the host factor Niemann–Pick C1 (NPC1), a lysosomal membrane protein that is involved in lysosomal cholesterol transport, as a critical factor in BTV infection. This finding prompted us to investigate the possibility of testing imipramine, an antidepressant drug known to inhibit NPC1 function by interfering with intracellular cholesterol trafficking. In this study, we evaluated the sensitivity of BTV to imipramine using in vitro assays. Our results demonstrate that imipramine pretreatment inhibited in vitro replication and progeny release of BTV-4, BTV-8, and BTV-16. Collectively, our findings highlight the importance of NPC1 for BTV infection and recommend the reprofiling of imipramine as a potential antiviral drug against BTV.

Abstract Although there is a strong association between osteoporosis and skeletal muscle atrophy/dysfunction, the functional relevance of a particular biological pathway that regulates synchronously bone and skeletal muscle physiopathology is still elusive. Receptor-activator of nuclear factor κB (RANK), its ligand RANKL and the soluble decoy receptor osteoprotegerin (OPG) are the key regulators of osteoclast differentiation and bone remodelling. We thus hypothesized that RANK/RANKL/OPG, which is a key pathway for bone regulation, is involved in Duchenne muscular dystrophy (DMD) physiopathology. Our results show that muscle-specific RANK deletion (mdx-RANK mko ) in dystrophin deficient mdx mice improves significantly specific force [54% gain in force] of EDL muscles with no protective effect against eccentric contraction-induced muscle dysfunction. In contrast, full-length OPG-Fc injections restore the force of dystrophic EDL muscles [162% gain in force], protect against eccentric contraction-induced muscle dysfunction ex vivo and significantly improve functional performance on downhill treadmill and post-exercise physical activity. Since OPG serves a soluble receptor for RANKL and as a decoy receptor for TRAIL, mdx mice were injected with anti-RANKL and anti-TRAIL antibodies to decipher the dual function of OPG. Injections of anti-RANKL and/or anti-TRAIL increase significantly the force of dystrophic EDL muscle [45% and 17% gains in force, respectively]. In agreement, truncated OPG-Fc that contains only RANKL domains produces similar gains, in terms of force production, than anti-RANKL treatments. To corroborate that full-length OPG-Fc also acts independently of RANK/RANKL pathway, dystrophin/RANK double-deficient mice were treated with full-length OPG-Fc for 10 days. Dystrophic EDL muscles exhibited a significant gain in force relative to untreated dystrophin/RANK double-deficient mice, indicating that the effect of full-length OPG-Fc is in part independent of the RANKL/RANK interaction. The sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) activity is significantly depressed in dysfunctional and dystrophic muscles and full-length OPG-Fc treatment increased SERCA activity and SERCA-2a expression. These findings demonstrate the superiority of full-length OPG-Fc treatment relative to truncated OPG-Fc, anti-RANKL, anti-TRAIL or muscle RANK deletion in improving dystrophic muscle function, integrity and protection against eccentric contractions. In conclusion, full-length OPG-Fc represents an efficient alternative in the development of new treatments for muscular dystrophy in which a single therapeutic approach may be foreseeable to maintain both bone and skeletal muscle functions.

The spleen plays an important role in protective immunity to bloodborne pathogens. Macrophages and dendritic cells (DCs) in the spleen marginal zone capture microbial antigens to trigger adaptive immune responses. Marginal zone macrophages (MZMs) can also act as a replicative niche for intracellular pathogens, providing a platform for mounting the immune response. Here, we describe a role for RANK in the coordinated function of antigen-presenting cells in the spleen marginal zone and triggering anti-viral immunity. Targeted deletion of RANK results in the selective loss of CD169+ MZMs, which provide a niche for viral replication, while RANK signaling in DCs promotes the recruitment and activation of anti-viral memory CD8 T cells. These studies reveal a role for the RANKL/RANK signaling axis in the orchestration of protective immune responses in the spleen marginal zone that has important implications for the host response to viral infection and induction of acquired immunity.

Abstract c-Jun N-terminal kinase (JNK) is a member of the mitogen-activated protein kinase family and controls various physiological processes including apoptosis. A specific upstream activator of JNKs is the mitogen-activated protein kinase kinase 7 (MKK7). It has been reported that MKK7-JNK signaling plays an important regulatory role in neural development, however, post-developmental functions in the nervous system have not been elucidated. In this study, we generated neuron-specific Mkk7 knockout mice (MKK7 cKO), which impaired constitutive activation of JNK in the nervous system. MKK7 cKO mice displayed impaired circadian behavioral rhythms and decreased locomotor activity. MKK7 cKO mice at 8 months showed motor dysfunctions such as weakness of hind-limb and gait abnormality in an age-dependent manner. Axonal degeneration in the spinal cord and muscle atrophy were also observed, along with accumulation of the axonal transport proteins JNK-interacting protein 1 and amyloid beta precursor protein in the brains and spinal cords of MKK7 cKO mice. Thus, the MKK7-JNK signaling pathway plays important roles in regulating circadian rhythms and neuronal maintenance in the adult nervous system.

Immunometabolism explores how the intracellular metabolic pathways in immune cells can regulate their function under different micro-environmental and (patho-)-physiological conditions (Pearce, 2010; Buck et al., 2015; O'Neill and Pearce, 2016). In the last decade great advances have been made in studying and manipulating metabolic programs in immune cells. Immunometabolism has primarily focused on glycolysis, the TCA cycle and oxidative phosphorylation (OXPHOS) as well as free fatty acid synthesis and oxidation. These pathways are important for providing the energy needs of cell growth, membrane rigidity, cytokine production and proliferation. In this review, we will however, highlight the specific role of iron metabolism at the cellular and organismal level, as well as how the bioavailability of this metal orchestrates complex metabolic programs in immune cell homeostasis and inflammation. We will also discuss how dysregulation of iron metabolism contributes to alterations in the immune system and how these novel insights into iron regulation can be targeted to metabolically manipulate immune cell function under pathophysiological conditions, providing new therapeutic opportunities for autoimmunity and cancer.

Long non-coding (lnc) RNAs are numerous and found throughout the mammalian genome, and many are thought to be involved in the regulation of gene expression. However, the majority remain relatively uncharacterised and of uncertain function making the use of model systems to uncover their mode of action valuable. Imprinted lncRNAs target and recruit epigenetic silencing factors to a cluster of imprinted genes on the same chromosome, making them one of the best characterized lncRNAs for silencing distant genes in cis. In this study we examined silencing of the distant imprinted gene Slc22a3 by the lncRNA Airn in the Igf2r imprinted cluster in mouse. Previously we proposed that imprinted lncRNAs may silence distant imprinted genes by disrupting promoter-enhancer interactions by being transcribed through the enhancer, which we called the enhancer interference hypothesis. Here we tested this hypothesis by first using allele-specific chromosome conformation capture (3C) to detect interactions between the Slc22a3 promoter and the locus of the Airn lncRNA that silences it on the paternal chromosome. In agreement with the model, we found interactions enriched on the maternal allele across the entire Airn gene consistent with multiple enhancer-promoter interactions. Therefore, to test the enhancer interference hypothesis we devised an approach to delete the entire Airn gene. However, the deletion showed that there are no essential enhancers for Slc22a2, Pde10a and Slc22a3 within the Airn gene, strongly indicating that the Airn RNA rather than its transcription is responsible for silencing distant imprinted genes. Furthermore, we found that silent imprinted genes were covered with large blocks of H3K27me3 on the repressed paternal allele. Therefore we propose an alternative hypothesis whereby the chromosome interactions may initially guide the lncRNA to target imprinted promoters and recruit repressive chromatin, and that these interactions are lost once silencing is established.

Summary: The HECT domain E3 ligase HACE1 has been identified as a tumor suppressor in multiple cancers. Here, we report that HACE1 is a central gatekeeper of TNFR1-induced cell fate. Genetic inactivation of HACE1 inhibits TNF-stimulated NF-κB activation and TNFR1-NF-κB-dependent pathogen clearance in vivo. Moreover, TNF-induced apoptosis was impaired in hace1 mutant cells and knockout mice in vivo. Mechanistically, HACE1 is essential for the ubiquitylation of the adaptor protein TRAF2 and formation of the apoptotic caspase-8 effector complex. Intriguingly, loss of HACE1 does not impair TNFR1-mediated necroptotic cell fate via RIP1 and RIP3 kinases. Loss of HACE1 predisposes animals to colonic inflammation and carcinogenesis in vivo, which is markedly alleviated by genetic inactivation of RIP3 kinase and TNFR1. Thus, HACE1 controls TNF-elicited cell fate decisions and exerts tumor suppressor and anti-inflammatory activities via a TNFR1-RIP3 kinase-necroptosis pathway. : Tortola et al. report that the E3 ubiquitin ligase HACE1 is a gatekeeper of TNFR1-mediated cell fate. Hace1 deficiency impairs TNF-driven NF-κB activation and apoptosis and predisposes cells to necroptosis. Consequently, hace1–/– mice show enhanced colitis and colon cancer, which can be reverted by inactivation of pro-necroptotic kinase RIP3 and TNFR1.