Continual improvement of CRISPR-induced multiplex mutagenesis in Arabidopsis.

CRISPR/Cas9 is currently the most powerful tool to generate mutations in plant genomes and more efficient tools are needed as the scale of experiments increases. In the model plant Arabidopsis, the choice of the promoter driving Cas9 expression is critical to generate germline mutations. Several optimal promoters have been reported. However, it is unclear which promoter is ideal as they have not been thoroughly tested side by side. Furthermore, most plant vectors still use one of the two Cas9 nuclear localization sequence (NLS) configurations initially reported. We genotyped more than 6000 Arabidopsis T2 plants to test seven promoters and six types of NLSs across 14 targets to systematically improve the generation of single and multiplex inheritable mutations. We found that the RPS5A promoter and bipartite NLS were individually the most efficient components. When combined, 99% of T2 plants contained at least one knockout (KO) mutation and 84% contained 4- to 7-plex KOs, the highest multiplexing KO rate in Arabidopsis to date. These optimizations will be useful to generate higher-order KOs in the germline of Arabidopsis and will likely be applicable to other CRISPR systems as well.

The antigenic landscape of human influenza N2 neuraminidases from 2009 until 2017.

Human H3N2 influenza viruses are subject to rapid antigenic evolution which translates into frequent updates of the composition of seasonal influenza vaccines. Despite these updates, the effectiveness of influenza vaccines against H3N2-associated disease is suboptimal. Seasonal influenza vaccines primarily induce hemagglutinin-specific antibody responses. However, antibodies directed against influenza neuraminidase (NA) also contribute to protection. Here, we analysed the antigenic diversity of a panel of N2 NAs derived from human H3N2 viruses that circulated between 2009 and 2017. The antigenic breadth of these NAs was determined based on the NA inhibition (NAI) of a broad panel of ferret and mouse immune sera that were raised by infection and recombinant N2 NA immunisation. This assessment allowed us to distinguish at least four antigenic groups in the N2 NAs derived from human H3N2 viruses that circulated between 2009 and 2017. Computational analysis further revealed that the amino acid residues in N2 NA that have a major impact on susceptibility to NAI by immune sera are in proximity of the catalytic site. Finally, a machine learning method was developed that allowed to accurately predict the impact of mutations that are present in our N2 NA panel on NAI. These findings have important implications for the renewed interest to develop improved influenza vaccines based on the inclusion of a protective NA antigen formulation.

Two proteins, the hemagglutinin and the neuraminidase, protrude from the surface of the influenza virus. Their detection by the immune system allows the host organism to mount defences against the viral threat. The virus evolves in response to this pressure, which manifests as changes in the appearance of its hemagglutinin and neuraminidase. This process, known as antigenic drift, leads to the proteins evading detection. It is also why flu vaccines require frequent updates, as they rely on 'training' the immune system to recognise the most important strains in circulation ­ primarily by exposing it to appropriate versions of hemagglutinin. While the antigenic drift of hemagglutinin has been extensively studied, much less is known about how the neuraminidase accumulates mutations, and how these affect the immune response. To investigate this question, Catani et al. selected 43 genetically distant neuraminidases from human viral samples isolated between 2009 and 2017. Statistical analyses were applied to define their relatedness, revealing that a group of closely related neuraminidases predominated from 2009 to 2015, before they were being taken over by a second group. A third group, which was identified in viruses isolated in 2013, was remarkably close to the neuraminidase of strains that circulated in the late 1990s. The fourth and final group of neuraminidases was derived from influenza viruses that normally circulate in pigs but can also occasionally infect humans. Next, Catani et al. examined the immune response that these 43 neuraminidases could elicit in mice, as well as in ferrets ­ the animal most traditionally used in influenza research. This allowed them to pinpoint which changes in the neuraminidase sequences were important to escape recognition by the host. Data obtained from the two model species were comparable, suggesting that these experiments could be conducted on mice going forward, which are easier to work with than ferrets. Finally, Catani et al. used machine learning to build a computational model that could predict how strongly the immune system would respond to a specific neuraminidase variant. These findings could help guide the development of new vaccines that include neuraminidases tailored to best prime and train the immune system against a larger variety of strains. This may aid the development of 'supra-seasonal' vaccines that protect against a broad range of influenza viruses, reducing the need for yearly updates.

Safety and efficacy of faecal microbiota transplantation in patients with mild to moderate Parkinson's disease (GUT-PARFECT): a double-blind, placebo-controlled, randomised, phase 2 trial.

Background: Dysregulation of the gut microbiome has been implicated in Parkinson's disease (PD). This study aimed to evaluate the clinical effects and safety of a single faecal microbiota transplantation (FMT) in patients with early-stage PD. Methods: The GUT-PARFECT trial, a single-centre randomised, double-blind, placebo-controlled trial was conducted at Ghent University Hospital between December 01, 2020 and December 12, 2022. Participants (aged 50-65 years, Hoehn and Yahr stage 2) were randomly assigned to receive nasojejunal FMT with either healthy donor stool or their own stool. Computer-generated randomisation was done in a 1:1 ratio through permutated-block scheduling. Treatment allocation was concealed for participants and investigators. The primary outcome measure at 12 months was the change in the Movement Disorders Society-Unified Parkinson's Disease Rating Scale (MDS-UPDRS) motor score obtained during off-medication evaluations. Intention-to-treat analysis was performed using a mixed model for repeated measures analysis. This completed trial is registered on ClinicalTrials.gov (NCT03808389). Findings: Between December 2020 and December 2021, FMT procedures were conducted on 46 patients with PD: 22 in the healthy donor group and 24 in the placebo group. Clinical evaluations were performed at baseline, 3, 6, and 12 months post-FMT. Full data analysis was possible for 21 participants in the healthy donor group and 22 in the placebo group. After 12 months, the MDS-UPDRS motor score significantly improved by a mean of 5.8 points (95% CI -11.4 to -0.2) in the healthy donor group and by 2.7 points (-8.3 to 2.9) in the placebo group (p = 0.0235). Adverse events were limited to temporary abdominal discomfort. Interpretation: Our findings suggested a single FMT induced mild, but long-lasting beneficial effects on motor symptoms in patients with early-stage PD. These findings highlight the potential of modulating the gut microbiome as a therapeutic approach and warrant a further exploration of FMT in larger cohorts of patients with PD in various disease stages. Funding: Flemish PD patient organizations (VPL and Parkili), Research Foundation Flanders (FWO), Biocodex Microbiota Foundation.

N-terminal proteoforms may engage in different protein complexes.

Alternative translation initiation and alternative splicing may give rise to N-terminal proteoforms, proteins that differ at their N-terminus compared with their canonical counterparts. Such proteoforms can have altered localizations, stabilities, and functions. Although proteoforms generated from splice variants can be engaged in different protein complexes, it remained to be studied to what extent this applies to N-terminal proteoforms. To address this, we mapped the interactomes of several pairs of N-terminal proteoforms and their canonical counterparts. First, we generated a catalogue of N-terminal proteoforms found in the HEK293T cellular cytosol from which 22 pairs were selected for interactome profiling. In addition, we provide evidence for the expression of several N-terminal proteoforms, identified in our catalogue, across different human tissues, as well as tissue-specific expression, highlighting their biological relevance. Protein-protein interaction profiling revealed that the overlap of the interactomes for both proteoforms is generally high, showing their functional relation. We also showed that N-terminal proteoforms can be engaged in new interactions and/or lose several interactions compared with their canonical counterparts, thus further expanding the functional diversity of proteomes.

A decoupled Virotrap approach to study the interactomes of N-terminal proteoforms.

Given that up to 20% of N-termini of human proteins differ from canonical N-termini as retrieved from sequence databases, a variety of N-terminal proteoforms exists in human cells. These N-terminal proteoforms arise through alternative translation initiation or alternative splicing among others. While such proteoforms diversify the biological functions of the proteome, they remain largely understudied. Recent studies showed that proteoforms expand protein interaction networks by interacting with different prey proteins. As a mass spectrometry-based method to study protein-protein interactions, Virotrap avoids cell lysis by trapping protein complexes in viral-like particles, thereby allowing for the identification of transient and less stable interactions. This chapter describes an adjusted version of Virotrap, decoupled Virotrap, that allows for the detection of interaction partners specific for N-terminal proteoforms.

Protein interactors of 3-O sulfated heparan sulfates in human MCI and age-matched control cerebrospinal fluid.

Heparan sulfates (HS) proteoglycans are commonly found on the cell surface and mediate many processes. Binding of HS ligands is determined by the sulfation code on the HS chain that can be N-/2-O/6-O- or 3-O-sulfated, generating heterogenous sulfation patterns. 3-O sulfated HS (3S-HS) play a role in several (patho)physiological processes such as blood coagulation, viral pathogenesis and binding and internalization of tau in Alzheimer's disease. However, few 3S-HS-specific interactors are known. Thus, our insight into the role of 3S-HS in health and disease is limited, especially in the central nervous system. Using human CSF, we determined the interactome of synthetic HS with defined sulfation patterns. Our affinity-enrichment mass spectrometry studies expand the repertoire of proteins that may interact with (3S-)HS. Validating our approach, ATIII, a known 3S-HS interactor, was found to require GlcA-GlcNS6S3S for binding, similar to what has been reported. Our dataset holds novel, potential HS and 3S-HS protein ligands, that can be explored in future studies focusing on molecular mechanisms that depend on 3S-HS in (patho)physiological conditions.

Anti-neuraminidase and anti-hemagglutinin immune serum can confer inter-lineage cross protection against recent influenza B.

Influenza B viruses (IBV) are responsible for a considerable part of the burden caused by influenza virus infections. Since their emergence in the 1980s, the Yamagata and Victoria antigenic lineages of influenza B circulate in alternate patterns across the globe. Furthermore, their evolutionary divergence and the appearance of new IBV subclades complicates the prediction of future influenza vaccines compositions. It has been proposed that the addition of the neuraminidase (NA) antigen could potentially induce a broader protection and compensate for hemagglutinin (HA) mismatches in the current vaccines. Here we show that anti-NA and -HA sera against both Victoria and Yamagata lineages have limited inter-lineage cross-reactivity. When transferred to mice prior to infection with a panel of IBVs, anti-NA sera were as potent as anti-HA sera in conferring protection against homologous challenge and, in some cases, conferred superior protection against challenge with heterologous IBV strains.

An Unexpected Encounter: Respiratory Syncytial Virus Nonstructural Protein 1 Interacts with Mediator Subunit MED25.

Human respiratory syncytial virus (RSV) is the leading cause of severe acute lower respiratory tract infections in infants worldwide. Nonstructural protein NS1 of RSV modulates the host innate immune response by acting as an antagonist of type I and type III interferon (IFN) production and signaling in multiple ways. Likely, NS1 performs this function by interacting with different host proteins. In order to obtain a comprehensive overview of the NS1 interaction partners, we performed three complementary protein-protein interaction screens, i.e., BioID, MAPPIT, and KISS. To closely mimic a natural infection, the BioID proximity screen was performed using a recombinant RSV in which the NS1 protein is fused to a biotin ligase. Remarkably, MED25, a subunit of the Mediator complex, was identified in all three performed screening methods as a potential NS1-interacting protein. We confirmed the interaction between MED25 and RSV NS1 by coimmunoprecipitation, not only upon overexpression of NS1 but also with endogenous NS1 during RSV infection. We also demonstrate that the replication of RSV can be enhanced in MED25 knockout A549 cells, suggesting a potential antiviral role of MED25 during RSV infection. Mediator subunits function as transcriptional coactivators and are involved in transcriptional regulation of their target genes. Therefore, the interaction between RSV NS1 and cellular MED25 might be beneficial for RSV during infection by affecting host transcription and the host immune response to infection. IMPORTANCE Innate immune responses, including the production of type I and III interferons, play a crucial role in the first line of defense against RSV infection. However, only a poor induction of type I IFNs is observed during RSV infection, suggesting that RSV has evolved mechanisms to prevent type I IFN expression by the infected host cell. A unique RSV protein, NS1, is largely responsible for this effect, probably through interaction with multiple host proteins. A better understanding of the interactions that occur between RSV NS1 and host proteins may help to identify targets for an effective antiviral therapy. We addressed this question by performing three complementary protein-protein interaction screens and identified MED25 as an RSV NS1-interacting protein. We propose a role in innate anti-RSV defense for this Mediator complex subunit.

A general approach to identify low-frequency variants within influenza samples collected during routine surveillance.

Influenza viruses exhibit considerable diversity between hosts. Additionally, different quasispecies can be found within the same host. High-throughput sequencing technologies can be used to sequence a patient-derived virus population at sufficient depths to identify low-frequency variants (LFV) present in a quasispecies, but many challenges remain for reliable LFV detection because of experimental errors introduced during sample preparation and sequencing. High genomic copy numbers and extensive sequencing depths are required to differentiate false positive from real LFV, especially at low allelic frequencies (AFs). This study proposes a general approach for identifying LFV in patient-derived samples obtained during routine surveillance. Firstly, validated thresholds were determined for LFV detection, whilst balancing both the cost and feasibility of reliable LFV detection in clinical samples. Using a genetically well-defined population of influenza A viruses, thresholds of at least 104 genomes per microlitre and AF of ≥5 % were established as detection limits. Secondly, a subset of 59 retained influenza A (H3N2) samples from the 2016-2017 Belgian influenza season was composed. Thirdly, as a proof of concept for the added value of LFV for routine influenza monitoring, potential associations between patient data and whole genome sequencing data were investigated. A significant association was found between a high prevalence of LFV and disease severity. This study provides a general methodology for influenza LFV detection, which can also be adopted by other national influenza reference centres and for other viruses such as SARS-CoV-2. Additionally, this study suggests that the current relevance of LFV for routine influenza surveillance programmes might be undervalued.

Association of Cell Death Markers With Tumor Immune Cell Infiltrates After Chemo-Radiation in Cervical Cancer.

Irradiation induces distinct cellular responses such as apoptosis, necroptosis, iron-dependent cell death (a feature of ferroptosis), senescence, and mitotic catastrophe. Several of these outcomes are immunostimulatory and may represent a potential for immunogenic type of cell death (ICD) induced by radiotherapy triggering abscopal effects. The purpose of this study is to determine whether intra-tumoral ICD markers can serve as biomarkers for the prediction of patient's outcomes defined as the metastasis status and survival over a 5-year period. Thirty-eight patients with locally advanced cervical cancer, treated with neoadjuvant chemoradiotherapy using cisplatin were included in this study. Pre-treatment tumor biopsy and post-treatment hysterectomy samples were stained for cell death markers and danger associated molecular patterns (DAMPs): cleaved caspase-3 (apoptosis), phosphorylated mixed lineage kinase domain like pseudokinase (pMLKL; necroptosis), glutathione peroxidase 4 (GPX4; ferroptosis) and 4-hydroxy-2-noneal (4-HNE; ferroptosis), high mobility group box 1 (HMGB1) and calreticulin. Although these markers could not predict the patient's outcome in terms of relapse or survival, many significantly correlated with immune cell infiltration. For instance, inducing ferroptosis post-treatment seems to negatively impact immune cell recruitment. Measuring ICD markers could reflect the impact of treatment on the tumor microenvironment with regard to immune cell recruitment and infiltration. One Sentence Summary: Cell death readouts during neoadjuvant chemoradiation in cervical cancer.

MLKL deficiency in BrafV600EPten-/- melanoma model results in a modest delay of nevi development and reduced lymph node dissemination in male mice.

Cancers acquire several capabilities to survive the multistep process in carcinogenesis. Resisting cell death is one of them. Silencing of the necroptosis initiator Ripk3 occurs in a wide variety of cancer types including melanoma. Little is known about the role of the necroptosis executioner MLKL in tumor development. Studies often indicate opposing roles for MLKL as a tumor-suppressing or a tumor-promoting protein. This study investigates the role of MLKL during melanoma initiation and progression using a tamoxifen-inducible melanoma mouse model driven by melanocyte-specific overexpression of mutated Braf and simultaneous deletion of Pten (BrafV600EPten-/-). In this model we observed a clear sex difference: melanoma initiation and progression were faster in females mice. Mlkl deficiency in male mice resulted in a modest but significant reduction of nevi growth rate compared to the littermate control. In these mice, infiltration and expansion of melanoma cells in the inguinal lymph node were also modestly decreased. This is likely to be a consequence of the delay in nevi development. No significant difference was observed in the Mlkl-deficient condition in female mice in which melanoma development was faster. Overall, our results indicate that in this genetic model MLKL has a minor role during melanoma initiation and progression.

Continuous Mapping Identifies Loci Associated With Weevil Resistance [Cosmopolites sordidus (Germar)] in a Triploid Banana Population.

The first step toward marker-assisted selection is linking the phenotypes to molecular markers through quantitative trait loci (QTL) analysis. While the process is straightforward in self-pollinating diploid (2x) species, QTL analysis in polyploids requires unconventional methods. In this study, we have identified markers associated with weevil Cosmopolites sordidus (Germar) resistance in bananas using 138 triploid (2n = 3x) hybrids derived from a cross between a tetraploid "Monyet" (2n = 4x) and a 2x "Kokopo" (2n = 2x) banana genotypes. The population was genotyped by Diversity Arrays Technology Sequencing (DArTSeq), resulting in 18,009 polymorphic single nucleotide polymorphisms (SNPs) between the two parents. Marker-trait association was carried out by continuous mapping where the adjusted trait means for the corm peripheral damage (PD) and total cross-section damage (TXD), both on the logit scale, were regressed on the marker allele frequencies. Forty-four SNPs that were associated with corm PD were identified on the chromosomes 5, 6, and 8, with 41 of them located on chromosome 6 and segregated in "Kokopo." Eleven SNPs associated with corm total TXD were identified on chromosome 6 and segregated in "Monyet." The additive effect of replacing one reference allele with the alternative allele was determined at each marker position. The PD QTL was confirmed using conventional QTL linkage analysis in the simplex markers segregating in "Kokopo" (AAAA × RA). We also identified 43 putative genes in the vicinity of the markers significantly associated with the two traits. The identified loci associated with resistance to weevil damage will be used in the efforts of developing molecular tools for marker-assisted breeding in bananas.

The genome of the extremophile Artemia provides insight into strategies to cope with extreme environments.

BACKGROUND: Brine shrimp Artemia have an unequalled ability to endure extreme salinity and complete anoxia. This study aims to elucidate its strategies to cope with these stressors. RESULTS AND DISCUSSION: Here, we present the genome of an inbred A. franciscana Kellogg, 1906. We identified 21,828 genes of which, under high salinity, 674 genes and under anoxia, 900 genes were differentially expressed (42%, respectively 30% were annotated). Under high salinity, relevant stress genes and pathways included several Heat Shock Protein and Leaf Embryogenesis Abundant genes, as well as the trehalose metabolism. In addition, based on differential gene expression analysis, it can be hypothesized that a high oxidative stress response and endocytosis/exocytosis are potential salt management strategies, in addition to the expression of major facilitator superfamily genes responsible for transmembrane ion transport. Under anoxia, genes involved in mitochondrial function, mTOR signalling and autophagy were differentially expressed. Both high salt and anoxia enhanced degradation of erroneous proteins and protein chaperoning. Compared with other branchiopod genomes, Artemia had 0.03% contracted and 6% expanded orthogroups, in which 14% of the genes were differentially expressed under high salinity or anoxia. One phospholipase D gene family, shown to be important in plant stress response, was uniquely present in both extremophiles Artemia and the tardigrade Hypsibius dujardini, yet not differentially expressed under the described experimental conditions. CONCLUSIONS: A relatively complete genome of Artemia was assembled, annotated and analysed, facilitating research on its extremophile features, and providing a reference sequence for crustacean research.

Exploration of Synergistic Action of Cell Wall-Degrading Enzymes against Mycobacterium tuberculosis.

The major global health threat tuberculosis is caused by Mycobacterium tuberculosis. M. tuberculosis has a complex cell envelope-a partially covalently linked composite of polysaccharides, peptidoglycan, and lipids, including a mycolic acid layer-which conveys pathogenicity but also protects against antibiotics. Given previous successes in treating Gram-positive and -negative infections with cell wall-degrading enzymes, we investigated such an approach for M. tuberculosis. In this study, we aimed to (i) develop an M. tuberculosis microtiter growth inhibition assay that allows undisturbed cell envelope formation to overcome the invalidation of results by typical clumped M. tuberculosis growth in surfactant-free assays, (ii) explore anti-M. tuberculosis potency of cell wall layer-degrading enzymes, and (iii) investigate the concerted action of several such enzymes. We inserted a bacterial luciferase operon in an auxotrophic M. tuberculosis strain to develop a microtiter assay that allows proper evaluation of cell wall-degrading anti-M. tuberculosis enzymes. We assessed growth inhibition by enzymes (recombinant mycobacteriophage mycolic acid esterase [LysB], fungal α-amylase, and human and chicken egg white lysozymes) and combinations thereof in the presence or absence of biopharmaceutically acceptable surfactant. Our biosafety level 2 assay identified both LysB and lysozymes as potent M. tuberculosis inhibitors but only in the presence of surfactant. Moreover, the most potent disruption of the mycolic acid hydrophobic barrier was obtained by the highly synergistic combination of LysB, α-amylase, and polysorbate 80. Synergistically acting cell wall-degrading enzymes are potently inhibiting M. tuberculosis, which sets the scene for the design of specifically tailored antimycobacterial (fusion) enzymes. Airway delivery of protein therapeutics has already been established and should be studied in animal models for active TB.

Cell-penetrating Alphabody protein scaffolds for intracellular drug targeting.

The therapeutic scope of antibody and nonantibody protein scaffolds is still prohibitively limited against intracellular drug targets. Here, we demonstrate that the Alphabody scaffold can be engineered into a cell-penetrating protein antagonist against induced myeloid leukemia cell differentiation protein MCL-1, an intracellular target in cancer, by grafting the critical B-cell lymphoma 2 homology 3 helix of MCL-1 onto the Alphabody and tagging the scaffold's termini with designed cell-penetration polypeptides. Introduction of an albumin-binding moiety extended the serum half-life of the engineered Alphabody to therapeutically relevant levels, and administration thereof in mouse tumor xenografts based on myeloma cell lines reduced tumor burden. Crystal structures of such a designed Alphabody in complex with MCL-1 and serum albumin provided the structural blueprint of the applied design principles. Collectively, we provide proof of concept for the use of Alphabodies against intracellular disease mediators, which, to date, have remained in the realm of small-molecule therapeutics.

Zilucoplan in patients with acute hypoxic respiratory failure due to COVID-19 (ZILU-COV): A structured summary of a study protocol for a randomised controlled trial.

OBJECTIVES: Zilucoplan (complement C5 inhibitor) has profound effects on inhibiting acute lung injury post COVID-19, and can promote lung repair mechanisms that lead to improvement in lung oxygenation parameters. The purpose of this study is to investigate the efficacy and safety of Zilucoplan in improving oxygenation and short- and long-term outcome of COVID-19 patients with acute hypoxic respiratory failure. TRIAL DESIGN: This is a phase 2 academic, prospective, 2:1 randomized, open-label, multi-center interventional study. PARTICIPANTS: Adult patients (≥18y old) will be recruited at specialized COVID-19 units and ICUs at 9 Belgian hospitals. The main eligibility criteria are as follows: 1) Inclusion criteria: a. Recent (≥6 days and ≤16 days) SARS-CoV-2 infection. b. Chest CT scan showing bilateral infiltrates within the last 2 days prior to randomisation. c. Acute hypoxia (defined as PaO2/FiO2 below 350 mmHg or SpO2 below 93% on minimal 2 L/min supplemental oxygen). d. Signs of cytokine release syndrome characterized by either high serum ferritin, or high D-dimers, or high LDH or deep lymphopenia or a combination of those. 2) Exclusion criteria: e. Mechanical ventilation for more than 24 hours prior to randomisation. f. Active bacterial or fungal infection. g. History of meningococcal disease (due to the known high predisposition to invasive, often recurrent meningococcal infections of individuals deficient in components of the alternative and terminal complement pathways). INTERVENTION AND COMPARATOR: Patients in the experimental arm will receive daily 32,4 mg Zilucoplan subcutaneously and a daily IV infusion of 2g of the antibiotic ceftriaxone for 14 days (or until hospital discharge, whichever comes first) in addition to standard of care. These patients will receive additional prophylactic antibiotics until 14 days after the last Zilucoplan dose: hospitalized patients will receive a daily IV infusion of 2g of ceftriaxone, discharged patients will switch to daily 500 mg of oral ciprofloxacin. The control group will receive standard of care and a daily IV infusion of 2g of ceftriaxone for 1 week (or until hospital discharge, whichever comes first), to control for the effects of antibiotics on the clinical course of COVID-19. MAIN OUTCOMES: The primary endpoint is the improvement of oxygenation as measured by mean and/or median change from pre-treatment (day 1) to post-treatment (day 6 and 15 or at discharge, whichever comes first) in PaO2/FiO2 ratio, P(A-a)O2 gradient and a/A PO2 ratio. (PAO2= Partial alveolar pressure of oxygen, PaO2=partial arterial pressure of oxygen, FiO2=Fraction of inspired oxygen). RANDOMISATION: Patients will be randomized in a 2:1 ratio (Zilucoplan: control). Randomization will be done using an Interactive Web Response System (REDCap). BLINDING (MASKING): In this open-label trial neither participants, caregivers, nor those assessing the outcomes will be blinded to group assignment. NUMBERS TO BE RANDOMISED (SAMPLE SIZE): A total of 81 patients will be enrolled: 54 patients will be randomized to the experimental arm and 27 patients to the control arm. TRIAL STATUS: ZILU-COV protocol Version 4.0 (June 10 2020). Participant recruitment started on June 23 2020 and is ongoing. Given the uncertainty of the pandemic, it is difficult to predict the anticipated end date. TRIAL REGISTRATION: The trial was registered on Clinical Trials.gov on May 11th, 2020 (ClinicalTrials.gov Identifier: NCT04382755 ) and on EudraCT (Identifier: 2020-002130-33 ). FULL PROTOCOL: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol.

Ionizing radiation results in a mixture of cellular outcomes including mitotic catastrophe, senescence, methuosis, and iron-dependent cell death.

Radiotherapy is commonly used as a cytotoxic treatment of a wide variety of tumors. Interestingly, few case reports underlined its potential to induce immune-mediated abscopal effects, resulting in regression of metastases, distant from the irradiated site. These observations are rare, and apparently depend on the dose used, suggesting that dose-related cellular responses may be involved in the distant immunogenic responses. Ionizing radiation (IR) has been reported to elicit immunogenic apoptosis, necroptosis, mitotic catastrophe, and senescence. In order to link a cellular outcome with a particular dose of irradiation, we performed a systematic study in a panel of cell lines on the cellular responses at different doses of X-rays. Remarkably, we observed that all cell lines tested responded in a similar fashion to IR with characteristics of mitotic catastrophe, senescence, lipid peroxidation, and caspase activity. Iron chelators (but not Ferrostatin-1 or vitamin E) could prevent the formation of lipid peroxides and cell death induced by IR, suggesting a crucial role of iron-dependent cell death during high-dose irradiation. We also show that in K-Ras-mutated cells, IR can induce morphological features reminiscent of methuosis, a cell death modality that has been recently described following H-Ras or K-Ras mutation overexpression.

Treatment of severely ill COVID-19 patients with anti-interleukin drugs (COV-AID): A structured summary of a study protocol for a randomised controlled trial.

OBJECTIVES: The purpose of this study is to test the safety and effectiveness of individually or simultaneously blocking IL-6, IL-6 receptor and IL-1 versus standard of care on blood oxygenation and systemic cytokine release syndrome in patients with COVID-19 coronavirus infection and acute hypoxic respiratory failure and systemic cytokine release syndrome. TRIAL DESIGN: A phase 3 prospective, multi-center, interventional, open label, 6-arm 2x2 factorial design study. PARTICIPANTS: Subjects will be recruited at the specialized COVID-19 wards and/or ICUs at 16 Belgian participating hospitals. Only adult (≥18y old) patients will be recruited with recent (≤16 days) COVID-19 infection and acute hypoxia (defined as PaO2/FiO2 below 350mmHg or PaO2/FiO2 below 280 on supplemental oxygen and immediately requiring high flow oxygen device or mechanical ventilation) and signs of systemic cytokine release syndrome characterized by high serum ferritin, or high D-dimers, or high LDH or deep lymphopenia or a combination of those, who have not been on mechanical ventilation for more than 24 hours before randomisation. Patients should have had a chest X-ray and/or CT scan showing bilateral infiltrates within the last 2 days before randomisation. Patients with active bacterial or fungal infection will be excluded. INTERVENTION AND COMPARATOR: Patients will be randomized to 1 of 5 experimental arms versus usual care. The experimental arms consist of Anakinra alone (anti-IL-1 binding the IL-1 receptor), Siltuximab alone (anti-IL-6 chimeric antibody), a combination of Siltuximab and Anakinra, Tocilizumab alone (humanised anti-IL-6 receptor antibody) or a combination of Anakinra with Tocilizumab in addition to standard care. Patients treated with Anakinra will receive a daily subcutaneous injection of 100mg for a maximum of 28 days or until hospital discharge, whichever comes first. Siltuximab (11mg/kg) or Tocilizumab (8mg/kg, with a maximum dose of 800mg) are administered as a single intravenous injection immediately after randomization. MAIN OUTCOMES: The primary end point is the time to clinical improvement defined as the time from randomization to either an improvement of two points on a six-category ordinal scale measured daily till day 28 or discharge from the hospital or death. This ordinal scale is composed of (1) Death; (2) Hospitalized, on invasive mechanical ventilation or ECMO; (3) Hospitalized, on non-invasive ventilation or high flow oxygen devices; (4) Hospitalized, requiring supplemental oxygen; (5) Hospitalized, not requiring supplemental oxygen; (6) Not hospitalized. RANDOMISATION: Patients will be randomized using an Interactive Web Response System (REDCap). A 2x2 factorial design was selected with a 2:1 randomization regarding the IL-1 blockade (Anakinra) and a 1:2 randomization regarding the IL-6 blockade (Siltuximab and Tocilizumab). BLINDING (MASKING): In this open-label trial neither participants, caregivers, nor those assessing the outcomes are blinded to group assignment. NUMBERS TO BE RANDOMISED (SAMPLE SIZE): A total of 342 participants will be enrolled: 76 patients will receive usual care, 76 patients will receive Siltuximab alone, 76 patients will receive Tocilizumab alone, 38 will receive Anakinra alone, 38 patients will receive Anakinra and Siltuximab and 38 patients will receive Anakinra and Tocilizumab. TRIAL STATUS: COV-AID protocol version 3.0 (15 Apr 2020). Participant recruitment is ongoing and started on April 4th 2020. Given the current decline of the COVID-19 pandemic in Belgium, it is difficult to anticipate the rate of participant recruitment. TRIAL REGISTRATION: The trial was registered on Clinical Trials.gov on April 1st, 2020 (ClinicalTrials.gov Identifier: NCT04330638) and on EudraCT on April 3rd 2020 (Identifier: 2020-001500-41). FULL PROTOCOL: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol.

Correction to: Treatment of severely ill COVID-19 patients with anti-interleukin drugs (COV-AID): A structured summary of a study protocol for a randomised controlled trial.

An amendment to this paper has been published and can be accessed via the original article.

Correction to: Sargramostim to treat patients with acute hypoxic respiratory failure due to COVID-19 (SARPAC): A structured summary of a study protocol for a randomised controlled trial.

An amendment to this paper has been published and can be accessed via the original article.