Uptake of oomycete RXLR effectors into host cells by clathrin-mediated endocytosis.

Filamentous (oomycete and fungal) plant pathogens deliver cytoplasmic effector proteins into host cells to facilitate disease. How RXLR effectors from the potato late blight pathogen Phytophthora infestans enter host cells is unknown. One possible route involves clathrin-mediated endocytosis (CME). Transient silencing of NbCHC, encoding clathrin heavy chain, or the endosome marker gene NbAra6 encoding a Rab GTPase in the model host Nicotiana benthamiana, attenuated P. infestans infection and reduced translocation of RXLR effector fusions from transgenic pathogen strains into host cells. By contrast, silencing PP1c isoforms, susceptibility factors not required for endocytosis, reduced infection but did not attenuate RXLR effector uptake. Endosome enrichment by ultracentrifugation and sucrose gradient fractionation revealed co-localization of RXLR effector Pi04314-RFP with clathrin-coated vesicles. Immunopurification of clathrin- and NbAra6-associated vesicles during infection showed that RXLR effectors Pi04314-RFP and AvrBlb1-RFP, but not apoplastic effector PiSCR74-RFP, were co-immunoprecipitated during infection with pathogen strains secreting these effectors. Tandem mass spectrometry analyses of proteins co-immunoprecipitated with NbAra6-GFP during infection revealed enrichment of host proteins associated with endocytic vesicles alongside multiple pathogen RXLR effectors, but not apoplastic effectors, including PiSCR74, which do not enter host cells. Our data show that the uptake of P. infestans RXLR effectors into plant cells occurs via CME.

RNA polymerase II promotes the organization of chromatin following DNA replication.

Understanding how chromatin organisation is duplicated on the two daughter strands is a central question in epigenetics. In mammals, following the passage of the replisome, nucleosomes lose their defined positioning and transcription contributes to their re-organisation. However, whether transcription plays a greater role in the organization of chromatin following DNA replication remains unclear. Here we analysed protein re-association with newly replicated DNA upon inhibition of transcription using iPOND coupled to quantitative mass spectrometry. We show that nucleosome assembly and the re-establishment of most histone modifications are uncoupled from transcription. However, RNAPII acts to promote the re-association of hundreds of proteins with newly replicated chromatin via pathways that are not observed in steady-state chromatin. These include ATP-dependent remodellers, transcription factors and histone methyltransferases. We also identify a set of DNA repair factors that may handle transcription-replication conflicts during normal transcription in human non-transformed cells. Our study reveals that transcription plays a greater role in the organization of chromatin post-replication than previously anticipated.

Triggering MSR1 promotes JNK-mediated inflammation in IL-4-activated macrophages.

Alternatively activated M2 macrophages play an important role in maintenance of tissue homeostasis by scavenging dead cells, cell debris and lipoprotein aggregates via phagocytosis. Using proteomics, we investigated how alternative activation, driven by IL-4, modulated the phagosomal proteome to control macrophage function. Our data indicate that alternative activation enhances homeostatic functions such as proteolysis, lipolysis and nutrient transport. Intriguingly, we identified the enhanced recruitment of the TAK1/MKK7/JNK signalling complex to phagosomes of IL-4-activated macrophages. The recruitment of this signalling complex was mediated through K63 polyubiquitylation of the macrophage scavenger receptor 1 (MSR1). Triggering of MSR1 in IL-4-activated macrophages leads to enhanced JNK activation, thereby promoting a phenotypic switch from an anti-inflammatory to a pro-inflammatory state, which was abolished upon MSR1 deletion or JNK inhibition. Moreover, MSR1 K63 polyubiquitylation correlated with the activation of JNK signalling in ovarian cancer tissue from human patients, suggesting that it may be relevant for macrophage phenotypic shift in vivo Altogether, we identified that MSR1 signals through JNK via K63 polyubiquitylation and provides evidence for the receptor's involvement in macrophage polarization.

Thermal proteome profiling of breast cancer cells reveals proteasomal activation by CDK4/6 inhibitor palbociclib.

Palbociclib is a CDK4/6 inhibitor approved for metastatic estrogen receptor-positive breast cancer. In addition to G1 cell cycle arrest, palbociclib treatment results in cell senescence, a phenotype that is not readily explained by CDK4/6 inhibition. In order to identify a molecular mechanism responsible for palbociclib-induced senescence, we performed thermal proteome profiling of MCF7 breast cancer cells. In addition to affecting known CDK4/6 targets, palbociclib induces a thermal stabilization of the 20S proteasome, despite not directly binding to it. We further show that palbociclib treatment increases proteasome activity independently of the ubiquitin pathway. This leads to cellular senescence, which can be counteracted by proteasome inhibitors. Palbociclib-induced proteasome activation and senescence is mediated by reduced proteasomal association of ECM29. Loss of ECM29 activates the proteasome, blocks cell proliferation, and induces a senescence-like phenotype. Finally, we find that ECM29 mRNA levels are predictive of relapse-free survival in breast cancer patients treated with endocrine therapy. In conclusion, thermal proteome profiling identifies the proteasome and ECM29 protein as mediators of palbociclib activity in breast cancer cells.

Wnt regulates amino acid transporter Slc7a5 and so constrains the integrated stress response in mouse embryos.

Amino acids are essential for cellular metabolism, and it is important to understand how nutrient supply is coordinated with changing energy requirements during embryogenesis. Here, we show that the amino acid transporter Slc7a5/Lat1 is highly expressed in tissues undergoing morphogenesis and that Slc7a5-null mouse embryos have profound neural and limb bud outgrowth defects. Slc7a5-null neural tissue exhibited aberrant mTORC1 activity and cell proliferation; transcriptomics, protein phosphorylation and apoptosis analyses further indicated induction of the integrated stress response as a potential cause of observed defects. The pattern of stress response gene expression induced in Slc7a5-null embryos was also detected at low level in wild-type embryos and identified stress vulnerability specifically in tissues undergoing morphogenesis. The Slc7a5-null phenotype is reminiscent of Wnt pathway mutants, and we show that Wnt/ß-catenin loss inhibits Slc7a5 expression and induces this stress response. Wnt signalling therefore normally supports the metabolic demands of morphogenesis and constrains cellular stress. Moreover, operation in the embryo of the integrated stress response, which is triggered by pathogen-mediated as well as metabolic stress, may provide a mechanistic explanation for a range of developmental defects.

Neural differentiation, selection and transcriptomic profiling of human neuromesodermal progenitor-like cells in vitro.

Robust protocols for directed differentiation of human pluripotent cells are required to determine whether mechanisms operating in model organisms are relevant to our own development. Recent work in vertebrate embryos has identified neuromesodermal progenitors as a bipotent cell population that contributes to paraxial mesoderm and spinal cord. However, precise protocols for in vitro differentiation of human spinal cord progenitors are lacking. Informed by signalling in amniote embryos, we show here that transient dual-SMAD inhibition, together with retinoic acid (dSMADi-RA), provides rapid and reproducible induction of human spinal cord progenitors from neuromesodermal progenitor-like cells. Using CRISPR-Cas9 to engineer human embryonic stem cells with a GFP-reporter for neuromesodermal progenitor-associated gene Nkx1.2 we facilitate selection of this cell population. RNA-sequencing was then used to identify human and conserved neuromesodermal progenitor transcriptional signatures, to validate this differentiation protocol and to reveal new pathways/processes in human neural differentiation. This optimised protocol, novel reporter line and transcriptomic data are useful resources with which to dissect molecular mechanisms regulating human spinal cord generation and allow the scaling-up of distinct cell populations for global analyses, including proteomic, biochemical and chromatin interrogation.

Kinetochores coordinate pericentromeric cohesion and early DNA replication by Cdc7-Dbf4 kinase recruitment.

Centromeres play several important roles in ensuring proper chromosome segregation. Not only do they promote kinetochore assembly for microtubule attachment, but they also support robust sister chromatid cohesion at pericentromeres and facilitate replication of centromeric DNA early in S phase. However, it is still elusive how centromeres orchestrate all these functions at the same site. Here, we show that the budding yeast Dbf4-dependent kinase (DDK) accumulates at kinetochores in telophase, facilitated by the Ctf19 kinetochore complex. This promptly recruits Sld3-Sld7 replication initiator proteins to pericentromeric replication origins so that they initiate replication early in S phase. Furthermore, DDK at kinetochores independently recruits the Scc2-Scc4 cohesin loader to centromeres in G1 phase. This enhances cohesin loading and facilitates robust pericentromeric cohesion in S phase. Thus, we have found the central mechanism by which kinetochores orchestrate early S phase DNA replication and robust sister chromatid cohesion at microtubule attachment sites.

How well do RNA-Seq differential gene expression tools perform in a complex eukaryote? A case study in Arabidopsis thaliana.

MOTIVATION: RNA-seq experiments are usually carried out in three or fewer replicates. In order to work well with so few samples, differential gene expression (DGE) tools typically assume the form of the underlying gene expression distribution. In this paper, the statistical properties of gene expression from RNA-seq are investigated in the complex eukaryote, Arabidopsis thaliana, extending and generalizing the results of previous work in the simple eukaryote Saccharomyces cerevisiae. RESULTS: We show that, consistent with the results in S.cerevisiae, more gene expression measurements in A.thaliana are consistent with being drawn from an underlying negative binomial distribution than either a log-normal distribution or a normal distribution, and that the size and complexity of the A.thaliana transcriptome does not influence the false positive rate performance of nine widely used DGE tools tested here. We therefore recommend the use of DGE tools that are based on the negative binomial distribution. AVAILABILITY AND IMPLEMENTATION: The raw data for the 17 WT Arabidopsis thaliana datasets is available from the European Nucleotide Archive (E-MTAB-5446). The processed and aligned data can be visualized in context using IGB (Freese et al., 2016), or downloaded directly, using our publicly available IGB quickload server at https://compbio.lifesci.dundee.ac.uk/arabidopsisQuickload/public_quickload/ under 'RNAseq>Froussios2019'. All scripts and commands are available from github at https://github.com/bartongroup/KF_arabidopsis-GRNA. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

EMSY expression affects multiple components of the skin barrier with relevance to atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is a common, complex, and highly heritable inflammatory skin disease. Genome-wide association studies offer opportunities to identify molecular targets for drug development. A risk locus on chromosome 11q13.5 lies between 2 candidate genes, EMSY and LRRC32 (leucine-rich repeat-containing 32) but the functional mechanisms affecting risk of AD remain unclear. OBJECTIVES: We sought to apply a combination of genomic and molecular analytic techniques to investigate which genes are responsible for genetic risk at this locus and to define mechanisms contributing to atopic skin disease. METHODS: We used interrogation of available genomic and chromosome conformation data in keratinocytes, small interfering RNA (siRNA)-mediated knockdown in skin organotypic culture and functional assessment of barrier parameters, mass spectrometric global proteomic analysis and quantitative lipid analysis, electron microscopy of organotypic skin, and immunohistochemistry of human skin samples. RESULTS: Genomic data indicate active promoters in the genome-wide association study locus and upstream of EMSY; EMSY, LRRC32, and intergenic variants all appear to be within a single topologically associating domain. siRNA-knockdown of EMSY in organotypic culture leads to enhanced development of barrier function, reflecting increased expression of structural and functional proteins, including filaggrin and filaggrin-2, as well as long-chain ceramides. Conversely, overexpression of EMSY in keratinocytes leads to a reduction in markers of barrier formation. Skin biopsy samples from patients with AD show greater EMSY staining in the nucleus, which is consistent with an increased functional effect of this transcriptional control protein. CONCLUSION: Our findings demonstrate an important role for EMSY in transcriptional regulation and skin barrier formation, supporting EMSY inhibition as a therapeutic approach.

Mechanisms mitigating problems associated with multiple kinetochores on one microtubule in early mitosis.

Proper chromosome segregation in mitosis relies on correct kinetochore interaction with spindle microtubules. In early mitosis, each kinetochore usually interacts with the lateral side of each microtubule and is subsequently tethered at the microtubule end. However, since eukaryotic cells carry multiple chromosomes, multiple kinetochores could occasionally interact with a single microtubule. The consequence of this is unknown. Here, we find that, although two kinetochores (two pairs of sister kinetochores) can interact with the lateral side of one microtubule, only one kinetochore can form a sustained attachment to the microtubule end in budding yeast (Saccharomyces cerevisiae). This leads to detachment of the other kinetochore from the microtubule end (or a location in its proximity). Intriguingly, in this context, kinetochore sliding along a microtubule towards a spindle pole delays and diminishes discernible kinetochore detachment. This effect expedites collection of the entire set of kinetochores to a spindle pole. We propose that cells are equipped with the kinetochore-sliding mechanism to mitigate problems associated with multiple kinetochores on one microtubule in early mitosis.

How many biological replicates are needed in an RNA-seq experiment and which differential expression tool should you use?

RNA-seq is now the technology of choice for genome-wide differential gene expression experiments, but it is not clear how many biological replicates are needed to ensure valid biological interpretation of the results or which statistical tools are best for analyzing the data. An RNA-seq experiment with 48 biological replicates in each of two conditions was performed to answer these questions and provide guidelines for experimental design. With three biological replicates, nine of the 11 tools evaluated found only 20%-40% of the significantly differentially expressed (SDE) genes identified with the full set of 42 clean replicates. This rises to >85% for the subset of SDE genes changing in expression by more than fourfold. To achieve >85% for all SDE genes regardless of fold change requires more than 20 biological replicates. The same nine tools successfully control their false discovery rate at ≲5% for all numbers of replicates, while the remaining two tools fail to control their FDR adequately, particularly for low numbers of replicates. For future RNA-seq experiments, these results suggest that at least six biological replicates should be used, rising to at least 12 when it is important to identify SDE genes for all fold changes. If fewer than 12 replicates are used, a superior combination of true positive and false positive performances makes edgeR and DESeq2 the leading tools. For higher replicate numbers, minimizing false positives is more important and DESeq marginally outperforms the other tools.

Quantitative proteome analysis of temporally resolved phagosomes following uptake via key phagocytic receptors.

Macrophages operate at the forefront of innate immunity and their discrimination of foreign versus "self" particles is critical for a number of responses including efficient pathogen killing, antigen presentation, and cytokine induction. In order to efficiently destroy the particles and detect potential threats, macrophages express an array of receptors to sense and phagocytose prey particles. In this study, we accurately quantified a proteomic time-course of isolated phagosomes from murine bone marrow-derived macrophages induced by particles conjugated to seven different ligands representing pathogen-associated molecular patterns, immune opsonins or apoptotic cell markers. We identified a clear functional differentiation over the three timepoints and detected subtle differences between certain ligand-phagosomes, indicating that triggering of receptors through a single ligand type has mild, but distinct, effects on phagosome proteome and function. Moreover, our data shows that uptake of phosphatidylserine-coated beads induces an active repression of NF-κB immune responses upon Toll-like receptor (TLR)-activation by recruitment of anti-inflammatory regulators to the phagosome. This data shows for the first time a systematic time-course analysis of bone marrow-derived macrophages phagosomes and how phagosome fate is regulated by the receptors triggered for phagocytosis.

High resolution imaging reveals heterogeneity in chromatin states between cells that is not inherited through cell division.

BACKGROUND: Genomes of eukaryotes exist as chromatin, and it is known that different chromatin states can influence gene regulation. Chromatin is not a static structure, but is known to be dynamic and vary between cells. In order to monitor the organisation of chromatin in live cells we have engineered fluorescent fusion proteins which recognize specific operator sequences to tag pairs of syntenic gene loci. The separation of these loci was then tracked in three dimensions over time using fluorescence microscopy. RESULTS: We established a work flow for measuring the distance between two fluorescently tagged, syntenic gene loci with a mean measurement error of 63 nm. In general, physical separation was observed to increase with increasing genomic separations. However, the extent to which chromatin is compressed varies for different genomic regions. No correlation was observed between compaction and the distribution of chromatin markers from genomic datasets or with contacts identified using capture based approaches. Variation in spatial separation was also observed within cells over time and between cells. Differences in the conformation of individual loci can persist for minutes in individual cells. Separation of reporter loci was found to be similar in related and unrelated daughter cell pairs. CONCLUSIONS: The directly observed physical separation of reporter loci in live cells is highly dynamic both over time and from cell to cell. However, consistent differences in separation are observed over some chromosomal regions that do not correlate with factors known to influence chromatin states. We conclude that as yet unidentified parameters influence chromatin configuration. We also find that while heterogeneity in chromatin states can be maintained for minutes between cells, it is not inherited through cell division. This may contribute to cell-to-cell transcriptional heterogeneity.

Statistical models for RNA-seq data derived from a two-condition 48-replicate experiment.

MOTIVATION: High-throughput RNA sequencing (RNA-seq) is now the standard method to determine differential gene expression. Identifying differentially expressed genes crucially depends on estimates of read-count variability. These estimates are typically based on statistical models such as the negative binomial distribution, which is employed by the tools edgeR, DESeq and cuffdiff. Until now, the validity of these models has usually been tested on either low-replicate RNA-seq data or simulations. RESULTS: A 48-replicate RNA-seq experiment in yeast was performed and data tested against theoretical models. The observed gene read counts were consistent with both log-normal and negative binomial distributions, while the mean-variance relation followed the line of constant dispersion parameter of ∼0.01. The high-replicate data also allowed for strict quality control and screening of 'bad' replicates, which can drastically affect the gene read-count distribution. AVAILABILITY AND IMPLEMENTATION: RNA-seq data have been submitted to ENA archive with project ID PRJEB5348. CONTACT: g.j.barton@dundee.ac.uk.

High-resolution quantitative proteome analysis reveals substantial differences between phagosomes of RAW 264.7 and bone marrow derived macrophages.

Macrophages are important immune cells operating at the forefront of innate immunity by taking up foreign particles and microbes through phagocytosis. The RAW 264.7 cell line is commonly used for experiments in the macrophage and phagocytosis field. However, little is known how its functions compare to primary macrophages. Here, we have performed an in-depth proteomics characterization of phagosomes from RAW 264.7 and bone marrow derived macrophages by quantifying more than 2500 phagosomal proteins. Our data indicate that there are significant differences for a large number of proteins including important receptors such as mannose receptor 1 and Siglec-1. Moreover, bone marrow derived macrophages phagosomes mature considerably faster by fusion with endosomes and the lysosome which we validated using fluorogenic phagocytic assays. We provide a valuable resource for researcher in the field and recommend careful use of the RAW 264.7 cell line when studying phagosome functions. All MS data have been deposited in the ProteomeXchange with identifier PXD001293 (http://proteomecentral.proteomexchange.org/dataset/PXD001293).

Live imaging of nascent RNA dynamics reveals distinct types of transcriptional pulse regulation.

Transcription of genes can be discontinuous, occurring in pulses or bursts. It is not clear how properties of transcriptional pulses vary between different genes. We compared the pulsing of five housekeeping and five developmentally induced genes by direct imaging of single gene transcriptional events in individual living Dictyostelium cells. Each gene displayed its own transcriptional signature, differing in probability of firing and pulse duration, frequency, and intensity. In contrast to the prevailing view from both prokaryotes and eukaryotes that transcription displays binary behavior, strongly expressed housekeeping genes altered the magnitude of their transcriptional pulses during development. These nonbinary "tunable" responses may be better suited than stochastic switch behavior for housekeeping functions. Analysis of RNA synthesis kinetics using fluorescence recovery after photobleaching implied modulation of housekeeping-gene pulse strength occurs at the level of transcription initiation rather than elongation. In addition, disparities between single cell and population measures of transcript production suggested differences in RNA stability between gene classes. Analysis of stability using RNAseq revealed no major global differences in stability between developmental and housekeeping transcripts, although strongly induced RNAs showed unusually rapid decay, indicating tight regulation of expression.

A periodicity of approximately 1 hour in X-ray emission from the active galaxy RE J1034+396.

Active galactic nuclei and quasars are thought to be scaled-up versions of Galactic black hole binaries, powered by accretion onto supermassive black holes with masses of 10(6)-10(9) M[symbol: see text], as opposed to the approximately 10 M [symbol: see text] in binaries (here M [symbol: see text] is the solar mass). One example of the similarities between these two types of systems is the characteristic rapid X-ray variability seen from the accretion flow. The power spectrum of this variability in black hole binaries consists of a broad noise with multiple quasi-periodic oscillations superimposed on it. Although the broad noise component has been observed in many active galactic nuclei, there have hitherto been no significant detections of quasi-periodic oscillations. Here we report the discovery of an approximately 1-hour X-ray periodicity in a bright active galaxy, RE J1034+396. The signal is highly statistically significant (at the 5.6 sigma level) and very coherent, with quality factor Q > 16. The X-ray modulation arises from the direct vicinity of the black hole.

A quantitative spatial proteomics analysis of proteome turnover in human cells.

Measuring the properties of endogenous cell proteins, such as expression level, subcellular localization, and turnover rates, on a whole proteome level remains a major challenge in the postgenome era. Quantitative methods for measuring mRNA expression do not reliably predict corresponding protein levels and provide little or no information on other protein properties. Here we describe a combined pulse-labeling, spatial proteomics and data analysis strategy to characterize the expression, localization, synthesis, degradation, and turnover rates of endogenously expressed, untagged human proteins in different subcellular compartments. Using quantitative mass spectrometry and stable isotope labeling with amino acids in cell culture, a total of 80,098 peptides from 8,041 HeLa proteins were quantified, and their spatial distribution between the cytoplasm, nucleus and nucleolus determined and visualized using specialized software tools developed in PepTracker. Using information from ion intensities and rates of change in isotope ratios, protein abundance levels and protein synthesis, degradation and turnover rates were calculated for the whole cell and for the respective cytoplasmic, nuclear, and nucleolar compartments. Expression levels of endogenous HeLa proteins varied by up to seven orders of magnitude. The average turnover rate for HeLa proteins was ~20 h. Turnover rate did not correlate with either molecular weight or net charge, but did correlate with abundance, with highly abundant proteins showing longer than average half-lives. Fast turnover proteins had overall a higher frequency of PEST motifs than slow turnover proteins but no general correlation was observed between amino or carboxyl terminal amino acid identities and turnover rates. A subset of proteins was identified that exist in pools with different turnover rates depending on their subcellular localization. This strongly correlated with subunits of large, multiprotein complexes, suggesting a general mechanism whereby their assembly is controlled in a different subcellular location to their main site of function.

Tmem79/Matt is the matted mouse gene and is a predisposing gene for atopic dermatitis in human subjects.

BACKGROUND: Atopic dermatitis (AD) is a major inflammatory condition of the skin caused by inherited skin barrier deficiency, with mutations in the filaggrin gene predisposing to development of AD. Support for barrier deficiency initiating AD came from flaky tail mice, which have a frameshift mutation in Flg and also carry an unknown gene, matted, causing a matted hair phenotype. OBJECTIVE: We sought to identify the matted mutant gene in mice and further define whether mutations in the human gene were associated with AD. METHODS: A mouse genetics approach was used to separate the matted and Flg mutations to produce congenic single-mutant strains for genetic and immunologic analysis. Next-generation sequencing was used to identify the matted gene. Five independently recruited AD case collections were analyzed to define associations between single nucleotide polymorphisms (SNPs) in the human gene and AD. RESULTS: The matted phenotype in flaky tail mice is due to a mutation in the Tmem79/Matt gene, with no expression of the encoded protein mattrin in the skin of mutant mice. Matt(ft) mice spontaneously have dermatitis and atopy caused by a defective skin barrier, with mutant mice having systemic sensitization after cutaneous challenge with house dust mite allergens. Meta-analysis of 4,245 AD cases and 10,558 population-matched control subjects showed that a missense SNP, rs6684514, [corrected] in the human MATT gene has a small but significant association with AD. CONCLUSION: In mice mutations in Matt cause a defective skin barrier and spontaneous dermatitis and atopy. A common SNP in MATT has an association with AD in human subjects.

SFX-01 in hospitalised patients with community-acquired pneumonia during the COVID-19 pandemic: a double-blind, randomised, placebo-controlled trial.

Introduction: Sulforaphane can induce the transcription factor, Nrf2, promoting antioxidant and anti-inflammatory responses. In this study, hospitalised patients with community-acquired pneumonia (CAP) were treated with stabilised synthetic sulforaphane (SFX-01) to evaluate impact on clinical status and inflammation. Methods: Double-blind, randomised, placebo-controlled trial of SFX-01 (300 mg oral capsule, once daily for 14 days) conducted in Dundee, UK, between November 2020 and May 2021. Patients had radiologically confirmed CAP and CURB-65 (confusion, urea >7 mmol·L-1, respiratory rate ≥30 breaths·min-1, blood pressure <90 mmHg (systolic) or ≤60 mmHg (diastolic), age ≥65 years) score ≥1. The primary outcome was the seven-point World Health Organization clinical status scale at day 15. Secondary outcomes included time to clinical improvement, length of stay and mortality. Effects on Nrf2 activity and inflammation were evaluated on days 1, 8 and 15 by measurement of 45 serum cytokines and mRNA sequencing of peripheral blood leukocytes. Results: The trial was terminated prematurely due to futility with 133 patients enrolled. 65 patients were randomised to SFX-01 treatment and 68 patients to placebo. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection was the cause of CAP in 103 (77%) cases. SFX-01 treatment did not improve clinical status at day 15 (adjusted OR 0.87, 95% CI 0.41-1.83; p=0.71), time to clinical improvement (adjusted hazard ratio (aHR) 1.02, 95% CI 0.70-1.49), length of stay (aHR 0.84, 95% CI 0.56-1.26) or 28-day mortality (aHR 1.45, 95% CI 0.67-3.16). The expression of Nrf2 targets and pro-inflammatory genes, including interleukin (IL)-6, IL-1ß and tumour necrosis factor-α, was not significantly changed by SFX-01 treatment. At days 8 and 15, respectively, 310 and 42 significant differentially expressed genes were identified between groups (false discovery rate adjusted p<0.05, log2FC >1). Conclusion: SFX-01 treatment did not improve clinical status or modulate key Nrf2 targets in patients with CAP primarily due to SARS-CoV-2 infection.