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.

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.

The location and development of Replicon Cluster Domains in early replicating DNA.

Background: It has been known for many years that in metazoan cells, replication origins are organised into clusters where origins within each cluster fire near-synchronously. Despite clusters being a fundamental organising principle of metazoan DNA replication, the genomic location of origin clusters has not been documented. Methods: We synchronised human U2OS by thymidine block and release followed by L-mimosine block and release to create a population of cells progressing into S phase with a high degree of synchrony. At different times after release into S phase, cells were pulsed with EdU; the EdU-labelled DNA was then pulled down, sequenced and mapped onto the human genome. Results: The early replicating DNA showed features at a range of scales. Wavelet analysis showed that the major feature of the early replicating DNA was at a size of 500 kb, consistent with clusters of replication origins. Over the first two hours of S phase, these Replicon Cluster Domains broadened in width, consistent with their being enlarged by the progression of replication forks at their outer boundaries. The total replication signal associated with each Replicon Cluster Domain varied considerably, and this variation was reproducible and conserved over time. We provide evidence that this variability in replication signal was at least in part caused by Replicon Cluster Domains being activated at different times in different cells in the population. We also provide evidence that adjacent clusters had a statistical preference for being activated in sequence across a group, consistent with the 'domino' model of replication focus activation order observed by microscopy. Conclusions: We show that early replicating DNA is organised into Replicon Cluster Domains that behave as expected of replicon clusters observed by DNA fibre analysis. The coordinated activation of different Replicon Cluster Domains can generate the replication timing programme by which the genome is duplicated.

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.

Acute depletion of the ARID1A subunit of SWI/SNF complexes reveals distinct pathways for activation and repression of transcription.

The ARID1A subunit of SWI/SNF chromatin remodeling complexes is a potent tumor suppressor. Here, a degron is applied to detect rapid loss of chromatin accessibility at thousands of loci where ARID1A acts to generate accessible minidomains of nucleosomes. Loss of ARID1A also results in the redistribution of the coactivator EP300. Co-incident EP300 dissociation and lost chromatin accessibility at enhancer elements are highly enriched adjacent to rapidly downregulated genes. In contrast, sites of gained EP300 occupancy are linked to genes that are transcriptionally upregulated. These chromatin changes are associated with a small number of genes that are differentially expressed in the first hours following loss of ARID1A. Indirect or adaptive changes dominate the transcriptome following growth for days after loss of ARID1A and result in strong engagement with cancer pathways. The identification of this hierarchy suggests sites for intervention in ARID1A-driven diseases.

Global ubiquitylation analysis of mitochondria in primary neurons identifies endogenous Parkin targets following activation of PINK1.

How activation of PINK1 and Parkin leads to elimination of damaged mitochondria by mitophagy is largely based on cell lines with few studies in neurons. Here, we have undertaken proteomic analysis of mitochondria from mouse neurons to identify ubiquitylated substrates of endogenous Parkin. Comparative analysis with human iNeuron datasets revealed a subset of 49 PINK1 activation­dependent diGLY sites in 22 proteins conserved across mouse and human systems. We use reconstitution assays to demonstrate direct ubiquitylation by Parkin in vitro. We also identified a subset of cytoplasmic proteins recruited to mitochondria that undergo PINK1 and Parkin independent ubiquitylation, indicating the presence of alternate ubiquitin E3 ligase pathways that are activated by mitochondrial depolarization in neurons. Last, we have developed an online resource to search for ubiquitin sites and enzymes in mitochondria of neurons, MitoNUb. These findings will aid future studies to understand Parkin activation in neuronal subtypes.

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.

Distinct signals and immune cells drive liver pathology and glomerulonephritis in ABIN1[D485N] mice.

We report that TLR7, IL-6, and the adaptive immune system are essential for autoimmunity and glomerulonephritis but not for liver pathology in mice expressing the ubiquitin-binding-defective ABIN1[D485N] mutant. The blood and organs of ABIN1[D485N] mice have exceptionally high numbers of patrolling monocytes (pMo), which develop independently of IL-6 and the adaptive immune system. They are detectable in the blood months before autoimmunity and organ pathology are seen and may have diagnostic potential. The splenic pMo, inflammatory monocytes (iMo), and neutrophils of ABIN1[D485N] mice expressed high levels of mRNAs encoding proteins released during NETosis, which together with the high numbers of monocyte-derived dendritic cells (MoDCs) may drive the liver pathology in ABIN1[D485N] mice, and contribute to the pathology of other organs. The splenic iMo of ABIN1[D485N] mice displayed high expression of mRNAs encoding proteins controlling cell division and were actively dividing; this may underlie the increased pMo and MoDC numbers, which are derived from iMo. An orally active IRAK4 inhibitor suppressed all facets of the disease phenotype and prevented the increase in pMo numbers.

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.

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.

Live imaging of marked chromosome regions reveals their dynamic resolution and compaction in mitosis.

When human cells enter mitosis, chromosomes undergo substantial changes in their organization to resolve sister chromatids and compact chromosomes. To comprehend the timing and coordination of these events, we need to evaluate the progression of both sister chromatid resolution and chromosome compaction in one assay. Here we achieved this by analyzing changes in configuration of marked chromosome regions over time, with high spatial and temporal resolution. This assay showed that sister chromatids cycle between nonresolved and partially resolved states with an interval of a few minutes during G2 phase before completing full resolution in prophase. Cohesins and WAPL antagonistically regulate sister chromatid resolution in late G2 and prophase while local enrichment of cohesin on chromosomes prevents precocious sister chromatid resolution. Moreover, our assay allowed quantitative evaluation of condensin II and I activities, which differentially promote sister chromatid resolution and chromosome compaction, respectively. Our assay reveals novel aspects of dynamics in mitotic chromosome resolution and compaction that were previously obscure in global chromosome assays.

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.

Proteome-wide analysis of protein abundance and turnover remodelling during oncogenic transformation of human breast epithelial cells.

Background: Viral oncogenes and mutated proto-oncogenes are potent drivers of cancer malignancy. Downstream of the oncogenic trigger are alterations in protein properties that give rise to cellular transformation and the acquisition of malignant cellular phenotypes. Developments in mass spectrometry enable large-scale, multidimensional characterisation of proteomes. Such techniques could provide an unprecedented, unbiased view of how oncogene activation remodels a human cell proteome. Methods: Using quantitative MS-based proteomics and cellular assays, we analysed how transformation induced by activating v-Src kinase remodels the proteome and cellular phenotypes of breast epithelial (MCF10A) cells. SILAC MS was used to comprehensively characterise the MCF10A proteome and to measure v-Src-induced changes in protein abundance across seven time-points (1-72 hrs). We used pulse-SILAC MS ( Boisvert et al., 2012), to compare protein synthesis and turnover in control and transformed cells. Follow-on experiments employed a combination of cellular and functional assays to characterise the roles of selected Src-responsive proteins. Results: Src-induced transformation changed the expression and/or turnover levels of ~3% of proteins, affecting ~1.5% of the total protein molecules in the cell. Transformation increased the average rate of proteome turnover and disrupted protein homeostasis. We identify distinct classes of protein kinetics in response to Src activation. We demonstrate that members of the polycomb repressive complex 1 (PRC1) are important regulators of invasion and migration in MCF10A cells. Many Src-regulated proteins are present in low abundance and some are regulated post-transcriptionally. The signature of Src-responsive proteins is highly predictive of poor patient survival across multiple cancer types. Open access to search and interactively explore all these proteomic data is provided via the EPD database ( www.peptracker.com/epd). Conclusions: We present the first comprehensive analysis measuring how protein expression and protein turnover is affected by cell transformation, providing a detailed picture at the protein level of the consequences of activation of an oncogene.

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.

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.

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.

Molecular mechanisms facilitating the initial kinetochore encounter with spindle microtubules.

The initial kinetochore (KT) encounter with a spindle microtubule (MT; KT capture) is one of the rate-limiting steps in establishing proper KT-MT interaction during mitosis. KT capture is facilitated by multiple factors, such as MT extension in various directions, KT diffusion, and MT pivoting. In addition, KTs generate short MTs, which subsequently interact with a spindle MT. KT-derived MTs may facilitate KT capture, but their contribution is elusive. In this study, we find that Stu1 recruits Stu2 to budding yeast KTs, which promotes MT generation there. By removing Stu2 specifically from KTs, we show that KT-derived MTs shorten the half-life of noncaptured KTs from 48-49 s to 28-34 s. Using computational simulation, we found that multiple factors facilitate KT capture redundantly or synergistically. In particular, KT-derived MTs play important roles both by making a significant contribution on their own and by synergistically enhancing the effects of KT diffusion and MT pivoting. Our study reveals fundamental mechanisms facilitating the initial KT encounter with spindle MTs.

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.

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.