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Leishmania are unicellular parasites that cause human and animal diseases. Like other kinetoplastids, they possess large transcriptional start regions (TSRs) which are defined by histone variants and histone lysine acetylation. Cellular interpretation of these chromatin marks is not well understood. Eight bromodomain factors, the reader modules for acetyl-lysine, are found across Leishmania genomes. Using L. mexicana, Cas9-driven gene deletions indicate that BDF1–5 are essential for promastigotes. Dimerisable, split Cre recombinase (DiCre)-inducible gene deletion of BDF5 show it is essential for both promastigotes and murine infection. ChIP-seq identifies BDF5 as enriched at TSRs. XL-BioID proximity proteomics shows the BDF5 landscape is enriched for BDFs, HAT2, proteins involved in transcriptional activity, and RNA processing; revealing a Conserved Regulators of Kinetoplastid Transcription (CRKT) Complex. Inducible deletion of BDF5 causes global reduction in RNA polymerase II transcription. Our results indicate the requirement of Leishmania to interpret histone acetylation marks through the bromodomain-enriched CRKT complex for normal gene expression and cellular viability.

Leishmania are unicellular parasites that cause human and animal disease. Alongside other organisms in kinetoplastida, they have evolved an unusual genome architecture that requires all RNA polymerase II transcribed genes to be expressed constitutively, with transcriptional start regions denoted by histone variants and histone lysine acetylation. However, the way these chromatin marks are interpreted by the cell is not understood. Seven predicted bromodomain factors (BDF1-7), the reader modules for acetyl-lysine, were identified across Leishmania genomes. Using L. mexicana as a model, Cas9-driven gene deletions indicate that BDF1-5 are essential for promastigote survival, whilst DiCre inducible gene deletion of the dual bromodomain factor BDF5 identified it to be essential for both promastigotes and amastigotes. ChIP-seq assessment of BDF5s genomic distribution revealed it as highly enriched at transcriptional start sites. Using an optimised proximity proteomic and phosphoproteomic technique, XL-BioID, we defined the BDF5-proximal environment to be enriched for other bromodomain factors, histone acetyltransferase 2, and proteins essential for transcriptional activity and RNA processing. Inducible deletion of BDF5, led to a disruption of pol II transcriptional activity and global defects in gene expression. Our results indicate the requirement of Leishmania to interpret histone acetylation marks for normal levels of gene expression and thus cellular viability.

Circulating microRNAs (miRNAs) are exceptional mechanism-based correlates of disease, yet their potential remains largely untapped in COVID-19. Here, we determined circulating miRNA and cytokine and chemokine (CC) profiles in 171 blood plasma samples from 58 hospitalised COVID-19 patients. Thirty-two miRNAs were differentially expressed in severe cases when compared to moderate and mild cases. These miRNAs and their predicted targets reflected key COVID-19 features including cell death and hypoxia. Compared to mild cases, moderate and severe cases were characterised by a global decrease in circulating miRNA levels. Partial least squares regression using miRNA and CC measurements allowed for discrimination of severe cases with greater accuracy (87%) than using miRNA or CC levels alone. Correlation analysis revealed severity group-specific associations between CC and miRNA levels. Importantly, the miRNAs that correlated with IL6 and CXCL10, two cardinal COVID-19-associated cytokines, were distinct between severity groups, providing a novel qualitative way to stratify patients with similar levels of proinflammatory cytokines but different disease severity. Integration of miRNA and CC levels with clinical parameters revealed severity-specific signatures associated with clinical hallmarks of COVID-19. Our study highlights the existence of severity-specific circulating CC/miRNA networks, providing insight into COVID-19 pathogenesis and a novel approach for monitoring COVID-19 progression.

Differentiation between distinct stages is fundamental for the life cycle of intracellular protozoan parasites and for transmission between hosts, requiring stringent spatial and temporal regulation. Here, we apply kinome-wide gene deletion and gene tagging in Leishmania mexicana promastigotes to define protein kinases with life cycle transition roles. Whilst 162 are dispensable, 44 protein kinase genes are refractory to deletion in promastigotes and are likely core genes required for parasite replication. Phenotyping of pooled gene deletion mutants using bar-seq and projection pursuit clustering reveal functional phenotypic groups of protein kinases involved in differentiation from metacyclic promastigote to amastigote, growth and survival in macrophages and mice, colonisation of the sand fly and motility. This unbiased interrogation of protein kinase function in Leishmania allows targeted investigation of organelle-associated signalling pathways required for successful intracellular parasitism., Protein kinases are fundamental in cellular signalling required for Leishmania survival throughout the life cycle. Here, Baker and Catta-Preta et al. report on a kinome-wide functional study in Leishmania mexicana to define protein kinases with roles in life cycle transition.

Inflammatory cytokines and chemokines (CC) drive COVID-19 pathology. Yet, patients with similar circulating CC levels present with different disease severity. Here, we determined 171 microRNAomes from 58 hospitalised COVID-19 patients (Cohort 1) and levels of 25 cytokines and chemokines (CC) in the same samples. Combining microRNA (miRNA) and CC measurements allowed for discrimination of severe cases with greater accuracy than using miRNA or CC levels alone. Severity group-specific associations between miRNAs and COVID-19-associated CC (e.g. IL6, CCL20) or clinical hallmarks of COVID-19 (e.g. neutrophilia, hypoalbuminemia) separated patients with similar CC levels but different disease severity. Critically, analysis of an independent cohort of 108 patients from a different centre (Cohort 2) demonstrated feasibility of CC/miRNA profiling in leftover blood samples with similar severe disease CC and miRNA profiles, and revealed CCL20, IL6, IL10, and miR-451a as key correlates of fatal COVID-19. These findings highlight that systemic miRNA/CC networks underpin severe COVID-19. Funding: The study was funded by the UKRI MRC/NIHR award to the UK Coronavirus Immunology Consortium (UK-CIC, MR/V028448/1). Sample collection at Manchester NHS Trusts was supported by the NIHR Manchester Biomedical Research Centre (TH and AS) and 3M Global Giving award (JRG and TH). Sample collection at York and Scarborough NHS Trust was supported by the Wellcome Trust (ISSF grant WT204829 through the Centre for Future Health at the University of York) and the Hull York Medical School. JRG is supported by a senior fellowship by The Kennedy Trust for Rheumatology Research. This report contains independent research supported by the North West Lung Centre Charity and the NIHR Manchester Clinical Research Facility at Wythenshawe Hospital. Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: For the cohort 1, ethics approval was obtained from the North West-Haydock Research Ethics Committee for ManARTS (reference 15/NW/0409) and from the Wales Research Ethics Committee 4 for NCARC (reference 18/WA/0368). For the cohort 2, ethics approval was obtained from Yorkshire & The Humber - Leeds West Research Ethics Committee (REC reference 19/YH/0394 with approved 26 SA002 amendment of IRAS project 269597).

Retinoids, such as all- trans-retinoic acid (ATRA), are endogenous signaling molecules derived from vitamin A that influence a variety of cellular processes through mediation of transcription events in the cell nucleus. Because of these wide-ranging and powerful biological activities, retinoids have emerged as therapeutic candidates of enormous potential. However, their use has been limited, to date, due to a lack of understanding of the complex and intricate signaling pathways that they control. We have designed and synthesized a family of synthetic retinoids that exhibit strong, intrinsic, solvatochromatic fluorescence as multifunctional tools to interrogate these important biological activities. We utilized the unique photophysical characteristics of these fluorescent retinoids to develop a novel in vitro fluorometric binding assay to characterize and quantify their binding to their cellular targets, including cellular retinoid binding protein II (CRABPII). The dihydroquinoline retinoid, DC360, exhibited particularly strong binding ( K

A comprehensive, quantified identification of the mRNA-binding and whole cell proteomes in the three main Leishmania lifecycle stages, the first such comparison in kinetoplastid parasites, demonstrates trans-regulator RBPs select distinct, specific mRNA target pools in a stage-regulated manner despite equivalent, constitutive transcript levels available. Results further indicate that in L. mexicana parasites, mRNA levels are not a strong predictor of whole cell expression or RNA binding potential of encoded proteins. Included are the first proteomes from the human-infective metacyclic promastigote stage., Graphical Abstract Highlights Comprehensive, quantified mRNA-binding and whole cell proteomes of the three main Leishmania spp. lifecycle stages. Decisive evidence that trans-regulator RBPs can selectively bind distinct mRNA target pools in a stage-regulated manner despite equivalent transcript levels. Quantified evidence that in L. mexicana parasites, mRNA levels are not a strong predictor of whole cell expression or RNA binding potential of encoded proteins. Use of Histone H4 as the first marker of procyclic stage cells in Leishmania spp., Leishmania parasite infections, termed the leishmaniases, cause significant global infectious disease burden. The lifecycle of the parasite embodies three main stages that require precise coordination of gene regulation to survive environmental shifts between sandfly and mammalian hosts. Constitutive transcription in kinetoplastid parasites means that gene regulation is overwhelmingly reliant on post-transcriptional mechanisms, yet strikingly few Leishmania trans-regulators are known. Using optimized crosslinking and deep, quantified mass spectrometry, we present a comprehensive analysis of 1400 mRNA binding proteins (mRBPs) and whole cell proteomes from the three main Leishmania lifecycle stages. Supporting the validity, although the crosslinked RBPome is magnitudes more enriched, the protein identities of the crosslinked and non-crosslinked RBPomes were nearly identical. Moreover, multiple candidate RBPs were endogenously tagged and found to associate with discrete mRNA target pools in a stage-specific manner. Results indicate that in L. mexicana parasites, mRNA levels are not a strong predictor of the whole cell expression or RNA binding potential of encoded proteins. Evidence includes a low correlation between transcript and corresponding protein expression and stage-specific variation in protein expression versus RNA binding potential. Unsurprisingly, RNA binding protein enrichment correlates strongly with relative replication efficiency of the specific lifecycle stage. Our study is the first to quantitatively define and compare the mRBPome of multiple stages in kinetoplastid parasites. It provides novel, in-depth insight into the trans-regulatory mRNA:Protein (mRNP) complexes that drive Leishmania parasite lifecycle progression.

Bone marrow mesenchymal stem/stromal cells (MSCs) maintain bone homeostasis and repair through the ability to expand in response to mitotic stimuli and differentiate into skeletal lineages. Signalling mechanisms that enable precise control of MSC function remain unclear. Here we report that by initially examining differences in signalling pathway expression profiles of individual MSC clones, we identified a previously unrecognised signalling mechanism regulated by epidermal growth factor (EGF) in primary human MSCs. We demonstrate that EGF is able to activate β-catenin, a key component of the canonical Wnt signalling pathway. EGF is able to induce nuclear translocation of β-catenin in human MSCs but does not drive expression of Wnt target genes or T cell factor (TCF) activity in MSC reporter cell lines. Using an efficient Design of Experiments (DoE) statistical analysis, with different combinations and concentrations of EGF and Wnt ligands, we were able to confirm that EGF does not influence the Wnt/β-catenin pathway in MSCs. We show that the effects of EGF on MSCs are temporally regulated to initiate early “classical” EGF signalling mechanisms (e.g via mitogen activated protein kinase) with delayed activation of β-catenin. By RNA-sequencing, we identified gene sets that were exclusively regulated by the EGF/β-catenin pathway, which were distinct from classical EGF-regulated genes. However, subsets of classical EGF gene targets were significantly influenced by EGF/β-catenin activation. These signalling pathways cooperate to enable EGF-mediated proliferation of MSCs by alleviating the suppression of cell cycle pathways induced by classical EGF signalling., Highlights • Epidermal growth factor (EGF) controls mesenchymal stem cell (MSC) proliferation. • EGF signals through β-catenin in MSCs but not in related fibroblastic cells. • Classical EGF and EGF/β-catenin cooperatively regulate distinct gene sets in MSCs. • EGF/β-catenin enables MSC proliferation by alleviating cell cycle suppression.

Determining the mechanisms that distinguish protective immunity from pathological chronic inflammation remains a fundamental challenge. miR‐132 has been shown to play largely immunoregulatory roles in immunity; however, its role in CD4 + T cell function is poorly understood. Here, we show that CD4 + T cells express high levels of miR‐132 and that T cell activation leads to miR‐132 up‐regulation. The transcriptomic hallmark of splenic CD4 + T cells lacking the miR‐132/212 cluster during chronic infection is an increase in mRNA levels of ribosomal protein (RP) genes. BTAF1, a co‐factor of B‐TFIID and novel miR‐132/212‐3p target, and p300 contribute towards miR‐132/212‐mediated regulation of RP transcription. Following infection with Leishmania donovani, miR‐132 −/− CD4 + T cells display enhanced expression of IL‐10 and decreased IFNγ. This is associated with reduced hepatosplenomegaly and enhanced pathogen load. The enhanced IL‐10 expression in miR‐132 −/− Th1 cells is recapitulated in vitro following treatment with phenylephrine, a drug reported to promote ribosome synthesis. Our results uncover that miR‐132/212‐mediated regulation of RP expression is critical for optimal CD4 + T cell activation and protective immunity against pathogens.

The inactive X chromosome (Xi) serves as a model for establishment and maintenance of repressed chromatin and the function of polycomb repressive complexes (PRC1/2). Here we show that Xi transiently relocates from the nuclear periphery towards the interior during its replication, in a process dependent on CIZ1. Compromised relocation of Xi in CIZ1-null primary mouse embryonic fibroblasts is accompanied by loss of PRC-mediated H2AK119Ub1 and H3K27me3, increased solubility of PRC2 catalytic subunit EZH2, and genome-wide deregulation of polycomb-regulated genes. Xi position in S phase is also corrupted in cells adapted to long-term culture (WT or CIZ1-null), and also accompanied by specific changes in EZH2 and its targets. The data are consistent with the idea that chromatin relocation during S phase contributes to maintenance of epigenetic landscape in primary cells, and that elevated soluble EZH2 is part of an error-prone mechanism by which modifying enzyme meets template when chromatin relocation is compromised., The inactive X chromosome (Xi) is a model for establishment and maintenance of repressed chromatin and the function of polycomb repressive complexes. Here the authors show that Xi transiently relocates from the nuclear periphery during replication in a CIZ1-dependent manner, which plays a role in maintaining PRC-mediated repressed chromatin.

Background Adult schistosomes have a well-developed alimentary tract comprising an oral sucker around the mouth, a short esophagus and a blind ending gut. The esophagus is not simply a muscular tube for conducting blood from the mouth to gut but is divided into compartments, surrounded by anterior and posterior glands, where processing of ingested blood is initiated. Self-cure of rhesus macaques from a Schistosoma japonicum infection appears to operate by blocking the secretory functions of these glands so that the worms cease feeding and slowly starve to death. Here we use subtractive RNASeq to characterise the genes encoding the principal secretory products of S. japonicum esophageal glands, preparatory to evaluating their relevance as targets of the self-cure process. Methodology/Principal findings The heads and a small portion of the rear end of male and female S. japonicum worms were separately enriched by microdissection, for mRNA isolation and library construction. The sequence reads were then assembled de novo using Trinity and those genes enriched more than eightfold in the head preparation were subjected to detailed bioinformatics analysis. Of the 62 genes selected from the male heads, more than one third comprised MEGs encoding secreted or membrane-anchored proteins. Database searching using conserved motifs revealed that the MEG-4 and MEG-8/9 families had counterparts in the bird schistosome Trichobilharzia regenti, indicating an ancient association with blood processing. A second group of MEGs, including a MEG-26 family, encoded short peptides with amphipathic properties that most likely interact with ingested host cell membranes to destabilise them. A number of lysosomal hydrolases, two protease inhibitors, a secreted VAL and a putative natterin complete the line-up. There was surprisingly little difference between expression patterns in males and females despite the latter processing much more blood. Significance/Conclusions The mixture of approximately 40 proteins specifically secreted by the esophageal glands is responsible for initiating blood processing in the adult worm esophagus. They comprise the potential targets for the self-cure process in the rhesus macaque, and thus represent a completely new cohort of secreted proteins that can be investigated as vaccine candidates., Author summary Schistosomes are parasitic flatworms inhabiting the human bloodstream, surrounded by and feeding on humoral and cellular components of the immune system. They are normally long-lived but the rhesus macaque is able to mount a self-cure response directed against the esophageal secretions of the adult Schistosoma japonicum so that they stop feeding and slowly starve to death. The worm esophagus is a short tube connecting mouth to gut surrounded by anterior and posterior glands and our aim in this study was to identify the genes encoding the gland secretions. For this purpose we isolated the messenger RNA from both male and female worm heads and tails and obtained many millions of sequences. These were assembled into gene coding sequences using bioinformatics and then genes differentially expressed in the head region were identified by a subtraction process. We then focused on those genes encoding proteins with a leader sequence indicating their secretory status. The result is an inventory of approximately 40 genes; some encode protein binding motifs while others encode a short helix with a hydrophobic face, which may interact with host cell membranes. Genes encoding enzymes, protease inhibitors and a venom-like protein were also found. These proteins are being evaluated for their interactions with the antibodies generated by macaques during the self-cure process.

The ability to recognize familiar faces across different viewing conditions contrasts with the inherent difficulty in the perception of unfamiliar faces across similar image manipulations. It is widely believed that this difference in perception and recognition is based on the neural representation for familiar faces being less sensitive to changes in the image than it is for unfamiliar faces. Here, we used an functional magnetic resonance-adaptation paradigm to investigate image invariance in face-selective regions of the human brain. We found clear evidence for a degree of image-invariant adaptation to facial identity in face-selective regions, such as the fusiform face area. However, contrary to the predictions of models of face processing, comparable levels of image invariance were evident for both familiar and unfamiliar faces. This suggests that the marked differences in the perception of familiar and unfamiliar faces may not depend on differences in the way multiple images are represented in core face-selective regions of the human brain.

Understanding verbs typically activates posterior temporal regions and, in some circumstances, motion perception area V5. However, the nature and role of this activation remains unclear: does language alone indeed activate V5? And are posterior temporal representations modality-specific motion representations, or supra-modal motion-independent event representations? Here, we address these issues by investigating human and object motion sentences compared to corresponding state descriptions. We adopted the blank screen paradigm, which is known to encourage visual imagery, and used a localizer to identify V5 and temporal structures responding to motion. Analyses in each individual brain suggested that language modulated activity in the posterior temporal lobe but not within V5 in most participants. Moreover, posterior temporal structures strongly responded to both motion sentences and human static sentences. These results suggest that descriptive language alone need not recruit V5 and instead engages more schematic event representations in temporal cortex encoding animacy and motion.