Myeloid malignancies, including acute myeloid leukaemia (AML), arise from the expansion of haematopoietic stem and progenitor cells that acquire somatic mutations. Bulk molecular profiling has ...suggested that mutations are acquired in a stepwise fashion: mutant genes with high variant allele frequencies appear early in leukaemogenesis, and mutations with lower variant allele frequencies are thought to be acquired later
. Although bulk sequencing can provide information about leukaemia biology and prognosis, it cannot distinguish which mutations occur in the same clone(s), accurately measure clonal complexity, or definitively elucidate the order of mutations. To delineate the clonal framework of myeloid malignancies, we performed single-cell mutational profiling on 146 samples from 123 patients. Here we show that AML is dominated by a small number of clones, which frequently harbour co-occurring mutations in epigenetic regulators. Conversely, mutations in signalling genes often occur more than once in distinct subclones, consistent with increasing clonal diversity. We mapped clonal trajectories for each sample and uncovered combinations of mutations that synergized to promote clonal expansion and dominance. Finally, we combined protein expression with mutational analysis to map somatic genotype and clonal architecture with immunophenotype. Our findings provide insights into the pathogenesis of myeloid transformation and how clonal complexity evolves with disease progression.
Biologic therapies have transformed the management of psoriasis, but clinical outcome is variable leaving an unmet clinical need for predictive biomarkers of response. Here we perform in-depth ...immunomonitoring of blood immune cells of 67 patients with psoriasis, before and during therapy with the anti-TNF drug adalimumab, to identify immune mediators of clinical response and evaluate their predictive value. Enhanced NF-κBp65 phosphorylation, induced by TNF and LPS in type-2 dendritic cells (DC) before therapy, significantly correlates with lack of clinical response after 12 weeks of treatment. The heightened NF-κB activation is linked to increased DC maturation in vitro and frequency of IL-17
T cells in the blood of non-responders before therapy. Moreover, lesional skin of non-responders contains higher numbers of dermal DC expressing the maturation marker CD83 and producing IL-23, and increased numbers of IL-17
T cells. Finally, we identify and clinically validate LPS-induced NF-κBp65 phosphorylation before therapy as a predictive biomarker of non-response to adalimumab, with 100% sensitivity and 90.1% specificity in an independent cohort. Our study uncovers important molecular and cellular mediators underpinning adalimumab mechanisms of action in psoriasis and we propose a blood biomarker for predicting clinical outcome.
A surface plasmon resonance (SPR)-based SELEX approach has been used to raise RNA aptamers against a structured RNA, derived from XBP1 pre-mRNA, that folds as two contiguous hairpins. Thanks to the ...design of the internal microfluidic cartridge of the instrument, the selection was performed during the dissociation phase of the SPR analysis by recovering the aptamer candidates directly from the target immobilized onto the sensor chip surface. The evaluation of the pools was performed by SPR, simultaneously, during the association phase, each time the amplified and transcribed candidates were injected over the immobilized target. SPR coupled with SELEX from the first to the last round allowed identifying RNA aptamers that formed highly stable loop–loop complexes (KD equal to 8nM) with the hairpin located on the 5′ side of the target. High throughput sequencing of two key rounds confirmed the evolution observed by SPR and also revealed the selection of hairpins displaying a loop not fully complementary to the loop of its target. These candidates were selected mainly because they bound 79 times faster to the target than those having a complementary loop. SELEX coupled with SPR is expected to speed up the selection process because selection and evaluation are performed simultaneously.
•An SPR-based SELEX approach was used to identify aptamer.•Aptamer candidates were recovered from the target thanks to the microfluidic.•Selection and evaluation by SPR were performed simultaneously.•High affinity aptamers were selected.•The best aptamer does not display a loop fully complementary to the target loop.
Psoriasis is an immune-mediated disease characterised by chronically elevated pro-inflammatory cytokine levels, leading to aberrant keratinocyte proliferation and differentiation. Although certain ...clinical phenotypes, such as plaque psoriasis, are well defined, it is currently unclear whether there are molecular subtypes that might impact on prognosis or treatment outcomes.
We present a pipeline for patient stratification through a comprehensive analysis of gene expression in paired lesional and non-lesional psoriatic tissue samples, compared with controls, to establish differences in RNA expression patterns across all tissue types. Ensembles of decision tree predictors were employed to cluster psoriatic samples on the basis of gene expression patterns and reveal gene expression signatures that best discriminate molecular disease subtypes. This multi-stage procedure was applied to several published psoriasis studies and a comparison of gene expression patterns across datasets was performed.
Overall, classification of psoriasis gene expression patterns revealed distinct molecular sub-groups within the clinical phenotype of plaque psoriasis. Enrichment for TGFb and ErbB signaling pathways, noted in one of the two psoriasis subgroups, suggested that this group may be more amenable to therapies targeting these pathways. Our study highlights the potential biological relevance of using ensemble decision tree predictors to determine molecular disease subtypes, in what may initially appear to be a homogenous clinical group. The R code used in this paper is available upon request.
Downregulation of the cylindromatosis (CYLD) tumor suppressor has been associated with breast cancer development and progression. Here, we report a critical role for CYLD in maintaining the phenotype ...of mammary epithelial cells in vitro and in vivo. CYLD downregulation or inactivation induced an epithelial to mesenchymal transition of mammary epithelial cells that was dependent on the concomitant activation of the transcription factors Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) and transforming growth factor beta (TGFβ)signaling. CYLD inactivation enhanced the nuclear localization of YAP/TAZ and the phosphorylation of Small Mothers Against Decapentaplegic (SMAD)2/3 proteins in confluent cell culture conditions. Consistent with these findings were the hyperplastic alterations of CYLD-deficient mouse mammary epithelia, which were associated with enhanced nuclear expression of the YAP/TAZ transcription factors. Furthermore, in human breast cancer samples, downregulation of CYLD expression correlates with enhanced YAP/TAZ-regulated target gene expression. Our results identify CYLD as a critical regulator of a signaling node that prevents the coordinated activation of YAP/TAZ and the TGFβ pathway in mammary epithelial cells, in order to maintain their phenotypic identity and homeostasis. Consequently, they provide a novel conceptual framework that supports and explains a causal implication of deficient CYLD expression in aggressive human breast cancers.
Skin immune homeostasis is a multi-faceted process where dermal dendritic cells (DDCs) are key in orchestrating responses to environmental stressors. We have previously identified CD141+CD14+ DDCs as ...a skin-resident immunoregulatory population that is vitamin-D3 (VitD3) inducible from monocyte-derived DCs (moDCs), termed CD141hi VitD3 moDCs. We demonstrate that CD141+ DDCs and CD141hi VitD3 moDCs share key immunological features including cell surface markers, reduced T cell stimulation, IL-10 production, and a common transcriptomic signature. Bioinformatic analysis identified the neuroactive ligand receptor pathway and the neuropeptide, urocortin 2 (UCN2), as a potential immunoregulatory candidate molecule. Incubation with VitD3 upregulated UCN2 in CD141+ DCs and UVB irradiation induced UCN2 in CD141+ DCs in healthy skin in vivo. Notably, CD141+ DDC generation of suppressive Tregs was dependent upon the UCN2 pathway as in vivo administration of UCN2 reversed skin inflammation in humanized mice. We propose the neuropeptide UCN2 as a novel skin DC-derived immunoregulatory mediator with a potential role in UVB and VitD3-dependent skin immune homeostasis.
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•CD141+ skin DDCs and VitD3 moDCs are functionally and transcriptomically similar•CD141+ DDCs and CD141hi VitD3 moDCs preferentially express the neuropeptide UCN2•Solar UVB irradiation and VitD3 induces UCN2 in CD141+ DDCs and moDCs, respectively•UCN2 protects from alloimmune cell-mediated human skin inflammation in vivo
Immunology
Cellular ATP levels are generated by glucose-stimulated mitochondrial metabolism and determine metabolic responses, such as glucose-stimulated insulin secretion (GSIS) from the β-cells of pancreatic ...islets. We describe an analysis of the evolutionary processes affecting the core enzymes involved in glucose-stimulated insulin secretion in mammals. The proteins involved in this system belong to ancient enzymatic pathways: glycolysis, the TCA cycle and oxidative phosphorylation.
We identify two sets of proteins, or protein coalitions, in this group of 77 enzymes with distinct evolutionary patterns. Members of the glycolysis, TCA cycle, metabolite transport, pyruvate and NADH shuttles have low rates of protein sequence evolution, as inferred from a human-mouse comparison, and relatively high rates of evolutionary gene duplication. Respiratory chain and glutathione pathway proteins evolve faster, exhibiting lower rates of gene duplication. A small number of proteins in the system evolve significantly faster than co-pathway members and may serve as rapidly evolving adapters, linking groups of co-evolving genes.
Our results provide insights into the evolution of the involved proteins. We find evidence for two coalitions of proteins and the role of co-adaptation in protein evolution is identified and could be used in future research within a functional context.
Sequence-based variation in gene expression is a key driver of disease risk. Common variants regulating expression in cis have been mapped in many expression quantitative trait locus (eQTL) studies, ...typically in single tissues from unrelated individuals. Here, we present a comprehensive analysis of gene expression across multiple tissues conducted in a large set of mono- and dizygotic twins that allows systematic dissection of genetic (cis and trans) and non-genetic effects on gene expression. Using identity-by-descent estimates, we show that at least 40% of the total heritable cis effect on expression cannot be accounted for by common cis variants, a finding that reveals the contribution of low-frequency and rare regulatory variants with respect to both transcriptional regulation and complex trait susceptibility. We show that a substantial proportion of gene expression heritability is trans to the structural gene, and we identify several replicating trans variants that act predominantly in a tissue-restricted manner and may regulate the transcription of many genes.
Recessive dystrophic epidermolysis bullosa (RDEB) is a hereditary blistering disorder due to a lack of type VII collagen. At present, treatment is mainly supportive.
To determine whether intravenous ...allogeneic bone marrow–derived mesenchymal stromal/stem cells (BM-MSCs) are safe in RDEB adults and if the cells improve wound healing and quality of life.
We conducted a prospective, phase I/II, open-label study recruiting 10 RDEB adults to receive 2 intravenous infusions of BM-MSCs (on day 0 and day 14; each dose 2-4 × 106 cells/kg).
BM-MSCs were well tolerated with no serious adverse events to 12 months. Regarding efficacy, there was a transient reduction in disease activity scores (8/10 subjects) and a significant reduction in itch. One individual showed a transient increase in type VII collagen.
Open-label trial with no placebo.
MSC infusion is safe in RDEB adults and can have clinical benefits for at least 2 months.
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