Transcriptional and Functional Analysis of CD1c+ Human Dendritic Cells Identifies a CD163+ Subset Priming CD8+CD103+ T Cells.

Dendritic cells (DCs) are antigen-presenting cells controlling T cell activation. In humans, the diversity, ontogeny, and functional capabilities of DC subsets are not fully understood. Here, we identified circulating CD88-CD1c+CD163+ DCs (called DC3s) as immediate precursors of inflammatory CD88-CD14+CD1c+CD163+FcεRI+ DCs. DC3s develop via a specific pathway activated by GM-CSF, independent of cDC-restricted (CDP) and monocyte-restricted (cMoP) progenitors. Like classical DCs but unlike monocytes, DC3s drove activation of naive T cells. In vitro, DC3s displayed a distinctive ability to prime CD8+ T cells expressing a tissue homing signature and the epithelial homing alpha-E integrin (CD103) through transforming growth factor ß (TGF-ß) signaling. In vivo, DC3s infiltrated luminal breast cancer primary tumors, and DC3 infiltration correlated positively with CD8+CD103+CD69+ tissue-resident memory T cells. Together, these findings define DC3s as a lineage of inflammatory DCs endowed with a strong potential to regulate tumor immunity.

A Novel Preclinical In Vitro 3D Model of Oral Carcinogenesis for Biomarker Discovery and Drug Testing.

This study aimed to develop an in vitro three-dimensional (3D) cell culture model of oral carcinogenesis for the rapid, scalable testing of chemotherapeutic agents. Spheroids of normal (HOK) and dysplastic (DOK) human oral keratinocytes were cultured and treated with 4-nitroquinoline-1-oxide (4NQO). A 3D invasion assay using Matrigel was performed to validate the model. RNA was extracted and subjected to transcriptomic analysis to validate the model and assess carcinogen-induced changes. The VEGF inhibitors pazopanib and lenvatinib were tested in the model and were validated by a 3D invasion assay, which demonstrated that changes induced by the carcinogen in spheroids were consistent with a malignant phenotype. Further validation was obtained by bioinformatic analyses, which showed the enrichment of pathways associated with hallmarks of cancer and VEGF signalling. Overexpression of common genes associated with tobacco-induced oral squamous cell carcinoma (OSCC), such as MMP1, MMP3, MMP9, YAP1, CYP1A1, and CYP1B1, was also observed. Pazopanib and lenvatinib inhibited the invasion of transformed spheroids. In summary, we successfully established a 3D spheroid model of oral carcinogenesis for biomarker discovery and drug testing. This model is a validated preclinical model for OSCC development and would be suitable for testing a range of chemotherapeutic agents.

Impact of Local Alloimmunity and Recipient Cells in Transplant Arteriosclerosis.

RATIONALE: Transplant arteriosclerosis is the major limitation to long-term survival of solid organ transplantation. Although both immune and nonimmune cells have been suggested to contribute to this process, the complex cellular heterogeneity within the grafts, and the underlying mechanisms regulating the disease progression remain largely uncharacterized. OBJECTIVE: We aimed to delineate the cellular heterogeneity within the allografts, and to explore possible mechanisms underlying this process. METHODS AND RESULTS: Here, we reported the transcriptional profiling of 11 868 cells in a mouse model of transplant arteriosclerosis by single-cell RNA sequencing. Unbiased clustering analyses identified 21 cell clusters at different stages of diseases, and focused analysis revealed several previously unknown subpopulations enriched in the allografts. Interestingly, we found evidence of the local formation of tertiary lymphoid tissues and suggested a possible local modulation of alloimmune responses within the grafts. Intercellular communication analyses uncovered a potential role of several ligands and receptors, including Ccl21a and Cxcr3, in regulating lymphatic endothelial cell-induced early chemotaxis and infiltration of immune cells. In vivo mouse experiments confirmed the therapeutic potential of CCL21 and CXCR3 neutralizing antibodies in transplant arteriosclerosis. Combinational use of genetic lineage tracing and single-cell techniques further indicate the infiltration of host-derived c-Kit+ stem cells as heterogeneous populations in the allografts. Finally, we compared the immune response between mouse allograft and atherosclerosis models in single-cell RNA-seq analysis. By analyzing susceptibility genes of disease traits, we also identified several cell clusters expressing genes associated with disease risk. CONCLUSIONS: Our study provides a transcriptional and cellular landscape of transplant arteriosclerosis, which could be fundamental to understanding the initiation and progression of this disease. CCL21/CXCR3 was also identified as important regulators of immune response and may serve as potential therapeutic targets in disease treatment.

Dynamic transcriptomic analysis reveals suppression of PGC1α/ERRα drives perturbed myogenesis in facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is a prevalent, incurable myopathy, linked to epigenetic derepression of D4Z4 repeats on chromosome 4q, leading to ectopic DUX4 expression. FSHD patient myoblasts have defective myogenic differentiation, forming smaller myotubes with reduced myosin content. However, molecular mechanisms driving such disrupted myogenesis in FSHD are poorly understood. We performed high-throughput morphological analysis describing FSHD and control myogenesis, revealing altered myogenic differentiation results in hypotrophic myotubes. Employing polynomial models and an empirical Bayes approach, we established eight critical time points during which human healthy and FSHD myogenesis differ. RNA-sequencing at these eight nodal time points in triplicate, provided temporal depth for a multivariate regression analysis, allowing assessment of interaction between progression of differentiation and FSHD disease status. Importantly, the unique size and structure of our data permitted identification of many novel FSHD pathomechanisms undetectable by previous approaches. For further analysis here, we selected pathways that control mitochondria: of interest considering known alterations in mitochondrial structure and function in FSHD muscle, and sensitivity of FSHD cells to oxidative stress. Notably, we identified suppression of mitochondrial biogenesis, in particular via peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC1α), the cofactor and activator of oestrogen-related receptor α (ERRα). PGC1α knock-down caused hypotrophic myotubes to form from control myoblasts. Known ERRα agonists and safe food supplements biochanin A, daidzein or genistein, each rescued the hypotrophic FSHD myotube phenotype. Together our work describes transcriptomic changes in high resolution that occur during myogenesis in FSHD ex vivo, identifying suppression of the PGC1α-ERRα axis leading to perturbed myogenic differentiation, which can effectively be rescued by readily available food supplements.

Single-Cell RNA-Sequencing and Metabolomics Analyses Reveal the Contribution of Perivascular Adipose Tissue Stem Cells to Vascular Remodeling.

OBJECTIVE: Perivascular adipose tissue (PVAT) plays a vital role in maintaining vascular homeostasis. However, most studies ascribed the function of PVAT in vascular remodeling to adipokines secreted by the perivascular adipocytes. Whether mesenchymal stem cells exist in PVAT and play a role in vascular regeneration remain unknown. Approach and Results: Single-cell RNA-sequencing allowed direct visualization of the heterogeneous PVAT-derived mesenchymal stem cells (PV-ADSCs) at a high resolution and revealed 2 distinct subpopulations, among which one featured signaling pathways crucial for smooth muscle differentiation. Pseudotime analysis of cultured PV-ADSCs unraveled their smooth muscle differentiation trajectory. Transplantation of cultured PV-ADSCs in mouse vein graft model suggested the contribution of PV-ADSCs to vascular remodeling through smooth muscle differentiation. Mechanistically, treatment with TGF-ß1 (transforming growth factor ß1) and transfection of microRNA (miR)-378a-3p mimics induced a similar metabolic reprogramming of PV-ADSCs, including upregulated mitochondrial potential and altered lipid levels, such as increased cholesterol and promoted smooth muscle differentiation. CONCLUSIONS: Single-cell RNA-sequencing allows direct visualization of PV-ADSC heterogeneity at a single-cell level and uncovers 2 subpopulations with distinct signature genes and signaling pathways. The function of PVAT in vascular regeneration is partly attributed to PV-ADSCs and their differentiation towards smooth muscle lineage. Mechanistic study presents miR-378a-3p which is a potent regulator of metabolic reprogramming as a potential therapeutic target for vascular regeneration.

Phase I/II open-label trial of intravenous allogeneic mesenchymal stromal cell therapy in adults with recessive dystrophic epidermolysis bullosa.

BACKGROUND: Recessive dystrophic epidermolysis bullosa (RDEB) is a hereditary blistering disorder due to a lack of type VII collagen. At present, treatment is mainly supportive. OBJECTIVES: 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. METHODS: 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). RESULTS: 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. LIMITATIONS: Open-label trial with no placebo. CONCLUSIONS: MSC infusion is safe in RDEB adults and can have clinical benefits for at least 2 months.

Decreased microglial Wnt/ß-catenin signalling drives microglial pro-inflammatory activation in the developing brain.

Microglia of the developing brain have unique functional properties but how their activation states are regulated is poorly understood. Inflammatory activation of microglia in the still-developing brain of preterm-born infants is associated with permanent neurological sequelae in 9 million infants every year. Investigating the regulators of microglial activation in the developing brain across models of neuroinflammation-mediated injury (mouse, zebrafish) and primary human and mouse microglia we found using analysis of genes and proteins that a reduction in Wnt/ß-catenin signalling is necessary and sufficient to drive a microglial phenotype causing hypomyelination. We validated in a cohort of preterm-born infants that genomic variation in the Wnt pathway is associated with the levels of connectivity found in their brains. Using a Wnt agonist delivered by a blood-brain barrier penetrant microglia-specific targeting nanocarrier we prevented in our animal model the pro-inflammatory microglial activation, white matter injury and behavioural deficits. Collectively, these data validate that the Wnt pathway regulates microglial activation, is critical in the evolution of an important form of human brain injury and is a viable therapeutic target.

Machine learning shows association between genetic variability in PPARG and cerebral connectivity in preterm infants.

Preterm infants show abnormal structural and functional brain development, and have a high risk of long-term neurocognitive problems. The molecular and cellular mechanisms involved are poorly understood, but novel methods now make it possible to address them by examining the relationship between common genetic variability and brain endophenotype. We addressed the hypothesis that variability in the Peroxisome Proliferator Activated Receptor (PPAR) pathway would be related to brain development. We employed machine learning in an unsupervised, unbiased, combined analysis of whole-brain diffusion tractography together with genomewide, single-nucleotide polymorphism (SNP)-based genotypes from a cohort of 272 preterm infants, using Sparse Reduced Rank Regression (sRRR) and correcting for ethnicity and age at birth and imaging. Empirical selection frequencies for SNPs associated with cerebral connectivity ranged from 0.663 to zero, with multiple highly selected SNPs mapping to genes for PPARG (six SNPs), ITGA6 (four SNPs), and FXR1 (two SNPs). SNPs in PPARG were significantly overrepresented (ranked 7-11 and 67 of 556,000 SNPs; P < 2.2 × 10-7), and were mostly in introns or regulatory regions with predicted effects including protein coding and nonsense-mediated decay. Edge-centric graph-theoretic analysis showed that highly selected white-matter tracts were consistent across the group and important for information transfer (P < 2.2 × 10-17); they most often connected to the insula (P < 6 × 10-17). These results suggest that the inhibited brain development seen in humans exposed to the stress of a premature extrauterine environment is modulated by genetic factors, and that PPARG signaling has a previously unrecognized role in cerebral development.

Association of MicroRNAs and YRNAs With Platelet Function.

RATIONALE: Platelets shed microRNAs (miRNAs). Plasma miRNAs change on platelet inhibition. It is unclear whether plasma miRNA levels correlate with platelet function. OBJECTIVE: To link small RNAs to platelet reactivity. METHODS AND RESULTS: Next-generation sequencing of small RNAs in plasma revealed 2 peaks at 22 to 23 and 32 to 33 nucleotides corresponding to miRNAs and YRNAs, respectively. Among YRNAs, predominantly, fragments of RNY4 and RNY5 were detected. Plasma miRNAs and YRNAs were measured in 125 patients with a history of acute coronary syndrome who had undergone detailed assessment of platelet function 30 days after the acute event. Using quantitative real-time polymerase chain reactions, 92 miRNAs were assessed in patients with acute coronary syndrome on different antiplatelet therapies. Key platelet-related miRNAs and YRNAs were correlated with platelet function tests. MiR-223 (rp=0.28; n=121; P=0.002), miR-126 (rp=0.22; n=121; P=0.016), and other abundant platelet miRNAs and YRNAs showed significant positive correlations with the vasodilator-stimulated phosphoprotein phosphorylation assay. YRNAs, miR-126, and miR-223 were also among the small RNAs showing the greatest dependency on platelets and strongly correlated with plasma levels of P-selectin, platelet factor 4, and platelet basic protein in the population-based Bruneck study (n=669). A single-nucleotide polymorphism that facilitates processing of pri-miR-126 to mature miR-126 accounted for a rise in circulating platelet activation markers. Inhibition of miR-126 in mice reduced platelet aggregation. MiR-126 directly and indirectly affects ADAM9 and P2Y12 receptor expression. CONCLUSIONS: Levels of platelet-related plasma miRNAs and YRNAs correlate with platelet function tests in patients with acute coronary syndrome and platelet activation markers in the general population. Alterations in miR-126 affect platelet reactivity.

Site-specific DICER and DROSHA RNA products control the DNA-damage response.

Non-coding RNAs (ncRNAs) are involved in an increasingly recognized number of cellular events. Some ncRNAs are processed by DICER and DROSHA RNases to give rise to small double-stranded RNAs involved in RNA interference (RNAi). The DNA-damage response (DDR) is a signalling pathway that originates from a DNA lesion and arrests cell proliferation3. So far, DICER and DROSHA RNA products have not been reported to control DDR activation. Here we show, in human, mouse and zebrafish, that DICER and DROSHA, but not downstream elements of the RNAi pathway, are necessary to activate the DDR upon exogenous DNA damage and oncogene-induced genotoxic stress, as studied by DDR foci formation and by checkpoint assays. DDR foci are sensitive to RNase A treatment, and DICER- and DROSHA-dependent RNA products are required to restore DDR foci in RNase-A-treated cells. Through RNA deep sequencing and the study of DDR activation at a single inducible DNA double-strand break, we demonstrate that DDR foci formation requires site-specific DICER- and DROSHA-dependent small RNAs, named DDRNAs, which act in a MRE11­RAD50­NBS1-complex-dependent manner (MRE11 also known as MRE11A; NBS1 also known as NBN). DDRNAs, either chemically synthesized or in vitro generated by DICER cleavage, are sufficient to restore the DDR in RNase-A-treated cells, also in the absence of other cellular RNAs. Our results describe an unanticipated direct role of a novel class of ncRNAs in the control of DDR activation at sites of DNA damage.

Chromatin-associated RNA interference components contribute to transcriptional regulation in Drosophila.

RNA interference (RNAi) pathways have evolved as important modulators of gene expression that operate in the cytoplasm by degrading RNA target molecules through the activity of short (21-30 nucleotide) RNAs. RNAi components have been reported to have a role in the nucleus, as they are involved in epigenetic regulation and heterochromatin formation. However, although RNAi-mediated post-transcriptional gene silencing is well documented, the mechanisms of RNAi-mediated transcriptional gene silencing and, in particular, the role of RNAi components in chromatin dynamics, especially in animal multicellular organisms, are elusive. Here we show that the key RNAi components Dicer 2 (DCR2) and Argonaute 2 (AGO2) associate with chromatin (with a strong preference for euchromatic, transcriptionally active, loci) and interact with the core transcription machinery. Notably, loss of function of DCR2 or AGO2 showed that transcriptional defects are accompanied by the perturbation of RNA polymerase II positioning on promoters. Furthermore, after heat shock, both Dcr2 and Ago2 null mutations, as well as missense mutations that compromise the RNAi activity, impaired the global dynamics of RNA polymerase II. Finally, the deep sequencing of the AGO2-associated small RNAs (AGO2 RIP-seq) revealed that AGO2 is strongly enriched in small RNAs that encompass the promoter regions and other regions of heat-shock and other genetic loci on both the sense and antisense DNA strands, but with a strong bias for the antisense strand, particularly after heat shock. Taken together, our results show that DCR2 and AGO2 are globally associated with transcriptionally active loci and may have a pivotal role in shaping the transcriptome by controlling the processivity of RNA polymerase II.

CAGE-defined promoter regions of the genes implicated in Rett Syndrome.

BACKGROUND: Mutations in three functionally diverse genes cause Rett Syndrome. Although the functions of Forkhead box G1 (FOXG1), Methyl CpG binding protein 2 (MECP2) and Cyclin-dependent kinase-like 5 (CDKL5) have been studied individually, not much is known about their relation to each other with respect to expression levels and regulatory regions. Here we analyzed data from hundreds of mouse and human samples included in the FANTOM5 project, to identify transcript initiation sites, expression levels, expression correlations and regulatory regions of the three genes. RESULTS: Our investigations reveal the predominantly used transcription start sites (TSSs) for each gene including novel transcription start sites for FOXG1. We show that FOXG1 expression is poorly correlated with the expression of MECP2 and CDKL5. We identify promoter shapes for each TSS, the predicted location of enhancers for each gene and the common transcription factors likely to regulate the three genes. Our data imply Polycomb Repressive Complex 2 (PRC2) mediated silencing of Foxg1 in cerebellum. CONCLUSIONS: Our analyses provide a comprehensive picture of the regulatory regions of the three genes involved in Rett Syndrome.

Synthesis and Antitumour Evaluation of Tricyclic Indole-2-Carboxamides against Paediatric Brain Cancer Cells.

Antitumour properties of some cannabinoids (CB) have been reported in the literature as early as 1970s, however there is no clear consensus to date on the exact mechanisms leading to cancer cell death. The indole-based WIN 55,212-2 and SDB-001 are both known as potent agonists at both CB1 and CB2 receptors, yet we demonstrate herein that only the former can exert in vitro antitumour effects when tested against a paediatric brain cancer cell line KNS42. In this report, we describe the synthesis of novel 3,4-fused tricyclic indoles and evaluate their functional potencies at both cannabinoid receptors, as well as their abilities to inhibit the growth or proliferation of KNS42 cells. Compared to our previously reported indole-2-carboxamides, these 3,4-fused tricyclic indoles had either completely lost activities, or, showed moderate-to-weak antagonism at both CB1 and CB2 receptors. Compound 23 displayed the most potent antitumour properties among the series. Our results further support the involvement of non-CB pathways for the observed antitumour activities of amidoalkylindole-based cannabinoids, in line with our previous findings. Transcriptomic analysis comparing cells treated or non-treated with compound 23 suggested the observed antitumour effects of 23 are likely to result mainly from disruption of the FOXM1-regulated cell cycle pathways.

Patient-derived xenografts and single-cell sequencing identifies three subtypes of tumor-reactive lymphocytes in uveal melanoma metastases.

Uveal melanoma (UM) is a rare melanoma originating in the eye's uvea, with 50% of patients experiencing metastasis predominantly in the liver. In contrast to cutaneous melanoma, there is only a limited effectiveness of combined immune checkpoint therapies, and half of patients with uveal melanoma metastases succumb to disease within 2 years. This study aimed to provide a path toward enhancing immunotherapy efficacy by identifying and functionally validating tumor-reactive T cells in liver metastases of patients with UM. We employed single-cell RNA-seq of biopsies and tumor-infiltrating lymphocytes (TILs) to identify potential tumor-reactive T cells. Patient-derived xenograft (PDX) models of UM metastases were created from patients, and tumor sphere cultures were generated from these models for co-culture with autologous or MART1-specific HLA-matched allogenic TILs. Activated T cells were subjected to TCR-seq, and the TCRs were matched to those found in single-cell sequencing data from biopsies, expanded TILs, and in livers or spleens of PDX models injected with TILs. Our findings revealed that tumor-reactive T cells resided not only among activated and exhausted subsets of T cells, but also in a subset of cytotoxic effector cells. In conclusion, combining single-cell sequencing and functional analysis provides valuable insights into which T cells in UM may be useful for cell therapy amplification and marker selection.

Mycobacterium tuberculosis WhiB4 regulates oxidative stress response to modulate survival and dissemination in vivo.

Host-generated oxidative stress is considered one of the main mechanisms constraining Mycobacterium tuberculosis (Mtb) growth. The redox-sensing mechanisms in Mtb are not completely understood. Here we show that WhiB4 responds to oxygen (O2) and nitric oxide (NO) via its 4Fe-4S cluster and controls the oxidative stress response in Mtb. The WhiB4 mutant (MtbΔwhiB4) displayed an altered redox balance and a reduced membrane potential. Microarray analysis demonstrated that MtbΔwhiB4 overexpresses the antioxidant systems including alkyl hydroperoxidase (ahpC-ahpD) and rubredoxins (rubA-rubB). DNA binding assays showed that WhiB4 [4Fe-4S] cluster is dispensable for DNA binding. However, oxidation of the apo-WhiB4 Cys thiols induced disulphide-linked oligomerization, DNA binding and transcriptional repression, whereas reduction reversed the effect. Furthermore, WhiB4 binds DNA with a preference for GC-rich sequences. Expression analysis showed that oxidative stress repressed whiB4 and induced antioxidants in Mtb, while their hyper-induction was observed in MtbΔwhiB4. MtbΔwhiB4 showed increased resistance to oxidative stress in vitro and enhanced survival inside the macrophages. Lastly, MtbΔwhiB4 displayed hypervirulence in the lungs of guinea pigs, but showed a defect in dissemination to their spleen. These findings suggest that WhiB4 systematically calibrates the activation of oxidative stress response in Mtb to maintain redox balance, and to modulate virulence.

Long non-coding RNA modifies chromatin: epigenetic silencing by long non-coding RNAs.

Common themes are emerging in the molecular mechanisms of long non-coding RNA-mediated gene repression. Long non-coding RNAs (lncRNAs) participate in targeted gene silencing through chromatin remodelling, nuclear reorganisation, formation of a silencing domain and precise control over the entry of genes into silent compartments. The similarities suggest that these are fundamental processes of transcription regulation governed by lncRNAs. These findings have paved the way for analogous investigations on other lncRNAs and chromatin remodelling enzymes. Here we discuss these common mechanisms and provide our view on other molecules that warrant similar investigations. We also present our concepts on the possible mechanisms that may facilitate the exit of genes from the silencing domains and their potential therapeutic applications. Finally, we point to future areas of research and put forward our recommendations for improvements in resources and applications of existing technologies towards targeted outcomes in this active area of research.

Single cell transcriptomics reveals cell type specific features of developmentally regulated responses to lipopolysaccharide between birth and 5 years.

Background: Human perinatal life is characterized by a period of extraordinary change during which newborns encounter abundant environmental stimuli and exposure to potential pathogens. To meet such challenges, the neonatal immune system is equipped with unique functional characteristics that adapt to changing conditions as development progresses across the early years of life, but the molecular characteristics of such adaptations remain poorly understood. The application of single cell genomics to birth cohorts provides an opportunity to investigate changes in gene expression programs elicited downstream of innate immune activation across early life at unprecedented resolution. Methods: In this study, we performed single cell RNA-sequencing of mononuclear cells collected from matched birth cord blood and 5-year peripheral blood samples following stimulation (18hrs) with two well-characterized innate stimuli; lipopolysaccharide (LPS) and Polyinosinic:polycytidylic acid (Poly(I:C)). Results: We found that the transcriptional response to LPS was constrained at birth and predominantly partitioned into classical proinflammatory gene upregulation primarily by monocytes and Interferon (IFN)-signaling gene upregulation by lymphocytes. Moreover, these responses featured substantial cell-to-cell communication which appeared markedly strengthened between birth and 5 years. In contrast, stimulation with Poly(I:C) induced a robust IFN-signalling response across all cell types identified at birth and 5 years. Analysis of gene regulatory networks revealed IRF1 and STAT1 were key drivers of the LPS-induced IFN-signaling response in lymphocytes with a potential developmental role for IRF7 regulation. Conclusion: Additionally, we observed distinct activation trajectory endpoints for monocytes derived from LPS-treated cord and 5-year blood, which was not apparent among Poly(I:C)-induced monocytes. Taken together, our findings provide new insight into the gene regulatory landscape of immune cell function between birth and 5 years and point to regulatory mechanisms relevant to future investigation of infection susceptibility in early life.

MECP2 genomic structure and function: insights from ENCODE.

MECP2, a relatively small gene located in the human X chromosome, was initially described with three exons transcribing RNA from which the protein MeCP2 was translated. It is now known to have four exons from which two isoforms are translated; however, there is also evidence of additional functional genomic structures within MECP2, including exons potentially transcribing non-coding RNAs. Accompanying the recognition of a higher level of intricacy within MECP2 has been a recent surge of knowledge about the structure and function of human genes more generally, to the extent that the definition of a gene is being revisited. It is timely now to review the published and novel functional elements within MECP2, which is proving to have a complexity far greater than was previously thought.

Genomic analysis in short- and long-term patients with malignant pleura mesothelioma treated with palliative chemotherapy.

BACKGROUND: Malignant pleural mesothelioma (MPM) is an aggressive tumour with poor prognosis. The aim of this study was to identify genetic mutations associated with poor or extended survival in patients who received palliative chemotherapy. METHODS: A total of 720 patients diagnosed with MPM between 2005 and 2015 were identified. Overall survival (OS) was longer than 30 months from diagnosis for 27 patients. Twelve of 27 (44%) of the pleural biopsies from long-term survivors were retrieved and matched with 12 biopsies from patients who survived less than 12 months; one biopsy was then excluded for poor DNA quality. RESULTS: A total of 11 patients had a mean OS of 5.5 months, whereas 12 patients lived more than 30 months (mean OS: 55.8 ± 25). Mutational analysis identified 428 alterations; of which, 148, classified as somatic and functional, were considered further. Among these, 85% were missense variants, 8% were variants causing a stop gain and 6% were splice variants. Loss-of-function mutations in UQCRC1 were significantly associated with reduced survival in patients with MPM (p = 0.027), while a higher frequency of mutations in MXRA5 and RAPGEF6 was registered in long-term survivors. CONCLUSION: This is the first study evaluating the relationship between the mutational profile and outcome in patients with MPM after palliative chemotherapy. UQCRC1 codes for cytochrome b-c1 complex subunit 1 which plays a fundamental role in normal mitochondrial functions and in cell metabolism. Recent studies described UQCRC1 deregulation in other cancers. Our results suggest a possible role for mitochondrial metabolism in the biology of mesothelioma.