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1.
EMBO J ; 2024 Aug 29.
Artículo en Inglés | MEDLINE | ID: mdl-39210146

RESUMEN

DNA i-motif structures are formed in the nuclei of human cells and are believed to provide critical genomic regulation. While the existence, abundance, and distribution of i-motif structures in human cells has been demonstrated and studied by immunofluorescent staining, and more recently NMR and CUT&Tag, the abundance and distribution of such structures in human genomic DNA have remained unclear. Here we utilise high-affinity i-motif immunoprecipitation followed by sequencing to map i-motifs in the purified genomic DNA of human MCF7, U2OS and HEK293T cells. Validated by biolayer interferometry and circular dichroism spectroscopy, our approach aimed to identify DNA sequences capable of i-motif formation on a genome-wide scale, revealing that such sequences are widely distributed throughout the human genome and are common in genes upregulated in G0/G1 cell cycle phases. Our findings provide experimental evidence for the widespread formation of i-motif structures in human genomic DNA and a foundational resource for future studies of their genomic, structural, and molecular roles.

2.
J Infect Dis ; 2024 Apr 24.
Artículo en Inglés | MEDLINE | ID: mdl-38655824

RESUMEN

BACKGROUND: Hepatitis C virus (HCV) and hepatitis B virus (HBV) cause chronic hepatitis with important clinical differences. HCV causes hepatic steatosis and insulin resistance, while HBV confers increased risk of liver cancer. We hypothesised these differences may be due to virus-specific effects on mitochondrial function. METHODS: Seahorse technology was utilised to investigate effects of virus infection on mitochondrial function. Cell based assays were used to measure mitochondrial membrane potential and quantify pyruvate and lactate. Mass spectrometry was performed on mitochondria isolated from HBV expressing, HCV infected and control cells cultured with isotope-labelled amino acids, to identify proteins with different abundance. Altered expression of key mitochondrial proteins was confirmed by real time PCR and western blot. RESULTS: Reduced mitochondrial function and ATP production were observed with HCV infection and HBV expression. HCV impairs glycolysis and reduces expression of genes regulating fatty acid oxidation, promoting lipid accumulation. HBV causes lactate accumulation by increasing expression of lactate dehydrogenase A, which converts pyruvate to lactate. In HBV expressing cells there was marked enrichment of pyruvate dehydrogenase kinase, inhibiting conversion of pyruvate to acetyl-CoA and thereby reducing its availability for mitochondrial oxidative phosphorylation. CONCLUSIONS: HCV and HBV impair mitochondrial function and reduce ATP production. HCV reduces acetyl-CoA availability for energy production by impairing fatty acid oxidation, causing lipid accumulation and hepatic steatosis. HBV has no effect on fatty oxidation but reduces acetyl-CoA availability by disrupting pyruvate metabolism. This promotes lactic acidosis and oxidative stress, increasing the risk of disease progression and liver cancer.

3.
Blood ; 138(16): 1391-1405, 2021 10 21.
Artículo en Inglés | MEDLINE | ID: mdl-33974080

RESUMEN

We performed a phase 1 clinical trial to evaluate outcomes in patients receiving donor-derived CD19-specific chimeric antigen receptor (CAR) T cells for B-cell malignancy that relapsed or persisted after matched related allogeneic hemopoietic stem cell transplant. To overcome the cost and transgene-capacity limitations of traditional viral vectors, CAR T cells were produced using the piggyBac transposon system of genetic modification. Following CAR T-cell infusion, 1 patient developed a gradually enlarging retroperitoneal tumor due to a CAR-expressing CD4+ T-cell lymphoma. Screening of other patients led to the detection, in an asymptomatic patient, of a second CAR T-cell tumor in thoracic para-aortic lymph nodes. Analysis of the first lymphoma showed a high transgene copy number, but no insertion into typical oncogenes. There were also structural changes such as altered genomic copy number and point mutations unrelated to the insertion sites. Transcriptome analysis showed transgene promoter-driven upregulation of transcription of surrounding regions despite insulator sequences surrounding the transgene. However, marked global changes in transcription predominantly correlated with gene copy number rather than insertion sites. In both patients, the CAR T-cell-derived lymphoma progressed and 1 patient died. We describe the first 2 cases of malignant lymphoma derived from CAR gene-modified T cells. Although CAR T cells have an enviable record of safety to date, our results emphasize the need for caution and regular follow-up of CAR T recipients, especially when novel methods of gene transfer are used to create genetically modified immune therapies. This trial was registered at www.anzctr.org.au as ACTRN12617001579381.


Asunto(s)
Inmunoterapia Adoptiva/efectos adversos , Linfoma/etiología , Receptores de Antígenos de Linfocitos T/uso terapéutico , Anciano , Elementos Transponibles de ADN , Regulación Neoplásica de la Expresión Génica , Técnicas de Transferencia de Gen , Humanos , Inmunoterapia Adoptiva/métodos , Leucemia de Células B/genética , Leucemia de Células B/terapia , Linfoma/genética , Linfoma de Células B/genética , Linfoma de Células B/terapia , Masculino , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/metabolismo , Transcriptoma , Transgenes
4.
Am J Hematol ; 98(1): 159-165, 2023 01.
Artículo en Inglés | MEDLINE | ID: mdl-35560045

RESUMEN

We designed a trial to simultaneously address the problems of graft versus host disease (GVHD), infection, and recurrence of malignancy after allogeneic stem cell transplantation. CD34+ stem cell isolation was used to minimize the development of acute and chronic GVHD. Two prophylactic infusions, one combining donor-derived cytomegalovirus, Epstein-Barr virus, and Aspergillus fumigatus specific T-cells and the other comprising donor-derived CD19 directed chimeric antigen receptor (CAR) bearing T-cells, were given 21-28 days after transplant. Two patients were transplanted for acute lymphoblastic leukemia from HLA identical siblings using standard doses of cyclophosphamide and total body irradiation without antilymphocyte globulin. Patients received no post-transplant immune suppression and were given no pre-CAR T-cell lymphodepletion. Neutrophil and platelet engraftment was prompt. Following adoptive T-cell infusions, there was rapid appearance of antigen-experienced CD8+ and to a lesser extent CD4+ T-cells. Tetramer-positive T-cells targeting CMV and EBV appeared rapidly after T-cell infusion and persisted for at least 1 year. CAR T-cell expansion occurred and persisted for up to 3 months. T-cell receptor tracking confirmed the presence of product-derived T-cell clones in blood targeting all three pathogens. Both patients are alive over 3 years post-transplant without evidence of GVHD or disease recurrence. Combining robust donor T-cell depletion with directed T-cell adoptive immunotherapy targeting infectious and malignant antigens permits independent modulation of GVHD, infection, and disease recurrence. The combination may separate GVHD from the graft versus tumor effect, accelerate immune reconstitution, and improve transplant tolerability.


Asunto(s)
Infecciones por Virus de Epstein-Barr , Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Linfocitos T , Infecciones por Virus de Epstein-Barr/complicaciones , Infecciones por Virus de Epstein-Barr/terapia , Trasplante Homólogo , Resultado del Tratamiento , Herpesvirus Humano 4 , Enfermedad Injerto contra Huésped/etiología , Enfermedad Injerto contra Huésped/prevención & control , Trasplante de Células Madre , Inmunoterapia , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia
5.
Int J Mol Sci ; 24(10)2023 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-37240132

RESUMEN

The hepatitis C virus (HCV) relies on cellular lipid pathways for virus replication and also induces liver steatosis, but the mechanisms involved are not clear. We performed a quantitative lipidomics analysis of virus-infected cells by combining high-performance thin-layer chromatography (HPTLC) and mass spectrometry, using an established HCV cell culture model and subcellular fractionation. Neutral lipid and phospholipids were increased in the HCV-infected cells; in the endoplasmic reticulum there was an ~four-fold increase in free cholesterol and an ~three-fold increase in phosphatidyl choline (p < 0.05). The increase in phosphatidyl choline was due to the induction of a non-canonical synthesis pathway involving phosphatidyl ethanolamine transferase (PEMT). An HCV infection induced expression of PEMT while knocking down PEMT with siRNA inhibited virus replication. As well as supporting virus replication, PEMT mediates steatosis. Consistently, HCV induced the expression of the pro-lipogenic genes SREBP 1c and DGAT1 while inhibiting the expression of MTP, promoting lipid accumulation. Knocking down PEMT reversed these changes and reduced the lipid content in virus-infected cells. Interestingly, PEMT expression was over 50% higher in liver biopsies from people infected with the HCV genotype 3 than 1, and three times higher than in people with chronic hepatitis B, suggesting that this may account for genotype-dependent differences in the prevalence of hepatic steatosis. PEMT is a key enzyme for promoting the accumulation of lipids in HCV-infected cells and supports virus replication. The induction of PEMT may account for virus genotype specific differences in hepatic steatosis.


Asunto(s)
Hígado Graso , Hepatitis C Crónica , Hepatitis C , Humanos , Hepacivirus/genética , Hepacivirus/metabolismo , Transferasas/metabolismo , Hepatitis C/genética , Hígado Graso/patología , Replicación Viral , Genotipo , Colesterol/metabolismo , Fosfatidilcolinas/metabolismo , Fenotipo , Fosfatidiletanolamina N-Metiltransferasa/genética
6.
Int J Mol Sci ; 23(19)2022 Sep 22.
Artículo en Inglés | MEDLINE | ID: mdl-36232428

RESUMEN

Rett syndrome (RTT) is a rare disorder and one of the most abundant causes of intellectual disabilities in females. Single mutations in the gene coding for methyl-CpG-binding protein 2 (MeCP2) are responsible for the disorder. MeCP2 regulates gene expression as a transcriptional regulator as well as through epigenetic imprinting and chromatin condensation. Consequently, numerous biological pathways on multiple levels are influenced. However, the exact molecular pathways from genotype to phenotype are currently not fully elucidated. Treatment of RTT is purely symptomatic as no curative options for RTT have yet to reach the clinic. The paucity of this is mainly due to an incomplete understanding of the underlying pathophysiology of the disorder with no clinically useful common disease drivers, biomarkers, or therapeutic targets being identified. With the premise of identifying universal and robust disease drivers and therapeutic targets, here, we interrogated a range of RTT transcriptomic studies spanning different species, models, and MECP2 mutations. A meta-analysis using RNA sequencing data from brains of RTT mouse models, human post-mortem brain tissue, and patient-derived induced pluripotent stem cell (iPSC) neurons was performed using weighted gene correlation network analysis (WGCNA). This study identified a module of genes common to all datasets with the following ten hub genes driving the expression: ATRX, ADCY7, ADCY9, SOD1, CACNA1A, PLCG1, CCT5, RPS9, BDNF, and MECP2. Here, we discuss the potential benefits of these genes as therapeutic targets.


Asunto(s)
Síndrome de Rett , Animales , Factor Neurotrófico Derivado del Encéfalo/genética , Cromatina , Femenino , Humanos , Proteína 2 de Unión a Metil-CpG/metabolismo , Ratones , Mutación , Síndrome de Rett/genética , Síndrome de Rett/metabolismo , Superóxido Dismutasa-1/genética
7.
FASEB J ; 34(7): 9547-9562, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32501591

RESUMEN

Circulating plasma TRAIL levels are suppressed in patients with cardiovascular and diabetic diseases. To identify novel targets in vascular metabolic diseases, genome-wide transcriptome of aortic tissue from Trail-/- versus Trail+/+ mice were interrogated. We found 861 genes differentially expressed with TRAIL deletion. Gene enrichment analyses showed many of these genes were related to inflammation, cell-to-cell cytoskeletal interactions, and transcriptional modulation. We identified vascular protective and pathological gene clusters, with Ifi205 as the most significantly reduced vascular protective gene, whereas Glut1, the most significantly increased pathological gene with TRAIL deletion. We hypothesized that therapeutic targets could be devised from such integrated analysis and validated our findings from vascular tissues of diabetic mice. From the differentially expressed gene targets, enriched transcription factor (TF) and microRNA binding motifs were identified. The top two TFs were Elk1 and Sp1, with enrichment to eight gene targets common to both. miR-520d-3p and miR-377-3p were the top enriched microRNAs with TRAIL deletion; with four overlapping genes enriched for both microRNAs. Our findings offer an alternate in silico approach for therapeutic target identification and present a deeper understanding of gene signatures and pathways altered with TRAIL suppression in the vasculature.


Asunto(s)
Diabetes Mellitus Experimental/complicaciones , Angiopatías Diabéticas/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Ligando Inductor de Apoptosis Relacionado con TNF/fisiología , Transcriptoma , Animales , Biología Computacional , Angiopatías Diabéticas/etiología , Angiopatías Diabéticas/patología , Humanos , Ratones , Ratones Noqueados , MicroARNs/genética
8.
Brain Behav Immun ; 94: 308-317, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33422639

RESUMEN

Although genetic variation is a major risk factor of neurodevelopmental disorders, environmental factors during pregnancy and early life are also important in disease expression. Animal models demonstrate that maternal inflammation causes fetal neuroinflammation and neurodevelopmental deficits, and brain transcriptomics of neurodevelopmental disorders in humans show upregulated differentially expressed genes are enriched in immune pathways. We prospectively recruited 200 sequentially referred children with tic disorders/obsessive-compulsive disorder (OCD), 100 autoimmune neurological controls, and 100 age-matched healthy controls. A structured interview captured the maternal and family history of autoimmune disease and other pro-inflammatory states. Maternal blood and published Tourette brain transcriptomes were analysed for overlapping enriched pathways. Mothers of children with tics/OCD had a higher rate of autoimmune disease compared with mothers of children with autoimmune neurological conditions (p = 0.054), and mothers of healthy controls (p = 0.0004). Autoimmunity was similarly elevated in first- and second-degree maternal relatives of children with tics/OCD (p < 0.0001 and p = 0.014 respectively). Other pro-inflammatory states were also more common in mothers of children with tics/OCD than controls (p < 0.0001). Upregulated differentially expressed genes in maternal autoimmune disease and Tourette brain transcriptomes were commonly enriched in innate immune processes. Pro-inflammatory states, including autoimmune disease, are more common in the mothers and families of children with tics/OCD. Exploratory transcriptome analysis indicates innate immune signalling may link maternal inflammation and childhood tics/OCD. Targeting inflammation may represent preventative strategies in pregnancy and treatment opportunities for children with neurodevelopmental disorders.


Asunto(s)
Trastorno Obsesivo Compulsivo , Trastornos de Tic , Tics , Autoinmunidad/genética , Niño , Femenino , Humanos , Inmunidad Innata/genética , Recién Nacido , Inflamación/genética , Trastorno Obsesivo Compulsivo/genética , Embarazo , Transcriptoma
9.
Int J Mol Sci ; 22(18)2021 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-34576118

RESUMEN

Rett Syndrome (RTT) is an X linked neurodevelopmental disorder caused by mutations in the methyl-CpG-binding protein 2 (MECP2) gene, resulting in severe cognitive and physical disabilities. Despite an apparent normal prenatal and postnatal development period, symptoms usually present around 6 to 18 months of age. Little is known about the consequences of MeCP2 deficiency at a molecular and cellular level before the onset of symptoms in neural cells, and subtle changes at this highly sensitive developmental stage may begin earlier than symptomatic manifestation. Recent transcriptomic studies of patient induced pluripotent stem cells (iPSC)-differentiated neurons and brain organoids harbouring pathogenic mutations in MECP2, have unravelled new insights into the cellular and molecular changes caused by these mutations. Here we interrogated transcriptomic modifications in RTT patients using publicly available RNA-sequencing datasets of patient iPSCs harbouring pathogenic mutations and healthy control iPSCs by Weighted Gene Correlation Network Analysis (WGCNA). Preservation analysis identified core gene pathways involved in translation, ribosomal function, and ubiquitination perturbed in some MECP2 mutant iPSC lines. Furthermore, differential gene expression of the parental fibroblasts and iPSC-derived neurons revealed alterations in genes in the ubiquitination pathway and neurotransmission in fibroblasts and differentiated neurons respectively. These findings might suggest that global translational dysregulation and proteasome ubiquitin function in Rett syndrome begins in progenitor cells prior to lineage commitment and differentiation into neural cells.


Asunto(s)
Redes Reguladoras de Genes , Complejo de la Endopetidasa Proteasomal/metabolismo , Biosíntesis de Proteínas/genética , Síndrome de Rett/genética , Ubiquitina/metabolismo , Análisis por Conglomerados , Bases de Datos Genéticas , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Proteína 2 de Unión a Metil-CpG/química , Proteína 2 de Unión a Metil-CpG/genética , Proteína 2 de Unión a Metil-CpG/metabolismo , Neuronas/metabolismo , Análisis de Componente Principal , Dominios Proteicos , Ubiquitina/genética
10.
Am J Hum Genet ; 101(2): 255-266, 2017 Aug 03.
Artículo en Inglés | MEDLINE | ID: mdl-28777932

RESUMEN

Breast cancer risk is strongly associated with an intergenic region on 11q13. We have previously shown that the strongest risk-associated SNPs fall within a distal enhancer that regulates CCND1. Here, we report that, in addition to regulating CCND1, this enhancer regulates two estrogen-regulated long noncoding RNAs, CUPID1 and CUPID2. We provide evidence that the risk-associated SNPs are associated with reduced chromatin looping between the enhancer and the CUPID1 and CUPID2 bidirectional promoter. We further show that CUPID1 and CUPID2 are predominantly expressed in hormone-receptor-positive breast tumors and play a role in modulating pathway choice for the repair of double-strand breaks. These data reveal a mechanism for the involvement of this region in breast cancer.


Asunto(s)
Neoplasias de la Mama/genética , Cromosomas Humanos Par 11/genética , Ciclina D1/genética , Reparación del ADN/genética , ARN Largo no Codificante/genética , Línea Celular Tumoral , Cromatina/metabolismo , Roturas del ADN de Doble Cadena , Daño del ADN/genética , Elementos de Facilitación Genéticos/genética , Estrógenos/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Predisposición Genética a la Enfermedad/genética , Humanos , Células MCF-7 , Polimorfismo de Nucleótido Simple/genética , Regiones Promotoras Genéticas/genética , Interferencia de ARN , ARN Guía de Kinetoplastida/genética , ARN Interferente Pequeño/genética
11.
Trends Genet ; 32(10): 620-637, 2016 10.
Artículo en Inglés | MEDLINE | ID: mdl-27592414

RESUMEN

Although a considerable portion of eukaryotic genomes is transcribed as long noncoding RNAs (lncRNAs), the vast majority are functionally uncharacterised. The rapidly expanding catalogue of mechanistically investigated lncRNAs has provided evidence for distinct functional subclasses, which are now ripe for exploitation as a general model to predict functions for uncharacterised lncRNAs. By utilising publicly-available genome-wide datasets and computational methods, we present several developed and emerging in silico approaches to characterise and predict the functions of lncRNAs. We propose that the application of these techniques provides valuable functional and mechanistic insight into lncRNAs, and is a crucial step for informing subsequent functional studies.


Asunto(s)
Genoma , ARN Largo no Codificante/genética , Biología Computacional , Bases de Datos Genéticas , ARN Largo no Codificante/química , ARN Largo no Codificante/aislamiento & purificación
12.
Bioinformatics ; 34(6): 920-927, 2018 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-29092009

RESUMEN

Motivation: The branchpoint element is required for the first lariat-forming reaction in splicing. However current catalogues of human branchpoints remain incomplete due to the difficulty in experimentally identifying these splicing elements. To address this limitation, we have developed a machine-learning algorithm-branchpointer-to identify branchpoint elements solely from gene annotations and genomic sequence. Results: Using branchpointer, we annotate branchpoint elements in 85% of human gene introns with sensitivity (61.8%) and specificity (97.8%). In addition to annotation, branchpointer can evaluate the impact of SNPs on branchpoint architecture to inform functional interpretation of genetic variants. Branchpointer identifies all published deleterious branchpoint mutations annotated in clinical variant databases, and finds thousands of additional clinical and common genetic variants with similar predicted effects. This genome-wide annotation of branchpoints provides a reference for the genetic analysis of splicing, and the interpretation of noncoding variation. Availability and implementation: Branchpointer is written and implemented in the statistical programming language R and is freely available under a BSD license as a package through Bioconductor. Contact: b.signal@garvan.org.au or t.mercer@garvan.org. Supplementary information: Supplementary data are available at Bioinformatics online.


Asunto(s)
Genoma Humano , Intrones , Aprendizaje Automático , Anotación de Secuencia Molecular , Empalme del ARN , Análisis de Secuencia de ADN/métodos , Variación Genética , Humanos , Sensibilidad y Especificidad , Programas Informáticos
14.
Exp Eye Res ; 168: 161-170, 2018 03.
Artículo en Inglés | MEDLINE | ID: mdl-29305299

RESUMEN

Keratolenticular dysgenesis (KLD) and ectopia lentis are congenital eye defects. The aim of this study is the identification of molecular genetic alterations responsible for those ocular anomalies with neurologic impairment in an individual with a de novo balanced chromosome translocation t(11;18)(q23.3;q11.2)dn. Disruption of OAF, the human orthologue of the Drosophila oaf, by the 11q23.3 breakpoint results in reduced expression of this transcriptional regulator. Furthermore, four most likely nonfunctional chimeric transcripts comprising up to OAF exon 3, derived from the der(11) allele, have also been identified. This locus has been implicated by publicly available genome-wide association data in corneal disease and corneal topography. The expression of the poliovirus receptor-related 1(PVRL1) or nectin cell adhesion molecule 1 (NECTIN1), a paralogue of nectin cell adhesion molecule 3 (PVRL3) associated with congenital ocular defects, situated 500 kb upstream from 11q23.3 breakpoint, is increased. The 18q11.2 breakpoint is localized between cutaneous T-cell lymphoma-associated antigen 1(CTAGE1) and retinoblastoma binding protein 8 (RBBP8) genes. Genomic imbalance that could contribute to the observed phenotype was excluded. Analysis of gene expression datasets throughout normal murine ocular lens embryogenesis suggests that OAF expression is significantly enriched in the lens from early stages of development through adulthood, whereas PVRL1 is lens-enriched until E12.5 and then down-regulated. This contrasts with the observation that the proposita's lymphoblastoid cell lines exhibit low OAF and high PVRL1 expression as compared to control, which offers further support that the alterations described above are most likely responsible for the clinical phenotype. Finally, gene interaction topology data for PVRL1 also agree with our proposal that disruption of OAF by the translocation breakpoint and misregulation of PVRL1 due to a position effect contribute to the observed ocular and neurological phenotype.


Asunto(s)
Segmento Anterior del Ojo/anomalías , Opacidad de la Córnea/genética , Desplazamiento del Cristalino/genética , Anomalías del Ojo/genética , Glicoproteínas de Membrana/genética , Nectinas/genética , Animales , Longitud Axial del Ojo/patología , Córnea/patología , Citocromo P-450 CYP1B1/genética , Perfilación de la Expresión Génica , Humanos , Cristalino/patología , Ratones , Translocación Genética
15.
Proc Natl Acad Sci U S A ; 112(52): 15898-903, 2015 Dec 29.
Artículo en Inglés | MEDLINE | ID: mdl-26578815

RESUMEN

We surveyed the "dark" proteome-that is, regions of proteins never observed by experimental structure determination and inaccessible to homology modeling. For 546,000 Swiss-Prot proteins, we found that 44-54% of the proteome in eukaryotes and viruses was dark, compared with only ∼14% in archaea and bacteria. Surprisingly, most of the dark proteome could not be accounted for by conventional explanations, such as intrinsic disorder or transmembrane regions. Nearly half of the dark proteome comprised dark proteins, in which the entire sequence lacked similarity to any known structure. Dark proteins fulfill a wide variety of functions, but a subset showed distinct and largely unexpected features, such as association with secretion, specific tissues, the endoplasmic reticulum, disulfide bonding, and proteolytic cleavage. Dark proteins also had short sequence length, low evolutionary reuse, and few known interactions with other proteins. These results suggest new research directions in structural and computational biology.


Asunto(s)
Biología Computacional/métodos , Bases de Datos de Proteínas , Proteínas/metabolismo , Proteoma/metabolismo , Algoritmos , Animales , Archaea/genética , Archaea/metabolismo , Bacterias/genética , Bacterias/metabolismo , Eucariontes/metabolismo , Humanos , Modelos Moleculares , Conformación Proteica , Proteínas/química , Proteínas/genética , Proteoma/química , Proteoma/genética , Virus/genética , Virus/metabolismo
16.
Biochim Biophys Acta ; 1859(1): 16-22, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26297315

RESUMEN

Over the last decade, long noncoding RNAs (lncRNAs) have emerged as a fundamental molecular class whose members play pivotal roles in the regulation of the genome. The observation of pervasive transcription of mammalian genomes in the early 2000s sparked a revolution in the understanding of information flow in eukaryotic cells and the incredible flexibility and dynamic nature of the transcriptome. As a molecular class, distinct loci yielding lncRNAs are set to outnumber those yielding mRNAs. However, like many important discoveries, the road leading to uncovering this diverse class of molecules that act through a remarkable repertoire of mechanisms, was not a straight one. The same characteristic that most distinguishes lncRNAs from mRNAs, i.e. their developmental-stage, tissue-, and cell-specific expression, was one of the major impediments to their discovery and recognition as potentially functional regulatory molecules. With growing numbers of lncRNAs being assigned to biological functions, the specificity of lncRNA expression is now increasingly recognized as a characteristic that imbues lncRNAs with great potential as biomarkers and for the development of highly targeted therapeutics. Here we review the history of lncRNA research and how technological advances and insight into biological complexity have gone hand-in-hand in shaping this revolution. We anticipate that as increasing numbers of these molecules, often described as the dark matter of the genome, are characterized and the structure-function relationship of lncRNAs becomes better understood, it may ultimately be feasible to decipher what these non-(protein)-coding genes encode. This article is part of a Special Issue entitled: Clues to long noncoding RNA taxonomy1, edited by Dr. Tetsuro Hirose and Dr. Shinichi Nakagawa.


Asunto(s)
Regulación de la Expresión Génica , Especificidad de Órganos/genética , ARN Largo no Codificante/genética , Transcriptoma/genética , Animales , Linaje de la Célula/genética , Secuencia Conservada/genética , Genoma , Humanos , ARN Largo no Codificante/biosíntesis , ARN Mensajero/genética
17.
Nucleic Acids Res ; 43(Database issue): D168-73, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25332394

RESUMEN

Despite the prevalence of long noncoding RNA (lncRNA) genes in eukaryotic genomes, only a small proportion have been examined for biological function. lncRNAdb, available at http://lncrnadb.org, provides users with a comprehensive, manually curated reference database of 287 eukaryotic lncRNAs that have been described independently in the scientific literature. In addition to capturing a great proportion of the recent literature describing functions for individual lncRNAs, lncRNAdb now offers an improved user interface enabling greater accessibility to sequence information, expression data and the literature. The new features in lncRNAdb include the integration of Illumina Body Atlas expression profiles, nucleotide sequence information, a BLAST search tool and easy export of content via direct download or a REST API. lncRNAdb is now endorsed by RNAcentral and is in compliance with the International Nucleotide Sequence Database Collaboration.


Asunto(s)
Bases de Datos de Ácidos Nucleicos , ARN Largo no Codificante/fisiología , Secuencia de Bases , Secuencia Conservada , Expresión Génica , Humanos , Internet , Proteínas/genética , ARN Largo no Codificante/química , ARN Largo no Codificante/metabolismo , Alineación de Secuencia
18.
J Immunol ; 192(4): 1404-14, 2014 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-24446516

RESUMEN

The cytokine IL-21 has been shown to influence immune responses through both costimulatory effects on effector T cells and opposing inhibitory effects on T regulatory cells (Tregs). To distinguish the effect of IL-21 on the immune system from that of its effect on Tregs, we analyzed the role of IL-21/IL-21R signaling in mice made genetically deficient in IL-2, which exhibit a deficit in IL-2-dependent Foxp3 regulatory T cells and suffer from a fatal multiorgan inflammatory disease. Our findings demonstrate that in the absence of IL-21/IL-21R signaling, Il2(-/-) mice retained a deficiency in Tregs yet exhibited a reduced and delayed inflammatory disease. The improved health of Il2(-/-)Il21r(-/-) mice was reflected in reduced pancreatitis and hemolytic anemia and this was associated with distinct changes in lymphocyte effector populations, including the reduced expansion of both T follicular helper cells and Th17 cells and a compensatory increase in IL-22 in the absence of IL-21R. IL-21/IL-21R interactions were also important for the expansion of effector and memory CD8(+) T cells, which were critical for the development of pancreatitis in Il2(-/-) mice. These findings demonstrate that IL-21 is a major target of immune system regulation.


Asunto(s)
Anemia Hemolítica/inmunología , Subunidad alfa del Receptor de Interleucina-21/metabolismo , Interleucinas/metabolismo , Pancreatitis/inmunología , Linfocitos T Reguladores/inmunología , Anemia Hemolítica/genética , Animales , Anticuerpos/sangre , Formación de Anticuerpos/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/inmunología , Factores de Transcripción Forkhead/deficiencia , Factores de Transcripción Forkhead/genética , Interleucina-2/genética , Subunidad alfa del Receptor de Interleucina-21/genética , Interleucinas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Pancreatitis/genética , Transducción de Señal/genética , Transducción de Señal/inmunología , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/metabolismo , Células Th17/citología , Células Th17/inmunología , Interleucina-22
20.
BMC Genomics ; 15: 476, 2014 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-24929644

RESUMEN

BACKGROUND: Free circulating DNA (fcDNA) has many potential clinical applications, due to the non-invasive way in which it is collected. However, because of the low concentration of fcDNA in blood, genome-wide analysis carries many technical challenges that must be overcome before fcDNA studies can reach their full potential. There are currently no definitive standards for fcDNA collection, processing and whole-genome sequencing. We report novel detailed methodology for the capture of high-quality methylated fcDNA, library preparation and downstream genome-wide Next-Generation Sequencing. We also describe the effects of sample storage, processing and scaling on fcDNA recovery and quality. RESULTS: Use of serum versus plasma, and storage of blood prior to separation resulted in genomic DNA contamination, likely due to leukocyte lysis. Methylated fcDNA fragments were isolated from 5 donors using a methyl-binding protein-based protocol and appear as a discrete band of ~180 bases. This discrete band allows minimal sample loss at the size restriction step in library preparation for Next-Generation Sequencing, allowing for high-quality sequencing from minimal amounts of fcDNA. Following sequencing, we obtained 37 × 10(6)-86 × 10(6) unique mappable reads, representing more than 50% of total mappable reads. The methylation status of 9 genomic regions as determined by DNA capture and sequencing was independently validated by clonal bisulphite sequencing. CONCLUSIONS: Our optimized methods provide high-quality methylated fcDNA suitable for whole-genome sequencing, and allow good library complexity and accurate sequencing, despite using less than half of the recommended minimum input DNA.


Asunto(s)
Metilación de ADN , ADN/sangre , ADN/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Anciano , Composición de Base , ADN/química , ADN/aislamiento & purificación , Contaminación de ADN , Femenino , Biblioteca de Genes , Voluntarios Sanos , Humanos , Persona de Mediana Edad , Reproducibilidad de los Resultados , Factores de Riesgo , Análisis de Secuencia de ADN
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