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Tools for genome-wide rapid identification of peptide-major histocompatibility complex targets of T-cell receptors (TCR) are not yet universally available. We present a new antigen screening method, the T-synapse (Tsyn) reporter system, which includes antigen-presenting cells (APC) with a Fas-inducible NF-κB reporter and T cells with a nuclear factor of activated T cells (NFAT) reporter. To functionally screen for target antigens from a cDNA library, productively interacting T cell-APC aggregates were detected by dual-reporter activity and enriched by flow sorting followed by antigen identification quantified by deep sequencing (Tsyn-seq). When applied to a previously characterized TCR specific for the E7 antigen derived from human papillomavirus type 16 (HPV16), Tsyn-seq successfully enriched the correct cognate antigen from a cDNA library derived from an HPV16-positive cervical cancer cell line. Tsyn-seq provides a method for rapidly identifying antigens recognized by TCRs of interest from a tumor cDNA library. See related Spotlight by Makani and Joglekar, p. 515.
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Sinapsis Inmunológicas , Receptores de Antígenos de Linfocitos T , Linfocitos T , Humanos , Células Presentadoras de Antígenos/inmunología , Línea Celular Tumoral , Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Papillomavirus Humano 16/inmunología , Papillomavirus Humano 16/genética , Sinapsis Inmunológicas/inmunología , Factores de Transcripción NFATC/metabolismo , Factores de Transcripción NFATC/inmunología , Proteínas E7 de Papillomavirus/inmunología , Proteínas E7 de Papillomavirus/genética , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/inmunologíaRESUMEN
Although targeting oxidative phosphorylation (OXPHOS) is a rational anticancer strategy, clinical benefit with OXPHOS inhibitors has yet to be achieved. Here we advanced IACS-010759, a highly potent and selective small-molecule complex I inhibitor, into two dose-escalation phase I trials in patients with relapsed/refractory acute myeloid leukemia (NCT02882321, n = 17) and advanced solid tumors (NCT03291938, n = 23). The primary endpoints were safety, tolerability, maximum tolerated dose and recommended phase 2 dose (RP2D) of IACS-010759. The PK, PD, and preliminary antitumor activities of IACS-010759 in patients were also evaluated as secondary endpoints in both clinical trials. IACS-010759 had a narrow therapeutic index with emergent dose-limiting toxicities, including elevated blood lactate and neurotoxicity, which obstructed efforts to maintain target exposure. Consequently no RP2D was established, only modest target inhibition and limited antitumor activity were observed at tolerated doses, and both trials were discontinued. Reverse translational studies in mice demonstrated that IACS-010759 induced behavioral and physiological changes indicative of peripheral neuropathy, which were minimized with the coadministration of a histone deacetylase 6 inhibitor. Additional studies are needed to elucidate the association between OXPHOS inhibition and neurotoxicity, and caution is warranted in the continued development of complex I inhibitors as antitumor agents.
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Antineoplásicos , Leucemia Mieloide Aguda , Neoplasias , Animales , Ratones , Antineoplásicos/efectos adversos , Inhibidores de Histona Desacetilasas/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/patología , Neoplasias/patología , Fosforilación Oxidativa , HumanosRESUMEN
PURPOSE: Cisplatin (CDDP)-based chemotherapy is a first-line treatment for patients with advanced head and neck squamous cell carcinomas (HNSCC), despite a high rate of treatment failures, acquired resistance, and subsequent aggressive behavior. The purpose of this study was to study the mechanism of CDDP resistance and metastasis in HNSCC. We investigated the role of NRF2 pathway activation as a driven event for tumor progression and metastasis of HNSCC. EXPERIMENTAL DESIGN: Human HNSCC cell lines that are highly resistant to CDDP were generated. Clonogenic survival assays and a mouse model of oral cancer were used to examine the impact of NRF2 activation in vitro and in vivo on CDDP sensitivity and development of metastasis. Western blotting, immunostaining, whole-exome sequencing, single-cell transcriptomic and epigenomic profiling platforms were performed to dissect clonal evolution and molecular mechanisms. RESULTS: Implantation of CDDP-resistant HNSCC cells into the tongues of nude mice resulted in a very high rate of distant metastases. The CDDP-resistant cells had significantly higher expression of NRF2 pathway genes in the presence of newly acquired KEAP1 mutations, or via epigenomic activation of target genes. Knockdown of NRF2 or restoration of the wild-type KEAP1 genes resensitized resistant cells to CDDP and decreased distant metastasis (DM). Finally, treatment with inhibitor of glutaminase-1, a NRF2 target gene, alleviated CDDP resistance. CONCLUSIONS: CDDP resistance and development of DM are associated with dysregulated and epigenetically reprogrammed KEAP1-NRF2 signaling pathway. A strategy targeting KEAP1/NRF2 pathway or glutamine metabolism deserves further clinical investigation in patients with CDDP-resistant head and neck tumors.
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Antineoplásicos , Neoplasias de Cabeza y Cuello , Factor 2 Relacionado con NF-E2 , Carcinoma de Células Escamosas de Cabeza y Cuello , Animales , Humanos , Ratones , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Cisplatino/farmacología , Cisplatino/uso terapéutico , Resistencia a Antineoplásicos/genética , Epigénesis Genética , Epigenómica , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/genética , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Ratones Desnudos , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Transducción de Señal , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Carcinoma de Células Escamosas de Cabeza y Cuello/genéticaRESUMEN
Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a family of RNA-binding proteins that modulate multiple aspects of gene activity and RNA processing, including transcription, splicing, localization, translation, and decay of RNA. Interaction of hnRNPs with RNA is a highly dynamic but regulated process. Poly(ADP-ribose) polymerase (PARP)-dependent PARylation of different hnRNPs is a well-known posttranslational modification that affects their interactions with RNA. Here, we described a protocol for in situ localization of RNA-binding proteins (RBPs) on giant polytene chromosomes in Drosophila larval salivary glands, which have been widely used to visualize the dynamic binding profiles of various RBPs and other transcription-related proteins at specific loci on chromosomes. This chapter also includes a stepwise description of RNA:RNA in situ hybridization, in conjunction with immunostaining, using polytene chromosome squashes or intact tissues. We also highlight advanced live cell imaging methods, including FRAP and FLIP, using transgenic lines that express fluorescent-tagged hnRNPs. These cytological approaches can be used to visualize the localization of RNA-binding proteins and their interacting RNAs under different cellular conditions.
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Proteínas de Drosophila , Drosophila , Animales , Drosophila/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas Nucleares Heterogéneas/genética , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Cromosomas Politénicos/genética , Cromosomas Politénicos/metabolismo , ARN/metabolismoRESUMEN
Neurodegenerative diseases are characterized by selective vulnerability of distinct cell populations; however, the cause for this specificity remains elusive. Here, we show that entorhinal cortex layer 2 (EC2) neurons are unusually vulnerable to prolonged neuronal inactivity compared with neighboring regions of the temporal lobe, and that reelin + stellate cells connecting EC with the hippocampus are preferentially susceptible within the EC2 population. We demonstrate that neuronal death after silencing can be elicited through multiple independent means of activity inhibition, and that preventing synaptic release, either alone or in combination with electrical shunting, is sufficient to elicit silencing-induced degeneration. Finally, we discovered that degeneration following synaptic silencing is governed by competition between active and inactive cells, which is a circuit refinement process traditionally thought to end early in postnatal life. Our data suggests that the developmental window for wholesale circuit plasticity may extend into adulthood for specific brain regions. We speculate that this sustained potential for remodeling by entorhinal neurons may support lifelong memory but renders them vulnerable to prolonged activity changes in disease.
Neurodegenerative conditions cause irreversible damage to the brain and have a devastating impact on quality of life. However, these diseases start gradually, meaning that the entire brain is not affected at once. For example, the initial signs of Alzheimer's disease appear only in specific areas. One of the first brain regions to degenerate in Alzheimer's is the entorhinal cortex. In healthy individuals, entorhinal neurons send electrical signals to the hippocampus, a part of the brain important for memory and learning. During Alzheimer's, hippocampal neurons also die off, leading to 'shrinkage' of this brain region and, ultimately, the memory problems that are a hallmark of the disease. Many neurons in the developing brain require electrical input from other cells to survive in other words, if they do not belong to an 'active circuit', they are eliminated. This is crucial for the connection between the entorhinal cortex and the hippocampus, where the circuit's development and maintenance require carefully controlled electrical activity. Abnormal electrical activity is also an early sign of diseases like Alzheimer's, but how this relates to degeneration is still poorly understood. By investigating these questions, Zhao, Grunke, Wood et al. uncovered a potential relationship between electrical activity and degeneration in the adult brain, long after the circuit between the hippocampus and the entorhinal cortex had matured. Mice were genetically engineered so that their entorhinal cortex would carry a protein designed to silence electrical signaling. The communication between the entorhinal cortex and the hippocampus could therefore be shut off by activating the protein with an injected drug. Remarkably, within just a few days of silencing, cells from the entorhinal cortex started to die off. Zhao, Grunke, Wood et al. went on to show that different silencing methods yielded the same results in other words, the degeneration of cells from the entorhinal cortex was not linked to a particular method. This vulnerability to electrical inactivity was also unique to the entorhinal cortex: when neighboring parts of the brain were silenced, the nerve cells in these areas did not die as readily. Interestingly, in one of their experiments, Zhao, Grunke, Wood et al. found that electrical activity of neighboring nerve cells participated in killing the silenced neurons, suggesting that nerve cells in these brain areas might compete to survive. Overall, this work highlights a direct link between electrical activity and nerve cell degeneration in a part of the brain severely affected by Alzheimer's. In the future, Zhao, Grunke, Wood et al. hope that these results will pave the way to a better understanding of the biological mechanisms underpinning such neurodegenerative diseases.
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Enfermedad de Alzheimer , Ratones , Animales , Enfermedad de Alzheimer/metabolismo , Neuronas/fisiología , Hipocampo/metabolismo , Corteza EntorrinalRESUMEN
Undifferentiated pleomorphic sarcoma (UPS) and malignant peripheral nerve sheath tumor (MPNST) are aggressive soft tissue sarcomas that do not respond well to current treatment modalities. The limited availability of UPS and MPNST cell lines makes it challenging to identify potential therapeutic targets in a laboratory setting. Understanding the urgent need for improved treatments for these tumors and the limited cellular models available, we generated additional cell lines to study these rare cancers. Patient-derived tumors were used to establish 4 new UPS models, including one radiation-associated UPS-UPS271.1, UPS511, UPS0103, and RIS620, one unclassified spindle cell sarcoma-USC060.1, and 3 new models of MPNST-MPNST007, MPNST3813E, and MPNST4970. This study examined the utility of the new cell lines as sarcoma models by assessing their tumorigenic potential and mutation status for known sarcoma-related genes. All the cell lines formed colonies and migrated in vitro. The in vivo tumorigenic potential of the cell lines and corresponding xenografts was determined by subcutaneous injection or xenograft re-passaging into immunocompromised mice. USC060.1 and UPS511 cells formed tumors in mice upon subcutaneous injection. UPS0103 and RIS620 tumor implants formed tumors in vivo, as did MPNST007 and MPNST3813E tumor implants. Targeted sequencing analysis of a panel of genes frequently mutated in sarcomas identified TP53, RB1, and ATRX mutations in a subset of the cell lines. These new cellular models provide the scientific community with powerful tools for detailed studies of tumorigenesis and for investigating novel therapies for UPS and MPNST.
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Neurofibrosarcoma , Sarcoma , Neoplasias de los Tejidos Blandos , Animales , Humanos , Ratones , Modelos Teóricos , Mutación , Neurofibrosarcoma/genética , Sarcoma/genética , Sarcoma/patología , Neoplasias de los Tejidos Blandos/genéticaRESUMEN
Breast cancer is the most common female malignancy in both the developed and developing world. Doxorubicin is one of the most commonly used chemotherapies for breast cancer. Unfortunately, up to 60% of survivors report long-term chemotherapy-induced cognitive dysfunction (CICD) characterized by deficits in working memory, processing speed and executive function. Currently, no therapeutic standard for treating CICD exists. Here, we hypothesized that treatment with a blood-brain barrier permeable histone deacetylase 6 (HDAC6) inhibitor can successfully reverse long-term doxorubicin-induced cognitive dysfunction. Methods: The puzzle box test and novel object/place recognition test were used to assess cognitive function following a therapeutic doxorubicin dosing schedule in female mice. Mitochondrial function and morphology in neuronal synaptosomes were evaluated using the Seahorse XF24 extracellular flux analyzer and transmission electron microscopy, respectively. Hippocampal postsynaptic integrity was evaluated using immunofluorescence. Hippocampal microglia phenotype was determined using advanced imaging techniques and single-nucleus RNA sequencing. Results: A 14-day treatment with a blood-brain barrier permeable HDAC6 inhibitor successfully reversed long-term CICD in the domains of executive function, working and spatial memory. No significant changes in mitochondrial function or morphology in neuronal synaptosomes were detected. Long-term CICD was associated with a decreased expression of postsynaptic PSD95 in the hippocampus. These changes were associated with decreased microglial ramification and alterations in the microglia transcriptome that suggest a stage 1 disease-associated microglia (DAM) phenotype. HDAC6 inhibition completely reversed these doxorubicin-induced alterations, indicating a restoration of microglial homeostasis. Conclusion: Our results show that decreased postsynaptic integrity and a neurodegenerative microglia phenotype closely resembling stage 1 DAM microglia contribute to long-term CICD. Moreover, HDAC6 inhibition shows promise as an efficacious pharmaceutical intervention to alleviate CICD and improve quality of life of breast cancer survivors.
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Disfunción Cognitiva/tratamiento farmacológico , Histona Desacetilasa 6/antagonistas & inhibidores , Microglía/efectos de los fármacos , Piridazinas/farmacología , Sinapsis/efectos de los fármacos , Animales , Disfunción Cognitiva/inducido químicamente , Homólogo 4 de la Proteína Discs Large/metabolismo , Relación Dosis-Respuesta a Droga , Doxorrubicina/antagonistas & inhibidores , Femenino , Hipocampo/citología , Hipocampo/efectos de los fármacos , Homeostasis/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Transcripción Genética/efectos de los fármacosRESUMEN
Although the RNA helicase Upf1 has hitherto been examined mostly in relation to its cytoplasmic role in nonsense mediated mRNA decay (NMD), here we report high-throughput ChIP data indicating genome-wide association of Upf1 with active genes in Schizosaccharomyces pombe. This association is RNase sensitive, correlates with Pol II transcription and mRNA expression levels. Changes in Pol II occupancy were detected in a Upf1 deficient (upf1Δ) strain, prevalently at genes showing a high Upf1 relative to Pol II association in wild-type. Additionally, an increased Ser2 Pol II signal was detected at all highly transcribed genes examined by ChIP-qPCR. Furthermore, upf1Δ cells are hypersensitive to the transcription elongation inhibitor 6-azauracil. A significant proportion of the genes associated with Upf1 in wild-type conditions are also mis-regulated in upf1Δ. These data envisage that by operating on the nascent transcript, Upf1 might influence Pol II phosphorylation and transcription.
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ARN Helicasas/metabolismo , ARN Polimerasa II/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Regulación Fúngica de la Expresión Génica , Genoma Fúngico , Fosforilación , ARN Helicasas/genética , ARN Polimerasa II/genética , Schizosaccharomyces , Proteínas de Schizosaccharomyces pombe/genética , Activación TranscripcionalRESUMEN
OBJECTIVE: Enhancer aberrations are beginning to emerge as a key epigenetic feature of colorectal cancers (CRC), however, a comprehensive knowledge of chromatin state patterns in tumour progression, heterogeneity of these patterns and imparted therapeutic opportunities remain poorly described. DESIGN: We performed comprehensive epigenomic characterisation by mapping 222 chromatin profiles from 69 samples (33 colorectal adenocarcinomas, 4 adenomas, 21 matched normal tissues and 11 colon cancer cell lines) for six histone modification marks: H3K4me3 for Pol II-bound and CpG-rich promoters, H3K4me1 for poised enhancers, H3K27ac for enhancers and transcriptionally active promoters, H3K79me2 for transcribed regions, H3K27me3 for polycomb repressed regions and H3K9me3 for heterochromatin. RESULTS: We demonstrate that H3K27ac-marked active enhancer state could distinguish between different stages of CRC progression. By epigenomic editing, we present evidence that gains of tumour-specific enhancers for crucial oncogenes, such as ASCL2 and FZD10, was required for excessive proliferation. Consistently, combination of MEK plus bromodomain inhibition was found to have synergistic effects in CRC patient-derived xenograft models. Probing intertumour heterogeneity, we identified four distinct enhancer subtypes (EPIgenome-based Classification, EpiC), three of which correlate well with previously defined transcriptomic subtypes (consensus molecular subtypes, CMSs). Importantly, CMS2 can be divided into two EpiC subgroups with significant survival differences. Leveraging such correlation, we devised a combinatorial therapeutic strategy of enhancer-blocking bromodomain inhibitors with pathway-specific inhibitors (PARPi, EGFRi, TGFßi, mTORi and SRCi) for EpiC groups. CONCLUSION: Our data suggest that the dynamics of active enhancer underlies CRC progression and the patient-specific enhancer patterns can be leveraged for precision combination therapy.
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Cromatina , Neoplasias Colorrectales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Elementos de Facilitación Genéticos/genética , Humanos , Proteínas Nucleares , Factores de Transcripción/genéticaRESUMEN
Histone methyltransferase KMT2D harbors frequent loss-of-function somatic point mutations in several tumor types, including melanoma. Here, we identify KMT2D as a potent tumor suppressor in melanoma through an in vivo epigenome-focused pooled RNAi screen and confirm the finding by using a genetically engineered mouse model (GEMM) based on conditional and melanocyte-specific deletion of KMT2D. KMT2D-deficient tumors show substantial reprogramming of key metabolic pathways, including glycolysis. KMT2D deficiency aberrantly upregulates glycolysis enzymes, intermediate metabolites, and glucose consumption rates. Mechanistically, KMT2D loss causes genome-wide reduction of H3K4me1-marked active enhancer chromatin states. Enhancer loss and subsequent repression of IGFBP5 activates IGF1R-AKT to increase glycolysis in KMT2D-deficient cells. Pharmacological inhibition of glycolysis and insulin growth factor (IGF) signaling reduce proliferation and tumorigenesis preferentially in KMT2D-deficient cells. We conclude that KMT2D loss promotes tumorigenesis by facilitating an increased use of the glycolysis pathway for enhanced biomass needs via enhancer reprogramming, thus presenting an opportunity for therapeutic intervention through glycolysis or IGF pathway inhibitors.
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N-Metiltransferasa de Histona-Lisina/metabolismo , Melanoma/genética , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Animales , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Genes Supresores de Tumor , Glucosa/metabolismo , Glucólisis/genética , Histona Metiltransferasas/genética , Histona Metiltransferasas/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Insulina/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Receptor IGF Tipo 1/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Transducción de Señal , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
The distribution of assembled, and potentially translating, ribosomes within cells can be visualised in Drosophila by using Bimolecular Fluorescence Complementation (BiFC) to monitor the interaction between tagged pairs of 40S and 60S ribosomal proteins (RPs) that are close neighbours across inter-subunit junctions in the assembled 80S ribosome. Here we describe transgenes expressing two novel RP pairs tagged with Venus-based BiFC fragments that considerably increase the sensitivity of this technique we termed Ribo-BiFC. This improved method should provide a convenient way of monitoring the local distribution of ribosomes in most Drosophila cells and we suggest that it could be implemented in other organisms. We visualised 80S ribosomes in different neurons, particularly photoreceptors in the larva, pupa and adult brain. Assembled ribosomes are most abundant in the various neuronal cell bodies, but they are also present along the full length of axons. They are concentrated in growth cones of developing photoreceptors and are apparent at the terminals of mature larval photoreceptors targeting the larval optical neuropil. Surprisingly, there is relatively less puromycin incorporation in the distal portion of axons in the larval optic stalk, suggesting that some of the ribosomes that have initiated translation may not be engaged in elongation in growing axons.This article has an associated First Person interview with the first author of the paper.
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Axones/metabolismo , Drosophila/genética , Drosophila/metabolismo , Imagen Molecular , Neuronas/metabolismo , Ribosomas/metabolismo , Animales , Técnica del Anticuerpo Fluorescente , Humanos , Imagen Molecular/métodos , Estructura Molecular , Células Fotorreceptoras/metabolismo , Proteínas Ribosómicas/metabolismo , Ribosomas/químicaRESUMEN
UPF1 is an RNA helicase that is required for nonsense-mediated mRNA decay (NMD) in eukaryotes, and the predominant view is that UPF1 mainly operates on the 3'UTRs of mRNAs that are directed for NMD in the cytoplasm. Here we offer evidence, obtained from Drosophila, that UPF1 constantly moves between the nucleus and cytoplasm by a mechanism that requires its RNA helicase activity. UPF1 is associated, genome-wide, with nascent RNAs at most of the active Pol II transcription sites and at some Pol III-transcribed genes, as demonstrated microscopically on the polytene chromosomes of salivary glands and by ChIP-seq analysis in S2 cells. Intron recognition seems to interfere with association and translocation of UPF1 on nascent pre-mRNAs, and cells depleted of UPF1 show defects in the release of mRNAs from transcription sites and their export from the nucleus.
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Proteínas de Drosophila/metabolismo , ARN Helicasas/metabolismo , ARN Mensajero/metabolismo , Transcripción Genética , Animales , DrosophilaRESUMEN
Mango is one of the most important fruits of tropical ecological region of the world, well known for its nutritive value, aroma and taste. Its world production is >45MT worth >200 billion US dollars. Genomic resources are required for improvement in productivity and management of mango germplasm. There is no web-based genomic resources available for mango. Hence rapid and cost-effective high throughput putative marker discovery is required to develop such resources. RAD-based marker discovery can cater this urgent need till whole genome sequence of mango becomes available. Using a panel of 84 mango varieties, a total of 28.6 Gb data was generated by ddRAD-Seq approach on Illumina HiSeq 2000 platform. A total of 1.25 million SNPs were discovered. Phylogenetic tree using 749 common SNPs across these varieties revealed three major lineages which was compared with geographical locations. A web genomic resources MiSNPDb, available at http://webtom.cabgrid.res.in/mangosnps/ is based on 3-tier architecture, developed using PHP, MySQL and Javascript. This web genomic resources can be of immense use in the development of high density linkage map, QTL discovery, varietal differentiation, traceability, genome finishing and SNP chip development for future GWAS in genomic selection program. We report here world's first web-based genomic resources for genetic improvement and germplasm management of mango.
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Mangifera/genética , Filogenia , Polimorfismo de Nucleótido Simple , Bases de Datos Genéticas , Frutas/genética , Genoma de Planta , Genómica , Internet , FilogeografíaRESUMEN
Although it is currently understood that the exon junction complex (EJC) is recruited on spliced mRNA by a specific interaction between its central protein, eIF4AIII, and splicing factor CWC22, we found that eIF4AIII and the other EJC core proteins Y14 and MAGO bind the nascent transcripts of not only intron-containing but also intronless genes on Drosophila polytene chromosomes. Additionally, Y14 ChIP-seq demonstrates that association with transcribed genes is also splicing-independent in Drosophila S2 cells. The association of the EJC proteins with nascent transcripts does not require CWC22 and that of Y14 and MAGO is independent of eIF4AIII. We also show that eIF4AIII associates with both polysomal and monosomal RNA in S2 cell extracts, whereas Y14 and MAGO fractionate separately. Cumulatively, our data indicate a global role of eIF4AIII in gene expression, which would be independent of Y14 and MAGO, splicing, and of the EJC, as currently understood.
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Drosophila melanogaster/genética , Factor 4A Eucariótico de Iniciación/genética , Cromosomas Politénicos/química , Precursores del ARN/genética , Empalme del ARN , ARN Mensajero/genética , Animales , Fraccionamiento Celular , Células Cultivadas , Mapeo Cromosómico , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Factor 4A Eucariótico de Iniciación/metabolismo , Exones , Intrones , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Cromosomas Politénicos/metabolismo , Unión Proteica , Precursores del ARN/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Unlike the telomerase-dependent mammalian telomeres, HeT-A, TART, and TAHRE (HTT) retroposon arrays regulate Drosophila telomere length. Cap prevents telomeric associations (TAs) and telomeric fusions (TFs). Our results suggest important roles of Hrb87F in telomeric HTT array and cap maintenance in Drosophila. All chromosome arms, except 2L, in Df(3R)Hrb87F homozygotes (Hrb87F-null) displayed significantly elongated telomeres with amplified HTT arrays and high TAs, all of which resolved without damage. Presence of FLAG-tagged Hrb87F (FLAG-Hrb87F) on cap and subtelomeric regions following hsFLAG-Hrb87F transgene expression in Df(3R)Hrb87F homozygotes suppressed TAs without affecting telomere length. A normal X-chromosome telomere expanded within five generations in Hrb87F-null background and displayed high TAs, but not when hsFLAG-Hrb87F was co-expressed. Tel (1) /Gaiano line or HP1 loss-of-function mutant-derived expanded telomeres carry Hrb87F on cap and HTT arrays while Hrb87F-null telomeres have HP1 and HOAP on caps and expanded HTT arrays. ISWI, seen only on cap on normal telomeres, was abundant on Hrb87F-null expanded HTT arrays. Extended telomeres derived from Tel (1) (Gaiano) or HP1-null mutation background interact with those from Hrb87F-null, since while the end association frequency was negligible in Df(3R)Hrb87F/+ nuclei, it increased significantly in co-presence of Tel (1) or HP1-null-based expanded telomere/s. Together, these suggest complex interactions between members of the proteome of telomere so that absence of any key member leads to telomere expansion and/or enhanced TAs/TFs. HTT expansion in Hrb87F-null condition is not developmental but a germline event presumably because absence of Hrb87F in germline may deregulate HTT retroposition/replication leading to telomere elongation.
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Proteínas de Drosophila/metabolismo , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Proteínas Nucleares/metabolismo , Homeostasis del Telómero/fisiología , Telómero/metabolismo , Cromosoma X/metabolismo , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster , Ribonucleoproteínas Nucleares Heterogéneas/genética , Proteínas Nucleares/genética , Telómero/genética , Cromosoma X/genéticaRESUMEN
A delayed organismic lethality was reported in Drosophila following heat shock when developmentally active and stress-inducible noncoding hsrω-n transcripts were down-regulated during heat shock through hs-GAL4-driven expression of the hsrω-RNAi transgene, despite the characteristic elevation of all heat shock proteins (Hsp), including Hsp70. Here, we show that hsrω-RNAi transgene expression prior to heat shock singularly prevents accumulation of Hsp70 in all larval tissues without affecting transcriptional induction of hsp70 genes and stability of their transcripts. Absence of the stress-induced Hsp70 accumulation was not due to higher levels of Hsc70 in hsrω-RNAi transgene-expressing tissues. Inhibition of proteasomal activity during heat shock restored high levels of the induced Hsp70, suggesting very rapid degradation of the Hsp70 even during the stress when hsrω-RNAi transgene was expressed ahead of heat shock. Unexpectedly, while complete absence of hsrω transcripts in hsrω (66) homozygotes (hsrω-null) did not prevent high accumulation of heat shock-induced Hsp70, hsrω-RNAi transgene expression in hsrω-null background blocked Hsp70 accumulation. Nonspecific RNAi transgene expression did not affect Hsp70 induction. These observations reveal that, under certain conditions, the stress-induced Hsp70 can be selectively and rapidly targeted for proteasomal degradation even during heat shock. In the present case, the selective degradation of Hsp70 does not appear to be due to down-regulation of the hsrω-n transcripts per se; rather, this may be an indirect effect of the expression of hsrω-RNAi transgene whose RNA products may titrate away some RNA-binding proteins which may also be essential for stability of the induced Hsp70.
Asunto(s)
Drosophila melanogaster/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Respuesta al Choque Térmico/genética , ARN Largo no Codificante/genética , Animales , Proteínas de Drosophila/biosíntesis , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas HSP70 de Choque Térmico/genética , Larva/genética , Larva/metabolismo , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Activación Transcripcional/genéticaRESUMEN
The nucleus limited long-noncoding hsrω-n transcripts, hnRNPs, and some other RNA processing proteins organize nucleoplasmic omega speckles in Drosophila. Unlike other nuclear speckles, omega speckles rapidly disappear following cell stress, while hnRNPs and other associated proteins move away from chromosome sites, nucleoplasm, and the disappearing speckles to get uniquely sequestered at hsrω locus. Omega speckles reappear and hnRNPs get redistributed to normal locations during recovery from stress. With a view to understand the dynamics of omega speckles and their associated proteins, we used live imaging of GFP tagged hnRNPs (Hrb87F, Hrb98DE, or Squid) in unstressed and stressed Drosophila cells. Omega speckles display size-dependent mobility in nucleoplasmic domains with significant colocalization with nuclear matrix Tpr/Megator and SAFB proteins, which also accumulate at hsrω gene site after stress. Instead of moving towards the nuclear periphery located hsrω locus following heat shock or colchicine treatment, omega speckles rapidly disappear within nucleoplasm while chromosomal and nucleoplasmic hnRNPs move, stochastically or, more likely, by nuclear matrix-mediated transport to hsrω locus in non-particulate form. Continuing transcription of hsrω during cell stress is essential for sequestering incoming hnRNPs at the site. While recovering from stress, the sequestered hnRNPs are released as omega speckles in ISWI-dependent manner. Photobleaching studies reveal hnRNPs to freely move between nucleoplasm, omega speckles, chromosome regions, and hsrω gene site although their residence periods at chromosomes and hsrω locus are longer. A model for regulation of exchange of hnRNPs between nuclear compartments by hsrω-n transcripts is presented.
Asunto(s)
Núcleo Celular/genética , Drosophila melanogaster/genética , Ribonucleoproteínas Nucleares Heterogéneas/genética , Calor , Animales , Núcleo Celular/fisiologíaRESUMEN
OBJECTIVE: The objective of this study was to compare image quality for abdominal computed tomographic (CT) images acquired at 200 and 50 mA s and reconstructed with image-based iterative reconstruction. MATERIALS AND METHODS: In this institutional review board-approved prospective study, 22 patients (mean [SD] age, 64.3 [14.4] years; male-female ratio, 12:10) gave informed consent for acquisition of additional abdominal CT images on 64-slice multi-detector CT (MDCT) (Siemens Definition Flash). Standard-dose images were acquired at 200 quality reference mA s, whereas low-dose images were acquired at 50 mA s (all series: 120 kV; 5-mm section thickness; pitch, 0.9:1). The low-dose images were reconstructed with a nonlinear 3-dimensional iterative image reconstruction (3D-IIR) (SafeCT; MedicVision, Tirat Carmel, Israel) (4 settings, namely, A1, A2, A3, and A4) and were assessed by 3 abdominal radiologists for lesion detection, image noise, and visibility of small structures. CATPHAN 500 was scanned at the respective doses to obtain noise spectral density and modulation transfer function. RESULTS: Subjective image noise was unacceptable at 50-mA s filtered back projection and improved to average in 50-mA s A1 and minimal or no noise in 50-mA s A4. However, the visibility of small structures was similar to standard-dose filtered back projection images on 50-mA s A2. Objective image noise was reduced to 66% for the 50-mA s 3D-IIR images (9.08 [2.3]/26.75 [6.8]). The modulation transfer function curve demonstrated resolution improvement in the low-dose images with the 3D-IIR technique, whereas the noise spectral density curve confirmed noise suppression in the 50-mA s 3D-IIR images. CONCLUSIONS: Three-dimensional iterative image reconstruction helps to lower image noise without affecting the visibility of small structures at "moderate" settings. Diagnostically acceptable abdominal CT examinations can be acquired at 75% lower-radiation dose with the help of the image-based iterative reconstruction technique.