RESUMO
Global warming is affecting plant phenology, with potential consequences on the dynamics of growth reactivation of sugar maple and the timings of maple syrup production. In this study, we assess the temperatures inducing the daily reactivation or cessation of sap production. We selected 19 sugarbushes across Quebec, Canada, using a tapping method associated with the tubing system, we recorded the daily timings of onset and ending of sap production during winter and spring 2018, and we associated the hourly temperatures at each site. Sap production occurred from mid-February to the end of April, starting on average between 10 and 11 AM, and ending from 6 to 8 PM. We observed a seasonal pattern in the onset and ending of sap production during spring, with the onset showing a greater change than the ending. Onset and ending of sap production occurred mostly under temperatures ranging between -2 and 2 °C. The production of sap in maple is closely related to circadian freeze-thaw cycles and occurs under nighttime and daytime temperatures fluctuating below and above 0 °C. The daily lengthening of the duration of sap production mirrors the changes in the timings of freeze and thaw events and can be explained by the physical properties of the water and the physiological processes occurring during growth reactivation. The ongoing warming will result in earlier and warmer springs, which may anticipate the cycles of freeze and thaw and advance sap production in sugar maple.
Assuntos
Acer , Quebeque , Estações do Ano , Canadá , CongelamentoRESUMO
Numerous genetic and epigenetic alterations render cancer cells selectively dependent on specific genes and regulatory pathways, and represent potential vulnerabilities that can be therapeutically exploited. Here we describe an RNA interference (RNAi)-based synthetic interaction screen to identify genes preferentially required for proliferation of p53-deficient (p53-) human cancer cells. We find that compared to p53-competent (p53+) human cancer cell lines, diverse p53- human cancer cell lines are preferentially sensitive to loss of the transcription factor ETV1 and the DNA damage kinase ATR. In p53- cells, RNAi-mediated knockdown of ETV1 or ATR results in decreased expression of the telomerase catalytic subunit TERT leading to growth arrest, which can be reversed by ectopic TERT expression. Chromatin immunoprecipitation analysis reveals that ETV1 binds to a region downstream of the TERT transcriptional start-site in p53- but not p53+ cells. We find that the role of ATR is to phosphorylate and thereby stabilize ETV1. Our collective results identify a regulatory pathway involving ETV1, ATR, and TERT that is preferentially important for proliferation of diverse p53- cancer cells.
Assuntos
Proteínas de Ciclo Celular , Proliferação de Células , Proteínas de Ligação a DNA , Proteínas Serina-Treonina Quinases , Telomerase , Fatores de Transcrição , Proteína Supressora de Tumor p53 , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Humanos , Neoplasias/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , Telomerase/genética , Telomerase/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genéticaRESUMO
The full details of a synthesis of the hetidine framework of the C20-diterpenoid alkaloids and its conversion to the atisine core structure are reported. The application of the hetidine framework to the synthesis of dihydronavirine, which is the formal reduction product of the natural product navirine, is also described. Key to the success of these studies is the use of a Ga(III)-catalyzed cycloisomerization reaction of alkynylindenes to prepare a [6-7-6] framework that was advanced to the hetidine skeleton. Furthermore, a Michael/aldol sequence was developed for the construction of the bicyclo[2.2.2] framework that is characteristic of the hetidines and atisines.
Assuntos
Alcaloides/síntese química , Alcinos/química , Produtos Biológicos/síntese química , Diterpenos/síntese química , Alcaloides/química , Produtos Biológicos/química , Catálise , Ciclização , Diterpenos/química , Estrutura Molecular , EstereoisomerismoRESUMO
Runt-related transcription factors (RUNX1, RUNX2, and RUNX3) are key lineage-specific regulators of progenitor cell growth and differentiation but also function pathologically as cancer genes that contribute to tumorigenesis. RUNX2 attenuates growth and stimulates maturation of osteoblasts during bone formation but is also robustly expressed in a subset of osteosarcomas, as well as in metastatic breast and prostate tumors. To assess the biological function of RUNX2 in osteosarcoma cells, we examined human genomic promoter interactions for RUNX2 using chromatin immunoprecipitation (ChIP)-microarray analysis in SAOS-2 cells. Promoter binding of both RUNX2 and RNA polymerase II was compared with gene expression profiles of cells in which RUNX2 was depleted by RNA interference. Many RUNX2-bound loci (1550 of 2339 total) exhibit promoter occupancy by RNA polymerase II and contain the RUNX consensus motif 5'-((T/A/C)G(T/A/C)GG(T/G). Gene ontology analysis indicates that RUNX2 controls components of multiple signaling pathways (e.g. WNT, TGFß, TNFα, and interleukins), as well as genes linked to cell motility and adhesion (e.g. the focal adhesion-related genes FAK/PTK2 and TLN1). Our results reveal that siRNA depletion of RUNX2, PTK2, or TLN1 diminishes motility of U2OS osteosarcoma cells. Thus, RUNX2 binding to diverse gene loci may support the biological properties of osteosarcoma cells.
Assuntos
Neoplasias Ósseas/metabolismo , Movimento Celular , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Genoma Humano , Proteínas de Neoplasias/metabolismo , Osteossarcoma/metabolismo , Elementos de Resposta , Neoplasias Ósseas/genética , Adesão Celular/genética , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Loci Gênicos , Humanos , Proteínas de Neoplasias/genética , Análise de Sequência com Séries de Oligonucleotídeos , Osteossarcoma/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismoRESUMO
BACKGROUND: Adult human fibroblasts grown in low oxygen and with FGF2 supplementation have the capacity to tip the healing outcome of skeletal muscle injury - by favoring regeneration response in vivo over scar formation. Here, we compare the transcriptomes of control adult human dermal fibroblasts and induced regeneration-competent (iRC) fibroblasts to identify transcriptional changes that may be related to their regeneration competence. RESULTS: We identified a unique gene-expression profile that characterizes FGF2-induced iRC fibroblast phenotype. Significantly differentially expressed genes due to FGF2 treatment were identified and analyzed to determine overrepresented Gene Ontology terms. Genes belonging to extracellular matrix components, adhesion molecules, matrix remodelling, cytoskeleton, and cytokines were determined to be affected by FGF2 treatment. CONCLUSIONS: Transcriptome analysis comparing control adult human fibroblasts with FGF2-treated fibroblasts identified functional groups of genes that reflect transcriptional changes potentially contributing to their regeneration competence. This comparative transcriptome analysis should contribute new insights into genes that characterize cells with greater regenerative potential.
Assuntos
Fator 2 de Crescimento de Fibroblastos/farmacologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Regeneração/genética , Transcriptoma/genética , Adulto , Moléculas de Adesão Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Citocinas/metabolismo , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Fibroblastos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Ontologia Genética , Humanos , Receptores de Citocinas/metabolismo , Regeneração/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transcriptoma/efeitos dos fármacos , Cicatrização/efeitos dos fármacos , Cicatrização/genéticaRESUMO
Two strategies for the synthesis of the icetexane diterpenoids icetexone and epi-icetexone that rely on Ga(III)-catalyzed cycloisomerization of alkynyl indene substrates to yield fused [6-7-6] tricycles have been explored. In the first approach, access to a tricycle bearing a gem-dimethyl group paved the way for explorations of C-H functionalization of one of the methyl groups in close proximity to a hydroxyl-directing group. This approach was ultimately unsuccessful and led only to ring cleaved products. In the second approach, an alkynyl indene substrate bearing a cyano substituent was utilized, which was effective in providing a functional handle to access the icetexone subclass of diterpenoids. A key epoxide opening/diazene rearrangement sequence was utilized to complete a formal synthesis of icetexone and epi-icetexone, which is discussed in detail. Furthermore, the cyano-containing substrate has been prepared in enantioenriched form using a Rh-catalyzed conjugate addition reaction, which now provides a route to the enantioselective synthesis of these natural products.
RESUMO
FlyFactorSurvey (http://pgfe.umassmed.edu/TFDBS/) is a database of DNA binding specificities for Drosophila transcription factors (TFs) primarily determined using the bacterial one-hybrid system. The database provides community access to over 400 recognition motifs and position weight matrices for over 200 TFs, including many unpublished motifs. Search tools and flat file downloads are provided to retrieve binding site information (as sequences, matrices and sequence logos) for individual TFs, groups of TFs or for all TFs with characterized binding specificities. Linked analysis tools allow users to identify motifs within our database that share similarity to a query matrix or to view the distribution of occurrences of an individual motif throughout the Drosophila genome. Together, this database and its associated tools provide computational and experimental biologists with resources to predict interactions between Drosophila TFs and target cis-regulatory sequences.
Assuntos
Bases de Dados Genéticas , Proteínas de Drosophila/metabolismo , Drosophila/genética , Elementos Reguladores de Transcrição , Fatores de Transcrição/metabolismo , Animais , Bactérias/genética , Sítios de Ligação , Software , Técnicas do Sistema de Duplo-Híbrido , Interface Usuário-ComputadorRESUMO
A comprehensive understanding of the C-H bond cleavage step by the concerted metalation-deprotonation (CMD) pathway is important in further development of cross-coupling reactions using different catalysts. Distortion-interaction analysis of the C-H bond cleavage over a wide range of (hetero)aromatics has been performed in an attempt to quantify the various contributions to the CMD transition state (TS). The (hetero)aromatics evaluated were divided in different categories to allow an easier understanding of their reactivity and to quantify activation characteristics of different arene substituents. The CMD pathway to the C-H bond cleavage for different classes of arenes is also presented, including the formation of pre-CMD intermediates and the analysis of bonding interactions in TS structures. The effects of remote C2 substituents on the reactivity of thiophenes were evaluated computationally and were corroborated experimentally with competition studies. We show that nucleophilicity of thiophenes, evaluated by Hammett σ(p) parameters, correlates with each of the distortion-interaction parameters. In the final part of this manuscript, we set the initial equations that can assist in the development of predictive guidelines for the functionalization of C-H bonds catalyzed by transition metal catalysts.
RESUMO
The challenge of achieving selective and predictable functionalizations at C-H bonds with complex poly(hetero)aromatic substrates was addressed by two different approaches. Site-selectivity can be obtained by applying various reaction conditions that are (hetero)arene specific to substrates that contain indoles, pyridine N-oxide, and polyfluorinated benzenes. An experimental classification of electron-rich heteroarenes based on their reactivity toward palladium-catalyzed C-H functionalization was established, the result of which correlated well with the order of reactivity predicted by the DFT-calculated concerted metalation-deprotonation (CMD) pathway. Model substrates containing two reactive heteroarenes were then reacted under general reaction conditions to demonstrate the applicability this reactivity chart in predicting the regioselectivity of the palladium-catalyzed direct arylation and benzylation reactions.
Assuntos
Compostos Heterocíclicos/química , Hidrocarbonetos Fluorados/química , Compostos Organometálicos/química , Paládio/química , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Estrutura Molecular , EstereoisomerismoRESUMO
BACKGROUND: Chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (ChIP-seq) or ChIP followed by genome tiling array analysis (ChIP-chip) have become standard technologies for genome-wide identification of DNA-binding protein target sites. A number of algorithms have been developed in parallel that allow identification of binding sites from ChIP-seq or ChIP-chip datasets and subsequent visualization in the University of California Santa Cruz (UCSC) Genome Browser as custom annotation tracks. However, summarizing these tracks can be a daunting task, particularly if there are a large number of binding sites or the binding sites are distributed widely across the genome. RESULTS: We have developed ChIPpeakAnno as a Bioconductor package within the statistical programming environment R to facilitate batch annotation of enriched peaks identified from ChIP-seq, ChIP-chip, cap analysis of gene expression (CAGE) or any experiments resulting in a large number of enriched genomic regions. The binding sites annotated with ChIPpeakAnno can be viewed easily as a table, a pie chart or plotted in histogram form, i.e., the distribution of distances to the nearest genes for each set of peaks. In addition, we have implemented functionalities for determining the significance of overlap between replicates or binding sites among transcription factors within a complex, and for drawing Venn diagrams to visualize the extent of the overlap between replicates. Furthermore, the package includes functionalities to retrieve sequences flanking putative binding sites for PCR amplification, cloning, or motif discovery, and to identify Gene Ontology (GO) terms associated with adjacent genes. CONCLUSIONS: ChIPpeakAnno enables batch annotation of the binding sites identified from ChIP-seq, ChIP-chip, CAGE or any technology that results in a large number of enriched genomic regions within the statistical programming environment R. Allowing users to pass their own annotation data such as a different Chromatin immunoprecipitation (ChIP) preparation and a dataset from literature, or existing annotation packages, such as GenomicFeatures and BSgenome, provides flexibility. Tight integration to the biomaRt package enables up-to-date annotation retrieval from the BioMart database.
Assuntos
Imunoprecipitação da Cromatina/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Software , Sítios de Ligação , GenomaRESUMO
Conditions for the palladium-catalyzed direct arylation of a wide range of heterocycles with aryl bromides are reported. Those conditions employ a stoichiometric ratio of both coupling partners, as well as a substoichiometric quantity of pivalic acid, which results in significantly faster reactions. An evaluation of the influence of the nature of the aryl halide has also been carried out.
Assuntos
Compostos Heterocíclicos/química , Compostos Heterocíclicos/síntese química , Hidrocarbonetos Bromados/química , Paládio/química , Catálise , Estrutura Molecular , EstereoisomerismoRESUMO
The concerted metalation-deprotonation mechanism predicts relative reactivity and regioselectivity for a diverse set of arenes spanning the entire spectrum of known palladium-catalyzed direct arylation coupling partners. An analysis following an active strain model provides a more complete portrayal of the important arene/catalyst parameters leading to a successful coupling. The breadth of arenes whose reactivity can be predicted by the CMD mechanism indicates that it may be far more widespread than previously imagined.
Assuntos
Paládio/química , Compostos de Sulfidrila/síntese química , Catálise , Modelos Químicos , Estrutura Molecular , Prótons , Estereoisomerismo , Compostos de Sulfidrila/química , Termodinâmica , Tiofenos/síntese química , Tiofenos/químicaRESUMO
The ordered expression of genes after growth factor stimulation in G(1) supportsthe onset of DNA replication. To characterize regulatory events during S-phase when cell cycle progression has become growth factor independent, we have profiled the expression of over 7,000 human genes using GeneChip DNA microarray analysis. HeLa cells were synchronized at the beginning of S-phase by thymidine/aphidicolin block, and RNA populations were analyzed throughout the S and G(2) phases. Expression of genes involved in DNA replication is maximal during early S-phase, whereas histone mRNAs peak at mid S-phase. Genes related to cell proliferation, including those encoding cyclins, oncoproteins, growth factors, proteins involved in signal transduction, and DNA repair proteins, follow distinct temporal patterns of expression that are functionally linked to initiation of DNA replication and progression through S-phase. The timing of expression for many genes in tumor-derived HeLa cells is highly conserved when compared with normal cells. In contrast, a number of genes show growth phenotype-related expression patterns that may directly reflect loss of stringent growth control in tumor cells. Our data reveal there is a core subset of cell growth-related genes that is fundamental to cycling cells irrespective of cell growth phenotype.
Assuntos
Proteínas de Ciclo Celular , Ciclo Celular/genética , Replicação do DNA/genética , Proteínas de Ligação a DNA , Regulação Leucêmica da Expressão Gênica , Nucleossomos/genética , Divisão Celular/genética , DNA/biossíntese , DNA/genética , Reparo do DNA/genética , Fatores de Transcrição E2F , Fase G1/genética , Perfilação da Expressão Gênica , Células HeLa , Histonas/genética , Humanos , Mitose/genética , Nucleossomos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RNA/genética , RNA/metabolismo , Fase S/genética , Fatores de Transcrição/genéticaRESUMO
Whole-genome techniques toward identification of microbial genes required for their survival and growth during infection have been useful for studies of bacterial pathogenesis. The advent of massively parallel sequencing platforms has created the opportunity to markedly accelerate such genome-scale analyses and achieve unprecedented sensitivity, resolution, and quantification. This chapter provides an overview of a genome-scale methodology that combines high-density transposon mutagenesis with a mariner transposon and deep sequencing to identify genes that are needed for survival in experimental models of pathogenesis. Application of this approach to a model pathogen, Haemophilus influenzae, has provided a comprehensive analysis of the relative role of each gene of this human respiratory pathogen in a murine pulmonary model. The method is readily adaptable to nearly any organism amenable to transposon mutagenesis.
Assuntos
Bactérias/genética , Bactérias/patogenicidade , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mutagênese Insercional/genética , Análise de Sequência de DNA/métodos , Animais , Biotina/metabolismo , Biotinilação , Cromossomos Bacterianos/genética , Primers do DNA/genética , Elementos de DNA Transponíveis/genética , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , DNA Bacteriano/metabolismo , Genoma Bacteriano/genética , Haemophilus influenzae/genética , Haemophilus influenzae/patogenicidade , Poliadenilação , Reprodutibilidade dos TestesRESUMO
Broadly applicable palladium-catalyzed heteroarene benzylation reactions are described with a focus on the most challenging heterocyclic classes under traditional benzylation techniques such as sulfur-containing heterocycles and those bearing functional groups that would be incompatible with reactions requiring Lewis acids and/or strong bases.
Assuntos
Compostos Heterocíclicos/síntese química , Paládio/química , Compostos de Benzil/química , Catálise , Ciclização , Estrutura Molecular , Enxofre/químicaRESUMO
A domino palladium-catalyzed Heck-intermolecular direct arylation reaction has been developed, giving access to a variety of dihydrobenzofurans, indolines, and oxindoles. A variety of sulfur-containing heterocycles such as thiazoles, thiophenes, and benzothiophene can be employed as the direct arylation coupling partner in yields up to 99%.
Assuntos
Paládio/química , Catálise , Compostos Heterocíclicos/química , Indicadores e Reagentes/química , Cinética , Compostos Organometálicos/químicaRESUMO
Runt-related transcription factor 2 (Runx2) controls lineage commitment, proliferation, and anabolic functions of osteoblasts as the subnuclear effector of multiple signaling axes (e.g. transforming growth factor-beta/BMP-SMAD, SRC/YES-YAP, and GROUCHO/TLE). Runx2 levels oscillate during the osteoblast cell cycle with maximal levels in G(1). Here we examined what functions and target genes of Runx2 control osteoblast growth. Forced expression of wild type Runx2 suppresses growth of Runx2(-/-) osteoprogenitors. Point mutants defective for binding to WW domain or SMAD proteins or the nuclear matrix retain this growth regulatory ability. Hence, key signaling pathways are dispensable for growth control by Runx2. However, mutants defective for DNA binding or C-terminal gene repression/activation functions do not block proliferation. Target gene analysis by Affymetrix expression profiling shows that the C terminus of Runx2 regulates genes involved in G protein-coupled receptor signaling (e.g. Rgs2, Rgs4, Rgs5, Rgs16, Gpr23, Gpr30, Gpr54, Gpr64, and Gna13). We further examined the function of two genes linked to cAMP signaling as follows: Gpr30 that is stimulated and Rgs2 that is down-regulated by Runx2. RNA interference of Gpr30 and forced expression of Rgs2 in each case inhibit osteoblast proliferation. Notwithstanding its growth-suppressive potential, our results surprisingly indicate that Runx2 may sensitize cAMP-related G protein-coupled receptor signaling by activating Gpr30 and repressing Rgs2 gene expression in osteoblasts to increase responsiveness to mitogenic signals.
Assuntos
Diferenciação Celular/fisiologia , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Regulação da Expressão Gênica/fisiologia , Osteoblastos/metabolismo , Receptores Acoplados a Proteínas G/biossíntese , Transdução de Sinais/fisiologia , Células-Tronco/metabolismo , Animais , Relógios Biológicos/fisiologia , Ciclo Celular/fisiologia , Linhagem Celular Transformada , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Perfilação da Expressão Gênica/métodos , Camundongos , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Osteoblastos/citologia , Estrutura Terciária de Proteína/fisiologia , Receptores Acoplados a Proteínas G/genética , Células-Tronco/citologiaRESUMO
During cell division, cessation of transcription is coupled with mitotic chromosome condensation. A fundamental biological question is how gene expression patterns are retained during mitosis to ensure the phenotype of progeny cells. We suggest that cell fate-determining transcription factors provide an epigenetic mechanism for the retention of gene expression patterns during cell division. Runx proteins are lineage-specific transcription factors that are essential for hematopoietic, neuronal, gastrointestinal, and osteogenic cell fates. Here we show that Runx2 protein is stable during cell division and remains associated with chromosomes during mitosis through sequence-specific DNA binding. Using siRNA-mediated silencing, mitotic cell synchronization, and expression profiling, we identify Runx2-regulated genes that are modulated postmitotically. Novel target genes involved in cell growth and differentiation were validated by chromatin immunoprecipitation. Importantly, we find that during mitosis, when transcription is shut down, Runx2 selectively occupies target gene promoters, and Runx2 deficiency alters mitotic histone modifications. We conclude that Runx proteins have an active role in retaining phenotype during cell division to support lineage-specific control of gene expression in progeny cells.
Assuntos
Cromossomos Humanos/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Epigênese Genética/fisiologia , Regulação da Expressão Gênica/fisiologia , Mitose/fisiologia , Western Blotting , Diferenciação Celular , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Ensaio de Desvio de Mobilidade Eletroforética , Epigênese Genética/genética , Perfilação da Expressão Gênica , Humanos , Microscopia de Fluorescência , Regiões Promotoras Genéticas/genética , Interferência de RNARESUMO
To better study early events in glioma genesis, markers that reliably denote landmarks in glioma development are needed. In the present study, we used microarray analysis to compare the gene expression patterns of magnetic resonance imaging (MRI)-localized N-ethyl-N-nitrosourea (ENU)-induced tumors in rat brains with those of uninvolved contralateral side and normal brains. Our analysis identified osteopontin (OPN) as the most up-regulated gene in glioma. Using immunohistochemistry we then confirmed OPN expression in every tumor examined (n = 17), including those with diameters as small as 300 mum. By contrast, no OPN immunostaining was seen in normal brain or in brains removed from ENU-exposed rats before the development of glioma. Further studies confirmed that OPN was co-localized exclusively in intratumoral glial fibrillary acidic protein-expressing cells and was notably absent from nestin-expressing ones. In conjunction with this, we confirmed that both normal neurosphere cells and ENU-im-mortalized subventricular zone/striatal cells produced negligible amounts of OPN compared to the established rat glioma cell line C6. Furthermore, inducing OPN expression in an immortalized cell line increased cell proliferation. Based on these findings, we conclude that OPN overexpression in ENU-induced gliomas occurs within a specific subset of intratumoral glial fibrillary acidic protein-positive cells and becomes evident at the stage of tumor progression.