Element profiling
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Monitoring of single cell signal transduction in leukemic cellular subsets has been proposed to provide deeper understanding of disease biology and prognosis, but has so far not been tested in a clinical trial of targeted therapy. We developed a complete mass cytometry analysis pipeline for characterization of intracellular signal transduction patterns in the major leukocyte subsets of chronic phase chronic myeloid leukemia. Changes in phosphorylated Bcr-Abl1 and the signaling pathways involved were readily identifiable in peripheral blood single cells already within three hours of the patient receiving oral nilotinib. The signal transduction profiles of healthy donors were clearly distinct from those of the patients at diagnosis. Furthermore, using principal component analysis, we could show that phosphorylated transcription factors STAT3 (Y705) and CREB (S133) within seven days reflected BCR-ABL1IS at three and six months. Analyses of peripheral blood cells longitudinally collected from patients in the ENEST1st clinical trial showed that single cell mass cytometry appears to be highly suitable for future investigations addressing tyrosine kinase inhibitor dosing and effect. (clinicaltrials.gov identifier: 01061177).
- MeSH
- analýza jednotlivých buněk metody MeSH
- bcr-abl fúzní proteiny metabolismus MeSH
- chronická fáze myeloidní leukemie farmakoterapie patologie MeSH
- fosforylace MeSH
- leukocyty metabolismus MeSH
- lidé MeSH
- protein vázající cAMP responzivní element metabolismus MeSH
- pyrimidiny farmakologie terapeutické užití MeSH
- signální transdukce účinky léků imunologie MeSH
- transkripční faktor STAT3 metabolismus MeSH
- tyrosinkinasy terapeutické užití MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- klinické zkoušky MeSH
- práce podpořená grantem MeSH
Insects often overcome unfavorable seasons in a hormonally regulated state of diapause during which their activity ceases, development is arrested, metabolic rate is suppressed, and tolerance of environmental stress is bolstered. Diapausing insects pass through a stereotypic succession of eco-physiological phases termed "diapause development." The phasing is varied in the literature, and the whole concept is sometimes criticized as being too artificial. Here we present the results of transcriptional profiling using custom microarrays representing 1,042 genes in the drosophilid fly, Chymomyza costata Fully grown, third-instar larvae programmed for diapause by a photoperiodic (short-day) signal were assayed as they traversed the diapause developmental program. When analyzing the gradual dynamics in the transcriptomic profile, we could readily distinguish distinct diapause developmental phases associated with induction/initiation, maintenance, cold acclimation, and termination by cold or by photoperiodic signal. Accordingly, each phase is characterized by a specific pattern of gene expression, supporting the physiological relevance of the concept of diapause phasing. Further, we have dissected in greater detail the changes in transcript levels of elements of several signaling pathways considered critical for diapause regulation. The phase of diapause termination is associated with enhanced transcript levels in several positive elements stimulating direct development (the 20-hydroxyecdysone pathway: Ecr, Shd, Broad; the Wnt pathway: basket, c-jun) that are countered by up-regulation in some negative elements (the insulin-signaling pathway: Ilp8, PI3k, Akt; the target of rapamycin pathway: Tsc2 and 4EBP; the Wnt pathway: shaggy). We speculate such up-regulations may represent the early steps linked to termination of diapause programming.
- MeSH
- cirkadiánní rytmus genetika MeSH
- diapauza hmyzu genetika MeSH
- diapauza genetika MeSH
- Drosophilidae genetika MeSH
- fotoperioda MeSH
- hmyz genetika MeSH
- hmyzí proteiny genetika MeSH
- larva metabolismus MeSH
- sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů metody MeSH
- stanovení celkové genové exprese metody MeSH
- transkriptom MeSH
- vývojová regulace genové exprese genetika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Thinning supplies of natural resources increase attention to sustainable microbial production of bio-based fuels. The strain Clostridium beijerinckii NRRL B-598 is a relatively well-described butanol producer regarding its genotype and phenotype under various conditions. However, a link between these two levels, lying in the description of the gene regulation mechanisms, is missing for this strain, due to the lack of transcriptomic data. RESULTS: In this paper, we present a transcription profile of the strain over the whole fermentation using an RNA-Seq dataset covering six time-points with the current highest dynamic range among solventogenic clostridia. We investigated the accuracy of the genome sequence and particular genome elements, including pseudogenes and prophages. While some pseudogenes were highly expressed, all three identified prophages remained silent. Furthermore, we identified major changes in the transcriptional activity of genes using differential expression analysis between adjacent time-points. We identified functional groups of these significantly regulated genes and together with fermentation and cultivation kinetics captured using liquid chromatography and flow cytometry, we identified basic changes in the metabolism of the strain during fermentation. Interestingly, C. beijerinckii NRRL B-598 demonstrated different behavior in comparison with the closely related strain C. beijerinckii NCIMB 8052 in the latter phases of cultivation. CONCLUSIONS: We provided a complex analysis of the C. beijerinckii NRRL B-598 fermentation profile using several technologies, including RNA-Seq. We described the changes in the global metabolism of the strain and confirmed the uniqueness of its behavior. The whole experiment demonstrated a good reproducibility. Therefore, we will be able to repeat the experiment under selected conditions in order to investigate particular metabolic changes and signaling pathways suitable for following targeted engineering.
- MeSH
- bakteriofágy genetika MeSH
- butanoly metabolismus MeSH
- Clostridium beijerinckii genetika metabolismus virologie MeSH
- DNA virů genetika MeSH
- fermentace MeSH
- genetická transkripce MeSH
- kinetika MeSH
- pseudogeny genetika MeSH
- sekvenční analýza RNA * MeSH
- stanovení celkové genové exprese * MeSH
- Publikační typ
- časopisecké články MeSH
Transcription factors (TFs) fine-tune the host defense transcriptome in response to pathogen invasions. No information is available on Zingiber zerumbet (Zz) TFs involved in defense response against Pythium myriotylum. Here, we provide a global identification, characterization, and temporal expression profiling of Zz TFs following an incompatible interaction with P. myriotylum using a transcriptome sequencing approach. We identified a total of 903 TFs belonging to 96 families based on their conserved domains. Evolutionary analysis clustered the Zz TFs according to their phylogenetic affinity, providing glimpses of their functional diversities. High throughput expression array analysis highlighted a complex interplay between activating and repressing transcription factors in fine-tuning Zz defense response against P. myriotylum. The high differential modulation of TFs involved in cell wall fortification, lignin biosynthesis, and SA/JA hormone crosstalk allows us to envisage that this mechanism plays a central role in restricting P. myriotylum proliferation in Zz. This study lays a solid foundation and provides valuable resources for the investigation of the evolutionary history and biological functions of Zz TF genes involved in defense response.
- MeSH
- fyziologický stres MeSH
- imunita rostlin * MeSH
- molekulární evoluce MeSH
- Pythium patogenita MeSH
- responzivní elementy MeSH
- rostlinné proteiny genetika metabolismus MeSH
- transkripční faktory genetika metabolismus MeSH
- transkriptom * MeSH
- zázvorníkovité genetika imunologie mikrobiologie MeSH
- Publikační typ
- časopisecké články MeSH
Molecular and biochemical studies have shown that gene contains single or combination of different cis-acting regulatory elements are actively controlling the transcriptional regulation of associated genes, downstream effects of these result in the modulation of various biological pathways such as biotic/abiotic stress responses, hormonal responses to growth and development processes and secondary metabolite production. Therefore, the identification of promoters and their cis-regulatory elements is one of intriguing area to study the dynamic complex regulatory network of genes activities by integrating computational, comparative, structural and functional genomics. Several bioinformatics servers or database have been established to predict the cis-acting elements present in the promoter region of target gene and their association with the expression profiles in the TFs. The aim of this study is to predict possible cis-acting regulatory elements that have putative role in the transcriptional regulation of a dynamic network of metabolite gene activities controlling prenylflavonoid and bitter acids biosynthesis in hop (Humulus lupulus). Recent release of hop draft genome enabled us to predict the possible cis-acting regulatory elements by extracting 2kbp of 5' regulatory regions of genes important for lupulin metabolome biosynthesis, using Plant CARE, PLACE and Genomatix Matinspector professional databases. The result reveals the plausible role of cis-acting regulatory elements in the regulation of gene expression primarily involved in lupulin metabolome biosynthesis including under various stress conditions.
- MeSH
- Humulus genetika MeSH
- promotorové oblasti (genetika) * MeSH
- regulace genové exprese u rostlin genetika MeSH
- regulační elementy transkripční genetika MeSH
- regulační oblasti nukleových kyselin genetika MeSH
- výpočetní biologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The genomic shock hypothesis suggests that allopolyploidy is associated with genome changes driven by transposable elements, as a response to imbalances between parental insertion loads. To explore this hypothesis, we compared three allotetraploids, Nicotiana arentsii, N. rustica and N. tabacum, which arose over comparable time frames from hybridisation between increasingly divergent diploid species. We used sequence-specific amplification polymorphism (SSAP) to compare the dynamics of six transposable elements in these allopolyploids, their diploid progenitors and in corresponding synthetic hybrids. We show that element-specific dynamics in young Nicotiana allopolyploids reflect their dynamics in diploid progenitors. Transposable element mobilisation is not concomitant with immediate genome merger, but occurs within the first generations of allopolyploid formation. In natural allopolyploids, such mobilisations correlate with imbalances in the repeat profile of the parental species, which increases with their genetic divergence. Other restructuring leading to locus loss is immediate, nonrandom and targeted at specific subgenomes, independently of cross orientation. The correlation between transposable element mobilisation in allopolyploids and quantitative imbalances in parental transposable element loads supports the genome shock hypothesis proposed by McClintock.
Iron is an essential micronutrient involved in many biological processes and is often limiting for primary production in large regions of the World Ocean. Metagenomic and physiological studies have identified clades or ecotypes of marine phytoplankton that are specialized in iron depleted ecological niches. Although less studied, eukaryotic picophytoplankton does contribute significantly to primary production and carbon transfer to higher trophic levels. In particular, metagenomic studies of the green picoalga Ostreococcus have revealed the occurrence of two main clades distributed along coast-offshore gradients, suggesting niche partitioning in different nutrient regimes. Here, we present a study of the response to iron limitation of four Ostreococcus strains isolated from contrasted environments. Whereas the strains isolated in nutrient-rich waters showed high iron requirements, the oceanic strains could cope with lower iron concentrations. The RCC802 strain, in particular, was able to maintain high growth rate at low iron levels. Together physiological and transcriptomic data indicate that the competitiveness of RCC802 under iron limitation is related to a lowering of iron needs though a reduction of the photosynthetic machinery and of protein content, rather than to cell size reduction. Our results overall suggest that iron is one of the factors driving the differentiation of physiologically specialized Ostreococcus strains in the ocean.
We describe a novel gene fusion, EWSR1-CREM, identified in 3 cases of clear cell carcinoma (CCC) using anchored multiplex polymerase chain reaction, a next-generation sequencing-based technique. CCC is a low-grade salivary tumor recently characterized to have EWSR1-ATF1 fusions in the majority of cases. Three cases of malignant tumor presenting in the base of tongue, lung, and nasopharynx were studied. All cases shared a clear cell morphology with hyalinized stroma, presence of mucin and p63 positivity and were initially diagnosed as mucoepidermoid carcinoma but were negative for evidence of any of the expected gene fusions. Anchored multiplex polymerase chain reaction demonstrated a EWSR1-CREM fusion in all 3 cases to confirm a diagnosis of CCC. This finding is biologically justified as CREM and ATF1 both belong to the CREB family of transcription factors. EWSR1-CREM fusions have not been previously reported in CCC and have only rarely been reported in other tumors. We show that the ability to discover novel gene variants with next-generation sequencing-based assays has clinical utility in the pathologic classification of fusion gene-associated tumors.
- MeSH
- adenokarcinom z jasných buněk genetika patologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- modulátor elementu responzivního pro cyklický AMP genetika MeSH
- nádory jazyka genetika patologie MeSH
- nádory nosohltanu genetika MeSH
- nádory plic genetika patologie MeSH
- onkogenní fúze MeSH
- protein EWS vázající RNA genetika MeSH
- senioři MeSH
- stanovení celkové genové exprese MeSH
- transkriptom MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- kazuistiky MeSH
Steroid hormone receptors represent a major target in drug discovery. As ligand inducible transcription factors, their activity can be modulated by small lipophilic molecules. Here we describe two panels of potent and selective luciferase reporter cell lines based on cells with low endogenous steroid receptor activity (U2OS). The panels contain reporter cell lines for estrogen receptors α and β, androgen, glucocorticoid, mineralocorticoid, and progesterone receptors. In the first panel, the activation of either synthetic, steroid response elements containing promoter or viral promoter is mediated by full-length steroid receptors. The second panel is based on the expression of the chimeric receptor, which was created by the replacement of the N-terminal part of the molecule by Gal4 DBD and that binds to multiple UAS sites in the reporter promoter. Both panels were extensively characterized by profiling 28 ligands in dose response manner in agonist and antagonist mode. We have analyzed and compared the responses to tested ligands from both panels and concluded that in general both systems generated similar qualitative response in terms of potency, efficacy, partial agonism/antagonism, mixed agonistic/antagonistic profiles and the rank of potencies was well conserved between both panels. However, we have also identified some artifacts introduced by the Gal4/LBD reporter assays in contrast to their full-length receptor reporter counterparts. Keeping in mind the advantages and drawbacks of each reporter format, these cell lines represent powerful and selective tools for profiling large compound libraries (HTS) and for detailed study of mechanisms by which compounds exert their biological effects.
- MeSH
- aktivace transkripce účinky léků MeSH
- genetické inženýrství metody MeSH
- knihovny malých molekul analýza MeSH
- lidé MeSH
- luciferasy genetika metabolismus MeSH
- nádorové buněčné linie MeSH
- objevování léků metody MeSH
- plazmidy genetika MeSH
- promotorové oblasti (genetika) účinky léků MeSH
- rekombinantní proteiny genetika metabolismus MeSH
- reportérové geny MeSH
- rychlé screeningové testy metody MeSH
- sekvenční delece MeSH
- steroidní receptory agonisté antagonisté a inhibitory genetika metabolismus MeSH
- steroidy farmakologie MeSH
- transfekce MeSH
- transkripční faktory genetika metabolismus MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
