Avian influenza has emerged as a devastating disease and may cross species barrier and adapt to a new host, causing enormous economic loss and great public health threats, and non-structural protein 1 (NS1) is a multifunctional non-structural protein of avian influenza virus (AIV) that counters cellular antiviral activities and is a virulence factor. RNA interference (RNAi) provides a powerful promising approach to inhibit viral infection specifically. To explore the possibility of using RNAi as a strategy against AIV infection, after the fusion protein expression plasmids pNS1-enhanced green fluorescent protein (EGFP), which contain the EGFP reporter gene and AIV NS1 as silencing target, were constructed and NS1-EGFP fusion protein expressing HEK293 cell lines were established, four small interfering RNAs (siRNAs) targeting NS1 gene were designed, synthesized, and used to transfect the stable cell lines. Flow cytometry, real-time quantitative polymerase chain reaction, and Western blot were performed to assess the expression level of NS1. The results suggested that sequence-dependent specific siRNAs effectively inhibited mRNA accumulation and protein expression of AIV NS1 in vitro. These findings provide useful information for the development of RNAi-based prophylaxis and therapy for AIV infection.

• MeSH
• buněčné linie MeSH
• faktory virulence antagonisté a inhibitory   genetika   MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• lidé MeSH
• malá interferující RNA * MeSH
• průtoková cytometrie MeSH
• rekombinantní fúzní proteiny analýza   genetika   MeSH
• reportérové geny MeSH
• umělá fúze genů MeSH
• virové nestrukturální proteiny antagonisté a inhibitory   genetika   MeSH
• virus chřipky A, podtyp H5N1 genetika   růst a vývoj   MeSH
• western blotting MeSH
• zelené fluorescenční proteiny analýza   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

KEY MESSAGE: Making use of wheat chromosomal resources, we developed 11 gene-associated markers for the region of interest, which allowed reducing gene interval and spanning it by four BAC clones. Positional gene cloning and targeted marker development in bread wheat are hampered by high complexity and polyploidy of its nuclear genome. Aiming to clone a Russian wheat aphid resistance gene Dn2401 located on wheat chromosome arm 7DS, we have developed a strategy overcoming problems due to polyploidy and enabling efficient development of gene-associated markers from the region of interest. We employed information gathered by GenomeZipper, a synteny-based tool combining sequence data of rice, Brachypodium, sorghum and barley, and took advantage of a high-density linkage map of Aegilops tauschii. To ensure genome- and locus-specificity of markers, we made use of survey sequence assemblies of isolated wheat chromosomes 7A, 7B and 7D. Despite the low level of polymorphism of the wheat D subgenome, our approach allowed us to add in an efficient and cost-effective manner 11 new gene-associated markers in the Dn2401 region and narrow down the target interval to 0.83 cM. Screening 7DS-specific BAC library with the flanking markers revealed a contig of four BAC clones that span the Dn2401 region in wheat cultivar 'Chinese Spring'. With the availability of sequence assemblies and GenomeZippers for each of the wheat chromosome arms, the proposed strategy can be applied for focused marker development in any region of the wheat genome.

• MeSH
• býložravci MeSH
• chromozomy rostlin MeSH
• DNA primery MeSH
• DNA rostlinná genetika   MeSH
• genetická vazba MeSH
• genetické markery MeSH
• genomika MeSH
• jednonukleotidový polymorfismus MeSH
• mapování chromozomů * MeSH
• mikrosatelitní repetice MeSH
• mšice * MeSH
• pšenice genetika   MeSH
• rostlinné geny * MeSH
• syntenie MeSH
• umělé bakteriální chromozomy MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Rusko MeSH

Accumulated evidence suggests that the endosymbiotic Trichomonasvirus (TVV) may play a role in the pathogenesis and drug susceptibility of Trichomonas vaginalis. Several reports have shown that extracellular vesicles (EVs) released from TVV-positive (TVV+) trichomonads can modulate the immune response in human vaginal epithelial cells and animal models. These results prompted us to examine whether EVs released from TVV+ isolates contained TVV. We isolated small extracellular vesicles (sEVs) from six T. vaginalis isolates that were either TVV free (ATCC 50143), harbored a single (ATCC 30236, ATCC 30238, T1), two (ATCC PRA-98), or three TVV subspecies (ATCC 50148). The presence of TVV subspecies in the six isolates was observed using reverse transcription-polymerase chain reaction (RT-PCR). Transmission electron microscopy (TEM) confirmed the presence of cup-shaped sEVs with a size range from 30-150 nm. Trichomonas vaginalis tetraspanin (TvTSP1; TVAG_019180), the classical exosome marker, was identified in all the sEV preparations. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed that all the sEVs isolated from TVV+ isolates contain viral capsid proteins derived from the same TVV subspecies in that isolate as demonstrated by RT-PCR. To provide more comprehensive information on the TVV subspecies population in other T. vaginalis isolates, we investigated the distribution of TVV subspecies in twenty-four isolates by mining the New-Generation Sequencing (NGS) RNAseq datasets. Our results should be beneficial for future studies investigating the role of TVV on the pathogenicity of T. vaginalis and the possible transmission of virus subspecies among different isolates via sEVs.

• MeSH
• chromatografie kapalinová MeSH
• dvouvláknová RNA MeSH
• extracelulární vezikuly * genetika   MeSH
• RNA-viry * genetika   MeSH
• tandemová hmotnostní spektrometrie MeSH
• Trichomonas vaginalis * genetika   MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Identification of mutants with impairments in auxin biosynthesis and dynamics by forward genetic screening is hindered by the complexity, redundancy and necessity of the pathways involved. Furthermore, although a few auxin-deficient mutants have been recently identified by screening for altered responses to shade, ethylene, N-1-naphthylphthalamic acid (NPA) or cytokinin (CK), there is still a lack of robust markers for systematically isolating such mutants. We hypothesized that a potentially suitable phenotypic marker is root curling induced by CK, as observed in the auxin biosynthesis mutant CK-induced root curling 1 / tryptophan aminotransferase of Arabidopsis 1 (ckrc1/taa1). Phenotypic observations, genetic analyses and biochemical complementation tests of Arabidopsis seedlings displaying the trait in large-scale genetic screens showed that it can facilitate isolation of mutants with perturbations in auxin biosynthesis, transport and signaling. However, unlike transport/signaling mutants, the curled (or wavy) root phenotypes of auxin-deficient mutants were significantly induced by CKs and could be rescued by exogenous auxins. Mutants allelic to several known auxin biosynthesis mutants were re-isolated, but several new classes of auxin-deficient mutants were also isolated. The findings show that CK-induced root curling provides an effective marker for discovering genes involved in auxin biosynthesis or homeostasis.

• MeSH
• Arabidopsis enzymologie   genetika   MeSH
• biologický transport účinky léků   MeSH
• cytokininy metabolismus   MeSH
• fenotyp MeSH
• kořeny rostlin enzymologie   genetika   růst a vývoj   MeSH
• kyseliny indoloctové metabolismus   farmakologie   MeSH
• mutace MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• regulátory růstu rostlin biosyntéza   MeSH
• semenáček účinky léků   růst a vývoj   metabolismus   MeSH
• signální transdukce účinky léků   MeSH
• tryptofantransaminasa genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Bread wheat (Triticum aestivum L.) is a staple food for a significant part of the world's population. The growing demand on its production can be satisfied by improving yield and resistance to biotic and abiotic stress. Knowledge of the genome sequence would aid in discovering genes and QTLs underlying these traits and provide a basis for genomics-assisted breeding. Physical maps and BAC clones associated with them have been valuable resources from which to generate a reference genome of bread wheat and to assist map-based gene cloning. As a part of a joint effort coordinated by the International Wheat Genome Sequencing Consortium, we have constructed a BAC-based physical map of bread wheat chromosome arm 7DS consisting of 895 contigs and covering 94% of its estimated length. By anchoring BAC contigs to one radiation hybrid map and three high resolution genetic maps, we assigned 73% of the assembly to a distinct genomic position. This map integration, interconnecting a total of 1713 markers with ordered and sequenced BAC clones from a minimal tiling path, provides a tool to speed up gene cloning in wheat. The process of physical map assembly included the integration of the 7DS physical map with a whole-genome physical map of Aegilops tauschii and a 7DS Bionano genome map, which together enabled efficient scaffolding of physical-map contigs, even in the non-recombining region of the genetic centromere. Moreover, this approach facilitated a comparison of bread wheat and its ancestor at BAC-contig level and revealed a reconstructed region in the 7DS pericentromere.

• MeSH
• Aegilops genetika   MeSH
• centromera genetika   MeSH
• chromozomy rostlin genetika   MeSH
• fyzikální mapování chromozomů metody   MeSH
• genom rostlinný MeSH
• hybridizace genetická MeSH
• klonování DNA MeSH
• pšenice genetika   MeSH
• rostlinné geny MeSH
• šlechtění rostlin MeSH
• umělé bakteriální chromozomy genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH

BACKGROUND: The large and complex genome of bread wheat (Triticum aestivum L., ~17 Gb) requires high resolution genome maps with saturated marker scaffolds to anchor and orient BAC contigs/ sequence scaffolds for whole genome assembly. Radiation hybrid (RH) mapping has proven to be an excellent tool for the development of such maps for it offers much higher and more uniform marker resolution across the length of the chromosome compared to genetic mapping and does not require marker polymorphism per se, as it is based on presence (retention) vs. absence (deletion) marker assay. METHODS: In this study, a 178 line RH panel was genotyped with SSRs and DArT markers to develop the first high resolution RH maps of the entire D-genome of Ae. tauschii accession AL8/78. To confirm map order accuracy, the AL8/78-RH maps were compared with:1) a DArT consensus genetic map constructed using more than 100 bi-parental populations, 2) a RH map of the D-genome of reference hexaploid wheat 'Chinese Spring', and 3) two SNP-based genetic maps, one with anchored D-genome BAC contigs and another with anchored D-genome sequence scaffolds. Using marker sequences, the RH maps were also anchored with a BAC contig based physical map and draft sequence of the D-genome of Ae. tauschii. RESULTS: A total of 609 markers were mapped to 503 unique positions on the seven D-genome chromosomes, with a total map length of 14,706.7 cR. The average distance between any two marker loci was 29.2 cR which corresponds to 2.1 cM or 9.8 Mb. The average mapping resolution across the D-genome was estimated to be 0.34 Mb (Mb/cR) or 0.07 cM (cM/cR). The RH maps showed almost perfect agreement with several published maps with regard to chromosome assignments of markers. The mean rank correlations between the position of markers on AL8/78 maps and the four published maps, ranged from 0.75 to 0.92, suggesting a good agreement in marker order. With 609 mapped markers, a total of 2481 deletions for the whole D-genome were detected with an average deletion size of 42.0 Mb. A total of 520 markers were anchored to 216 Ae. tauschii sequence scaffolds, 116 of which were not anchored earlier to the D-genome. CONCLUSION: This study reports the development of first high resolution RH maps for the D-genome of Ae. tauschii accession AL8/78, which were then used for the anchoring of unassigned sequence scaffolds. This study demonstrates how RH mapping, which offered high and uniform resolution across the length of the chromosome, can facilitate the complete sequence assembly of the large and complex plant genomes.

• MeSH
• chromozomy rostlin genetika   MeSH
• genom rostlinný * MeSH
• genotyp MeSH
• lipnicovité genetika   MeSH
• mapování chromozomů MeSH
• mapování pomocí radiačních hybridů metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

• MeSH
• karcinom z přechodných buněk chirurgie   MeSH
• lidé MeSH
• nádory močového měchýře * MeSH
• nádory močovodu chirurgie   MeSH
• nefrektomie MeSH
• nefroureterektomie * MeSH
• ureter MeSH
• urologické nádory chirurgie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• dopisy MeSH
• komentáře MeSH

• Publikační typ
• abstrakt z konference MeSH

Resistance to glucocorticoids (GC), the common agents for remission induction in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL), poses a significant therapeutic hurdle. Therefore, dissecting the mechanisms shaping GC resistance could lead to new treatment modalities. Here, we showed that CD9- BCP-ALL cells were preferentially resistant to prednisone and dexamethasone over other standard cytotoxic agents. Concordantly, we identified significantly more poor responders to the prednisone prephase among BCP-ALL patients with a CD9- phenotype, especially for those with adverse presenting features including older age, higher white cell count and BCR-ABL1. Furthermore, gain- and loss-offunction experiments dictated a definitive functional linkage between CD9 expression and GC susceptibility, as demonstrated by the reversal and acquisition of relative GC resistance in CD9low and CD9high BCP-ALL cells, respectively. Despite physical binding to the GC receptor NR3C1, CD9 did not alter its expression, phosphorylation or nuclear translocation but potentiated the induction of GC-responsive genes in GC-resistant cells. Importantly, the MEK inhibitor trametinib exhibited higher synergy with GC against CD9- than CD9+ lymphoblasts to reverse drug resistance in vitro and in vivo. Collectively, our results elucidate a previously unrecognized regulatory function of CD9 in GC sensitivity, and inform new strategies for management of children with resistant BCP-ALL.

• MeSH
• antigeny CD9 * metabolismus   genetika   MeSH
• chemorezistence * genetika   MeSH
• dexamethason farmakologie   MeSH
• dítě MeSH
• glukokortikoidy * farmakologie   terapeutické užití   MeSH
• lidé MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• pre-B-buněčná leukemie * farmakoterapie   metabolismus   genetika   patologie   MeSH
• předškolní dítě MeSH
• receptory glukokortikoidů metabolismus   genetika   MeSH
• zvířata MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Cowpea (Vigna unguiculata [L.] Walp.) is a major crop for worldwide food and nutritional security, especially in sub-Saharan Africa, that is resilient to hot and drought-prone environments. An assembly of the single-haplotype inbred genome of cowpea IT97K-499-35 was developed by exploiting the synergies between single-molecule real-time sequencing, optical and genetic mapping, and an assembly reconciliation algorithm. A total of 519 Mb is included in the assembled sequences. Nearly half of the assembled sequence is composed of repetitive elements, which are enriched within recombination-poor pericentromeric regions. A comparative analysis of these elements suggests that genome size differences between Vigna species are mainly attributable to changes in the amount of Gypsy retrotransposons. Conversely, genes are more abundant in more distal, high-recombination regions of the chromosomes; there appears to be more duplication of genes within the NBS-LRR and the SAUR-like auxin superfamilies compared with other warm-season legumes that have been sequenced. A surprising outcome is the identification of an inversion of 4.2 Mb among landraces and cultivars, which includes a gene that has been associated in other plants with interactions with the parasitic weed Striga gesnerioides. The genome sequence facilitated the identification of a putative syntelog for multiple organ gigantism in legumes. A revised numbering system has been adopted for cowpea chromosomes based on synteny with common bean (Phaseolus vulgaris). An estimate of nuclear genome size of 640.6 Mbp based on cytometry is presented.

• MeSH
• chromozomy rostlin genetika   MeSH
• délka genomu genetika   MeSH
• DNA rostlinná chemie   genetika   MeSH
• fazol genetika   MeSH
• genom rostlinný genetika   MeSH
• mapování chromozomů MeSH
• retroelementy genetika   MeSH
• rostlinné geny genetika   MeSH
• sekvenční analýza DNA metody   MeSH
• syntenie MeSH
• vigna genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH