The merit of RNASeq data relies heavily on correct normalization. However, most methods assume that the majority of transcripts show no differential expression between conditions. This assumption may not always be correct, especially when one condition results in overexpression. We present a new method (NormQ) to normalize the RNASeq library size, using the relative proportion observed from RT-qPCR of selected marker genes. The method was compared against the popular median-of-ratios method, using simulated and real-datasets. NormQ produced more matches to differentially expressed genes in the simulated dataset and more distribution profile matches for both simulated and real datasets.

• Publikační typ
• časopisecké články MeSH

BACKGROUND: Diapause is a developmental alternative to direct ontogeny in many invertebrates. Its primary adaptive meaning is to secure survival over unfavourable seasons in a state of developmental arrest usually accompanied by metabolic suppression and enhanced tolerance to environmental stressors. During photoperiodically triggered diapause of insects, the ontogeny is centrally turned off under hormonal control, the molecular details of this transition being poorly understood. Using RNAseq technology, we characterized transcription profiles associated with photoperiodic diapause induction in the larvae of the drosophilid fly Chymomyza costata with the goal of identifying candidate genes and processes linked to upstream regulatory events that eventually lead to a complex phenotypic change. RESULTS: Short day photoperiod triggering diapause was associated to inhibition of 20-hydroxy ecdysone (20-HE) signalling during the photoperiod-sensitive stage of C. costata larval development. The mRNA levels of several key genes involved in 20-HE biosynthesis, perception, and signalling were significantly downregulated under short days. Hormonal change was translated into downregulation of a series of other transcripts with broad influence on gene expression, protein translation, alternative histone marking by methylation and alternative splicing. These changes probably resulted in blockade of direct development and deep restructuring of metabolic pathways indicated by differential expression of genes involved in cell cycle regulation, metabolism, detoxification, redox balance, protection against oxidative stress, cuticle formation and synthesis of larval storage proteins. This highly complex alteration of gene transcription was expressed already during first extended night, within the first four hours after the change of the photoperiodic signal from long days to short days. We validated our RNAseq differential gene expression results in an independent qRT-PCR experiment involving wild-type (photoperiodic) and NPD-mutant (non-photoperiodic) strains of C. costata. CONCLUSIONS: Our study revealed several strong candidate genes for follow-up functional studies. Candidate genes code for upstream regulators of a complex change of gene expression, which leads to phenotypic switch from direct ontogeny to larval diapause.

• MeSH
• Drosophilidae embryologie   genetika   MeSH
• genetická transkripce * MeSH
• larva genetika   MeSH
• reprodukovatelnost výsledků MeSH
• sekvenční analýza RNA MeSH
• shluková analýza MeSH
• stanovení celkové genové exprese MeSH
• transkriptom MeSH
• vývojová regulace genové exprese MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Ticks salivate while feeding on their hosts. Saliva helps blood feeding through host anti-hemostatic and immunomodulatory components. Previous transcriptomic and proteomic studies revealed the complexity of tick saliva, comprising hundreds of polypeptides grouped in several multi-genic families such as lipocalins, Kunitz-domain containing peptides, metalloproteases, basic tail secreted proteins, and several other families uniquely found in ticks. These studies also revealed that the composition of saliva changes with time; expression of transcripts from the same family wax and wane as a function of feeding time. Here, we examined whether host immune factors could influence sialome switching by comparing sialomes of ticks fed naturally on a rabbit, to ticks artificially fed on defibrinated blood depleted of immune components. Previous studies were based on transcriptomes derived from pools of several individuals. To get an insight into the uniqueness of tick sialomes, we performed transcriptomic analyses of single salivary glands dissected from individual adult female I. ricinus ticks. Multivariate analysis identified 1,279 contigs differentially expressed as a function of time and/or feeding mode. Cluster analysis of these contigs revealed nine clusters of differentially expressed genes, four of which appeared consistently across several replicates, but five clusters were idiosyncratic, pointing to the uniqueness of sialomes in individual ticks. The disclosure of tick quantum sialomes reveals the unique salivary composition produced by individual ticks as they switch their sialomes throughout the blood meal, a possible mechanism of immune evasion.

• MeSH
• klíště genetika   metabolismus   MeSH
• králíci MeSH
• lidé MeSH
• sekvenční analýza RNA MeSH
• slinné žlázy metabolismus   MeSH
• sliny metabolismus   MeSH
• stanovení celkové genové exprese MeSH
• transkriptom * MeSH
• výpočetní biologie MeSH
• vysoce účinné nukleotidové sekvenování metody   MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• Publikační typ
• abstrakt z konference MeSH

In the past decade, single-cell transcriptomics has helped to uncover new cell types and states and led to the construction of a cellular compendium of health and disease. Despite this progress, some difficult-to-sequence cells remain absent from tissue atlases. Eosinophils-elusive granulocytes that are implicated in a plethora of human pathologies1-5-are among these uncharted cell types. The heterogeneity of eosinophils and the gene programs that underpin their pleiotropic functions remain poorly understood. Here we provide a comprehensive single-cell transcriptomic profiling of mouse eosinophils. We identify an active and a basal population of intestinal eosinophils, which differ in their transcriptome, surface proteome and spatial localization. By means of a genome-wide CRISPR inhibition screen and functional assays, we reveal a mechanism by which interleukin-33 (IL-33) and interferon-γ (IFNγ) induce the accumulation of active eosinophils in the inflamed colon. Active eosinophils are endowed with bactericidal and T cell regulatory activity, and express the co-stimulatory molecules CD80 and PD-L1. Notably, active eosinophils are enriched in the lamina propria of a small cohort of patients with inflammatory bowel disease, and are closely associated with CD4+ T cells. Our findings provide insights into the biology of eosinophils and highlight the crucial contribution of this cell type to intestinal homeostasis, immune regulation and host defence. Furthermore, we lay a framework for the characterization of eosinophils in human gastrointestinal diseases.

• MeSH
• analýza genové exprese jednotlivých buněk MeSH
• antigeny CD80 metabolismus   MeSH
• eozinofily * klasifikace   cytologie   imunologie   metabolismus   MeSH
• idiopatické střevní záněty imunologie   MeSH
• imunita * MeSH
• interferon gama MeSH
• interleukin 33 MeSH
• kolitida * imunologie   patologie   MeSH
• lidé MeSH
• myši MeSH
• proteom MeSH
• střeva * imunologie   patologie   MeSH
• T-lymfocyty MeSH
• transkriptom MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

• MeSH
• analýza genové exprese jednotlivých buněk metody   přístrojové vybavení   MeSH
• analýza přežití MeSH
• chimerické antigenní receptory * imunologie   terapeutické užití   MeSH
• doba přežití bez progrese choroby MeSH
• lidé MeSH
• maturační antigen B-buněk imunologie   účinky léků   MeSH
• mnohočetný myelom * terapie   MeSH
• nežádoucí účinky léčiv epidemiologie   MeSH
• syndrom uvolnění cytokinů chemicky indukované   epidemiologie   MeSH
• T-lymfocyty imunologie   MeSH
• výsledek terapie MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• klinická studie MeSH
• práce podpořená grantem MeSH

Tissue inhibitor of metalloprotease 4 (TIMP4) contributes to poor prognosis in breast and other tumours. However, the mechanisms of how TIMP4 influences breast cancer cell behaviour are unknown. Our aim was to explore the signalling pathways modulated by TIMP4 in breast cancer cells. Human recombinant TIMP4 was added to MCF7 breast cancer cells and RNASeq was performed. TIMP4 RNASeq results were validated by RT-PCR. Network analyses of TIMP4-exposed cells showed that ER-α, HIF1A and TGF-β signalling were activated, whereas FOXO3 signalling was downregulated. ER-α protein levels were increased and concordantly, promoters of TIMP4-upregulated genes were significantly enriched in oestrogen-binding sites. We concluded that TIMP4 modulates multiple signalling pathways relevant in cancer in MCF7 cells, including the ER-α cascade.

• MeSH
• alfa receptor estrogenů metabolismus   MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• lidé MeSH
• MFC-7 buňky MeSH
• nádory prsu patofyziologie   MeSH
• regulace genové exprese u nádorů účinky léků   genetika   MeSH
• rekombinantní proteiny farmakologie   MeSH
• signální transdukce * účinky léků   MeSH
• tkáňové inhibitory metaloproteinas genetika   metabolismus   farmakologie   MeSH
• western blotting MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

OBJECTIVE: One of the current hypotheses to explain the proinflammatory immune response in IBD is a dysregulated T cell reaction to yet unknown intestinal antigens. As such, it may be possible to identify disease-associated T cell clonotypes by analysing the peripheral and intestinal T-cell receptor (TCR) repertoire of patients with IBD and controls. DESIGN: We performed bulk TCR repertoire profiling of both the TCR alpha and beta chains using high-throughput sequencing in peripheral blood samples of a total of 244 patients with IBD and healthy controls as well as from matched blood and intestinal tissue of 59 patients with IBD and disease controls. We further characterised specific T cell clonotypes via single-cell RNAseq. RESULTS: We identified a group of clonotypes, characterised by semi-invariant TCR alpha chains, to be significantly enriched in the blood of patients with Crohn's disease (CD) and particularly expanded in the CD8+ T cell population. Single-cell RNAseq data showed an innate-like phenotype of these cells, with a comparable gene expression to unconventional T cells such as mucosal associated invariant T and natural killer T (NKT) cells, but with distinct TCRs. CONCLUSIONS: We identified and characterised a subpopulation of unconventional Crohn-associated invariant T (CAIT) cells. Multiple evidence suggests these cells to be part of the NKT type II population. The potential implications of this population for CD or a subset thereof remain to be elucidated, and the immunophenotype and antigen reactivity of CAIT cells need further investigations in future studies.

• MeSH
• CD8-pozitivní T-lymfocyty MeSH
• Crohnova nemoc * genetika   MeSH
• lidé MeSH
• NKT buňky * MeSH
• receptory antigenů T-buněk alfa-beta genetika   MeSH
• receptory antigenů T-buněk metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Stem rust is an important disease of wheat that can be controlled using resistance genes. The gene SuSr-D1 identified in cultivar 'Canthatch' suppresses stem rust resistance. SuSr-D1 mutants are resistant to several races of stem rust that are virulent on wild-type plants. Here we identify SuSr-D1 by sequencing flow-sorted chromosomes, mutagenesis, and map-based cloning. The gene encodes Med15, a subunit of the Mediator Complex, a conserved protein complex in eukaryotes that regulates expression of protein-coding genes. Nonsense mutations in Med15b.D result in expression of stem rust resistance. Time-course RNAseq analysis show a significant reduction or complete loss of differential gene expression at 24 h post inoculation in med15b.D mutants, suggesting that transcriptional reprogramming at this time point is not required for immunity to stem rust. Suppression is a common phenomenon and this study provides novel insight into suppression of rust resistance in wheat.

• MeSH
• Basidiomycota patogenita   MeSH
• chromozomy rostlin genetika   MeSH
• duplikace genu MeSH
• exprese genu MeSH
• fenotyp MeSH
• imunita rostlin genetika   MeSH
• lipnicovité klasifikace   genetika   MeSH
• mapování chromozomů MeSH
• mediátorový komplex genetika   MeSH
• mutace MeSH
• nemoci rostlin genetika   imunologie   mikrobiologie   MeSH
• odolnost vůči nemocem genetika   MeSH
• pšenice genetika   imunologie   mikrobiologie   MeSH
• regulace genové exprese u rostlin MeSH
• rostlinné geny genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

Between 2002- 2017 we analyzed gDNA of 123 unrelated index patiens with suspect progressive or recurrent intrahepatic cholestasis (PFIC or BRIC). Mutation analysis of ATP8B1, ABCB11, ABCB4 and TJP2 was run in 82 patiens with low GGT cholestasis, ABCB4 was sequenced in 41 patients with high GGT cholestasis. The diagnosis of PFIC1, PFIC2, BRIC, PFIC3 and mild forms of MDR3 deficiency were confirmed in 1, 14, 10, 5 and 8 patients, respectively. The diagnosis was established in 38 (30.8%) index patients. The diagnosis remains unclear in 85 (69.2%) patients. Our goal is to find molecular basis and mechanism of the disease in most (or at least some) of these 85 patients and in several patients expected to come in 2018-2021. To achieve this, we plan to perform whole exome sequencing and, when appropriate, genome sequencing, copy number analysis or RNAseq, in most of these patients and their family members. Expression of mutated proteins will be detected on immunohistology. Pathogenicity of novel mutations will be tested in transfected cultured cells. For expected results see Aims.

V letech 2002-2017 jsme analyzovali genomovou DNA od 123 nepříbuzných probandů se suspektní progresivní či rekurentní intrahepatální cholestázou (PFIC nebo BRIC). Mutační analýza ATP8B1, ABCB11, a TJP2 byla provedena u 82 nemocných s normální aktivitou GGT. ABCB4 byl sekvenován u 41 nemocných se zvýšenou aktivitou GGT. U 38 (30,8%) probandů se podařilo potvrdit dg PFIC1 (n=1), PFIC2 (n=14), BRIC (n=10), PFIC3 (n=5) a mírných forem deficitu MDR3 (n=8). U 85 (69,2%) nemocných je dg nejasná. Naším cílem je zjistit molekulární příčinu a mechanizmus choroby u většiny či alespoň u některých z těchto 85 pacientů. K jeho dosažení plánujeme provést sekvenování exomu a v odůvodněných případech i sekvenování genomu, analýzu počtu kopií či RNAseq u většiny z těchto 85 pacientů a jejich rodinných příslušníků. Exprese mutovaných proteinů bude studována imunohistologicky. Patogenita nových mutací bude testována na úrovni mRNA a proteinu na transfekovaných buněčných liniích. Očekávané výsledky viz Specifické cíle v sekci Cíle projektu.

• Klíčová slova
• intrahepatální cholestáza, žluč, sekvenování exomu, intrahepaticcholestasis, bile, whole exome sequencing,

• NLK Publikační typ
• závěrečné zprávy o řešení grantu AZV MZ ČR