Pelomyxa is a genus of anaerobic amoebae that live in consortia with multiple prokaryotic endosymbionts. Although the symbionts represent a large fraction of the cellular biomass, their metabolic roles have not been investigated. Using single-cell genomics and transcriptomics, we have characterized the prokaryotic community associated with P. schiedti, which is composed of two bacteria, Candidatus Syntrophus pelomyxae (class Deltaproteobacteria) and Candidatus Vesiculincola pelomyxae (class Clostridia), and a methanogen, Candidatus Methanoregula pelomyxae. Fluorescence in situ hybridization and electron microscopy showed that Ca. Vesiculincola pelomyxae is localized inside vesicles, whereas the other endosymbionts occur freely in the cytosol, with Ca. Methanoregula pelomyxae enriched around the nucleus. Genome and transcriptome-based reconstructions of the metabolism suggests that the cellulolytic activity of P. schiedti produces simple sugars that fuel its own metabolism and the metabolism of a Ca. Vesiculincola pelomyxae, while Ca. Syntrophus pelomyxae energy metabolism relies on degradation of butyrate and isovalerate from the environment. Both species of bacteria and the ameba use hydrogenases to transfer the electrons from reduced equivalents to hydrogen, a process that requires a low hydrogen partial pressure. This is achieved by the third endosymbiont, Ca. Methanoregula pelomyxae, which consumes H2 and formate for methanogenesis. While the bacterial symbionts can be successfully eliminated by vancomycin treatment without affecting the viability of the amoebae, treatment with 2-bromoethanesulfonate, a specific inhibitor of methanogenesis, killed the amoebae, indicating the essentiality of the methanogenesis for this consortium.
- MeSH
- Amoeba * MeSH
- anaerobióza MeSH
- Bacteria genetika MeSH
- hybridizace in situ fluorescenční MeSH
- methan metabolismus MeSH
- vodík metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- methan MeSH
- vodík MeSH
These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.
Association studies have linked microbiome alterations with many human diseases. However, they have not always reported consistent results, thereby necessitating cross-study comparisons. Here, a meta-analysis of eight geographically and technically diverse fecal shotgun metagenomic studies of colorectal cancer (CRC, n = 768), which was controlled for several confounders, identified a core set of 29 species significantly enriched in CRC metagenomes (false discovery rate (FDR) < 1 × 10-5). CRC signatures derived from single studies maintained their accuracy in other studies. By training on multiple studies, we improved detection accuracy and disease specificity for CRC. Functional analysis of CRC metagenomes revealed enriched protein and mucin catabolism genes and depleted carbohydrate degradation genes. Moreover, we inferred elevated production of secondary bile acids from CRC metagenomes, suggesting a metabolic link between cancer-associated gut microbes and a fat- and meat-rich diet. Through extensive validations, this meta-analysis firmly establishes globally generalizable, predictive taxonomic and functional microbiome CRC signatures as a basis for future diagnostics.
- MeSH
- adenom genetika mikrobiologie MeSH
- biologické modely MeSH
- databáze genetické MeSH
- druhová specificita MeSH
- feces mikrobiologie MeSH
- kohortové studie MeSH
- kolorektální nádory genetika mikrobiologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- metagenom * MeSH
- nádorové biomarkery metabolismus MeSH
- reprodukovatelnost výsledků MeSH
- senioři MeSH
- střevní mikroflóra genetika MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- metaanalýza MeSH
- Research Support, N.I.H., Extramural MeSH
- Názvy látek
- nádorové biomarkery MeSH
Here we describe a C-SWAT library for high-throughput tagging of Saccharomyces cerevisiae open reading frames (ORFs). In 5,661 strains, we inserted an acceptor module after each ORF that can be efficiently replaced with tags or regulatory elements. We validated the library with targeted sequencing and tagged the proteome with bright fluorescent proteins to quantify the effect of heterologous transcription terminators on protein expression and to localize previously undetected proteins.
- MeSH
- DNA fungální genetika MeSH
- genom fungální * MeSH
- genomová knihovna * MeSH
- místa se sekvenční adresou MeSH
- otevřené čtecí rámce MeSH
- proteom genetika MeSH
- proteomika MeSH
- Saccharomyces cerevisiae - proteiny genetika MeSH
- Saccharomyces cerevisiae genetika MeSH
- sekvenční analýza DNA MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- validační studie MeSH
- Názvy látek
- DNA fungální MeSH
- proteom MeSH
- Saccharomyces cerevisiae - proteiny MeSH
T-cell prolymphocytic leukemia (T-PLL) is a rare and aggressive neoplasm of mature T-cells with an urgent need for rationally designed therapies to address its notoriously chemo-refractory behavior. The median survival of T-PLL patients is <2 years and clinical trials are difficult to execute. Here we systematically explored the diversity of drug responses in T-PLL patient samples using an ex vivo drug sensitivity and resistance testing platform and correlated the findings with somatic mutations and gene expression profiles. Intriguingly, all T-PLL samples were sensitive to the cyclin-dependent kinase inhibitor SNS-032, which overcame stromal-cell-mediated protection and elicited robust p53-activation and apoptosis. Across all patients, the most effective classes of compounds were histone deacetylase, phosphoinositide-3 kinase/AKT/mammalian target of rapamycin, heat-shock protein 90 and BH3-family protein inhibitors as well as p53 activators, indicating previously unexplored, novel targeted approaches for treating T-PLL. Although Janus-activated kinase-signal transducer and activator of transcription factor (JAK-STAT) pathway mutations were common in T-PLL (71% of patients), JAK-STAT inhibitor responses were not directly linked to those or other T-PLL-specific lesions. Overall, we found that genetic markers do not readily translate into novel effective therapeutic vulnerabilities. In conclusion, novel classes of compounds with high efficacy in T-PLL were discovered with the comprehensive ex vivo drug screening platform warranting further studies of synergisms and clinical testing.
- MeSH
- buněčný cyklus genetika MeSH
- chemorezistence * MeSH
- chromozomální aberace MeSH
- cílená molekulární terapie MeSH
- exprese genu MeSH
- fenotyp MeSH
- inhibitory proteinkinas farmakologie MeSH
- Janus kinasy metabolismus MeSH
- lidé středního věku MeSH
- lidé MeSH
- mutace * MeSH
- nádorové biomarkery * MeSH
- nádorové buněčné linie MeSH
- oxazoly farmakologie MeSH
- protinádorové látky farmakologie terapeutické užití MeSH
- rychlé screeningové testy * MeSH
- screeningové testy protinádorových léčiv * MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- stanovení celkové genové exprese MeSH
- T-buněčná prolymfocytární leukemie farmakoterapie genetika metabolismus MeSH
- thiazoly farmakologie MeSH
- transkripční faktory STAT metabolismus MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- inhibitory proteinkinas MeSH
- Janus kinasy MeSH
- N-(5-(((5-(1,1-dimethylethyl)-2-oxazolyl)methyl)thio)-2-thiazolyl)-4-piperidinecarboxamide MeSH Prohlížeč
- nádorové biomarkery * MeSH
- oxazoly MeSH
- protinádorové látky MeSH
- thiazoly MeSH
- transkripční faktory STAT MeSH
Allogeneic stem cell transplantation (alloSCT) is used for treating patients with T-prolymphocytic leukemia (T-PLL). However, direct evidence of GvL activity in T-PLL is lacking. We correlated minimal residual disease (MRD) kinetics with immune interventions and T-cell receptor (TCR) repertoire diversity alterations in patients after alloSCT for T-PLL. Longitudinal quantitative MRD monitoring was performed by clone-specific real-time PCR of TCR rearrangements (n=7), and TCR repertoire diversity assessment by next-generation sequencing (NGS; n=3) Although post-transplant immunomodulation (immunosuppression tapering or donor lymphocyte infusions) resulted in significant reduction (>1 log) of MRD levels in 7 of 10 occasions, durable MRD clearance was observed in only two patients. In all three patients analyzed by TCR-NGS, MRD responses were reproducibly associated with a shift from a clonal, T-PLL-driven profile to a polyclonal signature. Novel clonotypes that could explain a clonal GvL effect did not emerge. In conclusion, TCR-based MRD quantification appears to be a suitable tool for monitoring and guiding treatment interventions in T-PLL. The MRD responses to immune modulation observed here provide first molecular evidence for GvL activity in T-PLL which, however, may be often only transient and reliant on a poly-/oligoclonal rather than a monoclonal T-cell response.
- MeSH
- buněčné klony imunologie MeSH
- dospělí MeSH
- genová přestavba T-lymfocytů genetika MeSH
- homologní transplantace MeSH
- imunomodulace * MeSH
- kinetika MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- lidé středního věku MeSH
- lidé MeSH
- reakce štěpu proti leukémii * MeSH
- receptory antigenů T-buněk analýza genetika MeSH
- reziduální nádor diagnóza genetika MeSH
- senioři MeSH
- T-buněčná prolymfocytární leukemie diagnóza terapie MeSH
- transplantace kmenových buněk metody MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- senioři MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- receptory antigenů T-buněk MeSH
