Recombination of antibody genes in B cells can involve distant genomic loci and contribute a foreign antigen-binding element to form hybrid antibodies with broad reactivity for Plasmodium falciparum. So far, antibodies containing the extracellular domain of the LAIR1 and LILRB1 receptors represent unique examples of cross-chromosomal antibody diversification. Here, we devise a technique to profile non-VDJ elements from distant genes in antibody transcripts. Independent of the preexposure of donors to malaria parasites, non-VDJ inserts were detected in 80% of individuals at frequencies of 1 in 104 to 105 B cells. We detected insertions in heavy, but not in light chain or T cell receptor transcripts. We classify the insertions into four types depending on the insert origin and destination: 1) mitochondrial and 2) nuclear DNA inserts integrated at VDJ junctions; 3) inserts originating from telomere proximal genes; and 4) fragile sites incorporated between J-to-constant junctions. The latter class of inserts was exclusively found in memory and in in vitro activated B cells, while all other classes were already detected in naïve B cells. More than 10% of inserts preserved the reading frame, including transcripts with signs of antigen-driven affinity maturation. Collectively, our study unravels a mechanism of antibody diversification that is layered on the classical V(D)J and switch recombination.
- MeSH
- B-lymfocyty * imunologie MeSH
- CD antigeny imunologie MeSH
- genomika MeSH
- geny pro imunoglobuliny * MeSH
- imunoglobulinový receptor leukocytů B1 imunologie MeSH
- inzerční mutageneze MeSH
- lehké řetězce imunoglobulinů genetika MeSH
- lidé MeSH
- Plasmodium falciparum MeSH
- protilátky protozoální genetika MeSH
- receptory antigenů T-buněk genetika MeSH
- receptory imunologické imunologie MeSH
- rozmanitost protilátek * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND/AIM: Per literature, patients with epidermal growth factor receptor (EGFR) exon-20 insertions respond poorly to tyrosine kinase inhibitors (TKIs). This study analyzed real-world data to examine the prognostic and predictive value of these mutations. PATIENTS AND METHODS: We conducted a retrospective cohort study using Czech TULUNG Registry data, with data on multiple mutation types, collected in 2011-2020. RESULTS: We analyzed 554 (95.85%) patients with EGFR exon-19 deletions or exon-21 L858R substitutions and 24 (4.15%) patients with exon-20 insertions who received first-line high-value therapies. We summarized clinical characteristics and outcomes in all patients and by cohort. The risk of progression was statistically significantly higher (86%) in the exon-20 insertion cohort compared to the cohort with other mutations. Although not statistically significant, the risk of death was 44% higher in patients with exon-20 insertions. CONCLUSION: Advanced NSCLC patients with rare EGFR exon-20 insertions have a high risk of progression.
- MeSH
- časové faktory MeSH
- chemorezistence MeSH
- dospělí MeSH
- erbB receptory genetika MeSH
- exony MeSH
- fenotyp MeSH
- genetická predispozice k nemoci MeSH
- inhibitory proteinkinas terapeutické užití MeSH
- inzerční mutageneze * MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- nádory plic farmakoterapie genetika mortalita patologie MeSH
- nemalobuněčný karcinom plic farmakoterapie genetika mortalita patologie MeSH
- progrese nemoci MeSH
- protinádorové látky terapeutické užití MeSH
- registrace MeSH
- retrospektivní studie MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- výsledek terapie MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
The plant-specific receptor-like cytoplasmic kinases (RLCKs) form a large, poorly characterized family. Members of the RLCK VI_A class of dicots have a unique characteristic: their activity is regulated by Rho-of-plants (ROP) GTPases. The biological function of one of these kinases was investigated using a T-DNA insertion mutant and RNA interference. Loss of RLCK VI_A2 function resulted in restricted cell expansion and seedling growth. Although these phenotypes could be rescued by exogenous gibberellin, the mutant did not exhibit lower levels of active gibberellins nor decreased gibberellin sensitivity. Transcriptome analysis confirmed that gibberellin is not the direct target of the kinase; its absence rather affected the metabolism and signalling of other hormones such as auxin. It is hypothesized that gibberellins and the RLCK VI_A2 kinase act in parallel to regulate cell expansion and plant growth. Gene expression studies also indicated that the kinase might have an overlapping role with the transcription factor circuit (PIF4-BZR1-ARF6) controlling skotomorphogenesis-related hypocotyl/cotyledon elongation. Furthermore, the transcriptomic changes revealed that the loss of RLCK VI_A2 function alters cellular processes that are associated with cell membranes, take place at the cell periphery or in the apoplast, and are related to cellular transport and/or cell wall reorganisation.
- MeSH
- Arabidopsis účinky léků enzymologie genetika růst a vývoj MeSH
- DNA bakterií genetika metabolismus MeSH
- DNA vazebné proteiny genetika metabolismus MeSH
- geneticky modifikované rostliny MeSH
- gibereliny metabolismus farmakologie MeSH
- hypokotyl účinky léků enzymologie genetika růst a vývoj MeSH
- inzerční mutageneze MeSH
- kotyledon účinky léků enzymologie genetika růst a vývoj MeSH
- kyseliny indoloctové metabolismus farmakologie MeSH
- protein-serin-threoninkinasy genetika metabolismus MeSH
- proteiny huseníčku genetika metabolismus MeSH
- regulace genové exprese u rostlin * MeSH
- regulátory růstu rostlin farmakologie MeSH
- semenáček účinky léků enzymologie genetika růst a vývoj MeSH
- stanovení celkové genové exprese MeSH
- transkripční faktory bHLH genetika metabolismus MeSH
- transkripční faktory genetika metabolismus MeSH
- transkriptom MeSH
- vývojová regulace genové exprese MeSH
- Publikační typ
- časopisecké články MeSH
The luteinizing hormone receptor (LHCGR) has a little studied polymorphic 6 bp insertion (rs4539842/insLQ). This study has evaluated the insLQ polymorphism in relation to potential associations with hormonal characteristics of human small antral follicles (hSAFs). In total, 310 hSAFs were collected from 86 women undergoing fertility preservation. Analysis included hormonal profile of 297 follicular fluid (FF) samples and 148 corresponding granulosa cells samples were evaluated by qPCR for selected genes. Significantly reduced and non-detectable mRNA levels of anti-Müllerian hormone receptor II (AMHR2) and LHCGR, respectively, were observed for insLQ/insLQ compared to -/insLQ and the -/- genotypes. Moreover, LHCGR and CYP19a1 together with oestradiol and inhibin-B were significantly increased in -/insLQ compared to the -/- genotype. The homozygous insLQ genotype showed strong significant associations to GC specific genes LHCGR and CYP19a1, which may translate into significant changes in FF hormone profiles and an altered LH signaling.
- MeSH
- dospělí MeSH
- folikulární buňky metabolismus MeSH
- folikulární tekutina metabolismus MeSH
- genotyp MeSH
- hormony metabolismus MeSH
- inzerční mutageneze * MeSH
- lidé MeSH
- messenger RNA genetika metabolismus MeSH
- mladiství MeSH
- mladý dospělý MeSH
- ovariální folikul metabolismus MeSH
- polymorfismus genetický * MeSH
- receptory LH genetika MeSH
- regulace genové exprese * MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
KEY MESSAGE : bZIP TF network in pollen. Transcriptional control of gene expression represents an important mechanism guiding organisms through developmental processes and providing plasticity towards environmental stimuli. Because of their sessile nature, plants require effective gene regulation for rapid response to variation in environmental and developmental conditions. Transcription factors (TFs) provide such control ensuring correct gene expression in spatial and temporal manner. Our work reports the interaction network of six bZIP TFs expressed in Arabidopsis thaliana pollen and highlights the potential functional role for AtbZIP18 in pollen. AtbZIP18 was shown to interact with three other pollen-expressed bZIP TFs-AtbZIP34, AtbZIP52, and AtbZIP61 in yeast two-hybrid assays. AtbZIP18 transcripts are highly expressed in pollen, and at the subcellular level, an AtbZIP18-GFP fusion protein was located in the nucleus and cytoplasm/ER. To address the role of AtbZIP18 in the male gametophyte, we performed phenotypic analysis of a T-DNA knockout allele, which showed slightly reduced transmission through the male gametophyte. Some of the phenotype defects in atbzip18 pollen, although observed at low penetrance, were similar to those seen at higher frequency in the T-DNA knockout of the interacting partner, AtbZIP34. To gain deeper insight into the regulatory role of AtbZIP18, we analysed atbzip18/- pollen microarray data. Our results point towards a potential repressive role for AtbZIP18 and its functional redundancy with AtbZIP34 in pollen.
- MeSH
- Arabidopsis cytologie metabolismus ultrastruktura MeSH
- dimerizace MeSH
- DNA rostlinná MeSH
- inzerční mutageneze MeSH
- proteiny huseníčku metabolismus MeSH
- pyl genetika růst a vývoj metabolismus ultrastruktura MeSH
- regulace genové exprese u rostlin MeSH
- rekombinantní fúzní proteiny metabolismus MeSH
- trans-aktivátory metabolismus MeSH
- transkripční faktory bZIP metabolismus MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
Maintenance of genome integrity via repair of DNA damage is a key biological process required to suppress diseases, including Fanconi anemia (FA). We generated loss-of-function human haploid cells for FA complementation group C (FANCC), a gene encoding a component of the FA core complex, and used genome-wide CRISPR libraries as well as insertional mutagenesis to identify synthetic viable (genetic suppressor) interactions for FA. Here we show that loss of the BLM helicase complex suppresses FANCC phenotypes and we confirm this interaction in cells deficient for FA complementation group I and D2 (FANCI and FANCD2) that function as part of the FA I-D2 complex, indicating that this interaction is not limited to the FA core complex, hence demonstrating that systematic genome-wide screening approaches can be used to reveal genetic viable interactions for DNA repair defects.
- MeSH
- buněčné linie MeSH
- CRISPR-Cas systémy MeSH
- DNA-helikasy genetika MeSH
- Fanconiho anemie genetika MeSH
- haploidie MeSH
- HEK293 buňky MeSH
- helikasy RecQ genetika MeSH
- inzerční mutageneze MeSH
- lidé MeSH
- NAD(P)H dehydrogenasa (chinon) genetika MeSH
- oprava DNA genetika MeSH
- poškození DNA MeSH
- protein FANCC genetika MeSH
- protein FANCD2 genetika MeSH
- proteiny FANC genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Myeloblastosis-associated virus 2 (MAV-2) is a highly tumorigenic simple avian retrovirus. Chickens infected in ovo with MAV-2 develop tumors in the kidneys, lungs, and liver with a short latency, less than 8 weeks. Here we report the results of molecular analyses of MAV-2-induced liver tumors that fall into three classes: hepatic hemangiosarcomas (HHSs), intrahepatic cholangiocarcinomas (ICCs), and hepatocellular carcinomas (HCCs). Comprehensive inverse PCR-based screening of 92 chicken liver tumors revealed that in ca. 86% of these tumors, MAV-2 provirus had integrated into one of four gene loci: HRAS, EGFR, MET, and RON Insertionally mutated genes correlated with tumor type: HRAS was hit in HHSs, MET in ICCs, RON mostly in ICCs, and EGFR mostly in HCCs. The provirus insertions led to the overexpression of the affected genes and, in the case of EGFR and RON, also to the truncation of exons encoding the extracellular ligand-binding domains of these transmembrane receptors. The structures of truncated EGFR and RON closely mimic the structures of oncogenic variants of these genes frequently found in human tumors (EGFRvIII and sfRON).IMPORTANCE These data describe the mechanisms of oncogenesis induced in chickens by the MAV-2 retrovirus. They also show that molecular processes converting cellular regulatory genes to cancer genes may be remarkably similar in chickens and humans. We suggest that the MAV-2 retrovirus-based model can complement experiments performed using mouse models and provide data that could translate to human medicine.
- MeSH
- cholangiokarcinom genetika virologie MeSH
- geny erbB-1 * MeSH
- hemangiosarkom genetika virologie MeSH
- hepatocelulární karcinom genetika virologie MeSH
- integrace viru MeSH
- inzerční mutageneze * MeSH
- karcinogeneze * MeSH
- kur domácí genetika MeSH
- lidé MeSH
- nádory jater genetika virologie MeSH
- onkogeny MeSH
- protoonkogenní proteiny c-met genetika MeSH
- proviry genetika fyziologie MeSH
- ptačí proteiny genetika MeSH
- tyrosinkinasové receptory genetika MeSH
- virus ptačí myeloblastózy genetika fyziologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Na(+)/Ca(2+) exchanger (NCX) proteins operate through the alternating access mechanism, where the ion-binding pocket is exposed in succession either to the extracellular or the intracellular face of the membrane. The archaeal NCX_Mj (Methanococcus jannaschii NCX) system was used to resolve the backbone dynamics in the inward-facing (IF) and outward-facing (OF) states by analyzing purified preparations of apo- and ion-bound forms of NCX_Mj-WT and its mutant, NCX_Mj-5L6-8. First, the exposure of extracellular and cytosolic vestibules to the bulk phase was evaluated as the reactivity of single cysteine mutants to a fluorescent probe, verifying that NCX_Mj-WT and NCX_Mj-5L6-8 preferentially adopt the OF and IF states, respectively. Next, hydrogen-deuterium exchange-mass spectrometry (HDX-MS) was employed to analyze the backbone dynamics profiles in proteins, preferentially adopting the OF (WT) and IF (5L6-8) states either in the presence or absence of ions. Characteristic differences in the backbone dynamics were identified between apo NCX_Mj-WT and NCX_Mj-5L6-8, thereby underscoring specific conformational patterns owned by the OF and IF states. Saturating concentrations of Na(+) or Ca(2+) specifically modify HDX patterns, revealing that the ion-bound/occluded states are much more stable (rigid) in the OF than in the IF state. Conformational differences observed in the ion-occluded OF and IF states can account for diversifying the ion-release dynamics and apparent affinity (Km ) at opposite sides of the membrane, where specific structure-dynamic elements can effectively match the rates of bidirectional ion movements at physiological ion concentrations.
- MeSH
- apoproteiny chemie genetika metabolismus MeSH
- archeální proteiny chemie genetika metabolismus MeSH
- buněčná membrána chemie MeSH
- cystein chemie MeSH
- interakční proteinové domény a motivy MeSH
- inzerční mutageneze MeSH
- kinetika MeSH
- konformace proteinů MeSH
- ligandy MeSH
- Methanocaldococcus metabolismus MeSH
- molekulární modely * MeSH
- mutace MeSH
- peptidové fragmenty chemie genetika metabolismus MeSH
- pumpa pro výměnu sodíku a vápníku chemie genetika metabolismus MeSH
- rekombinantní proteiny chemie metabolismus MeSH
- sodík metabolismus MeSH
- stabilita proteinů MeSH
- substituce aminokyselin MeSH
- vápník metabolismus MeSH
- vazebná místa MeSH
- vodík-deuteriová výměna MeSH
- výpočetní biologie MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
Weak toxin from Naja kaouthia (WTX) belongs to the group of nonconventional "three-finger" snake neurotoxins. It irreversibly inhibits nicotinic acetylcholine receptors and allosterically interacts with muscarinic acetylcholine receptors (mAChRs). Using site-directed mutagenesis, NMR spectroscopy, and computer modeling, we investigated the recombinant mutant WTX analogue (rWTX) which, compared with the native toxin, has an additional N-terminal methionine residue. In comparison with the wild-type toxin, rWTX demonstrated an altered pharmacological profile, decreased binding of orthosteric antagonist N-methylscopolamine to human M1- and M2-mAChRs, and increased antagonist binding to M3-mAChR. Positively charged arginine residues located in the flexible loop II were found to be crucial for rWTX interactions with all types of mAChR. Computer modeling suggested that the rWTX loop II protrudes to the M1-mAChR allosteric ligand-binding site blocking the entrance to the orthosteric site. In contrast, toxin interacts with M3-mAChR by loop II without penetration into the allosteric site. Data obtained provide new structural insight into the target-specific allosteric regulation of mAChRs by "three-finger" snake neurotoxins.
- MeSH
- Elapidae MeSH
- inzerční mutageneze MeSH
- jedy hadů čeledi Elapidae chemie MeSH
- konformace proteinů MeSH
- molekulární sekvence - údaje MeSH
- neurotoxiny chemie genetika metabolismus MeSH
- nukleární magnetická rezonance biomolekulární MeSH
- receptory muskarinové metabolismus MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
