The main aim of this study was to determine expanded sequence types (eSTs) of Ureaplasma species (U. spp.). DNA isolated from the amniotic fluid of pregnancies complicated by preterm prelabor rupture of membranes (PPROM) using an expanded multilocus sequence typing scheme. Additionally, the study sought to examine whether phylogenetic subgroups of U. spp. DNA differ with respect to maternal demographic and clinical parameters and selected aspects of short-term neonatal morbidity. This retrospective cohort study was focused on singleton pregnancies complicated by PPROM occurring between the gestational ages of 24+0 and 36+6 weeks, where amniocentesis was conducted to assess the intra-amniotic environment and the presence of U. spp. DNA in the amniotic fluid samples was confirmed. The stored aliquots of U. spp. DNA were used to assess differences in nucleotide sequences in six U. spp. genes (ftsH, rpL22, valS, thrS,ureG, and mba-np1) using the eMLST scheme. The expanded multilocus sequence typing scheme was performed in 73 samples of U. spp. DNA isolated from pregnancies complicated by PPROM. In total, 33 different U. spp. DNA eSTs were revealed, 21 (#20, 233-244, 248-251, 253, 255, 259, and 262) of which were novel. The most frequently identified eST was #41, identified in 18% (13/73) of the aliquots. Based on their genetic relationships, the U. spp. DNA was divided into two clusters and four subgroups [cluster I (U. parvum): A, 43% (n = 31); B, 15% (n = 11); and C, 26% (n = 19); cluster II (U. urealyticum): 1; 16% (n = 12)]. Cluster II had a higher rate of polymicrobial findings than cluster I (58% vs 16%; p = 0.005), while subgroup A had the highest rate of concomitant Mycoplasma hominis in the amniotic fluid samples (66%; p = 0.04). In conclusion, Ureaplasma spp. DNA obtained from PPROM consisted of 33 different eSTs of U. spp. DNA. No differences in maternal and neonatal characteristics were found among the phylogenetical subgroups of U. spp. DNA, except for a higher rate of polymicrobial amniotic fluid findings in those with U. urealyticumand the concomitant presence of M. hominis in the amniotic fluid in those with the presence of U. parvum.
- MeSH
- DNA bakterií analýza genetika MeSH
- dospělí MeSH
- fylogeneze MeSH
- gestační stáří MeSH
- infekční komplikace v těhotenství mikrobiologie MeSH
- lidé MeSH
- multilokusová sekvenční typizace * MeSH
- plodová voda * mikrobiologie MeSH
- předčasný odtok plodové vody * mikrobiologie MeSH
- retrospektivní studie MeSH
- těhotenství MeSH
- Ureaplasma * genetika izolace a purifikace MeSH
- ureaplasmatické infekce * mikrobiologie MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
The short oligodeoxynucleotide (ODN) probes are suitable for good discrimination of point mutations. However, the probes suffer from low melting temperatures. In this work, the strategy of using acridine-4-carboxamide intercalators to improve thermal stabilisation is investigated. The study of large series of acridines revealed that optimal stabilisation is achieved upon decoration of acridine by secondary carboxamide carrying sterically not demanding basic function bound through a two-carbon linker. Two highly active intercalators were attached to short probes (13 or 18 bases; designed as a part of HFE gene) by click chemistry into positions 7 and/or 13 and proved to increase the melting temperate (Tm) of the duplex by almost 8°C for the best combination. The acridines interact with both single- and double-stranded DNAs with substantially preferred interaction for the latter. The study of interaction suggested higher affinity of the acridines toward the GC- than AT-rich sequences. Good discrimination of two point mutations was shown in practical application with HFE gene (wild type, H63D C > G and S65C A > C mutations). Acridine itself can also serve as a fluorophore and also allows discrimination of the fully matched sequences from those with point mutations in probes labelled only with acridine.
Chronic lymphocytic leukemia is a disease with up-regulated expression of the transmembrane tyrosine-protein kinase ROR1, a member of the Wnt/planar cell polarity pathway. In this study, we identified COBLL1 as a novel interaction partner of ROR1. COBLL1 shows clear bimodal expression with high levels in chronic lymphocytic leukemia patients with mutated IGHV and approximately 30% of chronic lymphocytic leukemia patients with unmutated IGHV. In the remaining 70% of chronic lymphocytic leukemia patients with unmutated IGHV, COBLL1 expression is low. Importantly, chronic lymphocytic leukemia patients with unmutated IGHV and high COBLL1 have an unfavorable disease course with short overall survival and time to second treatment. COBLL1 serves as an independent molecular marker for overall survival in chronic lymphocytic leukemia patients with unmutated IGHV. In addition, chronic lymphocytic leukemia patients with unmutated IGHV and high COBLL1 show impaired motility and chemotaxis towards CCL19 and CXCL12 as well as enhanced B-cell receptor signaling pathway activation demonstrated by increased PLCγ2 and SYK phosphorylation after IgM stimulation. COBLL1 expression also changes during B-cell maturation in non-malignant secondary lymphoid tissue with a higher expression in germinal center B cells than naïve and memory B cells. Our data thus suggest COBLL1 involvement not only in chronic lymphocytic leukemia but also in B-cell development. In summary, we show that expression of COBLL1, encoding novel ROR1-binding partner, defines chronic lymphocytic leukemia subgroups with a distinct response to microenvironmental stimuli, and independently predicts survival of chronic lymphocytic leukemia with unmutated IGHV.
- MeSH
- analýza přežití MeSH
- chronická lymfatická leukemie klasifikace diagnóza genetika mortalita MeSH
- lidé MeSH
- mutace MeSH
- pohyb buněk MeSH
- polarita buněk MeSH
- prognóza MeSH
- signální dráha Wnt MeSH
- sirotčí receptory podobné receptoru tyrosinkinasy metabolismus MeSH
- těžké řetězce imunoglobulinů genetika MeSH
- transkripční faktory metabolismus MeSH
- variabilní oblast imunoglobulinu genetika MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Neutrophil gelatinase-associated lipocalin is an extracellular protein produced mostly in kidney. Recently, it has become a promising biomarker of renal damage in vivo. On the other hand, the validation of NGAL as a biomarker for nephrotoxicity estimation in vitro has not been characterized in detail yet. Since the HK-2 cells are frequently used human kidney cell line, we aimed to characterize the production of NGAL in these cells and to evaluate NGAL as a possible marker of cell impairment. We used heavy metals (mercury, cadmium), peroxide, drugs (acetaminophen, gentamicin) and cisplatin to mimic nephrotoxicity. HK-2 cells were incubated with selected compounds for 1-24h and cell viability was measured together with extracellular NGAL production. We proved that HK-2 cells possess a capacity to produce NGAL in amount of 2pg/ml/h. We found a change in cell viability after 24h incubation with all tested toxic compounds. The largest decrease of the viability was detected in mercury, acetaminophen, cisplatin and gentamicin. Unexpectedly, we found also a significant decrease in NGAL production in HK-2 cells treated with these toxins for 24h: to 11±5%, 54±5%, 57±6% and 76±9% respectively, compared with controls (=100%). Our results were followed with qPCR analysis when we found no significant increase in LCN2 gene expression after 24h incubation. We conclude that extracellular NGAL production negatively correlates with HK-2 cell impairment.
- MeSH
- akutní poškození ledvin chemicky indukované metabolismus MeSH
- biologické markery metabolismus MeSH
- buněčné linie MeSH
- cisplatina toxicita MeSH
- gentamiciny toxicita MeSH
- kadmium toxicita MeSH
- lidé MeSH
- lipokalin-2 genetika metabolismus MeSH
- paracetamol toxicita MeSH
- rtuť toxicita MeSH
- terc-butylhydroperoxid toxicita MeSH
- viabilita buněk účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The clinical course of chronic lymphocytic leukemia (CLL) is highly variable. Patients with unmutated IGHV (U-CLL) usually progress rapidly, whereas patients with mutated IGHV (M-CLL) have a more indolent disease. The expression of several genes correlates closely with the IGHV mutational status and could be used to assess prognosis in CLL. We analyzed the prognostic relevance of COBLL1, LPL, and ZAP70 gene expression, which correlated with IGHV mutational status (p < 0.0001), in 117 CLL patients and established a prognostic parameter dividing the tested cohort according to the disease aggressiveness. Our prognostic parameter was validated on an independent cohort of 161 CLL patients and achieved a high accuracy (94%). Patients divided according to the prognostic parameter differ in overall survival and time to first treatment (p < 0.0001, HR = 2.300/5.970, 95% CI: 1.587-3.450/4.621-15.86). Our approach provides a reliable alternative method to prognosis assessment via IGHV mutational status analysis.
- MeSH
- analýza přežití MeSH
- chronická lymfatická leukemie diagnóza genetika mortalita MeSH
- dospělí MeSH
- exprese genu MeSH
- lidé středního věku MeSH
- lidé MeSH
- lipoproteinlipasa genetika MeSH
- mutace * MeSH
- nádorové biomarkery MeSH
- prognóza MeSH
- progrese nemoci MeSH
- protein-tyrosinkináza ZAP-70 genetika MeSH
- reprodukovatelnost výsledků MeSH
- ROC křivka MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- těžké řetězce imunoglobulinů genetika MeSH
- transkripční faktory genetika MeSH
- variabilní oblast imunoglobulinu genetika MeSH
- Check Tag
- dospělí MeSH
- 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
Skin exposure to ultraviolet (UV) light evokes a complex stress response in keratinocytes. Keratin filament organization provides structural stability and mechanical integrity of keratinocytes. Involucrin is a transglutaminase substrate protein contributing to the formation of insoluble cornified envelopes. However, a more complex role for keratins and involucrin has been proposed, including the regulation of cell stress response. The aim was to evaluate modulations of keratin 1, 10 and involucrin expression in HaCaT in the light of the complex response of these cells to UV-B radiation, including effects on c-Jun and matrix metalloproteinase 1 (MMP-1) gene expression and production of interleukin (IL) 6 and 8. A UV-B (300±5 nm) dose of 10 mJ/cm(2) was selected since this dose resulted in a partial decrease in cell viability in contrast to higher UV-B doses, which induced complete cell death 48 h after treatment. The UV-B radiation induced significant expression of keratin 1 and 10 and decreased expression of involucrin. This was accompanied by increased expression of c-Jun and MMP-1 and IL-6 and IL-8 production. The data suggest that the expression of keratin 1, 10 and involucrin is modulated in HaCaT keratinocytes as a part of the complex stress response to UV radiation.
- Publikační typ
- časopisecké články MeSH
Decades of liver regeneration studies still left the termination phase least elucidated. However regeneration ending mechanisms are clinicaly relevant. We aimed to analyse the timing and transcriptional control of the latest phase of liver regeneration, both controversial. Male Wistar rats were subjected to 2/3 partial hepatectomy with recovery lasting from 1 to 14 days. Time-series microarray data were assessed by innovative combination of hierarchical clustering and principal component analysis and validated by real-time RT-PCR. Hierarchical clustering and principal component analysis in agreement distinguished three temporal phases of liver regeneration. We found 359 genes specifically altered during late phase regeneration. Gene enrichment analysis and manual review of microarray data suggested five pathways worth further study: PPAR signalling pathway; lipid metabolism; complement, coagulation and fibrinolytic cascades; ECM remodelling and xenobiotic biotransformation. Microarray findings pertinent for termination phase were substantiated by real-time RT-PCR. In conclusion, transcriptional profiling mapped late phase of liver regeneration beyond 5th day of recovery and revealed 5 pathways specifically acting at this time. Inclusion of longer post-surgery intervals brought improved coverage of regeneration time dynamics and is advisable for further works. Investigation into the workings of suggested pathways
- MeSH
- genetická transkripce MeSH
- hepatektomie MeSH
- játra * metabolismus MeSH
- krysa rodu rattus MeSH
- metabolismus lipidů MeSH
- modely nemocí na zvířatech MeSH
- nemoci jater * genetika metabolismus veterinární MeSH
- potkani Wistar MeSH
- receptory aktivované proliferátory peroxizomů metabolismus MeSH
- regenerace jater * MeSH
- regulace genové exprese MeSH
- transkriptom * MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
Most high-risk neuroblastomas develop resistance to cytostatics and therefore there is a need to develop new drugs. In previous studies, we found that ellipticine induces apoptosis in human neuroblastoma cells. We also investigated whether ellipticine was able to induce resistance in the UKF-NB-4 neuroblastoma line and concluded that it may be possible after long-term treatment with increasing concentrations of ellipticine. The aim of the present study was to investigate the mechanisms responsible for ellipticine resistance. To elucidate the mechanisms involved, we used the ellipticine-resistant subline UKF-NB-4(ELLI) and performed comparative genomic hybridization, multicolor and interphase FISH, expression microarray, real-time RT-PCR, flow cytometry and western blotting analysis of proteins. On the basis of our results, it appears that ellipticine resistance in neuroblastoma is caused by a combination of overexpression of Bcl-2, efflux or degradation of the drug and downregulation of topoisomerases. Other mechanisms, such as upregulation of enzymes involved in oxidative phosphorylation, cellular respiration, V-ATPases, aerobic respiration or spermine synthetase, as well as reduced growth rate, may also be involved. Some changes are expressed at the DNA level, including gains, amplifications or deletions. The present study demonstrates that resistance to ellipticine is caused by a combination of mechanisms.
- MeSH
- apoptóza účinky léků MeSH
- chemorezistence genetika MeSH
- DNA-topoisomerasy biosyntéza MeSH
- elipticiny metabolismus farmakologie MeSH
- hybridizace in situ fluorescenční MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- neuroblastom farmakoterapie genetika metabolismus patologie MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- protinádorové látky metabolismus farmakologie MeSH
- protoonkogenní proteiny c-bcl-2 metabolismus MeSH
- průtoková cytometrie MeSH
- sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
- srovnávací genomová hybridizace MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
