Anesthesia is regarded as an important milestone in medicine. However, the negative effect on memory and learning has been observed. In addition, the impact of anesthetics on postoperative cognitive functions is still discussed. In this work, in vivo experiment simulating a general anesthesia and ICU sedation was designed to assess the impact of two intravenous (midazolam, dexmedetomidine) and two inhalational (isoflurane, desflurane) agents on neuronal centers for cognition (neocortex), learning, and memory (hippocampus). More than 3600 proteins were quantified across both neocortex and hippocampus. Proteomic study revealed relatively mild effects of anesthetics, nevertheless, protein dysregulation uncovered possible different effect of isoflurane (and midazolam) compared to desflurane (and dexmedetomidine) to neocortical and hippocampal proteins. Isoflurane induced the upregulation of hippocampal NMDAR and other proteins of postsynaptic density and downregulation of GABA signaling, whereas desflurane and dexmedetomidine rather targeted mitochondrial VDAC isoforms and protein regulating apoptotic activity.
- Publikační typ
- časopisecké články MeSH
The formation of hematopoietic cells relies on the chromatin remodeling activities of ISWI ATPase SMARCA5 (SNF2H) and its complexes. The Smarca5 null and conditional alleles have been used to study its functions in embryonic and organ development in mice. These mouse model phenotypes vary from embryonic lethality of constitutive knockout to less severe phenotypes observed in tissue-specific Smarca5 deletions, e.g., in the hematopoietic system. Here we show that, in a gene dosage-dependent manner, the hypomorphic allele of SMARCA5 (S5tg) can rescue not only the developmental arrest in hematopoiesis in the hCD2iCre model but also the lethal phenotypes associated with constitutive Smarca5 deletion or Vav1iCre-driven conditional knockout in hematopoietic progenitor cells. Interestingly, the latter model also provided evidence for the role of SMARCA5 expression level in hematopoietic stem cells, as the Vav1iCre S5tg animals accumulate stem and progenitor cells. Furthermore, their hematopoietic stem cells exhibited impaired lymphoid lineage entry and differentiation. This observation contrasts with the myeloid lineage which is developing without significant disturbances. Our findings indicate that animals with low expression of SMARCA5 exhibit normal embryonic development with altered lymphoid entry within the hematopoietic stem cell compartment.
- MeSH
- adenosintrifosfatasy metabolismus MeSH
- buněčná diferenciace genetika MeSH
- hematopoetické kmenové buňky * metabolismus MeSH
- hematopoéza * genetika MeSH
- myši MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Stable isotope labeling by amino acids in cell culture (SILAC) and iodoacetyl tandem mass tag (iodoTMT) are well-implemented mass spectrometry-based approaches for quantification of proteins and for site-mapping of cysteine modification. We describe here a combination of SILAC and iodoTMT to assess ongoing changes in the global proteome and cysteine modification levels using liquid chromatography separation coupled with high-resolution mass spectrometry (LC-MS/MS).
The mechanisms by which myelodysplastic syndrome (MDS) cells resist the effects of hypomethylating agents (HMA) are currently the subject of intensive research. A better understanding of mechanisms by which the MDS cell becomes to tolerate HMA and progresses to acute myeloid leukemia (AML) requires the development of new cellular models. From MDS/AML cell lines we developed a model of 5-azacytidine (AZA) resistance whose stability was validated by a transplantation approach into immunocompromised mice. When investigating mRNA expression and DNA variants of the AZA resistant phenotype we observed deregulation of several cancer-related pathways including the phosphatidylinosito-3 kinase signaling. We have further shown that these pathways can be modulated by specific inhibitors that, while blocking the proliferation of AZA resistant cells, are unable to increase their sensitivity to AZA. Our data reveal a set of molecular mechanisms that can be targeted to expand therapeutic options during progression on AZA therapy.
- MeSH
- anotace sekvence MeSH
- azacytidin farmakologie MeSH
- biologické modely * MeSH
- chemorezistence * účinky léků genetika MeSH
- DNA nádorová genetika MeSH
- fosfatidylinositol-3-kinasy metabolismus MeSH
- myši SCID MeSH
- myši MeSH
- protoonkogenní proteiny c-akt metabolismus MeSH
- reprodukovatelnost výsledků MeSH
- signální transdukce účinky léků MeSH
- transkriptom genetika MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Preterm prelabour rupture of membranes beyond the 34th week of gestation (late PPROM) is frequently associated with the risk of the microbial invasion of the amniotic fluid (MIAC) and histological chorioamnionitis (HCA). Hence, we employed a Tandem Mass Tag-based approach to uncover amniotic fluid proteome response to the presence of MIAC and HCA in late PPROM. Protein dysregulation was associated with only five cases in the group of 15 women with confirmed MIAC and HCA. Altogether, 138 amniotic fluid proteins were changed in these five cases exclusively. These proteins were particularly associated with excessive neutrophil responses to infection, such as neutrophil degranulation and extracellular trap formation. We believe that the quantification of these proteins in amniotic fluid may assist in revealing women with the highest risk of excessive inflammatory response in late PPROM.
- MeSH
- chorioamnionitida metabolismus mikrobiologie MeSH
- dospělí MeSH
- infekční komplikace v těhotenství metabolismus MeSH
- kohortové studie MeSH
- lidé MeSH
- novorozenec MeSH
- předčasný odtok plodové vody metabolismus MeSH
- proteomika metody MeSH
- těhotenství MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- novorozenec MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Myelodysplastic syndromes (MDS) are a heterogeneous group of hematological malignancies with a high risk of transformation to acute myeloid leukemia (AML). MDS are associated with posttranslational modifications of proteins and variations in the protein expression levels. In this work, we present a novel interactomic diagnostic method based on both protein array and surface plasmon resonance biosensor technology, which enables monitoring of protein-protein interactions in a label-free manner. In contrast to conventional methods based on the detection of individual biomarkers, our presented method relies on measuring interactions between arrays of selected proteins and patient plasma. We apply this method to plasma samples obtained from MDS and AML patients, as well as healthy donors, and demonstrate that even a small protein array comprising six selected proteins allows the method to discriminate among different MDS subtypes and healthy donors.
- MeSH
- analýza hlavních komponent MeSH
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mapování interakce mezi proteiny * MeSH
- mladý dospělý MeSH
- myelodysplastické syndromy krev diagnóza MeSH
- povrchová plasmonová rezonance MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- vazba proteinů 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
- práce podpořená grantem MeSH
Under normal conditions, the cellular redox status is maintained in a steady state by reduction and oxidation processes. These redox alterations in the cell are mainly sensed by protein thiol residues of cysteines thus regulating protein function. The imbalance in redox homeostasis may therefore regulate protein turnover either directly by redox modulating of transcription factors or indirectly by the degradation of damaged proteins due to oxidation. A new analytical method capable of simultaneously assessing cellular protein expression and cysteine oxidation would provide a valuable tool for the field of cysteine-targeted biology. Here, we show a workflow based on protein quantification using metabolic labeling and determination of cysteine oxidation using reporter ion quantification. We applied this approach to determine protein and redox changes in cells after 5-min, 60-min and 32-h exposure to H2O2, respectively. Based on the functional analysis of our data, we confirmed a biological relevance of this approach and its applicability for parallel mapping of cellular proteomes and redoxomes under diverse conditions. In addition, we revealed a specific pattern of redox changes in peroxiredoxins in a short time-interval cell exposure to H2O2. Overall, our present study offers an innovative, versatile experimental approach to the multifaceted assessment of cellular proteome and its redox status, with broad implications for biomedical research towards a better understanding of organismal physiology and diverse disease conditions.
- MeSH
- chromatografie kapalinová MeSH
- cystein metabolismus MeSH
- oxidace-redukce * MeSH
- oxidační stres MeSH
- peroxid vodíku metabolismus MeSH
- peroxiredoxiny metabolismus MeSH
- proteom * MeSH
- proteomika * metody MeSH
- tandemová hmotnostní spektrometrie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Myelodysplastic syndromes (MDS) represent a heterogeneous group of pre-leukemic disorders, characterized by ineffective hematopoiesis and the abnormal blood cell development of one or more lineages. Oxidative stress, as an important factor in the carcinogenesis of onco-hematological diseases, is also one of the known factors involved in the pathogenesis of MDS. An increase of reactive oxygen species (ROS) may lead to the oxidation of DNA, lipids, and proteins, thereby causing cell damage. Protein carbonylation caused by ROS is defined as an irreversible post-translational oxidative modification of amino acid side chains, and could play an important role in signaling processes. The detection of protein carbonyl groups is a specific useful marker of oxidative stress. In this study, we examined 32 patients divided into three different subtypes of MDS according to the World Health Organization (WHO) classification criteria as refractory anemia with ringed sideroblasts (RARS), refractory cytopenia with multilineage dysplasia (RCMD), refractory anemia with excess blasts-1,2 (RAEB-1,2). We found significant differences in protein carbonylation between the group of all MDS patients and healthy controls (P=0.0078). Furthermore, carbonylated protein levels were significantly elevated in RARS patients compared to healthy donors (P=0.0013) and to RCMD patients (P=0.0277). We also found a significant difference in the total iron binding capacity (TIBC) between individual subgroups of MDS patients (P=0.0263). Moreover, TIBC was decreased in RARS patients compared to RCMD patients (P=0.0203). TIBC moderately negatively correlated with carbonyl levels (r=-0.5978, P=0.0054) in the MDS patients as a whole. Additionally we observed changes in the carbonylated proteins of RARS patients in comparison with healthy controls and their negative controls. Using tandem mass spectrometry (LC-MS/MS) we identified 27 uniquely carbonylated proteins of RARS patients, which were generated by ROS and could influence the pathophysiology of low-risk MDS. These data indicate that increased protein carbonylation is related with RARS as low-risk MDS subgroup. We suggest that this type of post-translational modification in MDS disease is not "only" a consequence of oxidative stress, but also plays an active role in the pathophysiology and iron metabolism within the RARS subgroup of MDS. Measurement of plasma carbonyl levels and the isolation of carbonylated plasma proteins, followed by their identification, could serve as a potential diagnostic and prognostic tool in MDS.
- MeSH
- dospělí MeSH
- karbonylace proteinů MeSH
- krevní proteiny metabolismus MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- myelodysplastické syndromy diagnóza metabolismus MeSH
- oxidační stres MeSH
- prognóza MeSH
- reaktivní formy kyslíku metabolismus MeSH
- refrakterní anemie MeSH
- senioři MeSH
- tandemová hmotnostní spektrometrie MeSH
- vazba proteinů MeSH
- železo metabolismus MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
We report an ultra-low fouling surface plasmon resonance imaging (SPRi) biosensor for the rapid simultaneous detection of multiple miRNAs in erythrocyte lysate (EL) at subpicomolar levels without need of RNA extraction. The SPRi chips were coated with ultra-low fouling functionalizable poly(carboxybetaine acrylamide) (pCBAA) brushes having optimized thicknesses and directly functionalized with amino-modified oligonucleotide probes. We have characterized the effect of the brush thickness on the probe loading capacity: a loading capacity of ~9.8×10(12) probes/cm(2) was achieved for pCBAA having a thickness of ~40 nm. The probe-functionalized sensor also exhibited a high resistance to fouling from ~90% EL samples (<2 ng/cm(2)). A two-step detection assay was employed for multiplexed miRNA detection in EL. Specifically, the assay consisted of (i) a sandwich-type hybridization of the probe-functionalized pCBAA with target miRNA in EL (bound to biotinylated oligonucleotides) and (ii) the capture of streptavidin-functionalized gold nanoparticles to the aforementioned biotinylated probes. We have demonstrated that this approach enables the detection of miRNAs in EL at concentrations as low as 0.5 pM. Finally, we have confirmed the detection of four endogenous miRNAs representing a set of potential miRNA biomarkers of myelodysplastic syndrome (MDS) in clinical EL samples (miR-16, miR-181, miR-34a, and miR-125b). The results revealed significantly higher levels of miR-16 in all the clinical EL samples compared to the other measured miRNAs.
- MeSH
- akrylamidy chemie MeSH
- analýza selhání vybavení MeSH
- biokompatibilní potahované materiály chemická syntéza MeSH
- biosenzitivní techniky přístrojové vybavení MeSH
- design vybavení MeSH
- frakcionace buněk MeSH
- komplexní směsi analýza MeSH
- mikro RNA analýza chemie genetika MeSH
- polymery chemie MeSH
- povrchová plasmonová rezonance přístrojové vybavení MeSH
- reprodukovatelnost výsledků MeSH
- senzitivita a specificita MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
