Testate Amoebae Examined by Confocal and Two-Photon Microscopy: Implications for Taxonomy and Ecophysiology

Testate amoebae (TA) are a group of free-living protozoa, important in ecology and paleoecology. Testate amoebae taxonomy is mainly based on the morphological features of the shell, as examined by means of light microscopy or (environmental) scanning electron microscopy (SEM/ESEM). We explored the potential applications of confocal laser scanning microscopy (CLSM), two photon excitation microscopy (TPEM), phase contrast, differential interference contrast (DIC Nomarski), and polarization microscopy to visualize TA shells and inner structures of living cells, which is not possible by SEM or environmental SEM. Images captured by CLSM and TPEM were utilized to create three-dimensional (3D) visualizations and to evaluate biovolume inside the shell by stereological methods, to assess the function of TA in ecosystems. This approach broadens the understanding of TA cell and shell morphology, and inner structures including organelles and endosymbionts, with potential implications in taxonomy and ecophysiolog

Use of germline genetic variability for prediction of chemoresistance and prognosis of breast cancer patients

The aim of our study was to set up a panel for targeted sequencing of chemoresistance genes and the main transcription factors driving their expression and to evaluate their predictive and prognostic value in breast cancer patients. Coding and regulatory regions of 509 genes, selected from PharmGKB and Phenopedia, were sequenced using massive parallel sequencing in blood DNA from 105 breast cancer patients in the testing phase. In total, 18,245 variants were identified of which 2565 were novel variants (without rs number in dbSNP build 150) in the testing phase. Variants with major allele frequency over 0.05 were further prioritized for validation phase based on a newly developed decision tree. Using emerging in silico tools and pharmacogenomic databases for functional predictions and associations with response to cytotoxic therapy or disease-free survival of patients, 55 putative variants were identified and used for validation in 805 patients with clinical follow up using KASP™ technology. In conclusion, associations of rs2227291, rs2293194, and rs4376673 (located in ATP7A, KCNAB1, and DFFB genes, respectively) with response to neoadjuvant cytotoxic therapy and rs1801160 in DPYD with disease-free survival of patients treated with cytotoxic drugs were validated and should be further functionally characterized. © 2018 by the authors. Licensee MDPI, Basel, Switzerland.Czech Medical Council [15-25618A]; Charles University [GAUK 1776218

Role of genetic variation in cytochromes P450 in breast cancer prognosis and therapy response

Breast cancer is the most frequent cancer in the female population worldwide. The role of germline genetic variability in cytochromes P450 (CYP) in breast cancer prognosis and individualized therapy awaits detailed elucidation. In the present study, we used the next-generation sequencing to assess associations of germline variants in the coding and regulatory sequences of all human CYP genes with response of the patients to the neoadjuvant cytotoxic chemotherapy and disease-free survival (n = 105). A total of 22 prioritized variants associating with a response or survival in the above evaluation phase were then analyzed by allelic discrimination in the large confirmation set (n = 802). Associations of variants in CYP1B1, CYP4F12, CYP4X1, and TBXAS1 with the response to the neoadjuvant cytotoxic chemotherapy were replicated by the confirmation phase. However, just association of variant rs17102977 in CYP4X1 passed the correction for multiple testing and can be considered clinically and statistically validated. Replicated associations for variants in CYP4X1, CYP24A1, and CYP26B1 with disease-free survival of all patients or patients stratified to subgroups according to therapy type have not passed a false discovery rate test. Although statistically not confirmed by the present study, the role of CYP genes in breast cancer prognosis should not be ruled out. In conclusion, the present study brings replicated association of variant rs17102977 in CYP4X1 with the response of patients to the neoadjuvant cytotoxic chemotherapy and warrants further research of genetic variation CYPs in breast cancer.Czech Medical Council [NV19-08-00113]; Czech Ministry of Education, Youth and Sports, INTER-EXCELLENCE LTA-USA grant [19032]; Charles University Research Fund [Q39]NV19-08-00113; Univerzita Karlova v Praze, UK; Ministerstvo Školství, Mládeže a Tělovýchovy, MŠMT: 1903

Effect of cholesterol depletion on signalling cascade initiated with receptors coupled to G protein class Gq/G11

Membrane domains are an important structure in plasamatic membrane. They concentrate various signaling molecules. Their main structural component is cholesterol and by its removal the membrane domains are disrupted. The aim of our work was to examine the effect of cholesterol depeletion on signaling initiated thyreothropin releasing hormone (TRH). Although its signaling cascade is located within membrane domains the receptor itself is not. We showed that cholesterol depletion by -cyclodextrin caused release of Gq/11 proteins and caveolin 2 from membrane domains. We also discovered that cholesterol depletion decreases potency of TRH to activate G proteins as well as induction of release of intracellular Ca2+ In the last part we investigated the effect of disruption of the cell membrane integrity by cholesterol depletion on thyrotropin-releasing hormone receptor (TRH-R) surface mobility and internalization in HEK293 cells stably expressing TRH-R-eGFP fusion protein. CLSM studies indicated that the internalization of receptor molecules initiated by TRH stimulation was significantly attenuated. The detailed analysis of recovery of TRH-R-eGFP fluorescence in bleached spots of different sizes indicated that cholesterol depletion results in an increase of overall receptor mobility. We suggest that migration of..

Effect of cholesterol depletion on signalling cascade initiated with receptors coupled to G protein class Gq/G11

Membrane domains are an important structure in plasamatic membrane. They concentrate various signaling molecules. Their main structural component is cholesterol and by its removal the membrane domains are disrupted. The aim of our work was to examine the effect of cholesterol depeletion on signaling initiated thyreothropin releasing hormone (TRH). Although its signaling cascade is located within membrane domains the receptor itself is not. We showed that cholesterol depletion by -cyclodextrin caused release of Gq/11 proteins and caveolin 2 from membrane domains. We also discovered that cholesterol depletion decreases potency of TRH to activate G proteins as well as induction of release of intracellular Ca2+ In the last part we investigated the effect of disruption of the cell membrane integrity by cholesterol depletion on thyrotropin-releasing hormone receptor (TRH-R) surface mobility and internalization in HEK293 cells stably expressing TRH-R-eGFP fusion protein. CLSM studies indicated that the internalization of receptor molecules initiated by TRH stimulation was significantly attenuated. The detailed analysis of recovery of TRH-R-eGFP fluorescence in bleached spots of different sizes indicated that cholesterol depletion results in an increase of overall receptor mobility. We suggest that migration of...Membránové domény jsou důležitou strukturou v plazmatické membráně. Koncentrují se v nich různé signální molekuly. Jejich hlavní strukturní složkou je cholesterol a jeho odstraněním dochází k jejich narušení. Předkládaná práce se zabývá vlivem, jaký má deplece cholesterolu na signalizaci iniciovanou thyreotropním hormonem (TRH), jehož signální kaskáda se pravděpodobně nachází v membránových doménách, ač on sám není jejich součástí. S pomocí látky -cyclodextrin jsme depletovali cholesterol z plazmatických membrán. Ukázali jsme, že deplece cholesterolu vede k uvolňování Gq/11 proteinů a kaveolinu 2 z membránových domén. Také jsme ukázali, že tato deplece vede ke snížení schopnosti TRH aktivovat trimerní G proteiny stejně jako indukovat výlev Ca2+ z intracelulárních zásob. V poslední části jsme prozkoumali efekt deplece cholesterolu na mobilitu TRH receptoru v plazmatické membráně a na jeho internalizaci indukovanou TRH. K tomuto účelu byla použita linie buněk HEK 293 stabilně exprimující fuzní protein TRH-R-eGFP. Studie s pomocí konfokálního mikroskopu ukázaly, že internalizace receptoru je výrazně potlačena. Detailní analýza návratu fluorescence TRH-R-eGFP do vybělených oblastí o různých velikostech ukázala, že deplece cholesterolu zvyšuje mobilitu receptoru. Jsme přesvědčeni, že návrat fluorescence probíhá...Department of PhysiologyKatedra fyziologieFaculty of SciencePřírodovědecká fakult

Effect of cholesterol depletion on signalling cascade initiated with receptors coupled to G protein class Gq/G11

Membrane domains are an important structure in plasamatic membrane. They concentrate various signaling molecules. Their main structural component is cholesterol and by its removal the membrane domains are disrupted. The aim of our work was to examine the effect of cholesterol depeletion on signaling initiated thyreothropin releasing hormone (TRH). Although its signaling cascade is located within membrane domains the receptor itself is not. We showed that cholesterol depletion by -cyclodextrin caused release of Gq/11 proteins and caveolin 2 from membrane domains. We also discovered that cholesterol depletion decreases potency of TRH to activate G proteins as well as induction of release of intracellular Ca2+ In the last part we investigated the effect of disruption of the cell membrane integrity by cholesterol depletion on thyrotropin-releasing hormone receptor (TRH-R) surface mobility and internalization in HEK293 cells stably expressing TRH-R-eGFP fusion protein. CLSM studies indicated that the internalization of receptor molecules initiated by TRH stimulation was significantly attenuated. The detailed analysis of recovery of TRH-R-eGFP fluorescence in bleached spots of different sizes indicated that cholesterol depletion results in an increase of overall receptor mobility. We suggest that migration of..

Role of Genetic Variation in Cytochromes P450 in Breast Cancer Prognosis and Therapy Response

Breast cancer is the most frequent cancer in the female population worldwide. The role of germline genetic variability in cytochromes P450 (CYP) in breast cancer prognosis and individualized therapy awaits detailed elucidation. In the present study, we used the next-generation sequencing to assess associations of germline variants in the coding and regulatory sequences of all human CYP genes with response of the patients to the neoadjuvant cytotoxic chemotherapy and disease-free survival (n = 105). A total of 22 prioritized variants associating with a response or survival in the above evaluation phase were then analyzed by allelic discrimination in the large confirmation set (n = 802). Associations of variants in CYP1B1, CYP4F12, CYP4X1, and TBXAS1 with the response to the neoadjuvant cytotoxic chemotherapy were replicated by the confirmation phase. However, just association of variant rs17102977 in CYP4X1 passed the correction for multiple testing and can be considered clinically and statistically validated. Replicated associations for variants in CYP4X1, CYP24A1, and CYP26B1 with disease-free survival of all patients or patients stratified to subgroups according to therapy type have not passed a false discovery rate test. Although statistically not confirmed by the present study, the role of CYP genes in breast cancer prognosis should not be ruled out. In conclusion, the present study brings replicated association of variant rs17102977 in CYP4X1 with the response of patients to the neoadjuvant cytotoxic chemotherapy and warrants further research of genetic variation CYPs in breast cancer

Single-Nucleotide Polymorphisms in MICA and MICB Genes Could Play a Role in the Outcome in AML Patients after HSCT

NKG2D and its ligands, MICA and MICB, are known as the key regulators of NK cells. NK cells are the first reconstituted cells after the allogeneic hematopoietic stem cell transplantation (HSCT); therefore, it is crucial to understand their role in HSCT outcome. In the presented study, we investigated the single amino acid changes across the exons 2–4 of MICA and MICB genes, and point mutations within the NKG2D gene, which defines the type of NKG2D haploblock (HNK/LNK) in the donors (n = 124), as well as in patients with acute myeloid leukemia (n = 78). In our cohort, we found that graft from a donor with at least one MICA allele containing glycine at position 14 (MICA-14Gly) is significantly associated with deterioration of a patient’s overall survival (OS) (p < 0.05). We also observed a negative effect of MICB-58 (Lys → Glu) polymorphism on relapse-free survival (RFS), although it was not statistically significant in multivariate analysis (p = 0.069). To our knowledge, this is the first work describing the role of MICA-14 and MICB-58 polymorphisms on HSCT outcome