BACKGROUND AND AIMS: Schistosoma mansoni infection is one of the worldwide leading causes of liver fibrosis and portal hypertension. The objective of this study was to evaluate whether polyhydroxylated bile acids (BAs), known to protect mice from the development of acquired cholestatic liver injury, counteract S. mansoni-induced inflammation and fibrosis. METHODS: Adult FVB/N wild type (WT) and Abcb11/Bsep-/- mice were infected with either 25 or 50 S. mansoni cercariae. Eight weeks post infection, effects on liver histology, serum biochemistry, gene expression profile of proinflammatory cytokines and fibrotic markers, hepatic hydroxyproline content and FACS analysis were performed. RESULTS: Bsep-/- mice infected with S. mansoni showed significantly less hepatic inflammation and tendentially less fibrosis compared to infected WT mice. Despite elevated alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase levels in infected Bsep-/- mice, inflammatory cells such as M2 macrophages and Mac-2/galectin-3+ cells were reduced in these animals. Accordingly, mRNA-expression levels of anti-inflammatory cytokines (IL-4 and IL-13) were increased in Bsep-/- mice upon infection. Furthermore, infected Bsep-/- mice exhibited decreased hepatic egg load and parasite fecundity, consequently affecting the worm reproduction rate. This outcome could arise from elevated serum BA levels and lower blood pH in Bsep-/- mice. CONCLUSIONS: The loss of Bsep and the resulting changes in bile acid composition and blood pH are associated with the reduction of parasite fecundity, thus attenuating the development of S. mansoni-induced hepatic inflammation and fibrosis.
- MeSH
- Cytokines metabolism MeSH
- Fertility MeSH
- Liver Cirrhosis prevention & control etiology MeSH
- Liver pathology MeSH
- Mice MeSH
- Parasites * MeSH
- Schistosoma mansoni MeSH
- Schistosomiasis mansoni * complications MeSH
- Inflammation pathology MeSH
- Bile Acids and Salts metabolism MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Current classifications (World Health Organization-HAEM5/ICC) define up to 26 molecular B-cell precursor acute lymphoblastic leukemia (BCP-ALL) disease subtypes by genomic driver aberrations and corresponding gene expression signatures. Identification of driver aberrations by transcriptome sequencing (RNA-Seq) is well established, while systematic approaches for gene expression analysis are less advanced. Therefore, we developed ALLCatchR, a machine learning-based classifier using RNA-Seq gene expression data to allocate BCP-ALL samples to all 21 gene expression-defined molecular subtypes. Trained on n = 1869 transcriptome profiles with established subtype definitions (4 cohorts; 55% pediatric / 45% adult), ALLCatchR allowed subtype allocation in 3 independent hold-out cohorts (n = 1018; 75% pediatric / 25% adult) with 95.7% accuracy (averaged sensitivity across subtypes: 91.1% / specificity: 99.8%). High-confidence predictions were achieved in 83.7% of samples with 98.9% accuracy. Only 1.2% of samples remained unclassified. ALLCatchR outperformed existing tools and identified novel driver candidates in previously unassigned samples. Additional modules provided predictions of samples blast counts, patient's sex, and immunophenotype, allowing the imputation in cases where these information are missing. We established a novel RNA-Seq reference of human B-lymphopoiesis using 7 FACS-sorted progenitor stages from healthy bone marrow donors. Implementation in ALLCatchR enabled projection of BCP-ALL samples to this trajectory. This identified shared proximity patterns of BCP-ALL subtypes to normal lymphopoiesis stages, extending immunophenotypic classifications with a novel framework for developmental comparisons of BCP-ALL. ALLCatchR enables RNA-Seq routine application for BCP-ALL diagnostics with systematic gene expression analysis for accurate subtype allocation and novel insights into underlying developmental trajectories.
- Publication type
- Journal Article MeSH
Nanodiamonds are innovative nanocrystalline carbon particles able to deliver chemically conjugated miRNAs. In oncology, the use of miRNA-based therapies may represent an advantage, based on their ability to simultaneously target multiple intracellular oncogenic targets. Here, nanodiamonds were tested and optimized to deliver miR-34a, a miRNA playing a key role in inhibiting tumor development and progression in many cancers. The physical-chemical properties of nanodiamonds were investigated suggesting electrical stability and uniformity of structure and size. Moreover, we evaluated nanodiamond cytotoxicity on two breast cancer cell models and confirmed their excellent biocompatibility. Subsequently, nanodiamonds were conjugated with miR-34a, using the chemical crosslinker polyethyleneimine; real-time PCR analysis revealed a higher level of miR-34a in cancer cells treated with the different formulations of nanodiamonds than with commercial transfectant. A significant and early nanodiamond-miR-34a uptake was recorded by FACS and fluorescence microscopy analysis in MCF7 and MDA-MB-231 cells. Moreover, nanodiamond-miR-34a significantly inhibited both cell proliferation and migration. Finally, a remarkable anti-tumor effect of miR-34a-conjugated nanodiamonds was observed in both heterotopic and orthotopic murine xenograft models. In conclusion, this study provides a rationale for the development of new therapeutic strategies based on use of miR-34a delivered by nanodiamonds to improve the clinical treatment of neoplasms.
- Publication type
- Journal Article MeSH
Sertoli cells (SCs) are the only somatic cells that reside in seminiferous tubules of testis. They directly interact with and support the development of germ cells, thus have an indispensable role in the process of spermatogenesis. SCs first appear in a proliferative state and then, with the initiation of the first wave of spermatogenesis, progress to a mature "nurturing" state which supports lifelong continuous sperm production. During this development, the SC transcriptome must adapt rapidly as obstacles in SC maturation often result in deficiencies in male fertility. Due to its importance in spermatogenesis, a reliable, rapid, and precise method for the isolation of high purity, viable and unadulterated SC has been largely missing. We have developed an improved method for the preparation of a testicular single cell suspension comprised of two alternative protocols to separate SCs from the rest of the testicular cells by FACS. The first sorting scheme is based on their co-expression of surface specific markers, FSHr and Occludin-1, while the second focuses on the co-staining of SCs with FSHr-specific antibody and Hoechst 33342, which discriminates DNA content of testicular cells. The entire procedure can be completed in less than 3 h which permits the analysis of the development-related transcriptional profile of these cells. Notably, our comparative study showed that this method resulted in a SC transcriptome that is largely comparable to SCs which were briskly isolated due to their cell-specific expression of fluorescent protein. Interestingly, we also show that SCs sorted as FSHr+Occludin+ cells contained a tangible portion of transcripts from all types of testicular germ cells. Sorting of SCs according to their 2C DNA content significantly reduced the presence of these transcripts, thus seems to be the most suitable approach for accurate determination of the SC transcriptome. We believe that these novel approaches for the isolation of SCs will assist researchers in the elucidation of their function as well as their role in spermatogenesis and disorders related to male infertility.
- Publication type
- Journal Article MeSH
We present here a new iPS cell line for modeling sporadic form of ALS. Cell line was generated by reprogramming skin fibroblasts isolated with explant culture technology from skin biopsy, donated by ALS patient. For reprogramming, polycistronic self-replicating RNA vector was used and derived iPS cells were characterized by immunocytochemistry and FACS (pluripotent factors expression), karyotyping, STR fingerprinting analysis and in vitro differentiation assay. New cell line showed normal (46, XY) karyotype and differentiated in vitro into cells from three germ layers. STR analysis proved the origin and originality of the cell line.
PURPOSE: To assess the efficacy of the novel clinical formulation of fenretinide (LAU-7b) for the treatment of allergic asthma. To study the association between LAU-7b treatment in allergic asthma and the modulation of very long chain ceramides (VLCC). METHODS: We used two allergens (OVA and HDM) to induce asthma in mouse models and we established a treatment protocol with LAU-7b. The severity of allergic asthma reaction was quantified by measuring the airway resistance, quantifying lung inflammatory cell infiltration (Haematoxylin and eosin stain) and mucus production (Periodic acid Schiff satin). IgE levels were measured by ELISA. Immunophenotyping of T cells was done using Fluorescence-activated cell sorting (FACS) analysis. The analysis of the specific species of lipids and markers of oxidation was performed using mass spectrometry. RESULTS: Our data demonstrate that 10 mg/kg of LAU-7b was able to protect OVA- and HDM-challenged mice against increase in airway hyperresponsiveness, influx of inflammatory cells into the airways, and mucus production without affecting IgE levels. Treatment with LAU-7b significantly increased percentage of regulatory T cells and CD4+ IL-10-producing T cells and significantly decreased percentage of CD4+ IL-4-producing T cells. Our data also demonstrate a strong association between the improvement in the lung physiology and histology parameters and the drug-induced normalization of the aberrant distribution of ceramides in allergic mice. CONCLUSION: 9 days of 10 mg/kg of LAU-7b daily treatment protects the mice against allergen-induced asthma and restores VLCC levels in the lungs and plasma.
- MeSH
- Allergens immunology MeSH
- Asthma drug therapy immunology metabolism MeSH
- Ceramides metabolism MeSH
- Fenretinide therapeutic use MeSH
- Clinical Protocols MeSH
- Methylcellulose chemistry MeSH
- Disease Models, Animal MeSH
- Mice MeSH
- Ovalbumin immunology MeSH
- Drug Compounding MeSH
- Pyroglyphidae immunology MeSH
- Animals MeSH
- Check Tag
- Male MeSH
- Mice MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Despite the development of several cultivation methods, the rate of discovery of microorganisms that are yet-to-be cultivated outpaces the rate of isolating and cultivating novel species in the laboratory. Furthermore, no current cultivation technique is capable of selectively isolating and cultivating specific bacterial taxa or phylogenetic groups independently of morphological or physiological properties. Here, we developed a new method to isolate living bacteria solely based on their 16S rRNA gene sequence. We showed that bacteria can survive a modified version of the standard fluorescence in situ hybridization (FISH) procedure, in which fixation is omitted and other factors, such as centrifugation and buffers, are optimized. We also demonstrated that labelled DNA probes can be introduced into living bacterial cells by means of chemical transformation and that specific hybridization occurs. This new method, which we call live-FISH, was then combined with fluorescence-activated cell sorting (FACS) to sort specific taxonomic groups of bacteria from a mock and natural bacterial communities and subsequently culture them. Live-FISH represents the first attempt to systematically optimize conditions known to affect cell viability during FISH and then to sort bacterial cells surviving the procedure. No sophisticated probe design is required, making live-FISH a straightforward method to be potentially used in combination with other single-cell techniques and for the isolation and cultivation of new microorganisms.
- MeSH
- Bacillus genetics MeSH
- Bacteria genetics MeSH
- RNA, Bacterial genetics MeSH
- DNA Probes MeSH
- Phylogeny MeSH
- In Situ Hybridization, Fluorescence * MeSH
- Microbiological Techniques * MeSH
- Flow Cytometry MeSH
- RNA, Ribosomal, 16S genetics MeSH
- Cell Separation * MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Závěrečná zpráva o řešení grantu Agentury pro zdravotnický výzkum MZ ČR
Nestr.
Defekty imunity se často vyznačují kombinací nedostatečné kontroly infekce a zároveň autoimunitními projevy způsobenými reakcí organismu proti vlastním antigenům. Patogenetickým podkladem mohu být poruchy ve funkci T a B lymfocytů a špatná regulace jejich vzájemné spolupráce. V navrhovaném projektu plánujeme pomocí nejmodernějších technik mnohobarevné průtokové cytometrie (MC-FACS) a molekulární genetiky podrobně charakterizovat složení, aberace a vzájemné systémové vztahy fyziologických i patologických subpopulací lymfocytů u všech primárních imunodeficitů (PID) diagnostikovaných na zúčastněných pracovištích v průběhu řešení projektu a u souboru pacientů s běžným variabilním imunodeficitem (CVID) a selektivním deficitem IgA. Na základě bioinformatické analýzy výsledků MC-FACS od přibližně 150 pacientů bude vypracováno v součinnosti s mezinárodní skupinou EuroFlow-PID diagnostické schéma pro jednotky s již známou genetickou příčinou a budou vytipovány skupiny pacientů s podobným laboratorním a klinickým obrazem k celogenomové analýze a validaci vytipovaných nových variací.; Immune defects are often a combination of inadequate infection control and autoimmune phenomena caused by overreaction to autologous antigens. Underlying cause may be found at impaired function of T and B cells and improper regulation of their cooperation. In the proposed project, we will characterize composition, abnormalities and systemic relationships of normal and abnormal subsets of lymphocytes using cutting edge techniques of multi-color flow cytometry (MC-FACS) and molecular genetics in all primary immunodeficiency (PID) patients in both centers and in patients with Common Variable Immunodeficiency (CVID) and selective IgA deficiency. Bioinformatic analysis of standardized MC-FACS data from 150 patients will serve us to develop a diagnostic scheme together with EuroFlow PID international group for PIDs with established molecular genetic lesion. Furthermore, cases with commonalities in MC-FACS and clinical presentation will be investigated by whole exome sequencing to elucidate new genetic variations leading to PID.
- MeSH
- Early Diagnosis MeSH
- Genetic Testing MeSH
- Immune System Phenomena MeSH
- Lymphocytes MeSH
- Molecular Biology MeSH
- Primary Immunodeficiency Diseases diagnosis MeSH
- Flow Cytometry MeSH
- Conspectus
- Patologie. Klinická medicína
- NML Fields
- alergologie a imunologie
- NML Publication type
- závěrečné zprávy o řešení grantu AZV MZ ČR
Identification of novel proteins with changed expression in resistant cancer cells could be helpful in elucidation mechanisms involved in the development of acquired resistance to paclitaxel. In this study, we carried out a 2D-PAGE using the mitochondrial-enriched fraction from paclitaxel-resistant MCF7/PacR cells compared to original paclitaxel-sensitive MCF7 breast cancer cells. Differentially expressed proteins were identified employing mass spectrometry. We found that lysosomal cathepsin D and mitochondrial abhydrolase-domain containing protein 11 (ABHD11) had decreased expression in MCF7/PacR cells. On the other hand, mitochondrial carbamoyl-phosphate synthetase 1 (CPS1) and ATPase family AAA-domain containing protein 3A and 3B (ATAD3A, ATAD3B) were overexpressed in MCF7/PacR cells. Further, we showed that there was no difference in localization of CPS1 in MCF7 and MCF7/PacR cells. We demonstrated a significant increase in the number of CPS1 positive MCF7/PacR cells, using FACS analysis, compared to the number of CPS1 positive MCF7 cells. Silencing of CPS1 expression by specific siRNA had no significant effect on the resistance of MCF7/PacR cells to paclitaxel. To summarize, we identified several novel proteins of a mitochondrial fraction whose role in acquired resistance to paclitaxel in breast cancer cells should be further assessed.
- MeSH
- Drug Resistance, Neoplasm * drug effects MeSH
- Cell Fractionation MeSH
- Carbamoyl-Phosphate Synthase (Ammonia) genetics metabolism MeSH
- Humans MeSH
- MCF-7 Cells MeSH
- Mitochondrial Proteins genetics metabolism MeSH
- Mitochondria genetics metabolism MeSH
- Breast Neoplasms drug therapy genetics metabolism MeSH
- Paclitaxel pharmacology MeSH
- Proteome MeSH
- Proteomics methods MeSH
- Gene Expression Regulation, Neoplastic MeSH
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization MeSH
- Tandem Mass Spectrometry MeSH
- Gene Silencing MeSH
- Check Tag
- Humans MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
Cieľ. Štúdia sa zaoberá analýzou tvárových výrazov vyvolaných súcitným podnetom v podobe krátkeho videa. Výskumný súbor a nastavenie. Výskumný súbor tvorilo 151 respondentov vo veku od 18 do 59 (M = 25,17; SD = 7,81), vybraných dostupným výberom. Respondenti si doma, v kontexte redukovanej sociálnej žiadúcnosti, pozreli súcitný video podnet a ich tváre boli počas pozerania nahrávané online, prostredníctvom webkamery. Výskumné otázky. Cieľom štúdie je popísať spontánny tvárový výraz súcitu vyvolaný súcitným videom prostredníctvom zmien v tvárovej muskulárnej aktivite – akčných jednotiek. Cieľom bolo tiež zistiť, ktoré primárne emócie (hnev, znechutenie, strach, radosť, smútok a prekvapenie) sú najviac zapájané v tvárovom výraze respondentov počas pozerania najsúcitnejšieho momentu videa v porovnaní s neutrálnym momentom. Štatistická analýza. Na štatistickú analýzu bol použitý štatistický softvér SPSS, verzia 20.0 a program R. Tvárové výrazy boli analyzované manuálne pomocou metódy Facial Action Coding System (FACS) štyrmi certifikovanými kódermi a tiež automaticky pomocou softvéru Emotion ID. Výsledky. Analýzy tvárových výrazov kódermi preukázala, že akčné jednotky 7 (sťahovač viečka), 12 (vyťahovač kútikov pier), 43 (zatváranie očí) a 56 (hlava naklonená doprava) sa objavovali signifikantne častejšie v najviac súcitnom momente videa v porovnaní s neutrálnym momentom. Analýza tvárových výrazov prostredníctvom softvéru Emotion ID preukázala signifikantne vyššiu pravdepodobnosť výskytu hnevu a smútku v najsúcitnejšom momente oproti neutrálnemu momentu. Výsledky FACS kódovania poukazujú na to, že súcitný tvárový výraz je spojený s určitým druhom „falošného“ úsmevu, mierne poklesnutými hornými viečkami, napnutými spodnými viečkami a hlavou naklonenou doprava. Limity. Hlavnými limitmi štúdie sú relatívne malý výskumný súbor v rámci jednej kultúry, a tiež dostupný výber respondentov. Taktiež sociálny kontext súcitu nebol zobraný do úvahy kvôli situácii navodenej video podnetom.
Objectives. The study focuses on the analysis of facial expressions elicited by a compassionate video stimulus. Sample and setting. There was a convenient sample of 151 respondents aged from 18 to 59 years old (M= 25.17; SD = 7.81). Respondents watched a compassionate video stimulus and their faces were recorded through webcams online at home in the context of reduced social desirability. Research questions. The goal of the study was to describe the spontaneous facial expression of compassion elicited by the compassionate video through changes in the facial muscular activity – action units. Additionally, next aim was to explore which basic emotions (anger, disgust, fear, happiness, sadness, and surprise) are the most involved in facial expression of participants while watching the most compassionate moment of the video compared to the baseline. Statistical analysis. The authors analyzed facial expressions simultaneously by the manual method the Facial Action Coding System (FACS) by four certified coders as well as automatically by Emotion ID software. Results. Analysis of facial expressions by coders demonstrated that action units 7 (lid tightener), 12 (lip corner puller), 43 (eyes closed) and 56 (head tilt right) appeared significantly more frequently in the most compassionate moment compared to the baseline moment. Analysis of facial expressions by Emotion ID software demonstrated that there was a significantly higher probability of appearance of anger and sadness in the most compassionate moment than in the baseline moment. In addition, the results of the study show that a compassionate facial expression is associated with some kind of “false” smile, mildly lowered upper eyelids, contracted lower eyelids and head tilted to the right as coded by FACS. Study limitation. The main limitations of the study are that research was conducted on a relatively small and not representative sample from one culture and also the social context of compassion was missing.
- MeSH
- Behavioral Research MeSH
- Adult MeSH
- Emotions MeSH
- Empathy * MeSH
- Data Interpretation, Statistical MeSH
- Middle Aged MeSH
- Humans MeSH
- Young Adult MeSH
- Facial Muscles MeSH
- Facial Recognition MeSH
- Data Collection MeSH
- Software MeSH
- Videotape Recording MeSH
- Facial Expression * MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Young Adult MeSH
