Nestin is a unique intermediate filament expressed for a short period in the developing heart. It was also documented in several cell types of the adult myocardium under pathological conditions such as myocardial infarction or fibrosis. However, circumstances of nestin re-occurrence in the diseased or aging heart have not been elucidated yet. In this work we immunohistochemically detected nestin to determine its expression and distribution pattern in the left ventricular myocardium of normotensive Wistar Kyoto (WKY) rats and in the hypertrophic ones of spontaneously hypertensive (SHR) rats, both at the age of 1 and 1.5 year. No nestin+ cells were identified in the intact myocardium of 1-year-old WKY rats, whereas in the aged 1.5-year-old WKY rats nestin+ endothelial cells in some blood vessels were discovered. In the hypertrophic myocardium of all SHR rats, nestin was rarely detected in desmin+ vimentin- cardiomyocytes and in some vimentin+ interstitial cells often accumulated in clusters, varying in intensity of desmin immunoreactivity. Moreover, nestin was infrequently expressed in the endothelial cells of some myocardial blood vessels in 1-year-old SHR rats, but not in 1.5-year-old ones. Quantitative image analysis of nestin expression in the myocardium confirmed significant increase in 1.5-year-old WKY rats and in SHR rats of both ages compared to the intact 1-year-old WKY rats. This study firstly documents nestin re-expression indicating cytoskeletal remodelling in different cell types of the aging intact and chronically pressure over-loaded hypertrophied myocardium. Our findings confirm nestin involvement in complex changes during myocardial hypertrophy and progressive aging.

• MeSH
• hypertenze metabolismus   patologie   MeSH
• kardiomyocyty metabolismus   patologie   MeSH
• krysa rodu rattus MeSH
• myokard * metabolismus   patologie   MeSH
• nestin * metabolismus   MeSH
• potkani inbrední SHR * MeSH
• potkani inbrední WKY * MeSH
• proteiny intermediálních filament metabolismus   MeSH
• proteiny nervové tkáně metabolismus   MeSH
• stárnutí * metabolismus   patologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Although some clinical studies have reported increased mitochondrial respiration in patients with fatty liver and early non‐alcoholic steatohepatitis (NASH), there is a lack of in vitro models of non‐alcoholic fatty liver disease (NAFLD) with similar findings. Despite being the most commonly used immortalized cell line for in vitro models of NAFLD, HepG2 cells exposed to free fatty acids (FFAs) exhibit a decreased mitochondrial respiration. On the other hand, the use of HepaRG cells to study mitochondrial respiratory changes following exposure to FFAs has not yet been fully explored. Therefore, the present study aimed to assess cellular energy metabolism, particularly mitochondrial respiration, and lipotoxicity in FFA‐treated HepaRG and HepG2 cells. HepaRG and HepG2 cells were exposed to FFAs, followed by comparative analyses that examained cellular metabolism, mitochondrial respiratory enzyme activities, mitochondrial morphology, lipotoxicity, the mRNA expression of selected genes and triacylglycerol (TAG) accumulation. FFAs stimulated mitochondrial respiration and glycolysis in HepaRG cells, but not in HepG2 cells. Stimulated complex I, II‐driven respiration and β‐oxidation were linked to increased complex I and II activities in FFA‐treated HepaRG cells, but not in FFA‐treated HepG2 cells. Exposure to FFAs disrupted mitochondrial morphology in both HepaRG and HepG2 cells. Lipotoxicity was induced to a greater extent in FFA‐treated HepaRG cells than in FFA‐treated HepG2 cells. TAG accumulation was less prominent in HepaRG cells than in HepG2 cells. On the whole, the present study demonstrates that stimulated mitochondrial respiration is associated with lipotoxicity in FFA‐treated HepaRG cells, but not in FFA‐treated HepG2 cells. These findings suggest that HepaRG cells are more suitable for assessing mitochondrial respiratory adaptations in the developed in vitro model of early‐stage NASH.

• MeSH
• buněčné linie MeSH
• buňky Hep G2 MeSH
• dýchání MeSH
• kyseliny mastné neesterifikované MeSH
• lidé MeSH
• mitochondrie MeSH
• nealkoholová steatóza jater * MeSH
• triglyceridy MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Although Holder pasteurization is the recommended method for processing breast milk, it does affect some of its nutritional and biological properties and is ineffective at inactivating spores. The aim of this study was to find and validate an alternative methodology for processing breast milk to increase its availability for newborn babies and reduce the financial loss associated with discarding milk that has become microbiologically positive. We prepared two series of breast milk samples inoculated with the Bacillus cereus (B. cereus) strain to verify the effectiveness of two high-pressure treatments: (1) 350 MPa/5 min/38 °C in four cycles and (2) cumulative pressure of 350 MPa/20 min/38 °C. We found that the use of pressure in cycles was statistically more effective than cumulative pressure. It reduced the number of spores by three to four orders of magnitude. We verified that the method was reproducible. The routine use of this method could lead to an increased availability of milk for newborn babies, and at the same time, reduce the amount of wasted milk. In addition, high-pressure treatment preserves the nutritional quality of milk.

• Publikační typ
• časopisecké články MeSH

The lack of physiological parity between 2D cell culture and in vivo culture has led to the development of more organotypic models, such as organoids. Organoid models have been developed for a number of tissues, including the liver. Current organoid protocols are characterized by a reliance on extracellular matrices (ECMs), patterning in 2D culture, costly growth factors and a lack of cellular diversity, structure, and organization. Current hepatic organoid models are generally simplistic and composed of hepatocytes or cholangiocytes, rendering them less physiologically relevant compared to native tissue. We have developed an approach that does not require 2D patterning, is ECM independent, and employs small molecules to mimic embryonic liver development that produces large quantities of liver-like organoids. Using single-cell RNA sequencing and immunofluorescence, we demonstrate a liver-like cellular repertoire, a higher order cellular complexity, presenting with vascular luminal structures, and a population of resident macrophages: Kupffer cells. The organoids exhibit key liver functions, including drug metabolism, serum protein production, urea synthesis and coagulation factor production, with preserved post-translational modifications such as N-glycosylation and functionality. The organoids can be transplanted and maintained long term in mice producing human albumin. The organoids exhibit a complex cellular repertoire reflective of the organ and have de novo vascularization and liver-like function. These characteristics are a prerequisite for many applications from cellular therapy, tissue engineering, drug toxicity assessment, and disease modeling to basic developmental biology.

• MeSH
• hepatocyty MeSH
• játra * MeSH
• kultivované buňky MeSH
• lidé MeSH
• myši MeSH
• organoidy * MeSH
• tkáňové inženýrství MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND & AIMS: Lymphedema cholestasis syndrome 1 or Aagenaes syndrome is a condition characterized by neonatal cholestasis, lymphedema, and giant cell hepatitis. The genetic background of this autosomal recessive disease was unknown up to now. METHODS: A total of 26 patients with Aagenaes syndrome and 17 parents were investigated with whole-genome sequencing and/or Sanger sequencing. PCR and western blot analyses were used to assess levels of mRNA and protein, respectively. CRISPR/Cas9 was used to generate the variant in HEK293T cells. Light microscopy, transmission electron microscopy and immunohistochemistry for biliary transport proteins were performed in liver biopsies. RESULTS: One specific variant (c.-98G>T) in the 5'-untranslated region of Unc-45 myosin chaperone A (UNC45A) was identified in all tested patients with Aagenaes syndrome. Nineteen were homozygous for the c.-98G>T variant and seven were compound heterozygous for the variant in the 5'-untranslated region and an exonic loss-of-function variant in UNC45A. Patients with Aagenaes syndrome exhibited lower expression of UNC45A mRNA and protein than controls, and this was reproduced in a CRISPR/Cas9-created cell model. Liver biopsies from the neonatal period demonstrated cholestasis, paucity of bile ducts and pronounced formation of multinucleated giant cells. Immunohistochemistry revealed mislocalization of the hepatobiliary transport proteins BSEP (bile salt export pump) and MRP2 (multidrug resistance-associated protein 2). CONCLUSIONS: c.-98G>T in the 5'-untranslated region of UNC45A is the causative genetic variant in Aagenaes syndrome. IMPACT AND IMPLICATIONS: The genetic background of Aagenaes syndrome, a disease presenting with cholestasis and lymphedema in childhood, was unknown until now. A variant in the 5'-untranslated region of the Unc-45 myosin chaperone A (UNC45A) was identified in all tested patients with Aagenaes syndrome, providing evidence of the genetic background of the disease. Identification of the genetic background provides a tool for diagnosis of patients with Aagenaes syndrome before lymphedema is evident.

• MeSH
• 5' nepřekládaná oblast genetika   MeSH
• cholestáza * genetika   MeSH
• HEK293 buňky MeSH
• intracelulární signální peptidy a proteiny * genetika   MeSH
• lidé MeSH
• lymfedém * diagnóza   genetika   metabolismus   MeSH
• myosiny genetika   metabolismus   MeSH
• novorozenec MeSH
• transportní proteiny genetika   MeSH
• Check Tag
• lidé MeSH
• novorozenec MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• Publikační typ
• abstrakt z konference MeSH

BACKGROUND: The endothelial glycocalyx (EG) plays a vital role in the physiology and pathophysiology of human microcirculation. Having relevant EG damage model would be important tool for testing new interventions aiming at EG protection and recovery. We describe the first in vivo EG damage model in pig. OBJECTIVE: To investigate the course of animal EG damage induced by specific enzymes. MATERIAL AND METHODS: Four anesthetized piglets received enzymes: 1g hyaluronidase and 25 IU heparanase I intravenously. Blood and urine samples were collected at baseline and 20/40/60/80/100/120 min for detecting markers of endothelial and EG function. Sublingual microcirculation and EG thickness were assessed by Side-stream Dark Field (SDF) imaging and Perfused Boundary Region (PBR) respectively. EG of the mesentery artery was visualized in fluorescent microscopy. RESULTS: Biochemical marker of EG damage syndecan-1 showed temporary increase with return to baseline and was reflected by PBR values. Albumin levels suggested brief period of capillary leakage (decrease in the serum, increase in the urine) with a trend to normalization. Urine glycosaminoglycans peaked at 120 minutes. Microcirculatory perfusion parameter showed significant alteration. Diffusion parameters were altered with no statistical significance. CONCLUSION: EG damage induced by specific enzymes was reflected by temporary changes of biochemical makers together with alteration of microcirculation and changes in fluorescent microscopy of EG layer. Our results support to further validate presented model of EG damage on a larger number of animals.

• MeSH
• glykokalyx * MeSH
• kapiláry MeSH
• mikrocirkulace MeSH
• pilotní projekty MeSH
• prasata MeSH
• trávení MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Autophagy inhibition through small-molecule inhibitors is one of the approaches to increase the efficiency of radiotherapy in oncological patients. A new inhibitor-Lys05-with the potential to accumulate within lysosomes and to block autophagy was discovered a few years ago. Several studies have addressed its chemosensitizing effects but nothing is known about its impact in the context of ionizing radiation (IR). To describe its role in radiosensitization, we employed radioresistant human non-small cell lung carcinoma cells (H1299, p53-negative). Combined treatment of H1299 cells by Lys05 together with IR decreased cell survival in the clonogenic assay and real-time monitoring of cell growth more than either Lys05 or IR alone. Immunodetection of LC3 and p62/SQSTM1 indicated that autophagy was inhibited, which correlated with increased SQSTM1 and decreased BNIP3 gene expression determined by qRT-PCR. Fluorescence microscopy and flow cytometry uncovered an accumulation of lysosomes. Similarly, transmission electron microscopy demonstrated the accumulation of autophagosomes confirming the ability of Lys05 to potentiate autophagy inhibition in H1299 cells. We report here for the first time that Lys05 could be utilized in combination with IR as a promising future strategy in the eradication of lung cancer cells.

• MeSH
• apoptóza účinky záření   MeSH
• fluorescenční mikroskopie MeSH
• ionizující záření * MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádory plic metabolismus   MeSH
• průtoková cytometrie MeSH
• transmisní elektronová mikroskopie MeSH
• western blotting MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The primary cilium is considered as a key component of morphological cellular stability. However, cancer cells are notorious for lacking primary cilia in most cases, depending upon the tumour type. Previous reports have shown the effect of starvation and cytostatics on ciliogenesis in normal and cancer cells although with limited success, especially when concerning the latter. In this study, we evaluated the presence and frequency of primary cilia in breast fibroblasts and in triple-negative breast cancer cells after treatment with cytostatics finding that, in the case of breast fibroblasts, primary cilia were detected at their highest incidence 72 hours after treatment with 120 nM doxorubicin. Further, multiciliated cells were also detected after treatment with 80 nM doxorubicin. On the other hand, treatment with taxol increased the number of ciliated cells only at low concentrations (1.25 and 3.25 nM) and did not induce multiciliation. Interestingly, triple-negative breast cancer cells did not present primary cilia after treatment with either doxorubicin or taxol. This is the first study reporting the presence of multiple primary cilia in breast fibroblasts induced by doxorubicin. However, the null effect of these cytostatics on primary cilia incidence in the evaluated triple negative breast carcinomas cell lines requires further research.

• MeSH
• cilie účinky léků   metabolismus   MeSH
• cytostatické látky toxicita   MeSH
• doxorubicin farmakologie   MeSH
• fibroblasty účinky léků   metabolismus   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• paclitaxel farmakologie   MeSH
• proliferace buněk účinky léků   MeSH
• viabilita buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The cellular components of the satellite cell niche participate in the regulation of skeletal muscle regeneration. Beside myogenic cells at different developmental stages, this niche is formed by cells of the immune system, the interstitial connective tissue and the vascular system. Unambiguous determination of the origin of these cell types could contribute to optimization of the cell-based therapy of skeletal muscle disorders. In our work, we intravenously transplanted mouse GFP+ unseparated bone marrow cells into whole-body lethally irradiated immunocompetent mice four weeks before cardiotoxin-induced injury of the recipients' skeletal muscles. Seven and 28 days after the toxin injection, the injured regenerating and contralateral intact muscles were examined for identification of GFP+ bone marrow-derived cells by direct fluorescence, protein immunohistochemistry and immunogold transmission electron microscopy. In both the intact and injured muscles, GFP positivity was determined in immune cells, mainly in macrophages, and in interstitial spindleshaped cells. Moreover, in the injured muscles, rare GFP+ endothelial cells of the blood vessels and newly formed myotubes and muscle fibres were present. Our results confirmed the ability of bone marrowderived cells to contribute to the cellular component of the satellite cell niche in the intact and regenerating skeletal muscle. These cells originated not only from haematopoietic stem cells, but obviously also from other stem or progenitor cells residing in the bone marrow, such as multipotent mesenchymal stromal cells and endothelial progenitors.

• MeSH
• buňky kostní dřeně cytologie   MeSH
• fluorescence MeSH
• kosterní svaly zranění   patologie   ultrastruktura   MeSH
• myši inbrední C57BL MeSH
• nika kmenových buněk * MeSH
• regenerace fyziologie   MeSH
• satelitní buňky kosterního svalu cytologie   MeSH
• zelené fluorescenční proteiny metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH