BACKGROUND: Diffuse midline glioma, H3 K27-altered (DMG) is a fatal tumour that arises in the midline structures of the brain. When located in the pons, it is more commonly referred to as diffuse intrinsic pontine glioma (DIPG). DMG/DIPG is usually diagnosed when children are < 10 years, and it has a median overall survival of < 12 months after diagnosis. Radiological imaging is still the gold standard for DIPG diagnosis while the use of biopsy procedures led to our knowledge on its biology, such as with the identification of the canonical histone H3K27M mutation. However, the need to improve survival encourages the development of non-invasive, fast and inexpensive assays on biofluids for optimizing molecular diagnoses in DMG/DIPG. Here, we propose a rapid, new, imaging and epigenetics-based approach to diagnose DMG/DIPG in the plasma of paediatric patients. METHODS: A total of 20 healthy children (mean age: 10.5 years) and 24 children diagnosed with DMG/DIPG (mean age: 8.5 years) were recruited. Individual histones (H2A, H2B, H3, H4, macroH2A1.1 and macroH2A1.2), histone dimers and nucleosomes were assayed in biofluids by means of a new advanced flow cytometry ImageStream(X)-adapted method. RESULTS: We report a significant increase in circulating histone dimers and tetramers (macroH2A1.1/H2B versus control: p value < 0.0001; macroH2A1.2/H2B versus control: p value < 0.0001; H2A/H2B versus control: p value < 0.0001; H3/H4 versus control: p value = 0.008; H2A/H2B/H3/H4 versus control: p value < 0.0001) and a significant downregulation of individual histones (H2B versus control: p value < 0.0001; H3 versus control: p value < 0.0001; H4 versus control: p value < 0.0001). Moreover, histones were also detectable in the cerebrospinal fluid (CSF) of patients with DMG/DIPG and in the supernatant of SF8628, OPBG-DIPG002 and OPBG-DIPG004 DMG/DIPG cell lines, with patterns mostly similar to each other, but distinct compared to blood plasma. CONCLUSIONS: In summary, we identified circulating histone signatures able to detect the presence of DMG/DIPG in biofluids of children, using a rapid and non-invasive ImageStream(X)-based imaging technology, which may improve diagnosis and benefit the patients.

• MeSH
• difuzní intrinsický pontinní gliom genetika   diagnóza   krev   MeSH
• dítě MeSH
• epigeneze genetická MeSH
• gliom genetika   diagnóza   krev   patologie   diagnostické zobrazování   MeSH
• histony * genetika   metabolismus   krev   MeSH
• lidé MeSH
• mladiství MeSH
• mutace MeSH
• nádorové biomarkery krev   MeSH
• nádory mozkového kmene genetika   diagnóza   krev   diagnostické zobrazování   patologie   metabolismus   MeSH
• předškolní dítě MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

INTRODUCTION: Polyunsaturated fatty acids (PUFAs) are essential nutrients that humans obtain from their diet, primarily through fish oil consumption. However, fish oil production is no longer sustainable. An alternative approach is to produce PUFAs through marine microalgae. Despite the potential of algae strains to accumulate high concentrations of PUFAs, including essential fatty acids (EFAs), many aspects of PUFA production by microalgae remain unexplored and their current production outputs are frequently suboptimal. METHODS: In this study, we optimized biomass and selected ω-3 PUFAs production in two strains of algae, Schizochytrium marinum AN-4 and Schizochytrium limacinum CO3H. We examined a broad range of cultivation conditions, including pH, temperature, stirring intensity, nutrient concentrations, and their combinations. RESULTS: We found that both strains grew well at low pH levels (4.5), which could reduce bacterial contamination and facilitate the use of industrial waste products as substrate supplements. Intensive stirring was necessary for rapid biomass accumulation but caused cell disruption during lipid accumulation. Docosahexaenoic acid (DHA) yield was independent of cultivation temperature within a range of 28-34°C. We also achieved high cell densities (up to 9 g/L) and stable DHA production (average around 0.1 g/L/d) under diverse conditions and nutrient concentrations, with minimal nutrients required for stable production including standard sea salt, glucose or glycerol, and yeast extract. DISCUSSION: Our findings demonstrate the potential of Schizochytrium strains to boost industrial-scale PUFA production and make it more economically viable. Additionally, these results may pave the way for smaller-scale production of essential fatty acids in a domestic setting. The development of a new minimal culturing medium with reduced ionic strength and antibacterial pH could further enhance the feasibility of this approach.

• Publikační typ
• časopisecké články MeSH

Introduction: Pediatric brain tumours (PBT) are one of the most common malignancies during childhood, with variable severity according to the location and histological type. Certain types of gliomas, such a glioblastoma and diffuse intrinsic pontine glioma (DIPG), have a much higher mortality than ependymoma and medulloblastoma. Early detection of PBT is essential for diagnosis and therapeutic interventions. Liquid biopsies have been demonstrated using cerebrospinal fluid (CSF), mostly restricted to cell free DNA, which display limitations of quantity and integrity. In this pilot study, we sought to demonstrate the detectability and robustness of cell free histones in the CSF. Methods: We collected CSF samples from a pilot cohort of 8 children with brain tumours including DIPG, medulloblastoma, glioblastoma, ependymoma and others. As controls, we collected CSF samples from nine children with unrelated blood malignancies and without brain tumours. We applied a multichannel flow imaging approach on ImageStream(X) to image indiviual histone or histone complexes on different channels. Results: Single histones (H2A, macroH2A1.1, macroH2A1.2 H2B, H3, H4 and histone H3 bearing the H3K27M mutation), and histone complexes are specifically detectable in the CSF of PBT patients. H2A and its variants macroH2A1.1/macroH2A1/2 displayed the strongest signal and abundance, together with disease associated H3K27M. In contrast, mostly H4 is detectable in the CSF of pediatric patients with blood malignancies. Discussion: In conclusion, free histones and histone complexes are detectable with a strong signal in the CSF of children affected by brain tumours, using ImageStream(X) technology and may provide additive diagnostic and predictive information.

• Publikační typ
• časopisecké články MeSH

• Klíčová slova
• pahýlové lůžko, transfemorální amputace dolní končetiny,
• MeSH
• amputace klasifikace   ošetřování   rehabilitace   MeSH
• geriatrické ošetřovatelství MeSH
• lidé MeSH
• pahýl po amputaci chirurgie   MeSH
• protézy a implantáty klasifikace   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• techniky fyzikální terapie MeSH
• Check Tag
• lidé MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• Publikační typ
• přehledy MeSH

Nonalcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease in children and adolescents, increasing the risk of its progression toward nonalcoholic steatohepatitis (NASH), cirrhosis, and cancer. There is an urgent need for noninvasive early diagnostic and prognostic tools such as epigenetic marks (epimarks), which would replace liver biopsy in the future. We used plasma samples from 67 children with biopsy-proven NAFLD, and as controls we used samples from 20 children negative for steatosis by ultrasound. All patients were genotyped for patatin-like phospholipase domain containing 3 (PNPLA3), transmembrane 6 superfamily member 2 (TM6SF2), membrane bound O-acyltransferase domain containing 7 (MBOAT7), and klotho-β (KLB) gene variants, and data on anthropometric and biochemical parameters were collected. Furthermore, plasma cell-free DNA (cfDNA) methylation was quantified using a commercially available kit, and ImageStream(X) was used for the detection of free circulating histone complexes and variants. We found a significant enrichment of the levels of histone macroH2A1.2 in the plasma of children with NAFLD compared to controls, and a strong correlation between cfDNA methylation levels and NASH. Receiver operating characteristic curve analysis demonstrated that combination of cfDNA methylation, PNPLA3 rs738409 variant, coupled with either high-density lipoprotein cholesterol or alanine aminotransferase levels can strongly predict the progression of pediatric NAFLD to NASH with area under the curve >0.87. Conclusion: Our pilot study combined epimarks and genetic and metabolic markers for a robust risk assessment of NAFLD development and progression in children, offering a promising noninvasive tool for the consistent diagnosis and prognosis of pediatric NAFLD. Further studies are necessary to identify their pathogenic origin and function.

• MeSH
• dítě MeSH
• histony genetika   MeSH
• lidé MeSH
• lipasa genetika   MeSH
• membránové proteiny genetika   MeSH
• metylace DNA genetika   MeSH
• mladiství MeSH
• nealkoholová steatóza jater * diagnóza   MeSH
• pilotní projekty MeSH
• volné cirkulující nukleové kyseliny * metabolismus   MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mladiství MeSH
• Publikační typ
• časopisecké články MeSH

Onemocnění jater je obrovskou zdravotní zátěží po celém světě. Mezi hlavní rizikové faktory patří obezita, alkohol a virová hepatitida. Jejich patologickým vyústěním potom může být i ničivý hepatocelulární karcinom (HCC), v současné době rezistentní vůči chemoterapii. Chemoterapeutické léky indukují buněčnou senescenci, zatímco samotný epigenetický mechanismus zůstává nejasný. Histon makroH2A1.1, varianta histonu H2A, je marker senescence, ale účastní se také opravných procesů souvisejících s poškozením DNA. Podle našich nedávných zjištění existuje epigenetická vazba mezi makroH2A1.1 a karcinogenezí jater. Důvodem je skutečnost, že nadměrná exprese tohoto proteinu, typická pro rakovinu jater, způsobuje tzv. únik HCC buněk z DNA de-methylačního účinku léčiva decitabinu. Nové hypo-methylačního léčivo, guadecitabin, však vykazuje mimořádnou aktivitu při léčbě rakoviny jater. Proto je naším záměrem porozumět, jak toto léčivo překonává pro-tumorigenní, anti-senescentní a chemorezistentní účinky proteinu makroH2A1.1 a zabraňuje tak progresi onemocnění jater.; Liver diseases are a tremendous health burden worldwide. Obesity, in addition to alcohol and viral hepatitis, are major risks for liver disease, the pathological endpoint of which is the devastating hepatocellular carcinoma (HCC), which is currently refractory to chemotherapy. Chemotherapeutic drugs induce cellular senescence, but the epigenetic basis underlying these therapeutic effects remains unclear. The histone macroH2A1.1, a variant of histone H2A, is a marker of senescence and it is also implicated in DNA damage repair. Recently, we found an epigenetic link between macroH2A1.1 and liver carcinogenesis, whereby overexpression of this protein, which occurs in liver cancer, triggers an escape from the effect of the DNA demethylating anti-cancer agent decitabine. However, guadecitabine, a novel generation hypomethylating agent, demonstrated a superior activity against liver cancer. We propose to study here how this new hypomethylating agent can overcome the pro-tumorigenic, anti-senescence and chemoresistant effect of macroH2A1.1, preventing the progression of liver diseases.

• MeSH
• chemorezistence MeSH
• epigenomika MeSH
• hepatocelulární karcinom genetika   terapie   MeSH
• histony MeSH
• karcinogeneze MeSH
• protinádorové látky MeSH
• stárnutí buněk MeSH

• Konspekt
• Patologie. Klinická medicína
• NLK Obory
• onkologie
• genetika, lékařská genetika
• hepatologie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu AZV MZ ČR

BACKGROUND: Although metabolic associate fatty liver disease (MAFLD) is associated with obesity, it can also occur in lean patients. MAFLD is more aggressive in lean patients compared to obese patients, with a higher risk of mortality. Specific biomarkers to diagnose differentially lean or overweight MAFLD are missing. Histones and nucleosomes are released in the bloodstream upon cell death. Here, we propose a new, fast, imaging and epigenetics based approach to investigate the severity of steatosis in lean MAFLD patients. RESULTS: A total of 53 non-obese patients with histologically confirmed diagnosis of MAFLD were recruited. Twenty patients displayed steatosis grade 1 (0-33%), 24 patients with steatosis grade 2 (34-66%) and 9 patients with steatosis grade 3 (67-100%). The levels of circulating nucleosomes were assayed using enzyme-linked immunosorbent assay, while individual histones or histone dimers were assayed in serum samples by means of a new advanced flow cytometry ImageStream(X)-adapted method. Circulating nucleosome levels associated poorly with MAFLD in the absence of obesity. We implemented successfully a multi-channel flow methodology on ImageStream(X), to image single histone staining (H2A, H2B, H3, H4, macroH2A1.1 and macroH2A1.2). We report here a significant depletion of the levels of histone variants macroH2A1.1 and macroH2A1.2 in the serum of lean MAFLD patients, either individually or in complex with H2B. CONCLUSIONS: In summary, we identified a new circulating histone signature able to discriminate the severity of steatosis in individuals with lean MAFLD, using a rapid and non-invasive ImageStream(X)-based imaging technology.

• MeSH
• dospělí MeSH
• histony krev   MeSH
• hubenost krev   MeSH
• lidé středního věku MeSH
• lidé MeSH
• metabolické nemoci krev   komplikace   MeSH
• stupeň závažnosti nemoci MeSH
• ztučnělá játra krev   komplikace   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

As treatment options for patients with incurable metastatic castration-resistant prostate cancer (mCRPC) are considerably limited, novel effective therapeutic options are needed. Checkpoint kinase 1 (CHK1) is a highly conserved protein kinase implicated in the DNA damage response (DDR) pathway that prevents the accumulation of DNA damage and controls regular genome duplication. CHK1 has been associated with prostate cancer (PCa) induction, progression, and lethality; hence, CHK1 inhibitors SCH900776 (also known as MK-8776) and the more effective SCH900776 analog MU380 may have clinical applications in the therapy of PCa. Synergistic induction of DNA damage with CHK1 inhibition represents a promising therapeutic approach that has been tested in many types of malignancies, but not in chemoresistant mCRPC. Here, we report that such therapeutic approach may be exploited using the synergistic action of the antimetabolite gemcitabine (GEM) and CHK1 inhibitors SCH900776 and MU380 in docetaxel-resistant (DR) mCRPC. Given the results, both CHK1 inhibitors significantly potentiated the sensitivity to GEM in a panel of chemo-naïve and matched DR PCa cell lines under 2D conditions. MU380 exhibited a stronger synergistic effect with GEM than clinical candidate SCH900776. MU380 alone or in combination with GEM significantly reduced spheroid size and increased apoptosis in all patient-derived xenograft 3D cultures, with a higher impact in DR models. Combined treatment induced premature mitosis from G1 phase resulting in the mitotic catastrophe as a prestage of apoptosis. Finally, treatment by MU380 alone, or in combination with GEM, significantly inhibited tumor growth of both PC339-DOC and PC346C-DOC xenograft models in mice. Taken together, our data suggest that metabolically robust and selective CHK1 inhibitor MU380 can bypass docetaxel resistance and improve the effectiveness of GEM in DR mCRPC models. This approach might allow for dose reduction of GEM and thereby minimize undesired toxicity and may represent a therapeutic option for patients with incurable DR mCRPC.

• MeSH
• buněčná smrt účinky léků   MeSH
• checkpoint kinasa 1 antagonisté a inhibitory   metabolismus   MeSH
• chemorezistence účinky léků   MeSH
• deoxycytidin analogy a deriváty   farmakologie   MeSH
• docetaxel farmakologie   MeSH
• lidé MeSH
• mitóza * účinky léků   MeSH
• myši SCID MeSH
• nádorové buněčné linie MeSH
• nádory prostaty patologie   MeSH
• piperidiny chemie   farmakologie   MeSH
• proliferace buněk účinky léků   MeSH
• pyrazoly chemie   farmakologie   MeSH
• pyrimidiny chemie   farmakologie   MeSH
• S fáze účinky léků   MeSH
• xenogenní modely - testy protinádorové aktivity MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The aim of the study was to identify the optimum cultivation conditions for the microalgal growth and lipid production of the oleaginous microalga Chlorella pyrenoidosa Chick (IPPAS C2). Moreover, an appropriate NO3- concentration in the cultivation medium for maximized lipid accumulation was determined. The experimental design involved a biphasic cultivation strategy with an initial biomass accumulating phase under optimized light (400 μmol/m2 per s), temperature (25 °C), and elevated CO2 concentration in the air mixture (3%), followed by a mid-elevated CO2 concentration (0.5%) for lipid induction. The highest lipid yields of 172.47 ± 18.1 and 179.65 ± 25.4 mg/L per day were detected for NO3- concentrations of 100 and 150 mg/L. The optimization approach presented here led not only to the maximization of lipid yield but also to the development of a biphasic cultivation strategy easily applicable to the cultivation process without the necessity for algal cell harvesting between the first and second cultivation phases.

• MeSH
• biomasa MeSH
• Chlorella růst a vývoj   metabolismus   MeSH
• dusičnany metabolismus   MeSH
• fotobioreaktory MeSH
• kultivační média metabolismus   MeSH
• lipidy biosyntéza   MeSH
• mikrořasy růst a vývoj   metabolismus   MeSH
• oxid uhličitý metabolismus   MeSH
• techniky vsádkové kultivace MeSH
• teplota MeSH
• Publikační typ
• časopisecké články MeSH