The immunohistochemical (IHC) or fluorescence/chromogenic in situ hybridization (FISH/CISH) assays for assessing HER2 are now recommended by the American Society of Clinical Oncologists and the College of American Pathologists, but there are an increasing number of published studies describing alternative diagnoses at the molecular level. Inspired by these studies, we established a laboratory-developed test (LDT) to analyze HER2 status not only at the gene expression level but also at the gene copy number. A precise copy number calculation was fulfilled including the Control Genomic DNA of known concentration, which allowed subsequent assay validation at the DNA level. The results were reported according to the concordant results of the DNA and RNA approaches. By comparing with IHC determination, completely identical results were found in ten blank samples, which underlines the legitimacy of molecular biological approaches in this diagnostic field. An equivocal sample that was positive by IHC and qPCR was found to be negative by the FISH and so it may change the choice of personalized medicine. The topic of this short communication will hopefully contribute to allowing IVD-certified diagnostics based on the HER2 gene expression profile or copy number to be tested in the Czech Republic as well.

• MeSH
• DNA genetika   metabolismus   MeSH
• genová dávka * MeSH
• hybridizace in situ fluorescenční * metody   MeSH
• imunohistochemie * metody   MeSH
• lidé MeSH
• nádorové biomarkery genetika   metabolismus   MeSH
• nádory prsu genetika   metabolismus   diagnóza   MeSH
• receptor erbB-2 * genetika   metabolismus   MeSH
• RNA metabolismus   genetika   analýza   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Our research has developed a highly sensitive and simple assay to detect small amounts of animal and human biological material in less than 40 min. The handheld SaLux19 device developed at the Max Planck Institute of Experimental Medicine in Göttingen, Germany, was used to validate our concept. The proposed system uses isothermal amplification of DNA in a rapid assay format. Our results show that the assay can detect Sus scrofa nucleic acids with very high sensitivity and specificity. This detection system has potential for forensic scenarios.

• Publikační typ
• časopisecké články MeSH

Epigenetic aberrations are well known to play an important role in carcinogenesis, and also have a great potential to serve as biomarkers in many types of cancers, including ovarian cancer in which sensitive and specific biomarkers and detection methods are critically needed. The aim of this study was to investigate methylation of cadherin genes CDH10, CDH13 and CDH18 in ovarian cancer tissue by comparison with control tissue. The study group consisted of 38 patients with ovarian cancer and 25 control patients. For detection of epigenetic events we used next generation sequencing, the most important data were confirmed using high-resolution melting analysis and real-time PCR. We observed significantly higher methylation in CDH13, sporadic methylation in CDH10 and loss of methylation in CDH18 in the ovarian cancer group compared with the control group. These observations suggest that changes in methylation of cadherin genes may be one of the major mechanisms associated with ovarian cancer progression. In addition, because of the high frequency of methylation of the CDH13 gene in the early stages of ovarian cancer, the analyzed CpG sites might be good targets for next study of potential ovarian cancer screening biomarkers.

• MeSH
• dospělí MeSH
• kadheriny genetika   MeSH
• lidé středního věku MeSH
• lidé MeSH
• metylace DNA * MeSH
• mladý dospělý MeSH
• mucinózní adenokarcinom genetika   patologie   MeSH
• nádorové biomarkery genetika   MeSH
• nádory endometria genetika   patologie   MeSH
• nádory vaječníků genetika   patologie   MeSH
• následné studie MeSH
• prognóza MeSH
• promotorové oblasti (genetika) MeSH
• regulace genové exprese u nádorů * MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• serózní cystadenokarcinom genetika   patologie   MeSH
• studie případů a kontrol MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

DNA methylation is well-known to be associated with ovarian cancer (OC) and has great potential to serve as a biomarker in monitoring response to therapy and for disease screening. The purpose of this study was to investigate methylation of HNF1B and GATA4 and correlate detected methylation with clinicopathological characteristic of OC patients. The study group consisted of 64 patients with OC and 35 control patients. To determine the most important sites of HNF1B and GATA4, we used next-generation sequencing. For further confirmation of detected methylation of selected regions, we used high-resolution melting analysis and methylation-specific real-time polymerase chain reaction (PCR). Selected regions of HNF1B and GATA4 were completely methylation free in all control samples, whereas methylation-positive pattern was observed in 32.8% (HNF1B) and 45.3% (GATA4) of OC samples. Evaluating both genes together, we were able to detect methylation in 65.6% of OC patients. We observed a statistically significant difference in HNF1B methylation between samples with different stages of OC. We also detected subtype specific methylation in GATA4 and a decrease of methylation in late stages of OC. The combination of unmethylated HNF1B and methylated GATA4 was associated with longer overall survival. In our study, we employed innovative approach of methylation analysis of HNF1B and GATA4 to search for possible epigenetic biomarkers. We confirmed the significance of the HNF1B and GATA4 hypermethylation with emphasis on the need of selecting the most relevant sites for analysis. We suggest selected CpGs to be further examined as a potential positive prognostic factor.

• MeSH
• dospělí MeSH
• hepatocytární jaderný faktor 1-beta genetika   metabolismus   MeSH
• Kaplanův-Meierův odhad MeSH
• lidé středního věku MeSH
• lidé MeSH
• metylace DNA * MeSH
• nádorové biomarkery * MeSH
• nádory vaječníků diagnóza   genetika   metabolismus   mortalita   MeSH
• následné studie MeSH
• prognóza MeSH
• promotorové oblasti (genetika) MeSH
• regulace genové exprese u nádorů MeSH
• senioři MeSH
• staging nádorů MeSH
• stupeň nádoru MeSH
• transkripční faktor GATA4 genetika   metabolismus   MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Kongenitální centrální hypoventilační syndrom (CCHS) je vzácná celoživotní porucha dechového centra, s genetickým podkladem v mutaci genu PHOX2B. Onemocnění se obvykle manifestuje v novorozeneckém věku hypoventilací/apnoí ve spánku. Klinická tíže hypoventilace a riziko rozvoje přidružených onemocnění (Hirschsprungova choroba, nádory neurální lišty, poruchy autonomního nervového systému) se liší dle typu mutace PHOX2B genu. U 90 % pa­cientů se jedná o heterozygotní mutaci typu PARMs (Polyalanin Repeat Mutations), u zbylých 10 % pa­cientů nacházíme heterozygotní mutaci typu missence, nonsense nebo frameshift (NON‑PARMs). U prezentovaných novorozenců s geneticky potvrzeným CCHS byla v prvním týdnu po narození nápadná hypoventilace/apnoe vedoucí k významné hyperkapnii a hypoxémii. Po velmi krátké potřebě umělé plicní ventilace se fyziologické krevní plyny dařilo udržovat pomocí neinvazivní spánkové ventilace. S moderními technikami pro domácí ventilaci a sledováním ve specializovaných centrech mají děti s CCHS dlouhodobě dobrou prognózu s nízkou mortalitou.

Congenital central hypoventilation syndrome (CCHS) is a rare, lifelong disorder of the breathing centre, resulting from a mutation of the PHOX2B gene. CCHS typically manifests in newborns with alveolar hypoventilation/apnea during sleep. Clinical severity of hypoventilation and a risk of associated conditions (Hirschsprung disease, tumors of neural crest and autonomic nervous system dysfunction) depend on the type of the PHOX2B gene mutation. Approximately 90% of individuals with the CCHS phenotype are heterozygous for the PARMs‑type mutation (Polyalanine Repeat Expansion Mutations), the remaining 10% of patients express heterozygous missense‑, nonsense‑ or frameshift‑type mutation (non‑PARMs). Significant hypoventilation leading to severe hypercapnia and hypoxemia was observed in two of our newborns with proved CCHS during the first week after birth. Following a very short period of mechanical ventilation, we succeeded in maintaining physiological blood gases with noninvasive ventilation during sleep. With modern techniques for home ventilation and follow up at specialized centres, children with CCHS have good long‑term prognosis and low mortality. Key words: central hypoventilation – Ondine‘s Curse The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manu­script met the ICMJE “uniform requirements” for biomedical papers.

• MeSH
• genotyp MeSH
• homeodoménové proteiny genetika   MeSH
• hypoventilace * diagnóza   genetika   patofyziologie   terapie   MeSH
• lidé MeSH
• novorozenec nedonošený MeSH
• novorozenec MeSH
• plicní ventilace MeSH
• posunová mutace genetika   MeSH
• sekvenční analýza DNA MeSH
• spánková apnoe centrální * diagnóza   genetika   patofyziologie   terapie   MeSH
• transkripční faktory genetika   MeSH
• trvalý přetlak v dýchacích cestách MeSH
• ventilace umělá s výdechovým přetlakem MeSH
• Check Tag
• lidé MeSH
• novorozenec MeSH
• ženské pohlaví MeSH
• Publikační typ
• kazuistiky MeSH
• práce podpořená grantem MeSH

Stress-induced fibroblast senescence is thought to contribute to skin aging. Ultraviolet light (UV) radiation is the most potent environmental risk factor in these processes. An Epilobium angustifolium (EA) extract was evaluated for its capacity to reverse the senescent response of normal human dermal fibroblasts (NHDF) in vitro and to exhibit skin photo-protection in vivo. The HPLC-UV-MS analysis of the EA preparation identified three major polyphenol groups: tannins (oenothein B), phenolic acids (gallic and chlorogenic acids) and flavonoids. EA extract increased the cell viability of senescent NHDF induced by serum deprivation. It diminished connective tissue growth factor and fibronectin gene expressions in senescent NHDF. Down-regulation of the UV-induced release of both matrix metalloproteinase-1 and -3 and the tissue inhibitor of matrix metalloproteinases-1 and -2, and also down-regulation of the gene expression of hyaluronidase 2 were observed in repeatedly UV-irradiated NHDF after EA extract treatment. Interestingly, EA extract diminished the down-regulation of sirtuin 1 dampened by UV-irradiation. The application of EA extract using a sub-irritating dose protected skin against UV-induced erythema formation in vivo. In summary, EA extract diminished stress-induced effects on NHDF, particularly on connective tissue growth factor, fibronectin and matrix metalloproteinases. These results collectively suggest that EA extract may possess anti-aging properties and that the EA polyphenols might account for these benefits.

• MeSH
• dítě MeSH
• dospělí MeSH
• down regulace účinky léků   účinky záření   MeSH
• Epilobium chemie   MeSH
• erytém farmakoterapie   etiologie   MeSH
• extracelulární matrix účinky léků   metabolismus   účinky záření   MeSH
• fenotyp MeSH
• fibroblasty cytologie   účinky léků   metabolismus   účinky záření   MeSH
• fibronektiny genetika   MeSH
• GPI-vázané proteiny genetika   MeSH
• hyaluronoglukosaminidasa genetika   MeSH
• kůže cytologie   účinky léků   účinky záření   MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• molekuly buněčné adheze genetika   MeSH
• radioprotektivní látky chemie   farmakologie   terapeutické užití   MeSH
• regulace genové exprese účinky léků   účinky záření   MeSH
• rostlinné extrakty chemie   farmakologie   terapeutické užití   MeSH
• růstový faktor pojivové tkáně genetika   MeSH
• sirtuin 1 genetika   MeSH
• stárnutí buněk účinky léků   účinky záření   MeSH
• stárnutí kůže účinky léků   účinky záření   MeSH
• ultrafialové záření škodlivé účinky   MeSH
• viabilita buněk účinky léků   účinky záření   MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Glucomannans belong to yeast and fungal cell wall polysaccharides with known immunostimulatory and radioprotective effects. However, glucomannan protective effects against pathological consequences of skin exposure to short wavelength solar light, ultraviolet (UV) radiation, are unclear. Herein, a highly branched glucomannan (GM) isolated from the cell wall of Candida utilis, a member of the alpha-(1-->6)-D-mannan group, was tested for its photoprotective effects in an in vitro model of UVB-irradiated human keratinocytes and an in vivo model of UV-induced erythema formation in human volunteers. GM suppressed the UVB-induced decrease of keratinocyte viability, which was connected with the suppression of UVB-induced keratinocyte apoptosis. GM reduced UVB-mediated caspase activation together with suppression of DNA fragment release into the cytoplasm. Furthermore, GM suppressed UVB-induced gene expression of pro-inflammatory markers including nuclear factor kappa B, inducible nitric oxide synthase, interleukins 8 and 1, together with suppression of prostaglandin E2 and interleukin 1alpha protein release. In vivo, GM decreased UV-induced skin erythema formation, which was correlated with a decrease of phosholipase A(2) activity within the stratum corneum. It could be concluded that GM isolated from C. utilis possesses significant photoprotective effects on human keratinocytes in vitro as well as in vivo.

• MeSH
• apoptóza účinky léků   účinky záření   MeSH
• biologické markery analýza   MeSH
• Candida chemie   MeSH
• erytém etiologie   prevence a kontrola   MeSH
• exprese genu MeSH
• financování organizované MeSH
• hodnocení léčiv MeSH
• keratinocyty účinky léků   účinky záření   MeSH
• kultivované buňky MeSH
• lidé MeSH
• mannany aplikace a dávkování   farmakologie   izolace a purifikace   MeSH
• radioprotektivní látky aplikace a dávkování   farmakologie   MeSH
• ultrafialové záření škodlivé účinky   MeSH
• viabilita buněk účinky léků   MeSH
• zánět MeSH
• Check Tag
• lidé MeSH

Hepatic stellate cells (HSC) and liver myofibroblasts (MFB) are two cell populations most likely responsible for the synthesis of most connective tissue components in fibrotic liver. They differ in their origin and location, and possibly in patterns of gene expression. Normal and carbon tetrachloride-cirrhotic livers from rats were used to isolate HSC. Liver was perfused with pronase and collagenase solutions, followed by centrifugation of the cell suspension on a density gradient. HSC were quiescent 2 days after plating on plastic but they became activated after another 5 days in culture. When the culture was passaged 5 times, its character changed profoundly as HSC were replaced by MFB. Microarray analysis was used to determine gene expression in quiescent HSC, activated HSC and MFB. The expression of 49 genes coding for connective tissue proteins, proteoglycans, metalloproteinases and their inhibitors, growth factors and cellular markers was determined. The pattern of gene expression changed during HSC activation and there were distinct differences between HSC and MFB. Little difference between normal cells and cells isolated from cirrhotic liver was found.

• MeSH
• experimentální cirhóza jater metabolismus   MeSH
• exprese genu MeSH
• extracelulární matrix chemie   MeSH
• fibroblasty cytologie   metabolismus   účinky léků   MeSH
• financování organizované MeSH
• imunohistochemie MeSH
• játra cytologie   metabolismus   účinky léků   MeSH
• krysa rodu rattus MeSH
• kultivované buňky MeSH
• messenger RNA biosyntéza   účinky léků   MeSH
• metaloproteasy genetika   metabolismus   MeSH
• myocyty hladké svaloviny cytologie   metabolismus   účinky léků   MeSH
• otrava chloridem uhličitým MeSH
• pojivová tkáň metabolismus   účinky léků   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• proteoglykany genetika   metabolismus   MeSH
• sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
• tkáňové inhibitory metaloproteinas MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH
• Publikační typ
• srovnávací studie MeSH

• MeSH
• exony MeSH
• hypertrofická kardiomyopatie genetika   MeSH
• lidé MeSH
• mutace MeSH
• plošný screening MeSH
• těžké řetězce myosinu analýza   genetika   MeSH
• Check Tag
• lidé MeSH