The scope of the study was to apply Phenotype Biolog MicroArray (PM) technology to test the antibiotic sensitivity of the bacterial strains isolated from on-site wastewater treatment facilities. In the first step of the study, the percentage values of resistant bacteria from total heterotrophic bacteria growing on solid media supplemented with various antibiotics were determined. In the untreated wastewater, the average shares of kanamycin-, streptomycin-, and tetracycline-resistant bacteria were 53, 56, and 42%, respectively. Meanwhile, the shares of kanamycin-, streptomycin-, and tetracycline-resistant bacteria in the treated wastewater were 39, 33, and 29%, respectively. To evaluate the antibiotic susceptibility of the bacteria present in the wastewater, using the phenotype microarrays (PMs), the most common isolates from the treated wastewater were chosen: Serratia marcescens ss marcescens, Pseudomonas fluorescens, Stenotrophomonas maltophilia, Stenotrophomonas rhizophila, Microbacterium flavescens, Alcaligenes faecalis ss faecalis, Flavobacterium hydatis, Variovorax paradoxus, Acinetobacter johnsonii, and Aeromonas bestiarum. The strains were classified as multi-antibiotic-resistant bacteria. Most of them were resistant to more than 30 antibiotics from various chemical classes. Phenotype microarrays could be successfully used as an additional tool for evaluation of the multi-antibiotic resistance of environmental bacteria and in preliminary determination of the range of inhibition concentration.

• MeSH
• antibakteriální látky farmakologie   MeSH
• Bacteria klasifikace   účinky léků   genetika   izolace a purifikace   MeSH
• bakteriální léková rezistence MeSH
• čištění vody přístrojové vybavení   MeSH
• mikročipová analýza metody   MeSH
• odpadní voda chemie   mikrobiologie   MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH

Epithelial-mesenchymal interaction between stromal fibroblasts and cancer cells influences the functional properties of tumor epithelium, including the tumor progression and spread. We compared fibroblasts prepared from stroma of squamous cell carcinoma and normal dermal fibroblasts concerning their biological activity toward normal keratinocytes assessed by immunocytochemistry and profiling of gene activation for growth factors/cytokines by microarray chip technology. IGF-2 and BMP-4 were determined as candidate factors responsible for tumor-associated fibroblast activity that influences normal epithelia. This effect was confirmed by addition of recombinant IGF-2 and BMP4, respectively, to the culture medium. This hypothesis was also verified by inhibition experiments where blocking antibodies were employed in the medium conditioned by cancer-associated fibroblast. Presence of these growth factors was also detected in tumor samples.

• MeSH
• buňky NIH 3T3 MeSH
• čipová analýza proteinů MeSH
• fenotyp MeSH
• fibroblasty metabolismus   MeSH
• imunohistochemie MeSH
• insulinu podobný růstový faktor II biosyntéza   MeSH
• keratinocyty cytologie   metabolismus   MeSH
• kostní morfogenetický protein 4 biosyntéza   MeSH
• lidé MeSH
• myši MeSH
• nádory hlavy a krku metabolismus   patologie   MeSH
• rekombinantní proteiny biosyntéza   MeSH
• spinocelulární karcinom metabolismus   patologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Understanding the control of gene expression is critical for our understanding of the relationship between genotype and phenotype. The need for reliable assessment of transcript abundance in biological samples has driven scientists to develop novel technologies such as DNA microarray and RNA-Seq to meet this demand. This review focuses on comparing the two most useful methods for whole transcriptome gene expression profiling. Microarrays are reliable and more cost effective than RNA-Seq for gene expression profiling in model organisms. RNA-Seq will eventually be used more routinely than microarray, but right now the techniques can be complementary to each other. Microarrays will not become obsolete but might be relegated to only a few uses. RNA-Seq clearly has a bright future in bioinformatic data collection.

• MeSH
• sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů metody   MeSH
• sekvenční analýza RNA metody   MeSH
• stanovení celkové genové exprese metody   MeSH
• výpočetní biologie * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• srovnávací studie MeSH

This study combines mRNA and protein analysis using cDNA and antibody microarray techniques, respectively. These create a novel, integrated perspective into cellular molecular profiles. The aims of this study were to establish a reliable way of integrating these two approaches in order to obtain complex molecular profiles of the cell and to find suitable methods to normalize the data obtained using these approaches.

Antibody microarray and cDNA microarray techniques were used to study expression alterations in HL-60 cells that were differentiated into granulocytes using all-trans retinoic acid (ATRA). We selected this model to evaluate this combined profiling technique because the expression levels of most of the mRNA and protein species in these cells are not altered; therefore it is easier to track and define those species that are changed. The proteins whose levels were altered included c-myc, c-jun, Pyk2, FAK, PKC, TRF1, NF-kappaB and certain caspase types. These proteins are involved in apoptosis and hematopoietic differentiation pathways, and some have also been reported to have oncogenic potential. We compared the results obtained using the two methods, verified them by immunoblotting analysis, and devised normalization approaches.

This is one of the first demonstrations that a combination of antibody microarray and cDNA microarray techniques is required for complex molecular profiling of cells based on multiple parameters. This approach allows a more detailed molecular phenotype of the given sample to be obtained. The results obtained using a combination of the two profiling methods are consistent with those from previous studies that used more traditional methods.

• MeSH
• čipová analýza proteinů MeSH
• financování organizované MeSH
• fokální adhezní kinasa 2 analýza   MeSH
• geny myc MeSH
• HL-60 buňky MeSH
• lidé MeSH
• messenger RNA analýza   MeSH
• protein TRF1 analýza   MeSH
• sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
• tretinoin farmakologie   MeSH
• Check Tag
• lidé MeSH

OBJECTIVE: Hematopoietic stem cells (enriched in fraction of CD34+ cells) have the ability to regenerate hematopoiesis in all of its lineages, and this potential is clinically used in transplanting bone marrow or peripheral blood stem cells. Our objective was to assemble a suitable method for evaluating gene expression in enriched populations of hematopoietic stem cells. We compared biologic properties of cells cultured ex vivo obtained using two different ways of immunomagnetic separation (positive selection of CD34+ cells and negative selection of Lin- cells) by means of a cDNA microarray technique. METHODS: CD34+ and Lin- cells were enriched from peripheral blood stem cell (PBSCs) grafts of patients with non-Hodgkin's lymphoma. Isolated cells were in the presence of cytokine PBSCs, Flt-3 ligand, interleukin-3, interleukin-6, and granulocyte colony-stimulating factor. At days 0, 4, 6, 8, 10, 12, and 14 cells were harvested and analyzed by cDNA microarrays. Total cell expansion, CD34+, colony-forming unit for granulocyte-macrophage and megakaryocytes expansion, vitality, and phenotype of cells were also analyzed. RESULTS: cDNA microarray analysis of cultured hematopoietic cells proved equivalence of the two enrichment methods for PBSC samples and helped us characterize differentiating cells cultured ex vivo. CONCLUSION: Our methodologic approach is helpful in characterizing cultured hematopoietic cells cultured ex vivo, but it is also suitable for more general purposes. Equivalence of CD34+ and Lin- selection methods from PBSC samples proved by cDNA microarray may have an implication for graft manipulation in an experimental setting of hematopoietic transplantation. Total cell expansion and colony formation and phenotype from CD34+ selected and from Lin- samples were comparable.

• MeSH
• antigeny CD34 imunologie   MeSH
• financování organizované MeSH
• imunofenotypizace MeSH
• imunomagnetická separace MeSH
• komplementární DNA MeSH
• lidé MeSH
• lymfom genetika   imunologie   terapie   MeSH
• sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
• transplantace kmenových buněk MeSH
• Check Tag
• lidé MeSH

Technologie DNA čipů je v současné době nejúčinnější a nejrozšířenější metodou studia genové exprese. Počet prací využívajících tuto technologii v posledních letech exponenciálně narůstá a možnosti jejího využití se výrazně posunuly z oblasti experimentální do oblasti klinické. Ve výzkumu kolorektálních karcinomů bylo za posledních pět let využito DNA čipů ve více než šedesáti studiích. Tyto práce popisovaly schopnost čipů odlišit nádorovou tkáň od zdravé střevní sliznice, rozdělit nádory podle histopatologického stádia, anatomické lokalizace, mikrosatelitní nestability, determinovat znaky regionálních a vzdálených metastáz v primárních nádorech, predikovat léčebnou odpověď a identifikovat prognostické skupiny pacientů. Srovnatelnost a reprodukovatelnost výsledků DNA čipových studií bohužel výrazně ovlivňuje jejich technologická rozmanitost. Přesto jsme nalezli několik desítek genů, potenciálních markerů kolorektální karcinogeneze, jejichž pozměněná exprese v nádorové tkáni byla pozorována ve dvou a více nezávislých studiích. Slibné výsledky přinesly práce zaměřené na využití profilů genové exprese ke stanovení prognózy. Průměrná senzitivita predikce relapsu nádorového onemocnění, vzdálenostní progrese a délky celkového přežití pomocí profilů genové exprese byla pro všechny tři parametry přibližně 80%. Dobré analytické vlastnosti prokázaly DNA čipy také v predikci léčebné odpovědi. Klinické využití profilů genové exprese bude znamenat zásadní krok vedoucí směrem k individualizaci léčby a dispenzarizace pacientů s kolorektálními karcinomy.

DNA microarray technology is currently the most effective and widespread technique used for gene expression studies. Over the last years the number of reports related to this technology exponentially increases and possibilities of DNA microarrays usage markedly drifted from basic to clinical research. DNA microarrays were used in more than sixty studies focused on colorectal cancer during last five years. This reports show efficiency of this technology to distinguish tumor from normal colonic tissue and classify tumors in order to their pathological grade, anatomic localization and microsatellite status. Subsequent papers demonstrate abilities of gene expression profiles to determinate molecular signature of metastatic disease in primary tumors and predict therapy response and disease prognosis. However, comparability and reproducibility of studies based on DNA microarrays are notably affected by their technological diversity. Anyway, we found several genes (potential markers of colorectal carcinogenesis) with altered expression in tumors identified by two or more independent studies. Promising results were reached by gene expression profiles in prediction of colorectal cancer prognosis. DNA microarrays showed good analytical ability also in therapy response prediction. Clinical application of gene expression profiles will be the important advance leading to individualized therapy and dispensarization of patients with colorectal cancer.

• MeSH
• exprese genu genetika   účinky léků   MeSH
• financování organizované MeSH
• kolorektální nádory diagnóza   etiologie   genetika   MeSH
• lékařská onkologie metody   trendy   MeSH
• lidé MeSH
• metastázy nádorů diagnóza   genetika   MeSH
• mikrosatelitní nestabilita MeSH
• nádory podle lokalizace MeSH
• prognóza MeSH
• progrese nemoci MeSH
• sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů metody   využití   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• přehledy MeSH

BACKGROUND: To strengthen research and differential diagnostics of mitochondrial disorders, we constructed and validated an oligonucleotide microarray (h-MitoArray) allowing expression analysis of 1632 human genes involved in mitochondrial biology, cell cycle regulation, signal transduction and apoptosis. Using h-MitoArray we analyzed gene expression profiles in 9 control and 13 fibroblast cell lines from patients with F1Fo ATP synthase deficiency consisting of 2 patients with mt9205deltaTA microdeletion and a genetically heterogeneous group of 11 patients with not yet characterized nuclear defects. Analysing gene expression profiles, we attempted to classify patients into expected defect specific subgroups, and subsequently reveal group specific compensatory changes, identify potential phenotype causing pathways and define candidate disease causing genes. RESULTS: Molecular studies, in combination with unsupervised clustering methods, defined three subgroups of patient cell lines--M group with mtDNA mutation and N1 and N2 groups with nuclear defect. Comparison of expression profiles and functional annotation, gene enrichment and pathway analyses of differentially expressed genes revealed in the M group a transcription profile suggestive of synchronized suppression of mitochondrial biogenesis and G1/S arrest. The N1 group showed elevated expression of complex I and reduced expression of complexes III, V, and V-type ATP synthase subunit genes, reduced expression of genes involved in phosphorylation dependent signaling along MAPK, Jak-STAT, JNK, and p38 MAP kinase pathways, signs of activated apoptosis and oxidative stress resembling phenotype of premature senescent fibroblasts. No specific functionally meaningful changes, except of signs of activated apoptosis, were detected in the N2 group. Evaluation of individual gene expression profiles confirmed already known ATP6/ATP8 defect in patients from the M group and indicated several candidate disease causing genes for nuclear defects. CONCLUSION: Our analysis showed that deficiency in the ATP synthase protein complex amount is generally accompanied by only minor changes in expression of ATP synthase related genes. It also suggested that the site (mtDNA vs nuclear DNA) and the severity (ATP synthase content) of the underlying defect have diverse effects on cellular gene expression phenotypes, which warrants further investigation of cell cycle regulatory and signal transduction pathways in other OXPHOS disorders and related pharmacological models.

• MeSH
• analýza hlavních komponent MeSH
• buněčné linie MeSH
• fenotyp MeSH
• fibroblasty MeSH
• financování organizované MeSH
• genom mitochondriální MeSH
• lidé MeSH
• mitochondriální DNA genetika   MeSH
• mitochondriální nemoci MeSH
• mitochondriální protonové ATPasy genetika   nedostatek   MeSH
• modely genetické MeSH
• sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů metody   statistika a číselné údaje   MeSH
• sekvenční delece MeSH
• shluková analýza MeSH
• stanovení celkové genové exprese metody   statistika a číselné údaje   MeSH
• Check Tag
• lidé MeSH

Under physiological conditions, human ovarian granulosa cells (GCs), are responsible for a number of processes associated with folliculogenesis and oogenesis. The primary functions of GCs in the individual phases of follicle growth are: Hormone production in response to follicle stimulating hormone (FSH), induction of ovarian follicle atresia through specific molecular markers and production of nexus cellular connections for communication with the oocyte. In recent years, interest in obtaining stem cells from particular tissues, including the ovary, has increased. Special attention has been paid to the novel properties of GCs during long‑term in vitro culture. It has been demonstrated that the usually recycled material in the form of follicular fluid can be a source of cells with stem‑like properties. The study group consisted of patients enrolled in the in vitro fertilization procedure. Total RNA was isolated from GCs at 4 time points (after 1, 7, 15 and 30 days of culture) and was used for microarray expression analysis (Affymetrix® Human HgU 219 Array). The expression of 22,480 transcripts was examined. The selection of significantly altered genes was based on a P‑value <0.05 and expression higher than two‑fold. The leucine rich repeat containing 17, collagen type I α1 chain, bone morphogenetic protein 4, twist family bHLH transcription factor 1, insulin like growth factor binding protein 5, GLI family zinc finger 2 and collagen triple helix repeat containing genes exhibited the highest changes in expression. Reverse‑transcription‑quantitative PCR was performed to validate the results obtained in the analysis of expression microarrays. The direction of expression changes was validated in the majority of cases. The presented results indicated that GCs have the potential of cells that can differentiate towards osteoblasts in long‑term in vitro culture conditions. Increased expression of genes associated with the osteogenesis process suggests a potential for uninduced change of GC properties towards the osteoblast phenotype. The present study, therefore, suggests that GCs may become an excellent starting material in obtaining stable osteoblast cultures. GCs differentiated towards osteoblasts may be used in regenerative and reconstructive medicine in the future.

• MeSH
• buněčná diferenciace * MeSH
• diferenciační antigeny biosyntéza   MeSH
• dospělí MeSH
• folikulární buňky metabolismus   patologie   MeSH
• lidé MeSH
• mladiství MeSH
• osteoblasty metabolismus   patologie   MeSH
• regulace genové exprese * MeSH
• sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů * MeSH
• stanovení celkové genové exprese * MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Whole exome sequencing is a powerful tool for the analysis of genetically heterogeneous conditions. The prioritization of variants identified often focuses on nonsense, frameshift and canonical splice site mutations, and highly deleterious missense variants, although other defects can also play a role. The definition of the phenotype range and course of rare genetic conditions requires long-term clinical follow-up of patients. CASE PRESENTATION: We report an adult female patient with severe intellectual disability, severe speech delay, epilepsy, autistic features, aggressiveness, sleep problems, broad-based clumsy gait and constipation. Whole exome sequencing identified a de novo mutation in the SYNGAP1 gene. The variant was located in the broader splice donor region of intron 10 and replaced G by A at position +5 of the splice site. The variant was predicted in silico and shown experimentally to abolish the regular splice site and to activate a cryptic donor site within exon 10, causing frameshift and premature termination. The overall clinical picture of the patient corresponded well with the characteristic SYNGAP1-associated phenotype observed in previously reported patients. However, our patient was 31 years old which contrasted with most other published SYNGAP1 cases who were much younger. Our patient had a significant growth delay and microcephaly. Both features normalised later, although the head circumference stayed only slightly above the lower limit of the norm. The patient had a delayed puberty. Her cognitive and language performance remained at the level of a one-year-old child even in adulthood and showed a slow decline. Myopathic facial features and facial dysmorphism became more pronounced with age. Although the gait of the patient was unsteady in childhood, more severe gait problems developed in her teens. While the seizures remained well-controlled, her aggressive behaviour worsened with age and required extensive medication. CONCLUSIONS: The finding in our patient underscores the notion that the interpretation of variants identified using whole exome sequencing should focus not only on variants in the canonical splice dinucleotides GT and AG, but also on broader splice regions. The long-term clinical follow-up of our patient contributes to the knowledge of the developmental trajectory in individuals with SYNGAP1 gene defects.

• MeSH
• dospělí MeSH
• exom MeSH
• fenotyp MeSH
• genetická variace MeSH
• genomika MeSH
• karyotypizace MeSH
• lidé MeSH
• mentální retardace diagnóza   genetika   MeSH
• mikrocefalie diagnóza   genetika   MeSH
• mikročipová analýza MeSH
• mutace MeSH
• následné studie MeSH
• proteiny aktivující GTPasu ras genetika   MeSH
• sekvence nukleotidů MeSH
• sekvenční analýza DNA MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH

DiGeorgeův syndrom (velokardiofaciální syndrom, VCFS, 22q11DS) je způsobený mikrodelecemi přibližně 40 genů na jedné kopii chromosomu 22. Projevem syndromu je variabilní kombinace více než 190 fenotypových příznaků, mezi nejčastější patří vrozené vady srdce, orofaciální rozštěp a velofaryngeální insuficience. Končetinové defekty v oblasti rukou a nohou nepatří do klasického klinického obrazu DiGeorgeova syndromu. Cílem práce bylo u pacienta s typickým fenotypovým projevem 22q11DS a současně s raritním nálezem těžké ektrodaktylie na všech čtyřech končetinách zhodnotit rozsah genomické odchylky metodou celogenomových SNP arrayí za účelem vysvětlení končetinové vady. Bylo provedeno vyšetření metodou FISH, sekvenace genu TP63 a celogenomová SNP array pomocí kitu HumanCytoSNP-12 DNA Analysis Beadchip (Illumina Inc.). Metodou FISH byl potvrzen DiGeorgeův syndrom, sekvenace genu TP63 neodhalila patogenní mutaci, vyšetření metodou SNP arrayí potvrdilo deleci v oblasti 22q11.2 obvyklého rozsahu 2,9 Mb. Nebyla potvrzena původní hypotéza, že by se u pacienta mohlo jednat o deleci 22q11.2 většího rozsahu, než je obvyklé, která by zahrnovala gen asociovaný s vývojem končetin a vysvětlila těžký končetinový defekt. Ačkoli končetinové defekty nepředstavují typickou patologii v rámci DiGeorgeova syndromu, je nutno myslet na tuto diagnózu u každého novorozence s vrozenou srdeční vadou, rozštěpem obličeje a anomáliemi končetin. Přestože je u DiGeorgeova syndromu běžná značná variabilní expresivita, nelze u našeho pacienta vyloučit také náhodnou koincidenci dvou nezávislých genetických vad, DiGeorgeova syndromu v důsledku mikrodelece 22q11.2 a ektrodaktylie způsobené mutací jiného/jiných genů.

DiGeorge syndrome (velocardiofacial syndrome, VCFS, 22q11DS) is caused by a microdeletion of approximately 40 genes from one copy of chromosome 22. Expression of the syndrome is a variable combination of more than 190 phenotypic characteristics, the most common are congenital heart disease, cleft palate and velopharyngeal insufficiency. Limb anomalies of hands and feets are not a typical clinical symptoms of DiGeorge syndrome. The aim of the study was to evaluate the extent of genomic changes in a 4.5 years old child with classical phenotypic features of 22q11DS in combination with severe ectrodactyly of all four limbs in addition; using microarray based single nucleotide polymorphism techniques in order to explain the origin of limb anomaly defects. FISH analysis, TP63 gene sequencing and whole genome SNP array analysis using HumanCytoSNP-12 DNA Analysis Beadchip (Illumina Inc.) were performed. FISH method revealed DiGeorge syndrome, TP63 gene sequencing did not detect any deleterious mutation, microarray SNP analysis established microdeletion of 22q11.2 region in standard defined range of 2,9 Mb. The original possible hypothesis, that in a patient it could exist a much more genomic changes than only these related to 22q11.2 region, involving genes associated with limb development and formation and thus explaining devastating limbs damage in patient, unfortunately was not confirmed. Although limb defects do not belong to the typical pathogenic signs of DiGeorge syndrome, this diagnosis should be considered in all neonate with congenital heart defect, cleft palate and limb anomalies. Even though variable phenotypical expressivity in DiGeorge syndrome is frequent, we can not exclude the possibility of coincidence of two independent genetic defects in our patient; DiGeorge syndrome due to 22q11.2 microdeletion and ectrodactyly which results of another gene/s mutation.

• Klíčová slova
• SNP microarray, ektrodaktylie, ruka s rozštěpem, klepeto, split hand/split foot malformation,
• MeSH
• chromozomální delece MeSH
• cytogenetické vyšetření MeSH
• DiGeorgeův syndrom * diagnóza   genetika   MeSH
• fenotyp MeSH
• hybridizace in situ fluorescenční MeSH
• lidé MeSH
• lidské chromozomy, pár 22 MeSH
• nádorové supresorové proteiny genetika   MeSH
• novorozenec MeSH
• předškolní dítě MeSH
• rozštěp patra MeSH
• rozštěp rtu MeSH
• sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
• syndaktylie MeSH
• transkripční faktory genetika   MeSH
• vrozené deformity nohy (od hlezna dolů) MeSH
• vrozené deformity ruky MeSH
• vrozené srdeční vady MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• novorozenec MeSH
• předškolní dítě MeSH
• Publikační typ
• kazuistiky MeSH
• práce podpořená grantem MeSH