Cíl studie: Seznámení s preimplantační diagnostikou (PGD) metodou fluorescenční in situ hybridizace (FISH) a s některými faktory, které zásadně ovlivňují výsledky získané touto technikou z pohledu molekulárního cytogenetika. Jsou shrnuty jednotlivé kritické kroky práce s jednou buňkou při PGD-FISH jak z literárních údajů, tak z vlastních experimentálních poznatků. Jednotlivé kroky jsou analyzovány a jsou uvedeny možnosti jejich řešení. Typ studie: Přehledový článek. Název a sídlo pracoviště: Ústav lékařské genetiky a fetální medicíny FN a LF UP, Olomouc, Centrum asistované reprodukce při Porodnicko-gynekologické klinice FN, Olomouc. Předmět a metoda studie: Úvod do problematiky PGD. Přehled a rozbor faktorů, na kterých závisí úspěšnost metody FISH při PGD. – Výběr embryí pro experimentální fázi PGD-FISH. – Volba fixační metody a předzpracování (pretreatment), použití vhodných sond. – Zaměření fixované buňky. – Interpretace získaných výsledků. Navržení způsobu jejich úspěšného řešení. Závěr: V práci je podáno přehledné zpracování metody PGD-FISH. Jsou zdůrazněny a zhodnoceny některé kritické aspekty analýzy jednobuněčného preparátu a navrženy možnosti a postupy jejich optimálního řešení.

Objective: Information on preimplantation diagnostics (PGD) by means of the method of fluorescent in situ hybridization (FISH) and some factors, which principally influence the results obtained with this technique from the point of view of a molecular cytogeneticist. Critical steps of work with one cell in PGD-FISH are summarized based on data from literature as well as from the authors, own experimental experience. The individual steps are analyzed and possibilities of execution are presented. Type of study: A review. Setting: Institute of Medical Genetics and Fetal Medicine of Faculty Hospital and Medical Faculty of Palacky University, Center of Assisted Reproduction at Obstetric-Gynecological Clinic, Faculty Hospital, Olomouc. Subject and Methods of Study: Introduction into the PGD problem. A survey and analysis of factors decisive for the success of the FISH method for PGD. – Selection of embrya for the experimental phase of PGD-FISH. – Selection of fixation method and pretreatment, application of suitable probes. – Localization of the fixed cell. – Interpretation of the results obtained. Recommendation of the mode of their successful solution. Conclusion: The paper presents a survey of the PGD-FISH method. Some critical aspects in the analysis of unicellular preparation are pointed out and evaluated and possibilities and procedures of the optimal solution are suggested.

• MeSH
• aneuploidie MeSH
• blastomery MeSH
• embryo savčí patologie   MeSH
• fertilizace in vitro MeSH
• finanční podpora výzkumu jako téma MeSH
• fixativa chemie   MeSH
• hybridizace in situ fluorescenční metody   MeSH
• lidé MeSH
• preimplantační diagnóza metody   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• srovnávací studie 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 genetika   MeSH
• Bacteria genetika   MeSH
• bakteriální RNA genetika   MeSH
• DNA sondy MeSH
• fylogeneze MeSH
• hybridizace in situ fluorescenční * MeSH
• mikrobiologické techniky * MeSH
• průtoková cytometrie MeSH
• RNA ribozomální 16S genetika   MeSH
• separace buněk * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

In endemic species that co-occur with widespread congeners, hybridization can lead to an influx of novel and beneficial genetic variation, but high rates of introgression may cause genetic swamping of the endemic species and have detrimental effects on its survival potential. This study examines hybridization between sympatric populations of the Carpathian barbel Barbus carpathicus, a recently discovered cryptic species with a restricted range, and the widespread common barbel Barbus barbus. Based on six diagnostic allozyme loci, a microsatellite locus and mtDNA, hybrids were found to be present at multiple localities within the Vistula River drainage (Baltic Sea) as well as in the Tisza River system of the Danube River drainage (Black Sea). However, the numbers of hybrids were very low; four individuals of 230 fish sampled from the Vistula drainage. Bayesian assessment of their nuclear genotypes suggested that two hybrids in the Vistula drainage and nine in the Tisza system were F1 generation, and one in the Vistula drainage and one in the Tisza system were backcrosses (BC) to B. barbus, while no F2 or BC to B. carpathicus were detected. No hybrid carried B. carpathicus mtDNA and cytonuclear linkage disequilibria showed significant positive associations between hybrid genotypes and B. barbus mtDNA, suggesting unidirectionality in the interspecific mating with a disproportionate contribution of B. barbus mothers. Despite geographically broad occurrence of hybrids, these data provide evidence of strong constraints on hybridization in the native breeding habitats and the lack of introgression towards B. carpathicus.

• MeSH
• alely MeSH
• chiméra MeSH
• Cyprinidae genetika   MeSH
• genetická variace MeSH
• genotyp MeSH
• hybridizace genetická MeSH
• izoenzymy analýza   MeSH
• mikrosatelitní repetice MeSH
• mitochondriální DNA genetika   MeSH
• multilokusová sekvenční typizace MeSH
• populační genetika MeSH
• řeky MeSH
• sekvenční analýza DNA MeSH
• vazebná nerovnováha MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Slovenská republika MeSH

Hybridization and introgression can impact the evolution of natural populations. Several wild canid species hybridize in nature, sometimes originating new taxa. However, hybridization with free-ranging dogs is threatening the genetic integrity of grey wolf populations (Canis lupus), or even the survival of endangered species (e.g., the Ethiopian wolf C. simensis). Efficient molecular tools to assess hybridization rates are essential in wolf conservation strategies. We evaluated the power of biparental and uniparental markers (39 autosomal and 4 Y-linked microsatellites, a melanistic deletion at the β-defensin CBD103 gene, the hypervariable domain of the mtDNA control-region) to identify the multilocus admixture patterns in wolf x dog hybrids. We used empirical data from 2 hybrid groups with different histories: 30 presumptive natural hybrids from Italy and 73 Czechoslovakian wolfdogs of known hybrid origin, as well as simulated data. We assessed the efficiency of various marker combinations and reference samples in admixture analyses using 69 dogs of different breeds and 99 wolves from Italy, Balkans and Carpathian Mountains. Results confirmed the occurrence of hybrids in Italy, some of them showing anomalous phenotypic traits and exogenous mtDNA or Y-chromosome introgression. Hybridization was mostly attributable to village dogs and not strictly patrilineal. The melanistic β-defensin deletion was found only in Italian dogs and in putative hybrids. The 24 most divergent microsatellites (largest wolf-dog FST values) were equally or more informative than the entire panel of 39 loci. A smaller panel of 12 microsatellites increased risks to identify false admixed individuals. The frequency of F1 and F2 was lower than backcrosses or introgressed individuals, suggesting hybridization already occurred some generations in the past, during early phases of wolf expansion from their historical core areas. Empirical and simulated data indicated the identification of the past generation backcrosses is always uncertain, and a larger number of ancestry-informative markers is needed.

• MeSH
• beta-defensiny genetika   MeSH
• chromozom Y MeSH
• genetická variace MeSH
• genetické markery * MeSH
• genotyp MeSH
• hybridizace genetická * MeSH
• mikrosatelitní repetice MeSH
• mitochondriální DNA MeSH
• molekulární evoluce MeSH
• multilokusová sekvenční typizace * MeSH
• populační genetika MeSH
• psi MeSH
• shluková analýza MeSH
• vlci MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• psi MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Itálie MeSH

Complex chromosomal rearrangements (CCR) represent rare structural chromosome abnormalities frequently associated with infertility. In this study, meiotic segregation in spermatozoa of an infertile normospermic carrier of a 4-breakpoint t(1;3;6) CCR was analysed. A newly developed array comparative genomic hybridization protocol was used, and all chromosomes in 50 single sperm cells were simultaneously examined. Three-colour FISH was used to analyse chromosome segregation in 1557 other single sperm cells. It was also used to measure an interchromosomal effect; sperm chromatin structure assay was used to measure chromatin integrity. A high-frequency of unbalanced spermatozoa (84%) was observed, mostly arising from the 3:3 symmetrical segregation mode. Array comparative genomic hybridization was used to detect additional aneuploidies in two out of 50 spermatozoa (4%) in chromosomes not involved in the complex chromosome rearrangement. Significantly increased rates of diploidy and XY disomy were found in the CCR carrier compared with the control group (P < 0.001). Defective condensation of sperm chromatin was also found in 22.7% of spermatozoa by sperm chromatin structure assay. The results indicate that the infertility in the man with CCR and normal spermatozoa was caused by a production of chromosomally unbalanced, XY disomic and diploid spermatozoa and spermatozoa with defective chromatin condensation.

• MeSH
• analýza jednotlivých buněk MeSH
• body zlomu chromozomu * MeSH
• dospělí MeSH
• genová přestavba * MeSH
• heterozygot MeSH
• hybridizace in situ fluorescenční MeSH
• lidé MeSH
• mužská infertilita etiologie   MeSH
• poruchy sexuálního vývoje s karyotypem 46, XY diagnóza   genetika   patologie   patofyziologie   MeSH
• profáze meiózy I MeSH
• segregace chromozomů * MeSH
• spermie patologie   MeSH
• srovnávací genomová hybridizace MeSH
• translokace genetická * MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH

• MeSH
• blastomery MeSH
• embryo savčí cytologie   MeSH
• hybridizace in situ fluorescenční MeSH
• implantace embrya MeSH
• Publikační typ
• kongresy MeSH

In the current study, the sensitivity and specificity of methods of HER2 status detection were studied in 55 patients presenting with gastric/gastroesophageal junction carcinoma (30 intestinal and 25 diffuse), in small biopsy (endoscopy; n=33) and resection specimens (n=22). The primary objective of the present study was to compare various methods for the assessment of HER2 status, with regards to the sensitivity and specificity of each method, as well as their concordance. In all cases, the status of HER2 was determined using immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), silver in situ hybridization (SISH), and quantitative polymerase chain reaction (qPCR). The concordance rate between IHC and ISH was 100% for IHC 0 and 3+. The concordance rate for IHC 1+ was 100% between IHC and SISH, and 92.9% between IHC and FISH. The concordance rate among different FISH methods was 100%, between FISH and SISH it was 96.2%, and between qPCR and ISH methods it was 88.5%. Thus, the results demonstrate that different in situ hybridization methods are comparable and that none were superior. Furthermore, the IHC and FISH methods were found to be comparable and the concordance rate was particularly good. qPCR analysis correlated well with the other methods and appears to be a possible alternative tool for detection of the HER2 status. However, the concordance rate of qPCR with other methods was identified to be lower in the diffuse carcinoma group of endoscopy biopsy specimens; therefore investigation of further cases is required.

• MeSH
• adenokarcinom diagnóza   metabolismus   MeSH
• diagnostické techniky molekulární MeSH
• dospělí MeSH
• gastroezofageální junkce patologie   MeSH
• hybridizace in situ fluorescenční MeSH
• imunohistochemie MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• lidé středního věku MeSH
• lidé MeSH
• nádory žaludku diagnóza   metabolismus   MeSH
• receptor erbB-2 genetika   metabolismus   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

Although the chromosome 18 alpha-satellite probe is considered to have a very low polymorphism rate, the routine use of this probe in prenatal diagnosis revealed rare variants in size and copy number of these sequences. A polymorphic signal was detected in preimplantation genetic diagnosis (PGD) for aneuploidy, in a patient with repeated early miscarriages. A third small signal of chromosome 18 alpha-satellite probe was observed in two of four evaluated embryos. Hybridization to the woman's metaphasic lymphocytes revealed that the small signal was localized in the pericentromeric region of chromosome 1. Reanalysis of blastomeres with telomeric probes for chromosome 18q confirmed the presence of only two copies of chromosome 18. Options for verifying PGD analysis results, to prevent misdiagnosis in cases of suspected polymorphism, are discussed. Although some authors speculate about a possible role of heterochromatin polymorphism in infertility, this rare polymorphism of 18 alpha-satellite sequences is in itself probably a normal variant. This is the third report of a cross-hybridization of the chromosome 18 alpha-satellite probe and the first report of the localization of the polymorphic 18 alpha-satellite signal to chromosome 1.

• MeSH
• aneuploidie MeSH
• DNA sondy genetika   MeSH
• dospělí MeSH
• hybridizace in situ fluorescenční MeSH
• lidé MeSH
• lidské chromozomy, pár 1 genetika   MeSH
• lidské chromozomy, pár 18 genetika   MeSH
• mozaicismus MeSH
• polymorfismus genetický MeSH
• preimplantační diagnóza MeSH
• satelitní DNA genetika   MeSH
• těhotenství MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• Publikační typ
• kazuistiky MeSH
• práce podpořená grantem MeSH

The aim of the study was to determine whether or not the tyrosine kinase receptor ERBB2 is overexpressed in synovial sarcomas (SSs). We also focused on the cell cycle-related nuclear protein-Ki-67. Thirty-two samples were available for immunohistochemistry and only 1 case revealed a weak diffuse membrane ERBB-2 staining. The remaining cases showed either no staining (20 cases) or weak focal membrane staining (9 cases). In our 3 highly overexpressed ERBB2 mRNA samples, fluorescence in situ hybridization showed no amplification of the ERBB2 gene. ERBB2 mRNA expression was present in all samples of SSs at a comparable level to that in breast carcinoma control group, with a 2+ or 3+ immunopositivity. The high level of ERBB2 mRNA expression correlated with a high level of Ki-67 mRNA. The level of Ki-67 mRNA correlated with Ki-67 protein expression. The study shows that ERBB2 mRNA expression is very strong in SSs, but the membrane ERBB-2 protein expression is practically absent.

• MeSH
• antigen Ki-67 analýza   genetika   metabolismus   MeSH
• dítě MeSH
• dospělí MeSH
• financování organizované MeSH
• hybridizace in situ fluorescenční MeSH
• imunohistochemie MeSH
• kojenec MeSH
• lidé středního věku MeSH
• lidé MeSH
• messenger RNA analýza   metabolismus   MeSH
• mladiství MeSH
• předškolní dítě MeSH
• proliferace buněk MeSH
• receptor erbB-2 analýza   genetika   metabolismus   MeSH
• synoviom patologie   MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• kojenec MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH

Gekkotan lizards are a highly specious (∼1600 described species) clade of squamate lizards with nearly cosmopolitan distribution in warmer areas. The clade is primarily nocturnal and forms an ecologically dominant part of the world nocturnal herpetofauna. However, molecular cytogenetic methods to study the evolution of karyotypes have not been widely applied in geckos. Our aim here was to uncover the extent of chromosomal rearrangements across the whole group Gekkota and to search for putative synapomorphies supporting the newly proposed phylogenetic relationships within this clade. We applied cross-species chromosome painting with the recently derived whole-chromosomal probes from the gekkonid species Gekko japonicus to members of the major gekkotan lineages. We included members of the families Diplodactylidae, Carphodactylidae, Pygopodidae, Eublepharidae, Phyllodactylidae and Gekkonidae. Our study demonstrates relatively high chromosome conservatism across the ancient group of gekkotan lizards. We documented that many changes in chromosomal shape across geckos can be attributed to intrachromosomal rearrangements. The documented rearrangements are not totally in agreement with the recently newly erected family Phyllodactylidae. The results also pointed to homoplasy, particularly in the reuse of chromosome breakpoints, in the evolution of gecko karyotypes.

• MeSH
• chromozomy * MeSH
• fylogeneze MeSH
• hybridizace in situ fluorescenční MeSH
• ještěři klasifikace   genetika   MeSH
• karyotyp MeSH
• malování chromozomů MeSH
• metafáze genetika   MeSH
• molekulární evoluce MeSH
• rekombinace genetická * MeSH
• translokace genetická * MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH