Tumor biomarkers increasingly provide information for predicting outcomes with chemotherapeutic regimens (personalized medicine). Topo2A is a DNA helicase targeted by anthracyclines, cytotoxic therapeutics used in both adjuvant and palliative treatments of breast cancer. TOP2A gene amplification/deletion is implicated in response to anthracycline-based chemotherapy. We describe an approach for analyzing formalin-fixed, paraffin-embedded breast tumors on tissue microarrays with TOP2A fluorescence in situ hybridization coupled with cytokeratin immunofluorescence to target tumor cells. Stained tissue from patient specimens was imaged and analyzed using Metafer/Metacyte (Metasystems, Waltham, MA, USA), including customized image classifiers. TOP2A/CEN17 ratios of 2.0 or greater (amplified) and 0.8 or less (deleted) were observed for 10.0% and 6.1% of the patients, respectively. Patient outcomes for adjuvant chemotherapy (cyclophosphamide-epirubicin-fluorouracil, cyclophosphamide-methotrexate-fluorouracil, no chemotherapy) were evaluated. No statistical significance was achieved for clinical end points regarding TOP2A status in anthracycline-treated patients. However, patients with TOP2A aberrations receiving methotrexate-based therapy exhibited a significant decrease in 5-year distant disease-free survival and breast cancer-specific overall survival, especially for patients with TOP2A deletions (disease-free survival: hazard ratio, 5.31 [P = .001], and breast cancer-specific overall survival: hazard ratio, 6.45 [P < .001]). No significant differences were seen in patients included in the no-chemotherapy group. Topo2A protein levels were assessed by immunohistochemistry with no correlative statistical relevance to immunofluorescence/fluorescence in situ hybridization-based prognosis for cyclophosphamide-epirubicin-fluorouracil or cyclophosphamide-methotrexate-fluorouracil groups. Interestingly, aberrant (under)expressing patients in the no-chemotherapy group exhibited better 5-year distant disease-free survival (hazard ratio, 0.39; P = .004), trending toward more favorable breast cancer-specific overall survival (hazard ratio, 0.61; P = .11). Our results indicate a strategy by which fluorescence in situ hybridization scoring targeted to cytokeratin-positive tumor cells may provide a tool for added precision and efficiency in TOP2A evaluation from tumor tissue. Copyright 2012 Elsevier Inc. All rights reserved.

• MeSH
• adjuvantní chemoterapie MeSH
• antigeny nádorové * genetika   MeSH
• cyklofosfamid terapeutické užití   MeSH
• DNA vazebné proteiny * genetika   MeSH
• DNA-topoisomerasy typu II * genetika   MeSH
• dospělí MeSH
• fluoruracil terapeutické užití   MeSH
• hybridizace in situ fluorescenční MeSH
• keratiny * genetika   MeSH
• lidé středního věku MeSH
• lidé MeSH
• methotrexát terapeutické užití   MeSH
• nádory prsu * farmakoterapie   genetika   patologie   MeSH
• přežití bez známek nemoci MeSH
• protokoly protinádorové kombinované chemoterapie terapeutické užití   MeSH
• senioři MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• senioři MeSH
• ženské pohlaví MeSH

Leiomyosarcoma with adipocytic differentiation or lipoleiomyosarcoma is an uncommon sarcoma of the female genital tract with only a few individual reports in the literature. We therefore performed a morphologic, immunohistochemical, MDM2 gene amplification and RNA and DNA sequencing analysis of a series of gynecologic lipoleiomyosarcoma to better define the clinicopathologic spectrum. Six tumors from 6 patients were identified and classified as spindled lipoleiomyosarcoma (n = 2), mixed spindled and myxoid lipoleiomyosarcoma (n = 1), epithelioid lipoleiomyosarcoma with focal myxoid features (n = 1) and mixed spindled and epithelioid lipoleiomyosarcoma (n = 2). Patient age ranged from 41 to 64 years (mean: 49; median: 50). Primary location included uterine corpus (3), uterine corpus/cervix (2) and broad ligament (1). Tumor size ranged from 4.5 to 22 cm (mean: 11.2; median: 9.8). Four patients had metastasis at presentation or subsequently developed recurrent or distant disease. Patient status was known for 5: 2 dead of disease, 2 alive with disease and 1 alive without evidence of disease. Immunohistochemical expression of smooth muscle markers, ER, PR and WT-1 showed patterns similar to non-adipocytic gynecologic leiomyosarcomas. MDM2 amplification fluorescence in situ hybridization performed on 2 tumors was negative in 1 and equivocal in 1. Sequencing studies performed on 3 tumors found TP53 mutations in 3, with 1 tumor also having an ATRX alteration. No gene fusions were identified. Although lipoleiomyosarcomas have a diverse morphologic spectrum, our findings suggest the smooth muscle component shares morphologic and immunohistochemical features with female genital tract non-adipocytic leiomyosarcomas. Lipoleiomyosarcomas also have genetic alterations associated with non-adipocytic gynecologic leiomyosarcomas.

• MeSH
• dospělí MeSH
• hybridizace in situ fluorescenční MeSH
• imunohistochemie MeSH
• leiomyosarkom * patologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• molekulární biologie MeSH
• nádor z hladké svalové tkáně * patologie   MeSH
• nádorové biomarkery genetika   analýza   MeSH
• protoonkogenní proteiny c-mdm2 genetika   MeSH
• ženské pohlavní orgány chemie   patologie   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Successful derivation and cultivation of primordial germ cells (PGCs) opened the way to efficient transgenesis and genome editing in the chicken. Furthermore, implantation of male PGCs from non-chicken galliform species into the chicken embryos resulted in cross-species germline chimeras and viable offspring. We have recently improved the PGC technology by demonstrating that chicken male PGCs transplanted into the testes of adult cockerel recipients mature into functional sperms. However, the availability of this orthotopic transplantation for cross-species transfer remains to be explored. Here we tested the capacity of genetically distant male PGCs to mature in the microenvironment of adult testes. We derived PGCs from the Chinese black-bone Silkie and transplanted them into infertile White Leghorn cockerels. Within 15-18 weeks after transplantation, we observed restoration of spermatogenesis in recipient cockerels and production of healthy progeny derived from the transplanted PGCs. Our findings also indicate the possibility of cross-species orthotopic transplantation of PGCs. Thus, our results might contribute to the preservation of endangered avian species and maintaining the genetic variability of the domestic chicken.

• MeSH
• chiméra genetika   MeSH
• chov metody   MeSH
• křížení genetické MeSH
• kultivované buňky MeSH
• kur domácí * klasifikace   genetika   MeSH
• kuřecí embryo MeSH
• ohrožené druhy MeSH
• spermatogeneze fyziologie   MeSH
• spermie cytologie   transplantace   MeSH
• testis cytologie   MeSH
• transplantace heterologní veterinární   MeSH
• zachování plodnosti metody   veterinární   MeSH
• zachování přírodních zdrojů * metody   MeSH
• zárodečné buňky transplantace   MeSH
• zvířata MeSH
• Check Tag
• kuřecí embryo MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Oral cancer is a paradigm of Slaughter's concept of field cancerization, where tumors are thought to originate within an area of cells containing genetic alterations that predispose to cancer development. The field size is unclear but may represent a large area of tissue, and the origin of mutations is also unclear. Here, we analyzed whole exome and transcriptome features in contralateral tumor-distal tongue (i.e. distant from the tumor, not tumor-adjacent) and corresponding tumor tissues of 15 patients with squamous cell carcinoma of the oral tongue. The number of point mutations ranged from 41 to 237 in tumors and from one to 78 in tumor-distal samples. Tumor-distal samples showed mainly clock-like (associated with aging) or tobacco smoking mutational signatures. Tumors additionally showed mutations that associate with cytidine deaminase AID/APOBEC enzyme activities or a UV-like signature. Importantly, no point mutations were shared between a tumor and the matched tumor-distal sample in any patient. TP53 was the most frequently mutated gene in tumors (67%), whereas a TP53 mutation was detected in only one tumor-distal sample, and this mutation was not shared with the matched tumor. Arm-level copy number variation (CNV) was found in 12 tumors, with loss of chromosome (Chr) 8p or gain of 8q being the most frequent events. Two tumor-distal samples showed a gain of Chr8, which was associated with increased expression of Chr8-located genes in these samples, although gene ontology did not show a role for these genes in oncogenic processes. In situ hybridization revealed a mixed pattern of Chr8 gain and neutral copy number in both tumor cells and adjacent nontumor epithelium in one patient. We conclude that distant field cancerization exists but does not present as tumor-related mutational events. The data are compatible with etiologic field effects, rather than classical monoclonal field cancerization theory. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

• MeSH
• jazyk patologie   MeSH
• lidé MeSH
• nádory jazyka * genetika   MeSH
• nádory úst * patologie   MeSH
• spinocelulární karcinom * patologie   MeSH
• variabilita počtu kopií segmentů DNA MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

During mitotic prophase, chromosomes of the pathogenic unicellular eukaryote Giardia intestinalis condense in each of the cell's two nuclei. In this study, Giardia chromosomes were investigated using light microscopy, high-resolution field emission scanning electron microscopy, and in situ hybridization. For the first time, we describe the overall morphology, condensation stages, and mitotic segregation of these chromosomes. Despite the absence of several genes involved in the cohesion and condensation pathways in the Giardia genome, we observed chromatin organization similar to those found in eukaryotes, i.e., 10-nm nucleosomal fibrils, 30-nm fibrils coiled to chromomeres or in parallel arrangements, and closely aligned sister chromatids. DNA molecules of Giardia terminate with telomeric repeats that we visualized on each of the four chromatid endings of metaphase chromosomes. Giardia chromosomes lack primary and secondary constrictions, thus preventing their classification based on the position of the centromere. The anaphase poleward segregation of sister chromatids is atypical in orientation and tends to generate lagging chromatids between daughter nuclei. In the Giardia genome database, we identified two putative members of the kleisin family thought to be responsible for condensin ring establishment. Thus far, Giardia chromosomes (300 nm to 1.5 μm) are the smallest chromosomes that were analyzed at the ultrastructural level. This study complements the existing molecular and sequencing data on Giardia chromosomes with cytological and ultrastructural information.

• MeSH
• adenosintrifosfatasy analýza   MeSH
• buněčné jádro ultrastruktura   MeSH
• chromozomy fyziologie   ultrastruktura   MeSH
• DNA vazebné proteiny analýza   MeSH
• Giardia lamblia genetika   ultrastruktura   MeSH
• mitóza MeSH
• multiproteinové komplexy analýza   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Phlebotomus orientalis Parrot (Diptera: Psychodidae) is the main vector of visceral leishmaniasis (VL) caused by Leishmania donovani in East Africa. Here we report on life cycle parameters and susceptibility to L. donovani of two P. orientalis colonies originating from different sites in Ethiopia: a non-endemic site in the lowlands - Melka Werer (MW), and an endemic focus of human VL in the highlands - Addis Zemen (AZ). METHODOLOGY/PRINCIPAL FINDINGS: Marked differences in life-cycle parameters between the two colonies included distinct requirements for larval food and humidity during pupation. However, analyses using Random Amplified Polymorphic DNA (RAPD) PCR and DNA sequencing of cytB and COI mitochondrial genes did not reveal any genetic differences. F1 hybrids developed successfully with higher fecundity than the parental colonies. Susceptibility of P. orientalis to L. donovani was studied by experimental infections. Even the lowest infective dose tested (2×10(3) per ml) was sufficient for successful establishment of L. donovani infections in about 50% of the P. orientalis females. Using higher infective doses, the infection rates were around 90% for both colonies. Leishmania development in P. orientalis was fast, the presence of metacyclic promastigotes in the thoracic midgut and the colonization of the stomodeal valve by haptomonads were recorded in most P. orientalis females by day five post-blood feeding. CONCLUSIONS: Both MW and AZ colonies of P. orientalis were highly susceptible to Ethiopian L. donovani strains. As the average volume of blood-meals taken by P. orientalis females are about 0.7 µl, the infective dose at the lowest concentration was one or two L. donovani promastigotes per sand fly blood-meal. The development of L. donovani was similar in both P. orientalis colonies; hence, the absence of visceral leishmaniasis in non-endemic area Melka Werer cannot be attributed to different susceptibility of local P. orientalis populations to L. donovani.

• MeSH
• Leishmania donovani patogenita   MeSH
• Phlebotomus genetika   parazitologie   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Etiopie MeSH

In most eukaryotes, telomeres consist of tandem arrays of a short repetitive DNA sequence. Insect telomeres are generally constituted by a (TTAGG)n repeat motif. Usually, telomeres are maintained by telomerase, a specialized reverse transcriptase that adds this sequence to chromosome ends. We examined telomerase activity in 15 species across Insecta. Telomerase activity was revealed in Isoptera, Blattaria, Lepidoptera, Hymenoptera, Trichoptera, Coleoptera, and Sternorrhyncha. In contrast, we were not able to detect telomerase activity in Orthoptera, Zygentoma, and Phasmida. Because we found telomerase activity in phylogenetically distant species, we conclude that a distribution pattern of (TTAGG)n sequence in Insecta is generally consistent with that of telomerase activity. Thus, the TTAGG-telomerase system is functional across the Insecta. Using real-time quantitative telomeric repeat amplification protocol (RTQ-TRAP) system, we quantified telomerase activity in different developmental stages and different tissues of a cockroach, Periplaneta americana. We show that telomerase is upregulated in young instars and gradually declines during development. In adults, it is most active in testes and ovaries. Thus, the telomerase activity of hemimetabolous insects seems to be associated with cell proliferation and organismal development.

• MeSH
• bourec genetika   MeSH
• fylogeneze * MeSH
• hmyz genetika   MeSH
• hybridizace in situ fluorescenční MeSH
• replikace DNA genetika   MeSH
• tandemové repetitivní sekvence genetika   MeSH
• telomerasa genetika   MeSH
• telomery genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Next-generation sequencing (NGS) provides a powerful tool for the discovery of important genes and alleles in crop plants and their wild relatives. Despite great advances in NGS technologies, whole-genome shotgun sequencing is cost-prohibitive for species with complex genomes. An attractive option is to reduce genome complexity to a single chromosome prior to sequencing. This work describes a strategy for studying the genomes of distant wild relatives of wheat by isolating single chromosomes from addition or substitution lines, followed by chromosome sorting using flow cytometry and sequencing of chromosomal DNA by NGS technology. We flow-sorted chromosome 5M(g) from a wheat/Aegilops geniculata disomic substitution line [DS5M(g) (5D)] and sequenced it using an Illumina HiSeq 2000 system at approximately 50 × coverage. Paired-end sequences were assembled and used for structural and functional annotation. A total of 4236 genes were annotated on 5M(g) , in close agreement with the predicted number of genes on wheat chromosome 5D (4286). Single-gene FISH indicated no major chromosomal rearrangements between chromosomes 5M(g) and 5D. Comparing chromosome 5M(g) with model grass genomes identified synteny blocks in Brachypodium distachyon, rice (Oryza sativa), sorghum (Sorghum bicolor) and barley (Hordeum vulgare). Chromosome 5M(g) -specific SNPs and cytogenetic probe-based resources were developed and validated. Deletion bin-mapped and ordered 5M(g) SNP markers will be useful to track 5M-specific introgressions and translocations. This study provides a detailed sequence-based analysis of the composition of a chromosome from a distant wild relative of bread wheat, and opens up opportunities to develop genomic resources for wild germplasm to facilitate crop improvement.

• MeSH
• Brachypodium genetika   MeSH
• chromozomy rostlin genetika   MeSH
• genom rostlinný genetika   MeSH
• hybridizace in situ fluorescenční MeSH
• ječmen (rod) genetika   MeSH
• jednonukleotidový polymorfismus MeSH
• lipnicovité klasifikace   genetika   MeSH
• mapování chromozomů MeSH
• molekulární evoluce MeSH
• pořadí genů MeSH
• pšenice genetika   MeSH
• rostlinné geny genetika   MeSH
• rýže (rod) genetika   MeSH
• Sorghum genetika   MeSH
• vysoce účinné nukleotidové sekvenování metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

BACKGROUND: Genetically divergent cryptic species are frequently detected by molecular methods. These discoveries are often a byproduct of molecular barcoding studies in which fragments of a selected marker are used for species identification. Highly divergent mitochondrial lineages and putative cryptic species are even detected in intensively studied animal taxa, such as the crustacean genus Daphnia. Recently, eleven such lineages, exhibiting genetic distances comparable to levels observed among well-defined species, were recorded in the D. longispina species complex, a group that contains several key taxa of freshwater ecosystems. We tested if three of those lineages represent indeed distinct species, by analyzing patterns of variation of ten nuclear microsatellite markers in six populations. RESULTS: We observed a discordant pattern between mitochondrial and nuclear DNA, as all individuals carrying one of the divergent mitochondrial lineages grouped at the nuclear level with widespread, well-recognized species coexisting at the same localities (Daphnia galeata, D. longispina, and D. cucullata). CONCLUSIONS: A likely explanation for this pattern is the introgression of the mitochondrial genome of undescribed taxa into the common species, either in the distant past or after long-distance dispersal. The occurrence of highly divergent but rare mtDNA lineages in the gene pool of widespread species would suggest that hybridization and introgression in the D. longispina species complex is frequent even across large phylogenetic distances, and that discoveries of such distinct clades must be interpreted with caution. However, maintenance of ancient polymorphisms through selection is another plausible alternative that may cause the observed discordance and cannot be entirely excluded.

• MeSH
• buněčné jádro genetika   MeSH
• Daphnia genetika   MeSH
• druhová specificita MeSH
• fylogeneze * MeSH
• genetická variace * MeSH
• hybridizace genetická MeSH
• mikrosatelitní repetice genetika   MeSH
• mitochondriální DNA genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Methylation systems have been conserved during the divergence of plants and animals, although they are regulated by different pathways and enzymes. However, studies on the interactions of the epigenomes among evolutionarily distant organisms are lacking. To address this, we studied the epigenetic modification and gene expression of plant chromosome fragments (~30 Mb) in a human-Arabidopsis hybrid cell line. The whole-genome bisulfite sequencing results demonstrated that recombinant Arabidopsis DNA could retain its plant CG methylation levels even without functional plant methyltransferases, indicating that plant DNA methylation states can be maintained even in a different genomic background. The differential methylation analysis showed that the Arabidopsis DNA was undermethylated in the centromeric region and repetitive elements. Several Arabidopsis genes were still expressed, whereas the expression patterns were not related to the gene function. We concluded that the plant DNA did not maintain the original plant epigenomic landscapes and was under the control of the human genome. This study showed how two diverging genomes can coexist and provided insights into epigenetic modifications and their impact on the regulation of gene expressions between plant and animal genomes.

• MeSH
• Arabidopsis genetika   MeSH
• buněčné linie MeSH
• chromozomy rostlin genetika   MeSH
• DNA rostlinná genetika   MeSH
• epigeneze genetická genetika   MeSH
• epigenom genetika   MeSH
• epigenomika metody   MeSH
• genom rostlinný genetika   MeSH
• hybridní buňky fyziologie   MeSH
• lidé MeSH
• methyltransferasy genetika   MeSH
• metylace DNA genetika   MeSH
• repetitivní sekvence nukleových kyselin genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH