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The great efforts spent in the maintenance of past diversity in genebanks are rationalized by the potential role of plant genetic resources (PGR) in future crop improvement-a concept whose practical implementation has fallen short of expectations. Here, we implement a genomics-informed prebreeding strategy for wheat improvement that does not discriminate against nonadapted germplasm. We collect and analyze dense genetic profiles for a large winter wheat collection and evaluate grain yield and resistance to yellow rust (YR) in bespoke core sets. Breeders already profit from wild introgressions but PGR still offer useful, yet unused, diversity. Potential donors of resistance sources not yet deployed in breeding were detected, while the prebreeding contribution of PGR to yield was estimated through 'Elite × PGR' Fsub1/subcrosses. Genomic prediction within and across genebanks identified the best parents to be used in crosses with elite cultivars whose advanced progenies can outyield current wheat varieties in multiple field trials.

Purpose In advanced tumor stages, pediatric rhabdomyosarcoma (RMS) shows an intrinsic resistance to standard chemotherapy, which is associated with a dismal prognosis. Alternative therapeutic approaches and optimization of already existent treatment protocols are urgently needed in these conditions. The µ-opioid receptor (OPRM1) agonist, D,L-methadone is frequently used for analgesia in oncological patients. Recent evidence has shown that D,L-methadone in combination with chemotherapeutic agents may enhance their cytotoxic effect against cancer cells. There are no related data in pediatric rhabdomyosarcoma (RMS). Methods Antitumor effects of combined D,L-methadone and doxorubicin, carboplatin, and vincristine on RMS cell lines RD and RH30 were analyzed using following outcome data: expression of the OPRM1 receptor (Western blot), cell growth inhibition (MTT assay), cell migration (wound-healing assay), apoptosis induction (caspase-3/7 assay), and reactive oxygen species (ROS) production (flow cytometry). Results In both cell lines, OPRM1 expression was significantly increased after combined treatment of D,L-methadone with all three cytotoxic drugs tested, which resulted in suppression of tumor cell growth and increase of apoptosis rates. These effects were mediated by increased ROS production and up-regulation of caspase-3/7 activity. Doxorubicin combined with D,L-methadone significantly reduced cell migration in both cell lines. Carboplatin or vincristine in combination with D,L-methadone had only an impact on cell migration in RH30 cells. Conclusions This new therapeutic approach in RMS provides strong antitumor effects in vitro. The combination of standard chemotherapy and D,L-methadone requires further investigation. Especially advanced tumors with a limited effectiveness of conventional treatment regimens seem a potential target of this approach.

Purpose ESIN (elastic stable intramedullary nailing) is considered the gold standard for various pediatric fractures. The aim of this study was to analyze the incidence and type of complications during or after TEN (titanium elastic nail) removal. Methods A retrospective data analysis was performed. Metal removal associated complications and preoperative extraosseous length/outlet angle of TENs as possible causes of complications were assessed. Results The complication rate in 384 TEN removals was 3.1% (n = 12). One major complication (rupture of M. extensor pollicis brevis) was documented. One refracture at the forearm occurred, however, remodeling prior TEN removal was completed. Ten minor complications were temporary or without irreversible restrictions (3 infections, 5 scaring/granuloma, 2 temporary paraesthesia). In 38 cases (16 forearms, 10 femora, 9 humeri, 3 lower legs), intra-operative fluoroscopy had to be used to locate the implants. In patients with forearm fractures, extraosseous implant length was relatively shorter than in cases without fluoroscopy (p = 0.01), but outlet angle of TENs was not significantly different in these two groups (28.5° vs 25.6°). In patients with femur fractures, extraosseous implant length and outlet angle were tendentially shorter, respectively, lower, but this did not reach statistical significance. Conclusion Removal of TENs after ESIN is a safe procedure with a low complication rate. Technically inaccurate TEN implantation makes removal more difficult and complicated. To prevent an untimely removal and patient discomfort, nail ends must be exactly positioned and cut. Intraoperative complications may be minimized with removal of TENs before signs of overgrowth. Evidence Level III, retrospective.

Background Injury to the artery of Adamkiewicz (AKA) during surgery may lead to spinal cord ischemia and severe neurologic complications. Posterior mediastinal tumors may be adjacent to AKA, but data on preoperative visualization of AKA in children are rare. This study analyzed the importance of identifying the AKA preoperatively by spinal digital subtraction angiography (DSA) in children with posterior mediastinal tumors for therapeutic procedure. Methods Between 2002 and 2021, 36 children with posterior mediastinal tumors were evaluated for surgery at the authors’ clinic. In 10 children with left-sided or bilateral tumor located at vertebral levels T8 to L1, spinal DSA was performed during preoperative workup to assess AKA. The patient and tumor characteristics as well as the diagnostic and therapeutic procedures were analyzed. Results The median age of the 10 children at examination was 69 months (range, 16–217 months). Three of the children were younger than 2 years. The tumor entities were neuroblastoma, ganglioneuroblastoma, ganglioneuroma, local relapse of a hepatocellular carcinoma, and neurofibroma. The AKA was identified in all cases, and proximity to the tumor was detected in four patients, three of whom had their planned surgery changed to irradiation. No complications occurred during spinal DSA or surgery. Conclusions In posterior mediastinal pediatric tumors, spinal DSA is a safe and reliable method for preoperative visualization of the AKA. It can show proximity to the tumor and guide the local therapy, thereby avoiding critical intra- and postoperative situations.

Background The benefit of adjuvant therapy in synovial sarcoma (SS) treatment is under debate. Long-term follow-up data are missing. Methods SS patients treated in the consecutive trials CWS-81, CWS-86, CWS-91, CWS-96, CWS-2002-P, and the SoTiSaR-registry till 2013 were analyzed. Results Median age of 185 patients was 13.9 years (0.1–56)—with median follow-up of 7.4 years for 163 survivors. Most tumors (76%) were located in extremities. Size was 10 cm in 13 (7%) (13 missing). In 84 (45%) tumors, first excision was complete (R0 corresponding to IRS-I-group) and in 101 (55%) marginal (R1 corresponding to IRS-II-group). In a subsequent surgical intervention during chemotherapy, R0-status was accomplished in 23 additional IRS-II-group patients with secondary surgery. Radiotherapy was administered to 135 (73%), thereof 62 with R0-status and 67 R1-status (6 missing information). Adjuvant chemotherapy was administered to all but six patients. 5-year event-free (EFS) and overall survival (OS) was 82.9% ± 5.7 (95%CI) and 92.5% ± 3.9. Local and metastatic relapse-free survival was 91.3% ± 4.3 and 92.3% ± 4.1 at 5 years, respectively. In the multivariate analysis, tumor size and no chemotherapy were independently associated with EFS. Size and site were associated with OS. In a detailed analysis of local and metastatic events, tumor size was associated with an independent risk for developing metastases. No independent factor for suffering local recurrence could be identified. Discussion Omission of chemotherapy in a non-stratified way seems not justified. Size governs survival due to high linear association with risk of suffering metastatic recurrence in a granular classification.

Purpose Natural products are generally regarded as safe and have been shown to mediate anticancer activities against a variety of cell types. Zerumbone is a natural cyclic sesquiterpene derived from the rhizome of Zingiber zerumbet, which has attracted extensive attention in the recent decade for anticancer activities. The present study investigates the in vitro effect of zerumbone on rhabdomyosarcoma cells. Methods Two rhabdomyosarcoma cell lines (RD and RH30) were used as the model system. The growth inhibition of zerumbone was measured by MTT-assay, apoptosis via flow cytometry, gene expression by real-time PCR, the migration by transwell assay, and intracellular signaling by Western blotting. Results Zerumbone shows anticancer effects on RD and RH30 cells in a dose-dependent manner via cell growth inhibition and induction of apoptosis. Exposure of RD and RH30 cells on zerumbone also resulted in a decrease of migration and downregulation of the hedgehog pathway. Conclusions Taken together, our study provided the first evidence that zerumbone imparted strong inhibitory and apoptotic effects on pediatric rhabdomyosarcoma cell lines and merit further investigation as a promising candidate for the anticancer therapy.

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. In 7% of the cases it is localized at the chest wall or intrathoracically. The aim of this study was to analyze the multimodal treatment concepts and outcomes of children suffering from intrathoracic and chest wall RMS treated within three different Cooperative Soft Tissue Sarcoma (CWS) trials and one registry (Soft Tissue Sarcoma Registry, SoTiSaR). Data of 51 patients with thoracic RMS enrolled in three different CWS trials (CWS-86, -91, -2002P) and one registry (SoTiSaR) were analyzed retrospectively. Surgery and its influence on outcome were assessed. Median follow-up was 37.5 months (0.9–152.5). Median age of the patients was 8.8 years (range 0–19 years). The 5-year overall survival rate (OS) was 57% (95%-CI 49–65) and the 5-year event–free survival rate (EFS) was 45% (38–52). Thirty-five patients had tumors located at the chest wall (EFS: 51%, 43–59), and 16 patients had intrathoracic tumors (EFS: 26%, 13–39). Seventeen patients with tumors ≤ 5 cm had a better outcome (EFS: 64%, 52–76) compared to patients with tumors larger than 5 cm (EFS: 36%, 27–45). Radiotherapy (RT) significantly improved the survival of patients with alveolar RMS compared to patients with embryonal histology (EFS: 66%, 52–80 vs. 32%, 21–43 p = 0.02). Complete tumor excision during delayed surgery was the main prognostic factor for survival (p = 0.045). Thoracic RMS is a rare tumor entity. Completeness of tumor resection significantly improved survival of the patients.

Our understanding of the evolution of plant sex chromosomes is increasing rapidly due to high-throughput sequencing data and phylogenetic and molecular-cytogenetic approaches that make it possible to infer the evolutionary direction and steps leading from homomorphic to heteromorphic sex chromosomes. Here, we focus on four species of Coccinia, a genus of 25 dioecious species, including Coccinia grandis, the species with the largest known plant Y chromosome. Based on a phylogeny for the genus, we selected three species close to C. grandis to test the distribution of eight repetitive elements including two satellites, and several plastid and mitochondrial probes, that we had previously found to have distinct accumulation patterns in the C. grandis genome. Additionally, we determined C-values and performed immunostaining experiments with (peri-)centromere-specific antibodies on two species (for comparison with C. grandis). In spite of no microscopic chromosomal heteromorphism, single pairs of chromosomes in male cells of all three species accumulate some of the very same repeats that are enriched on the C. grandis Y chromosome, pointing to either old (previous) sex chromosomes or incipient (newly arising) ones, that is, to sex chromosome turnover. A 144-bp centromeric satellite repeat (CgCent) that characterizes all C. grandis chromosomes except the Y is highly abundant in all centromeric regions of the other species, indicating that the centromeric sequence of the Y chromosome diverged very recently.

Duckweeds are small, free-floating, largely asexual and highly neotenous organisms. They display the most rapid growth among flowering plants and are of growing interest in aquaculture and genome biology. Genomic and chromosomal data are still rare. Applying flow-cytometric genome size measurement, microscopic determination of frond, cell and nucleus morphology, as well as fluorescence in situ hybridization (FISH) for localization of ribosomal DNA (rDNA), we compared eleven species, representative for the five duckweed genera to search for potential correlations between genome size, cell and nuclei volume, simplified body architecture (neoteny), chromosome numbers and rDNA loci. We found a ~14-fold genome size variation (from 160 to 2203 Mbp), considerable differences in frond size and shape, highly variable guard cell and nucleus size, chromosome number (from 2n = 36 to 82) and number of 5S and 45S rDNA loci. In general, genome size is positively correlated with guard cell and nucleus volume (p Spirodela and Landoltia.

Modern treatment concepts for bladder/prostate rhabdomyosarcoma (BPRMS) are designed to improve survival, to reduce therapy intensity, and to increase bladder preservation rates. Nevertheless, treatment is not optimal. The purpose of this study was to analyze BPRMS patients treated within the CWS-2002P trial regarding outcome, treatment modalities, complications, and to compare the data with the precursor trial CWS-96.Fifty children with localized embryonal BPRMS were analyzed. Eight patients were excluded. Patients received neoadjuvant chemotherapy. At week 9, reassessment using MRI scan was performed. Depending on tumor size, age, and response, local therapy consisting of radiotherapy and/or surgery was initiated. After local therapy, systemic therapy was continued.Patients' median age was 35.6 months. Median follow-up was 59 months. The 5-year OS was 84.5 % and the 5-year ES 79.9 %. Ten patients underwent combined radiochemotherapy and tumor resection (5-year ES: 87.5 %). Six patients were treated solely with radiochemotherapy (5-year ES: 60 %). Twenty-six patients received preoperative chemotherapy followed by tumor resection (ES: 80.8). One patient was treated with chemotherapy only and survived. The bladder preservation rate was 80.9 %.The outcome within the CWS-2002P trial regarding OS and ES seemed to be better than in the precursor trial CWS-96 due to a reduction of protocol violations, but there was no statistical significant difference possibly due to low numbers. Radiotherapy was used less frequently, and the bladder preservation rate was slightly higher. Novel concepts will be required in the future to improve bladder preservation rates.

In situ hybridisation is a powerful tool to investigate the genome and chromosome architecture. Nick translation (NT) is widely used to label DNA probes for fluorescence in situ hybridisation (FISH). However, NT is limited to the use of long double-stranded DNA and does not allow the labelling of single-stranded and short DNA, e.g. oligonucleotides. An alternative technique is the copper(I)-catalysed azide-alkyne cycloaddition (CuAAC), at which azide and alkyne functional groups react in a multistep process catalysed by copper(I) ions to give 1,4-distributed 1,2,3-triazoles at a high yield (also called 'click reaction'). We successfully applied this technique to label short single-stranded DNA probes as well as long PCR-derived double-stranded probes and tested them by FISH on plant chromosomes and nuclei. The hybridisation efficiency of differently labelled probes was compared to those obtained by conventional labelling techniques. We show that copper(I)-catalysed azide-alkyne cycloaddition-labelled probes are reliable tools to detect different types of repetitive sequences on chromosomes opening new promising routes for the detection of single copy gene. Moreover, a combination of FISH using such probes with other techniques, e.g. immunohistochemistry (IHC) and cell proliferation assays using 5-ethynyl-deoxyuridine, is herein shown to be easily feasible.

Package leaflets of medicines distributed within the European Union should use the template headings and standard texts created by the Working Group on the Quality Review of Documents (QRD). The following study investigated how the QRD template is implemented in this patient information.All English-language package leaflets available on the European Medicines Agency website for centralized authorization procedures were downloaded 3 times, separated by a time period of 1 year. A catalog of criteria was applied to analyze QRD template text use.Because of the rapid implementation of updates, the template text used in each package leaflet (N = 565) increased from an average of 444 words in the first download to 565 words during the 2 subsequent years. With template version 7, the fraction of template text per leaflet was 19.7% in 2011, which increased to 21.5% in 2013 with implementation of version 9.Limitation to mandatory contents through stricter use of the QRD template's bracketing convention, which would reduce package leaflets' text without loss of essential information, is suggested. In addition, making the current QRD template more concise is strongly recommended.

In children, ureteropelvic junction obstruction (UPJO) is mostly caused by intrinsic factors (IUPJO); extrinsic UPJO are rare and often due to crossing vessels (CVs). We retrospectively reviewed all data of children with UPJO that underwent surgery in our institution from 2004 to 2011. Analyses included age at surgery, gender, preoperative and postoperative results of ultrasound and renal scans [differential renal function (DRF); signs of obstruction], and pathology reports. Available histological specimens of cases with CV were compared to a random selection of intrinsic cases in a blinded fashion. After additional Masson’s trichrome staining, the specimens were scored for fibrosis, muscular hypertrophy, and chronic inflammation. Out of 139 patients with UPJO, 39 cases were associated with CV. Median age at surgery was 68 months (range 2–194) in the CV group and 11.5 months (range 0–188) in IUPJO group. Laparoscopic dismembered pyeloplasty (LDMP) was carried out in 134 and open DMP in five patients. Preoperative ultrasound identified 28/39 cases with CV. DRF below 40 % was more frequently seen in CV patients (p = 0.020). Histological analyses revealed no differences between the CV and IUPJO specimens in total. CV patients with higher grades of muscular hypertrophy had lower preoperative DRF, compared to those with higher preoperative DRF (p = 0.026). Functional recovery after (L)DMP was excellent in both groups. We could not find any significant histological differences between CV and IUPJO in children. To obtain excellent functional recovery, surgical procedures with a definite correction of the UPJ should be preferred in paediatric patients with CV.

Species with holocentric chromosomes are often characterized by a rapid karyotype evolution. In contrast to species with monocentric chromosomes where acentric fragments are lost during cell division, breakage of holocentric chromosomes creates fragments with normal centromere activity. To decipher the mechanism that allows holocentric species an accelerated karyotype evolution via chromosome breakage, we analyzed the chromosome complements of irradiated Luzula elegans plants. The resulting chromosomal fragments and rearranged chromosomes revealed holocentromere-typical CENH3 and histone H2AThr120ph signals as well as the same mitotic mobility like unfragmented chromosomes. Newly synthesized telomeres at break points become detectable 3 weeks after irradiation. The presence of active telomerase suggests a telomerase-based mechanism of chromosome healing. A successful transmission of holocentric chromosome fragments across different generations was found for most offspring of irradiated plants. Hence, a combination of holokinetic centromere activity and the fast formation of new telomeres at break points enables holocentric species a rapid karyotype evolution involving chromosome fissions and rearrangements.

Surgery for rhabdomyosarcoma is challenging due to a lack of clear delineation between tumor and surrounding tissue. Mutilating surgery can be necessary in difficult tumor localizations. Therefore, novel diagnostic and therapeutic modalities are required. The aim of this study was to evaluate the in vivo tumor detection of RMS using fluorescence laparoscopy and to analyze the efficacy of hypericin-induced photodynamic therapy in a mouse model. Seventeen NOD/LtSz-scid IL2Rγnull-mice were divided into four groups. In group 1, mCherry-expressing tumor cells and in group 2–4 non-transfected tumor cells were xenotransplanted. Three weeks later, one fluorochrome per group (ICG, ICG-cetuximab, hypericin) was injected. Fluorescence laparoscopy was carried out and tumors were resected using fluorescence guidance. In the hypericin group, photodynamic therapy was performed using blue light and apoptosis was evaluated by TUNEL test. A clear discrimination between healthy and tumor tissue was feasible by fluorescending properties with mCherry expressing tumor cells and after injection of hypericin. No fluorescence was detected in mice injected with ICG and ICG-labeled cetuximab. Hypericin photodynamic therapy induced apoptosis of tumor cells after exposure to blue light. Intraoperative photodynamic diagnosis was feasible using mCherry-transfected tumor cells or hypericin. Additionally, intraoperative photodynamic therapy was possible and effective.

Using amplification of marker sequences with DNA from a set of distinct microdissected Vicia faba L. chromosomes covering the entire genome, we could unambiguously show that the linkage group I.B, which includes the pseudogene of legumin B4 (ψ1) and was previously ascribed to the metacentric chromosome I, actually belongs to chromosome IV. By considering the breakpoints of the translocated BKH chromosomes III and IV, even the subchromosomal position of loci LG085 and CNGC4 could be inferred. Anchoring all linkage groups to distinct faba bean chromosomes will facilitate quantitative trait locus fine mapping and gene identification using synteny, and will boost the development of efficient markers for selection in breeding programs.

Sexual reproduction is ubiquitous among eukaryotes, and fully asexual lineages are extremely rare. Prominent among ancient asexual lineages are the arbuscular mycorrhizal fungi (AMF), a group of plant symbionts with a multinucleate cytoplasm. Genomic divergence among co-existing nuclei was proposed to drive the evolutionary success of AMF in the absence of sex(1), but this hypothesis has been contradicted by recent genome analyses that failed to find significant genetic diversity within an AMF isolate(2,3). Here, we set out to resolve issues surrounding the genome organization and sexual potential of AMF by exploring the genomes of five isolates of Rhizophagus irregularis, a model AMF. We find that genetic diversity in this species varies among isolates and is structured in a homo-dikaryon-like manner usually linked with the existence of a sexual life cycle. We also identify a putative AMF mating-type locus, containing two genes with structural and evolutionary similarities with the mating-type locus of some Dikarya. Our analyses suggest that this locus may be multi-allelic and that AMF could be heterothallic and bipolar. These findings reconcile opposing views on the genome organization of these ubiquitous plant symbionts and open avenues for strain improvement and environmental application of these organisms.

Multimodality treatment approaches provide high local control and satisfying overall survival (OS) for children with localized bladder and/or prostate rhabdomyosarcoma (BP-RMS). However, current strategies including surgery and conventional radiotherapy are compromised by high rates of long-term genitourinary adverse effects. Therefore, a planning study combining organ preserving surgery with three different innovative adjuvant radiotherapy approaches was performed. A case of a 21-month-old boy with BP-RMS treated with polychemotherapy according to the CWS 2002-P protocol, prostatectomy, partial cystectomy, and adjuvant high dose rate brachytherapy (HDR-BT) was used to perform a planning study comparing HDR-BT with intensity-modulated radiotherapy (IMRT) and intensity-modulated proton therapy (IMPT) planning. All modalities provide good coverage of the target volume and spare critical normal tissues. Rectum doses could be reduced by 2/3 using IMPT and by 1/3 using BT compared to IMRT. In terms of sparing the pelvis growth plates, BT and IMPT are also superior to IMRT. All modalities provide good sparing of normal tissue. BT and IMPT are superior to IMRT with regard to doses on rectum and growth plates. BT is equivalent to IMPT in adequately selected tumors.

Fluorescent chromatin tagging by the lacO operator/lac repressor system in Arabidopsis thaliana is useful to trace distinct chromatin domains in living cells. Nevertheless, the tandem repeats of the tagging system may alter the spatial organisation of chromatin within nuclei by increasing homologous pairing as well as association with heterochromatin. Efficient homologous pairing occurs if lacO repeat arrays of ∼10 kb are present at two loci, either on the same chromosome or on different chromosomes. DNA hypomethylation of lacO repeats results in reduced homologous pairing. Because, in plants, DNA methylation can serve as a signal for H3-lysine9-dimethylation (H3K9me2), and subsequently for non-CG-context DNA methylation, SET-domain histone methyltransferase and chromodomain dna methyltransferase 3 (cmt3) mutations were introgressed. In suvh4 suvh5 suvh6 and cmt3 mutants, H3K9me2 associated with lacO repeats is diminished, but homologous pairing persists. Thus, neither H3K9me2 nor CMT3-mediated non-CG methylation are required at wild-type level for homologous pairing of lacO repeat loci.

Sugar beet (Beta vulgaris) chromosomes consist of large heterochromatic blocks in pericentromeric, centromeric, and intercalary regions comprised of two different highly abundant DNA satellite families. To investigate DNA methylation at single base resolution at heterochromatic regions, we applied a method for strand-specific bisulfite sequencing of more than 1,000 satellite monomers followed by statistical analyses. As a result, we uncovered diversity in the distribution of different methylation patterns in both satellite families. Heavily methylated CG and CHG (H=A, T, or C) sites occur more frequently in intercalary heterochromatin, while CHH sites, with the exception of CAA, are only sparsely methylated, in both intercalary and pericentromeric/centromeric heterochromatin. We show that the difference in DNA methylation intensity is correlated to unequal distribution of heterochromatic histone H3 methylation marks. While clusters of H3K9me2 were absent from pericentromeric heterochromatin and restricted only to intercalary heterochromatic regions, H3K9me1 and H3K27me1 were observed in all types of heterochromatin. By sequencing of a small RNA library consisting of 6.76 million small RNAs, we identified small interfering RNAs (siRNAs) of 24 nucleotides in size which originated from both strands of the satellite DNAs. We hypothesize an involvement of these siRNAs in the regulation of DNA and histone methylation for maintaining heterochromatin.

Arabidopsis accessions Col-0 and C24 and their reciprocal hybrids were employed as a model system to investigate the potential relationship between changes in DNA methylation, chromatin structure, endopolyploidization and gene expression in heterotic genotypes. Nucleolus size, endopolyploidization level and distribution of DNA and histone H3 methylation at the microscopic level does not differ between parents and their hybrids. Methylation sensitive amplified polymorphism revealed a largely constant pattern of DNA methylation (97% of signals analyzed) after intraspecific crosses. The parental expression profile of selected genes was maintained in hybrid offspring. No correlation was found between expression pattern and DNA methylation levels at restriction sites within 5' regulatory regions. Thus, the results revealed only minor changes of chromatin properties and other nuclear features in response to intraspecific hybridization in Arabidopsis thaliana.

In contrast to yeast, plant interphase nuclei often display incomplete alignment (cohesion) along sister chromatid arms. Sister chromatid cohesion mediated by the multi-subunit cohesin complex is essential for correct chromosome segregation during nuclear divisions and for DNA recombination repair. The cohesin complex consists of the conserved proteins SMC1, SMC3, SCC3, and an alpha-kleisin subunit. Viable homozygous mutants could be selected for the Arabidopsis thaliana alpha-kleisins SYN1, SYN2, and SYN4, which can partially compensate each other. For the kleisin SYN3 and for the single-copy genes SMC1, SMC3, and SCC3, only heterozygous mutants were obtained that displayed between 77% and 97% of the wild-type transcript level. Compared to wild-type nuclei, sister chromatid alignment was significantly decreased along arms in 4C nuclei of the homozygous syn1 and syn4 and even of the heterozygous smc1, smc3, scc3, and syn3 mutants. Knocking out SYN1 and SYN4 additionally impaired sister centromere cohesion. Homozygous mutants of SWITCH1 (required for meiotic sister chromatid alignment) displayed sterility and decreased sister arm alignment. For the cohesin loading complex subunit SCC2, only heterozygous mutants affecting sister centromere alignment were obtained. Defects of the alpha-kleisin SYN4, which impair sister chromatid alignment in 4C differentiated nuclei, do apparently not disturb alignment during prometaphase nor cause aneuploidy in meristematic cells. The syn2, 3, 4 scc3 and swi1 mutants display a high frequency of anaphases with bridges (~10% to20% compared to 2.6% in wild type). Our results suggest that (a) already a slight reduction of the average transcript level in heterozygous cohesin mutants may cause perturbation of cohesion, at least in some leaf cells at distinct loci; (b) the decreased sister chromatid alignment in cohesin mutants can obviously not fully be compensated by other cohesion mechanisms such as DNA concatenation; (c) some cohesin genes, in addition to cohesion, might have further essential functions (e.g., for genome stability, apparently by facilitating correct recombination repair of double-strand breaks).

The occurrence and extent of genome size variation within species is controversially discussed and thorough analyses are rare. Given the large morphological variation in Lagenaria siceraria (bottle gourds) and its wide distribution in Africa we here analysed (1) the genome size variation within cultivars of L. siceraria, (2) the correlation between genome size and morphological traits, and (3) the geographical patterns of DNA content within the species. We measured 2C-values of 366 individuals from 117 accessions of L. siceraria (2n = 22) from Africa, America and Asia via flow cytometry with propidium iodide as DNA stain. We found that 2C-value in L. siceraria (0.683–0.776 pg/2C) is about two times lower than previously reported and varies by about 12% among all accessions. Moreover, our results indicated a clear correlation of genome size with two different seed or usage types and with growing elevations in West Africa. Within the seed types genome size varies by 6.6 and 7.5%, respectively. The genome size differences in seed types of L. siceraria might indicate differences in their evolutionary history and necessitates a re-evaluation of the phylogenetic relationships within L. siceraria while the correlation between 2C-value and the elevation of the collecting sites might indicate an adaptation of genome size to an unknown ecological parameter connected to altitude.

The chromosomal distribution of seven histone methylation marks (H3K4me2, H3K9me1,2,3 and H3K27me1,2,3) was analysed in the gymnosperm species Pinus sylvestris and Picea abies. Similarly to the situation in other investigated eukaryotes, dimethylation of lysine 4 of histone H3 is restricted to euchromatin in gymnosperms. Surprisingly, also H3K9me1-a mark classified as heterochromatin-specific in angiosperms-labels the euchromatin in P. sylvestris and P. abies. The other investigated methylation marks are either equally distributed along the chromosomes, as H3K9me2 and H3K27me1 (in both species) and H3K9me3 (in P. abies), or enriched at specific types of heterochromatin, as H3K9me3 (in P. sylvestris) and H3K27me2 and H3K27me3 in both species. Although the methylation marks themselves are apparently conserved, their functional specificity within the frame of the 'epigenetic code' might have diverged during evolution.

Ten of the 17 species of the taxonomically difficult Andean mint genus Minthostachys (Lamiaceae) were submitted to flow cytometric measurements of nuclear DNA content to test the hypothesis of the occurrence of different ploidy levels within the genus. Nuclear DNA content was found to vary from 1.643 to 1.775 pg, i.e by only ca. 8% between individual accessions, thus providing no evidence for polyploidy in Minthostachys. While these results do not preclude the possibility that the genus contains polyploid species nor the occurrence of heteroploidy with nearly identical nuclear DNA contents, they suggest that polyploidy did not play a major role in its diversification.

By flow cytometric experiments in Cyclamen persicum both with propidium iodide (PI) and DAPI (4′,6-Diamidino-2-phenylindol)-staining we were able to present (1) a new estimation for the absolute DNA content in the range of 3.17 pg DNA/2C and (2) ploidy abnormalities which were detected in the pathway of somatic embryogenesis. These aberrations might have arisen from ploidy mutations or abnormal polyploidization processes. Morphologically normal somatic embryos contained the smallest proportion of individuals with ploidy abnormalities, but also a regenerated plant in the green house displayed a significantly elevated DNA content. The results are discussed in the framework of future application of in vitro propagation techniques based on regeneration from fastly growing undifferentiated cells like long-term callus and suspension cultures.

The prognosis of rhabdomyosarcoma (RMS) in advanced stages is still sobering. Therapy is limited due to local tumor recurrence, development of metastases and multidrug resistance. The aim of this study was to investigate the development of multidrug resistance in cell lines and in xenografts of alveolar and embryonal RMS treated according to the German Soft Tissue Sarcoma Study (CWS). Alveolar and embryonal RMS cell lines were treated with Vincristine, Topotecan, Carboplatin, Actinomycin D, or Ifosfamide. Expression levels of resistance-associated genes were assessed using Real time-PCR. Nude mice (NMRI nu/nu, n = 10 per group) underwent xenotransplantation of human embryonal or alveolar RMS. Animals were treated with standard chemotherapeutic drugs Vincristine, Topotecan, Carboplatin, Actinomycin D, or Ifosfamide according to treatment schedules of the CWS-study. Tumor sizes were measured and relative tumor volumes were calculated. Animals were sacrified after 20 days and standard histology, Real-time-PCR for MDR1-, MRP-, LRP- and MDM2-gene as well as immunhistochemistry for MDR1-, LRP-, and MRP-protein were performed. In the cell lines, an up-regulation of MDR-1 gene was found in alveolar rhabdomyosarcoma. In embryonal rhabdomyosarcoma, an up-regulation of LRP and MRP was found. Standard chemotherapy of alveolar rhabdomyosarcoma resulted in a significant reduction of tumor growth (P < 0.05) in all groups. In embryonal rhabdomyosarcoma strongest effects were found after treatment with Ifosfamide, Vincristine and Carboplatin (P < 0.05). RT-PCR revealed a MDR1-dependent mechanism in alveolar rhabdomyosarcoma. In embryonal rhabdomyosarcoma, MDR1 occurred to a lower degree. Immunhistochemistry revealed correlating expression levels of multidrug resistance-associated proteins. The use of established chemotherapy on human RMS in vivo had strong effects on xenografts compared to their controls. In all cases, there was only a reduction of tumor growth, but not a complete eradication of the tumors. Chemotherapy seemed to upregulate the expression of resistance-associated genes in vitro and in vivo. The mechanism of multidrug resistance depends on the tumor subtype. Therefore, further investigations will be required to evaluate multidrug resistance in patients and to investigate new modalities for a reversal of multidrug resistance.

Gene targeting is currently of distinct interest as an innovative additive treatment option in various malignancies. Its role in pediatric liver tumors has not yet been evaluated thoroughly. For the first time the authors systematically analyzed both lipid-based transfection as well as transduction with adenovirus vectors (Ad) and Sendai virus vectors (SeVV) in order to optimize gene transfer into hepatoblastoma (HB) cells. Two HB cell lines were infected with Ad or SeVV coding for green fluorescent protein (Ad-GFP, SeVV-GFP); transduction efficiencies and apoptosis were assessed using flow cytometry. Furthermore, lipofection of HB cell lines with plasmid-constructs comprising liver-specific promoters was performed using Lipofectamine™ 2000 and FuGENE 6™; lipofection efficiency was monitored by flow cytometry, microscopy, and luciferase activity. The Ad-GFP showed higher transduction rates (61–86%) than the SeVV-GFP (4–24%) depending on the HB cell line used. Infections with first generation SeVV vectors (SeVV-GFP) led to increased target cell apoptosis (7–43%) compared to Ad-GFP (4–16%). The Lipofectamine™ 2000 revealed a higher transfection efficiency than the FuGENE 6™ for both HB cell lines tested. The liver-specific promoters were found to be differently active in the HB cell lines. This study delineates recombinant adenovirus vectors as a promising tool for gene transduction in the HB cells. Furthermore, enhanced activity of the liver-specific promoters in HUH6 cells compared to HepT1 cells supports the observation of varying biological behavior in histologically differing HB tissues.

Differential painting of all five chromosome pairs of Arabidopsis thaliana revealed for the first time the interphase chromosome arrangement in a euploid plant. Side-by-side arrangement of heterologous chromosome territories and homologous association of chromosomes 1, 3 and 5 (on average in 35-50% of nuclei) are in accordance with the random frequency predicted by computer simulations. Only the nucleolus organizing region (NOR)-bearing chromosome 2 and 4 homologs associate more often than randomly, since NORs mostly attach to a single nucleolus. Somatic pairing of homologous approximately 100 kb segments occurs less frequently than homolog association, not significantly more often than expected at random and not simultaneously along the homologs. Thus, chromosome arrangement in Arabidopsis differs from that in Drosophila (characterized by somatic pairing of homologs), in spite of similar genome size, sequence organization and chromosome number. Nevertheless, in up to 31.5% of investigated Arabidopsis nuclei allelic sequences may share positions close enough for homologous recombination.

The subfamily of the Lemnoideae belongs to a different order than other monocotyledonous species that have been sequenced and comprises aquatic plants that grow rapidly on the water surface. Here we select Spirodela polyrhiza for whole-genome sequencing. We show that Spirodela has a genome with no signs of recent retrotranspositions but signatures of two ancient whole-genome duplications, possibly 95 million years ago (mya), older than those in Arabidopsis and rice. Its genome has only 19,623 predicted protein-coding genes, which is 28% less than the dicotyledonous Arabidopsis thaliana and 50% less than monocotyledonous rice. We propose that at least in part, the neotenous reduction of these aquatic plants is based on readjusted copy numbers of promoters and repressors of the juvenile-to-adult transition. The Spirodela genome, along with its unique biology and physiology, will stimulate new insights into environmental adaptation, ecology, evolution and plant development, and will be instrumental for future bioenergy applications., Spirodela, or duckweed, is a basal monocotyledonous plant with both pharmaceutical and commercial value. Here, the authors sequence the genome of Spirodela polyrhiza, suggesting its genome has evolved by neotenous reduction and clonal propagation, and provide a platform for future comparative genomic studies in angiosperms.

The current 'state-of-art' as to chromosome painting in plants is reviewed. We define different situations described as painting so far: i) Genomic in situ hybridisation (GISH) with total genomic DNA to distinguish alien chromosomes on the basis of divergent dispersed repeats, ii) 'Chromosomal in situ suppression' (CISS) hybridisation with chromosome-derived DNA probes and blocking of interchromosomally dispersed repeats by total genomic or C0t-1 DNA in excess, iii) exceptional cases of single chromosome painting by probes containing chromosome-specific dispersed repeats, and iv) Fluorescence in situ hybridisation (FISH) with extended contigs of large insert clones for painting of those chromosomes of a euploid complement which harbour the cloned sequences. While GISH was successfully applied in most plant hybrids and/or their derivatives, painting of individual chromosomes by CISS hybridisations of chromosome-specific DNA probes have so far not revealed convincing results in plants. The reason for this failure and the use of possible alternative approaches are discussed. At least for small plant genomes, painting by large insert single sequence clones provides a promising alternative tool to solve cytogenetic questions, which up to now could not be tackled otherwise. An example of such a painting is described in detail for Arabidopsis thaliana.

The multisubunit protein complex cohesin is required to establish cohesion between sister chromatids during S phase and to maintain it during G2 and M phases. Cohesin is essential for mitosis, and even partial defects cause very high rates of chromosome loss. In budding yeast, cohesin associates with specific sites which are distributed along the entire length of a chromosome but are more dense in the vicinity of the centromere. Real-time imaging of individual centromeres tagged with green fluorescent protein suggests that cohesin bound to centromeres is important for bipolar attachment to microtubules. This cohesin is, however, incapable of resisting the consequent force, which leads to sister centromere splitting and chromosome stretching. Meanwhile, cohesin bound to sequences flanking the centromeres prevents sister chromatids from completely unzipping and is required to pull back together sister centromeres that have already split. Cohesin therefore has a central role in generating a dynamic tension between microtubules and sister chromatid cohesion at centromeres, which lasts until chromosome segregation is finally promoted by separin-dependent cleavage of the cohesin subunit Scc1p.

The resolution of the chromosomal positions of six high- and one low-copy sequences by oligonucleotide-primed in situ (PRINS) labelling was compared with corresponding data obtained after fluorescent in situ hybridization (FISH) on field-bean and barley chromosomes. While PRINS proved to be suitable for the rapid detection of high-copy tandem repeats at the same loci as those revealed by FISH, no clear PRINS signal was obtained for the low-copy family of vicilin genes at their locus on field-bean chromosome II. This indicates that localization of short target sequences by primer extension via Taq polymerase in situ does not yet provide a resolution equal, or superior, to FISH on plant chromosomes. Therefore, the use of a cocktail of chromosome-specific single-copy sequences as primers for PRINS is no alternative for the not as yet feasible chromosome painting in plants.

A comprehensive survey of the molecular-cytogenetic features of the Vicia faba chromosome complement (2n = 12) is given. It includes previous as well as new original data. Various Giemsa, restriction endonuclease and fluorochrome banding patterns, azacytidine-mediated segment extension, replication patterns, lateral A/T asymmetry and sequence localization data for tandemly arranged simple sequence repeats, dispersed repeats and coding sequences as well as histone acetylation patterns are considered. This allows not only to distinguish and characterize telomeres, subtelomeres, centromeres and the NOR, but also the structure of the 5S rRNA gene loci and two main types of interstitial heterochromatin. Additionally, it offers physical landmarks within euchromatic areas. Thus, the field bean genome, exemplified by the reconstructed karyotype ACB, belongs to the cytogenetically best investigated plant genomes.

The karyotype of H. annuus was analysed by computer-aided image processing with respect to the chromosome length, arm ratio, occurrence and chromosomal position of intercalary heterochromatin and the location of 18S/25S and 5S ribosomal RNA genes. The karyotype was subdivided into a group of four acrocentric chromosome pairs, of which two were distinguishable by HKG (HCl, KOH, Giemsa) banding and a group of 13 meta- to submetacentric pairs. The latter could be subdivided into seven pairs with one and six pairs with two HKG bands. Three pairs of submetacentric satellite chromosomes revealed 18S/25S rDNA loci after fluorescence in situ hybridization (FISH) and silver staining. A fourth, smaller and possibly inactive, locus occurred in the terminal position on a metacentric pair. One submetacentric satellite chromosome pair revealed a 5S rRNA gene locus in the pericentromeric position; a second locus marked a submetacentric pair with one HKG band. The C-banding technique marks exclusively centromeric heterochromatin. Measurements of chromosomes in combination with Giemsa banding and FISH enabled the discrimination of most chromosome pairs of the sunflower.

In situ localization of short low- or single-copy sequences is still difficult in plants. One solution to this problem could be the use of large yeast artificial chromosomes (YACs) for fluorescence in situ hybridization. Two YACs specific for a single copy marker on the long arm of the NOR-chromosome 2 of tomato (Lycopersicon esculentum) were selected. Both probes hybridized exclusively to this chromosome, although one produced a slightly dispersed hybridization signal. Hybridization of these YACs onto potato chromosome showed a clear single locus on the homoeologous potato chromosome in both cases.

The positions of 18/25S rRNA genes, 5S RNA genes and of Arabidopsis-type telomeric repeats were localized by fluorescent in situ hybridization (FISH) on the chromosomes of three coniferous species; Picea abies, Larix decidua and Pinus sylvestris, each with 2n=24 chromosomes. Computer-aided chromosome analysis was performed on the basis of the chromosome length, the arm length ratio and the position of the hybridization signals. This enabled the chromosomes of the Norway spruce, 4 chromosomes of the European larch and 3 of the karyotype of the Scots pine to be individually distinguished. With respect to the chromosomal positions of rDNA and 5S rDNA loci, chromosome pair I of P. sylvestris is suggested to be homoeologous to pair II of P. abies, while another chromosome pair of P. sylvestris might be homoeologous to chromosome pair III of L. decidua.

Using fluorescence in situ hybridization (FISH), four different seed protein genes were physically mapped on metaphase chromosomes of Vicia faba L. dropped on slides. FISH with a 2.8 kb genomic probe of a legumin B4 gene resulted in reproducible signals on the long arm of chromosome III near the centromere. The same clone cross-hybridized at a lower frequency to the short arm of chromosome II, where the closely related legumin B3 gene family is located. The locus for legumin A-genes could be detected in the distal half of the long arm of chromosome V using a 1.7 kb cDNA clone. The locus of an unknown seed protein gene was mapped to the long arm of chromosome I using a mixture of polymerase chain reaction-amplified DNA fragments of the coding region of up to 1 kb in size.

Data for chromosomal localization of theArabidopsis-type of telomeric sequence repeats (TTTAGGG)n are compiled for 44 species belonging to 14 families of angiosperms, gymnosperms and bryophytes. For 23 species and seven families this is the first report. Species of all families, except theAlliaceae, revealed these sequences at their chromosome termini. This indicates thatArabidopsis-type telomeric repeats are highly conserved. It is inferred that they represent the basic telomere sequence of higher plant phyla. In theAlliaceae, a deviating sequence (and mechanism?) for the stabilization of chromosome termini has possibly evolved secondarily. Nine species revealed interstitial telomeric sequences in addition to the terminal ones, in three species (Vicia faba, Pinus elliottii, P. sylvestris) also at centromeric positions. Interstitial telomeric sequences may indicate karyotype reconstructions, in particular alterations of chromosome numbers by chromosome fusion — or inversions with one breakpoint within the terminal array of repeats. They may contribute to stabilization of chromosome breaks, especially centric fissions, and increase the frequency of meiotic and illegitimate recombination.

The localization of rRNA genes was studied by fluorescent in situ hybridization (FISH) on chromosomes of the cultivated apple, M.×domestica ‘Pinova’ (2n=34). The 18S/25S rRNA loci were detected in terminal positions of the short arms of two submetacentric and two metacentric chromosome pairs. One 5S rRNA gene locus was found in the proximal region of the short arm of a small metacentric chromosome pair.