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Abstract The genomes of many plants, animals, and fungi frequently comprise dispensable B chromosomes that rely upon various chromosomal drive mechanisms to counteract the tendency of non-essential genetic elements to be purged over time. The B chromosome of rye – a model system for nearly a century – undergoes targeted nondisjunction during first pollen mitosis, favouring segregation into the generative nucleus, thus increasing their numbers over generations. However, the genetic mechanisms underlying this process are poorly understood. Here, using a newly-assembled, ~430 Mb-long rye B chromosome pseudomolecule, we identify five candidate genes whose role as trans-acting moderators of the chromosomal drive is supported by karyotyping, chromosome drive analysis and comparative RNA-seq. Among them, we identify DCR28, coding a microtubule-associated protein related to cell division, and detect this gene also in the B chromosome of Aegilops speltoides. The DCR28 gene family is neo-functionalised and serially-duplicated with 15 B chromosome-located copies that are uniquely highly expressed in the first pollen mitosis of rye.

Background: Perianal rhabdomyosarcoma ((P)RMS) are rare and have a poor prognosis. Data in young children are limited and local therapy is not well defined. Combined brachytherapy and surgery is routinely being used for RMS at other sites in children as it provides good oncologic outcomes and allows for organ-sparing surgery. The objective of this study was to evaluate this combination treatment for local tumor control and organ-sparing surgery in young children with (P)RMS. Methods: A retrospective review of the medical records of all children who underwent surgery and brachytherapy for (P)RMS at our institution since 2009 was conducted. Results: Surgery for (P)RMS was performed in 6 patients at a median age of 19 months (range 8–50). Embryonal RMS was diagnosed in 4 patients and alveolar RMS in 2 patients, of which 1 patient had FOXO1 fusion-positive RMS. All patients underwent postoperative high-dose rate (HDR) brachytherapy. Organ-preserving surgery was achieved in 5 of 6 patients (83 %). In 1 patient, the entire sphincter was infiltrated, making organ-preserving resection impossible. 5 of 6 patients (83 %) exhibited an event-free and overall survival at a median follow-up of 26 months (range 8–107). One patient died due to locoregional recurrence. Complications were urethral leakage in 1 patient followed by urethral stenosis and delayed wound healing and vaginal stenosis in another patient. No patient reported fecal incontinence. Conclusions: Combined treatment with surgery and HDR brachytherapy is feasible in very young children with (P)RMS and leads to a favorable oncologic outcome. Preliminary data show a good functional preservation.

Abstract The presence of incompatibility alleles in primary amphidiploids constitutes a reproductive barrier in newly synthesized wheat-rye hybrids. To overcome this barrier, the genome stabilization process includes large-scale chromosome rearrangements. In incompatible crosses resulting in fertile amphidiploids, the elimination of one of the incompatible alleles Eml-A1 or Eml-R1b can occur already in the somatic tissue of the wheat × rye hybrid embryo. We observed that the interaction of incompatible loci Eml-A1 of wheat and Eml-R1b of rye after overcoming embryo lethality leads to hybrid sterility in primary triticale. During subsequent seed reproductions (R1, R2 or R3) most of the chromosomes of A, B, D and R subgenomes undergo rearrangement or eliminations to increase the fertility of the amphidiploid by natural selection. Genotyping-by-sequencing (GBS) coverage analysis showed that improved fertility is associated with the elimination of entire and partial chromosomes carrying factors that either cause the disruption of plant development in hybrid plants or lead to the restoration of the euploid number of chromosomes (2n = 56) in the absence of one of the incompatible alleles. Highly fertile offspring obtained in compatible and incompatible crosses can be successfully adapted for the production of triticale pre-breeding stocks.

Wilms tumors (WT) are characterized by variable contributions of blastemal, epithelial and stromal elements, reflecting their diverse cellular origins and genetic drivers. In vitro models remain rare, despite a growing need to better characterize tumor biology and evaluate new treatments. Using three approaches, we have now established a large collection of long-term cultures that represent this diversity. Adherent WT cultures are predominated by stromal cells, 3D spheroids model blastema, and patient-derived organoid cultures of both tumor and healthy kidney tissue result in the preferential growth of epithelial cells. Adherent, spheroid and organoid cultures are also clearly distinguishable by their transcriptome. Preclinical drug screening experiments revealed sensitivity to a range of inhibitors, that are highly effective in other childhood solid tumors. Sensitivity was related to MYCN status, a marker associated with adverse outcome across human cancers including WT. The combination of the three culture techniques represents a promising tool to both explore tumor heterogeneity in vitro and to facilitate characterization of candidate driver genes, in order to improve treatment regimens in the future.

Introduction: Pancreaticopleural fistula (PPF) is a very rare complication of pancreatitis. The symptoms are non-specific and the diagnosis is often missed. Case presentation: A 14-year-old adolescent was admitted to the department of pediatric surgery with several readmissions due to recurrent left pleural effusion and chest pain. The patient also had a history of a left-sided congenital diaphragmatic hernia (CDH), which was surgically corrected via laparotomy at the age of two. The pleural effusion was explained in the context of pneumonia, but elevated serum levels of amylase and lipase prompted imaging. Computer tomography (CT) and standard magnetic resonance imaging (MRI) showed a small gap in the diaphragm and the pancreatic tail nearby leading to the diagnosis of recurrent CDH. Explorative thoracoscopy was performed, but showed the diaphragm intact. Pleurodesis was realized to treat the effusion. Two months later, recurrence of the pleural effusion in the left hemithorax and pancreatitis occurred. Magnetic resonance cholangiopancreatography (MRCP) showed a small subphrenic cyst close to the pancreatic tail and multiple supradiaphragmal cysts for the first time. Persistently, the small gap in the diaphragm close to the pancreatic tail was detected and the now suspected diagnosis was PPF. Subsequently, laparoscopic resection of the pancreatic tail was performed including the closure of the diaphragmatic gap. The course was uneventful, the pleural effusion disappeared, and the levels of serum pancreatic enzymes normalized during follow-up. Conclusion: PPF is rare and various entities may masquerade the diagnosis. A massive pleural effusion in combination with pancreatitis may indicate this serious condition.

Abstract Circular RNAs (circRNAs) are a regulatory RNA class. While cancer-driving functions have been identified for single circRNAs, how they modulate gene expression in cancer is not well understood. We investigate circRNA expression in the pediatric malignancy, neuroblastoma, through deep whole-transcriptome sequencing in 104 primary neuroblastomas covering all risk groups. We demonstrate that MYCN amplification, which defines a subset of high-risk cases, causes globally suppressed circRNA biogenesis directly dependent on the DHX9 RNA helicase. We detect similar mechanisms in shaping circRNA expression in the pediatric cancer medulloblastoma implying a general MYCN effect. Comparisons to other cancers identify 25 circRNAs that are specifically upregulated in neuroblastoma, including circARID1A. Transcribed from the ARID1A tumor suppressor gene, circARID1A promotes cell growth and survival, mediated by direct interaction with the KHSRP RNA-binding protein. Our study highlights the importance of MYCN regulating circRNAs in cancer and identifies molecular mechanisms, which explain their contribution to neuroblastoma pathogenesis.

Abstract The centromere is the chromosome region where microtubules attach during cell division. In contrast to monocentric chromosomes with one centromere, holocentric species usually distribute hundreds of centromere units along the entire chromatid. We assembled the chromosome-scale reference genome and analyzed the holocentromere and (epi)genome organization of the lilioid Chionographis japonica. Remarkably, each of its holocentric chromatids consists of only 7 to 11 evenly spaced megabase-sized centromere-specific histone H3-positive units. These units contain satellite arrays of 23 and 28 bp-long monomers capable of forming palindromic structures. Like monocentric species, C. japonica forms clustered centromeres in chromocenters at interphase. In addition, the large-scale eu- and heterochromatin arrangement differs between C. japonica and other known holocentric species. Finally, using polymer simulations, we model the formation of prometaphase line-like holocentromeres from interphase centromere clusters. Our findings broaden the knowledge about centromere diversity, showing that holocentricity is not restricted to species with numerous and small centromere units.

Abstract Cell type-specific mapping of element distribution is critical to fully understand how roots partition nutrients and toxic elements with aboveground parts. In this study, we developed a method that combines fluorescence-activated cell sorting (FACS) with inductively coupled plasma mass spectrometry (ICP-MS) to assess the ionome of different cell populations within Arabidopsis thaliana roots. The method reveals that most elements exhibit a radial concentration gradient increasing from the rhizodermis to inner cell layers, and detected previously unknown ionomic changes resulting from perturbed xylem loading processes. With this approach, we also identify a strong accumulation of manganese in trichoblasts of iron-deficient roots. We demonstrate that confining manganese sequestration in trichoblasts but not in endodermal cells efficiently retains manganese in roots, therefore preventing toxicity in shoots. These results indicate the existence of cell type-specific constraints for efficient metal sequestration in roots. Thus, our approach opens an avenue to investigate element compartmentation and transport pathways in plants.

Background: Paediatric adrenocortical carcinomas (ACC) are highly aggressive malignancies with a dismal prognosis in advanced and metastatic diseases. Little is known about outcome of patients with refractory and relapsed (r/r) disease. Procedure: National retrospective multicentre study including r/r ACC diagnosed in patients aged

A spontaneous mutant of the duckweed Lemna gibba clone no. 7796 (known as strain G3, WT) was discovered. In this mutant clone, L. gibba clone no. 9602 (mt), the morphological parameters (frond length, frond width, root length, root diameter) indicated an enlarged size. A change in the frond shape was indicated by the decreased frond length/width ratio, which could have taxonomic consequences. Several different cell types in both the frond and the root were also enlarged. Flow cytometric measurements disclosed the genome size of the WT as 557 Mbp/1C and that of the mt strain as 1153 Mbp/1C. This represents the results of polyploidisation of a diploid clone to a tetraploid one. The mutant clone flowered under the influence of long day-treatment in half-strength Hutner’s medium in striking contrast to the diploid WT. Low concentration of salicylic acid (CAB, RBCS) showed higher abundance in light and less dramatic decline in darkness in the mt than in WT, while this was not the case with plastid-encoded genes (RBCL, PSAA, PSBA, PSBC).

Angiosperm genome sizes (GS) range ~2400-fold and comprise genes and their regulatory regions, repeats, semi-degraded repeats, and ‘dark matter’. The latter represents repeats so degraded that they can no longer be recognised as repetitive. In exploring whether the histone modifications associated with chromatin packaging of these contrasting genomic components are conserved across the diversity of GS in angiosperms, we compared immunocytochemistry data for two species whose GS differ ~286-fold. We compared published data for Arabidopsis thaliana with a small genome (GS = 157 Mbp/1C) with newly generated data from Fritillaria imperialis, which has a giant genome (GS = 45,000 Mbp/1C). We compared the distributions of the following histone marks: H3K4me1, H3K4me2, H3K9me1, H3K9me2, H3K9me3, H3K27me1, H3K27me2, and H3K27me3. Assuming these histone marks are associated with the same genomic features across all species, irrespective of GS, our comparative analysis enables us to suggest that while H3K4me1 and H3K4me2 methylation identifies genic DNA, H3K9me3 and H3K27me3 marks are associated with ‘dark matter’, H3K9me1 and H3K27me1 mark highly homogeneous repeats, and H3K9me2 and H3K27me2 mark semi-degraded repeats. The results have implications for our understanding of epigenetic profiles, chromatin packaging and the divergence of genomes, and highlight contrasting organizations of the chromatin within the nucleus depending on GS itself.

Nigella is a small genus belonging to the Ranunculaceae family which is presumably originated and distributed in Aegean and the adjacent Western-Irano-Turanian region. Comparative repeat analysis of N. sativa, N. damascena and N. bucharica was performed using low-pass Illumina genomic reads followed by karyotyping and FISH mapping of seven Nigella species using the in silico identified repeats and ribosomal DNA (rDNA) probes. High- and moderate-copy repeat sequences occupy 57.52, 59.01, and 64.73% of N. sativa, N. damascena and N. bucharica genomes, respectively. Roughly, half of the genomes are retrotransposons (class I transposons), while DNA transposons (class II transposons) contributed to only about 2% of the genomes. The analyzed Nigella species possess large genomes of about 7.4 to 12.4 Gbp/1C. Only two satellite repeats in N. sativa, one in N. damascena and four in N. bucharica were identified, which were mostly (peri)centromeric and represented about 1% of each genome. A high variation in number and position of 45S rDNA loci were found among Nigella species. Interestingly, in N. hispanica, each chromosome revealed at least one 45S rDNA site and one of them occurs in hemizygous condition. Based on the chromosome numbers, genome size and (peri)centromeric satellites, three karyotype groups were observed: Two with 2n = 2x = 12 and a karyotype formula of 10m + 2t (including N. sativa, N. arvensis, N. hispanica as the first group and N. damascena and N. orientalis as the second group) and a more distant group with 2n = 2x = 14 and a karyotype formula of 8m + 2st + 4t (including N. integrifolia and N. bucharica). These karyotype groups agreed with the phylogenetic analysis using ITS and rbcL sequences. We conclude that variation in (peri)centromeric sequences, number and localization of rDNA sites as well as chromosome number (dysploidy) are involved in the diversification of the genus Nigella.

Abstract Background To assess the feasibility and possible value of semi-automated diffusion weighted imaging (DWI) volumetry of whole neuroblastic tumors with apparent diffusion coefficient (ADC) map evaluation after neoadjuvant chemotherapy. Methods Pediatric patients who underwent surgical resection of neuroblastic tumors at our institution from 2013 to 2019 and who received a preoperative MRI scan with DWI after chemotherapy were included. Tumor volume was assessed with a semi-automated approach in DWI using a dedicated software prototype. Quantitative ADC values were calculated automatically of the total tumor volume after manual exclusion of necrosis. Manual segmentation in T1 weighted and T2 weighted sequences was used as reference standard for tumor volume comparison. The Student’s t test was used for parametric data while the Wilcoxon rank sum test and the Kruskal-Wallis test were applied for non-parametric data. Results Twenty seven patients with 28 lesions (neuroblastoma (NB): n = 19, ganglioneuroblastoma (GNB): n = 7, ganglioneuroma (GN): n = 2) could be evaluated. Mean patient age was 4.5 ± 3.2 years. Median volume of standard volumetry (T1w or T2w) was 50.2 ml (interquartile range (IQR): 91.9 ml) vs. 45.1 ml (IQR: 98.4 ml) of DWI (p = 0.145). Mean ADC values (× 10− 6 mm2/s) of the total tumor volume (without necrosis) were 1187 ± 301 in NB vs. 1552 ± 114 in GNB/GN (p = 0.037). The 5th percentile of ADC values of NB (614 ± 275) and GNB/GN (1053 ± 362) provided the most significant difference (p = 0.007) with an area under the curve of 0.848 (p

The heterochromatic knob (hk4S) on Arabidopsis chromosome 4 prevents the recombination between accessions with and without hk4S. Here, via egg-cell specific expression of the Cas9 nuclease, the authors demonstrate targeted reversal of the 1.1 Mb long hk4S-inversion in Col-0 and restore the crossovers with Ler-1.

The freshwater plant water lettuce (Pistia stratiotes L.) grows in warm climatic zones and is used for phytoremediation and biomass production. P. stratiotes belongs to the Araceae, an ecologically and structurally diverse early monocot family, but the phylogenetic relationships among Araceae members are poorly understood. Ribosomal DNAs (rDNAs), including the 35S and 5S rDNA, encode the RNA components of ribosomes and are widely used in phylogenetic and evolutionary studies of various plant taxa. Here, we comprehensively characterized the chromosomal locations and molecular organization of 35S and 5S rDNA genes in water lettuce using karyological and molecular methods. Fluorescence in situ hybridization revealed a single location for the 35S and 5S rDNA loci, each on a different pair of the species’ 28 chromosomes. Molecular cloning and nucleotide sequencing of 35S rDNA of P. stratiotes, the first representative Araceae sensu stricto in which such a study was performed, displayed typical structural characteristics. The full-length repeat showed high sequence conservation of the regions producing the 18S, 5.8S, and 25S rRNAs and divergence of the internal transcribed spacers ITS1 and ITS2 as well as the large intergenic spacer (IGS). Alignments of the deduced sequence of 18S rDNA with the sequences available for other Araceae and representatives of other clades were used for phylogenetic analysis. Examination of 11 IGS sequences revealed significant intra-genomic length variability due to variation in subrepeat number, with four types of units detected within the 35S rDNA locus of the P. stratiotes genome (estimated size 407 Mb/1C). Similarly, the 5S rDNA locus harbors gene units comprising a conserved 119-bp sequence encoding 5S rRNA and two types of non-transcribed spacer (NTS) sequences. Type I was classified into four subtypes, which apparently originated via progressive loss of subrepeats within the duplicated NTS region containing the 3’ part of the 5S rRNA gene. The minor Type II NTS is shorter than Type I and differs in nucleotide composition. Some DNA clones containing two or three consecutive 5S rDNA repeats harbored 5S rDNA genes with different types of NTSs, confirming the mosaic composition of the 5S rDNA locus.

Some eukaryotes exhibit dramatic genome size differences between cells of different organs, resulting from the programmed elimination of chromosomes. Aegilops speltoides is an annual diploid species from the Poaceae family, with a maximum number of eight B chromosomes (Bs) in addition to its inherent seven pairs of standard A chromosomes (As). The Bs of this species undergo precise elimination in roots early in embryo development. In areal parts of the plant, the number of Bs is stable. To affect the root restricted process of B chromosome elimination, we employed X-ray mutagenesis, and different types of restructured Bs were identified. Standard Bs were observed in all analyzed shoots of mutagenized plants, while B-A translocations were only observed in 35.7% of F1 plants. In total 40 different B variants inconsistently escaped the elimination process in roots. As a result, mosaicism of B chromosome variants was found in roots. Only a small B chromosome fragment fused to an A chromosome was stably maintained in roots and shoots across F1 to F3 generations. The absence of B-A translocation chromosomes possessing a derived B centromere in root cells implies that the centromere of the B is a key component of the chromosome elimination process.

BackgroundLoss of available central vein access sites for parenteral nutrition delivery represents one of the main indications for intestinal transplantation in children with intestinal failure. Placement of central venous catheters can be challenging in advanced loss of patent venous pathways. We recently described the hybrid technique (interventional plus surgical approach) of central line placement in children. The aim of this study was to describe and analyze the interventions used during the hybrid procedures regarding feasibility, safety and outcome.MethodsWe retrospectively analyzed the course of all children in our intestinal rehabilitation program undergoing hybrid central line placement. We evaluated patients' conditions, interventional techniques and surgical peculiarities as well as outcome.Results203 children were treated in our intestinal rehabilitation program between 2010 and 2021. Due to loss of venous access, hybrid technique was performed in 53 children during 76 interventions. In 40 cases the same vessel was reused via Seldinger technique. Among the 30 ultrasound-guided new vessel punctures, 12 were performed by puncture of collateral vessels. Extended interventions due to thoracic central venous obstruction and/or thrombosis requiring additional access via a femoral vein for rehabilitation of the vascular system was performed during 29 procedures including catheter extraction (1), angioplasties (18), stent placement (1), revascularization (5) and thrombectomy (4). Placement of a central line was not possible in 6 children which eventually underwent extended thoracic/vascular surgery: in three children the previously placed catheter could not be removed, in one child, placement of a thrombectomy-catheter was not possible because of inferior vena cava occlusion, and in two children, revascularization failed. Intestinal transplantation was considered in one patient because of impending loss of vascular access. Two self-limiting minor extravasations and one intervention-associated pericardial effusion occurred.ConclusionsHybrid interventions for central venous catheter placement and vascular rehabilitation enable a high success rate in children with intestinal failure and end-stage vascular access, circumventing the need for intestinal transplantation or advanced surgery. The relevant procedures are complex and require a foresighted and individualized approach with a wide range of interventional techniques. If performed with expertise, this combined interventional/surgical approach is feasible and safe.

Polyploidization is an evolutionary event leading to structural changes of the genome(s), particularly allopolyploidization, which combines different genomes of distinct species. The tetraploid species, Sorghum halepense, is assumed an allopolyploid species formed by hybridization between diploid S. bicolor and S. propinquum. The repeat profiles of S. bicolor, S. halepense, and their relatives were compared to elucidate the repeats’ role in shaping their genomes. The repeat frequencies and profiles of the three diploid accessions (S. bicolor, S. bicolor ssp. verticilliflorum, and S. bicolor var. technicum) and two tetraploid accessions (S. halepense) are similar. However, the polymorphic distribution of the subtelomeric satellites preferentially enriched in the tetraploid S. halepense indicates drastic genome rearrangements after the allopolyploidization event. Verified by CENH3 chromatin immunoprecipitation (ChIP)-sequencing and fluorescence in situ hybridization (FISH) analysis the centromeres of S. bicolor are mainly composed of the abundant satellite SorSat137 (CEN38) and diverse CRMs, Athila of Ty3_gypsy and Ty1_copia-SIRE long terminal repeat (LTR) retroelements. A similar centromere composition was found in S. halepense. The potential contribution of S. bicolor in the formation of tetraploid S. halepense is discussed.

Abstract Background Biliary rhabdomyosarcoma (RMS) is the most common biliary tumor in children. The management of affected patients contains unique challenges because of the rarity of this tumor entity and its critical location at the porta hepatis, which can make achievement of a radical resection very difficult. Methods In a retrospective chart analysis we analysed children suffering from biliary RMS who were registered in three different CWS trials (CWS-96, CWS-2002P, and SoTiSaR registry). Results Seventeen patients (12 female, 5 male) with a median age of 4.3 years were assessed. The median follow-up was 42.2 months (10.7–202.5). The 5-year overall (OS) and event free survival (EFS) rates were 58% (45–71) and 47% (34–50), respectively. Patients > 10 years of age and those with alveolar histology had the worst prognosis (OS 0%). Patients with botryoid histology had an excellent survival (OS 100%) compared to those with non-botryoid histology (OS 38%, 22–54, p = 0.047). Microscopic complete tumor resection was achieved in almost all patients who received initial tumor biopsy followed by chemotherapy and delayed surgery. Conclusion Positive predictive factors for survival of children with biliary RMS are age ≤ 10 years and botryoid tumor histology. Primary surgery with intention of tumor resection should be avoided.

Abstract Background To evaluate optimal therapy and potential risk factors. Methods Data of DSRCT patients

Usually, chromosome sets (karyotypes) and genome sizes are rather stable for distinct species and therefore of diagnostic value for taxonomy. In combination with (cyto)genomics, both features provide essential cues for genome evolution and phylogenetic relationship studies within and between taxa above the species level. We present for the first time a survey on chromosome counts and genome size measurement for one or more accessions from all 36 duckweed species and discuss the evolutionary impact and peculiarities of both parameters in duckweeds.

Biomarkers allowing characterization of pediatric rhabdomyosarcoma (RMS) are lacking. Epitope detection in monocytes (EDIM) is a novel method focused on detection of the biomarkers TKTL1 (transketolase-like protein 1) and Apo10 (epitope of DNaseX) in activated monocytes (CD14+/CD16+) from patient’s blood. We investigated the expression of these biomarkers in RMS cell lines, tumor material, and peripheral blood from RMS patients. Expression levels of TKTL1 and DNaseX/Apo10 in RMS cell lines (RH30, RD) and tumor samples were analyzed by RT-PCR and flow cytometry. Blood samples of 29 RMS patients were measured and compared to 27 healthy individuals. The percentages of activated CD14+/CD16+ monocytes harboring TKTL1 and Apo10 were determined. EDIM-TKTL1 and EDIM-Apo10 expression scores were calculated. The relationship between TKTL1 expression and DNA-hypomethylation was evaluated. Both RMS cell lines and tumor samples showed significantly higher expression levels of TKTL1 and DNaseX/Apo10 compared to skeletal muscle cells (SkMC). EDIM-TKTL1 and EDIM-Apo10 scores were positive in 96.5% of patients with RMS. All healthy controls had negative corresponding scores. RMS cell lines show increased expression levels of the biomarkers TKTL1 and DNaseX/Apo10. The sensitivity of the EDIM blood test indicates that this assay might serve as an additional tool in pediatric RMS.

ObjectiveRadiotherapy (RT) is an integral part of the interdisciplinary treatment of patients with high-risk neuroblastoma (NB). With the continuous improvements of outcome, the interest in local treatment strategies that reduce treatment-related side effects while achieving optimal oncological results is growing. Proton beam therapy (PBT) represents a promising alternative to conventional photon irradiation with regard to the reduction of treatment burden.MethodRetrospective analysis of children with high or intermediate risk NB receiving PBT of the primary tumor site during first-line therapy between 2015 and 2020 was performed. Data from the prospective in-house registry Standard Protonentherapie WPE – Kinder- (KiProReg) with respect to tumor control and treatment toxicity were analyzed. Adverse events were classified according to CTCAE Version 4 (V4.0) before, during, and after PBT.ResultsIn total, 44 patients (24 male, 20 female) with high (n = 39) or intermediate risk NB (n = 5) were included in the analysis. Median age was 3.4 years (range, 1.4–9.9 years). PBT doses ranged from 21.0 to 39.6 Gray (Gy) (median 36.0 Gy). Five patients received PBT to the MIBG-avid residual at the primary tumor site at time of PBT according to the NB-2004 protocol. In 39 patients radiation was given to the pre-operative tumor bed with or without an additional boost in case of residual tumor. After a median follow-up (FU) of 27.6 months, eight patients developed progression, either local recurrence (n = 1) or distant metastases (n = 7). Four patients died due to tumor progression. At three years, the estimated local control, distant metastatic free survival, progression free survival, and overall survival was 97.7, 84.1, 81.8, and 90.9%, respectively. During radiation, seven patients experienced higher-grade (CTCAE ≥ °3) hematologic toxicity. No other higher grade acute toxicity occurred. After PBT, one patient developed transient myelitis while receiving immunotherapy. No higher grade long-term toxicity was observed up to date.ConclusionPBT was a well tolerated and effective local treatment in children with high and intermediate risk NB. The role of RT in an intensive multidisciplinary treatment regimen remains to be studied in the future in order to better define timing, doses, target volumes, and general need for RT in a particularly sensitive cohort of patients.

Evidence for the existence of dikaryote-like strains, low nuclear sequence diversity and inter-nuclear recombination in arbuscular mycorrhizal fungi has been recently reported based on single nucleus sequencing data. Here, we aimed to support evidence of inter-nuclear recombination using an approach that filters SNP calls more conservatively, keeping only positions that are exclusively single copy and homozygous, and with at least five reads supporting a given SNP. This methodology recovers hundreds of putative inter-nucleus recombination events across publicly available sequence data from individual nuclei. Challenges related to the acquisition and analysis of sequence data from individual nuclei are highlighted and discussed, and ways to address these issues in future studies are presented.

In citrus, a classical method of studying crossovers and segregation distortion (SD) is the genetic analysis of progenies. A new strategy combining fluorescence-activated cell sorting and whole genome amplification of haploid pollen nuclei with a large set of molecular markers, offers the opportunity to efficiently determine the frequency of crossovers and the identification of SD without the need to generate segregating populations. Here we have analyzed meiotic crossover events in a pollen nuclei population from “Eureka” lemon and the allelic SD was evaluated in a pollen nuclei population from a clementine × sweet orange hybrid (“CSO”). Data obtained from the “CSO” pollen nuclei population were compared to those obtained from genotyping of a segregating population (“RTSO”) arising from a hand-made sexual hybridization between diploid non apomictic selected tangor (mandarin × sweet orange; “RTO” tangor) as female parent pollinated with “CSO” tangor as male parent. The analysis of crossovers rates on chromosome 1 revealed the presence of up to five crossovers events on one arm and four on the corresponding other arm, with an average of 1.97 crossovers per chromosome while no crossover events were observed in five “Eureka” lemon pollen nuclei. The rate of SD observed in “CSO” pollen nuclei (13.8%) was slightly lower than that recovered in the “RTSO” population (20.7%). In the pollen nuclei population, SD was found on linkage group (LG) 2, while the “RTSO” population showed SD on LGs 2 and 7. Potential male gametic selection mechanisms were distinguished in pollen grains, while in the population, mechanisms of gametophytic selection and/or zygotic selection were observed. This methodology is a very useful tool to facilitate research focused on the reproductive biology of citrus and study the mechanisms that affect crossovers and SD.

Abstract Background Total colonic aganglionosis (TCA) is a rare variant of Hirschsprung’s disease occurring in 3–10% of the cases. Only few studies reported the long-term clinical and metabolic outcomes of patients with TCA. The aim of this study was to evaluate the functional and metabolic long-term outcomes of children undergoing surgical treatment for TCA. Methods A 15-year retrospective study was performed. Blood chemistry tests and stool analysis performed at the last follow-up visit were recorded. Height and weight development were assessed using the corresponding percentiles for age. Faecal continence and quality of life were evaluated using a detailed questionnaire. Results Eleven patients were included in the study. The median age at surgery was 6 months (range: 3–72 months). After histological confirmation, all patients underwent a total colectomy. Ileoanal anastomosis (n = 6), ileorectal anastomosis (n = 1), J-pouch (n = 1) and Duhamel procedure (n = 3) were performed. Temporary ileostomy was closed after a median of 8 weeks in 10/11 patients. After a median follow-up of 78 months (range: 27–199 months), all evaluated patients were continent. Height and weight were appropriate for age in only 5 patients. Vitamin B12 and folic acid serum levels were normal in all examined patients. Ten patients had normal hemoglobin serum levels. Seven patients had low transferrin saturation in serum. Hemoccult tests were negative in all examined patients. Despite complex postoperative courses in some cases, patients and parents showed good overall satisfaction in terms of quality of life. Conclusion The majority of patients reported a good quality of life. This can result from the adaptation of the patients to certain disease states. The failure to thrive seems to be related with the extent of aganglionosis. The inclusion of these patients in interdisciplinary long-term follow-up care, in which pediatric surgeons, gastroenterologists, and dieticians are involved, is essential.

Background/Aims: Rhabdomyosarcoma, the most common pediatric soft tissue sarcoma, may show an intrinsic refractoriness to standard chemotherapy in advanced tumor stages, which is associated with poor prognosis. Cellular mechanisms conferring tumor cell survival and therapy resistance in many tumor types include the serum & glucocorticoid inducible kinase (SGK) 1 pathway, a kinase expressed ubiquitously with particularly strong expression in skeletal muscle and some tumor types. The present study explored whether SGK1 is expressed in rhabdomyosarcoma and, if so, whether this kinase impacts on tumor cell survival, proliferation and migration. Multiple in vitro techniques were used to study the role of SGK1 in rhabdomyosarcoma. Methods: The Gene Chip® Human Genome U133 Plus 2.0 Array were employed to examine SGK1 transcriptional activity in healthy muscle and rhabdomyosarcoma tissue. SGK1 transcript levels were quantified in rhabdomyosarcoma cell lines RD (embryonal subtype) and RH30 (alveolar subtype) by RT-PCR, cell viability was measured using MTT assays. Clonal cell growth was assessed via colony forming assays and migration experiments were performed in a transwell system. Results: SGK1 is expressed in embryonal and alveolar rhabdomyosarcoma tissue samples and in RD and RH30 rhabdomyosarcoma cell lines. Administration of EMD638683 – an inhibitor specific for SGK1 - decreased viability of RD and RH30 cells, enhanced the effects of the cytotoxic drug doxorubicin leading to reduced migration and decreased cell proliferation. Conclusions: SGK1 is expressed in rhabdomyosarcoma cells where it contributes to survival, therapy resistance, cell proliferation and migration. Thus, SGK1 inhibitors may be considered a therapeutic option for the treatment of therapy-resistant rhabdomyosarcoma.

INTRODUCTION:Esophageal atresia with or without tracheoesophageal fistula (EA/TEF) occurs approximately 1 in 3.500 live births representing the most common malformation of the upper digestive tract. Only half a century ago, EA/TEF was fatal among affected newborns suggesting that the steady birth prevalence might in parts be due to mutational de novo events in genes involved in foregut development. METHODS:To identify mutational de novo events in EA/TEF patients, we surveyed the exome of 30 case-parent trios. Identified and confirmed de novo variants were prioritized using in silico prediction tools. To investigate the embryonic role of genes harboring prioritized de novo variants we performed targeted analysis of mouse transcriptome data of esophageal tissue obtained at the embryonic day (E) E8.5, E12.5, and postnatal. RESULTS:In total we prioritized 14 novel de novo variants in 14 different genes (APOL2, EEF1D, CHD7, FANCB, GGT6, KIAA0556, NFX1, NPR2, PIGC, SLC5A2, TANC2, TRPS1, UBA3, and ZFHX3) and eight rare de novo variants in eight additional genes (CELSR1, CLP1, GPR133, HPS3, MTA3, PLEC, STAB1, and PPIP5K2). Through personal communication during the project, we identified an additional EA/TEF case-parent trio with a rare de novo variant in ZFHX3. In silico prediction analysis of the identified variants and comparative analysis of mouse transcriptome data of esophageal tissue obtained at E8.5, E12.5, and postnatal prioritized CHD7, TRPS1, and ZFHX3 as EA/TEF candidate genes. Re-sequencing of ZFHX3 in additional 192 EA/TEF patients did not identify further putative EA/TEF-associated variants. CONCLUSION:Our study suggests that rare mutational de novo events in genes involved in foregut development contribute to the development of EA/TEF.

Flow cytometry is widely used to determine genome size and ploidy level in plants. This technique, when coupled with fluorescence-activated cell sorting (FACS), whole genome amplification and genotyping (WGA), opens up new opportunities for genetic studies of individualized nuclei. This strategy was used to analyze the genetic composition of single pollen nuclei of different citrus species. The flow cytometry and microscope observations allowed us to differentiate the populations of pollen nuclei present in the diploid and euploid genotypes analyzed, showing that citrus has binuclear pollen. We have identified in the “CSO” tangor an additional nuclei population composed by the vegetative plus generative nuclei. Genotyping of this nuclei population revealed that vegetative and generative nuclei show the same genetic configuration. In addition, we have demonstrated the presence of unreduced gametes in the diploid genotype “Mexican lime.” Genomic amplification is a robust method for haploid nuclei genotyping with several molecular markers, whereas in diploid nuclei using heterozygous markers showed a bias towards one of the two alleles, limiting the use of this tool in this type of nuclei. We further discuss the importance and applications of single pollen genotyping in citrus genetic studies.

Meiotic recombination is a fundamental mechanism to generate novel allelic combinations which can be harnessed by breeders to achieve crop improvement. The recombination landscape of many crop species, including the major crop barley, is characterized by a dearth of recombination in 65% of the genome. In addition, segregation distortion caused by selection on genetically linked loci is a frequent and undesirable phenomenon in double haploid populations which hampers genetic mapping and breeding. Here, we present an approach to directly investigate recombination at the DNA sequence level by combining flow-sorting of haploid pollen nuclei of barley with single-cell genome sequencing. We confirm the skewed distribution of recombination events toward distal chromosomal regions at megabase resolution and show that segregation distortion is almost absent if directly measured in pollen. Furthermore, we show a bimodal distribution of inter-crossover distances, which supports the existence of two classes of crossovers which are sensitive or less sensitive to physical interference. We conclude that single pollen nuclei sequencing is an approach capable of revealing recombination patterns in the absence of segregation distortion.