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Objective: Vestibular symptoms are seen variably in patients with otosclerosis, both in patients with the disease and in patients after treatment with stapes surgery. The goal of this study was to objectify any changes of the bone-conducted (BC) ocular vestibular evoked myogenic potentials (oVEMPs) in patients with otosclerosis before and after stapes surgery. Study Design: Twenty-six healthy subjects and 27 patients with otosclerosis were tested in a tertiary care center, and 11 of the patients were tested preoperatively and postoperatively. The ears of patients were divided in 4 groups: normal ears, otosclerotic ears, ears after primary stapes surgery, and ears after revision surgery. The stimulus used in oVEMP testing was a BC stimulus delivered by a mini-shaker at stimulus frequencies of 250 and 500 Hz. oVEMP characteristics were the n1 and p1 latencies, n1 Y p1 amplitude, and threshold. Results: No significant differences were seen in all oVEMP characteristics between the 4 groups of ears without surgery nor in the ears that were tested preoperatively and postoperatively. Conclusion: No or undetectably little damage to the utricle is caused by both otosclerotic disease and stapes surgery. There is no additive value of screening with BC oVEMPs perioperatively in otosclerotic patients. Key Words: Bone-conducted stimuliVOcular vestibular evoked myogenic potentialVOtolith organsVOtosclerosisVStapes surgeryVStapedotomyVVestibular disorders. Otol Neurotol 34:1109Y1114, 2013.

The science of osteoimmunology, a relatively new field of research, reveals the important interactions between the immune system and skeletal system. Interactions occur between prostaglandin metabolism, inflammatory proteins and bone metabolism. Systemic as well as local sources of inflammation appear to be actively involved in both bone formation and resorption. Non Steroidal Anti-Inflammatory Drugs (NSAIDs) can play a detrimental role in bone fractures, opposing the aim of the intervention, and can have such a negative impact on the synthesis of prostaglandins that they could even promote bone resorption. When used for a prolonged time, NSAIDs can also cause the development of an inflammatory cascade starting from the gastro-intestinal system, possibly resulting in bone resorption. Several studies show that the use of either selective or non-selective NSAIDs are intimately related to disturbances in immunological allostasis, bone metabolism and the inhibition or impediment of bone healing.

Off-target or random integration of exogenous DNA hampers precise genomic engineering and presents a safety risk in clinical gene therapy strategies. Genetic definition of random integration has been lacking for decades. Here, we show that the A-family DNA polymerase θ (Pol θ) promotes random integration, while canonical non-homologous DNA end joining plays a secondary role; cells double deficient for polymerase θ and canonical non-homologous DNA end joining are devoid of any integration events, demonstrating that these two mechanisms define random integration. In contrast, homologous recombination is not reduced in these cells and gene targeting is improved to 100% efficiency. Such complete reversal of integration outcome, from predominately random integration to exclusively gene targeting, provides a rational way forward to improve the efficacy and safety of DNA delivery and gene correction approaches., Random off-target integration events can impair precise gene targeting and poses a safety risk for gene therapy. Here the authors show that repression of polymerase θ and classical non-homologous recombination eliminates random integration.

Patients with high-risk subgroup medulloblastoma (MB) show poor overall survival rates despite aggressive treatment while survivors suffer from important treatment-associated morbidity. Particularly poor outcome is observed in patients with tumors of the molecular subgroup Group 3 with an amplification and high levels of the transcription factor MYC. We have previously shown that Group 3 MB cell lines with MYC amplification are highly susceptible to pan- and class I-histone deacetylase (HDAC) inhibitor treatment. We here explore the molecular interaction of the well known oncogenic driver MYC and class I HDAC2. Six established MB cell lines were used for experiments in cell culture, including MYC amplified cell lines. The functional interaction between MYC and HDAC2 was investigated by enzymatic inhibition of class I HDACs using class I-selective HDAC inhibitors (HDACi) and by siRNA-mediated knockdown of HDAC2. Observed protein degradation was rescued with proteasome inhibition using small molecule proteasome inhibitor MG132. The direct interaction of HDAC2 and MYC was analyzed in co-immunoprecipitation and mass spectrometry studies. The pharmacological inhibition of class I HDACs including HDAC2 as well as artificial HDAC2 protein depletion lead to reduced MYC protein levels, most likely due to increased proteasomal degradation. MYC and HDAC2 are localized in a protein complex together with other potential drug targets. Our data suggest that class I HDAC inhibition leads to destabilization of MYC protein depriving the cells from one of their main oncogenic drivers. The MYC/HDAC2 multi-protein complex represents an attractive target for novel combinatorial treatment approaches. Funding: MYCNet (BMBF Germany), Verein fur krebskranke Kinder Odenwald e.V.

Bronchopulmonary dysplasia (BPD) is characterized by abnormal alveolar septation and microvascular development. Certain types of monocytes and macrophages have been implicated in epithelial growth and angiogenesis, while others play a role in immune defense and inflammation. Aims: To investigate whether monocyte/macrophage profiles and their products correlate with BPD development. Methods: We investigated lungs of neonatal mice and tracheal aspirates (TAs) from preterm born infants. Results: We found that M2-MDSC like monocytes are abundantly present in the lungs of neonatal mice. These monocytes also showed high expression of proangiogenic genes (a.o. EMMPRIN, ENA78, GDF15, VEGFR2 and IGF-1) and strong immune suppressive capacity i n vitro , supporting a role in maintaining tissue homeostasis and lung growth. Upon repetitive exposure to LPS the monocytes lost their M2-profile and suppressive capacity and upregulated inflammatory markers (IL-8, IL-1b and IL-6 but not TNFa). We also found monocytes/macrophages in TAs from preterm born infants. Proteome profiling these TAs revealed high concentrations of both M2 and inflammatory monocyte/macrophage derived factors and angiogenic mediators. Addition of TAs to pericyte-endothelial cell co-cultures either enhanced or inhibited angiogenesis. Luminex analysis of TAs of preterm infants with (n=22) and without (n=23) BPD showed significant lower concentrations of the angiogenic factors EMMPRIN, ENA78, MCP1, GDF15, TNF-α, Angiopoietin-1, -2 and HGF in those infants who developed BPD. Conclusion: Our data indicate that monocyte/macrophage profiles in TAs of premature children correlate with BPD development and may be used to identify new preventive or therapeutic targets.

A newborn had a tight, dry skin with multiple fissures, an eclabium and ectropion. Breathing was compromised and regulation of body temperature was impaired. We described the patient as a 'collodion baby'. After genetic investigation the diagnosis congenital ichtyosis, recessive type 1 was made. At 12 months of age, the skin symptoms were significantly diminished.

Patients with congenital diaphragmatic hernia (CDH) suffer from severe pulmonary hypertension attributable to altered development of the pulmonary vasculature, which is often resistant to vasodilator therapy. Present treatment starts postnatally even though significant differences in the pulmonary vasculature are already present early during pregnancy. We examined the effects of prenatal treatment with the phosphodiesterase-5 inhibitor sildenafil on pulmonary vascular development in experimental CDH starting at a clinically relevant time. The well-established, nitrofen-induced CDH rodent model was treated daily with 100 mg/kg sildenafil from day 17.5 until day 20.5 of gestation (E17.5–20.5). Importantly, this timing perfectly corresponds to the developmental stage of the lung at 20 wk of human gestation, when CDH is detectable by 2D-ultrasonography and/or MRI. At E21.5 pups were delivered by caesarean section and euthanized by lethal injection of pentobarbital. The lungs were isolated and subsequently analyzed using immunostaining, real-time PCR, and volume measurements. Prenatal treatment with sildenafil improved lung morphology and attenuated vascular remodeling with reduced muscularization of the smaller vessels. Pulmonary vascular volume was not affected by sildenafil treatment. We show that prenatal treatment with sildenafil within a clinically relevant period improves pulmonary vascular development in an experimental CDH model. This may have important implications for the management of this disease and related pulmonary vascular diseases in human.

Atypical teratoid/rhabdoid tumors (AT/RT) are among the most common malignant brain tumors in infants. Comprehensive genomic studies revealed three distinct molecular subgroups (AT/RT-TYR, -MYC and -SHH). As primary cilia (PC) have already been shown to play a central role in other tumor entities, we aimed to characterize the distribution of PC across AT/RT subgroups and to target primary ciliogenesis in these tumors with dismal prognosis. We performed immunofluorescence to detect PC in AT/RT cell lines (n=2) and primary tumor sections (n=13). The functional role of PC was investigated by knockdown of KIF3A and pharmacological inhibition with Ciliobrevin D in vitro. The relevance of primary ciliogenesis in AT/RT biology was further elucidated using a fly model of SMARCB1 deficiency. We detected PC in both AT/RT cell lines and all primary tumors investigated in this study. Notably, we observed a significant subgroup-specific difference in the proportion of PC-positive cells. Specifically, AT/RT-TYR demonstrated the highest percentage of ciliated cells. Knockdown of KIF3A significantly reduced cell proliferation and stem cell properties. Additionally, death receptor 5 signaling was significantly up-regulated. Pharmacological inhibition by Ciliobrevin D phenocopied these results, specifically after induction of primary ciliogenesis through serum starvation. In a fly model of SMARCB1 deficiency, concomitant knockdown of several cilia-associated genes resulted in a substantial shift of the lethal phenotype with >20% of flies reaching adulthood. In conclusion, these results implicate PC as a key feature of AT/RT biology and suggest primary ciliogenesis as a potential therapeutic target, especially in AT/RT-TYR.

Although SMARCB1 mutations represent the only recurrent genetic aberration in atypical teratoid/rhabdoid tumors (ATRT), a comprehensive assessment of the epigenetic effect of SMARCB1 loss has not been attempted so far. To achieve this, we performed ChIP-sequencing for six histone marks (H3K4me1, H3K4me3, H3K27me3, H3K27Ac, H3K9me3, H3K36me3) and EZH2 in 11 primary ATRTs, encompassing all three molecular subgroups of ATRT. Chromatin segmentation analyses revealed that across all three subgroups a high percentage of ATRT epigenomes resides in a quiescent state and active enhancer states are significantly underrepresented as compared to non-neoplastic brain. We validated these findings in a SMARCB1-reexpressing ATRT cell line and also found an overall deincrease of H3K27Ac and H3K27me3 upon SMARCB1 reexpression. Interestingly, by comparing the genome wide distribution of EZH2 and H3K27me3, we also identified classes of ATRT-specific active genes bound by EZH2, but lackingH3K27me3, pointing at a non-canonical role of EZH2 in gene activation. ChIP-Sequencing of SMARCA4 in these samples revealed that many of these „EZH2 only” genes display a high signal of of this protein. Many of these genes (such as e.g. CCND2) were downregulated upon knock-down of SMARCA4 in ATRT cell lines. Adding to the overall characterization of the epigenome, we have shown that a number of genes - despite being bound by EZH2- are maintained in an active state by residual SWI/SNF complex binding. This sheds new light on the role of residual SWI/SNFSMARCA4 still being present in AT/RT and elucidates a mechanism by which AT/RT keep important tumor-genes in an active state.

Item does not contain fulltext The International Classification of Functioning, Disability and Health (ICF) provides a common language to understand what health means. An ICF core set, a list of ICF categories affected by a certain disease, is useful to objectify the content validity of a health status measurement. This study aims to identify the potential items of a gout specific 'ICF core set'. A three-round Delphi exercise was conducted, using web-based questionnaires. Health professionals, specialized in gout, nominated and subsequently rated the relevance of life areas divided into ICF categories. Agreement was determined by using the UCLA/RAND criteria. Simultaneously, a systematic review of gout measure outcomes was conducted. The results of these studies were compared using the second level of the ICF categories. In the Delphi study, consensus was found for 136 relevant ICF categories. The literature study extracted 134 different ICF categories in 149 articles. Three hundred and ten were non-defined outcomes. A large number of ICF categories were deemed to be relevant for people with gout. Only 29.7 % (19/64) of the level 2 categories, deemed to be relevant by health professionals, had been assessed as relevant in at least 5 % of gout outcome studies. Conversely, 70 % (19/27) of level 2 ICF categories assessed in at least 5 % of outcome studies were deemed relevant by health professionals. These ICF codes, which are found relevant in both studies, should be considered as mandatory in further research to a validated and practical core set of ICF categories. Published gout outcomes research fails to evaluate many life areas that are thought relevant by health professionals.

Soil has been identified as a possible carbon (C) sink to mitigate increasing atmospheric CO2 concentration. However, several recent studies have suggested that the potential of soil to sequester C is limited and that soil may become saturated with C under increasing CO2 levels. To test this concept of soil C saturation, we studied a gley and organic soil at a grassland site near a natural CO2 spring. Total and aggregate-associated soil organic C (SOC) concentration showed a significant increase with atmospheric CO2 concentration. An asymptotic function showed a better fit of SOC and aggregation with CO2 level than a linear model. There was a shift in allocation of total C from smaller size fractions to the largest aggregate fraction with increasing CO2 concentration. Litter inputs appeared to be positively related to CO2 concentration. Based on modeled function parameters and the observed shift in the allocation of the soil C from small to large aggregate-size classes, we postulate that there is a hierarchy in C saturation across different SOC pools. We conclude that the asymptotic response of SOC concentration at higher CO2 levels indicates saturation of soil C pools, likely because of a limit to physical protection of SOC.

Damage DNA binding protein 2 (DDB2) has a high affinityfor UV-damaged DNA and has been implicated in the initialsteps of global genome nucleotide excision repair (NER) inmammals. DDB2 binds to CUL4A and forms an E3ubiquitin ligase. In this study, we have analyzed theproperties of DDB2 and CUL4A in vivo. The majority ofDDB2 and CUL4A diffuse in the nucleus with a diffusionrate consistent with a high molecular mass complex.Essentially all DDB2 binds to UV-induced DNA damage,where each molecule resides for ~2 minutes. After theinduction of DNA damage, DDB2 is proteolyticallydegraded with a half-life that is two orders of magnitudelarger than its residence time on a DNA lesion. Thisindicates that binding to damaged DNA is not the primarytrigger for DDB2 breakdown. The bulk of DDB2 binds toand dissociates from DNA lesions independently ofdamage-recognition protein XPC. Moreover, the DDB2-containing E3 ubiquitin ligase is bound to many moredamaged sites than XPC, suggesting that there is littlephysical interaction between the two proteins. We proposea scenario in which DDB2 prepares UV-damagedchromatin for assembly of the NER complex.

In this paper we investigate Dutch corporate liquidity management in general, and target adjustment behaviour in particular. To this purpose, we use a simple error correction model of corporate liquidity holdings applied to firm-level data for the period 1977-1997. We confirm the existence of long-run liquidity targets at the firm level. We also find that changes in liquidity holdings are driven by short-run shocks as well as the urge to converge towards targeted liquidity levels. The rate of target convergence is higher when we include more firm-specific information in the target. This result supports the idea that increased precision in defining liquidity targets associates with a faster observed rate of target convergence. It also suggests that the slow speeds of adjustment obtained in many macro studies on money demand are artefacts of aggregation bias.

BACKGROUND: (blind field). METHODS: Expression profiling, molecular subgrouping and analysis of somatic copy number alterations were conducted on multiple independent cohorts of patient tumour samples to examine intermediates of the MET signaling pathway in medulloblastoma. To examine the in vitro and in vivo effects of foretinib treatment; MET signalling biochemical analysis; migration and invasion assays; and foretinib pharmacokinetic studies were performed. Medulloblastoma xenografts and transgenic mouse models were used to evaluate foretinib treatment in vivo. RESULTS: We analyzed three large non-overlapping cohorts of medulloblastoma patients (discovery cohort, n = 199; validation cohort 1, n = 439; validation cohort 2, n = 285) and demonstrated that cMET, known to be involved in tumor progression and dissemination, is a marker of sonic hedgehog (SHH) medulloblastoma. Importantly, immunohistochemical analysis of activated cMET (phosphorylated cMET) in another independent patient cohort (n = 385) revealed that cMET activation correlates with increased tumor relapse and a poor survival in pediatric patients with SHH medulloblastomas, thus defining a subset of patients that may benefit from cMET targeted therapy. We show that foretinib, an FDA approved inhibitor of cMET, suppresses cMET activation, decreases proliferation and induces apoptosis, both in medulloblastoma cell lines and in SHH medulloblastoma xenografts. Furthermore foretinib penetrates the blood-brain barrier and is effective both in the primary and in the metastatic compartments. Treatment of mouse xenografts and of an aggressive transgenic model of metastatic SHH medulloblastoma with foretinib reduced primary medulloblastoma growth, decreased the incidence of metastases by 36% and increased survival by 45%. CONCLUSIONS: Our results provide strong rationale for advancing foretinib into clinical trials for SHH-driven medulloblastomas. SECONDARY CATEGORY: Tumor Biology.

Genomic studies have shown that multiple molecular subtypes of pediatric brain tumors exist that are biologically and clinically highly distinct. These findings ask for novel subtype specific treatments. To develop these we need more and better preclinical models that correctly reflect the proper tumor (sub)type. Orthotopic patient-derived xenograft (PDX) models generated by intracranial injection of primary patient material into the brain of NSG mice offer the unique possibility to test novel substances in primary patient tissue in an in vivo environment. Prior to drug selection and testing, extensive molecular characterizations of PDX and matching primary tumor/blood (DNA methylation, DNA sequencing, and gene expression) are needed to see how the PDX represents the original disease and to learn about targetable oncogenic drivers in each model. In collaboration with several groups around the world we have generated and fully characterized thus far 75 PDX models reflecting 15 distinct subtypes of pediatric brain cancer. PDX models always retain their molecular subtype and in the vast majority of cases also mutations and copy number alterations compared to matching primary tumors. Most aggressive tumors, harboring MYC(N) amplifications, are overrepresented in the cohort, but also subtypes which have not been available for preclinical testing before due to lack of genetically engineered mouse models or suitable cell lines, such as Group 4 medulloblastoma, are included. All models and corresponding molecular data will become available for the community for preclinical research. Examples of such preclinical experiments will be presented. PDX models of pediatric brain tumors are still quite rare. Our repertoire of PDX models and corresponding molecular characterizations allow researchers all over the world to find the right models for their specific scientific questions. It will provide an unprecedented resource to study tumor biology and pave the way for improving treatment strategies for children with malignant brain tumors.

BACKGROUND: This study aimed to prospectively evaluate clinical, histopathological and molecular variables for outcome prediction in medulloblastoma patients. METHODS: Patients from the HIT2000 cooperative clinical trial were prospectively enrolled based on the availability of sufficient tumor material and complete clinical information. This revealed a cohort of 184 patients (median age 7.6 years), which was randomly split at a 2:1 ratio into a training (n = 127), and a validation (n = 57) dataset. All samples were subjected to thorough histopathological investigation, CTNNB1 mutation analysis, quantitative PCR, MLPA and FISH analyses for cytogenetic variables, and methylome analysis. RESULTS: By univariable analysis, clinical factors (M-stage), histopathological variables (large cell component, endothelial proliferation, synaptophysin pattern), and molecular features (chromosome 6q status, MYC amplification, TOP2A copy-number, subgrouping) were found to be prognostic. Molecular consensus subgrouping (WNT, SHH, Group 3, Group 4) was validated as an independent feature to stratify patients into different risk groups. When comparing methods for the identification of WNT-driven medulloblastoma, this study identified CTNNB1 sequencing and methylation profiling to most reliably identify these patients. After removing patients with particularly favorable (CTNNB1 mutation, extensive nodularity) or unfavorable (MYC amplification) markers, a risk score for the remaining “intermediate molecular risk” population dependent on age, M-stage, pattern of synaptophysin expression, and MYCN copy-number status was identified and validated, with speckled synaptophysin expression indicating worse outcome. CONCLUSIONS: Methylation subgrouping and CTNNB1 mutation status represent robust tools for the risk-stratification of medulloblastoma. A simple clinico-pathological risk score for “intermediate molecular risk” patients was identified, which deserves further validation. SECONDARY CATEGORY: Pediatrics.

Atypical teratoid rhabdoid tumor of the brain is a rare embryonal tumor of infancy which has become more widely recognized due to specific immunostaining now more routinely available. These tumors still carry a poor prognosis and no standard therapy is currently available. However, the recent development of aggressive multimodality strategies seems to be associated with improved survival rates. In this chapter, we provide a review of our current knowledge on the demographics and epidemiology, pathology, and molecular characteristics. We review each individual therapeutic modality used (surgery, conventional chemotherapy, high-dose chemotherapy with stem cell rescue, intrathecal chemotherapy, and radiation) and the contribution of each to outcome when available. We provide an overview of the most recent therapeutic trials published and discuss the future options in development.

Genetic events leading to the loss of heterozygosity (LOH) have been shown to play a crucial role in the development of cancer. However, LOH events do not occur only in genetically unstable cancer cells but also have been detected in normal somatic cells of mouse and man. Mice, in which one of the alleles for adenine phosphoribosyltransferase ( Aprt ) has been disrupted by gene targeting, were used to investigate the potency of carcinogens to induce LOH in vivo . After 7,12-dimethyl-1,2-benz[ a ]anthracene (DMBA) exposure, a 3-fold stronger mutagenic response was detected at the autosomal Aprt gene than at the X chromosomal hypoxantine-guanine phosphoribosyltransferase ( Hprt) gene in splenic T-lymphocytes. Allele-specific PCR analysis showed that the normal, nontargeted Aprt allele was lost in 70% of the DMBA-induced Aprt mutants. Fluorescence in situ hybridization analysis demonstrated that the targeted allele had become duplicated in almost all DMBA-induced mutants that displayed LOH at Aprt . These results indicate that the main mechanisms by which DMBA caused LOH were mitotic recombination or chromosome loss and duplication but not deletion. However, after treatment with the alkylating agent N -ethyl- N -nitrosourea, Aprt had a similar mutagenic response to Hprt while the majority (90%) of N -ethyl- N -nitrosourea-induced Aprt mutants had retained both alleles. Unexpectedly, irradiation with x-rays, which induce primarily large deletions, resulted in a significant increase of the mutant frequency at Hprt but not at Aprt . This in vivo study clearly indicates that, in normal somatic cells, carcinogen exposure can result in the induction of LOH events that are compatible with cell survival and may represent an initiating event in tumorigenesis.

Methane is an important greenhouse gas and the most abundant hydrocarbon in the Earth's atmosphere. Methanotrophic microorganisms can use methane as their sole energy source and play a crucial role in the mitigation of methane emissions in the environment. “ Candidatus Methylomirabilis oxyfera” is a recently described intra-aerobic methanotroph that is assumed to use nitric oxide to generate internal oxygen to oxidize methane via the conventional aerobic pathway, including the monooxygenase reaction. Previous genome analysis has suggested that, like the verrucomicrobial methanotrophs, “ Ca. Methylomirabilis oxyfera” encodes and transcribes genes for the Calvin-Benson-Bassham (CBB) cycle for carbon assimilation. Here we provide multiple independent lines of evidence for autotrophic carbon dioxide fixation by “ Ca. Methylomirabilis oxyfera” via the CBB cycle. The activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), a key enzyme of the CBB cycle, in cell extracts from an “ Ca. Methylomirabilis oxyfera” enrichment culture was shown to account for up to 10% of the total methane oxidation activity. Labeling studies with whole cells in batch incubations supplied with either 13 CH 4 or [ 13 C]bicarbonate revealed that “ Ca. Methylomirabilis oxyfera” biomass and lipids became significantly more enriched in 13 C after incubation with 13 C-labeled bicarbonate (and unlabeled methane) than after incubation with 13 C-labeled methane (and unlabeled bicarbonate), providing evidence for autotrophic carbon dioxide fixation. Besides this experimental approach, detailed genomic and transcriptomic analysis demonstrated an operational CBB cycle in “ Ca. Methylomirabilis oxyfera.” Altogether, these results show that the CBB cycle is active and plays a major role in carbon assimilation by “ Ca. Methylomirabilis oxyfera” bacteria. Our results suggest that autotrophy might be more widespread among methanotrophs than was previously assumed and implies that a methanotrophic community in the environment is not necessarily revealed by 13 C-depleted lipids.

The adult lung contains several distinct stem cells, although their properties and full potential are still being sorted out. We previously showed that ectopic Sox2 expression in the developing lung manipulated the fate of differentiating cells. Here, we addressed the question whether fully differentiated cells could be redirected towards another cell type. Therefore, we used transgenic mice to express an inducible Sox2 construct in type II pneumocytes, which are situated in the distal, respiratory areas of the lung. Within three days after the induction of the transgene, the type II cells start to proliferate and form clusters of cuboidal cells. Prolonged Sox2 expression resulted in the reversal of the type II cell towards a more embryonic, precursor-like cell, being positive for the stem cell markers Sca1 and Ssea1. Moreover, the cells started to co-express Spc and Cc10, characteristics of bronchioalveolar stem cells. We demonstrated that Sox2 directly regulates the expression of Sca1. Subsequently, these cells expressed Trp63, a marker for basal cells of the trachea. So, we show that the expression of one transcription factor in fully differentiated, distal lung cells changes their fate towards proximal cells through intermediate cell types. This may have implications for regenerative medicine, and repair of diseased and damaged lungs.

Xanthans occurring in different levels of disordered conformation were enzymatically hydrolyzed to their six pentamer repeating units (RUs). The RUs present in the enzyme digests were analyzed using LC–MS. As only disordered xanthan segments are degraded by cellulases, the influence of the six different RUs on the transitional behavior of xanthan could be studied. The results indicate that especially xanthan segments rich in RUs that are acetylated on the outer mannose unit stabilize the xanthan conformation. Acetylation of the inner mannose did not show to have a clear stabilizing effect on the xanthan conformation. As the enzymatic release of pyruvylated RUs gradually increased with increasing levels of disordered xanthan segments, it is concluded that the distribution of these RUs is random. On the contrary, single and double acetylated RUs are distributed over specific xanthan segments as these RUs were instantly hydrolyzed.

Purpose Reports detailing the prognostic impact of TP53 mutations in medulloblastoma offer conflicting conclusions. We resolve this issue through the inclusion of molecular subgroup profiles. Patients and Methods We determined subgroup affiliation, TP53 mutation status, and clinical outcome in a discovery cohort of 397 medulloblastomas. We subsequently validated our results on an independent cohort of 156 medulloblastomas. Results TP53 mutations are enriched in wingless (WNT; 16%) and sonic hedgehog (SHH; 21%) medulloblastomas and are virtually absent in subgroups 3 and 4 tumors (P < .001). Patients with SHH/TP53 mutant tumors are almost exclusively between ages 5 and 18 years, dramatically different from the general SHH distribution (P < .001). Children with SHH/TP53 mutant tumors harbor 56% germline TP53 mutations, which are not observed in children with WNT/TP53 mutant tumors. Five-year overall survival (OS; ± SE) was 41% ± 9% and 81% ± 5% for patients with SHH medulloblastomas with and without TP53 mutations, respectively (P < .001). Furthermore, TP53 mutations accounted for 72% of deaths in children older than 5 years with SHH medulloblastomas. In contrast, 5-year OS rates were 90% ± 9% and 97% ± 3% for patients with WNT tumors with and without TP53 mutations (P = .21). Multivariate analysis revealed that TP53 status was the most important risk factor for SHH medulloblastoma. Survival rates in the validation cohort mimicked the discovery results, revealing that poor survival of TP53 mutations is restricted to patients with SHH medulloblastomas (P = .012) and not WNT tumors. Conclusion Subgroup-specific analysis reconciles prior conflicting publications and confirms that TP53 mutations are enriched among SHH medulloblastomas, in which they portend poor outcome and account for a large proportion of treatment failures in these patients.

Differently modified xanthans, varying in degree of acetylation and/or pyruvylation were incubated with the experimental cellulase mixture C1-G1 from Myceliophthora thermophila C1. The ionic strength and/or temperature of the xanthan solutions were varied, to obtain different xanthan conformations. The exact conformation at the selected incubation conditions was determined by circular dichroism. The xanthan degradation was analyzed by size exclusion chromatography. It was shown that at a fixed xanthan conformation, the backbone degradation by cellulases is equal for each type of xanthan. Complete backbone degradation is only obtained at a fully disordered conformation, indicating that only the secondary xanthan structure influences the final degree of hydrolysis by cellulases. It is thereby shown that, independently on the degree of substitution, xanthan can be completely hydrolyzed to oligosaccharides. These oligosaccharides can be used to further investigate the primary structure of different xanthans and to correlate the molecular structure to the xanthan functionalities.

The recently described bacterium “ Candidatus Methylomirabilis oxyfera” couples the oxidation of the important greenhouse gas methane to the reduction of nitrite. The ecological significance of “ Ca . Methylomirabilis oxyfera” is still underexplored, as our ability to identify the presence of this bacterium is thus far limited to DNA-based techniques. Here, we investigated the lipid composition of “ Ca . Methylomirabilis oxyfera” to identify new, gene-independent biomarkers for the environmental detection of this bacterium. Multiple “ Ca . Methylomirabilis oxyfera” enrichment cultures were investigated. In all cultures, the lipid profile was dominated up to 46% by the fatty acid (FA) 10-methylhexadecanoic acid (10MeC 16:0 ). Furthermore, a unique FA was identified that has not been reported elsewhere: the monounsaturated 10-methylhexadecenoic acid with a double bond at the Δ7 position (10MeC 16:1Δ7 ), which comprised up to 10% of the total FA profile. We propose that the typical branched fatty acids 10MeC 16:0 and 10MeC 16:1Δ7 are key and characteristic components of the lipid profile of “ Ca . Methylomirabilis oxyfera.” The successful detection of these fatty acids in a peatland from which one of the enrichment cultures originated supports the potential of these unique lipids as biomarkers for the process of nitrite-dependent methane oxidation in the environment.

Source determination of nitrous oxide (N(2)O) from soils has so far been complicated by methodological constraints: the frequently used (15)N tracer method could not differentiate between pathways related to nitrification, that is, nitrifier nitrification (NN), nitrifier denitrification (ND), and nitrification-coupled denitrification (NCD). To overcome this problem, a dual isotope method using both (15)N and (18)O was proposed. However, O exchange between nitrogen oxides and water has been found to disturb such a method. We here explain in detail a novel dual isotope method that allows to quantify O exchange in denitrification and to differentiate N(2)O production from NN, ND, NCD, and fertilizer denitrification (FD). The method has already been applied to a range of soils with good success. Potential of and scope for further improvement of the method are discussed.

Continued association of RPA with sites of incomplete nucleotide excision repair averts further incision events until repair is completed., Single-stranded DNA gaps that might arise by futile repair processes can lead to mutagenic events and challenge genome integrity. Nucleotide excision repair (NER) is an evolutionarily conserved repair mechanism, essential for removal of helix-distorting DNA lesions. In the currently prevailing model, NER operates through coordinated assembly of repair factors into pre- and post-incision complexes; however, its regulation in vivo is poorly understood. Notably, the transition from dual incision to repair synthesis should be rigidly synchronized as it might lead to accumulation of unprocessed repair intermediates. We monitored NER regulatory events in vivo using sequential UV irradiations. Under conditions that allow incision yet prevent completion of repair synthesis or ligation, preincision factors can reassociate with new damage sites. In contrast, replication protein A remains at the incomplete NER sites and regulates a feedback loop from completion of DNA repair synthesis to subsequent damage recognition, independently of ATR signaling. Our data reveal an important function for replication protein A in averting further generation of DNA strand breaks that could lead to mutagenic and recombinogenic events.