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Age research in vertebrates is often limited by the longevity of available models. The teleost fish Nothobranchius furzeri has an exceptionally short lifespan with 3.5 months for the laboratory strain GRZ and about 6 months for the wild-derived strain MZM-0403. Here we have investigated telomere length in muscle and skin tissue of young and old fish of both strains using different methods. We found age-dependent telomere shortening in the MZM-0403 strain with the longer lifespan, whereas the short-lived GRZ strain showed no significant telomere shortening with advanced age. Sequencing of the two main telomerase genes Tert and Terc revealed that both genes are highly conserved between the N. furzeri strains while there is little conservation to other fish species and humans. Both genes are ubiquitously expressed in N. furzeri and expression levels of Tert and Terc correlate with telomerase activity in a tissue-specific manner. Unexpectedly, the expression level of Tert is increased in aged muscle and skin tissue of MZM-0403 suggesting that telomeres shorten upon ageing despite increased Tert expression and hence high telomerase activity. We further conclude that the extremely short lifespan of the GRZ strain is not caused by diminished telomerase activity or accelerated telomere shortening.

Recent technical developments have facilitated the application of cone-beam computed tomography (CBCT) for interventional and intraoperative imaging. The aim of this study was to compare the radiation doses and image quality in CBCT with those of conventional multislice spiral computed tomography (MSCT) for abdominal and genitourinary imaging.Different CBCT and MSCT protocols for imaging soft tissues and hard-contrast objects at different dose levels were investigated in this study. Local skin and organ doses were measured with thermoluminescent dosimeters placed in an anthropomorphic phantom. Moreover, the contrast-to-noise ratio, the noise-power spectrum, and the high-contrast resolution derived from the modulation transfer function were determined in a phantom with the same absorption properties as those of anthropomorphic phantom.The effective dose of the examined abdominal/genitourinary CBCT protocols ranged between 0.35 mSv and 18.1 mSv. As compared with MSCT, the local skin dose of CBCT examinations could locally reach much higher doses up to 190 mGy. The effective dose necessary to realize the same contrast-to-noise ratio with CBCT and MSCT depended on the MSCT convolution kernel: the MSCT dose was smaller than the corresponding CBCT dose for a soft kernel but higher than that for a hard kernel. The noise-power spectrum of the CBCT images at tube voltages of 85/90 kV(p) is at least half of that of images measured at 103/115 kV(p) at any arbitrarily chosen spatial frequency. Although the pixel size and slice thickness of CBCT were half of those of the MSCT images, high-contrast resolution was inferior to the MSCT images reconstructed with a hard convolution kernel.As compared with MSCT using a medium-hard convolution kernel, CBCT produces images at medium noise levels and, simultaneously, medium spatial resolution at approximately the same dose. It is well suited for visualizing hard-contrast objects in the abdomen with relatively low image noise and patient dose. For the detection of low-contrast objects at standard tube voltages of approximately 120 kV(p), however, MSCT should be preferred.

SummaryThe p53 tumor suppressor coordinates a series of antiproliferative responses that restrict the expansion of malignant cells, and as a consequence, p53 is lost or mutated in the majority of human cancers. Here, we show that p53 restricts expression of the stem and progenitor-cell-associated protein nestin in an Sp1/3 transcription-factor-dependent manner and that Nestin is required for tumor initiation in vivo. Moreover, loss of p53 facilitates dedifferentiation of mature hepatocytes into nestin-positive progenitor-like cells, which are poised to differentiate into hepatocellular carcinomas (HCCs) or cholangiocarcinomas (CCs) in response to lineage-specific mutations that target Wnt and Notch signaling, respectively. Many human HCCs and CCs show elevated nestin expression, which correlates with p53 loss of function and is associated with decreased patient survival. Therefore, transcriptional repression of Nestin by p53 restricts cellular plasticity and tumorigenesis in liver cancer.

SummaryAim: Reinvestigation of the radiation exposure of patients undergoing whole-body [18F]FDG-PET/CT examinations pursuant to the revised recommendations of the ICRP. Methods: Conversion coefficients for equivalent organ doses were determined for realistic anthropomorphic phantoms of reference persons. Based on these data, conversion coefficients for the effective dose were calculated using the revised tissue-weighting factors that account for the different radiation susceptibilities of organs and tissues, and the redefinition of the group ‘remainder tissues’. Results: Despite the markedly changed values of the equivalent organ doses estimated for FDG and of the tissue-weighting factors, the conversion coefficient for the effective dose resulting from FDG administration decreases only slightly by 10 %. For whole-body CT scans it remains even unchanged. Conclusion: The updated dose coefficients provide a valuable tool to easily assess the generic radiation risk of patients undergoing whole- body PET/CT (or PET/MRI) examinations and can be used, amongst others, for protocol optimization.

In chronic lymphocytic leukemia (CLL) short telomeres are associated with other adverse prognostic factors and poor survival. We and others have described association of telomere length with genomic complexity and clonal evolution in CLL. To understand if telomere shortening rather than being only a marker of cell proliferation, could also functionally contribute to disease progression, we generated Terc knock out in the Eµ-TCL1 murine CLL model (Eµ-TCL1 mTerc-/- mice). Comparison of the Eµ-TCL1 mTerc-/- mice from the generations G1, G2 and G3 with that of Eµ-TCL1 (TCL1+) did not show a difference in disease initiation, progression as well as survival even though a significant decrease in telomere length of tumor cells was observed with increasing generations. Of interest, the Eµ-TCL1 mTerc-/- G3 tumors (n=8; G3) more frequently showed defective DNA damage response compared to TCL1+ tumors (n=8), as analysed by changes in phosphorylation of gamma-H2AX, ATM and p53 measured by FACS at 1, 3, 6, 16 and 24 hours after gamma irradiation. Despite this predisposition to undergo genomic complexity, the G3 mice showed no difference in disease development compared to TCL1+ mice. Therefore we investigated if cell extrinsic factors in Terc-/- mice could affect tumor development. The Terc-/- microenvironment is known to be restrictive to B and T-lymphopoiesis and could hence inhibit CLL development. To study the impact of Terc-/- microenvironment, splenic tumors from the Eµ-TCL1 mTerc-/- G3 (n=11; G3) mice were transferred into syngeneic Terc+/+ C57Bl6 mice and compared with that of TCL1+ (n=11). No significant difference in disease burden and survival was observed between these 2 cohorts, indicating that there is no adverse influence of the Terc-/- microenvironment on the tumor growth in the Eµ-TCL1 mTerc-/- mice. Further, we hypothesized that the telomere length in the G3 mice may not be short enough to induce genomic instability and enhance disease aggressiveness. The mice were thus crossed to obtain the Eµ-TCL1 mTerc-/- generation G4 (G4). Though the survival of these mice were similar to that of Eµ-TCL1 controls (median survival :49 vs. 51 weeks; P=0.301), the G4 mice showed significantly decreased disease burden as measured by spleen weight, liver weights and tumor cell fraction. The G4 mice showed decreased fertility and hence further crosses to generate G5 were not performed. However, further shortening of telomeres was achieved by serially transplanting the tumors from G3 into syngeneic recipient mice for 2 generations. Telomere length of the tumor cells analysed by Q-PCR showed a significant decrease with increasing transfer rounds, compared to primary TCL1+ tumors. Interestingly, the 2nd round transfer of the G3 tumors led to a less severe disease and significantly longer survival of the recipient mice compared to 2nd round TCL1+ tumors transplants (median survival from date of transplantation: 15 vs. 7 weeks; P=0.030), indicating that cell intrinsic factors in the G3 tumors hamper proliferation of these cells. We finally analyzed if absence of telomerase could mask disease aggressiveness of the G3 tumors by crossing the G3 mice with wildtype Terc+/+ mice. The resulting TCL1+ mTerc+/- G4 mice showed an increased telomerase activity but had significantly shorter telomere length compared to TCL1+ mice. Strikingly, the TCL1+ mTerc+/- G4 mice showed faster disease development and significantly shorter survival (median survival: 42 vs. 51 weeks; p In summary, the TCL1+ mTerc-/- mice crossed through generations G1 to G4 did not show a difference in disease initiation, progression or survival despite significant shortening of telomeres. However, the tumors from G3 had defective DDR, indicating a potential for accumulating genetic aberrations and clonal evolution. But the absence of functional telomerase decreased the growth potential of these genomic instable tumors. Reconstitution of telomerase in G3 mice resulted in aggressive tumors with short telomere length and could therefore be a valuable murine model for genomic instability and aggressive CLL. Disclosures Tausch: Roche: Consultancy, Honoraria, Speakers Bureau; AbbVie: Consultancy, Honoraria, Other: travel support, Speakers Bureau. Schneider:Celgene: Other: travel grant. Döhner:AbbVie, Agios, Amgen, Astellas, Astex, Celator, Janssen, Jazz, Seattle Genetics: Consultancy, Honoraria; AROG, Bristol Myers Squibb, Pfizer: Research Funding; Celgene, Novartis, Sunesis: Honoraria, Research Funding. Stilgenbauer:AbbVie, AstraZeneca, Celgene, Gilead Sciences, Inc., GSK, Hoffmann La-Roche, Janssen, Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Background and aims The cyclin-dependent kinase inhibitor p21 has been implicated as a tumour suppressor. Moreover, recent genetic studies suggest that p21 might be a potential therapeutic target to improve regeneration in chronic diseases. The aim of this study was to delineate the role of p21 in chronic liver injury and to specify its role in hepatocarcinogenesis in a mouse model of chronic cholestatic liver injury. Methods The degree of liver injury, regeneration and tumour formation was assessed in Mdr2 −/− mice and compared with Mdr2/ p21 −/− mice. Moreover, the role of p21 was evaluated in hepatoma cells in vitro and in human hepatocellular carcinoma (HCC). Results Mdr2 −/− mice developed HCCs as a consequence of chronic inflammatory liver injury. In contrast, tumour development was profoundly delayed in Mdr2/ p21 −/− mice. Delayed tumour development was accompanied by markedly impaired liver regeneration in Mdr2/ p21 −/− mice. Moreover, the regenerative capacity of the Mdr2/ p21 −/− livers in response to partial hepatectomy declined with age in these mice. Hepatocyte transplantation experiments revealed that impaired liver regeneration was due to intrinsic factors within the cells and changes in the Mdr2/ p21 −/− microenvironment. In human HCCs, a subset of tumours expressed p21, which was associated with a significant shorter patient survival. Conclusions We provide experimental evidence that p21 is required for sustained liver regeneration and tumour development in chronic liver injury indicating that p21 needs to be tightly regulated in order to balance liver regeneration and cancer risk. Moreover, we identify p21 as a negative prognostic marker in human HCC.

Telomerase inhibition causes progressive telomere shortening and cellular senescence, which constitutes a universal barrier to tumor growth and therefore an attractive target for tumor therapy. To expand our previous studies, we investigated the global effects of telomere dysfunction on the proteome of tumor cells in order to find novel senescence biomarkers. Telomerase-deficient HCT-116 cell clones were analyzed by a quantitative proteomic approach using isotope-coded protein labeling (ICPL) and nanoflow-HPLC-MS/MS. Stringent reduction of the extensive proteomic data from this tumor cell model revealed a list of 59 markers including proteins identified in our former studies and a number of novel proteins involved in tumorigenesis and metastasis such as SFN, S100A4, ANXA2, and LGALS1. A loss of the chromatin protein HMGB2 was demonstrated not only in various telomerase-inhibited clones of different tumor cell lines, but also in normal human fibroblasts undergoing replicative senescence and in aging telomerase knockout mice. Impressively, a coherent and dense network of protein–protein interactions for the bulk of the markers and their implementation in signaling pathways involving key regulators for tumorigenesis were revealed. These results have an impact on the understanding of telomere- and senescence-related signal transduction in tumor cells in consideration of the general lack of senescence markers. Biological significance Induction of cellular senescence constitutes a potent concept for tumor therapy which interferes with immortalization and additional hallmarks of cancer. The application of a powerful quantitative proteomic approach using isotope-coded protein labeling to an approved model for senescence represented by telomerase inhibited tumor cells led to the identification of novel candidate biomarkers for telomere dysfunction and replicative senescence. Thereby, the identified markers not only fit in the context of the investigated processes with a relevance for additional hallmarks of cancer but are also involved in a strong interaction network and integrated in canonical pathways centered around key cancer-relevant proteins. These potential markers alone or in combination will significantly extend the view on telomere-associated signal transduction in tumor cells and contribute to the field of cellular senescence and aging in consideration of the general lack of biomarkers in this regard.

The three carbon atoms of allene moieties allow unique transformations and rapid generation of complexity. Not surprisingly, allenes became extremely versatile building blocks in organic synthesis. Transition-metal-catalyzed reactions of these cumulene π-systems have been particularly successful, and many applications in the synthesis of complex products have been reported. This review summarizes the palladium-catalyzed transformation of allenes published during the last decade. Many of the examples presented are impressive multicomponent processes or cascade reactions involving two or more steps leading to molecular complexity in simple one-pot operations. Consequently, several reactions have been developed with the goal of delivering new synthetic routes to natural products.

High telomerase activity is detected in nearly all human cancers but most human cells are devoid of telomerase activity. There is well-documented evidence that reactivation of telomerase occurs during cellular transformation. In humans, tumors can rely in reactivation of telomerase or originate in a telomerase positive stem/progenitor cell, or rely in alternative lengthening of telomeres, a telomerase-independent telomere-length maintenance mechanism. In this review, we will focus on the telomerase positive tumors. In this context, the recent findings that telomerase reverse transcriptase (TERT) promoter mutations represent the most common non-coding mutations in human cancer have flared up the long-standing discussion whether cancer originates from telomerase positive stem cells or telomerase reactivation is a final step in cellular transformation. Here, we will discuss the pros and cons of both concepts in the context of telomere length-dependent and telomere length-independent functions of telomerase. Together, these observations may provoke a re-evaluation of telomere and telomerase based therapies, both in telomerase inhibition for cancer therapy and telomerase activation for tissue regeneration and anti-ageing strategies.

Liver tumours are the sixth most common tumour entity worldwide and the second most common cause of cancer-related deaths. Hepatocellular carcinoma (HCC) is the most frequent liver tumour and is mainly related to viral infections, including HBV and HCV, alcohol consumption, metabolic syndrome and aflatoxin B exposure. Hepatocarcinogenesis is described as a multistep process, which starts mostly by a chronic disease and on the basis of cirrhosis formation. In the recent years, precursor lesions are well described by their morphological characterisation and there is evidence that those precursor lesions are involved in HCC formation by passing through a sequence of molecular events in hepatic nodules.1 Two different types of dysplastic nodules are specified, low-grade dysplastic nodules (LGDNs) and high-grade dysplastic nodules (HGDNs), which delineate the border between premalignant lesion and malignant transformation. LGDNs are morphologically characterised by a mild increase of cell density, and the presence of peripheral fibrous scar distinct from the surrounding cirrhotic liver. HGDNs may be distinctly or vaguely nodular in the background of a cirrhotic liver, and often show an increase in cell density, with an irregular trabecular pattern. LGDN is described as non-malignant, whereas HGDN is the precancerous lesion for the transformation in HCC. The detection between early HCCs and HGDN is more difficult for pathologists. A recent report describes the classification …

Classification and scoring of phospho-α-synuclein and PK-PET Blot. (A) Classification of phospho-α-synuclein staining into four different scores. Representative pictures for scoring. Score 0: no p-α-synuclein staining, score 1: little staining in brainstem and DpMe, score 2: strong staining in brainstem and DpMe, score 3: strong p-α-synuclein staining in brainstem, DpMe, and cerebellum indicating severe disease progression. (B) Scoring to classify PK-PET Blot. Score 0: no PK resistant aggregates, score 1: light aggregates in brainstem and Deep Mesencepahlic nucleus (DpMe), score 2: clear PK resistant aggregates in brainstem and DpMe, score 3: dominant aggregates in brainstem and DpMe. Score 4: Prominent aggregates in brainstem, DpMe and cerebellum. (PDF 125 kb)

Mating scheme for the generation of mouse cohorts. (A) Breeding scheme for generating 3rd generation telomerase knockout and α-synuclein transgenic mice (αSYNtg/tg G3Terc-/-) and corresponding control cohorts. Heterozygous α-synuclein (αSYN) mice were crossed with heterozygous telomerase knockout mice (Terc) to generate double transgenic αSYNtg/tg G1Terc-/- and single transgenic αSYNtg/tg, G1Terc-/- and Terc+/+ mice (G1 = first generation of telomerase knockout). These double transgenic αSYNtg/tg G1Terc-/- mice were crossed with each other to produce αSYNtg/tg G2Terc-/- mice and finally αSYNtg/tg G3Terc-/- mice. (B) Telomere length was measured in neurons of the brainstem using qFISH telomere staining double-stained with Cy5-Neuron N dye. The graph represents telomere length in brainstem in 75 weeks old mice (n = 5 mice per group). Analyzed were αSYNtg/tg G3Terc-/- in comparison to αSYNtg/tg mice (P = 0.0012) and G3Terc-/- in comparison to Terc+/+ mice (P = 0.0079) as well as αSYNtg/tg G3Terc-/- compared with G3Terc-/- (P = 0.03). (PDF 29 kb)

Exploratory behavior and string agility. Exploratory behavior of the mice has been analysed using the Open Field System. Several parameters, e.g. the activity, duration and the totally travelled distance were measured in three different zones of the open field: Border, intermediate and center of the open field. 7 to 10 mice of the indicated genotypes were recorded each for 10 min. (A) Activity in the open field and (B) duration of stay in the three indicated zones. αSYNtg/tg G3Terc-/- mice did not show a difference in comparison to αSYNtg/tg mice and the two control groups. Although a trend can be seen in duration, αSYNtg/tg and αSYNtg/tg G3Terc-/- stay even shorter in the intermediate and the center zone and prefer the border zone in comparison to Terc+/+ or G3Terc-/- mice. (C) String agility test shows no difference between the different groups. (D) Activated astrocytes were measured using GFAP immunohistochemistry. The histogram depicts GFAP positive astrocytes of the brainstem (n = 4–6 mice). αSYNtg/tg mice show a significant increase in astrocyte activation (62.35 ± 5.656) in comparison to αSYNtg/tg G3Terc-/- mice (39 ± 2.383, P = 0.0159). Furthermore, αSYNtg/tg mice with phenotype (85 weeks) show a significant activation (62.35 ± 5.65) if compared with non-phenotypic mice (75 weeks, 33.37 ± 4.086, P = 0.0095). Terc+/+ (14.3 ± 7.6) and G3Terc-/- mice (10.07 ± 7.10) show low activation levels. (PDF 33 kb)

Primer sequences which were used for RT-PCR. (DOC 40 kb)

Schematic representation of the parameter analyzed in 3D-reconstructed microglia. Automated analysis of each structural characteristic was performed using Imaris Bitplane software. (PDF 50 kb)

During attempts to prepare functionalized 5-iodopyridine derivatives the unexpected formation of iodo[1,3]dioxolo[4,5-c]pyridines was discovered. The conversion of 3-alkoxypyridin-4-ols into the corresponding 5-iodo compounds was achieved by reaction with one equivalent of iodine or tetramethylammonium dichloroiodate under basic conditions. When three equivalents of iodine were used in chlorinated solvents, after 5-iodination, subsequent reaction of the 3-alkoxy group took place to form a 1,3-dioxolane ring with the 4-hydroxyl group. Generation of the resulting iodo[1,3]dioxolo[4,5-c]pyridines is explained by a radical process known as the Hofmann–Loffler–Freytag reaction. Two 6-ethynylpyridine derivatives were examined in the iodination process to establish a route to pyridine-containing macrocycles. The pentasubstituted 5-iodopyridine derivative 21 could be prepared; however, attempts to achieve cyclotrimerization of this building block under different conditions were not successful. Reaction of 21 with copper chloride allowed isolation of a copper acetylide 22, which aggregates to a triangular trimeric complex containing four copper(I) ions such as [23·Cu]+ as monitored by ESI mass spectrometry.

SummaryCheckpoints that limit stem cell self-renewal in response to DNA damage can contribute to cancer protection but may also promote tissue aging. Molecular components that control stem cell responses to DNA damage remain to be delineated. Using in vivo RNAi screens, we identified basic leucine zipper transcription factor, ATF-like (BATF) as a major component limiting self-renewal of hematopoietic stem cells (HSCs) in response to telomere dysfunction and γ-irradiation. DNA damage induces BATF in a G-CSF/STAT3-dependent manner resulting in lymphoid differentiation of HSCs. BATF deletion improves HSC self-renewal and function in response to γ-irradiation or telomere shortening but results in accumulation of DNA damage in HSCs. Analysis of bone marrow from patients with myelodysplastic syndrome supports the conclusion that DNA damage-dependent induction of BATF is conserved in human HSCs. Together, these results provide experimental evidence that a BATF-dependent differentiation checkpoint limits self-renewal of HSCs in response to DNA damage.

The purpose of this study was to assess the radiation doses of different coronary CTA (CTA) protocols: second-generation dual-source 128-MDCT, first-generation dual-source 64-MDCT, and single-source 64-MDCT.Thermoluminescent dosimetry was used to determine scanner-specific dose coefficients for standard coronary CTA of an anthropomorphic phantom. These coefficients were used to estimate the effective doses (EDs) of retrospectively gated, prospectively triggered, and prospectively triggered high pitch coronary CTA performed at 100 and 120 kV. The coronary CTA protocols used in imaging of 43 patients undergoing dual-source 128-MDCT were analyzed for ED, image quality, and signal-to-noise ratio.Regardless of coronary CTA protocol and CT system, imaging at 100 kV lowered the ED 40-50%. In retrospectively gated 120-kV coronary CTA, the ED ranged from 5.7 to 10.7 mSv and was approximately 50% lower with single-source 64-MDCT than with either DSCT protocol. In prospectively triggered 120-kV coronary CTA, the ED ranged from 3.8 to 4.0 mSv. The lowest ED of all protocols (1.3 mSv) was observed in prospectively triggered high-pitch 100-kV coronary CTA performed with dual-source 128-MDCT. Patient measurements showed similar dose reductions for prospective triggering and low voltage settings without an influence on signal-to-noise ratio or image quality.A combination of prospective triggering with low voltage settings is an effective measure for reducing the ED of coronary CTA to values of 2-4 mSv independent of scanner system. Further dose reduction to nearly 1 mSv can be achieved with high-pitch prospectively triggered coronary CTA.

Telomere shortening impairs liver regeneration in mice and is associated with cirrhosis formation in humans with chronic liver disease. In humans, telomerase mutations have been associated with familial diseases leading to bone marrow failure or lung fibrosis. It is currently unknown whether telomerase mutations associate with cirrhosis induced by chronic liver disease. The telomerase RNA component (TERC) and the telomerase reverse transcriptase (TERT) were sequenced in 1,121 individuals (521 patients with cirrhosis induced by chronic liver disease and 600 noncirrhosis controls). Telomere length was analyzed in patients carrying telomerase gene mutations. Functional defects of telomerase gene mutations were investigated in primary human fibroblasts and patient-derived lymphocytes. An increased incidence of telomerase mutations was detected in cirrhosis patients (allele frequency 0.017) compared to noncirrhosis controls (0.003, P value 0.0007; relative risk [RR] 1.859; 95% confidence interval [CI] 1.552-2.227). Cirrhosis patients with TERT mutations showed shortened telomeres in white blood cells compared to control patients. Cirrhosis-associated telomerase mutations led to reduced telomerase activity and defects in maintaining telomere length and the replicative potential of primary cells in culture. Conclusion: This study provides the first experimental evidence that telomerase gene mutations are present in patients developing cirrhosis as a consequence of chronic liver disease. These data support the concept that telomere shortening can represent a causal factor impairing liver regeneration and accelerating cirrhosis formation in response to chronic liver disease. (HEPATOLOGY 2011;)

Liver cholestasis is a chronic liver disease and a major health problem worldwide. Cholestasis is characterised by a decrease in bile flow due to impaired secretion by hepatocytes or by obstruction of bile flow through intra- or extrahepatic bile ducts. Thereby cholestasis can induce ductal proliferation, hepatocyte injury and liver fibrosis. Notch signalling promotes the formation and maturation of bile duct structures. Here we investigated the liver regeneration process in the context of cholestasis induced by disruption of the Notch signalling pathway. Liver-specific deletion of recombination signal binding protein for immunoglobulin kappa j region (Rbpj), which represents a key regulator of Notch signalling, induces severe cholestasis through impaired intra-hepatic bile duct (IHBD) maturation, severe necrosis and increased lethality. Deregulation of the biliary compartment and cholestasis are associated with the change of several signalling pathways including a Kyoto Encyclopedia of Genes and Genomes (KEGG) gene set representing the Hippo pathway, further yes-associated protein (YAP) activation and upregulation of SRY (sex determining region Y)-box 9 (SOX9), which is associated with transdifferentiation of hepatocytes. SOX9 upregulation in cholestatic liver injury in vitro is independent of Notch signalling. We could comprehensively address that in vivo Rbpj depletion is followed by YAP activation, which influences the transdifferentiation of hepatocytes and thereby contributing to liver regeneration.

Overexpression of survivin, a crucial regulator of the mitotic spindle checkpoint with additional antiapoptotic effects, is associated with poor prognosis in neuroblastoma (NB) and other cancers. Thus, survivin is a promising therapeutic target in NB. Transcriptional inhibitors of survivin show preclinical efficacy and have been tested in clinical trials. The results of these trials, however, have been disappointing, due to modest efficacy and unexpected off-target effects. We thus investigated the consequences of marked, yet incomplete decrease of survivin on NB in a novel transgenic mouse model. To this end, we crossed mice haploinsufficient for survivin with TH-MYCN mice to generate BIRC5+/- MYCN tg/+ mice. These mice developed NB with markedly decreased mRNA and protein expression of survivin. Surprisingly, incidence and latency of the NB that developed in the BIRC5+/- MYCN tg/+ mice were not different from those in the TH-MYCN mice. Growth of tumors was not decreased in BIRC5+/- MYCN tg/+ mice, as assessed by serial MRI imaging, and survival of the mice was not increased. Immunohistochemistry of the NB did not show decreased proliferation (Ki67), increased apoptosis (activated caspase 3) or a change in vascularity (CD31). No difference in copy number variations between the two NB mouse models was discernible using CGH arrays. To determine whether dysfunctional T cells described in mice haploinsufficient for survivin could have masked any loss of aggressiveness of NB cells haploinsufficient for survivin, NB of BIRC5+/- MYCN tg/+ mice were retransplanted into survivin sufficient mice. No difference in tumor incidence, latency and growth was detected. Taken together, despite a marked decrease in the expression of BIRC5/survivin by haploinsufficiency development and growth of NB were not inhibited. While a further decrease of survivin beyond that seen in haploinsufficiency will inhibit NB, it most likely will also elicit marked side effects, given that survivin is essential for normal cells. Our results cast doubt on the efficacy of inhibitors of survivin expression in NB and call for targeting survivin by alternative approaches, such as inhibiting protein-protein interactions of survivin. Citation Format: Carmen Dorneburg, Volker Rasche, Andre Lechel, Sarah-Fee Katz, Klaus-Michael Debatin, Christian Beltinger. Marked decrease of BIRC5/Survivin by haploinsufficiency does not inhibit neuroblastoma in transgenic mice: Implications for survivin as a therapeutic target in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4136.

Telomere shortening limits the proliferation of primary human fibroblasts by the induction of senescence, which is mediated by ataxia telangiectasia mutated-dependent activation of p53. Here, we show that CHK2 deletion impairs the induction of senescence in mouse and human fibroblasts. By contrast, CHK2 deletion did not improve the stem-cell function, organ maintenance and lifespan of telomere dysfunctional mice and did not prevent the induction of p53/p21, apoptosis and cell-cycle arrest in telomere dysfunctional progenitor cells. Together, these results indicate that CHK2 mediates the induction of senescence in fibroblasts, but is dispensable for the induction of telomere dysfunction checkpoints at the stem and progenitor cell level in vivo.

β-Alkoxy-β-ketoenamides and hydroxylamine hydrochloride smoothly provide pyrimidine-N-oxides under very mild conditions. Alkyl, aryl, and heteroaryl substituents are compatible with this new method. By treatment with acetic acid anhydride these pyrimidine-N-oxides undergo a rearrangement furnishing highly functionalized 6-acetoxymethyl-substituted pyrimidine derivatives in good yields. Our method, which is based on a three-component reaction of alkoxyallenes, nitriles, and carboxylic acids, therefore constitutes a highly flexible route to densely substituted pyrimidine derivatives.

Objective: New generation Dual Source computed tomography (CT) scanners offer different x-ray spectra for Dual Energy imaging. Yet, an objective, manufacturer independent verification of the dose required for the different spectral combinations is lacking. The aim of this study was to assess dose and image noise of 2 different Dual Energy CT settings with reference to a standard chest scan and to compare image noise and contrast to noise ratios (CNR). Also, exact effective dose length products (E/DLP) conversion factors were to be established based on the objectively measured dose. Materials and Methods: An anthropomorphic Alderson phantom was assembled with thermoluminescent detectors (TLD) and its chest was scanned on a Dual Source CT (Siemens Somatom Definition) in dual energy mode at 140 and 80 kVp with 14 × 1.2 mm collimation. The same was performed on another Dual Source CT (Siemens Somatom Definition Flash) at 140 kVp with 0.8 mm tin filter (Sn) and 100 kVp at 128 × 0.6 mm collimation. Reference scans were obtained at 120 kVp with 64 × 0.6 mm collimation at equivalent CT dose index of 5.4 mGy * cm. Syringes filled with water and 17.5 mg iodine/mL were scanned with the same settings. Dose was calculated from the TLD measurements and the dose length products of the scanner. Image noise was measured in the phantom scans and CNR and spectral contrast were determined in the iodine and water samples. E/DLP conversion factors were calculated as ratio between the measured dose form the TLDs and the dose length product given in the patient protocol. Results: The effective dose measured with TLDs was 2.61, 2.69, and 2.70 mSv, respectively, for the 140/80 kVp, the 140 Sn/100 kVp, and the standard 120 kVp scans. Image noise measured in the average images of the phantom scans was 11.0, 10.7, and 9.9 HU (P > 0.05). The CNR of iodine with optimized image blending was 33.4 at 140/80 kVp, 30.7 at 140Sn/100 kVp and 14.6 at 120 kVp. E/DLP conversion factors were 0.0161 mSv/mGy * cm for the 140/80 kVp protocol, 0.0181 mSv/mGy * cm for the Sn140/100 kVp mode and 0.0180 mSv/mGy * cm for the 120 kVp examination. Conclusion: Dual Energy CT is feasible without additional dose. There is no significant difference in image noise, while CNR can be doubled with optimized dual energy CT reconstructions. A restriction in collimation is required for dose-neutrality at 140/80 kVp, whereas this is not necessary at 140 Sn/100 kVp. Thus, CT can be performed routinely in Dual Energy mode without additional dose or compromises in image quality.

Lithiated alkoxyallenes are very versatile components for the synthesis of heterocycles such as furans, pyrroles, and 1,2-oxazines, easily allowing the preparation of natural products via these heterocyclic intermediates. A surprising three-component synthesis of N-acylated enaminones allowed the synthesis of highly functionalized 4-hydroxypyridines, 5-acetyloxazoles, and pyrimidines. All these heterocyclic products are ready for further functionalizations, in particular for palladium-catalyzed reactions, leading to libraries of new interesting heterocycles.

A series of highly substituted β-alkoxy β-keto enamides were prepared by a multi-component reaction combining lithiated alkoxyallenes, nitriles and carboxylic acids. Apt conditions were developed for their conversion into 5-alkoxypyrimidine derivatives. This synthesis is highly flexible with respect to the substitution pattern at C-2 and C-4 of the pyrimidine core. The corresponding pyrimidin-5-yl nonaflates allowed subsequent transformations at C-5 by palladium-catalyzed couplings such as Sonogashira and Suzuki reactions. The Sonogashira coupling of a pyrimidinyl alkyne with a pyridinyl nonaflate and a subsequent cyclization led to an efficient access to pyrimidinyl-substituted furo[2,3-c]pyridine derivatives. The C-6 methyl group which is present in all of the prepared pyrimidines can easily be converted into formyl, carboxylic or alkynyl moieties which allow the synthesis of additional pyrimidine derivatives.

X-ray procedures have a substantial impact not only on patient care but also on man-made radiation exposure. Since a reliable risk-benefit analysis of medical X-rays can only be performed for diagnosis-related groups of patients, we determined specific exposure data for patients with the ten most common types of cancer. For all patients with the considered cancers undergoing medical X-ray procedures in a maximum-care hospital between 2000 and 2005, patient- and examination-specific data were retrieved from the hospital/radiology information system. From this data, the cumulative 5-year effective dose was estimated for each patient as well as the mean annual effective dose per patient and the mean patient observation time for each cancer site. In total, 151,439 radiographic, fluoroscopic, and CT procedures, carried out in 15,866 cancer patients (age, 62 ± 13 years), were evaluated. The mean 5-year cumulative dose varied between 8.6 mSv (prostate cancer) and 68.8 mSv (pancreas cancer). Due to an increasing use of CT scans, the mean annual effective dose per patient increased from 13.6 to 18.2 mSv during the 6-year period. Combining the results obtained in this study for a particular hospital with cancer incidence data for Germany, we estimated that cancer patients having X-ray studies constitute at least 1% of the population but receive more than 10% of the total effective dose related to all medical X-ray procedures performed nationwide per year. A large fraction of this dose is radiobiologically ineffective due to the reduced life expectancy of cancer patients.

Telomere dysfunction limits the proliferative capacity of human cells and induces organismal aging by activation of p53 and p21. Although deletion of p21 elongates the lifespan of telomere-dysfunctional mice, a direct analysis of p53 in telomere-related aging has been hampered by early tumor formation in p53 knockout mice. Here we analyzed the functional consequences of conditional p53 deletion. Intestinal deletion of p53 shortened the lifespan of telomere-dysfunctional mice without inducing tumor formation. In contrast to p21 deletion, the deletion of p53 impaired the depletion of chromosomal-instable intestinal stem cells in aging telomere-dysfunctional mice. These instable stem cells contributed to epithelial regeneration leading to an accumulation of chromosomal instability, increased apoptosis, altered epithelial cell differentiation and premature intestinal failure. Together, these results provide the first experimental evidence for an organ system in which p53-dependent mechanisms prevent tissue destruction in response to telomere dysfunction by depleting genetically instable stem cells.

By a flexible three-component synthesis, alkoxy-substituted enamides are easily available from lithiated alkoxyallenes, nitriles and carboxylic acids (see scheme). The treatment of these versatile intermediates with trifluoroacetic acid provided 5-acetyloxazoles in moderate to good yields. Different substituents are possible at C-2 and C-5 and the 5-acetyl group is a suitable handle for further synthetic transformations.

Substituted enamides were prepared by a three-component reaction of lithiated alkoxyallenes, nitriles, and carboxylic acids. Their subsequent condensation with ammonium salts provided alkoxy-substituted pyrimidine derivatives. This two-step method e is highly flexible with respect to the substitution pattern at C-2 and it C-6. The C-4 and C-5 positions can smoothly be functionalized employing either Pd-catalyzed couplings or oxidation methods.

A series of 4-pyridyl nonaflates was coupled with several terminal alkynes to efficiently provide new 4-alkynyl-substituted 3-alkoxypyridine derivatives. Apt conditions were developed for their conversion into furo[2,3-c]pyridines. Sonogashira reactions of 4-alkoxy-substituted 3-pyridyl nonaflates allowed an access to regioisomeric furo[3,2-c]pyridines. For both types of alkynyl-substituted alkoxypyridinesan alternative method for cyclization employing iodine monochloride furnished iodinated furo[2,3-c]- or furo[3,2-c]pyridines, which can undergo a second palladium-catalyzed step. Iodination of 4-hydroxypyridine derivative 24 with iodine afforded a pentasubstituted pyridine which after Sonogashira reaction immediately undergoes a cyclization to furo-pyridine 25. Thus, three different types of furo-pyridines can be prepared starting from one precursor. Several compounds prepared are fluorescent and show strong Stokes shifts. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008)

PURPOSE: For nonsuperimposed and three-dimensional imaging of jaws and teeth, multislice computer tomography (MSCT) can be performed, or alternatively digital volume tomography as a cone beam technique can be applied. The radiation dose of both procedures should be evaluated with different methods of dose assessment. MATERIALS AND METHODS: A 4-row MSCT (Volume Zoom Siemens®) and a cone beam CT (NewTom QR-DVT 9000®) were compared regarding the radiation exposure of the patient during a dental examination. Organ dose and effective dose were estimated by thermoluminescence dosimetry (TLD) using an Alderson-Rando phantom for both devices. In addition the effective dose of MSCT was calculated from the CTDI vol -value at scanner display and by CT-Expo® program. RESULTS: The effective dose of MSCT was 0.33 mSv for women (w) and 0.32 mSv for men (m) measured with TLD in the Alderson-Rando phantom, 0.39 / 0.35 mSv (w/m) by CTDI calculation and 0.39 / 0.33 mSv by CT-Expo® program. The effective dose of NewTom® QR-DVT 9000 from TLD measurement was 0.095 / 0.093 mSv (w/m). CONCLUSION: The radiation exposure of a typical dental examination with a NewTom® cone beam DVT is about one third of the MSCT dose. Both techniques, however, moderate patient doses. Dosimetry methods as routinely used for MSCT cannot be applied to cone beam DVT.

Purpose To compare the effective dose applied by sequential CT (SEQ), spiral CT (SCT), electron beam CT (EBT) and coronary angiography for investigations of the chest, abdomen and the heart. Methods The Alderson Phantom was used to compare the effective dose for all modalities. In addition, the effective dose for conventional CT (SEQ and SCT) was estimated with a mathematical phantom. Results For CT investigation of the chest and abdomen the dose was highest for the EBT (11 mSv and 25 mSv, respectively) and slightly lower for the SEQ (7.8 mSv and 21.5 mSv, respectively), whereas spiral CT required the least dose (5.3 mSv and 8.8 mSv, respectively). For coronary calcium screening (0.8 mSv) and EBT coronary angiography (1.7 mSv) the dose was lower than for coronary catheter angiography (3.3 mSv). For conventional CT the difference between the effective dose derived by the mathematical phantom and by the Alderson phantom was 2% to 20%. Conclusions For investigations of the chest and abdomen the effective dose applied by SCT is significantly lower than that with EBT and SEQ. For investigation of the coronary arteries the effective dose applied by EBT is lower than that for coronary catheter angiography.