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Quercetin, a dietary flavonoid found in fruits and vegetables, has been described as a substance with many anti-cancer properties in a variety of preclinical investigations. In the present study, we demonstrate that 2D and 3D melanoma models exhibit not only different sensitivities to quercetin, but also opposite, cancer-promoting effects when metastatic melanoma spheroids are treated with quercetin. Higher concentrations of quercetin reduce melanoma growth in three tested cell lines, whereas low concentrations induce the opposite effect in metastatic melanoma spheroids but not in the non-metastatic cell line. High (&gt, 12.5 &micro, M) or low (&lt, 6.3 &micro, M) quercetin concentrations decrease or enhance cell viability, spheroid size, and cell proliferation, respectively. Additionally, melanoma cells cultivated in 2D already show significant caspase 3 activity at very low concentrations (&gt, 0.4 &micro, M), whereas in 3D spheroids apoptotic cells, caspase 3 activity can only be detected in concentrations &ge, M. Further, we show that the tumor promoting or repressing effect in the 3D metastatic melanoma spheroids are likely to be elicited by a precisely controlled regulation of Nrf2/ARE-mediated cytoprotective genes, as well as ERK and NF-&kappa, B phosphorylation. According to the results obtained here, further studies are needed to better characterize the mechanisms of action underlying the pro- and anti-carcinogenic effects of quercetin on human melanomas.

Specific gene promoter DNA methylation is becoming a powerful epigenetic biomarker in cancer diagnostics. Five genes (CDH1, CDKN2Ap16, RASSF1A, TERT, and WT1) were selected based on their frequently published potential as epigenetic markers. Diagnostic promoter methylation assays were generated based on bisulfite-converted DNA pyrosequencing. The methylation patterns of 144 non-small-cell lung cancer (NSCLC) and 7 healthy control formalin-fixed paraffin-embedded (FFPE) samples were analyzed to evaluate the applicability of the putative diagnostic markers. Statistically significant changes in methylation levels are shown for TERT and WT1. Furthermore, 12 NSCLC and two benign lung cell lines were characterized for promoter methylation. The in vitro tests involved a comparison of promoter methylation in 2D and 3D cultures, as well as therapeutic tests investigating the impact of CDH1/CDKN2Ap16/RASSF1A/TERT/WT1 promoter methylation on sensitivity to tyrosine kinase inhibitor (TKI) and DNA methyl-transferase inhibitor (DNMTI) treatments. We conclude that the selected markers have potential and putative impacts as diagnostic or even predictive marker genes, although a closer examination of the resulting protein expression and pathway regulation is needed.

An energy of $362\:\text{MJ}$ is stored in each of the two LHC proton beams for nominal beam parameters. This will be further increased to about $700\:\text{MJ}$ in the future High Luminosity LHC (HL-LHC) and uncontrolled beam losses represent a significant hazard for the integrity and safe operation of the machine. In this paper, a number of failure mechanisms that can lead to a fast increase of beam losses are analyzed. Most critical are failures in the magnet protection system, namely the quench heaters and a novel protection system called Coupling-Loss Induced Quench (CLIQ). An important outcome is that magnet protection has to be evaluated for its impact on the beam and designed accordingly. In particular, CLIQ, which is to protect the new HL-LHC triplet magnets, constitutes the fastest known failure in the LHC if triggered spuriously. A schematic change of CLIQ to mitigate the hazard is presented. A loss of the Beam-Beam Kick due to the extraction of one beam is another source of beam losses with a fast onset. A significantly stronger impact is expected in the upcoming LHC Run III and HL-LHC as compared to the current LHC, mainly due to the increased bunch intensity. Its criticality and mitigation methods are discussed. It is shown that symmetric quenches in the superconducting magnets for the final focusing triplet can have a significant impact on the beam on short timescales. The impact on the beam due to failures of the Beam-Beam Compensating Wires as well as coherent excitations by the transverse beam damper are also discussed., Comment: 28 pages, 23 figures. To be published in Physical Review Accelerators and Beams

Micrometer-sized dust particles present in the Large Hadron Collider (LHC) beam pipe are believed to have caused many thousands of sporadic beam loss events around the LHC. These so-called unidentified falling objects (UFOs) have been under continuous study since the start of high intensity beam operation in the LHC due to their impact on the LHC availability: 139 beam dumps and 12 magnet quenches during Run II (2015–2018) alone. To mitigate the impact of UFOs on future accelerators such as the High Luminosity LHC and the Future Circular Collider, it is fundamental to foster a better understanding of these beam loss events. In this paper, key observations made since the start of LHC operation are summarized and the prevailing UFO hypothesis is confronted by a compilation of observations acquired during Run II. In particular, it is shown that UFOs must carry an initial negative charge before entering the proton beam, or that they are by some other means accelerated toward the beam not only by gravity. A simulation model for the dynamics of the dust particles and their interaction with the beam was developed over the years. This model is improved and validated by measurements. It is however also shown that a subset of observed beam losses, which contain a positive time profile skewness, cannot be explained by it. This work was supported by the High Luminosity Large Hadron Collider project.

// Nico Jacobi 1 , Rita Seeboeck 2 , Elisabeth Hofmann 2 , Helmut Schweiger 1 , Veronika Smolinska 1 , Thomas Mohr 3 , Alexandra Boyer 1 , Wolfgang Sommergruber 4 , Peter Lechner 5 , Corina Pichler-Huebschmann 5 , Kamil Onder 6, 7 , Harald Hundsberger 2 , Christoph Wiesner 2 and Andreas Eger 1, 2 1 IMC University of Applied Sciences Krems, Department Life Sciences, Research Institute for Applied Bioanalytics and Drug Development, A-3500 Krems, Austria 2 IMC University of Applied Sciences Krems, Department Life Sciences, Institute of Medical and Pharmaceutical Biotechnology, A-3500 Krems, Austria 3 Medical University of Vienna, Institute for Cancer Research, A-1090 Vienna, Austria 4 Boehringer Ingelheim, RCV GmbH and Co KG, A-1121 Vienna, Austria 5 University Clinic Tulln, Department Surgery, A-3430 Tulln, Austria 6 Research Program for Rational Drug Design in Dermatology and Rheumatology, Department of Dermatology, Paracelsus Medical University of Salzburg, A-5020 Salzburg, Austria 7 ProComCure Biotech, A-5081 Anif, Austria Correspondence to: Andreas Eger, email: andreas.eger@fh-krems.ac.at Keywords: drug discovery; 3D models; ERBB signaling; targeted drugs; oncogene addiction Received: August 25, 2017 Accepted: October 27, 2017 Published: November 17, 2017 ABSTRACT Complex three-dimensional (3D) in vitro models that recapitulate human tumor biology are essential to understand the pathophysiology of the disease and to aid in the discovery of novel anti-cancer therapies. 3D organotypic cultures exhibit intercellular communication, nutrient and oxygen gradients, and cell polarity that is lacking in two-dimensional (2D) monolayer cultures. In the present study, we demonstrate that 2D and 3D cancer models exhibit different drug sensitivities towards both targeted inhibitors of EGFR signaling and broad acting cytotoxic agents. Changes in the kinase activities of ErbB family members and differential expression of apoptosis- and survival-associated genes before and after drug treatment may account for the differential drug sensitivities. Importantly, EGFR oncoprotein addiction was evident only in the 3D cultures mirroring the effect of EGFR inhibition in the clinic. Furthermore, targeted drug efficacy was strongly increased when incorporating cancer-associated fibroblasts into the 3D cultures. Taken together, we provide conclusive evidence that complex 3D cultures are more predictive of the clinical outcome than their 2D counterparts. In the future, 3D cultures will be instrumental for understanding the mode of action of drugs, identifying genotype-drug response relationships and developing patient-specific and personalized cancer treatments.

Background: Malignant melanoma is an aggressive type of skin cancer with high risk for metastasis and chemoresistance. Disruption of tightly regulated processes such as cell cycle, cell adhesion, cell differentiation and cell death are predominant in melanoma development. So far, conventional treatment options have been insufficient to treat metastatic melanoma and survival rates are poor. Anthraquinone compounds have been reported to have anti-tumorigenic potential by DNA-interaction, promotion of apoptosis and suppression of proliferation in various cancer cells. Methods: In the current study, the racemic tetrahydroanthraquinone derivative (±)-4-deoxyaustrocortilutein (4-DACL) was synthesized and the cytotoxic activity against melanoma cells and melanoma spheroids determined by CellTiter-Blue viability Assay and phase contrast microscopy. Generation of reactive oxygen species (ROS) was determined with CellROX Green and Deep Red Reagent kit and microplate-based fluorometry. Luciferase reporter gene assays for nuclear factor kappa B (NF-κB) and p53 activities and western blotting analysis were carried out to detect the expression of anti-proliferative or pro-apoptotic (p53, p21, p27, MDM2, and GADD45M) and anti-apoptotic (p65, IκB-α, IKK) proteins. Cell cycle distribution and apoptosis rate were detected by flow cytometry, the morphological changes visualized by fluorescence microscopy and the activation of different caspase cascades distinguished by Caspase Glo 3/7, 8 and 9 Assays. Results: We demonstrated that 4-DACL displayed high activity against different malignant melanoma cells and melanoma spheroids and only low toxicity to melanocytes and other primary cells. In particular, 4-DACL treatment induced mitochondrial ROS, reduced NF-κB signaling activity and increased up-regulation of the cell cycle inhibitors cyclin-dependent kinase inhibitor p21 (p21WAF1/Cip1) and the tumor suppressor protein p53 in a dose-dependent manner, which was accompanied by decreased cell proliferation and apoptosis via the intrinsic pathway. Conclusion: According to these results, we suggest that 4-DACL may be a promising therapeutic agent for the treatment of malignant melanoma.

NUCLEAR PHYSICS IN ASTROPHYSICS CONFERENCE (NPA VII), York, England, 15 May 2015 - 22 May 2015; Journal of physics / Conference Series 940, 012024 (2018). doi:10.1088/1742-6596/940/1/012024, Published by IOP Publ., Bristol

In bladder cancer (BCa) patients undergoing radical cystoprostatectomy (RCPx), concomitant prostate cancer (PCa) is a common finding. Up to now there is no clear evidence to suggest that concomitant PCa is a predictor of outcome in these patients. Aim of this study was to assess incidence and clinicopathologic characteristics of concomitant PCa in RCPx specimen and correlate it to survival parameters from a single-centre material over two decades. All men who had undergone RCPx for BCa at our institution between 1994 and 2013 were included in this study. Clinicopathologic parameters for BCa and PCa were evaluated and correlated with outcome parameters. Survival analysis was performed for the subgroup of nonmetastatic organ-confined BCa to evaluate the role of concomitant Gleason Score (GS) ≥7 PCa. Of 945 men who had undergone RCPx for BCa, concomitant PCa was present in 237 patients (25.1 %). There was a significant increase in PCa incidence from 18.9 to 32.3 % between 1994 and 2013 (p = 0.009). Concomitant PCa represented a more aggressive phenotype at the end of the study (p = 0.037). In nonmetastatic organ-confined BCa, concomitant GS ≥7 PCa (HR 3.09; p = 0.0001) and age > 68 (HR 1.80; p = 0.0004) were independent negative predictors for overall survival. Concomitant PCa in RCPx specimen of BCa patients is a common finding. The incidence of concomitant PCa has significantly increased within 2 decades, presenting a more aggressive phenotype. Age and in particular concomitant GS ≥7 PCa are independent prognosticators for poor survival in patients with nonmetastatic organ-confined BCa.

Introduction: To analyze the primary stone free rate (pSFR) of flexible ureterorenoscopy (fURS) in the treatment of renal stones and to identify clinical predictors for the primary freedom from renal stones. Materials and Methods: Two hundred and seventy five patients, who underwent fURS for kidney stones were analyzed. Results: Index stone size was 6 mm. The stone was located in the lower calyx in 48%. Ureteral access sheath was used in 97%. Operation time was 35 min and primary stone clearance was 83%. pSFR increased from 74% in 2012 to 83% in 2013 and 90% in 2014 (p = 0.001). Preoperative stenting, index stone size, cumulative stone size, lithotripsy, ureteral access sheath and operation time were significantly correlated with the pSFR by univariate analysis. Multivariate regression analysis showed index stone size, cumulative stone size, ureteral access sheath and operation time as independent parameters for pSFR. Conclusions: fURS for kidney stones is safe with a high pSFR. Clinical parameters for pSFR are stone size, use of ureteral access sheath and operation time. In future, the effective use of fURS for the removal of kidney stones needs to be checked by prospective randomized trials.

Celiac disease (CD) is a chronic inflammatory condition caused by the ingestion of gliadin-containing food in genetically susceptible individuals. Undigested peptides of gliadin exert various effects, including increased intestinal permeability and inflammation in the small intestine. Although many therapeutic approaches are in development, a gluten-free diet is the only effective treatment for CD. Affecting at least 1% of the population in industrialized countries, it is important to generate therapeutic options against CD. Here, we describe the establishment of a high-throughput screening (HTS) platform based on AlphaLISA and electrical cell-substrate impedance sensing (ECIS) technology for the identification of anti-inflammatory and barrier-protective compounds in human enterocytes after pepsin-trypsin-digested gliadin (PT-gliadin) treatment. Our results show that the combination of these HTS technologies enables fast, reliable, simple, and label-free screening of IgY antibodies against PT-gliadin. Using this platform, we have identified a new chicken anti-PT-gliadin IgY antibody as a potential anti-CD agent.

The methanol extract of the Vietnamese freshwater cyanobacterium Nostoc sp. CAVN2 exhibited cytotoxic effects against MCF-7 and 5637 cancer cell lines as well as against nontumorigenic FL and HaCaT cells and was active against methicillin-resistant Staphylococcus aureus (MRSA) and Streptococcus pneumoniae. High-resolution mass spectrometric analysis indicated the presence of over 60 putative cyclophane-like compounds in an antimicrobially active methanol extract fraction. A paracyclophanes-focusing extraction and separation methodology led to the isolation of 5 new carbamidocyclophanes (1-5) and 11 known paracyclophanes (6-16). The structures and their stereochemical configurations were elucidated by a combination of spectrometric and spectroscopic methods including HRMS, 1D and 2D NMR analyses and detailed comparative CD analysis. The newly described monocarbamoylated [7.7]paracyclophanes (1, 2, 4 and 5) differ by a varying degree of chlorination in the side chains. Carbamidocyclophane J (3) is the very first reported carbamidocyclophane bearing a single halogenation in both butyl residues. Based on previous studies a detailed phylogenetic examination of cyclophane-producing cyanobacteria was carried out. The biological evaluation of 1-16 against various clinical pathogens highlighted a remarkable antimicrobial activity against MRSA with MICs of 0.1-1.0 μM, and indicated that the level of antibacterial activity is related to the presence of carbamoyl moieties.

Chronic inflammation is at least partially mediated by the chemokine-mediated attraction and by the adhesion molecule-directed binding of leukocytes to the activated endothelium. Therefore, it is therapeutically important to identify anti-inflammatory compounds able to control the interaction between leukocytes and the endothelial compartments of the micro- and macrocirculation. When testing novel drug candidates, it is, however, of the utmost importance to detect side effects, such as potential cytotoxic and barrier-disruptive activities. Indeed, minor changes in the endothelial monolayer integrity may increase the permeability of small blood vessels and capillaries, which, in extreme cases, can lead to edema development. Here, we describe the development of a high-throughput screening (HTS) platform, based on AlphaLISA technology, able to identify anti-inflammatory nontoxic natural or synthetic compounds capable of reducing tumor necrosis factor (TNF)-induced chemokine (interleukin [IL]-8) and adhesion molecule (ICAM-1) expression in human lung microvascular endothelial cells. Quantification of cell membrane-expressed ICAM-1 and of cell culture supernatant-associated levels of IL-8 was analyzed in HTS. In parallel, we monitored monolayer integrity and endothelial cell viability using the electrical cell substrate impedance sensing method. This platform allowed us to identify natural secondary metabolites from cyanobacteria, capable of reducing ICAM-1 and IL-8 levels in TNF-activated human microvascular endothelial cells in the absence of endothelial monolayer barrier disruption.

A 2MeV proton beam will produce a quasi-Maxwellian neutron spectrum of around 30 keV by the 7Li(p, n)7Be reaction. The experiments are mainly focused on the measurement of differential neutron capture cross sections relevant for the astrophysical s-process in nuclear synthesis. Moreover, proton capture cross sections for the astrophysical p-process can be measured directly with the proton beam. For an efficient time of flight measurement of the neutron energies along the 0.7 m long drift from the Li-target to the sample, 1ns short, intense proton pulses are needed at the target. Additionally, to reach $ 10^{7}$ n/cm2/s at the sample, a pulse repetition rate of 250 kHz is intended. After completion and successful running in, FRANZ will become a user facility with internal and external users. The 120 kV injector terminal and the 200mA proton source as well as the low-energy beam transport section and the FRANZ cave have been realized successfully. The 1.9 MV RF accelerator consists of a combined 4-Rod-RFQ/IH-DTL-resonator and is in the RF tuning and power testing phase. The 2 MeV transport and rebuncher section is ready for installation. In a first step FRANZ will offer experimental areas for neutron activation experiments and for proton beam experiments, as mentioned above. From the accelerator physics point of view, FRANZ will be an excellent facility for high current beam investigations and for beam wall interaction studies.

The transport of intense ion beams is affected by the collective behavior of this kind of multi-particle and multi-species system. The space charge expressed by the generalized perveance dominates the dynamical process of thermalisation, which leads to emittance growth. To prevent changes of intrinsic beam properties and to reduce the intensity dependent focusing forces, space charge compensation seems to be an adequate solution. In the case of positively charged ion beams, electrons produced by residual gas ionization and secondary electrons provide the space charge compensation. The influence of the compensation particles on the beam transport and the local degree of space charge compensation is given by different beam properties as well as the ion beam optics. Especially for highly charged ion beams, space charge compensation in combination with poor vacuum conditions leads to recombination processes and therefore increased beam losses. Strategies for providing a compensation-electron reservoir at very low residual gas pressures will be discussed.

In this study, the influence of halide ions on [7.7]paracyclophane biosynthesis in the cyanobacterium Nostoc sp. CAVN2 was investigated. In contrast to KI and KF, supplementation of the culture medium with KCl or KBr resulted not only in an increase of growth but also in an up-regulation of carbamidocyclophane production. LC-MS analysis indicated the presence of chlorinated, brominated, but also non-halogenated derivatives. In addition to 22 known cylindrocyclophanes and carbamidocyclophanes, 27 putative congeners have been detected. Nine compounds, carbamidocyclophanes M-U, were isolated, and their structural elucidation by 1D and 2D NMR experiments in combination with HRMS and ECD analysis revealed that they are brominated analogues of chlorinated carbamidocyclophanes. Quantification of the carbamidocyclophanes showed that chloride is the preferably utilized halide, but incorporation is reduced in the presence of bromide. Evaluation of the antibacterial activity of 30 [7.7]paracyclophanes and related derivatives against selected pathogenic Gram-positive and Gram-negative bacteria exhibited remarkable effects especially against methicillin- and vancomycin-resistant staphylococci and Mycobacterium tuberculosis. For deeper insights into the mechanisms of biosynthesis, the carbamidocyclophane biosynthetic gene cluster in Nostoc sp. CAVN2 was studied. The gene putatively coding for the carbamoyltransferase has been identified. Based on bioinformatic analyses, a possible biosynthetic assembly is discussed. peerReviewed

Background Chemokines play a critical role in tumor initiation, progression, and metastasis and have been associated with poor prognosis in diverse malignancies. The prognostic impact of chemokines for renal cell cancer (RCC) remains to be defined. Methods Patients diagnosed with RCC and operated between 07/07 and 05/11 were differentially assessed for expression profiles of a series of chemokines and their receptors by RT-qPCR and Western Blot analysis (tumor and adjacent normal tissue, n=37) and by Luminex for corresponding serum expression levels. Results were statistically correlated with clinicopathologic parameters. Results Gene expression of CCL2, CCR7, CXCL12, CXCR3, CXCR5 and CX3CL1 chemokines was significantly down-regulated in tumor compared to normal tissue. The gene profile for CCR6 was positively correlated with tumor size and stage. A positive linear correlation was found between CXCL12 and tumor stage as well as between CX3CR1 and C-reactive protein. In contrast to clear cell RCCs those of a chromophobe type showed a significantly down-regulated gene expression for CCR6, CCL20, and CXCL12. The CXCR7 serum level was significantly increased in patients with tumor-related mortality during postoperative follow-up. Conclusions Chemokines may serve as novel diagnostic and prognostic biomarkers for RCC. Studies on larger collectives are required for further assessment of potential clinical application.

The influence of the bisphosphonate zoledronic acid (ZA) on prostate cancer (PC) growth, adhesion and invasive behavior was investigated.PC-3, DU-145 and LNCaP cells were treated with ZA, and tumor-cell growth was then investigated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Furthermore, tumor-cell adhesion to vascular endothelium or to immobilized extracellular matrix proteins, as well as migratory properties of the cells, was evaluated. Integrin β subtypes, integrin-dependent signaling, as well as cell-cycle regulating proteins, were analyzed by western blots.ZA dose-dependently reduced tumor-cell growth but did not impair tumor-endothelium and tumor-matrix interaction. However, ZA significantly inhibited tumor migration and invasive activity. Cyclin E was reduced by ZA in LNCaP and DU-145, and p21 was elevated in LNCaP cells. p27 was upregulated in all tumor cell lines, compared with the controls. ZA elevated β1-integrin in PC-3 and diminished β4-integrin in PC-3 and DU-145 cells.ZA inhibits PC growth and motility but does not influence the mechanical contact between tumor cells and the vascular wall.

The growth potential of PC3 prostate cancer cells, sensible (PC3(par)) or resistant (PC3(res)) to the mTOR inhibitor everolimus (RAD001) was investigated. Cell growth and proliferation of PC3(res) was similar to that of PC3(par), and late apoptosis increased in PC3(par) but decreased in PC3(res) following treatment with low dosed everolimus. PC3(res) accumulated in the G2/M-phase, accompanied by cdk1, cdk2 and cyclin B elevation. Knocking down cdk1 or cyclin B distinctly blocked the growth activity of PC3(res). One reason for everolimus resistance may be up-regulation of the cdk1-cyclin B complex in prostate cancer cells, leading to enhanced progression towards G2/M.

Study Type – Diagnostic (exploratory cohort) Level of Evidence 2b What’s known on the subject? and What does the study add? Clinically relevant GSU in the prostatectomy specimen is a common phenomenon. Clinically relevant GSU occurs in one of three patients with clinically ‘very’ low-risk PCa, and a low number of biopsy cores is the key negative predictor. OBJECTIVE • To evaluate clinical predictors for Gleason score upgrade (GSU) in radical prostatectomy (RP) specimen, especially in patients with ‘very’ low risk PCA (T1c and biopsy Gleason score ≤6 and PSA

The concept of molecular tumor targeting might provide new hope in the treatment of advanced prostate cancer. We evaluated metastasis blocking properties of the histone deacetylase (HDAC) inhibitor valproic acid (VPA) and the mammalian target of rapamycin (mTOR) inhibitor RAD001 on prostate cancer cell lines. RAD001 or VPA were applied to PC-3 or LNCaP cells, either separately or in combination. Adhesion to vascular endothelium or to immobilized collagen, fibronectin or laminin was quantified. Migration and invasion were explored by a modified Boyden chamber assay. Integrin α and β subtypes were analyzed by flow cytometry, western blotting and RT-PCR. Effects of drug treatment on integrin related signaling, Akt and p70S6kinase activation, histone H3 and H4 acetylation were also determined. Separate application of RAD001 or VPA distinctly reduced tumor cell adhesion, migration and invasion, accompanied by elevated Akt activation and p70S6kinase de-activation. Integrin subtype expression was altered significantly by both compounds (VPA > RAD001). When both drugs were used in concert additive effects were observed on the migratory and invasive behavior but not on tumor-endothelium and tumor-matrix interaction. Separate mTOR or HDAC inhibition slows processes related to tumor metastasis. The RAD001-VPA combination showed advantage over VPA monotreatment with particular respect to migration and invasion. Ongoing studies are required to assess the relevance of VPA monotherapy versus VPA-RAD001 combination on tumor cell motility.

Aims To analyze the combined impact of the histone deacetylase (HDAC) inhibitor valproic acid (VPA) and the mammalian target of rapamycin (mTOR) inhibitor RAD001 on prostate cancer cell growth. Main methods PC-3, DU-145 and LNCaP cells were treated with RAD001, VPA or with an RAD001–VPA combination for 3 or 5 days. Tumor cell growth, cell cycle progression and cell cycle regulating proteins were then investigated by MTT assay, flow cytometry and Western blotting, respectively. Effects of drug treatment on cell signaling pathways were determined. Key findings Separate application of RAD001 or VPA distinctly reduced tumor cell growth and impaired cell cycle progression. Significant additive effects were evoked when both drugs were used in concert. Particularly, the cell cycle regulating proteins cdk1, cdk2, cdk4 and cyclin B were reduced, whereas p21 and p27 were enhanced by the RAD001–VPA combination. Signaling analysis revealed deactivation of EGFr, ERK1/2 and p70S6k. Phosphorylation of Akt was diminished in DU-145 but elevated in PC-3 and LNCaP cells. Significance The RAD001–VPA combination exerted profound antitumor properties on a panel of prostate cancer cell lines. Therefore, simultaneous blockage of HDAC and mTOR related pathways should be considered when designing novel therapeutic strategies for treating prostate carcinoma.

OBJECTIVE •To analyze the impact of a delayed radical cystectomy (rCx) and clinical variables on cancer-specific survival (CSS) in patients presenting 'high-risk' carcinoma not invading bladder muscle (nmiBCA). PATIENTS AND METHODS •Between 1989 and 2006, 278 patients who presented 'high-risk' nmiBCA finally underwent rCx in our institution. •CSS was correlated with clinical variables such as the number of transurethral resections of the bladder (TURBs), interval between first TURB and rCx, adjuvant therapies, tumour upstaging at rCx, tumour stage and lymph node (LN) status. RESULTS •The overall 5- and 10-year CSS was 82% and 76%, respectively. Significant correlations were found between the 5-year CSS and categorized number of TURBs (≤2 vs >2: 88% vs 71%; P= 0.001), interval between first TURB and rCx (≤4 months vs >4 months: 86% vs 77%; P= 0.04), adjuvant therapies (no vs yes: 86% vs 66%; P= 0.001), tumour upstaging at rCx (no vs yes: 89% vs 67%; P < 0.001), tumour stage at rCx (bladder confined vs non-confined: 88% vs 56%; P < 0.001) and LN status (no vs yes: 88% vs 36%; P < 0.001). •Multivariate analysis identified categorized number of TURBs (hazard ratio, HR, 0.14; 95% CI, 0.07-0.44; P < 0.001), categorized interval between first TURB and rCx (HR, 3.27; 95% CI, 1.24-8.59; P= 0.017), LN status (HR, 0.13; 95% CI, 0.06-0.26; P < 0.001) and tumour stage at rCx (HR, 0.49; 95% CI, 0.26-0.92; P= 0.03) as independent risk factors for CSS. CONCLUSION •Delay of rCx in 'high-risk' nmiBCA deteriorates CSS and should be avoided. The number of TURBs and the interval between first TURB and rCx are causative factors for delayed rCx and are independently correlated with CSS.

Background: Metastasis is associated with poor prognosis for melanoma. The formation of metastases is a multi-step process, in which cancer cells can subsequently acquire the potential to intravasate into the blood or lymph vessels, disseminate through the circulation, extravasate through the endothelium and invade the connective tissue. There is increasing evidence that chemokines have a pivotal role in the dissemination and establishment of melanoma metastasis. Methods: We isolated melanoma cells from melanoma metastasis and performed different migration assays and transendothelial resistance measurements of endothelial monolayers co-cultured with melanoma cells, in order to monitor barrier function and diapedesis and confirmed these results by confocal microscopy. Results: We observed that tumour endothelial cells (ECs) secrete high levels of CXCL9 in all, and CXCL10 in most melanoma metastases. Migration studies revealed that low concentrations of these chemokines induce chemotaxis, whereas high concentrations induce spontaneous migration of melanoma cells (chemokinesis/chemorepulsion) and the disruption of the endothelial barrier, resulting in an accelerated transendothelial migration (TEM). Addition of anti-CXCL9 or anti-CXCR3 antibodies to the co-cultures delayed the TEM of melanoma cells. Conclusion: Our data represent novel mechanisms by which tumour cells in melanoma metastases might use the chemokine-expressing endothelium to leave the tumour and eventually to form additional metastases at distinct sites.

Study Type – Diagnostic (exploratory cohort) Level of Evidence 2b What’s known on the subject? and What does the study add? Bladder cancer is initially diagnosed by white light cystoscopy followed by histopathological evaluation after transurethral resection of tissue suspicious for cancer. Difficulties may occur especially in the assessment of flat lesions or in discrimination between CIS and inflammatory disease. Confocal endomicroscopy during diagnostic colonoscopy has been a valuable tool for in vivo microscopic visualization and detection of colonic neoplasias and has contributed to optimized detection rates of up to 99%. We evaluated resected bladder urothelium of 18 patients by confocal endomicroscopy and correlated microscopic findings with standard histopathology. Typical tumour growth patterns such as altered nuclear to cytoplasmic ratio, pseudopapillar tissue stratification and neoangiogenesis were readily visible. As nuclear and subnuclear architecture of healthy bladder tissue could be discriminated against neoplastic tissue, confocal endomicroscopy is a promising novel tool for the in vivo microscopic visualization of bladder cancer. OBJECTIVE • To evaluate bladder urothelium by confocal laser endomicroscopy (CLE) and correlate microscopic findings with standard histopathology. PATIENTS AND METHODS • Fresh bladder urothelium tissue specimens were topically stained with acriflavine for instantaneous microscopic imaging. • A single-line laser in a handheld CLE probe delivered an excitation wavelength of 488 nm providing a high resolution of 0.7 µm and an adjustable imaging depth of 0–250 µm. • Resection specimens of 18 patients were investigated with 1000-fold magnification and ex vivo findings were compared with targeted histopathology (haematoxylin and eosin staining). RESULTS • Typical tumour growth patterns such as altered nuclear to cytoplasmic ratio, pseudopapillar tissue stratification and neoangiogenesis were readily visible. • Nuclear and subnuclear architecture of healthy bladder tissue could be discriminated against neoplastic tissue. CONCLUSIONS • In addition to white light cystoscopy, CLE is a promising novel tool for the in vivo microscopic visualization of bladder cancer; first results of the present study show its potential to define microscopic characteristics of bladder cancer tissue. • Further in vivo studies are necessary to determine sensitivity and specificity of the technique.

OBJECTIVE To investigate the associations between different overall or topographically restricted lymph node (LN) variables and cancer-specific survival (CSS) after radical cystectomy (RC) and extended LN dissection (LND) with curative intent in patients with LN-positive bladder cancer. PATIENTS AND METHODS Between 2001 and 2006, 152 patients had RC with standardized extended LND for bladder cancer with curative intent. Patients with positive LNs were stratified according to the median of the LN variables (LNs removed, number of positive LNs, LN density). CSS was related to overall and topographically restricted LN variables, e.g. different levels of LND, and relationships were tested by univariate and multivariate analyses. Level 1 LND comprised the regions of the external and internal iliac LNs and of the obturator LNs, level 2 the templates of common iliac and presacral LNs, and level 3 the para-aortic and paracaval LNs up to the inferior mesenteric artery. The mean (range) follow-up was 22 (1–84) months. RESULTS LN metastases were diagnosed in 46 of the 152 patients (30%) with extended LND. In these 46 patients, the median number of removed LNs was 33 (level 1, 15.5; level 2, 9.0; level 3, 7.0), the median number of positive LNs was 3 (1.5, 0.5 and 0.0, respectively) and the median LN density was 0.11 (0.10, 0.02 and 0.0, respectively). The CSS was 76% at 1 year and 23% at 3 years. There were significant correlations between the 3-year CSS and the overall LN density (≤0.11 vs >0.11; 34% vs 8%, P = 0.008), and the total number of positive LNs (≤3 vs >3; 33% vs 8%; P = 0.05). Overall LN density (hazard ratio 0.33, 95% confidence interval 0.15–0.72; P = 0.006) was an independent predictor for CSS in multivariate analysis. CONCLUSIONS Overall LN density is an independent predictor of survival after RC and extended LND with curative intent. Evaluation of topographically restricted LN positivity and density for different regions and levels of LND does not improve the prediction of CSS compared with overall LN positivity and density. A low incidence of level 3 LN positivity questions the clinical relevance of removing para-aortic and paracaval LNs. However, our data need to be confirmed by a prospective randomized trial.

Fur Docetaxel-refraktare Patienten mit metastasiertem hormonrefraktaren Prostatakarzinom (mHRPC) existiert bisher kein etabliertes Therapieregime. Deshalb berichten wir hier uber unsere Erfahrungen mit Mitoxantron plus Prednison (MP) als Zweitlinenchemotherapie. Zehn Docetaxel-refraktare mHRPC-Patienten wurden mit MP behandelt. Untersucht wurden PSA-Abfall, progressionsfreies Intervall und Schmerzstatus unter Chemotherapie. Unter MP zeigten 2 Patienten eine partielle PSA-Remission, 3 ein „stable disease“ und 5 waren progredient. Das progressionsfreie Intervall bei partieller PSA-Remission lag im Mittel bei 8 Monaten und bei „stable disease“ im Median bei 2 Monaten. Eine Schmerzreduktion wurde in 4 von 7 Patienten unter MP beobachtet. Eine Grad-4-Neutropenie trat in nur bei einem Patienten auf. Patienten mit PSA-Abfall unter Erst- und Zweitlinientherapie hatten ein progressionsfreies Intervall im Median von 11,5 Monaten. Zum gegenwartigen Zeitpunkt wird die Indikation fur MP als Zweitlinienchemotherapie fur Docetaxel-refraktare mHRPC-Patienten gesehen, die nicht in eine Studie eingeschlossen werden konnen. Trotz der masigen Rate von partiellen PSA-Remissionen wird bei jedem zweiten Patienten eine Schmerzreduktion erzielt. Ein progressionsfreies Intervall von mehreren Monaten kann erreicht werden. Die therapiebedingten Nebenwirkungen sind vergleichsweise gering.

The tyrosine kinase receptor c-kit and its ligand stem cell factor (SCF) have not been explored in prostate cancer (PC) bone metastasis. Herein, we found that three human PC cell lines and bone marrow stromal cells express a membrane-bound SCF isoform and release a soluble SCF. Bone marrow stromal cells revealed strong expression of c-kit, whereas PC cells showed very low levels of the receptor or did not express it all. Using an experimental model of PC bone metastasis, we found that intraosseous bone tumors formed by otherwise c-kit–negative PC3 cells strongly expressed c-kit, as demonstrated using immunohistochemical and Western blot analyses. Subcuta-neous PC3 tumors were, however, c-kit–negative. Both bone and subcutaneous PC3 tumors were positive for SCF. Immunohistochemical analysis of human specimens revealed that the expression frequency of c-kit in epithelial cells was of 5% in benign prostatic hyperplasia, 14% in primary PC, and 40% in PC bone metastases, suggesting an overall trend of increased c-kit expression in clinical PC progression. Stem cell factor expression frequency was more than 80% in all the cases. Our data suggest that the bone microenvironment up-regulates c-kit expression on PC cells, favoring their intraosseous expansion.

Objective To study clinical and histopathologic parameters after cystectomy and lymphadenectomy in non-muscle-invasive transitional cell carcinoma (TCC) of the bladder and their association with the prevalence of lymph node metastases (N+). Patients and methods Of 866 patients treated with radical cystectomy and lymphadenectomy, 219 had non-muscle-invasive TCC of the bladder. The prevalence of N+ was related to parameters such as gender, age, number of transurethral resections of the bladder (TURBs), intervals between first TURB and cystectomy, adjuvant therapy, maximum histopathologic tumor stage and grade at TURB, and tumor upstaging in the cystectomy specimen by univariate and multivariate analysis. Results A total of 33 patients (15%) had N+. By multivariate analyses, tumor upstaging and the number of TURBs were independent predictors of N+ at cystectomy. The number of TURBs increased the prevalence of N+ from 8% (one TURB) to 24% (two to four TURBs). Tumor upstaging in the cystectomy specimen increased the prevalence of N+ from 4% to 36%. Conclusion Inappropriate delay and staging errors of"high risk" non-muscle-invasive TCC of the bladder contribute to an increased prevalence of N+ and should be avoided. In our series, the number of TURBs and tumor upstaging in the cystectomy specimen were independent predictors for N+ by multivariate analysis.

OBJECTIVE To assess primary tumours of the urethra in males. PATIENTS AND METHODS We retrospectively reviewed our database from 1986 to 2006 for primary tumours of the male urethra; nine patients with primary tumours of the urethra were analysed and follow-up information was obtained. RESULTS Three patients had tumours of the prostatic urethra, two of which had proliferating focal inflammation and one a low-grade, superficial urothelial cancer. All patients were treated successfully with transurethral resection. Six patients had carcinoma of the bulbar or penile urethra, including two with previous local percutaneous radiotherapy for prostate cancer. All had primary surgical excision that was adapted to tumour location and extension. One patient had adjuvant chemotherapy after surgery. All but one patient remain recurrence-free after a median follow-up of 20 months. CONCLUSION Primary carcinoma of the male urethra is a rare entity. Previous radiotherapy might be a predisposing factor. Local surgical tumour control is essential for long-term survival, but the extent of surgery depends on tumour location and stage. Multimodal therapy might be required to obtain an optimum oncological outcome.

Acute lung injury is accompanied by an increased endothelial chemokine production and adhesion molecule expression, which may result in an extensive neutrophil infiltration. Moreover, a destruction of the alveolar epithelium and capillary endothelium may result in permeability edema. As such, the search for novel anti-inflammatory substances, able to downregulate these parameters as well as the tissue damage holds therapeutic promise. We therefore describe here the use of human endothelial cell-based in vitro assays for the detection of anti-inflammatory and wound-healing metabolites from cyanobacteria.

Rho GTPases regulate diverse cellular functions including adhesion, cytokinesis and motility, as well as the activity of the transcription factors NF-kappaB, serum response factor and C/EBP. alpha-Catulin, an alpha-catenin-related protein that shares structural similarities with cytoskeletal linker proteins, facilitates Rho signalling by serving as a scaffold for the Rho-specific guanine nucleotide exchange factor Lbc. We report here that alpha-catulin also interacts with a key component of the NF-kappaB signalling pathway, namely the IkappaB kinase (IKK)-beta. In co-immunoprecipitations, alpha-catulin can bind IKK-beta and Lbc. Ectopic expression of alpha-catulin augmented NF-kappaB activity, promoted cell migration and increased resistance to apoptosis, whereas knockdown experiments showed the opposite effects. Together, these features suggest that alpha-catulin has tumorigenic potential.

Authors from Mainz, Germany present the long-term follow-up of submucosal tunnel and serosa-lined extramural tunnel ureter implantation in the ileocaecal continent cutaneous urinary diversion first described in that department, called the Mainz-Pouch I. OBJECTIVE To assess upper urinary tract complications and renal function in patients with a submucosal tunnel and serosa-lined extramural tunnel ureter implantation during the long-term follow-up of ileocaecal continent cutaneous urinary diversion (Mainz pouch I). PATIENTS AND METHODS In all, 458 patients who had diversion with the ileocaecal pouch were analysed in a retrospective follow-up study. Uretero-intestinal implantation was done using a submucosal tunnel (ST) in 809 reno-ureteric units (RUs) and by the serosa-lined extramural tunnel (ET) technique in 74 RUs. The median age of the patients at the time of surgery was 47.1 years, and the median follow-up was 89.0 months. RESULTS For the ST, there was anastomotic obstruction in 59 RUs (7.3%) at a median of 16.8 months after diversion; the obstruction-free intervals at 1, 5 and 10 years were 97%, 93% and 91%, respectively. Obstruction rates were 13.9% for previously dilated upper tracts and 17.1% in patients with a neurogenic bladder. Serum creatinine levels were ≤1.6 mg/dL in 97% of the patients at the latest follow-up. For ET, there was anastomotic obstruction in three RUs (4.1%) at a median of 17.2 months after diversion. Obstruction-free intervals at 1, 5 and 10 years were 100%, 96% and 96%. Preoperative dilation of the upper tracts did not reduce the obstruction rate (3.1%), but it was 7.1% in patients with a neurogenic bladder. Serum creatinine levels were ≤1.6 mg/dL in 98% of the patients at the latest follow-up. CONCLUSIONS The ET gives lower obstruction rates than the ST, especially when upper tracts are dilated and in patients with a neurogenic bladder. Renal function remained stable with both techniques in the long term.

Die zunehmende Uberalterung unserer Gesellschaft erhoht kontinuierlich den Anteil alterer und polymorbider Patienten innerhalb der Inkontinenzklientel. Finanzielle und personelle Ressourcenknappheit zwingt zu rationellem und teleologisch orientiertem Umgang mit dem vorhandenen diagnostischen Instrumentarium. Die bei einem alten Menschen mit Harninkontinenz durchzufuhrenden Untersuchungen sollten einem nach Invasivitat gestaffelten Algorithmus folgen und sich auf den fur eine primar konservative Therapieplanung notwendigen Mindestumfang beschranken. Sinnvolle Diagnostik objektiviert beklagte Beschwerden, dient der Subgruppendifferenzierung, der Ursachenforschung und der Schweregradeinteilung, ermoglicht die Beurteilung von Komorbiditaten, hilft bei der Therapieplanung und dokumentiert Therapieverlauf und mogliche Behandlungskomplikationen. Dabei ist sie bei hinreichend detailliert dokumentierten Untersuchungsergebnissen so wenig invasiv und einfach wie moglich. Die Harninkontinenzbasisdiagnostik umfasst Anamnese, korperliche Untersuchung, Urinanalyse, freie Harnstrahlmessung mit sonographischer Restharnbestimmung, Miktionstagebuch und die Bestimmung des mentalen Status. Auf diese Weise ist die Harninkontinenz fast aller alteren Patienten ausreichend genau beurteilbar, um einen konservativen Behandlungsversuch zu beginnen. Nur eine kleine Minderheit mit inkongruenten Untersuchungsergebnissen oder therapierefraktare Rezidivfalle sollten einer invasiveren Zusatzdiagnostik (Zysturethroskopie, Urodynamik, morphologische und funktionelle Rontgendiagnostik, Perineal-/Introitussonographie) unterzogen werden, wenn sich aus ihr therapeutische Konsequenzen ableiten liesen. Ohne diese Therapierelevanz ist eine kostenintensive Spezialdiagnostik bei alteren Menschen auch wegen der nicht unerheblichen Untersuchungsmorbiditat abzulehnen.

Nowadays, male stress urinary incontinence is rare and almost always of iatrogenic origin (radiotherapy, pelvic surgery). However, the prognosis of urinary incontinence following surgery is good and can be improved by pelvic floor muscle exercises in combination with biofeedback systems. For the remaining patient cohort with persistent urinary incontinence, several established surgical treatment options are available. Suburothelial injections of bulking agents can easily be performed in an ambulatory setting. However, regardless of the material used, long-term results are disappointing. Moreover, the residual urethral function deteriorates due to cicatrization of the suburothelial plexus with consequent loss of urethral elasticity. The fascial sling procedure in males has to be performed in preoperated areas and is as technically demanding for the surgeon as it is burdening for the patient. Alloplastic material is not used, thus minimizing risks for arrosion or infection. Since the sling tension can neither be standardized nor postoperatively readjusted, the risk of overcorrection is considerable and the success of the procedure is heavily dependent on the surgeon's experience. Despite wear and high revision rates, the technically mature artificial sphincter produces excellent continence results and has become the gold standard in the therapy of male stress urinary incontinence. The circumferential and continuous urethral compression by the cuff is highly effective, but at the price of an almost inevitable urethral atrophy. To overcome this problem, various surgical techniques have been developed (tandem cuff, cuff downsizing, transcorporal cuff placement). However, the expensive artificial sphincter is not a nostrum for every incontinent man, since it requires certain minimal cognitive and manual capabilities. Therefore, the search for less demanding treatment alternatives seems to be necessary, even if one has to accept lower continence rates.