Insights into Sex Chromosome Evolution and Aging from the Genome of a Short-Lived Fish.

The killifish Nothobranchius furzeri is the shortest-lived vertebrate that can be bred in the laboratory. Its rapid growth, early sexual maturation, fast aging, and arrested embryonic development (diapause) make it an attractive model organism in biomedical research. Here, we report a draft sequence of its genome that allowed us to uncover an intra-species Y chromosome polymorphism representing-in real time-different stages of sex chromosome formation that display features of early mammalian XY evolution "in action." Our data suggest that gdf6Y, encoding a TGF-ß family growth factor, is the master sex-determining gene in N. furzeri. Moreover, we observed genomic clustering of aging-related genes, identified genes under positive selection, and revealed significant similarities of gene expression profiles between diapause and aging, particularly for genes controlling cell cycle and translation. The annotated genome sequence is provided as an online resource (http://www.nothobranchius.info/NFINgb).

CD44 Contributes to the Regulation of MDR1 Protein and Doxorubicin Chemoresistance in Osteosarcoma.

Osteosarcoma is the most common type of pediatric bone tumor. Despite great advances in chemotherapy during the past decades, the survival rates of osteosarcoma patients remain unsatisfactory. Drug resistance is one of the main reasons, leading to treatment failure and poor prognosis. Previous reports correlated expression of cluster of differentiation 44 (CD44) with drug resistance and poor survival of osteosarcoma patients, however the underlying mechanisms are poorly defined. Here, we investigated the role of CD44 in the regulation of drug chemoresistance, using osteosarcoma cells isolated from mice carrying a mutation of the tumor suppressor neurofibromatosis type 2 (Nf2) gene. CD44 expression was knocked-down in the cells using CRISPR/Cas9 approach. Subsequently, CD44 isoforms and mutants were re-introduced to investigate CD44-dependent processes. Sensitivity to doxorubicin was analyzed in the osteosarcoma cells with modified CD44 expression by immunoblot, colony formation- and WST-1 assay. To dissect the molecular alterations induced by deletion of Cd44, RNA sequencing was performed on Cd44-positive and Cd44-negative primary osteosarcoma tissues isolated from Nf2-mutant mice. Subsequently, expression of candidate genes was evaluated by quantitative reverse transcription PCR (qRT-PCR). Our results indicate that CD44 increases the resistance of osteosarcoma cells to doxorubicin by up-regulating the levels of multidrug resistance (MDR) 1 protein expression, and suggest the role of proteolytically released CD44 intracellular domain, and hyaluronan interactions in this process. Moreover, high throughput sequencing analysis identified differential regulation of several apoptosis-related genes in Cd44-positive and -negative primary osteosarcomas, including p53 apoptosis effector related to PMP-22 (Perp). Deletion of Cd44 in osteosarcoma cells led to doxorubicin-dependent p53 activation and a profound increase in Perp mRNA expression. Overall, our results suggest that CD44 might be an important regulator of drug resistance and suggest that targeting CD44 can sensitize osteosarcoma to standard chemotherapy.

The Effect of Water on the 2-Propanol Oxidation Activity of Co-Substituted LaFe1- Cox O3 Perovskites.

Perovskites are interesting oxidation catalysts due to their chemical flexibility enabling the tuning of several properties. In this work, we synthesized LaFe1-x Cox O3 catalysts by co-precipitation and thermal decomposition, characterized them thoroughly and studied their 2-propanol oxidation activity under dry and wet conditions to bridge the knowledge gap between gas and liquid phase reactions. Transient tests showed a highly active, unstable low-temperature (LT) reaction channel in conversion profiles and a stable, less-active high-temperature (HT) channel. Cobalt incorporation had a positive effect on the activity. The effect of water was negative on the LT channel, whereas the HT channel activity was boosted for x>0.15. The boost may originate from a slower deactivation rate of the Co3+ sites under wet conditions and a higher amount of hydroxide species on the surface comparing wet to dry feeds. Water addition resulted in a slower deactivation for Co-rich catalysts and higher activity in the HT channel state.

Cluster of differentiation 44 promotes osteosarcoma progression in mice lacking the tumor suppressor Merlin.

Merlin is a versatile tumor suppressor protein encoded by the NF2 gene. Several lines of evidence suggest that Merlin exerts its tumor suppressor activity, at least in part, by forming an inhibitory complex with cluster of differentiation 44 (CD44). Consistently, numerous NF2 mutations in cancer patients are predicted to perturb the interaction of Merlin with CD44. We hypothesized that disruption of the Merlin-CD44 complex through loss of Merlin, unleashes putative tumor- or metastasis-promoting functions of CD44. To evaluate the relevance of the Merlin-CD44 interaction in vivo, we compared tumor growth and progression in Cd44-positive and Cd44-negative Nf2-mutant mice. Heterozygous Nf2-mutant mice were prone to developing highly metastatic osteosarcomas. Importantly, while the absence of the Cd44 gene had no effect on the frequency of primary osteosarcoma development, it strongly diminished osteosarcoma metastasis formation in the Nf2-mutant mice. In vitro assays identified transendothelial migration as the most prominent cellular phenotype dependent on CD44. Adhesion to endothelial cells was blocked by interfering with integrin α4ß1 (very late antigen-4, VLA-4) on osteosarcoma cells and CD44 upregulated levels of integrin VLA-4 ß1 subunit. Among other putative functions of CD44, which may contribute to the metastatic behavior, the passage through the endothelial cells also appears to be critical in vivo, as CD44 significantly promoted formation of lung metastasis upon intravenous injection of osteosarcoma cells into immunocompromised mice. Altogether, our results strongly suggest that CD44 plays a metastasis-promoting role in the absence of Merlin.

Application of patient-derived liver cancer cells for phenotypic characterization and therapeutic target identification.

Primary liver cancer (PLC) ranks among the most lethal solid cancers worldwide due to lack of effective biomarkers for early detection and limited treatment options in advanced stages. Development of primary culture models that closely recapitulate phenotypic and molecular diversities of PLC is urgently needed to improve the patient outcome. Long-term cultures of 7 primary liver cancer cell lines of hepatocellular and cholangiocellular origin were established using defined culture conditions. Morphological and histological characteristics of obtained cell lines and xenograft tumors were analyzed and compared to original tumors. Time course analyses of transcriptomic and genomic changes were performed using next-generation sequencing (NGS). Key oncogenic alterations were identified by targeted NGS and cell lines carrying potentially actionable mutations were treated with corresponding specific inhibitors. PDCL fully resembled morphological features of the primary cancers in vitro and in vivo over extended period in culture. Genomic alterations as well as transcriptome profiles showed high similarity with primary tumors and remained stable during long-term culturing. Targeted-NGS confirmed that key oncogenic mutations such as TP53, KRAS, CTNNB1 as well as actionable mutations (e.g. MET, cKIT, KDR) were highly conserved in PDCL and amenable for individualized therapeutic approaches. Integrative genomic and transcriptomic approaches further demonstrated that PDCL more closely resemble molecular and prognostic features of PLC than established cell lines and are valuable tool for direct target evaluation. Our integrative analysis demonstrates that PDCL represents refined model for discovery of relevant molecular subgroups and exploration of precision medicine approaches for the treatment of this deadly disease.

Alterations in ERBB2 and BRCA and microsatellite instability as new personalized treatment options in small bowel carcinoma.

BACKGROUND: Carcinomas of the small bowel are rare tumors usually with dismal prognosis. Most recently, some potentially treatable molecular alterations were described. We emphasize the growing evidence of individualized treatment options in small bowel carcinoma. METHODS: We performed a DNA- based multi-gene panel using ultra-deep sequencing analysis (including 14 genes with up to 452 amplicons in total; KRAS, NRAS, HRAS, BRAF, DDR2, ERBB2, KEAP1, NFE2L2, PIK3CA, PTEN, RHOA, BRCA1, BRCA2 and TP53) as well as an RNA-based gene fusion panel including ALK, BRAF, FGFR1, FGFR2, FGFR3, MET, NRG1, NTRK1, NTRK2, NTRK3, RET and ROS1 on eleven formalin fixed and paraffin embedded small bowel carcinomas. Additionally, mismatch-repair-deficiency was analyzed by checking the microsatellite status using the five different mononucleotide markers BAT25, BAT26, NR-21, NR-22 and NR-27 and loss of mismatch repair proteins using four different markers (MLH1, MSH6, MSH2, PMS2). RESULTS: In five out of eleven small bowel carcinomas we found potentially treatable genetic alterations. Three patients demonstrated pathogenic (class 5) BRCA1 or BRCA2 mutations - one germline-related in a mixed neuroendocrine-non neuroendocrine neoplasm (MiNEN). Two additional patients revealed an activating ERBB2 mutation or PIK3CA mutation. Furthermore two tumors were highly microsatellite-instable (MSI-high), in one case associated to Lynch-syndrome. We did not find any gene fusions. CONCLUSION: Our results underscore, in particular, the relevance of potentially treatable molecular alterations (like ERBB2, BRCA and MSI) in small bowel carcinomas. Further studies are needed to proof the efficacy of these targeted therapies in small bowel carcinomas.

Laser-Induced Functionalization of Organo/Carbon Interfaces for Selective Adsorption of Au Nanoparticles in Microsized Domains.

Laser microprocessing of highly oriented pyrolytic graphite (HOPG) in conjunction with chemical functionalization routines is used to fabricate functional microsized domains. Infrared and Auger electron spectroscopy, contact angle measurements, and electron microscopy are used for characterization of laser-fabricated structures. HOPG samples are coated with alkylsiloxane monolayers. Laser-induced bromination of coated HOPG samples in gaseous bromine is carried out using a microfocused laser beam at a wavelength of 514 nm and 1/e2 laser spot diameter of about 2 µm. Subsequent azidation and amination results in functional domains with sizes in the range of 1.2 to 40 µm and more. At low laser powers and irradiation times fully functionalized circular-shaped structures are formed. At high laser powers and irradiation times laser processing results in decomposition of the organic monolayer and substrate in the center of the structures yielding donut-shaped structures. After laser processing and chemical transformation Au nanoparticles are selectively adsorbed onto the functional domains. This provides an opportunity to build up functional nanoparticle microarrays on carbon-based materials, e.g., for applications in sensing and electrocatalysis.

Poly(N,N-dimethylaminoethyl methacrylate) Brushes: pH-Dependent Switching Kinetics of a Surface-Grafted Thermoresponsive Polyelectrolyte.

The temperature-dependent switching behavior of poly(N,N-dimethylaminoethyl methacrylate) brushes in alkaline, neutral, and acidic solutions is examined. A novel microscopic laser temperature-jump technique is employed in order to study characteristic thermodynamic and kinetic parameters. Static laser micromanipulation experiments allow one to determine the temperature-dependent variation of the swelling ratio. The data reveal a strong shift of the volume phase transition of the polymer brushes to higher temperatures when going from pH = 10 to pH = 4. Dynamic laser micromanipulation experiments offer a temporal resolution on a submillisecond time scale and provide a means to determine the intrinsic rate constants. Both the swelling and the deswelling rates strongly decrease in acidic solutions. Complementary experiments using in situ atomic force microscopy show an increased polymer layer thickness at these conditions. The data are discussed on the basis of pH-dependent structural changes of the polymer brushes including protonation of the amine groups and conformational rearrangements. Generally, repulsive electrostatic interactions and steric effects are assumed to hamper and slow down temperature-induced switching in acidic solutions. This imposes significant restrictions for smart polymer surfaces, sensors, and devices requiring fast response times.

Photothermal laser fabrication of micro- and nanostructured chemical templates for directed protein immobilization.

Photothermal patterning of poly(ethylene glycol) terminated organic monolayers on surface-oxidized silicon substrates is carried out using a microfocused beam of a CW laser operated at a wavelength of 532 nm. Trichlorosilane and trimethoxysilane precursors are used for coating. Monolayers from trimethoxysilane precursors show negligible unspecific protein adsorption in the background, i.e., provide platforms of superior protein repellency. Laser patterning results in decomposition of the monolayers and yields chemical templates for directed immobilization of proteins at predefined positions. Characterization is carried out via complementary analytical methods including fluorescence microscopy, atomic force microscopy, and scanning electron microscopy. Appropriate labeling techniques (fluorescent markers and gold clusters) and substrates (native and thermally oxidized silicon substrates) are chosen in order to facilitate identification of protein adsorption and ensure high sensitivity and selectivity. Variation of the laser parameters at a 1/e(2) spot diameter of 2.8 µm allows for fabrication of protein binding domains with diameters on the micrometer and nanometer length scale. Minimum domain sizes are about 300 nm. In addition to unspecific protein adsorption on as-patterned monolayers, biotin-streptavidin coupling chemistry is exploited for specific protein binding. This approach represents a novel facile laser-based means for fabrication of protein micro- and nanopatterns. The routine is readily applicable to femtosecond laser processing of glass substrates for the fabrication of transparent templates.

Photothermal laser microsintering of nanoporous gold.

Photothermal processing of nanoporous gold using a microfocused continuous-wave laser at a wavelength of 532 nm and a 1/e(2) spot diameter of 2.9 µm has been studied. In addition, complementary experiments have been carried out via conventional annealing. Scanning electron microscopy has been used for characterization. Local laser irradiation at distinct laser powers and pulse lengths results in coarsening of the porous gold structures. During laser processing the pore size of the native nanoporous gold increases to maximum values in the range of 0.25-3 µm. The affected areas exhibit lateral dimensions in the range of 2-10 µm. Overall two regions are distinguished. An inner region, where large pores and ligaments are formed and an outer region, where the pore size and ligament size gradually change and approach the feature sizes of the native material. A qualitative thermokinetic model allows one to reproduce the experimentally observed dependence of the laser-induced morphologies on the laser parameters. On the basis of this model the underlying processes are attributed to sintering and melting of the gold structures. The presented results demonstrate the prospects of photothermal laser processing in engineering porous gold with spatially varying porosities on micrometer to nanometer length scales.

A novel targeted NGS panel identifies numerous homologous recombination deficiency (HRD)-associated gene mutations in addition to known BRCA mutations.

Deleterious mutations in the BRCA1 and BRCA2 genes have significant therapeutic relevance in clinical settings regarding personalized therapy approaches. BRCA1 and BRCA2 play a pivotal role in homologous recombination (HR) and thus are sensitive for PARP inhibitors (PARPi). Beyond the narrow scope of evaluating only the BRCA mutation status, PARPi can be beneficial for HR deficient (HRD) patients, who harbor mutations in other HR-associated genes. In the present retrospective study, a novel targeted HRD gene panel was validated and implemented for use with FFPE tissue. Samples of patients with ovarian, breast, pancreatic and prostate cancer were included. Variants were robustly detected with various DNA input amounts and the use of test samples showed complete concordance between previously known mutations and HRD panel results. From all the 90 samples included in this cohort, TP53 was the most frequently altered gene (73%). Deleterious BRCA1/2 mutations were found in 20 (22%) of all samples. New pathogenic or likely pathogenic mutations in additional HR-associated genes were identified in 22 (24%) patients. Taken together, the present study proves the feasibility of a new HRD gene panel with reliable panel performance and offers the possibility to easily screen for resistance mutations acquired over treatment time.Mutations in HR-associated genes, besides BRCA1/2, might represent promising potential targets for synthetic lethality approaches. Thus, a substantial number of patients may benefit from expanding the scope of therapeutic agents like PARPi.

The transcript catalogue of the short-lived fish Nothobranchius furzeri provides insights into age-dependent changes of mRNA levels.

BACKGROUND: The African annual fish Nothobranchius furzeri has over recent years been established as a model species for ageing-related studies. This is mainly based on its exceptionally short lifespan and the presence of typical characteristics of vertebrate ageing. To substantiate its role as an alternative vertebrate ageing model, a transcript catalogue is needed, which can serve e.g. as basis for identifying ageing-related genes. RESULTS: To build the N. furzeri transcript catalogue, thirteen cDNA libraries were sequenced using Sanger, 454/Roche and Solexa/Illumina technologies yielding about 39 Gb. In total, 19,875 protein-coding genes were identified and annotated. Of these, 71% are represented by at least one transcript contig with a complete coding sequence. Further, transcript levels of young and old fish of the strains GRZ and MZM-0403, which differ in lifespan by twofold, were studied by RNA-seq. In skin and brain, 85 differentially expressed genes were detected; these have a role in cell cycle control and proliferation, inflammation and tissue maintenance. An RNA-seq experiment for zebrafish skin confirmed the ageing-related relevance of the findings in N. furzeri. Notably, analyses of transcript levels between zebrafish and N. furzeri but also between N. furzeri strains differed largely, suggesting that ageing is accelerated in the short-lived N. furzeri strain GRZ compared to the longer-lived strain MZM-0403. CONCLUSIONS: We provide a comprehensive, annotated N. furzeri transcript catalogue and a first transcriptome-wide insight into N. furzeri ageing. This data will serve as a basis for future functional studies of ageing-related genes.

Monitoring of adenovirus serotypes in environmental samples by combined PCR and melting point analyses.

BACKGROUND: Human adenoviruses are promising candidates for addressing health risks associated with enteric viruses in environmental waters. Relatively harmless but common, these DNA viruses persist within the population and are generally considered extremely stable, remaining infectious in water for long periods of time. Group-specific or single species detection of human adenoviruses in environmental samples is usually based on polymerase chain reaction assays. Simultaneous identification of specific species or serotypes needs additional processing. Here we present a simple molecular approach for the monitoring of serotypic diversity in the human adenovirus populations in contaminated water sites. METHODS: Diversity patterns of human adenoviruses in environmental samples, collected in an outdoor artificial stream and pond simulation system, were analyzed using a closed tube polymerase chain reaction method with subsequent melting point analysis. RESULTS: Human adenovirus serotype 41 was the most prominent adenovirus serotype detected in environmental water samples, but melting point analyses indicated the presence of additional adenovirus serotypes. CONCLUSIONS: Based on investigations with spiked and environmental samples, a combination of qPCR and melting point analysis was shown to identify adenovirus serotypes in sewage contaminated water.

Hofmeister effect of sodium halides on the switching energetics of thermoresponsive polymer brushes.

A laser temperature-jump technique is used to probe the impact of sodium halides on the temperature-dependent switching kinetics and thermodynamics of poly(N-isopropylacrylamide) brushes. An analysis on the basis of a two-state model reveals van't Hoff enthalpy and entropy changes. Sodium halides increase the endothermicity and the entropic gain of the switching process below and above Tc following the Hofmeister series: NaCl > NaBr > NaI. In contrast, enthalpic and entropic changes at Tc remain virtually unaffected. This provides an unprecedented insight into the underlying switching energetics of this classic stimuli-responsive polymer. Because of its model character, these results represent an essential reference on the way to unpuzzle the molecular driving forces of the Hofmeister effect.

Mutations in the polyglutamine binding protein 1 gene cause X-linked mental retardation.

We found mutations in the gene PQBP1 in 5 of 29 families with nonsyndromic (MRX) and syndromic (MRXS) forms of X-linked mental retardation (XLMR). Clinical features in affected males include mental retardation, microcephaly, short stature, spastic paraplegia and midline defects. PQBP1 has previously been implicated in the pathogenesis of polyglutamine expansion diseases. Our findings link this gene to XLMR and shed more light on the pathogenesis of this common disorder.

A microinjection protocol for the generation of transgenic killifish (Species: Nothobranchius furzeri).

BACKGROUND: A challenge in age research is the absence of short-lived vertebrate model organisms. The turquoise killifish Nothobranchius furzeri has an exceptionally short lifespan of 4-10 months depending on the strain. Thus, it possesses the shortest known maximum lifespan of a vertebrate species that can be bred in captivity. RESULTS: Here we show the successful introduction of DNA and RNA molecules into the one-cell embryo of N. furzeri. For this purpose, we adapted existing microinjection protocols to inject through the remarkably thick and robust chorion of N. furzeri's eggs. The injected DNA transgene was integrated into the genome and transmitted to subsequent generations as indicated by the expression of the fluorophore enhanced green fluorescent protein (EGFP). Furthermore, we could confirm a special phase during embryonic development in which embryogenesis occurs within a re-aggregated mass of previously dispersed cells as it has been described for other related cyprinodont fish species. CONCLUSIONS: The transgenesis protocol described here provides a basis for a variety of genetic manipulations including overexpression of genes and determining their effects on lifespan and longevity. The feasibility to perform transgenesis is an important step to establish N. furzeri as a new model in age research.

N-Doped porous carbons obtained from chitosan and spent coffee as electrocatalysts with tuneable oxygen reduction reaction selectivity for H2O2 generation.

Nitrogen-containing porous carbons prepared by the pyrolysis of adequate biopolymer-based precursors have shown potential in several electrochemical energy-related applications. However, it is still of crucial interest to find the optimal precursors and process conditions which would allow the preparation of carbons with adequate porous structure as well as suitable nitrogen content and distribution of functional groups. In the present work we suggested a straightforward approach to prepare N-doped porous carbons by direct pyrolysis under nitrogen of chitosan : coffee blends of different compositions and using KOH for simultaneous surface activation. The synthetized carbon materials were tested for the electrochemical oxygen reduction to hydrogen peroxide (H2O2). A higher fraction of chitosan in the precursor led to a decrease in meso- and nano-porosity of the formed porous carbons, while their activity towards H2O2 generation increased. The nitrogen species derived from chitosan seem to play a very important role. Out of the synthesized catalysts the one with the largest content of pyridinic nitrogen sites exhibited the highest faradaic efficiency. The faradaic efficiencies and current densities of the synthesized materials were comparable with the ones of other commercially available carbons obtained from less renewable precursors.

Deletion of Cd44 Inhibits Metastasis Formation of Liver Cancer in Nf2-Mutant Mice.

Primary liver cancer is the third leading cause of cancer-related death worldwide. An increasing body of evidence suggests that the Hippo tumor suppressor pathway plays a critical role in restricting cell proliferation and determining cell fate during physiological and pathological processes in the liver. Merlin (Moesin-Ezrin-Radixin-like protein) encoded by the NF2 (neurofibromatosis type 2) gene is an upstream regulator of the Hippo signaling pathway. Targeting of Merlin to the plasma membrane seems to be crucial for its major tumor-suppressive functions; this is facilitated by interactions with membrane-associated proteins, including CD44 (cluster of differentiation 44). Mutations within the CD44-binding domain of Merlin have been reported in many human cancers. This study evaluated the relative contribution of CD44- and Merlin-dependent processes to the development and progression of liver tumors. To this end, mice with a liver-specific deletion of the Nf2 gene were crossed with Cd44-knockout mice and subjected to extensive histological, biochemical and molecular analyses. In addition, cells were isolated from mutant livers and analyzed by in vitro assays. Deletion of Nf2 in the liver led to substantial liver enlargement and generation of hepatocellular carcinomas (HCCs), intrahepatic cholangiocarcinomas (iCCAs), as well as mixed hepatocellular cholangiocarcinomas. Whilst deletion of Cd44 had no influence on liver size or primary liver tumor development, it significantly inhibited metastasis formation in Nf2-mutant mice. CD44 upregulates expression of integrin ß2 and promotes transendothelial migration of liver cancer cells, which may facilitate metastatic spreading. Overall, our results suggest that CD44 may be a promising target for intervening with metastatic spreading of liver cancer.

Approach to risk stratification for papillary thyroid carcinoma based on molecular profiling: institutional analysis.

BACKGROUND: Currently, treatment recommendations for papillary thyroid carcinoma are not based on the genetic background causing tumourigenesis. The aim of the present study was to correlate the mutational profile of papillary thyroid carcinoma with clinical parameters of tumour aggressiveness, to establish recommendations for risk-stratified surgical treatment. METHOD: Papillary thyroid carcinoma tumour tissue of patients undergoing thyroid surgery at the University Medical Centre Mainz underwent analysis of BRAF, TERT promoter and RAS mutational status as well as potential RET and NTRK rearrangements. Mutation status was correlated with clinical course of disease. RESULTS: One hundred and seventy-one patients operated for papillary thyroid carcinoma were included. The median age was 48 years (range 8-85) and 69 per cent (118/171) of patients were females. One hundred and nine papillary thyroid carcinomas were BRAF-V600E mutant, 16 TERT promotor mutant and 12 RAS mutant; 12 papillary thyroid carcinomas harboured RET rearrangements and two papillary thyroid carcinomas showed NTRK rearrangements. TERT promoter mutant papillary thyroid carcinomas had a higher risk of distant metastasis (OR 51.3, 7.0 to 1048.2, P < 0.001) and radioiodine-refractory disease (OR 37.8, 9.9 to 169.5, P < 0.001). Concomitant BRAF and TERT promoter mutations increased the risk of radioiodine-refractory disease in papillary thyroid carcinoma (OR 21.7, 5.6 to 88.9, P < 0.001). RET rearrangements were associated with a higher count of tumour-affected lymph nodes (OR 7950.9, 233.7 to 270495.7, P < 0.001) but did not influence distant metastasis or radioiodine-refractory disease. CONCLUSIONS: Papillary thyroid carcinoma with concomitant BRAF-V600E and TERT promoter mutations demonstrated an aggressive course of disease, suggesting the need for a more extensive surgical strategy. RET rearrangement-positive papillary thyroid carcinoma did not affect the clinical outcome, potentially obviating the need for prophylactic lymphadenectomy.

Combination of crossflow ultrafiltration, monolithic affinity filtration, and quantitative reverse transcriptase PCR for rapid concentration and quantification of model viruses in water.

We present a rapid and effective adsorption-elution method based on monolithic affinity filtration (MAF) for the concentration and purification of waterborne viruses. The MAF column consists of a hydrolyzed macroporous epoxy-based polymer. High recoveries were achieved by columns for the bacterial virus (bacteriophage) MS2 110 (±19)%, as model organism, as well as for human adenoviruses 42.4 (±3.4)% and murine noroviruses 42.6 (±1.9)%. This new concentration and purification method was combined with crossflow ultrafiltration (CUF). Because of the adsorption of the examined viruses to the macroporous surface of the MAF column at pH 3, concentrated matrix components by CUF can be removed. Bacteriophages MS2 were spiked in tap water and concentrated with the new CUF-MAF concentration method by a volumetric factor of 10(4) within 33 min. Furthermore, the detection limit for quantification of bacteriophage MS2 by quantitative reverse transcriptase PCR (qRT-PCR) could be improved from 79.47 to 0.0056 GU mL(-1) by a factor of 1.4 × 10(4). In a first study, we have shown that this method could also be applied for river water containing naturally MS2 and MS2-like phages.