Synthetic Compound Libraries Displayed on the Surface of Encoded Bacteriophage

AbstractWe describe a technology for attaching libraries of synthetic compounds to coat proteins of bacteriophage particles such that the identity of the chemical structure is encoded in the genome of the phage, analogous to peptides displayed on phage surfaces by conventional phage-display techniques. This format allows a library of synthetic compounds to be screened very efficiently as a single pool. Encoded phage serve as extremely robust reporters of the presence of each compound, providing exquisite sensitivity for identification of active compounds engaged in complex biological processes such as receptor-mediated endocytosis and transcytosis. To evaluate this approach, we constructed a library of 980 analogs of folic acid displayed on T7 phage, and demonstrated rapid identification of compounds that bind to folate receptor and direct endocytosis of associated phage particles into cells that express the targeted receptor

Brf1 loss and not overexpression disrupts tissues homeostasis in the intestine, liver and pancreas

RNA polymerase III (Pol-III) transcribes tRNAs and other small RNAs essential for protein synthesis and cell growth. Pol-III is deregulated during carcinogenesis; however, its role in vivo has not been studied. To address this issue, we manipulated levels of Brf1, a Pol-III transcription factor that is essential for recruitment of Pol-III holoenzyme at tRNA genes in vivo. Knockout of Brf1 led to embryonic lethality at blastocyst stage. In contrast, heterozygous Brf1 mice were viable, fertile and of a normal size. Conditional deletion of Brf1 in gastrointestinal epithelial tissues, intestine, liver and pancreas, was incompatible with organ homeostasis. Deletion of Brf1 in adult intestine and liver induced apoptosis. However, Brf1 heterozygosity neither had gross effects in these epithelia nor did it modify tumorigenesis in the intestine or pancreas. Overexpression of BRF1 rescued the phenotypes of Brf1 deletion in intestine and liver but was unable to initiate tumorigenesis. Thus, Brf1 and Pol-III activity are absolutely essential for normal homeostasis during development and in adult epithelia. However, Brf1 overexpression or heterozygosity are unable to modify tumorigenesis, suggesting a permissive, but not driving role for Brf1 in the development of epithelial cancers of the pancreas and gut

An evolutionary approach to systematic discovery of novel deubiquitinases, applied to Legionella

Deubiquitinating enzymes (DUBs) are important regulators of the posttranslational protein ubiquitination system. Mammalian genomes encode about 100 different DUBs, which can be grouped into seven different classes. Members of other DUB classes are found in pathogenic bacteria, which use them to target the host defense. By combining bioinformatical and experimental approaches, we address the question if the known DUB families have a common evolutionary ancestry and share conserved features that set them apart from other proteases. By systematically comparing family-specific hidden Markov models, we uncovered distant relationships between established DUBs and other cysteine protease families. Most DUB families share a conserved aromatic residue linked to the active site, which restricts the cleavage of substrates with side chains at the S2 position, corresponding to Gly-75 in ubiquitin. By applying these criteria to Legionella pneumophila ORFs, we identified lpg1621 and lpg1148 as deubiquitinases, characterized their cleavage specificities, and confirmed the importance of the aromatic gatekeeper motif for substrate selection

A family of unconventional deubiquitinases with modular chain specificity determinants

Deubiquitinating enzymes (DUBs) regulate ubiquitin signaling by trimming ubiquitin chains or removing ubiquitin from modified substrates. Similar activities exist for ubiquitin-related modifiers, although the enzymes involved are usually not related. Here, we report human ZUFSP (also known as ZUP1 and C6orf113) and fission yeast Mug105 as founding members of a DUB family different from the six known DUB classes. The crystal structure of human ZUFSP in covalent complex with propargylated ubiquitin shows that the DUB family shares a fold with UFM1- and Atg8-specific proteases, but uses a different active site more similar to canonical DUB enzymes. ZUFSP family members differ widely in linkage specificity through differential use of modular ubiquitin-binding domains (UBDs). While the minimalistic Mug105 prefers K48 chains, ZUFSP uses multiple UBDs for its K63-specific endo-DUB activity. K63 specificity, localization, and protein interaction network suggest a role for ZUFSP in DNA damage response

MIPAS IMK/IAA CFC-11 (CCl3F) and CFC-12 (CCl2F2) measurements: accuracy, precision and long-term stability

Profiles of CFC-11 (CCl3F) and CFC-12 (CCl2F2) of the Michelson Interferometer for Passive Atmospheric Sounding (MIPAS) aboard the European satellite Envisat have been retrieved from versions MIPAS/4.61 to MIPAS/4.62 and MIPAS/5.02 to MIPAS/5.06 level-1b data using the scientific level-2 processor run by Karlsruhe Institute of Technology (KIT), Institute of Meteorology and Climate Research (IMK) and Consejo Superior de Investigaciones Científicas (CSIC), Instituto de Astrofísica de Andalucía (IAA). These profiles have been compared to measurements taken by the balloon-borne cryosampler, Mark IV (MkIV) and MIPAS-Balloon (MIPAS-B), the airborne MIPAS-STRatospheric aircraft (MIPAS-STR), the satellite-borne Atmospheric Chemistry Experiment Fourier transform spectrometer (ACE-FTS) and the High Resolution Dynamic Limb Sounder (HIRDLS), as well as the ground-based Halocarbon and other Atmospheric Trace Species (HATS) network for the reduced spectral resolution period (RR: January 2005–April 2012) of MIPAS. ACE-FTS, MkIV and HATS also provide measurements during the high spectral resolution period (full resolution, FR: July 2002–March 2004) and were used to validate MIPAS CFC-11 and CFC-12 products during that time, as well as profiles from the Improved Limb Atmospheric Spectrometer, ILAS-II. In general, we find that MIPAS shows slightly higher values for CFC-11 at the lower end of the profiles (below  ∼  15 km) and in a comparison of HATS ground-based data and MIPAS measurements at 3 km below the tropopause. Differences range from approximately 10 to 50 pptv ( ∼  5–20 %) during the RR period. In general, differences are slightly smaller for the FR period. An indication of a slight high bias at the lower end of the profile exists for CFC-12 as well, but this bias is far less pronounced than for CFC-11 and is not as obvious in the relative differences between MIPAS and any of the comparison instruments. Differences at the lower end of the profile (below  ∼  15 km) and in the comparison of HATS and MIPAS measurements taken at 3 km below the tropopause mainly stay within 10–50 pptv (corresponding to  ∼  2–10 % for CFC-12) for the RR and the FR period. Between  ∼  15 and 30 km, most comparisons agree within 10–20 pptv (10–20 %), apart from ILAS-II, which shows large differences above  ∼  17 km. Overall, relative differences are usually smaller for CFC-12 than for CFC-11. For both species – CFC-11 and CFC-12 – we find that differences at the lower end of the profile tend to be larger at higher latitudes than in tropical and subtropical regions. In addition, MIPAS profiles have a maximum in their mixing ratio around the tropopause, which is most obvious in tropical mean profiles. Comparisons of the standard deviation in a quiescent atmosphere (polar summer) show that only the CFC-12 FR error budget can fully explain the observed variability, while for the other products (CFC-11 FR and RR and CFC-12 RR) only two-thirds to three-quarters can be explained. Investigations regarding the temporal stability show very small negative drifts in MIPAS CFC-11 measurements. These instrument drifts vary between  ∼  1 and 3 % decade−1. For CFC-12, the drifts are also negative and close to zero up to  ∼  30 km. Above that altitude, larger drifts of up to  ∼  50 % decade−1 appear which are negative up to  ∼  35 km and positive, but of a similar magnitude, above

Integrated Genomic Characterization of Papillary Thyroid Carcinoma

Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer. Here, we describe the genomic landscape of 496 PTCs. We observed a low frequency of somatic alterations (relative to other carcinomas) and extended the set of known PTC driver alterations to include EIF1AX, PPM1D, and CHEK2 and diverse gene fusions. These discoveries reduced the fraction of PTC cases with unknown oncogenic driver from 25% to 3.5%. Combined analyses of genomic variants, gene expression, and methylation demonstrated that different driver groups lead to different pathologies with distinct signaling and differentiation characteristics. Similarly, we identified distinct molecular subgroups of BRAF-mutant tumors, and multidimensional analyses highlighted a potential involvement of onco-miRs in less-differentiated subgroups. Our results propose a reclassification of thyroid cancers into molecular subtypes that better reflect their underlying signaling and differentiation properties, which has the potential to improve their pathological classification and better inform the management of the diseaseclose6