Coordination of Developmental Timing and Maturation by the F-box Protein DRE-1/FBXO11

Spatiotemporal control of cellular events is key to developmental progression in all organisms. Accordingly the abundance of regulatory proteins must be tightly controlled in a highly time- and tissue-specific manner. Pioneering work in the small roundworm Caenorhabditis elegans (C. elegans) has uncovered a network of genes, termed the heterochronic loci, which govern temporal coordination of developmental processes. Importantly this heterochronic network is remarkably conserved across species. There are currently over 30 identified heterochronic loci, which encode various transcriptional, translational and post-translational regulators including the first discovered microRNAs lin-4 and let-7. In 2007 our laboratory discovered a novel heterochronic gene, dre-1, which specifies late larval development in the C. elegans epidermis and gonad. Interestingly dre-1 encodes a highly conserved F-box protein; F-box proteins typically function as substrate recognition components of SCF E3-ubiquitin ligase complexes, thus conferring substrate specificity to the ubiquitin proteasome system (UPS). The initial analysis of DRE-1 and its mammalian homolog FBXO11 revealed their interaction with SCF complex components, suggesting conservation also on the functional level. Mutation of C. elegans dre-1 leads to a diverse array of developmental alterations including precocious terminal differentiation of epidermal blast cells, molting defects and alterations in gonadal outgrowth. Likewise, haploinsuffiency of Fbxo11 has been linked to craniofacial malformations such as cleft palates, as well as otitis media, an inflammation of the middle ear, in both mice and men. Furthermore, FBXO11 was recently shown to function as a tumor suppressor in lymphoid malignancies by controlling turnover of the oncoprotein BCL6. However, the DRE-1/FBXO11 substrate(s) coordinating developmental progression still remain to be elucidated. Thus the central questions of my thesis are: what are the substrates of DRE-1/FBXO11 in the heterochronic circuit, and how do they illuminate developmental timing events? In an effort to identify novel substrates of the SCFDRE-1/FBXO11 E3-ubiquitin ligase complex involved in developmental timing, I pursued an RNAi-based suppressor approach in C. elegans. I reasoned that client substrates should aberrantly accumulate in dre-1 mutants and cause developmental alterations across tissues. If so, then knockdown of such substrates would ameliorate related phenotypes. Here, I identified the highly conserved zinc-finger transcriptional repressor BLMP-1 as a new substrate of the SCFDRE-1 complex. Several lines of evidence reveal that BLMP-1 is a substrate of the SCFDRE-1 complex in developmental timing circuits. First, blmp-1 loss of function strongly suppressed dre-1 heterochronic phenotypes in epidermis and gonad and blmp-1 mutation itself exhibited developmental timing defects opposite to dre-1. blmp-1 also opposed dre-1 for other life history traits such as entry into the dauer diapause and adult longevity. Second, BLMP-1 protein was strikingly elevated upon depletion of dre-1 and other SCF complex components, as well as by inhibition of the proteasome. Moreover, BLMP-1 was regulated in a time- and tissue-specific manner by dre-1, consistent with corresponding phenotypes. Third, DRE-1 and BLMP-1 physically interacted in vivo in C. elegans and in vitro in a cell-based system. Importantly the role of DRE-1 in regulating BLMP-1 stability is evolutionary conserved, as direct protein interaction and degradation function was also observed for the human counterparts FBXO11 and BLIMP-1. In addition to blmp-1, I also investigated genetic interactions of dre-1 and cdt-2 in C. elegans, since CDT2 was recently identified as a FBXO11 substrate in mammalian systems. I found cdt-2 to genetically interact with dre-1 for specific processes in the epidermis. In particular gaps in an adult-specific cuticular structure occurring in dre-1 mutants could arise from elevated CDT-2 levels and a subsequent failure of cell cycle exit of epidermal blast cells, which synthesize these structures. My findings are in line with mammalian studies that established CDT2, which itself is part of an E3-ubiqitin ligase complex, as a critical cell cycle regulator by targeting substrates such as the CDK inhibitor p21 for proteasomal degradation. Altogether I conclude that dre-1 mutants’ diverse developmental alterations arise from aberrant accumulation of distinct target proteins. In particular, my work shows that spatiotemporal control of BLMP-1 protein by DRE-1 coordinates developmental timing and other life history traits in C. elegans. DRE-1, on the one hand, restrains BLMP-1 abundance in the epidermis to prevent precocious terminal differentiation and, on the other hand, triggers BLMP-1 degradation in distal tip cells to initiate dorso-ventral migratory events. The notion that DRE-1/BLMP-1 work together for multiple processes suggests these proteins comprise a two-protein module that may mediate related metazoan maturation processes. Taken together, my work fundamentally contributes to the understanding of developmental timing coordination by DRE-1/FBXO11 in C. elegans and might help to shed light on related processes in higher organisms

Quantification of trace element contents in frozen fluid inclusions by UV-fs-LA-ICP-MS analysis

We have developed a new analytical setup for the determination of trace element concentrations in fluid inclusions by UV-fs-LA-ICP-MS. Laser ablation was performed at a low temperature of -40 degrees C by using a modified heating-freezing stage as the ablation cell. With this method it was possible to successfully analyse 53 of 55 frozen synthetic NaCl-H2O fluid inclusions in quartz, covering a size range between 8 mu m and 25 mu m down to a depth of 50 mu m. The high success rate could be achieved as the 194 nm UV-fs-laser allows excellent control over the opening procedure of frozen fluid inclusions. Trace element analyses were performed with a fast scanning magnetic sector field ICP-MS. The lower limits of detection for fluid inclusion analysis vary from 0.1 mu g g(-1) (for Bi-209) to 10 mu g g(-1) (for K-39). The typical analytical uncertainty, depending on the element and respective concentration level, ranges between 10% and 30% (1RSD), based on the reproducibility of experimentally synthesized fluid inclusions. All elements from a stock solution, which behaved inert during the HP/HT experiments (B, K, Cd, Te, Tl, Pb and Bi), could be recovered in the synthetic inclusions at concentrations that correspond within their specific analytical uncertainties to their original concentration of 53 mu g g(-1). The method represents a highly efficient tool for the determination of accurate trace element data on low concentration levels in small fluid inclusions with a high success rate of >90%. The latter is particularly advantageous considering the commonly time consuming characterization of fluid inclusions.NTH Graduate School GeoFluxe

GenomeRNAi: a database for cell-based RNAi phenotypes

RNA interference (RNAi) has emerged as a powerful tool to generate loss-of-function phenotypes in a variety of organisms. Combined with the sequence information of almost completely annotated genomes, RNAi technologies have opened new avenues to conduct systematic genetic screens for every annotated gene in the genome. As increasing large datasets of RNAi-induced phenotypes become available, an important challenge remains the systematic integration and annotation of functional information. Genome-wide RNAi screens have been performed both in Caenorhabditis elegans and Drosophila for a variety of phenotypes and several RNAi libraries have become available to assess phenotypes for almost every gene in the genome. These screens were performed using different types of assays from visible phenotypes to focused transcriptional readouts and provide a rich data source for functional annotation across different species. The GenomeRNAi database provides access to published RNAi phenotypes obtained from cell-based screens and maps them to their genomic locus, including possible non-specific regions. The database also gives access to sequence information of RNAi probes used in various screens. It can be searched by phenotype, by gene, by RNAi probe or by sequence and is accessible a

Experimental tests on achieving equilibrium in synthetic fluid inclusions: Results for scheelite, molybdenite, and gold solubility at 800 °C and 200 MPa

Synthetic fluid inclusions formed in high P-T experiments, which are subsequently analyzed with LA-ICP-MS, enable us to collect thermodynamic data to constrain metal transport in aqueous fluids as well as partitioning of metals between coexisting phases. The most essential prerequisite for such studies is to ensure that equilibrium conditions between liquid and solid phases are reached prior to the formation of synthetic fluid inclusions in the host mineral. Various methods have been proposed by different authors to achieve this goal, but to this point our knowledge on the best approach to synthesize equilibrated fluid inclusions under constrained pressure, temperature, and compositional (P, T, and X) conditions remains poor. In addition, information on the time needed to reach equilibrium metal concentrations in the fluid as well as on the timing of the onset of fluid inclusion formation in the host mineral are scarce. The latter has been tested in a series of time-dependent experiments at 800 °C and 200 MPa using scheelite (CaWO4), molybdenite (MoS2) and metallic gold as dissolving phases and using different approaches to optimize the formation of equilibrated fluid inclusions. Both Embedded Image and Embedded Image were fixed during all experiments using the pyrite-pyrrhotite-magnetite buffer (PPM). As an intermediate in situ quenching of the sample charge plays an important role in the synthesis of fluid inclusions, we further tested the efficiency of such an intermediate quench for re-opening fluid inclusions formed at 600 °C and 200 MPa. Our results reveal that fluid inclusions start forming almost instantaneously and that equilibrium between fluid and solid phases occurs in the timescale of less than two hours for molybdenite and gold up to ca. 10 h for scheelite. The best approach to synthesize equilibrated fluid inclusions at 800 °C was obtained by using an intermediate quench on a previously unfractured quartz host. Experiments at 600 °C showed similar results and illustrate that this should be the method of choice down to this temperature. Below 600 °C pre-treatment of the quartz host (HF etching and/or thermal fracturing) becomes important to produce large enough fluid inclusions for the analyses via LA-ICP-MS and special care must be taken to prevent premature entrapment of the fluid. Fluids with 8 wt% NaCl in equilibrium with scheelite, molybdenite and gold at 800 °C and 200 MPa have concentrations of ca. 7300 ppm W, 1300 ppm Mo, and 300 ppm Au, respectively, which is in good agreement with results from other studies or extrapolation from lower temperatures. It can be concluded that the formation of synthetic fluid inclusions from an equilibrated fluid is possible, but different experimental designs are required, depending on the investigated temperature. In general, dissolution of solid phases seems to be much faster than previously assumed, so that experimental run durations can be designed considerably shorter, which is of great advantage when using fast-consuming mineral buffers.State of Lower SaxonyGraduate School GeoFluxesLeibniz Universität Hannove

Regulation of the CRL4(Cdt2) ubiquitin ligase and cell-cycle exit by the SCF(Fbxo11) ubiquitin ligase

F-box proteins and DCAF proteins are the substrate binding subunits of the Skp1-Cul1-F-box protein (SCF) and Cul4-RING protein ligase (CRL4) ubiquitin ligase complexes, respectively. Using affinity purification and mass spectrometry, we determined that the F-box protein FBXO11 interacts with CDT2, a DCAF protein that controls cell-cycle progression, and recruits CDT2 to the SCF(FBXO11)complex to promote its proteasomal degradation. In contrast to most SCF substrates, which exhibit phosphodegron-dependent binding to F-box proteins, CDK-mediated phosphorylation of Thr464 present in the CDT2 degron inhibits recognition by FBXO11. Finally, our results show that the functional interaction between FBXO11 and CDT2 is evolutionary conserved from worms to humans and plays an important role in regulating the timing of cell-cycle exit.Fil: Rossi, Mario. University Of New York; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires; ArgentinaFil: Duan, Shanshan. University Of New York; Estados Unidos. Howard Hughes Medical Institute; Estados UnidosFil: Jeong, Yeon Tae. University Of New York; Estados UnidosFil: Horn, Moritz. Max Planck Institute for Biology of Ageing; Alemania. University of Cologne; AlemaniaFil: Saraf, Anita. The Stowers Institute for Medical Research; Estados UnidosFil: Florens, Laurence. The Stowers Institute for Medical Research; Estados UnidosFil: Washburn, Michael P.. The Stowers Institute for Medical Research; Estados Unidos. University of Kansas; Estados UnidosFil: Antebi, Adam. Max Planck Institute for Biology of Ageing; Alemania. University of Cologne; AlemaniaFil: Pagano, Michele. University Of New York; Estados Unidos. Howard Hughes Medical Institute; Estados Unido

Surgical Treatment of Carcinomas of the Oral Minor Salivary Glands : Oncological Outcome in Dependence of Tumor Entity and Therapeutic Strategies

The aim of this study was to analyze the clinical outcomes of three types of minor salivary gland carcinomas (adenoid-cystic carcinomas (ACC), adeno carcinomas not otherwise specified (AC-NOS), and mucoepidermoid carcinomas (MEC)) after primary surgical therapy. A retrospective cohort study was designed and patients with cancer of the minor oral salivary glands treated in our department in the years 2011 to 2022 were included. Clinicopathological data were evaluated to compare overall survival and progression-free survival between the entities. Eighty-one patients were included. The rates of cervical metastases were 38.9% for ACC, 25% for MEC, and 9.1% for AC-NOS. ACC exhibited significantly higher rates of local and systemic disease recurrence (p = 0.02), and the presence of neck node metastases was confirmed as an independent prognostic factor for progressionfree survival (p = 0.014). Treatment success in terms of oncological outcome varied significantly between the different entities and implies different treatment regimens for each tumor entity

SMoTherSpectre: exploiting speculative execution through port contention

Spectre, Meltdown, and related attacks have demonstrated that kernels, hypervisors, trusted execution environments, and browsers are prone to information disclosure through micro-architectural weaknesses. However, it remains unclear as to what extent other applications, in particular those that do not load attacker-provided code, may be impacted. It also remains unclear as to what extent these attacks are reliant on cache-based side channels. We introduce SMoTherSpectre, a speculative code-reuse attack that leverages port-contention in simultaneously multi-threaded processors (SMoTher) as a side channel to leak information from a victim process. SMoTher is a fine-grained side channel that detects contention based on a single victim instruction. To discover real-world gadgets, we describe a methodology and build a tool that locates SMoTher-gadgets in popular libraries. In an evaluation on glibc, we found hundreds of gadgets that can be used to leak information. Finally, we demonstrate proof-of-concept attacks against the OpenSSH server, creating oracles for determining four host key bits, and against an application performing encryption using the OpenSSL library, creating an oracle which can differentiate a bit of the plaintext through gadgets in libcrypto and glibc