Metabolic turnover and dynamics of modified ribonucleosides by ¹³C labeling

Tandem mass spectrometry (MS/MS) is an accurate tool to assess modified ribonucleosides and their dynamics in mammalian cells. However, MS/MS quantification of lowly abundant modifications in non-ribosomal RNAs is unreliable, and the dynamic features of various modifications poorly understood. Here, we developed a 13C labeling approach, called 13C-dynamods, to quantify the turnover of base modifications in newly transcribed RNA. This turnover-based approach helped to resolve mRNA from ncRNA modifications in purified RNA or free ribonucleoside samples, and showed the distinct kinetics of the N6-methyladenosine (m6A) versus 7-methylguanosine (m7G) modification in polyA+-purified RNA. We uncovered that N6,N6-dimethyladenosine (m62A) exhibits distinct turnover in small RNAs and free ribonucleosides when compared to known m62A-modified large rRNAs. Finally, combined measurements of turnover and abundance of these modifications informed on the transcriptional versus posttranscriptional sensitivity of modified ncRNAs and mRNAs, respectively, to stress conditions. Thus, 13C-dynamods enables studies of the origin of modified RNAs at steady-state and subsequent dynamics under non-stationary conditions. These results open new directions to probe the presence and biological regulation of modifications in particular RNAs

3D Thermal and CFD Simulations of the Divertor Magnetic Coils for ITER

Magnetic diagnostics for the new generation fusion reactor “ITER” are required to be extremely reliable since they provide measurements essential for reactor operation and protection, plasma control and for measurement of several parameters fundamental to the plasma operation, such as plasma current and shape, disruptions and high frequency macro instabilities. ITER magnetic diagnostics consist of various sets of inductive coils and loops mounted on the inner wall, outside the vacuum vessel and in some of the divertor cassettes [1]. All these probes measure magnetic field or flux variations with respect to time, requiring a precise integration of the signals to recover the absolute values of the field components. They operate in a harsh reactor environment, subjected to nuclear heat loads mainly due to the neutron radiation, generated by the burning plasma. Difficult or impossible access after assembly requires reliability, especially in the area of wiring, connections and vacuum feed-throughs and in choosing margin against radiation damage and extreme transient electrical loads. Additional disturbing effects can arise when both a strong transient magnetic field and thermal gradient occur within the coil structure. All these aspects set a serial of strict design requirements and imply a serious technical challenge. This paper is focused on the design, simulation and optimization of the ITER divertor magnetic tangential coils. The divertor is one of the components exposed to the highest heat load in a fusion reactor, with a surface thermal peak load of 20 MW/m2. About 15 % of the energy produced by fusion reactions is absorbed in the divertor region. The radially-oriented divertor cassettes are exposed to inhomogeneous and time-dependent neutron flux. Six similar divertor cassettes are instrumented for magnetic measurements. Six pairs of equilibrium coils (normal and tangential to the mounting surface) are mounted within each of these cassettes. Of those, pairs near the top region of divertor dome will be exposed to the highest nuclear heating of all magnetic sensors, 2.5 MW/m3. The most critical issue for the divertor coils is to minimise Radiation Induced Thermo-Electric Sensitivity (RITES) [2] and Thermally Induced Electromagnetic Force (TIEMF) [3] by combining a proper choice of conductor with low temperature variation in the coil. Instead of Mineral Insulated Cable (MIC), which was foreseen as the preferred winding for the magnetic coils, a winding made of ceramic-coated steel wire was recently proposed [4]. It is thought that, for this wire, maintaining a temperature variation in the wiring below 10K will be sufficient to allow long-pulse operation. Variations of the divertor coil design have been investigated and simulated with the help of ANSYS programme. The aim was to keep the temperature variation in the winding pack within this limit. The optimisation of the coil, based only on a cooling by conduction was not sufficient to meet the 10 K target. Therefore, an actively water cooled coil was designed and simulated by the CFD code – ANSYS CFX

Cyclin D mediates tolerance of genome-doubling in cancers with functional p53

BACKGROUND: Aneuploidy and chromosomal instability (CIN) are common features of human malignancy that fuel genetic heterogeneity. Although tolerance to tetraploidization, an intermediate state that further exacerbates CIN, is frequently mediated by TP53 dysfunction, we find that some genome-doubled tumours retain wild-type TP53. We sought to understand how tetraploid cells with a functional p53/p21-axis tolerate genome-doubling events. METHOD: We performed quantitative proteomics in a diploid/tetraploid pair within a system of multiple independently derived TP53 wild-type tetraploid clones arising spontaneously from a diploid progenitor. We characterized adapted and acute tetraploidization in a variety of flow cytometry and biochemical assays and tested our findings against human tumours through bioinformatics analysis of the TCGA dataset. RESULTS: Cyclin D1 was found to be specifically overexpressed in early but not late passage tetraploid clones, and this overexpression was sufficient to promote tolerance to spontaneous and pharmacologically induced tetraploidy. We provide evidence that this role extends to D-type cyclins and their overexpression confers specific proliferative advantage to tetraploid cells. We demonstrate that tetraploid clones exhibit elevated levels of functional p53 and p21 but override the p53/p21 checkpoint by elevated expression of cyclin D1, via a stoichiometry-dependent and CDK activity-independent mechanism. Tetraploid cells do not exhibit increased sensitivity to abemaciclib, suggesting that cyclin D-overexpressing tumours might not be specifically amenable to treatment with CDK4/6 inhibitors. CONCLUSION: Our study suggests that D-type cyclin overexpression is an acute event, permissive for rapid adaptation to a genome-doubled state in TP53 wild-type tumours and that its overexpression is dispensable in later stages of tumour progression

Prototyping Conventionally Wound High-Frequency Magnetic Sensors for ITER

The high-frequency (HF) magnetic sensors for ITER are currently based on a conventional, Mirnov-type, pick-up coil, with an effective area in the range 0.03<(NA)EFF[m2]<0.1; the sensor is required to provide measurements of magnetic instabilities with magnitude around |deltaB/BPOL|~10-4 in the 10kHz to 2MHz frequency range. The physical, mechanical and electrical properties of one representative ITER HF pick-up coil design have been analyzed with particular attention to the manufacturing and assembly process for the winding pack, as its integrity was found to be of concern when performing a coupled electro-magnetic, structural and thermal analysis of the sensor. Three different options for the guiding grooves in that design have been tested, using copper and tungsten for the winding pack, but none of them has been convincing enough due to the likelihood of breakages of the thin grooving and of the tungsten wire itself. Hence, alternative designs still based on a conventional Mirnov-type pick-up coil have been explored, and a non-conventional Mirnov-type pick-up coil was produced using direct laser-cutting of a stainless steel hollow tube, avoiding the difficulties encountered during the winding operations for the conventional Mirnov-type sensors. This process manufacturing appears to be acceptable for HF magnetic sensors of Mirnov-type design in ITER, and it is recommended for future prototyping studies, as the effective area of our first prototype (NA)EFF~0.01m2 was well below the ITER requirement. The electrical characteristics and the frequency response of all these prototypes were evaluated up to 8MHz, with the results in good agreement with model calculations. The conventional Mirnov-type prototypes behave as expected in terms of their main electrical properties, and should satisfy the present measurement performance requirements. Finally, a direct measurement of the effective area of these sensors has shown that the geometrical value is a sufficiently correct estimate of its actual value at low frequencies (<10kHz) when the winding pack closely follows the nominal shape of the coil itself

The fidelity of synaptonemal complex assembly is regulated by a signaling mechanism that controls early meiotic progression

© 2014 Elsevier Inc.Proper chromosome segregation during meiosis requires the assembly of the synaptonemal complex (SC) between homologous chromosomes. However, the SC structure itself is indifferent to homology, andpoorly understood mechanisms that depend on conserved HORMA-domain proteins prevent ectopic SC assembly. Although HORMA-domain proteins are thought to regulate SC assembly as intrinsic components of meiotic chromosomes, here we uncover a key role for nuclear soluble HORMA-domain protein HTP-1 in the quality control of SC assembly. We show that a mutant form of HTP-1 impaired in chromosome loading provides functionality of an HTP-1-dependent checkpoint that delays exit from homology search-competent stages until all homolog pairs are linked by the SC. Bypassing of this regulatory mechanism results in premature meiotic progression and licensing of homology-independent SC assembly. These findings identify nuclear soluble HTP-1 as a regulator of early meiotic progression, suggesting parallels with the mode of action of Mad2 in the spindle assembly checkpoint

Identification and biochemical analysis of a novel APOB mutation that causes autosomal dominant hypercholesterolemia

Patients with autosomal dominant hypercholesterolemia (ADH) have a high risk of developing cardiovascular disease that can be effectively treated using statin drugs. Molecular diagnosis and family cascade screening is recommended for early identification of individuals at risk, but up to 40% of families have no mutation detected in known genes. This study combined linkage analysis and exome sequencing to identify a novel variant in exon 3 of APOB (Arg50Trp). Mass spectrometry established that low-density lipoprotein (LDL) containing Arg50Trp APOB accumulates in the circulation of affected individuals, suggesting defective hepatic uptake. Previously reported mutations in APOB causing ADH have been located in exon 26. This is the first report of a mutation outside this region causing this phenotype, therefore, more extensive screening of this large and highly polymorphic gene may be necessary in ADH families. This is now feasible due to the high capacity of recently available sequencing platforms

Baseline System Design And Prototyping For The Iter High-Frequency Magnetic Diagnostics Set

This paper reports the mechanical and electrical tests performed for the prototyping of the ITER high-frequency magnetic sensor and the analysis of the measurement performance of this diagnostic. The current design for the sensor is not suitable for manufacturing for ITER due to the high likelihood of breakages of the un-guided tungsten wire during the winding. A number of alternative designs and manufacturing processes have been investigated, with the Low Temperature Co-fired Ceramic technology giving the best results. The measurement performance of the baseline system design for the high-frequency magnetic diagnostic cannot meet the intended ITER requirements due to its intrinsic spatial periodicities