Structure-function analysis of USP1: insights into the role of Ser313 phosphorylation site and the effect of cancer-associated mutations on autocleavage

Background: In complex with its cofactor UAF1, the USP1 deubiquitinase plays an important role in cellular processes related to cancer, including the response to DNA damage. The USP1/UAF1 complex is emerging as a novel target in cancer therapy, but several aspects of its function and regulation remain to be further clarified. These include the role of the serine 313 phosphorylation site, the relative contribution of different USP1 sequence motifs to UAF1 binding, and the potential effect of cancer-associated mutations on USP1 regulation by autocleavage. Methods: We have generated a large set of USP1 structural variants, including a catalytically inactive form (C90S), non-phosphorylatable (S313A) and phosphomimetic (S313D) mutants, deletion mutants lacking potential UAF1 binding sites, a mutant (GG/AA) unable to undergo autocleavage at the well-characterized G670/G671 diglycine motif, and four USP1 mutants identified in tumor samples that cluster around this cleavage site (G667A, L669P, K673T and A676T). Using cell-based assays, we have determined the ability of these mutants to bind UAF1, to reverse DNA damage-induced monoubiquitination of PCNA, and to undergo autocleavage. Results: A non-phosphorylatable S313A mutant of USP1 retained the ability to bind UAF1 and to reverse PCNA ubiquitination in cell-based assays. Regardless of the presence of a phosphomimetic S313D mutation, deletion of USP1 fragment 420-520 disrupted UAF1 binding, as determined using a nuclear relocation assay. The UAF1 binding site in a second UAF1-interacting DUB, USP46, was mapped to a region homologous to USP1(420-520). Regarding USP1 autocleavage, co-expression of the C90S and GG/AA mutants did not result in cleavage, while the cancer-associated mutation L669P was found to reduce cleavage efficiency. Conclusions: USP1 phosphorylation at S313 is not critical for PCNA deubiquitination, neither for binding to UAF1 in a cellular environment. In this context, USP1 amino acid motif 420-520 is necessary and sufficient for UAF1 binding. This motif, and a homologous amino acid segment that mediates USP46 binding to UAF1, map to the Fingers sub-domain of these DUBs. On the other hand, our results support the view that USP1 autocleavage may occur in cis, and can be altered by a cancer-associated mutation.

Avaliação da função pulmonar em indivíduos obesos ou com sobrepeso e síndrome metabólica antes, três e seis meses após a colocação de balão intragástrico

Neste trabalho, buscou-se avaliar se o uso do balão intragástrico (BI) durante um período de seis meses por pacientes obesos ou com sobrepeso e com síndrome metabólica (SM) traz melhora nos parâmetros de função pulmonar, distribuição da gordura corporal e SM. Trata-se de um estudo longitudinal e intervencionista com indivíduos adultos que foram submetidos à avaliação antropométrica, da bioquímica sérica, dos parâmetros de função pulmonar e do padrão de distribuição da gordura corporal, antes da instalação do BI, durante o acompanhamento de seis meses e após a sua retirada. Nos dados obtidos três meses após a colocação do BI, os pacientes apresentaram aumento da capacidade de difusão ao monóxido de carbono com correlação positiva entre esta e o percentual total de gordura corporal (rs=0,39; p=0,05), o padrão ginoide (rs=0,41; p=0,05) e o padrão torácico (rs=0,42; p=0,01). Também foi observado que, após três meses da colocação do BI, houve redução significativa do índice de massa corpórea (IMC) (p=0,0001) e da força muscular inspiratória (p=0,009). Também houve aumento significativo da capacidade vital forçada (CVF) (p=0,0001), da capacidade pulmonar total (CPT) (p=0,001) e do volume de reserva expiratório (VRE) (p=0,0001). Ao fim do estudo, foi observada elevação estatisticamente significante da CPT (p=0,0001), capacidade residual funcional (p=0,0001), volume residual (VR) (p=0,0005) e VRE (p=0,0001). Também foi observada redução significativa do IMC, cuja mediana passou de 39,1 kg/m2 no início da avaliação para 34,5 kg/m2 no final dos seis meses (p=0,0001). Ao fim do estudo, 31 pacientes (77,5%) não apresentavam mais critérios diagnósticos para SM. Em relação aos parâmetros de distribuição da gordura corporal, também houve mudanças importantes com redução significante (p=0,0001) do percentual de gordura nos quatro padrões analisados (tronco, androide, ginoide e total). Houve correlação significante entre o delta da CPT e o delta da circunferência abdominal (ρ=-0,34; p=0,03), entre o delta da CRF e o delta do IMC (ρ=-0,39; p=0,01) e entre o delta do VRE e os deltas do IMC (ρ=-0,44; p=0,005) e do colesterol HDL (ρ=-0,37; p=0,02). Também houve correlação significante entre o delta do VRE com os deltas das gorduras de tronco (ρ=-0,51; p=0,004), androide (ρ=-0,46; p=0,01), ginoide (ρ=-0,55; p=0,001) e total (ρ=-0,59; p=0,0005).

The effect of work processes on the casing heat transfer of a transonic turbine

This paper considers the effect of the rotor tip on the casing heat load of a transonic axial flow turbine. The aim of the research is to understand the dominant causes of casing heat-transfer. Experimental measurements were conducted at engine-representative Mach number, Reynolds number and stage inlet to casing wall temperature ratio. Time-resolved heat-transfer coefficient and gas recovery temperature on the casing were measured using an array of heat-transfer gauges. Time-resolved static pressure on the casing wall was measured using Kulite pressure transducers. Time-resolved numerical simulations were undertaken to aid understanding of the mechanism responsible for casing heat load. The results show that between 35% and 60% axial chord the rotor tip-leakage flow is responsible for more than 50% of casing heat transfer. The effects of both gas recovery temperature and heat transfer coefficient were investigated separately and it is shown that an increased stagnation temperature in the rotor tip gap dominates casing heat-transfer. In the tip gap the stagnation temperature is shown to rise above that found at stage inlet (combustor exit) by as much as 35% of stage total temperature drop. The rise in stagnation temperature is caused by an isentropic work input to the tip-leakage fluid by the rotor. The size of this mechanism is investigated by computationally tracking fluid path-lines through the rotor tip gap to understand the unsteady work processes that occur. Copyright © 2005 by ASME.

Salinity measurements and use of the Practical Salinity Scale (PSS)

Salinity, temperature and pressure are parameters which govern the oceanographic state of a marine water body and together they make up density of seawater. In this contribution we will focus our interest on one of these parameters, the salinity: accuracy in relation to different purposes as well as observation technique and instrumentation. We will also discuss the definition of salinity. For example most of the Indian Ocean waters are within the salinity range from 34.60-34.80, which emphasize the importance of careful observations and clear definitions of salinity, in such a way that it is possible to define water masses and predict their movements. In coastal waters the salinity usually features much larger variation in time and space and thus less accuracy is sometimes needed. Salinity has been measured and defined in several ways over the past century. While early measurements were based on the amount of salt in a sea water sample, today the salinity of seawater is most often determined from its conductivity. As conductivity is a function of salinity and temperature, determination involves also measurement of the density of seawater is now more precisely estimated and thus the temperature. As a result of this method the Practical Salinity Scale (PSS) was developed. The best determination of salinity from conductivity and the temperature measurements gives salinity with resolution of 0.001 psu, while the accuracy of titration method was about ± 0.02‰. Because of that, even calculation of movements in the ocean is also improved.

Evolution of conserved secondary structures and their function in transcriptional regulation networks

Background: Many conserved secondary structures have been identified within conserved elements in the human genome, but only a small fraction of them are known to be functional RNAs. The evolutionary variations of these conserved secondary structures in h

Phenotype and Function of Monocyte-Derived Dendritic Cells from Chinese Rhesus Macaques

Dendritic cells (DCs) play a pivotal role in linking the innate immunity and acquired immunity in responses to pathogen. Non-human primates such as Chinese Rhesus Macaque (CRM) are the favorable models for preclinical study of potential therapeutic drugs,

Contact and channel modelling using part and function libraries in a function-based design approach

To support the development and analysis of engineering designs at the embodiment stage, designers work iteratively with representations of those designs as they consider the function and form of their constituent parts. Detailed descriptions of "what a machine does" usually include flows of forces and active principles within the technical system, and their localization within parts and across the interfaces between them. This means that a representation should assist a designer in considering form and function at the same time and at different levels of abstraction. This paper describes a design modelling approach that enables designers to break down a system architecture into its subsystems and parts, while assigning functions and flows to parts and the interfaces between them. In turn, this may reveal further requirements to fulfil functions in order to complete the design. The approach is implemented in a software tool which provides a uniform, computable language allowing the user to describe functions and flows as they are iteratively discovered, created and embodied. A database of parts allows the user to search for existing design solutions. The approach is illustrated through an example: modelling the complex mechanisms within a humanoid robot. Copyright © 2010 by ASME.

Measurements of triggering and transient growth in a model lean-premixed gas turbine combustor

Instability triggering and transient growth of thermoacoustic oscillations were experimentally investigated in combination with linear/nonlinear flame transfer function (FTF) methodology in a model lean-premixed gas turbine combustor operated with CH 4 and air at atmospheric pressure. A fully premixed flame with 10kW thermal power and an equivalence ratio of 0.60 was chosen for detailed characterization of the nonlinear transient behaviors. Flame transfer functions were experimentally determined by simultaneous measurements of inlet velocity fluctuations and heat release rate oscillations using a constant temperature anemometer and OH */CH * chemiluminescence emissions, respectively. The phase-resolved variation of the local flame structure at a limit cycle was measured by planar laser-induced fluorescence of OH. Simultaneous measurements of inlet velocity, OH */CH * emission, and acoustic pressure were performed to investigate the temporal evolution of the system from a stable to a limit cycle operation. This measurement allows us to describe an unsteady instability triggering event in terms of several distinct stages: (i) initiation of a small perturbation, (ii) exponential amplification, (iii) saturation, (iv) nonlinear evolution of the perturbations towards a new unstable periodic state, (v) quasi-steady low-amplitude periodic oscillation, and (vi) fully-developed high-amplitude limit cycle oscillation. Phase-plane portraits of instantaneous inlet velocity and heat release rate clearly show the presence of two different attractors. Depending on its initial position in phase space at infinitesimally small amplitude, the system evolves towards either a high-amplitude oscillatory state or a low-amplitude oscillatory state. This transient phenomenon was analyzed using frequency- and amplitude-dependent damping mechanisms, and compared to subcritical and supercritical bifurcation theories. The results presented in this paper experimentally demonstrate the hypothesis proposed by Preetham et al. based on analytical and computational solutions of the nonlinear G-equation [J. Propul. Power 24 (2008) 1390-1402]. Good quantitative agreement was obtained between measurements and predictions in terms of the conditions for the onset of triggering and the amplitude of triggered combustion instabilities. © 2011 The Combustion Institute.

Cinematographic OH-PLIF measurements of two interacting turbulent premixed flames with and without acoustic forcing

This paper describes an experimental investigation into the interactions that occur between two lean turbulent premixed flames stabilised on conical bluff-bodies when they are moved closer together. Cinematographic OH-PLIF measurements were acquired to investigate adjacent flame front interactions as a function of flame separation distance (S). Flame surface density (FSD) and curvature were determined to characterise the unforced flames. Acoustic forcing was then applied to explore the amplitude dependent thermo-acoustic response. Phase-averaged FSD and global heat release measurements in the form of OH * chemiluminescence were obtained for a range of forcing frequencies (f) and amplitudes (A) as a function of S. As the flames were brought closer together the adjacent annular jets were found to merge into a single jet structure. This caused adjacent flame fronts to merge above the wake region between the two flames at a location determined by the jet efflux (flame angle) and S. This region of flame-flame interaction we refer to as 'interacting region'. In the unforced flames, a trend of increasingly negative curvature for decreasing S produced a small net increase in flame surface area via cusp formation. When subjected to acoustic forcing, S-dependent regimes were found in the global heat release response as a function A. The overall trend showed that the occurrence of jet/flame merging reduces the value of A at which non-linear response occurs. In support of previous findings for flames stabilised along shear layers, the phase-averaged FSD showed that the flame dynamics that drive the thermo-acoustic response result from the roll-up of vortices which generate large-scale vortex-flame interactions. Compared with axisymmetric flames, the occurrence of jet merging alters the vortex-flame interactions resulting in an asymmetric contribution to the heat release between the wall and interacting regions. The majority of the heat release was found to occur in the interacting region through the rapid production and destruction of flame surface area. The occurrence of jet merging and large-scale interactions between adjacent flames result in different physical mechanisms that drive the thermo-acoustic response compared with single axisymmetric flames. © 2011.

Imaging the femoral cortex: thickness, density and mass from clinical CT.

There is growing evidence that focal thinning of cortical bone in the proximal femur may predispose a hip to fracture. Detecting such defects in clinical CT is challenging, since cortices may be significantly thinner than the imaging system's point spread function. We recently proposed a model-fitting technique to measure sub-millimetre cortices, an ill-posed problem which was regularized by assuming a specific, fixed value for the cortical density. In this paper, we develop the work further by proposing and evaluating a more rigorous method for estimating the constant cortical density, and extend the paradigm to encompass the mapping of cortical mass (mineral mg/cm(2)) in addition to thickness. Density, thickness and mass estimates are evaluated on sixteen cadaveric femurs, with high resolution measurements from a micro-CT scanner providing the gold standard. The results demonstrate robust, accurate measurement of peak cortical density and cortical mass. Cortical thickness errors are confined to regions of thin cortex and are bounded by the extent to which the local density deviates from the peak, averaging 20% for 0.5mm cortex.

Imaging the femoral cortex: Thickness, density and mass from clinical CT

There is growing evidence that focal thinning of cortical bone in the proximal femur may predispose a hip to fracture. Detecting such defects in clinical CT is challenging, since cortices may be significantly thinner than the imaging system's point spread function. We recently proposed a model-fitting technique to measure sub-millimetre cortices, an ill-posed problem which was regularized by assuming a specific, fixed value for the cortical density. In this paper, we develop the work further by proposing and evaluating a more rigorous method for estimating the constant cortical density, and extend the paradigm to encompass the mapping of cortical mass (mineral mg/cm 2) in addition to thickness. Density, thickness and mass estimates are evaluated on sixteen cadaveric femurs, with high resolution measurements from a micro-CT scanner providing the gold standard. The results demonstrate robust, accurate measurement of peak cortical density and cortical mass. Cortical thickness errors are confined to regions of thin cortex and are bounded by the extent to which the local density deviates from the peak, averaging 20% for 0.5mm cortex. © 2012 Elsevier B.V.

Robust F0 estimation based on a multi-microphone periodicity function for distant-talking speech

This work addresses the problem of deriving F0 from distanttalking speech signals acquired by a microphone network. The method here proposed exploits the redundancy across the channels by jointly processing the different signals. To this purpose, a multi-microphone periodicity function is derived from the magnitude spectrum of all the channels. This function allows to estimate F0 reliably, even under reverberant conditions, without the need of any post-processing or smoothing technique. Experiments, conducted on real data, showed that the proposed frequency-domain algorithm is more suitable than other time-domain based ones.

Magnetophonon resonance in graphite: High-field Raman measurements and electron-phonon coupling contributions

We perform Raman scattering experiments on natural graphite in magnetic fields up to 45 T, observing a series of peaks due to interband electronic excitations over a much broader magnetic field range than previously reported. We also explore electron-phonon coupling in graphite via magnetophonon resonances. The Raman G peak shifts and splits as a function of magnetic field, due to the magnetically tuned coupling of the E 2g optical phonons with the K- and H-point inter-Landau-level excitations. The analysis of the observed anticrossing behavior allows us to determine the electron-phonon coupling for both K- and H-point carriers. In the highest field range (>35 T) the G peak narrows due to suppression of electron-phonon interaction. © 2012 American Physical Society.

The structure of turbulent stratified and premixed methane/air flames I: Non-swirling flows

A series of flames in a turbulent methane/air stratified swirl burner is presented. The degree of stratification and swirl are systematically varied to generate a matrix of experimental conditions, allowing their separate and combined effects to be investigated. Non-swirling flows are considered in the present paper, and the effects of swirl are considered in a companion paper (Part II). A mean equivalence ratio of φ=0.75 is used, with φ for the highest level of stratification spanning 0.375-1.125. The burner features a central bluff-body to aid flame stabilization, and the influence of the induced recirculation zone is also considered. The current work focuses on non-swirling flows where two-component particle image velocimetry (PIV) measurements are sufficient to characterize the main features of the flow field. Scalar data obtained from Rayleigh/Raman/CO laser induced fluorescence (CO-LIF) line measurements at 103μm resolution allow the behavior of key combustion species-CH 4, CO 2, CO, H 2, H 2O and O 2-to be probed within the instantaneous flame front. Simultaneous cross-planar OH-PLIF is used to determine the orientation of the instantaneous flame normal in the scalar measurement window, allowing gradients in temperature and progress variable to be angle corrected to their three dimensional values. The relationship between curvature and flame thickness is investigated using the OH-PLIF images, as well as the effect of stratification on curvature.The main findings are that the behavior of the key combustion species in temperature space is well captured on the mean by laminar flame calculations regardless of the level of stratification. H 2 and CO are significant exceptions, both appearing at elevated levels in the stratified flames. Values for surface density function and by extension thermal scalar dissipation rate are found to be substantially lower than laminar values, as the thickening of the flame due to turbulence dominates the effect of increased strain. These findings hold for both premixed and stratified flames. The current series of flames is proposed as an interesting if challenging set of test cases for existing and emerging turbulent flame models, and data are available on request. © 2012 The Combustion Institute.

Experimental investigation of turbine stator well rim seal, reingestion and interstage seal flows using gas concentration techniques and displacement measurements

Gas turbine engine performance requires effective and reliable internal cooling over the duty cycle of the engine. Life predictions for rotating components subject to the main gas path temperatures are vital. This demands increased precision in the specification of the internal air system flows which provide turbine stator well cooling and sealing. This in turn requires detailed knowledge of the flow rates through rim seals and interstage labyrinth seals. Knowledge of seal movement and clearances at operating temperatures is of great importance when prescribing these flows. A test facility has been developed at the University of Sussex, incorporating a two stage turbine rated at 400 kW with an individual stage pressure ratio of 1.7:1. The mechanical design of the test facility allows internal cooling geometry to be rapidly re-configured, while cooling flow rates of between 0.71 CW, ENT and 1.46 C W, ENT, may be set to allow ingress or egress dominated cavity flows. The main annulus and cavity conditions correspond to in cavity rotational Reynolds numbers of 1.71×106< Reφ <1.93×106. Displacement sensors have been used to establish hot running seal clearances over a range of stator well flow conditions, allowing realistic flow rates to be calculated. Additionally, gas seeding techniques have been developed, where stator well and main annulus flow interactions are evaluated by measuring changes in gas concentration. Experiments have been performed which allow rim seal and re-ingestion flows to be quantified. It will be shown that this work develops the measurement of stator well cooling flows and provides data suitable for the validation of improved thermo-mechanical and CFD codes, beneficial to the engine design process. Copyright © 2011 by Rolls-Royce plc.