Annotating Protein Functional Residues by Coupling High-Throughput Fitness Profile and Homologous-Structure Analysis.

Identification and annotation of functional residues are fundamental questions in protein sequence analysis. Sequence and structure conservation provides valuable information to tackle these questions. It is, however, limited by the incomplete sampling of sequence space in natural evolution. Moreover, proteins often have multiple functions, with overlapping sequences that present challenges to accurate annotation of the exact functions of individual residues by conservation-based methods. Using the influenza A virus PB1 protein as an example, we developed a method to systematically identify and annotate functional residues. We used saturation mutagenesis and high-throughput sequencing to measure the replication capacity of single nucleotide mutations across the entire PB1 protein. After predicting protein stability upon mutations, we identified functional PB1 residues that are essential for viral replication. To further annotate the functional residues important to the canonical or noncanonical functions of viral RNA-dependent RNA polymerase (vRdRp), we performed a homologous-structure analysis with 16 different vRdRp structures. We achieved high sensitivity in annotating the known canonical polymerase functional residues. Moreover, we identified a cluster of noncanonical functional residues located in the loop region of the PB1 β-ribbon. We further demonstrated that these residues were important for PB1 protein nuclear import through the interaction with Ran-binding protein 5. In summary, we developed a systematic and sensitive method to identify and annotate functional residues that are not restrained by sequence conservation. Importantly, this method is generally applicable to other proteins about which homologous-structure information is available.ImportanceTo fully comprehend the diverse functions of a protein, it is essential to understand the functionality of individual residues. Current methods are highly dependent on evolutionary sequence conservation, which is usually limited by sampling size. Sequence conservation-based methods are further confounded by structural constraints and multifunctionality of proteins. Here we present a method that can systematically identify and annotate functional residues of a given protein. We used a high-throughput functional profiling platform to identify essential residues. Coupling it with homologous-structure comparison, we were able to annotate multiple functions of proteins. We demonstrated the method with the PB1 protein of influenza A virus and identified novel functional residues in addition to its canonical function as an RNA-dependent RNA polymerase. Not limited to virology, this method is generally applicable to other proteins that can be functionally selected and about which homologous-structure information is available

Exosomes: Versatile Nano Mediators of Immune Regulation.

One of many types of extracellular vesicles (EVs), exosomes are nanovesicle structures that are released by almost all living cells that can perform a wide range of critical biological functions. Exosomes play important roles in both normal and pathological conditions by regulating cell-cell communication in cancer, angiogenesis, cellular differentiation, osteogenesis, and inflammation. Exosomes are stable in vivo and they can regulate biological processes by transferring lipids, proteins, nucleic acids, and even entire signaling pathways through the circulation to cells at distal sites. Recent advances in the identification, production, and purification of exosomes have created opportunities to exploit these structures as novel drug delivery systems, modulators of cell signaling, mediators of antigen presentation, as well as biological targeting agents and diagnostic tools in cancer therapy. This review will examine the functions of immunocyte-derived exosomes and their roles in the immune response under physiological and pathological conditions. The use of immunocyte exosomes in immunotherapy and vaccine development is discussed

A comprehensive functional map of the hepatitis C virus genome provides a resource for probing viral proteins.

UnlabelledPairing high-throughput sequencing technologies with high-throughput mutagenesis enables genome-wide investigations of pathogenic organisms. Knowledge of the specific functions of protein domains encoded by the genome of the hepatitis C virus (HCV), a major human pathogen that contributes to liver disease worldwide, remains limited to insight from small-scale studies. To enhance the capabilities of HCV researchers, we have obtained a high-resolution functional map of the entire viral genome by combining transposon-based insertional mutagenesis with next-generation sequencing. We generated a library of 8,398 mutagenized HCV clones, each containing one 15-nucleotide sequence inserted at a unique genomic position. We passaged this library in hepatic cells, recovered virus pools, and simultaneously assayed the abundance of mutant viruses in each pool by next-generation sequencing. To illustrate the validity of the functional profile, we compared the genetic footprints of viral proteins with previously solved protein structures. Moreover, we show the utility of these genetic footprints in the identification of candidate regions for epitope tag insertion. In a second application, we screened the genetic footprints for phenotypes that reflected defects in later steps of the viral life cycle. We confirmed that viruses with insertions in a region of the nonstructural protein NS4B had a defect in infectivity while maintaining genome replication. Overall, our genome-wide HCV mutant library and the genetic footprints obtained by high-resolution profiling represent valuable new resources for the research community that can direct the attention of investigators toward unidentified roles of individual protein domains.ImportanceOur insertional mutagenesis library provides a resource that illustrates the effects of relatively small insertions on local protein structure and HCV viability. We have also generated complementary resources, including a website (http://hangfei.bol.ucla.edu) and a panel of epitope-tagged mutant viruses that should enhance the research capabilities of investigators studying HCV. Researchers can now detect epitope-tagged viral proteins by established antibodies, which will allow biochemical studies of HCV proteins for which antibodies are not readily available. Furthermore, researchers can now quickly look up genotype-phenotype relationships and base further mechanistic studies on the residue-by-residue information from the functional profile. More broadly, this approach offers a general strategy for the systematic functional characterization of viruses on the genome scale

Obscuration-dependent evolution of Active Galactic Nuclei

We aim to constrain the evolution of AGN as a function of obscuration using an X-ray selected sample of $\sim2000$ AGN from a multi-tiered survey including the CDFS, AEGIS-XD, COSMOS and XMM-XXL fields. The spectra of individual X-ray sources are analysed using a Bayesian methodology with a physically realistic model to infer the posterior distribution of the hydrogen column density and intrinsic X-ray luminosity. We develop a novel non-parametric method which allows us to robustly infer the distribution of the AGN population in X-ray luminosity, redshift and obscuring column density, relying only on minimal smoothness assumptions. Our analysis properly incorporates uncertainties from low count spectra, photometric redshift measurements, association incompleteness and the limited sample size. We find that obscured AGN with $N_{H}>{\rm 10^{22}\, cm^{-2}}$ account for ${77}^{+4}_{-5}\%$ of the number density and luminosity density of the accretion SMBH population with $L_{{\rm X}}>10^{43}\text{ erg/s}$, averaged over cosmic time. Compton-thick AGN account for approximately half the number and luminosity density of the obscured population, and ${38}^{+8}_{-7}\%$ of the total. We also find evidence that the evolution is obscuration-dependent, with the strongest evolution around $N_{H}\thickapprox10^{23}\text{ cm}^{-2}$. We highlight this by measuring the obscured fraction in Compton-thin AGN, which increases towards $z\sim3$, where it is $25\%$ higher than the local value. In contrast the fraction of Compton-thick AGN is consistent with being constant at $\approx35\%$, independent of redshift and accretion luminosity. We discuss our findings in the context of existing models and conclude that the observed evolution is to first order a side-effect of anti-hierarchical growth.Comment: Published in Ap

Planck Sunyaev-Zel'dovich Cluster Mass Calibration using Hyper Suprime-Cam Weak Lensing

Using $\sim$140 deg$^2$ Subaru Hyper Suprime-Cam (HSC) survey data, we stack the weak lensing (WL) signal around five Planck clusters found within the footprint. This yields a 15$\sigma$ detection of the mean Planck cluster mass density profile. The five Planck clusters span a relatively wide mass range, $M_{\rm WL,500c} = (2-30)\times10^{14}\,M_\odot/h$ with a mean mass of $M_{\rm WL,500c} = (4.15\pm0.61)\times10^{14}\,M_\odot/h$. The ratio of the stacked Planck Sunyaev-Zel'dovich (SZ) mass to the stacked WL mass is $\langle M_{\rm SZ}\rangle/\langle M_{\rm WL}\rangle = 1-b = 0.80\pm0.14$. This mass bias is consistent with previous WL mass calibrations of Planck clusters within the errors. We discuss the implications of our findings for the calibration of SZ cluster counts and the much discussed tension between Planck SZ cluster counts and Planck $\Lambda$CDM cosmology.Comment: 12 pages, 2 tables, 7 figures, accepted to PASJ special issu

The Threads of Biosystems Engineering

The core concepts, or threads, of Biosystems Engineering (BSEN) are variously understood by those within the discipline, but have never been unequivocally defined due to its early stage of development. This makes communication and teaching difficult compared to other well established engineering subjects. Biosystems Engineering is a field of Engineering which int egrates engineering science and design with applied biological, environmental and agricultural sciences. It represents an evolution of the Agricultural Engineering discipline applied to all living organisms not including biomedical applications. The basic key element for the emerging EU Biosystems Engineering program of studies is to ensure that it offers essential minimum fundamental engine ering knowledge and competences . A core curriculum developed by Erasmus Thematic Networks is used as benchmark for Agr icultural and Biosystems Engineering studies in Europe. The common basis of the core curriculum for the discipline across the Atlantic , including a minimum of competences comprising the Biosystems Engineering core competencies, has been defined by an Atlan tis project , but this needs to be taken further by defining the threads linking courses together. This paper presents a structured approach to define the Threads of BSEN . The definition of the mid-level competences and the associated learning outcomes has been one of the objectives of the Atlantis programme TABE.NET. The mid-level competences and learning outcomes for each of six specializations of BSEN are defined while the domain-specific knowledge to be acquired for each outcome is proposed. Once the proposed definitions are adopted, these threads will be available for global development of the BSEN

Vertical transport and electroluminescence in InAs/GaSb/InAs structures: GaSb thickness and hydrostatic pressure studies

We have measured the current-voltage (I-V) of type II InAs/GaSb/InAs double heterojunctions (DHETs) with 'GaAs like' interface bonding and GaSb thickness between 0-1200 \AA. A negative differential resistance (NDR) is observed for all DHETs with GaSb thickness $>$ 60 \AA below which a dramatic change in the shape of the I-V and a marked hysteresis is observed. The temperature dependence of the I-V is found to be very strong below this critical GaSb thickness. The I-V characteristics of selected DHETs are also presented under hydrostatic pressures up to 11 kbar. Finally, a mid infra-red electroluminescence is observed at 1 bar with a threshold at the NDR valley bias. The band profile calculations presented in the analysis are markedly different to those given in the literature, and arise due to the positive charge that it is argued will build up in the GaSb layer under bias. We conclude that the dominant conduction mechanism in DHETs is most likely to arise out of an inelastic electron-heavy-hole interaction similar to that observed in single heterojunctions (SHETs) with 'GaAs like' interface bonding, and not out of resonant electron-light-hole tunnelling as proposed by Yu et al. A Zener tunnelling mechanism is shown to contribute to the background current beyond NDR.Comment: 8 pages 12 fig

A practical implementation of the Overlap-Dirac operator

A practical implementation of the Overlap-Dirac operator ${{1+\gamma_5\epsilon(H)}\over 2}$ is presented. The implementation exploits the sparseness of $H$ and does not require full storage. A simple application to parity invariant three dimensional SU(2) gauge theory is carried out to establish that zero modes related to topology are exactly reproduced on the lattice.Comment: Y-axis label in figure correcte

Responses of Multipotent Retinal Stem Cells to IL-1 β

Purpose. To investigate how multipotent retinal stem cells (RSCs) isolated from mice respond to the proinflammatory signaling molecules, IL-1β, IL-18, and IL-17A. Materials and Methods. RSCs were cultured in a specific culture medium and were treated with these cytokines. Cell viability was detected by MTT assay; ultrastructure was evaluated by transmission electron microscopy; expression of IL-17rc and proapoptotic proteins was detected by immunocytochemistry and expression of Il-6 and Il-17a was detected by quantitative RT-PCR. As a comparison, primary mouse retinal pigment epithelium (RPE) cells were also treated with IL-1β, IL-18, or IL-17A and analyzed for the expression of Il-6 and Il-17rc. Results. Treatment with IL-1β, IL-18, or IL-17A decreased RSC viability in a dose-dependent fashion and led to damage in cellular ultrastructure including pyroptotic and/or necroptotic cells. IL-1β and IL-18 could induce proapoptotic protein expression. All treatments induced significantly higher expression of Il-6 and Il-17rc in both cells. However, neither IL-1β nor IL-18 could induce Il-17a expression in RSCs. Conclusions. IL-1β, IL-18, and IL-17A induce retinal cell death via pyroptosis/necroptosis and apoptosis. They also provoke proinflammatory responses in RSCs. Though IL-1β and IL-18 could not induce Il-17a expression in RSCs, they both increase Il-17rc expression, which may mediate the effect of Il-17a