Phenotypic lentivirus screens to identify functional single domain antibodies

Manipulation of proteins is key in assessing their in vivo function. Although genetic ablation is straightforward, reversible and specific perturbation of protein function remains a challenge. Single domain antibody fragments, such as camelid-derived VHHs, can serve as inhibitors or activators of intracellular protein function, but functional testing of identified VHHs is laborious. To address this challenge, we have developed a lentiviral screening approach to identify VHHs that elicit a phenotype when expressed intracellularly. We identified 19 antiviral VHHs that protect human A549 cells from lethal infection with influenza A virus (IAV) or vesicular stomatitis virus (VSV), respectively. Both negative-sense RNA viruses are vulnerable to VHHs uniquely specific for their respective nucleoproteins. Antiviral VHHs prevented nuclear import of viral ribonucleoproteins or mRNA transcription, respectively, and may provide clues for novel antiviral reagents. In principle, the screening approach described here should be applicable to identify inhibitors of any pathogen or biological pathway. To identify the proteins essential to a biological pathway, small-molecule inhibitors or activators may be used to manipulate protein function transiently. Alternatively, screens involving mutagenesis, a reduction in levels or complete elimination of gene products are common. As applied to mammalian cells, these methods usually seek to achieve the removal of a protein from its normal biological context. Many proteins are multifunctional, or are components of multi-subunit complexes. Depletion of any single component may cause unexpected phenotypes due to the collapse of entire protein complexes. Small-molecule inhibitors often lack specificity and at best can target a fraction of all the proteins of interest. The screening of chemically diverse libraries must be paired with sophisticated methods to identify the molecular targets of any hit identified. Antibodies have been used as intracellular perturbants of protein function after microinjection or cytosolic expression of single-chain variable antibody fragments, but technical challenges have limited their application to few selected cases. In addition to conventional antibodies, the immune system of camelids generates heavy-chain-only antibodies. Their antigen binding site only consists of the variable domain of the heavy chain. This domain can be expressed on its own and is referred to as a VHH or nanobody, an entity that can retain its function in the reducing environment of the cytosol, independent of glycosylation. Many VHHs bind to their targets with affinities comparable to conventional antibodies. VHHs expressed in the cytosol can therefore act as molecular perturbants by occluding the interfaces involved in protein–protein interactions, by binding in the active sites of enzymes, or through the recognition or stabilization of distinct conformations of their targets. Both phage and yeast display, as well as mass spectrometry in combination with high-throughput sequencing, allow the identification of VHHs based on their binding properties. However, the identification of inhibitory VHHs remains a time-consuming process. VHHs obtained through biochemical screening methods must be expressed individually in the relevant cell type to test for the functional consequences of VHH expression. To address this challenge, we developed a phenotypic VHH screening method in living cells.National Institutes of Health (U.S.) (Health Pioneer Award

Three-dimensional parton distribution functions g1Tg_{1T} and h1L⊥h_{1L}^\perp in the polarized proton-antiproton Drell-Yan process

We present predictions of the unweighted and weighted double spin asymmetries related to the transversal helicity distribution $g_{1T}$ and the longitudinal transversity distribution $h_{1L}^\perp$, two of eight leading-twist transverse momentum dependent parton distributions (TMDs) or three-dimensional parton distribution functions (3dPDFs), in the polarized proton-antiproton Drell-Yan process at typical kinematics on the Facility for Antiproton and Ion Research (FAIR). We conclude that FAIR is ideal to access the new 3dPDFs towards a detailed picture of the nucleon structure.Comment: 6 latex pages, 5 figures, version for publication in EPJ

A precision study of the fine tuning in the DiracNMSSM

Recently the DiracNMSSM has been proposed as a possible solution to reduce the fine tuning in supersymmetry. We determine the degree of fine tuning needed in the DiracNMSSM with and without non-universal gaugino masses and compare it with the fine tuning in the GNMSSM. To apply reasonable cuts on the allowed parameter regions we perform a precise calculation of the Higgs mass. In addition, we include the limits from direct SUSY searches and dark matter abundance. We find that both models are comparable in terms of fine tuning, with the minimal fine tuning in the GNMSSM slightly smaller.Comment: 20 pages + appendices, 10 figure

Mass Suppression in Octet Baryon Production

There is a striking suppression of the cross section for production of octet baryons in $e^+ e ^-$ annihilation, as the mass of the produced hadron increases. We present a simple parametrization for the fragmentation functions into octet baryons guided by two input models: the SU(3) flavor symmetry part is given by a quark-diquark model, and the baryon mass suppression part is inspired by the string model. We need only eight free parameters to describe the fragmentation functions for all octet baryons. These free parameters are determined by a fit to the experimental data of octet baryon production in $e^+ e ^-$ annihilation. Then we apply the obtained fragmentation functions to predict the cross section of the octet baryon production in charged lepton DIS and find consistency with the available experimental data. Furthermore, baryon production in $pp$ collisions is suggested to be an ideal domain to check the predicted mass suppression.Comment: 20 pages, 5 figure

Variation in Tropical Reef Symbiont Metagenomes Defined by Secondary Metabolism

The complex evolution of secondary metabolism is important in biology, drug development, and synthetic biology. To examine this problem at a fine scale, we compared the genomes and chemistry of 24 strains of uncultivated cyanobacteria, Prochloron didemni, that live symbiotically with tropical ascidians and that produce natural products isolated from the animals. Although several animal species were obtained along a >5500 km transect of the Pacific Ocean, P. didemni strains are >97% identical across much of their genomes, with only a few exceptions concentrated in secondary metabolism. Secondary metabolic gene clusters were sporadically present or absent in identical genomic locations with no consistent pattern of co-occurrence. Discrete mutations were observed, leading to new chemicals that we isolated from animals. Functional cassettes encoding diverse chemicals are exchanged among a single population of symbiotic P. didemni that spans the tropical Pacific, providing the host animals with a varying arsenal of secondary metabolites

The Higgs Working Group: Summary Report (2001)

Report of the Higgs working group for the Workshop `Physics at TeV Colliders', Les Houches, France, 21 May - 1 June 2001. It contains 7 separate sections: A. Theoretical Developments B. Higgs Searches at the Tevatron C. Experimental Observation of an invisible Higgs Boson at LHC D. Search for the Standard Model Higgs Boson using Vector Boson Fusion at the LHC E. Study of the MSSM channel $A/H \to \tau \tau$ at the LHC F. Searching for Higgs Bosons in $t\bar t H$ Production G. Studies of Charged Higgs Boson Signals for the Tevatron and the LHCComment: 120 pages, latex, many figures, proceedings of the Workshop `Physics at TeV Colliders', Les Houches, France, 21 May - 1 June 2001, full Author list included in paper. Typos corrected, author list and acknowledgements completed. Convernors: D. Cavalli, A. Djouadi, K. Jakobs, A. Nikitenko, M. Spira, C.E.M. Wagner, W.-M. Ya

Gluon Virtuality and Heavy Sea Quark Contributions to the Spin-Dependent g_1 Structure Function

We analyze the quark mass dependence of photon gluon fusion in polarized deep inelastic scattering for both the intrinsic and extrinsic gluon distributions of the nucleon. We calculate the effective number of flavors for each of the heavy and light quark photon gluon fusion contributions to the first moment of the spin-dependent structure function $g_1(x)$.Comment: LaTex, 19 page

Approach for Predicting Production Scenarios Focused on Cross Impact Analysis

AbstractOne of the most consistent challenges in business is anticipating what the future holds and what impact it may have on current production systems. The scenario technique is a well-established method for developing and forecasting multiple future development paths for companies. However, this method is mostly employed to develop and to support strategic long-term decisions. The core idea of the approach introduced in this paper is to convey the future impact of today's decisions on production systems to employees involved in production planning processes. With the help of immersive visualization, performed in virtual reality (VR) systems, planning participants can perceive how the factory must adapt to fit future demands.In this paper, the focus is on the fourth phase of the scenario technique – so called scenario development – and, in particular, the cross impact analysis. With this methodology, the interrelations, or cross impacts of the different basic elements are determined. The cross impact analysis results serve as a basis for the development of a standardized tool that can be used to create probable production scenarios out of given production systems. This standardized tool will facilitate the usage of the scenario technique for factory planning projects, as it focuses the immense diversity of future uncertainties companies are faced with on the factory level

Meta-analysis of genome-wide association studies from the CHARGE consortium identifies common variants associated with carotid intima media thickness and plaque

Carotid intima media thickness (cIMT) and plaque determined by ultrasonography are established measures of subclinical atherosclerosis that each predicts future cardiovascular disease events. We conducted a meta-analysis of genome-wide association data in 31,211 participants of European ancestry from nine large studies in the setting of the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) Consortium. We then sought additional evidence to support our findings among 11,273 individuals using data from seven additional studies. In the combined meta-analysis, we identified three genomic regions associated with common carotid intima media thickness and two different regions associated with the presence of carotid plaque (P < 5 × 10 -8). The associated SNPs mapped in or near genes related to cellular signaling, lipid metabolism and blood pressure homeostasis, and two of the regions were associated with coronary artery disease (P < 0.006) in the Coronary Artery Disease Genome-Wide Replication and Meta-Analysis (CARDIoGRAM) consortium. Our findings may provide new insight into pathways leading to subclinical atherosclerosis and subsequent cardiovascular events