Fast algorithm for calculating two-photon absorption spectra

We report a numerical calculation of the two-photon absorption coefficient of electrons in a binding potential using the real-time real-space higher-order difference method. By introducing random vector averaging for the intermediate state, the task of evaluating the two-dimensional time integral is reduced to calculating two one-dimensional integrals. This allows the reduction of the computation load down to the same order as that for the linear response function. The relative advantage of the method compared to the straightforward multi-dimensional time integration is greater for the calculation of non-linear response functions of higher order at higher energy resolution.Comment: 4 pages, 2 figures. It will be published in Phys. Rev. E on 1, March, 199

MEERCAT: Multiplexed Efficient Cell Free Expression of Recombinant QconCATs For Large Scale Absolute Proteome Quantification

A major challenge in proteomics is the absolute accurate quantification of large numbers of proteins. QconCATs, artificial proteins that are concatenations of multiple standard peptides, are well established as an efficient means to generate standards for proteome quantification. Previously, QconCATs have been expressed in bacteria, but we now describe QconCAT expression in a robust, cell-free system. The new expression approach rescues QconCATs that previously were unable to be expressed in bacteria and can reduce the incidence of proteolytic damage to QconCATs. Moreover, it is possible to cosynthesize QconCATs in a highly-multiplexed translation reaction, coexpressing tens or hundreds of QconCATs simultaneously. By obviating bacterial culture and through the gain of high level multiplexing, it is now possible to generate tens of thousands of standard peptides in a matter of weeks, rendering absolute quantification of a complex proteome highly achievable in a reproducible, broadly deployable system. One of the major challenges in proteomics is absolute quantification of individual proteins. The predominant technology in large scale protein quantification is MS of (usually tryptic) peptides derived from proteolysis of the proteome in vitro and it is well understood that although mass spectrometers can deliver linearity of response over many orders of magnitude, the response factor (signal intensity per mol of peptide) varies considerably among individual peptides (1, 2). One outcome is that commonly used “label-free” methods that sum the precursor ion intensities for the peptides derived from a single protein, are excellent for relative quantification, but are less satisfactory for absolute quantification. MS-based absolute quantification of proteins can be supported by external standards that are analyzed before and/or after the analyte or by stable-isotope labeled internal standards that are coanalyzed and which define the response factor for each peptide (3). These peptides can be individually synthesized and quantified (4) and there have been some remarkable large-scale studies. However, large numbers of accurately quantified peptides are costly. Further, a commercially produced, accurately quantified standard peptide is a finite resource and is hence best focused on low numbers of assays of a small number of target proteins. Intact protein standards (5⇓–7), or large fragments (8) provide multiple potential peptides for quantification of the targets. In 2005, a novel approach to the creation of standard peptides by biosynthesis was proposed in the form of QconCATs (9⇓⇓⇓–13). QconCATs are artificial proteins that are concatenations of standard peptides from multiple natural proteins, sometimes interspersed by short peptides to recapitulate the primary sequence context of the natural counterpart (14, 15). Peptides suitable for quantification are referred to as Q-peptides, and are not synonymous with proteotypic peptides, as the latter term refers to peptides, unique to one protein, that drive protein identification, not quantification. QconCATs genes are synthesized de novo and are routinely expressed in E. coli cultured in media supplemented with appropriate stable isotope labeled amino acids, such that peptides derived from QconCATs are discriminable from natural peptides within the mass spectrometer. The purified QconCATs are mixed with the biological analyte sample and coproteolyzed to generate a mixture of labeled (standard) and unlabeled (analyte) peptide pairs that can be analyzed by liquid chromatography coupled to MS to yield absolute quantification of the analyte proteins. QconCATs have the added advantage that with appropriate control of proteolysis (11) all standards are, by definition, in a 1:1 ratio, rendering independent quantification of each standard unnecessary; a single common peptide can function to quantify the QconCAT (13). However, successful expression of novel QconCATs in E. coli is not always guaranteed. In a large-scale quantification project that used over 100 independently designed and expressed QconCATs, we discovered that ∼1 in 10 of the concatamers would fail to express, even when a range of expression conditions were explored. Further, at a low frequency, some QconCATs were prone to proteolysis in the bacterial cell or during purification, rendering them of reduced value for quantification. Effective QconCAT deployment across large scale proteome quantification studies would require a high level of confidence in expression of every new construct. In addition, living-cell based synthesis systems are not ideal for high-throughput preparation of multiple QconCATs and many mass spectrometry laboratories are not equipped for the basic molecular biology that would be needed to subclone and express recombinant proteins. To enhance the potential of QconCAT technology for large-scale proteome quantification, we here focus on a wheat germ cell-free protein synthesis system (WGCFS)1 as a major enhancement to the workflow of high throughput QconCAT synthesis. WGCFS, which uses the powerful translation system for germination stored in wheat germ, realizes the highest yield of translation among commercially available eukaryotic derived cell-free systems (16⇓⇓⇓–20). Using WGCFS, we previously demonstrated the feasibility of synthesis of single, small QconCATs, typically 25 kDa (21). In the present study, we first assessed whether WGCFS could be used to express more typical QconCATs at approx. 60 kDa (for quantification of ∼25 proteins at two peptides per target protein), whether WGCFS would rescue “failed” QconCATs and whether this cell free system was able to reduce the risk of proteolytic degradation. Further, we established whether an additional step in efficiency could be derived from coexpression of multiple QconCATs in a single WGCFS reaction

Distinct cellular pathways select germline-encoded and somatically mutated antibodies into immunological memory

One component of memory in the antibody system is long-lived memory B cells selected for the expression of somatically mutated, high-affinity antibodies in the T cell-dependent germinal center (GC) reaction. A puzzling observation has been that the memory B cell compartment also contains cells expressing unmutated, low-affinity antibodies. Using conditional Bcl6 ablation, we demonstrate that these cells are generated through proliferative expansion early after immunization in a T cell-dependent but GC-independent manner. They soon become resting and long-lived and display a novel distinct gene expression signature which distinguishes memory B cells from other classes of B cells. GC-independent memory B cells are later joined by somatically mutated GC descendants at roughly equal proportions and these two types of memory cells efficiently generate adoptive secondary antibody responses. Deletion of T follicular helper (Tfh) cells significantly reduces the generation of mutated, but not unmutated, memory cells early on in the response. Thus, B cell memory is generated along two fundamentally distinct cellular differentiation pathways. One pathway is dedicated to the generation of high-affinity somatic antibody mutants, whereas the other preserves germ line antibody specificities and may prepare the organism for rapid responses to antigenic variants of the invading pathogen

Measurement of a small atmospheric νμ/νe\nu_\mu/\nu_e ratio

From an exposure of 25.5~kiloton-years of the Super-Kamiokande detector, 900 muon-like and 983 electron-like single-ring atmospheric neutrino interactions were detected with momentum $p_e > 100$ MeV/$c$, $p_\mu > 200$ MeV/$c$, and with visible energy less than 1.33 GeV. Using a detailed Monte Carlo simulation, the ratio $(\mu/e)_{DATA}/(\mu/e)_{MC}$ was measured to be $0.61 \pm 0.03(stat.) \pm 0.05(sys.)$, consistent with previous results from the Kamiokande, IMB and Soudan-2 experiments, and smaller than expected from theoretical models of atmospheric neutrino production.Comment: 14 pages with 5 figure

Observation of the east-west anisotropy of the atmospheric neutrino flux

The east-west anisotropy, caused by the deflection of primary cosmic rays in the Earth's magnetic field, is observed for the first time in the flux of atmospheric neutrinos. Using a 45 kt-year exposure of the Super-Kamiokande detector, 552 e-like and 633 mu-like horizontally-going events are selected in the momentum range between 400 and 3000 MeV/c. The azimuthal distribution of e-like and mu-like events agrees with the expectation from atmospheric neutrino flux calculations that account for the geomagnetic field, verifying that the geomagnetic field effects in the production of atmospheric neutrinos in the GeV energy range are well understood.Comment: 8 pages,3 figures revtex, submitted to PR

Measurement of radon concentrations at Super-Kamiokande

Radioactivity from radon is a major background for observing solar neutrinos at Super-Kamiokande. In this paper, we describe the measurement of radon concentrations at Super-Kamiokande, the method of radon reduction, and the radon monitoring system. The measurement shows that the current low-energy event rate between 5.0 MeV and 6.5 MeV implies a radon concentration in the Super-Kamiokande water of less than 1.4 mBq/m$^3$.Comment: 11 pages, 4 figure