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Single-site glycine-specific labeling of proteins.
Labeling of native proteins invites interest from diverse segments of science. However, there remains the significant unmet challenge in precise labeling at a single site of a protein. Here, we report the site-specific labeling of natural or easy-to-engineer N-terminus Gly in proteins with remarkable efficiency and selectivity. The method generates a latent nucleophile from N-terminus imine that reacts with an aldehyde to deliver an aminoalcohol under physiological conditions. It differentiates N-Gly as a unique target amongst other proteinogenic amino acids. The method allows single-site labeling of proteins in isolated form and extends to lysed cells. It administers an orthogonal aldehyde group primed for late-stage tagging with an affinity tag, 19F NMR probe, and a fluorophore. A user-friendly protocol delivers analytically pure tagged proteins. The mild reaction conditions do not alter the structure and function of the protein. The cellular uptake of fluorophore-tagged insulin and its ability to activate the insulin-receptor mediated signaling remains unperturbed
Azimuthal anisotropy and correlations at large transverse momenta in and Au+Au collisions at = 200 GeV
Results on high transverse momentum charged particle emission with respect to
the reaction plane are presented for Au+Au collisions at =
200 GeV. Two- and four-particle correlations results are presented as well as a
comparison of azimuthal correlations in Au+Au collisions to those in at
the same energy. Elliptic anisotropy, , is found to reach its maximum at
GeV/c, then decrease slowly and remain significant up to
-- 10 GeV/c. Stronger suppression is found in the back-to-back
high- particle correlations for particles emitted out-of-plane compared to
those emitted in-plane. The centrality dependence of at intermediate
is compared to simple models based on jet quenching.Comment: 4 figures. Published version as PRL 93, 252301 (2004
Azimuthal anisotropy in Au+Au collisions at sqrtsNN = 200 GeV
The results from the STAR Collaboration on directed flow (v_1), elliptic flow
(v_2), and the fourth harmonic (v_4) in the anisotropic azimuthal distribution
of particles from Au+Au collisions at sqrtsNN = 200 GeV are summarized and
compared with results from other experiments and theoretical models. Results
for identified particles are presented and fit with a Blast Wave model.
Different anisotropic flow analysis methods are compared and nonflow effects
are extracted from the data. For v_2, scaling with the number of constituent
quarks and parton coalescence is discussed. For v_4, scaling with v_2^2 and
quark coalescence is discussed.Comment: 26 pages. As accepted by Phys. Rev. C. Text rearranged, figures
modified, but data the same. However, in Fig. 35 the hydro calculations are
corrected in this version. The data tables are available at
http://www.star.bnl.gov/central/publications/ by searching for "flow" and
then this pape
In Silico Identification of Specialized Secretory-Organelle Proteins in Apicomplexan Parasites and In Vivo Validation in Toxoplasma gondii
Apicomplexan parasites, including the human pathogens Toxoplasma gondii and Plasmodium falciparum, employ specialized secretory organelles (micronemes, rhoptries, dense granules) to invade and survive within host cells. Because molecules secreted from these organelles function at the host/parasite interface, their identification is important for understanding invasion mechanisms, and central to the development of therapeutic strategies. Using a computational approach based on predicted functional domains, we have identified more than 600 candidate secretory organelle proteins in twelve apicomplexan parasites. Expression in transgenic T. gondii of eight proteins identified in silico confirms that all enter into the secretory pathway, and seven target to apical organelles associated with invasion. An in silico approach intended to identify possible host interacting proteins yields a dataset enriched in secretory/transmembrane proteins, including most of the antigens known to be engaged by apicomplexan parasites during infection. These domain pattern and projected interactome approaches significantly expand the repertoire of proteins that may be involved in host parasite interactions
Rapidity and Centrality Dependence of Proton and Anti-proton Production from Au+Au Collisions at sqrt(sNN) = 130GeV
We report on the rapidity and centrality dependence of proton and anti-proton
transverse mass distributions from Au+Au collisions at sqrt(sNN) = 130GeV as
measured by the STAR experiment at RHIC. Our results are from the rapidity and
transverse momentum range of |y|<0.5 and 0.35 <p_t<1.00GeV/c. For both protons
and anti-protons, transverse mass distributions become more convex from
peripheral to central collisions demonstrating characteristics of collective
expansion. The measured rapidity distributions and the mean transverse momenta
versus rapidity are flat within |y|<0.5. Comparisons of our data with results
from model calculations indicate that in order to obtain a consistent picture
of the proton(anti-proton) yields and transverse mass distributions the
possibility of pre-hadronic collective expansion may have to be taken into
account.Comment: 4 pages, 3 figures, 1 table, submitted to PR
Germline Genetic Variants Disturbing the Let-7/LIN28 Double-Negative Feedback Loop Alter Breast Cancer Susceptibility
Previous studies have shown that let-7 can repress the post-transcriptional translation of LIN28, and LIN28 in turn could block the maturation of let-7, forming a double-negative feedback loop. In this study, we investigated the effect of germline genetic variants on regulation of the homeostasis of the let-7/LIN28 loop and breast cancer risk. We initially demonstrated that the T/C variants of rs3811463, a single nucleotide polymorphism (SNP) located near the let-7 binding site in LIN28, could lead to differential regulation of LIN28 by let-7. Specifically, the C allele of rs3811463 weakened let-7–induced repression of LIN28 mRNA, resulting in increased production of LIN28 protein, which could in turn down-regulate the level of mature let-7. This effect was then validated at the tissue level in that the normal breast tissue of individuals with the rs3811463-TC genotype expressed significantly lower levels of let-7 and higher levels of LIN28 protein than those individuals with the rs3811463-TT genotype. Because previous in vitro and ex vivo experiments have consistently suggested that LIN28 could promote cellular transformation, we then systematically evaluated the relationship between rs3811463 as well as other common LIN28 SNPs and the risk of breast cancer in a stepwise manner. The first hospital-based association study (n = 2,300) demonstrated that two SNPs were significantly associated with breast cancer risk, one of which was rs3811463, while the other was rs6697410. The C allele of the rs3811463 SNP corresponded to an increased risk of breast cancer with an odds ratio (OR) of 1.25 (P = 0.0091), which was successfully replicated in a second independent study (n = 1,156) with community-based controls. The combined P-value of the two studies was 8.0×10−5. Taken together, our study demonstrates that host genetic variants could disturb the regulation of the let-7/LIN28 double-negative feedback loop and alter breast cancer risk
MFN1 structures reveal nucleotide-triggered dimerization critical for mitochondrial fusion
Mitochondria are double-membraned organelles with variable shapes influenced by metabolic conditions, developmental stage, and environmental stimuli. Their dynamic morphology is a result of regulated and balanced fusion and fission processes. Fusion is crucial for the health and physiological functions of mitochondria, including complementation of damaged mitochondrial DNAs and the maintenance of membrane potential. Mitofusins are dynamin-related GTPases that are essential for mitochondrial fusion. They are embedded in the mitochondrial outer membrane and thought to fuse adjacent mitochondria via combined oligomerization and GTP hydrolysis. However, the molecular mechanisms of this process remain unknown. Here we present crystal structures of engineered human MFN1 containing the GTPase domain and a helical domain during different stages of GTP hydrolysis. The helical domain is composed of elements from widely dispersed sequence regions of MFN1 and resembles the ‘neck’ of the bacterial dynamin-like protein. The structures reveal unique features of its catalytic machinery and explain how GTP binding induces conformational changes to promote GTPase domain dimerization in the transition state. Disruption of GTPase domain dimerization abolishes the fusogenic activity of MFN1. Moreover, a conserved aspartate residue trigger was found to affect mitochondrial elongation in MFN1, probably through a GTP-loading-dependent domain rearrangement. Thus, we propose a mechanistic model for MFN1-mediated mitochondrial tethering, and our results shed light on the molecular basis of mitochondrial fusion and mitofusin-related human neuromuscular disorders
Glutamate regulation of calcium and IP3 oscillating and pulsating dynamics in astrocytes
Recent years have witnessed an increasing interest in neuron-glia
communication. This interest stems from the realization that glia participates
in cognitive functions and information processing and is involved in many brain
disorders and neurodegenerative diseases. An important process in neuron-glia
communications is astrocyte encoding of synaptic information transfer: the
modulation of intracellular calcium dynamics in astrocytes in response to
synaptic activity. Here, we derive and investigate a concise mathematical model
for glutamate-induced astrocytic intracellular Ca2+ dynamics that captures the
essential biochemical features of the regulatory pathway of inositol
1,4,5-trisphosphate (IP3). Starting from the well-known two-state Li-Rinzel
model for calcium-induced-calcium release, we incorporate the regulation of the
IP3 production and phosphorylation. Doing so we extended it to a three-state
model (referred as the G-ChI model), that could account for Ca2+ oscillations
triggered by endogenous IP3 metabolism as well as by IP3 production by external
glutamate signals. Compared to previous similar models, our three-state models
include a more realistic description of the IP3 production and degradation
pathways, lumping together their essential nonlinearities within a concise
formulation. Using bifurcation analysis and time simulations, we demonstrate
the existence of new putative dynamical features. The cross-couplings between
IP3 and Ca2+ pathways endows the system with self-consistent oscillator
properties and favor mixed frequency-amplitude encoding modes over pure
amplitude modulation ones. These and additional results of our model are in
general agreement with available experimental data and may have important
implications on the role of astrocytes in the synaptic transfer of information.Comment: 42 pages, 16 figures, 1 table. Figure filenames mirror figure order
in the paper. Ending "S" in figure filenames stands for "Supplementary
Figure". This article was selected by the Faculty of 1000 Biology: "Genevieve
Dupont: Faculty of 1000 Biology, 4 Sep 2009" at
http://www.f1000biology.com/article/id/1163674/evaluatio
Interim Design Report
The International Design Study for the Neutrino Factory (the IDS-NF) was established by the community at the ninth International Workshop on Neutrino Factories, super-beams, and beta- beams which was held in Okayama in August 2007. The IDS-NF mandate is to deliver the Reference Design Report (RDR) for the facility on the timescale of 2012/13. In addition, the mandate for the study [3] requires an Interim Design Report to be delivered midway through the project as a step on the way to the RDR. This document, the IDR, has two functions: it marks the point in the IDS-NF at which the emphasis turns to the engineering studies required to deliver the RDR and it documents baseline concepts for the accelerator complex, the neutrino detectors, and the instrumentation systems. The IDS-NF is, in essence, a site-independent study. Example sites, CERN, FNAL, and RAL, have been identified to allow site-specific issues to be addressed in the cost analysis that will be presented in the RDR. The choice of example sites should not be interpreted as implying a preferred choice of site for the facility
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