Proceedings of the National Academy of Sciences

Published by National Academy of Sciences
Online ISSN: 1091-6490
Oxygen-atom vacancy on rutile TiO 2 (110) surface. The PBE approximation to the energy functional predicts a delocalized electronic state with 
Reaction and activation energy (eV) for some silanes
Schematic contour plot of an energy surface showing an initial state minimum, R, and fi ve possible fi nal state minima, P 1 – P 5 . Maxima are denoted with fi lled circles, and saddle points are denoted by × . In the harmonic approximation (the case illustrated here), the dividing surfaces are hyperplanar segments containing a fi rst-order saddle point and oriented with the normal pointing along the unstable mode. ( A ) A reactive classical trajectory requires impossibly long simulations of vibrations in the initial state until a transition is observed (blue line). ( B ) In the WKE procedure, a short trajectory containing the essential information is generated in the following way. A statistical estimate is made of the time needed to reach a point (denoted by an open blue circle) in the transition state, a short trajectory from that point forward in time is generated to determine the fi nal state (blue line), and a short trajectory backward in time is generated to verify that it originated in the initial state (red line); otherwise, a new trajectory is generated. The con fi guration space is partitioned into subspaces by dividing surfaces (straight red lines), and long time-scale simulation using the adaptive kinetic Monte Carlo (AKMC) method tracks the advance of the system from one subspace to another. 
An AKMC simulation of the diffusion of a pentamer water cluster on a Pt(111) surface using a model potential (39). Without coarse graining, the simulation would simply show repeated transitions between the initial state (A) and the two shoulder states (B and C). With coarse graining, states A and B are merged after a little over 2,000 iterations, and states A-C have been merged after about 3,000 iterations. After that, states D and E are revealed and then, A′ and F. Although one possible diffusion mechanism involves going from one ground state conguration to another (A-A′, etc.), a faster mechanism involves the rapid diffusion between metastable pentamer ring states (F-F′, etc), in between occasional visits to a ground state conguration.
Computer simulations of surface processes can reveal unexpected insight regarding atomic-scale structure and transitions. Here, the strengths and weaknesses of some commonly used approaches are reviewed as well as promising avenues for improvements. The electronic degrees of freedom are usually described by gradient-dependent functionals within Kohn-Sham density functional theory. Although this level of theory has been remarkably successful in numerous studies, several important problems require a more accurate theoretical description. It is important to develop new tools to make it possible to study, for example, localized defect states and band gaps in large and complex systems. Preliminary results presented here show that orbital density-dependent functionals provide a promising avenue, but they require the development of new numerical methods and substantial changes to codes designed for Kohn-Sham density functional theory. The nuclear degrees of freedom can, in most cases, be described by the classical equations of motion; however, they still pose a significant challenge, because the time scale of interesting transitions, which typically involve substantial free energy barriers, is much longer than the time scale of vibrations--often 10 orders of magnitude. Therefore, simulation of diffusion, structural annealing, and chemical reactions cannot be achieved with direct simulation of the classical dynamics. Alternative approaches are needed. One such approach is transition state theory as implemented in the adaptive kinetic Monte Carlo algorithm, which, thus far, has relied on the harmonic approximation but could be extended and made applicable to systems with rougher energy landscape and transitions through quantum mechanical tunneling.
We argue that given even an infinitely long data sequence, it is impossible (with any test statistic) to distinguish perfectly between linear and nonlinear processes (including slightly noisy chaotic processes). Our approach is to consider the set of moving-average (linear) processes and study its closure under a suitable metric. We give the precise characterization of this closure, which is unexpectedly large, containing nonergodic processes, which are Poisson sums of independent and identically distributed copies of a stationary process. Proofs of these results will appear elsewhere.
Top 10 firms from 1979 to 1983 and from 2003 to 2007 
Complex social networks have received increasing attention from researchers. Recent work has focused on mechanisms that produce scale-free networks. We theoretically and empirically characterize the buyer-supplier network of the US economy and find that purely scale-free models have trouble matching key attributes of the network. We construct an alternative model that incorporates realistic features of firms' buyer-supplier relationships and estimate the model's parameters using microdata on firms' self-reported customers. This alternative framework is better able to match the attributes of the actual economic network and aids in further understanding several important economic phenomena.
How do we perceive light? We assume that we have to absorb a photon to perceive light. When we set out to confirm this assumption experimentally then we find that everything we knew about light is wrong. We can perceive light wherever it is, provided light is in the perceivable range of our eyes. More than one photon detector can simultaneously detect a single photon. Obviously, a single photon cannot be absorbed by more than one detector. We can see a laser beam refract as it enters water from air but we find no refraction when we view the laser beam from the side of the container. It is possible only if we can see the light without having to absorb the light. Perception of the total solar eclipse as it occurs confirms this observation. All light sources emit only energy, light is produced by the object that absorbs this energy. Only difference between a luminous object and an illuminated object is that luminous objects generate their own energy to radiate light whereas illuminated objects need energy from an external source to radiate light. The observations are conclusively validated in several other advanced experiments. These experiments also show that the idea of motion cannot be associated with the light; illusion of motion of light is created due to movement of energy through the medium that produces light. These experiments conclusively invalidate theory of relativity, standard model of cosmology, and standard model of particle physics.
The HDN and the DGN. (a) In the HDN, each node corresponds to a distinct disorder, colored based on the disorder class to which it belongs, the name of the 22 disorder classes being shown on the right. A link between disorders in the same disorder class is colored with the corresponding dimmer color and links connecting different disorder classes are gray. The size of each node is proportional to the number of genes participating in the corresponding disorder (see key), and the link thickness is proportional to the number of genes shared by the disorders it connects. We indicate the name of disorders with 10 associated genes, as well as those mentioned in the text. For a complete set of names, see SI Fig. 13. (b) In the DGN, each node is a gene, with two genes being connected if they are implicated in the same disorder. The size of each node is proportional to the number of disorders in which the gene is implicated (see key). Nodes are light gray if the corresponding genes are associated with more than one disorder class. Genes associated with more than five disorders, and those mentioned in the text, are indicated with the gene symbol. Only nodes with at least one link are shown.
Characterizing the disease modules. (a) Number of observed physical interactions between the products of genes within the same disorder (red arrow) and the distribution of the expected number of interactions for the random control (blue) (P 10 6 ). (b) Distribution of the tissue-homogeneity of a disorder (red). Random control (blue) with the same number of genes chosen randomly is shown for comparison. (c) The distribution of PCC ij values of the expression profiles of each disease gene pair that belongs to the same disorder (red) and the control (blue), representing the PCC distribution between all gene pairs (P 10 6 ). (d) Distribution of the average PCC between expression profiles of all genes associated with the same disorder (red) is also shifted toward higher values than the random control (blue) with the same number of genes chosen randomly (P 10 6 ).
Next Section In fission yeast, the RNAi pathway is required for centromeric heterochromatin assembly. siRNAs derived from centromeric transcripts are incorporated into the RNA-induced transcriptional silencing (RITS) complex and direct it to nascent homologous transcripts. The RNA-induced transcriptional silencing-bound nascent transcripts further recruit the RNA-directed RNA polymerase complex (RDRC) to promote dsRNA synthesis and siRNA production. Heterochromatin coated with Swi6/Heterochromain Protein 1 is then formed following recruitment of chromatin modification machinery. Swi6 is also required for the upstream production of siRNA, although the mechanism for this has remained obscure. Here, we demonstrate that Swi6 recruits RDRC to heterochromatin through Ers1, an RNAi factor intermediate. An ers1⁺ mutant allele (ers1-C62) was identified in a genetic screen for mutants that alleviate centromeric silencing, and this phenotype was suppressed by overexpression of either the Hrr1 RDRC subunit or Clr4 histone H3-K9 methyltransferase. Ers1 physically interacts with Hrr1, and loss of Ers1 impairs RDRC centromeric localization. Although Ers1 failed to bind Clr4, a direct interaction with Swi6 was detected, and centromeric localization of Swi6 was enhanced by Clr4 overexpression in ers1-C62 cells. Consistent with this, deletion of swi6⁺ reduced centromeric localization of Ers1 and RDRC. Moreover, tethering of Ers1 or Hrr1 to centromeric heterochromatin partially bypassed Swi6 function. These findings demonstrate an alternative mechanism for RDRC recruitment and explain the essential role of Swi6/Heterochromain Protein 1 in RNAi-directed heterochromatin assembly.
The number of clusters, N C ID (olive) and N Cas ID (include asymmetrical links (light green), drops with %ID. The weighted chain count, N W ID (magenta), has 
Structural data growth since the inception of the PDB. (a) Growth of protein structural data deposited in the PDB since its inception in 1972. The average growth rate is 28.0% per year for PDB files (N PDB; gray) and 28.4% for nonidentical chains (N CHA; brown). Clustering or down-weighting sequence redundancy at the 25% identity level gives a lower growth rate, very similar for three measures: 24.4% for clustered chains (N C 25 ; olive), 23.0% for weighted
Sizes of SCOP categories depend on number of weighted chains and number of PDB files. ( a ) Sizes of the SCOP categories (families in blue, superfamilies in green, and folds in red) vary linearly (correlation Ͼ 0.998) with the number of files released in the PDB ( N PDB ). Large circles show the historical data for sizes from 13 releases of SCOP since October 1997 (see . 
Growth of sizes of SCOP categories with and without structural genomic data since 2000. ( a ) Influence of the worldwide SG initiative on the growth of protein structural data. With the SG data, growth is more rapid but even this growth rate has fallen recently (Fig. 2 b ). The white dots show the real SCOP count before October 2004; the solid lines and filled circles show estimates of the growth of families, superfamilies, and folds from the growth in N W 25 or N W(SG 25 Ϫ ) . Release dates are used here. ( b ) Percentage of nonredundant sequence data ( N W 25 ) in PDB files deposited in the preceding year that comes or 
Contrary to popular assumption, the rate of growth of structural data has slowed, and the Protein Data Bank (PDB) has not been growing exponentially since 1995. Reaching such a dramatic conclusion requires careful measurement of growth of novel structures, which can be achieved by clustering entry sequences, or by using a novel index to down-weight entries with a higher number of sequence neighbors. These measures agree, and growth rates are very similar for entire PDB files, clusters, and weighted chains. The overall sizes of Structural Classification of Proteins (SCOP) categories (number of families, superfamilies, and folds) appear to be directly proportional to the number of deposited PDB files. Using our weighted chain count, which is most correlated to the change in the size of each SCOP category in any time period, shows that the rate of increase of SCOP categories is actually slowing down. This enables the final size of each of these SCOP categories to be predicted without examining or comparing protein structures. In the last 3 years, structures solved by structural genomics (SG) initiatives, especially the United States National Institutes of Health Protein Structure Initiative, have begun to redress the slowing growth of the PDB. Structures solved by SG are 3.8 times less sequence-redundant than typical PDB structures. Since mid-2004, SG programs have contributed half the novel structures measured by weighted chain counts. Our analysis does not rely on visual inspection of coordinate sets: it is done automatically, providing an accurate, up-to-date measure of the growth of novel protein structural data.
We define and calculate surface areas.
Recent experimental and theoretical studies on the dynamics of the reactions of methane with F and Cl atoms have modified our understanding of mode-selective chemical reactivity. The O + methane reaction is also an important candidate to extend our knowledge on the rules of reactivity. Here, we report a unique full-dimensional ab initio potential energy surface for the O((3)P) + methane reaction, which opens the door for accurate dynamics calculations using this surface. Quasiclassical trajectory calculations of the angular and vibrational distributions for the ground state and CH stretching excited O + CHD(3)(v(1) = 0,1) → OH + CD(3) reactions are in excellent agreement with the experiment. Our theory confirms what was proposed experimentally: The mechanistic origin of the vibrational enhancement is that the CH-stretching excitation enlarges the reactive cone of acceptance.
Large and small tumor (T)antigens of simian virus 40 were synthesized in vitro with L-cell extracts that had been treated by the method of Palmiter to prevent amino-terminal acetylation of nascent proteins. Partial amino-terminal amino acid sequences of both forms of T-antigen were determined and found to be identical. Methionine residues were located at positions 1 and 14, a lysine residue at position 3, and leucine residues at positions 5, 11, 13,16, 17, and 19. These amino acid sequence data match perfectly the amino acid sequence predicted from a sequence of nucleotides in the E strand of simian virus 40 DNA which begins near the junction between HindII/III fragments A and C at about 0.65 map units. This strongly suggests that the sequence coding for the amino terminus of both proteins is located at this position. Furthermore, the data are consistent with a model for the synthesis of both forms of T-antigen that predicts that (i) small T-antigen is coded for by a sequence of nucleotides from the 5' end of the early region and (ii) large T-antigen is coded for by nucleotide sequences from two noncontiguous regions of simian virus 40 DNA.
Fig. S3C. The Fermi wavenumber í µí±˜ í µí°¹ = í µí±ž í µí°¹ 2 ⁄ = 0.067(í µí¼‹ í µí±Ž 0 ⁄ ) is then determined and the Fermi velocity í µí±£ derived from the following dispersion relation 
Figure S2 
To achieve and use the most exotic electronic phenomena predicted for the surface states of 3D topological insulators (TIs), it is necessary to open a "Dirac-mass gap" in their spectrum by breaking time-reversal symmetry. Use of magnetic dopant atoms to generate a ferromagnetic state is the most widely applied approach. However, it is unknown how the spatial arrangements of the magnetic dopant atoms influence the Dirac-mass gap at the atomic scale or, conversely, whether the ferromagnetic interactions between dopant atoms are influenced by the topological surface states. Here we image the locations of the magnetic (Cr) dopant atoms in the ferromagnetic TI Cr0.08(Bi0.1Sb0.9)1.92Te3. Simultaneous visualization of the Dirac-mass gap Δ(r) reveals its intense disorder, which we demonstrate is directly related to fluctuations in n(r), the Cr atom areal density in the termination layer. We find the relationship of surface-state Fermi wavevectors to the anisotropic structure of Δ(r) not inconsistent with predictions for surface ferromagnetism mediated by those states. Moreover, despite the intense Dirac-mass disorder, the anticipated relationship Δ(r) ∝ n(r) is confirmed throughout and exhibits an electron-dopant interaction energy J* = 145 meV·nm(2). These observations reveal how magnetic dopant atoms actually generate the TI mass gap locally and that, to achieve the novel physics expected of time-reversal symmetry breaking TI materials, control of the resulting Dirac-mass gap disorder will be essential.
The primary light-induced charge separation in reaction centers from Rhodopseudomonas sphaeroides R-26 has been investigated after excitation with laser pulses of 150 fsec duration within the longwave absorption band of the primary donor at 850 nm. An excited state of the primary donor, characterized by a broad absorption spectrum extending over the whole spectral range investigated (545-1240 nm), appeared within 100 fsec and gave rise to stimulated emission in the 870- to 1000-nm region with a 2.8-psec lifetime. The photooxidation of the primary donor, as measured at 1240 nm, and the photoreduction of the bacteriopheophytin acceptor, monitored at 545 nm and 675 nm, have been found to proceed simultaneously with a time constant of 2.8 +/- 0.2 psec. Kinetics of absorbance changes at other probe wavelengths gave no indication that an accessory bacteriochlorophyll is involved as a transient electron acceptor.
The synthesis of a photosensitive blocking group for the carboxyl function of neurotransmitters, in this case glycine, is reported. The compound, 2-methoxy-5-nitrophenyl glycine ester (caged glycine), is photolyzed by a laser pulse at 308 or 337 nm within 3 microseconds and with a product quantum yield of 0.2. The compound is hydrolyzed in water with a time constant tau of 6.1 min at pH 7.1 and 3 hr at pH 4.0. Mouse cerebral cortical neurons containing glycine receptors were used in biological assays. A cell-flow device, in which solutions of caged glycine at pH 4.0 were mixed with buffer to give a final pH of 7.1, was used to equilibrate the compound with receptors on the cell surface. Neither the caged compound nor the 2-methoxy-5-nitrophenol photolysis product affected the glycine receptors or modified their response to glycine. When cells equilibrated with caged glycine are irradiated by a laser pulse at 337 nm, glycine receptor channels are opened, as detected in whole-cell current recordings. The approach described may be used in the synthesis and characterization of photolabile precursors of neurotransmitters and other compounds that contain carboxyl groups and for kinetic investigations of neurotransmitter receptors in central nervous system cells in the microsecond time domain.
Thermoelectric power generation is one of the most promising techniques to use the huge amount of waste heat and solar energy. Traditionally, high thermoelectric figure-of-merit, ZT, has been the only parameter pursued for high conversion efficiency. Here, we emphasize that a high power factor (PF) is equivalently important for high power generation, in addition to high efficiency. A new n-type Mg2Sn-based material, Mg2Sn0.75Ge0.25, is a good example to meet the dual requirements in efficiency and output power. It was found that Mg2Sn0.75Ge0.25 has an average ZT of 0.9 and PF of 52 μW⋅cm(-1)⋅K(-2) over the temperature range of 25-450 °C, a peak ZT of 1.4 at 450 °C, and peak PF of 55 μW⋅cm(-1)⋅K(-2) at 350 °C. By using the energy balance of one-dimensional heat flow equation, leg efficiency and output power were calculated with Th = 400 °C and Tc = 50 °C to be of 10.5% and 6.6 W⋅cm(-2) under a temperature gradient of 150 °C⋅mm(-1), respectively.
Analysis of electron diffraction photographs of grains of Mn(2)Al(7), Fe(2)Al(7), and (Mn(0.7)Fe(0.3))(2)Al(7) leads to the conclusion that they are 5-fold twins of a 1664-atom orthorhombic crystal with a = 32.86 A, b = 31.23 A, and c = 24.80 A and with 16 icosahedral clusters of 104 atoms in positions shifted by small amounts from those of the cubic beta-tungsten structure.
Two different mutations that greatly diminish the rate of synthesis of the gene 0.3 protein of bacteriophage T7 have been characterized. One is in the initiator triplet for the 0.3 protein, changing it from AUG to ACG. This mutation was found to have little effect on binding of ribosomes to the 0.3 mRNA in vitro, although 0.3 protein synthesis was greatly depressed in vitro as well as in vivo. A suppressor mutation that partially restores the wild-type rate of synthesis was found to lie within the 0.3 RNA but not close to the mutant ACG (more than 64 nucleotides away). The second mutation is a G-to-A transition located 11 bases to the 5' side of the initiator AUG. This change eliminates a possible five-base pairing with a sequence near the 3' end of 16S ribosomal RNA, an interaction previous workers have proposed to be important for initiation of protein synthesis. This mutation causes the site of ribosome binding to shift about 15 bases to the 3' side, centering on an internal AUG, but this new site has only a poor potential interaction with 16S RNA. A suppressor mutation that restores the rate of 0.3 protein synthesis to essentially wild-type levels and also restores wild-type ribosome-binding behavior was found to lie adjacent to the original mutation, creating a new four-base complementarity with 16S RNA. These results provide strong support for the idea that a pairing interaction between mRNA and 16S RNA is involved in specific initiation of protein synthesis in Escherichia coli and indicate that this interaction may be important in selecting the site in mRNA at which the ribosomes bind.
Electron diffraction intensity data were collected from a 2:3 binary solid solution of two homologous phosphatidylethanolamines (1,2-dimyristoyl-sn-glycerophosphoethanolamine and 1,2-dipalmitoyl-sn-glycerophosphoethanolamine) epitaxially oriented by cocrystallization with naphthalene. The layer packing was determined directly by predicting the value of 12 of the 17 phases from sigma 1- and sigma 2-triplet invariants in space group P1. A reverse Fourier transform of the resulting potential maps provides estimates for three other phases and the two remaining ones were found by generating maps for the 2(2) = 4 possible phase combinations and then testing the smoothness of the potential profile of the hydrocarbon chain packing. The same phase solution can be found by translating a molecular model (based on the known x-ray crystal structure of a shorter homologue) past the unit cell origin. The solid solution is found to retain a stable polar group packing while the statistical occupancy of two terminal-chain carbons is expressed by a reduced potential profile at the nonpolar interface at the bilayer center.
Low-dose, high-resolution electron microscopy combined with conventional direct-phasing methods based on the estimates of triplet-structure invariants are used to determine phase values for all observed electron-diffraction-structure factor magnitudes from epitaxially oriented multilamellar paracrystals of the phosphospholipid 1,2-dihexadecyl-sn-glycerophosphoethanolamine. The reverse Fourier transform of these phase-structure factors is a one-dimensional electrostatic potential map that strongly resembles the electron-density maps calculated from similar x-ray-diffraction data. Determination of the phase values for the electron-diffraction data with structure invariants alone is nearly as successful as the combined use of two separate methods, assigning values to 13 of the 16 reflections--i.e., the electrostatic potential map closely resembles the one calculated with all data.
PFGE of recent Papua New Guinean isolates of P. falciparum designated 1934, 1904, 1916, 1917, 1775, 1703, 1933, and 1935 and cloned lines of P.falciparum designated 3D7, C10, and B9. (A) Hybridization with the chromosome 9-specific probe, the gene encoding merozoite surface antigen (MSA-1) (21), showed size variation (1.8 and 1.5 Mb) in chromosome 9 within recent isolates during adaptation to in vitro culture and in the cloned line 3D7. (B) Hybridization with the chromosome 2-specific probe, the gene coding for MSA-2 (16), showed no variation in size of chromosome 2 within any of the recent isolates. The cloned line 3D7 showed two sizes of chromosome 2. (C) Maps of chromosome 9 from parent isolate 1776 and the clone derived from this isolate designated C10. The maps from ref. 21 have been revised. The presence of additional Bgl or Bssh sites near the telomeres cannot be excluded. Apa I (A), Bgl (Bg), and Bssh2 (Bs) are indicated together with the markers shown. The presence of the 7H8/6 sequences is inferred from studies on other isolates. Cytoadherence and gametocytogenesis results for each clone are given as follows: isolate 1934-40 IRBC/100 C32MC, 9 GC/100 IRBC; isolate 1904-2 IRBC/100 C32MC; 0 GC/100 IRBC; isolate 1916-384 IRBC/100 C32MC, 46 GC/100 IRBC; isolate 1917-328 IRBC/100 C32MC, 38 GC/100 IRBC; isolate 1775-346 IRBC/100 C32MC, 56 GC/100 IRBC; isolate 1703-660  
PFGE analysis of chromosome 9 of the original and selected lines of HB3 and 1934 as well as isolate 1776 and clone C10. Chromosome 9 is identified with the chromosome 9-specific probei.e., the gene for merozoite surface antigen (MSA-1) (21). Bands 1.8 and 1.5 indicate chromosome size in Mb.
Virulence of the human malaria parasite Plasmodium falciparum is believed to relate to adhesion of parasitized erythrocytes to postcapillary venular endothelium (asexual cytoadherence). Transmission of malaria to the mosquito vector involves a switch from asexual to sexual development (gametocytogenesis). Continuous in vitro culture of P. falciparum frequently results in irreversible loss of asexual cytoadherence and gametocytogenesis. Field isolates and cloned lines differing in expression of these phenotypes were karyotyped by pulse-field gel electrophoresis. This analysis showed that expression of both phenotypes mapped to a 0.3-Mb subtelomeric deletion of chromosome 9. This deletion frequently occurs during adaptation of parasite isolates to in vitro culture. Parasites with this deletion did not express the variant surface agglutination phenotype and the putative asexual cytoadherence ligand designated P. falciparum erythrocyte membrane protein 1, which has recently been shown to undergo antigenic variation. The syntenic relationship between asexual cytoadherence and gametocytogenesis suggests that expression of these phenotypes is genetically linked. One explanation for this linkage is that both developmental pathways share a common cytoadherence mechanism. This proposed biological and genetic linkage between a virulence factor (asexual cytoadherence) and transmissibility (gametocytogenesis) would help explain why a high degree of virulence has evolved and been maintained in falciparum malaria.
Bacteriophages take over host resources primarily via the activity of proteins expressed early in infection. One of these proteins, produced by the Escherichia coli phage T7, is gene product (Gp) 0.4. Here, we show that Gp0.4 is a direct inhibitor of the E. coli filamenting temperature-sensitive mutant Z division protein. A chemically synthesized Gp0.4 binds to purified filamenting temperature-sensitive mutant Z protein and directly inhibits its assembly in vitro. Consequently, expression of Gp0.4 in vivo is lethal to E. coli and results in bacteria that are morphologically elongated. We further show that this inhibition of cell division by Gp0.4 enhances the bacteriophage's competitive ability. This division inhibition is thus a fascinating example of a strategy in bacteriophages to maximize utilization of their hosts' cell resources.
Recently, we reported (1) that the stoichiometry of proton pumping is about 0.4–0.5 proton per electron for members of the C family of respiratory oxygen reductases, also called the cbb3-type cytochrome c oxidases. This amount (0.5) is about half of the value obtained with the canonical A family of respiratory oxygen reductases, which includes the mitochondrial cytochrome c oxidases. We pointed out that the low stoichiometry correlates with the absence of the D channel for proton input and speculated that these properties may be an evolutionary adaptation to aerobic respiration under conditions of low oxygen. The measurements were obtained with intact cells at pH values near pH 6, using the electron donor, N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD). However, TMPD has a pKa of 6.5 (2) and, hence, oxidation of TMPD at pH 6 will result in proton release. This proton release could be misinterpreted as proton pumping, leading to a high apparent proton/electron stoichiometry. It is also possible that protonated TMPD could act as an uncoupler and result in an apparent low value of proton-pumping stoichiometry. The validity of our reported proton-pumping stoichiometries has been properly questioned by Rauhamaki et al. (3). We have, therefore, repeated the proton-pumping measurements at pH values greater than 7. For the caa3 A-family oxygen reductase from Thermus thermophilus YC1001, the measured proton/electron stoichiometry is decreased from about 1.1 (pH 5.8) to 0.7 ± 0.6 (n = 2) (pH 7.5). This value (0.7) is somewhat lower than the expected value of 1 for this enzyme. Measurements with the cbb3 C-family oxygen reductase from Rhodobacter capsulatus (KZ1) show the same proton pump stoichiometry for pH > 7 as was previously reported at pH 6: H+/e− = 0.4 ± 0.15 (n = 9). Hence, we confirm that proton pump stoichiometry of the cbb3-type oxygen reductase, at least from R capsulatus, is about 0.5 proton per electron as previously reported (1) and is about half of the value obtained with the A-family oxygen reductases. Possibly, the artifacts at pH 6 fortuitously cancel out.
Long-range stripe structure in real-space. (A) Crystal structure of bilayer manganite La 2−2x Sr 1þ2x Mn 2 O 7 with La∕Sr (purple) atoms between MnO 6 octahedra (Mn: blue, O: red). (B) Phase diagram of La 2−2x Sr 1þ2x Mn 2 O 7 , from refs. 15, 16. (C) CE ordering at x ¼ 0.50 and (D) Bistripe ordering at x ¼ 0.60. Red arrows indicate electron hopping paths, which are broken in the x ¼ 0.60 bi-stripe phase. The doping and temperature of our study is indicated by the blue arrow in box B, with x ∼ 0.59 and T c ∼ 160 K.
Electronic structure measurements of La 2 − 2 x Sr 1 þ 2 x Mn 2 O 7 ( x ∼ 0 . 59 ). ( A ) A schematic Fermi surface plot. ( B , C ) Energy vs. momentum dispersive band taken at T ∼ 20 K along the blue and red cuts in box A . ( D , E ) Stacked EDCs from the two cuts at various temperatures. ( F , G ) Stacked EDCs of x 1⁄4 0 . 5 compound, 
Temperature dependence of electronic structure. (A-C) EDC cuts 1, 2, and 3, indicated by the white dashed lines in Fig. 2B, taken at various temperatures. (D) Differential EDCs obtained by subtracting EDC cut 3 from EDC cut 2. (E) EDCs taken at a position similar to cut 3 from an x ¼ 0.38 sample.
Temperature-dependent spectral weight from ARPES and superlattice counts from X-ray scattering (ref. 26) of La 2−2x Sr 1þ2x Mn 2 O 7 (x ∼ 0.59). Red and blue squares represent the total (entire occupied band) and the near-E F (−0.1 eV to þ0.05 eV) spectral weight respectively. Black dots show the temperature-induced change in the nondispersive/localized weight, obtained by integrating the EDCs of Fig. 3C from −1.4 eV to −0.4 eV and scaling such that it extrapolates to zero at 0 K. All curves are scaled independently so that the change in weight can be qualitatively estimated.
Electronic phases with stripe patterns have been intensively investigated for their vital roles in unique properties of correlated electronic materials. How these real-space patterns affect the conductivity and other properties of materials (which are usually described in momentum space) is one of the major challenges of modern condensed matter physics. By studying the electronic structure of La(2-2x)Sr(1+2x)Mn(2)O(7) (x ∼ 0.59) and in combination with earlier scattering measurements, we demonstrate the variation of electronic properties accompanying the melting of so-called bi-stripes in this material. The static bi-stripes can strongly localize the electrons in the insulating phase above T(c) ∼ 160 K, while the fraction of mobile electrons grows, coexisting with a significant portion of localized electrons when the static bi-stripes melt below T(c). The presence of localized electrons below T(c) suggests that the melting bi-stripes exist as a disordered or fluctuating counterpart. From static to melting, the bi-stripes act as an atomic-scale electronic valve, leading to a "colossal" metal-insulator transition in this material.
Four-stranded guanine tetraplexes in RNA have been identified to be involved in crucial biological functions, such as dimerization of retroviral RNA, translational repression, and mRNA turnover. However, the structural basis for these biological processes is still largely unknown. Here we report the RNA tetraplex structure (UGGGGU)(4) at ultra-high resolution (0.61 A). The space group is P42(1)2, and cell constants are a = b = 36.16 A and c = 74.09 A. The structure was solved by the multiple-wavelength anomalous dispersion method using a set of three-wavelength data of the isomorphous bromo derivative (br)UGGGGU and refined to 0.61-A resolution. Each of the four strands in the asymmetric unit forms a parallel tetraplex with symmetry-related molecules. The tetraplex molecules stack on one another in opposite polarity (head-to-head or tail-to-tail) to form a pseudocontinuous column. All of the 5'-end uridines rotate around the backbone of G2, swing out, and form unique octaplexes with the neighboring G tetraplexes, whereas the 3'-end uridines are stacked-in and form uridine tetrads. All of the bases are anti, and the riboses are in the mixed C2'- and C3'-puckering mode. Strontium ions are observed in every other guanine tetrad plane, sitting on the fourfold axis and associated to the eight O6 atoms of neighboring guanine bases in a bipyramidal-antiprism geometry. The hydrogens are clearly observed in the structure.
Overall structure of bovine mitochondrial FB Gly-3–Glu mutant at 0.96-Å resolution. ( A ) The model includes all of the 175 amino acid residues of the mature bovine polypeptide. In slate is the N-terminal helix ␣ 1, and in blue is the remainder of the N-terminal domain, which includes the Mg 2 ϩ coordination site. In gray is the LRR domain, which contains four LRRs and the nuclear-export signal motif in human FB (highlighted in blue tint). FB is proposed to bind to the matrix side of the mitochondrial inner membrane in the shown orientation, with ␣ 1 helix dipping into the membrane interfacial phase. ( B ) View from the matrix side normal to the membrane, Ϸ 90° rotated about the horizontal with respect to A . 
Data collection and refinement statistics
Metal binding site in FB. The magnesium ion is coordinated by the carbonyl oxygens of Gly-34 and Thr-68 and four water molecules. No magnesium was added to the crystallization condition, which contains 1 mM EDTA, suggesting a high-affinity site for magnesium or other divalent metal in vivo. The electron density (2 F O FC) map is contoured at 1 (blue).
Coupling factor B (FB) is a mitochondrial inner membrane polypeptide that facilitates the energy-driven catalysis of ATP synthesis in animal mitochondria by blocking a proton leak across the membrane. Here, we report the crystal structure of the bovine mitochondrial FB mutant with Gly-3–Glu substitution determined at a resolution of 0.96 Å and that of the WT polypeptide at a resolution of 2.9 Å. The structure reveals an oblong, oval-shaped molecule with a unique globular N-terminal domain that is proposed to be the membrane anchor domain and the capping region to the C-terminal leucine-rich repeats domain. A short N-terminal α-helix, which extends away from the molecule's body, is suggestive of functioning as an anchor for FB to the matrix side of the mitochondrial inner membrane. Identification of a bound Mg²⁺ ion reveals that FB is a metalloprotein. We also report the cocrystal structures of FB bound with phenylarsine oxide and Cd²⁺, two known inhibitors of the FB coupling activity. • ATP synthase • energy coupling • leucine-rich repeat • mitochondria • x-ray crystallography
Observed climate sensitivities for all 43,710 model runs where it was calculable plotted against those predicted by the optimal regression tree on the basis of their parameter, hardware, and software values (Fig. 2 and SI Table 3). 
Frequency distributions for CS as calculated by taking the difference of average global mean temperature for the latter half of the control and doubled CO 2 phases. (A) For the 43,677 model runs where a fitted CS as used for all other analyses was obtained. The relationship of these sensitivities to the fitted sensitivities is shown in SI Fig. 9. (B) For the 13,313 model runs where an adequate fitted sensitivity could not be obtained (26 outliers in B fall outside the range graphed).
Regression tree for percentage CV in CS among model runs with identical parameters and starting conditions. The tree is read from top to bottom in the same way as Fig. 2, starting with the 4,712 parameter and starting condition sets where a CV could be calculated. Full details are given in SI Table 5. 
In complex spatial models, as used to predict the climate response to greenhouse gas emissions, parameter variation within plausible bounds has major effects on model behavior of interest. Here, we present an unprecedentedly large ensemble of >57,000 climate model runs in which 10 parameters, initial conditions, hardware, and software used to run the model all have been varied. We relate information about the model runs to large-scale model behavior (equilibrium sensitivity of global mean temperature to a doubling of carbon dioxide). We demonstrate that effects of parameter, hardware, and software variation are detectable, complex, and interacting. However, we find most of the effects of parameter variation are caused by a small subset of parameters. Notably, the entrainment coefficient in clouds is associated with 30% of the variation seen in climate sensitivity, although both low and high values can give high climate sensitivity. We demonstrate that the effect of hardware and software is small relative to the effect of parameter variation and, over the wide range of systems tested, may be treated as equivalent to that caused by changes in initial conditions. We discuss the significance of these results in relation to the design and interpretation of climate modeling experiments and large-scale modeling more generally. • classification and regression trees • climate change • distributed computing • general circulation models • sensitivity analysis
Energies for the formation of D and L-defects in a 6 bilayer slab (see Fig. S2 for further details). The left hand figure shows the energies required to move a D-defect into the bulk and out to the opposite surface. The right hand figure continues the calculation and moves an L-defect across the slab to annihilate the D-defect. Missing points are caused by barrier-free relaxation of the defects to nearby layers. The reference state is the most stable ideal surface calculated. 
The local environment of layer 1 ( A ) D and ( B , C ) L-defects. In this case the D-defect has two neighboring dangling protons, while the L-defect has four. For clarity only water molecules in the upper bilayer are shown in A , B , and C . A is a view across the surface askew to the surface normal. B is a view atop the surface. C is a side view of the surface, and D is a multilayer cross-sectional view of the reconstructed D-defect in the third layer. The top half of the bilayer is red, the lower half blue. The oxygen atom of the defect itself is green. 
Sampling of D (left) and L (right) formation energies as a function of number of dangling protons neighboring the site. Circles are the energies to form the near-surface defects (i.e., layer 1), triangles layer 3 defects, and red squares the energies to form the bulk-like (layer 5) defects. The different layer 3 D-defect types are detailed in the text. When the D-defect is surrounded by three protons, the formation energy in the surface layer 1 is 0.06 eV and 0.52 eV in bulk layer 5. For the case where the L-defect has three neighboring protons, the formation energy in layer 1 is 0.42 eV and 0.70 eV in layer 5. 
Surface segregation energies of ionic defects at the ice surface as function of neighboring dangling protons. H 3 O þ squares and OH − as circles. 
Geometry of hydronium and hydroxide surface defects. Only the upper bilayer or water molecules is shown for clarity. The bond length of the defect to its neighboring second layer water molecules are shown (in Å). For comparison bulk hydrogen bond distances are ≈1.70 Å. Top of the bilayer is coloured red while the lower layer is blue.
Using density functional theory we investigate whether intrinsic defects in ice surface segregate. We predict that hydronium, hydroxide, and the Bjerrum L- and D-defects are all more stable at the surface. However, the energetic cost to create a D-defect at the surface and migrate it into the bulk crystal is smaller than its bulk formation energy. Absolute and relative segregation energies are sensitive to the surface structure of ice, especially the spatial distribution of protons associated with dangling hydrogen bonds. It is found that the basal plane surface of hexagonal ice increases the bulk concentration of Bjerrum defects, strongly favoring D-defects over L-defects. Dangling protons associated with undercoordinated water molecules are preferentially injected into the crystal bulk as Bjerrum D-defects, leading to a surface dipole that attracts hydronium ions. Aside from the disparity in segregation energies for the Bjerrum defects, we find the interactions between defect species to be very finely balanced; surface segregation energies for hydronium and hydroxide species and trapping energies of these ionic species with Bjerrum defects are equal within the accuracy of our calculations. The mobility of the ionic hydronium and hydroxide species is greatly reduced at the surface in comparison to the bulk due to surface sites with high trapping affinities. We suggest that, in pure ice samples, the surface of ice will have an acidic character due to the presence of hydronium ions. This may be important in understanding the reactivity of ice particulates in the upper atmosphere and at the boundary layer.
B (Ti, Al) cite atom-oxygen (O) atom displacements in the unstrained (squares) and 1.2% compressively strained (circles) ðLaAlO 3 Þ 3 ∕ ðSrTiO 3 Þ 5 structure. 
Recently a metallic state was discovered at the interface between insulating oxides, most notably LaAlO(3) and SrTiO(3). Properties of this two-dimensional electron gas (2DEG) have attracted significant interest due to its potential applications in nanoelectronics. Control over this carrier density and mobility of the 2DEG is essential for applications of these unique systems, and may be achieved by epitaxial strain. However, despite the rich nature of strain effects on oxide materials properties, such as ferroelectricity, magnetism, and superconductivity, the relationship between the strain and electrical properties of the 2DEG at the LaAlO(3)/SrTiO(3) heterointerface remains largely unexplored. Here, we use different lattice constant single-crystal substrates to produce LaAlO(3)/SrTiO(3) interfaces with controlled levels of biaxial epitaxial strain. We have found that tensile-strained SrTiO(3) destroys the conducting 2DEG, while compressively strained SrTiO(3) retains the 2DEG, but with a carrier concentration reduced in comparison to the unstrained LaAlO(3)/SrTiO(3) interface. We have also found that the critical LaAlO(3) overlayer thickness for 2DEG formation increases with SrTiO(3) compressive strain. Our first-principles calculations suggest that a strain-induced electric polarization in the SrTiO(3) layer is responsible for this behavior. The polarization is directed away from the interface and hence creates a negative polarization charge opposing that of the polar LaAlO(3) layer. This behavior both increases the critical thickness of the LaAlO(3) layer, and reduces carrier concentration above the critical thickness, in agreement with our experimental results. Our findings suggest that epitaxial strain can be used to tailor 2DEGs properties of the LaAlO(3)/SrTiO(3) heterointerface.
Products desorbing during TPD after formamide adsorption at 300 K over the (011)-faceted TiO 2 (001) single crystal surface in dark conditions.
TPD after formamide adsorption at 300 K over TiO2 (001) surface and 15-min UV irradiation at 10 9 torr.
The same TPD as in Fig. 3 showing the smaller masses attributed to other fragments of nucleotides as detailed in the text.
We report the conversion of a large fraction of formamide (NH(2)CHO) to high-molecular-weight compounds attributed to nucleoside bases on the surface of a TiO(2) (001) single crystal in ultra-high vacuum conditions. If true, we present previously unreported evidence for making biologically relevant molecules from a C1 compound on any single crystal surface in high vacuum and in dry conditions. An UV light of 3.2 eV was necessary to make the reaction. This UV light excites the semiconductor surface but not directly the adsorbed formamide molecules or the reaction products. There thus is no need to use high energy in the form of photons or electrical discharge to make the carbon-carbon and carbon-nitrogen bonds necessary for life. Consequently, the reaction products may accumulate with time and may not be subject to decomposition by the excitation source. The formation of these molecules, by surface reaction of formamide, is proof that some minerals in the form of oxide semiconductors are active materials for making high-molecular-weight organic molecules that may have acted as precursors for biological compounds required for life in the universe.
Glutamic acid decarboxylase isoform 2 (GAD65; EC has been identified as a key target autoantigen of insulin-dependent diabetes mellitus (IDDM). IDDM is genetically associated with the major histocompatibility complex (MHC), and particular alleles from the HLA-DQ and HLA-DR loci contribute to disease. Among DR4 subtypes, HLA-DRB1*0401, HLA-DRB1*0402, and HLA-DRB1*0405 alleles lend susceptibility, while HLA-DRB1*0403 confers protection. We have utilized HLA-DR(alpha1*0101,beta1*0401) (hereafter referred to as DR0401), human CD4, murine class II null triple transgenic mice and recombinant GAD65 to generate T cell hybridomas, and we have used overlapping sets of peptides to map the immunodominant epitopes of this autoantigen. We have identified 10 immunogenic regions for GAD65, of which 6 are recognized by multiple hybridomas. These epitopes are also generated by human antigen-presenting cells and their presentation is DR0401 restricted, as shown by the use of typed human lymphoblastoid cell lines and antibody blocking experiments. Immunodominant GAD65 epitopes defined in transgenic mice correspond to GAD65 regions previously shown to elicit T cell responses specifically in DR0401 IDDM patients, underscoring the validity of this approach. Interestingly, although the major epitopes contain DR0401 binding motifs, one of the epitopes contains a DR0405 motif.
Coordinates from x-ray structures of HLA-A*6801, HLA-A*0201, and HLA-B*2705 were analyzed to examine the basis for their selectivity in peptide binding. The pocket that binds the side chain of the peptide's second amino acid residue (P2 residue) shows a preference for Val, Leu, and Arg in these three HLA subtypes, respectively. The Arg-specific pocket of HLA-B*2705 differs markedly from those of HLA-A*0201 and HLA-A*6801, as a result of numerous differences in the side chains that form the pocket's surface. The cause of the specificity differences between HLA-A*0201 and HLA-A*6801 is more subtle and depends both on a change in conformation of pocket residue Val-67 and on a sequence difference at residue 9. The Val-67 conformational change appears to be caused by a shift in the position of the alpha 1-domain alpha-helix relative to the beta-sheet in the cleft and may, in fact, depend on amino acid differences remote from the P2 pocket. Analysis of the stereochemistry of the P2 side chain interacting with its binding pocket permits an estimate to be made of its contribution to the free-energy change of peptide binding.
Identification of HLA-A*0201-restricted peptide epitope VP35#1. (A) Vaccinia virus polypeptides selected for analysis of peptides with highest probability for HLA-A*0201 binding are shown. Incubation of splenocytes from vaccinated HHD mice with peptide VP35#1 results in specific secretion of IFN-. T cell response is depicted as percentage of cytokine-secreting CD8 cells (%CD8). An irrelevant HLA-A*0201-binding peptide (huTyr 369) was used as a negative control. The numbers marked by an asterisk refer to results from two independent experiments. (B) Alignment of VP35#1-peptide sequences derived from poxvirus gene products homologous to vaccinia virus H3L protein: vaccinia virus strains MVA (VV-MVA), Copenhagen (VV-Cop), WR (VV-WR), and Tian Tan (VV-Tan); monkeypox virus Zaire-96-I-16 (MPXV-ZRE); camelpox virus M-96 (CPXV-KM96); ectromelia virus Naval (EV); variola major viruses Bangladesh-1975 (VAR-BSH) and India-1967 (VAR-IND); variola minor virus Garcia-1966 (VMN-GAR); yaba-like disease virus (YAB-YLD); molluscum contagiosum virus 1 (MC1); fowlpox virus (FPV-FCV); orf viruses NZ2 (Orf-NZ2), OV20 (Orf-OV20), and OV7 (Orf-OV7); myxoma virus Lausanne (MYXLAU); lumpy skin disease virus Neethling 2490 (LSD-NEE); and swinepox virus 17077-99 (SPV-KAS).
Isolation and expansion of human CD8 VP35#1-specific T cells from peripheral blood and lysis of VP35#1-presenting human target cells. Human HLA-A*0201 PBMCs were stimulated with autologous dendritic cells pulsed with VP35#1. T cells were stained with HLA-A2VP35#1 multimers after two in vitro stimulations and multimer-guided sorting (A). Numbers in the upper right quadrant represent the percentage of multimer-binding CD8 T cells gated on propidium iodide-negative PBMCs. T2 targets pulsed with either the VP35#1 peptide (s) or HIV-1 RT 476-484 peptide () at 1 M (B) or human A375H3L (s), A375LV (), or A375 cells (F) (C) were tested against the human A*0201restricted, VP35#1-specific T cells as effector cells at the indicated ET ratio.
Despite worldwide eradication of naturally occurring variola virus, smallpox remains a potential threat to both civilian and military populations. New, safe smallpox vaccines are being developed, and there is an urgent need for methods to evaluate vaccine efficacy after immunization. Here we report the identification of an immunodominant HLA-A*0201-restricted epitope that is recognized by cytotoxic CD8(+) T cells and conserved among Orthopoxvirus species including variola virus. This finding has permitted analysis and monitoring of epitope-specific T cell responses after immunization and demonstration of the identified T cell specificity in an A*0201-positive human donor. Vaccination of transgenic mice allowed us to compare the immunogenicity of several vaccinia viruses including highly attenuated, replication-deficient modified vaccinia virus Ankara (MVA). MVA vaccines elicited levels of CD8(+) T cell responses that were comparable to those induced by the replication-competent vaccinia virus strains. Finally, we demonstrate that MVA vaccination is fully protective against a lethal respiratory challenge with virulent vaccinia virus strain Western Reserve. Our data provide a basis to rationally estimate immunogenicity of safe, second-generation poxvirus vaccines and suggest that MVA may be a suitable candidate.
Initial studies suggested that major histocompatibility complex class I-restricted viral epitopes could be predicted by the presence of particular residues termed anchors. However, recent studies showed that nonanchor positions of the epitopes are also significant for class I binding and recognition by cytotoxic T lymphocytes (CTLs). We investigated if changing nonanchor amino acids could increase class I affinity, complex stability, and T-cell recognition of a natural viral epitope. This concept was tested by using the HLA-A 0201-restricted human immunodeficiency virus type 1 epitope from reverse transcriptase (pol). Position 1 (P1) amino acid substitutions were emphasized because P1 alterations may not alter the T-cell receptor interaction. The peptide with the P1 substitution of tyrosine for isoleucine (I1Y) showed a binding affinity for HLA-A 0201 similar to that of the wild-type pol peptide in a cell lysate assembly assay. Surprisingly, I1Y significantly increased the HLA-A 0201-peptide complex stability at the cell surface. I1Y sensitized HLA-A 0201-expressing target cells for wild-type pol-specific CTL lysis as well as wild-type pol. Peripheral blood lymphocytes from three HLA-A2 HIV-seropositive individuals were stimulated in vitro with I1Y and wild-type pol. I1Y stimulated a higher wild-type pol-specific CTL response than wild-type pol in all three donors. Thus, I1Y may be an "improved" epitope for use as a CTL-based human immunodeficiency virus vaccine component. The design of improved epitopes has important ramifications for prophylaxis and therapeutic vaccine development.
Target cell tropism of enveloped viruses is regulated by interactions between viral and cellular factors during transmission, dissemination, and replication within the host. Binding of viral envelope glycoproteins to specific cell-surface receptors determines susceptibility to viral entry. However, a number of cell-surface molecules bind viral envelope glycoproteins without mediating entry. Instead, they serve as capture receptors that disseminate viral particles to target organs or susceptible cells. We and others recently demonstrated that the C type lectins L-SIGN and DC-SIGN capture hepatitis C virus (HCV) by specific binding to envelope glycoprotein E2. In this study, we use an entry assay to demonstrate that HCV pseudoviruses captured by L-SIGN+ or DC-SIGN+ cells efficiently transinfect adjacent human liver cells. Virus capture and transinfection require internalization of the SIGN-HCV pseudovirus complex. In vivo, L-SIGN is largely expressed on endothelial cells in liver sinusoids, whereas DC-SIGN is expressed on dendritic cells. Capture of circulating HCV particles by these SIGN+ cells may facilitate virus infection of proximal hepatocytes and lymphocyte subpopulations and may be essential for the establishment of persistent infection.
Brain metastases are a serious obstacle in the treatment of patients with human epidermal growth factor receptor-2 (HER2)-amplified breast cancer. Although extracranial disease is controlled with HER2 inhibitors in the majority of patients, brain metastases often develop. Because these brain metastases do not respond to therapy, they are frequently the reason for treatment failure. We developed a mouse model of HER2-amplified breast cancer brain metastasis using an orthotopic xenograft of BT474 cells. As seen in patients, the HER2 inhibitors trastuzumab and lapatinib controlled tumor progression in the breast but failed to contain tumor growth in the brain. We observed that the combination of a HER2 inhibitor with an anti-VEGF receptor-2 (VEGFR2) antibody significantly slows tumor growth in the brain, resulting in a striking survival benefit. This benefit appears largely due to an enhanced antiangiogenic effect: Combination therapy reduced both the total and functional microvascular density in the brain xenografts. In addition, the combination therapy led to a marked increase in necrosis of the brain lesions. Moreover, we observed even better antitumor activity after combining both trastuzumab and lapatinib with the anti-VEGFR2 antibody. This triple-drug combination prolonged the median overall survival fivefold compared with the control-treated group and twofold compared with either two-drug regimen. These findings support the clinical development of this three-drug regimen for the treatment of HER2-amplified breast cancer brain metastases.
Molecular analysis of HLA-DRBI alleles in IBD patients and healthy controls 
The role of inflammatory T cells in Crohn's disease suggests that inherited variations in major histocompatibility complex (MHC) class II genes may be of pathogenetic importance in inflammatory bowel disease. The absence of consistent and strong associations with MHC class II genes in Caucasian patients with inflammatory bowel disease probably reflects the use of less precise typing approaches and the failure to type certain loci by any means. A PCR-sequence-specific oligonucleotide-based approach was used to type individual alleles of the HLA class II DRB1, DRB3, DRB4, and DRB5 loci in 40 patients with ulcerative colitis, 42 Crohn's disease patients, and 93 ethnically matched healthy controls. Detailed molecular typing of the above alleles has previously not been reported in patients with inflammatory bowel disease. A highly significant positive association with the HLA-DRB3*0301 allele was observed in patients with Crohn's disease (P = 0.0004) but not in patients with ulcerative colitis. The relative risk for this association was 7.04. Other less significant HLA class II associations were also noted in patients with Crohn's disease. One of these associations involved the HLA-DRB1*1302 allele, which is known to be in linkage disequilibrium with HLA-DRB3*0301. These data suggest that a single allele of an infrequently typed HLA class II locus is strongly associated with Crohn's disease and that MHC class II molecules may be important in its pathogenesis.
Ocular cicatricial pemphigoid (OCP) is an autoimmune blistering disease that affects the conjunctiva and multiple mucous membranes. Class I and II and complement genetic markers of the major histocompatibility complex were studied in 20 Caucasian OCP patients and members of their families. Frequencies of individual alleles and common fixed or extended haplotypes in the patients were compared with those in normal family control haplotypes and with overall normal Caucasian haplotypes. The most striking increase compared with overall controls was noted in HLA-DQw3 (P = 0.006), unassociated with any extended haplotype. All but 1 of the 20 patients carried DQw3 in linkage with HLA-DR4 (increased significantly with P = 0.042 compared with overall normal genotype controls) or DR5. The DQw3, on analysis by restriction fragment length polymorphism in genomic DNA, was, in every instance, DQw7 (3.1, DQB1*0301). The frequency of DQB1*0301 in patient haplotypes compared with overall normal DR4 and DR5 DQw3-bearing haplotypes was statistically significantly increased (P less than 0.003, relative risk = 9.6). The distribution of homozygotes and heterozygotes for DQB1*0301 among the patients was consistent with dominant but not recessive inheritance of DQB1*0301 or a gene, probably a class II allele, in linkage disequilibrium with it as the major histocompatibility complex susceptibility gene for OCP.
MHC class II haplotypes in patients with BP 
Statistical comparison of MHC class II DRB1, DQB1 alleles in BP 
Bullous pemphigoid (BP) is an autoimmune subepidermal blistering disease seen primarily in elderly persons. It is characterized clinically by the development of tense bullae and by the presence of an antibasement membrane antibody. In BP, the antigens involved in the autoimmunity are epidermal basement membrane peptides BPAg1 and BPAg2. We have compared high resolution typing of major histocompatibility complex class II loci (HLA-DRB1, DQB1) in 21 patients with BP, 17 with ocular cicatricial pemphigoid (OCP), and 22 with oral pemphigoid (OP) to a panel of 218 haplotypes of normal individuals. We found that the three diseases (BP, OCP, and OP) have significant association with DQB1*0301 (P = 0.005, P < 0.0001, and P = 0.001, respectively). The frequencies of alleles DQB1*0302, 0303, and 06, which share a specific amino acid sequence from position 71 to 77 (Thr-Arg-Ala-Glu-Leu-Val-Thr) were also increased (P = 0.01). We suggest that an identical major histocompatibility complex class II allele (DQB1*0301) is a common marker for enhanced susceptibility and that the same amino acid residues in positions 71-77 (DQB1*0301, -0302, -0305, -0602, -0603 alleles) are found in patients with BP, OCP and OP. Our findings propose that the autoimmune response in the three different clinical variants of pemphigoid, involves the recognition by T cells of a class II region of DQB1, bound to a peptide from the basement membrane of conjunctiva, oral mucosa, and skin.
HLA class I molecules bind peptides derived from proteins degraded in the cytoplasm and display them for surveillance by the immune system. The recognition of HLA class I molecules by natural killer (NK) cells generally inhibits the lytic process. To investigate the role of peptides in the interaction between HLA class I molecules and NK receptors, we first had to identify representative endogenous peptides. Individual peptides bound to HLA-Cw*0304 were isolated and sequenced by tandem mass spectrometry. These peptides ranged in length from 8 to 11 residues and shared an alanine at position 2 and a C-terminal leucine. The murine transporters associated with antigen processing (TAP)-deficient cell line RMA-S was transfected with HLA-Cw*0304 to test whether HLA molecules loaded with a single peptide could deliver the inhibitory signal to NK cells expressing p58.2, which is a killer cell inhibitory receptor known to interact with HLA molecules bearing the HLA-Cw3 public epitope. We found that, in the absence of exogenous peptides, the HLA-Cw*0304 transfectants were killed at levels comparable to untransfected RMA-S cells whereas protection from lysis required both HLA-Cw*0304 heavy chain expression and an exogenously added HLA-Cw*0304-binding peptide. Importantly, not only were HLA-Cw*0304-binding peptides required for protection, but the ability of individual peptides to provide protection differed widely. These studies indicate that the ability to distinguish between subsets of peptides may be a general feature of HLA class I recognition by NK cells.
Top-cited authors
Rob Knight
  • University of California, San Diego
Era L Pogosova-Agadjanyan
  • Fred Hutchinson Cancer Research Center
Christian Beckmann
  • Radboud University Medical Centre (Radboudumc)
Bert Vogelstein
  • Johns Hopkins Medicine
Irving Weissman
  • Stanford Medicine