bluuebunny/crossref_metadata_2025 huggingface dataset:
Dependence of charge trapping and tunneling on the silicon-nitride (Si3N4) thickness for tunnel barrier engineered nonvolatile memory applications
Authors: Jung Myung-Ho , Kim Kwan-Su , Park Goon-Ho , Cho Won-Ju
Published: February 2009

Charge trapping and tunneling characteristics of silicon-nitride (Si3N4) layer with various thicknesses were investigated for applications of tunnel barrier engineered nonvolatile memory (NVM). A critical thickness of Si3N4 layer for suppressing the charge trapping and enhancing the tunneling sensitivity of tunnel barrier were developed. Also, the charge trap centroid and charge trap density were extracted by constant current stress method. As a result, the optimization of Si3N4 thickness considerably improved the performances of NVM.
High sensitivity pulsed laser vibrometer and its application as a laser microphone
Authors: Wang Chen-Chia , Trivedi Sudhir , Jin Feng , Swaminathan V. , Rodriguez Ponciano , Prasad Narasimha S.
Published: February 2009

We experimentally demonstrate a high sensitivity pulsed laser vibrometer that is capable of detecting optically rough surfaces vibrating with the displacement value of 75 pm as well as its application as a laser microphone. By directing the probe light beam repeatedly onto the vibrating diaphragm and/or pressure sensing interface, the sensitivity of the pulsed laser vibrometer in detecting the displacement of the vibrating diaphragm is significantly improved down to an estimated value of less than 4 pm In this paper, we present the principles of operation of this new kind of laser microphone together with experimental validations.
Monolithic integration of quantum dot containing microdisk microcavities coupled to air-suspended waveguides
Authors: Koseki Shinichi , Zhang Bingyang , De Greve Kristiaan , Yamamoto Yoshihisa
Published: February 2009

GaAs microdisk microcavities coupled to monolithic air-suspended waveguide structures are fabricated with out-of-plane light coupling achieved via the grating couplers monolithically integrated in the input and output ports. Photoluminescence signal of the whispering gallery modes is extracted from the grating couplers through the waveguide. Quality factors of modes are obtained through a transmission measurement with quality factors of up to 9500 and transmission depth of ΔT=35%.
Reducing nanoparticles in metal ablation plumes produced by two delayed short laser pulses
Authors: Noël Sylvie , Hermann Jörg
Published: February 2009

Using fast imaging and atomic force microscopy, we demonstrate that the fraction of nanoparticles in ablation plumes produced by short pulse laser irradiation of metals is strongly altered when a second laser pulse of sufficiently large delay is applied. Comparing the results obtained for gold and copper, it is shown that a significant nanoparticle reduction is only observed if the delay between both laser pulses exceeds the characteristic time of electron-lattice thermalization. We propose the reduced electronic heat transport at large lattice temperature as the dominant mechanisms for the observed nanoparticle reduction.
Rapid passage effects in nitrous oxide induced by a chirped external cavity quantum cascade laser
Authors: van Helden J. H. , Peverall R. , Ritchie G. A. D. , Walker R. J.
Published: February 2009

A widely tunable pulsed external cavity quantum cascade laser operating around 8 μm has been used to make rotationally resolved measurements of rapid passage effects in the absorption spectrum of N2O. Rapid passage signals as a function of laser power and N2O pressure are presented. Comparisons are drawn with measurements performed on the same transition with a standard distributed feedback quantum cascade laser. The initial observations on rapid passage effects induced with an external cavity quantum cascade laser show that such high power, widely tunable radiation sources may find applications in both nonlinear optics and optical sensing experiments.
Anisotropic optical response of InAs/InP quantum dot avalanche photodiodes
Authors: Chia C. K. , Dong J. R. , Ng B. K.
Published: February 2009

Polarization resolved photocurrents in InAs/InP quantum dot (QD) avalanche photodiodes (APDs) comprising of three InAs QD layers have been determined under normal incidence. The responsivity of the InAs/InP APD from 1550 nm illumination was found to increase rapidly with increasing avalanche gain. Energy separation between the heavy-hole ground-state (hh1-e1) and heavy-hole excited-state (hh2-e2) absorptions was found to increase linearly with increasing applied electric field. The difference between the photocurrents corresponds to the hh1-e1 and hh2-e2 transitions, which increases exponentially after the onset of multiplication, suggesting the TE and TM polarization effects on the photoresponse of the InAs/InP APDs are amplified by the avalanche multiplication process.
Ab initio tensile experiment on a model of an intergranular glassy film in β-Si3N4 with prismatic surfaces
Authors: Ching W. Y. , Rulis Paul , Ouyang Lizhi , Misra A.
Published: February 2009

We report the results of a large-scale ab initio simulation of an intergranular glassy film (IGF) model in β-Si3N4. It is shown that the stress-strain behavior under uniaxial load in the model with prismatic surfaces and few defective bonds is very different from an earlier IGF model with basal planes. The results are explained by the fundamental electronic structure of the model.
The Perceptual Contrast of Impossible Shadow Edges
Authors: Soranzo Alessandro , Galmonte Alessandra , Agostini Tiziano
Published: February 2009

Luminance ratios along shadow edges remain the same even when they cross reflectance borders. According to Gilchrist (1988, Perception & Psychophysics43 415–424) this so-called ratio-invariance property is a crucial factor in the perception of shadows. However, Soranzo and Agostini (2004, Perception33 1359–1368) suggested that in some conditions (named ‘impossible shadows’), a luminance pattern might still be perceived as a shadow even if the ratio-invariance property along its edge is violated. This can occur when an edge is collinear with another edge (contextual edge) which incorporates it, shares the same polarity, and generates a larger ratio. The hypothesis that impossible shadows are actually perceived as shadows is here tested by comparing the perceptual contrast of a luminance edge in the absence of a contextual edge (control condition) to that of both possible shadow edges (where the contextual and mediating edge share the same ratio) and impossible shadow edges (where the ratio of the contextual edge is larger rather than that at the mediating edge). We found that the perceived contrast of luminance edges shrinks in both possible and impossible shadow conditions rather than in the control condition. This evidence supports the hypothesis that a luminance pattern might be perceived as a shadow even when the ratio-invariance property is violated.
β-Arrestin links endothelin A receptor to β-catenin signaling to induce ovarian cancer cell invasion and metastasis
Authors: Rosanò Laura , Cianfrocca Roberta , Masi Stefano , Spinella Francesca , Di Castro Valeriana , Biroccio Annamaria , Salvati Erica , Nicotra Maria Rita , Natali Pier Giorgio , Bagnato Anna
Published: February 2009

The activation of endothelin-A receptor (ETAR) by endothelin-1 (ET-1) has a critical role in ovarian tumorigenesis and progression. To define the molecular mechanism in ET-1-induced tumor invasion and metastasis, we focused on β-arrestins as scaffold and signaling proteins of G protein-coupled receptors. Here, we demonstrate that, in ovarian cancer cells, β-arrestin is recruited to ETAR to form two trimeric complexes: one through the interaction with Src leading to epithelial growth factor receptor (EGFR) transactivation and β-catenin Tyr phosphorylation, and the second through the physical association with axin, contributing to release and inactivation of glycogen synthase kinase (GSK)-3β and β-catenin stabilization. The engagement of β-arrestin in these two signaling complexes concurs to activate β-catenin signaling pathways. We then demonstrate that silencing of both β-arrestin-1 and β-arrestin-2 inhibits ETAR-driven signaling, causing suppression of Src, mitogen-activated protein kinase (MAPK), AKT activation, as well as EGFR transactivation and a complete inhibition of ET-1-induced β-catenin/TCF transcriptional activity and cell invasion. ETAR blockade with the specific ETAR antagonist ZD4054 abrogates the engagement of β-arrestin in the interplay between ETAR and the β-catenin pathway in the invasive program. Finally, ETAR is expressed in 85% of human ovarian cancers and is preferentially co-expressed with β-arrestin-1 in the advanced tumors. In a xenograft model of ovarian metastasis, HEY cancer cells expressing β-arrestin-1 mutant metastasize at a reduced rate, highlighting the importance of this molecule in promoting metastases. ZD4054 treatment significantly inhibits metastases, suggesting that specific ETAR antagonists, by disabling multiple signaling activated by ETAR/β-arrestin, may represent new therapeutic opportunities for ovarian cancer.
Structural basis for the inhibition of the essential
Plasmodium falciparum
M1 neutral aminopeptidase
Authors: McGowan Sheena , Porter Corrine J. , Lowther Jonathan , Stack Colin M. , Golding Sarah J. , Skinner-Adams Tina S. , Trenholme Katharine R. , Teuscher Franka , Donnelly Sheila M. , Grembecka Jolanta , Mucha Artur , Kafarski Pawel , DeGori Ross , Buckle Ashley M. , Gardiner Donald L. , Whisstock James C. , Dalton John P.
Published: February 2009

Plasmodium falciparum
parasites are responsible for the major global disease malaria, which results in >2 million deaths each year. With the rise of drug-resistant malarial parasites, novel drug targets and lead compounds are urgently required for the development of new therapeutic strategies. Here, we address this important problem by targeting the malarial neutral aminopeptidases that are involved in the terminal stages of hemoglobin digestion and essential for the provision of amino acids used for parasite growth and development within the erythrocyte. We characterize the structure and substrate specificity of one such aminopeptidase,
Pf
A-M1, a validated drug target. The X-ray crystal structure of
Pf
A-M1 alone and in complex with the generic inhibitor, bestatin, and a phosphinate dipeptide analogue with potent in vitro and in vivo antimalarial activity, hPheP[CH
2
]Phe, reveals features within the protease active site that are critical to its function as an aminopeptidase and can be exploited for drug development. These results set the groundwork for the development of antimalarial therapeutics that target the neutral aminopeptidases of the parasite.
Absence of autophagy results in reactive oxygen species-dependent amplification of RLR signaling
Authors: Tal Michal Caspi , Sasai Miwa , Lee Heung Kyu , Yordy Brian , Shadel Gerald S. , Iwasaki Akiko
Published: February 2009

Autophagy is a highly conserved process that maintains homeostasis by clearing damaged organelles and long-lived proteins. The consequences of deficiency in autophagy manifest in a variety of pathological states including neurodegenerative diseases, inflammatory disorders, and cancer. Here, we studied the role of autophagy in the homeostatic regulation of innate antiviral defense. Single-stranded RNA viruses are recognized by the members of the RIG-I-like receptors (RLRs) in the cytosol. RLRs signal through IPS-1, resulting in the production of the key antiviral cytokines, type I IFNs. Autophagy-defective Atg5
−/−
cells exhibited enhanced RLR signaling, increased IFN secretion, and resistance to infection by vesicular stomatitis virus. In the absence of autophagy, cells accumulated dysfunctional mitochondria, as well as mitochondria-associated IPS-1. Reactive oxygen species (ROS) associated with the dysfunctional mitochondria were largely responsible for the enhanced RLR signaling in Atg5
−/−
cells, as antioxidant treatment blocked the excess RLR signaling. In addition, autophagy-independent increase in mitochondrial ROS by treatment of cells with rotenone was sufficient to amplify RLR signaling in WT cells. These data indicate that autophagy contributes to homeostatic regulation of innate antiviral defense through the clearance of dysfunctional mitochondria, and revealed that ROS associated with mitochondria play a key role in potentiating RLR signaling.
Rational stabilization of enzymes by computational redesign of surface charge–charge interactions
Authors: Gribenko Alexey V. , Patel Mayank M. , Liu Jiajing , McCallum Scott A. , Wang Chunyu , Makhatadze George I.
Published: February 2009

Here, we report the application of a computational approach that allows the rational design of enzymes with enhanced thermostability while retaining full enzymatic activity. The approach is based on the optimization of the energy of charge–charge interactions on the protein surface. We experimentally tested the validity of the approach on 2 human enzymes, acylphosphatase (AcPh) and Cdc42 GTPase, that differ in size (98 vs. 198-aa residues, respectively) and tertiary structure. We show that the designed proteins are significantly more stable than the corresponding WT proteins. The increase in stability is not accompanied by significant changes in structure, oligomerization state, or, most importantly, activity of the designed AcPh or Cdc42. This success of the design methodology suggests that it can be universally applied to other enzymes, on its own or in combination with the other strategies based on redesign of the interactions in the protein core.
Perforin-mediated suppression of B-cell lymphoma
Authors: Bolitho Paul , Street Shayna E. A. , Westwood Jennifer A. , Edelmann Winfried , MacGregor Duncan , Waring Paul , Murray William K. , Godfrey Dale I. , Trapani Joseph A. , Johnstone Ricky W. , Smyth Mark J.
Published: February 2009

In the present study, we have examined the effect of perforin (pfp) deficiency in 4 models of mouse B-cell lymphomagenesis. We have examined pfp loss on the background of either Mlh1 tumor suppressor allele loss or oncogene expression [Ig heavy chain (Eμ)-v-Abl, Eμ-myc, and vav-bcl2]. Pfp was shown to act as a suppressor of B-cell malignancies characteristically driven by v-Abl or bcl-2, whereas Mlh loss cooperated in accelerating spontaneous B-cell lymphomas characteristic of pfp loss. No protective role for pfp was observed in the more aggressive Eμ-myc model of B-cell lymphoma. These transgenic models have allowed us to distinguish the role of pfp in surveillance of B-cell lymphomagenesis, as opposed to its loss simply driving the onset of a spontaneous lymphoma characteristic of pfp deficiency.
The
miR-17
∼
92
cluster collaborates with the Sonic Hedgehog pathway in medulloblastoma
Authors: Uziel Tamar , Karginov Fedor V. , Xie Suqing , Parker Joel S. , Wang Yong-Dong , Gajjar Amar , He Lin , Ellison David , Gilbertson Richard J. , Hannon Gregory , Roussel Martine F.
Published: February 2009

Medulloblastomas (MBs) are the most common brain tumors in children. Some are thought to originate from cerebellar granule neuron progenitors (GNPs) that fail to undergo normal cell cycle exit and differentiation. Because microRNAs regulate numerous aspects of cellular physiology and development, we reasoned that alterations in miRNA expression might contribute to MB. We tested this hypothesis using 2 spontaneous mouse MB models with specific initiating mutations,
Ink4c
−/−;
Ptch1
+/− and
Ink4c
−/−;
p53
−/−. We found that 26 miRNAs showed increased expression and 24 miRNAs showed decreased expression in proliferating mouse GNPs and MBs relative to mature mouse cerebellum, regardless of genotype. Among the 26 overexpressed miRNAs, 9 were encoded by the
miR-17
∼
92
cluster family, a group of microRNAs implicated as oncogenes in several tumor types. Analysis of human MBs demonstrated that 3
miR-17
∼
92
cluster miRNAs (
miR-92
,
miR-19a
, and
miR-20
) were also overexpressed in human MBs with a constitutively activated Sonic Hedgehog (SHH) signaling pathway, but not in other forms of the disease. To test whether the
miR-17
∼
92
cluster could promote MB formation, we enforced expression of these miRNAs in GNPs isolated from cerebella of postnatal (P) day P6
Ink4c
−/−;
Ptch1
+/− mice. These, but not similarly engineered cells from
Ink4c
−/−;
p53
−/− mice, formed MBs in orthotopic transplants with complete penetrance. Interestingly, orthotopic mouse tumors ectopically expressing
miR-17
∼
92
lost expression of the wild-type
Ptch1
allele. Our findings suggest a functional collaboration between the
miR-17
∼
92
cluster and the SHH signaling pathway in the development of MBs in mouse and man.
Association of
FOXO3A
variation with human longevity confirmed in German centenarians
Authors: Flachsbart Friederike , Caliebe Amke , Kleindorp Rabea , Blanché Hélène , von Eller-Eberstein Huberta , Nikolaus Susanna , Schreiber Stefan , Nebel Almut
Published: February 2009

The human forkhead box O3A gene (
FOXO3A
) encodes an evolutionarily conserved key regulator of the insulin–IGF1 signaling pathway that is known to influence metabolism and lifespan in model organisms. A recent study described 3 SNPs in the
FOXO3A
gene that were statistically significantly associated with longevity in a discovery sample of long-lived men of Japanese ancestry [Willcox
et al.
(2008)
Proc Natl Acad Sci USA
105:13987–13992]. However, this finding required replication in an independent population. Here, we have investigated 16 known
FOXO3A
SNPs in an extensive collection of 1,762 German centenarians/nonagenarians and younger controls and provide evidence that polymorphisms in this gene were indeed associated with the ability to attain exceptional old age. The
FOXO3A
association was considerably stronger in centenarians than in nonagenarians, highlighting the importance of centenarians for genetic longevity research. Our study extended the initial finding observed in Japanese men to women and indicates that both genders were likely to be equally affected by variation in
FOXO3A
. Replication in a French centenarian sample generated a trend that supported the previous results. Our findings confirmed the initial discovery in the Japanese sample and indicate
FOXO3A
as a susceptibility gene for prolonged survival in humans.
Genome-wide association study identifies
NRG1
as a susceptibility locus for Hirschsprung's disease
Authors: Garcia-Barcelo Maria-Mercè , Tang Clara Sze-man , Ngan Elly Sau-wai , Lui Vincent Chi-hang , Chen Yan , So Man-ting , Leon Thomas Yuk-yu , Miao Xiao-ping , Shum Cathy Ka-yee , Liu Feng-qin , Yeung Ming-yiu , Yuan Zhen-wei , Guo Wei-hong , Liu Lei , Sun Xiao-bing , Huang Liu-ming , Tou Jin-fa , Song You-qiang , Chan Danny , Cheung Kenneth M. C. , Wong Kenneth Kak-yuen , Cherny Stacey S. , Sham Pak-chung , Tam Paul Kwong-hang
Published: February 2009

Hirschsprung's disease (HSCR), or aganglionic megacolon, is a congenital disorder characterized by the absence of enteric ganglia in variable portions of the distal intestine.
RET
is a well-established susceptibility locus, although existing evidence strongly suggests additional loci contributing to sporadic HSCR. To identify these additional genetic loci, we carried out a genome-wide association study using the
Affymetrix
500K marker set. We successfully genotyped 293,836 SNPs in 181 Chinese subjects with sporadic HSCR and 346 ethnically matched control subjects. The SNPs most associated with HSCR were genotyped in an independent set of 190 HSCR and 510 control subjects. Aside from SNPs in
RET
, the strongest overall associations in plausible candidate genes were found for 2 SNPs located in intron 1 of the neuregulin1 gene (
NRG1
) on 8p12, with rs16879552 and rs7835688 yielding odds ratios of 1.68 [CI
95%
:(1.40, 2.00),
P
= 1.80 × 10
−8
] and 1.98 [CI
95%
:(1.59, 2.47),
P
= 1.12 × 10
−9
], respectively, for the heterozygous risk genotypes under an additive model. There was also a significant interaction between
RET
and
NRG1
(
P
= 0.0095), increasing the odds ratio 2.3-fold to 19.53 for the
RET
rs2435357 risk genotype (TT) in the presence of the
NRG1
rs7835688 heterozygote, indicating that
NRG1
is a modifier of HSRC penetrance. Our highly significant association findings are backed-up by the important role of NRG1 as regulator of the development of the enteric ganglia precursors. The identification of
NRG1
as an additional HSCR susceptibility locus not only opens unique fields of investigation into the mechanisms underlying the HSCR pathology, but also the mechanisms by which a discrete number of loci interact with each other to cause disease.
Principal determinants leading to transition state formation of a protein–protein complex, orientation trumps side-chain interactions
Authors: Horn James R. , Sosnick Tobin R. , Kossiakoff Anthony A.
Published: February 2009

The binding transition state (TS) is the rate-limiting step for transient molecular interactions. This important step in the molecular recognition process, however, is largely understood only at a qualitative level. To establish a more quantitative picture of the TS structure, we exploit a set of biophysical techniques that have provided major insights in protein folding applications. As a model system representing the large class of “weakly charged” protein–protein interactions, we examine the binding of a variety of human growth hormone (hGH) variants to the human growth hormone receptor (hGHR) and the human prolactin receptor (hPRLR). hGH variants were chosen to probe different features of the TS structure, based on their highly reengineered interfaces. Both Eyring and urea (
m
value) analyses suggest that the majority of binding surface burial occurs after TS. A comprehensive φ analysis showed that individual hGH interface residues do not contribute energetically to the stability of the TS, but there is a TS “hot spot” in the receptor. Zinc dependence studies that take advantage of an endogenous tetracoordinated interfacial metal binding demonstrate that surfaces of the molecules have attained a high orientational complementarity by the time the TS is reached. The model that best fits these data are that a “knobs-into-holes” process precisely aligns the two molecular interfaces in forming the TS structure. Surprisingly, most of the thermodynamic character of the binding reaction is focused in the fine-tuning process occurring after TS.
Structure of a trimeric variant of the Epstein–Barr virus glycoprotein B
Authors: Backovic Marija , Longnecker Richard , Jardetzky Theodore S.
Published: February 2009

Epstein–Barr virus (EBV) is a herpesvirus that is associated with development of malignancies of lymphoid tissue. EBV infections are life-long and occur in >90% of the population. Herpesviruses enter host cells in a process that involves fusion of viral and cellular membranes. The fusion apparatus is comprised of envelope glycoprotein B (gB) and a heterodimeric complex made of glycoproteins H and L. Glycoprotein B is the most conserved envelope glycoprotein in human herpesviruses, and the structure of gB from Herpes simplex virus 1 (HSV-1) is available. Here, we report the crystal structure of the secreted EBV gB ectodomain, which forms 16-nm long spike-like trimers, structurally homologous to the postfusion trimers of the fusion protein G of vesicular stomatitis virus (VSV). Comparative structural analyses of EBV gB and VSV G, which has been solved in its pre and postfusion states, shed light on gB residues that may be involved in conformational changes and membrane fusion. Also, the EBV gB structure reveals that, despite the high sequence conservation of gB in herpesviruses, the relative orientations of individual domains, the surface charge distributions, and the structural details of EBV gB differ from the HSV-1 protein, indicating regions and residues that may have important roles in virus-specific entry.
Contrasting size evolution in marine and freshwater diatoms
Authors: Litchman E. , Klausmeier C. A. , Yoshiyama K.
Published: February 2009

Diatoms are key players in the global carbon cycle and most aquatic ecosystems. Their cell sizes impact carbon sequestration and energy transfer to higher trophic levels. We report fundamental differences in size distributions of marine and freshwater diatoms, with marine diatoms significantly larger than freshwater species. An evolutionary game theoretical model with empirical allometries of growth and nutrient uptake shows that these differences can be explained by nitrogen versus phosphorus limitation, nutrient fluctuations and mixed layer depth differences. Constant and pulsed phosphorus supply select for small sizes, as does constant nitrogen supply. In contrast, intermediate frequency nitrogen pulses common in the ocean select for large sizes or the evolutionarily stable coexistence of large and small sizes. Size-dependent sinking interacts with mixed layer depth (MLD) to further modulate optimal sizes, with smaller sizes selected for by strong sinking and shallow MLD. In freshwaters, widespread phosphorus limitation, together with strong sinking and shallow MLD produce size distributions with smaller range, means and upper values, compared with the ocean. Shifting patterns of nutrient limitation and mixing may alter diatom size distributions, affecting global carbon cycle and the structure and functioning of aquatic ecosystems.
Counting the number of releasable synaptic vesicles in a presynaptic terminal
Authors: Ikeda Kaori , Bekkers John M.
Published: February 2009

Synaptic transmission depends on the continued availability of neurotransmitter-filled synaptic vesicles (SVs) for triggered release from presynaptic boutons. Surprisingly, small boutons in the brain, that already contain comparatively few SVs, are thought to retain the majority of these SVs in a “reserve” pool that is not mobilized under physiological conditions. Why such a scarce synaptic resource is normally inaccessible has been a matter of debate. Here, we readdress this issue by developing an electrophysiological approach for counting SVs released from boutons formed by a single, isolated neuron on itself (“autapses”). We show that, after treatment with Bafilomycin A1 to prevent reloading of discharged SVs with glutamate, each SV is counted only once on first-time release. Hence, by integrating all autaptic currents as they run down over time, we can estimate the total number of SVs released by a single neuron. This total can be normalized to the number of boutons on the neuron, giving the mean number of SVs released per bouton. We estimate that up to ≈130 vesicles can be released per bouton over ≈10 min of stimulation at 0.2 Hz. This number of vesicles represents a substantial proportion of the total number of SVs (100–200) that have been counted in these boutons by using electron microscopy. Thus, mild electrical stimulation, when maintained for sufficient time, causes the eventual release of many of the SVs in a bouton, including those in the putative reserve pool. This result suggests that SVs are functionally homogeneous in that the majority can contribute to basal synaptic transmission.
Dislocation theory of chirality-controlled nanotube growth
Authors: Ding Feng , Harutyunyan Avetik R. , Yakobson Boris I.
Published: February 2009

The periodic makeup of carbon nanotubes suggests that their formation should obey the principles established for crystals. Nevertheless, this important connection remained elusive for decades and no theoretical regularities in the rates and product type distribution have been found. Here we contend that any nanotube can be viewed as having a screw dislocation along the axis. Consequently, its growth rate is shown to be proportional to the Burgers vector of such dislocation and therefore to the chiral angle of the tube. This is corroborated by the ab initio energy calculations, and agrees surprisingly well with diverse experimental measurements, which shows that the revealed kinetic mechanism and the deduced predictions are remarkably robust across the broad base of factual data.
Selective translocation of intracellular Smoothened to the primary cilium in response to Hedgehog pathway modulation
Authors: Wang Yu , Zhou Zhe , Walsh Christopher T. , McMahon Andrew P.
Published: February 2009

Smoothened (Smo), a 7-pass transmembrane protein, is essential for transduction of a Hedgehog (Hh) signal across the cell membrane. Smo is also the principle therapeutic target for several candidate drugs in the treatment of Hh-related diseases. Mammalian Smo translocates to the primary cilium in response to Sonic hedgehog (Shh) ligand-mediated signaling. A mechanistic understanding of Smo translocation and its interactions with drug candidates is pivotal to our understanding of Hh signaling and the design, development and application of successful drugs. We established a system in which Smo was dual-labeled with GFP and a 12-aa tag whose recognition by an enzymatic process enables the posttranslational labeling of Smo in the cell membrane within the living cell. These tools enable the simultaneous visualization of all cellular Smo, and more specifically, the cell membrane restricted subpopulation. Using this system, we demonstrate that cyclopamine, a widely used Hh antagonist, induces a cilial translocation of Smo similar to that reported for Shh ligand and several Hh agonists. In contrast, other antagonists abrogate the Shh-induced, cilial translocation of Smo. We present evidence that the majority of cilial-localized Smo originates from an intracellular source and may traffic to the primary cilium through an intraflagellar transport (IFT) pathway.
Molecular simulations elucidate the substrate translocation pathway in a glutamate transporter
Authors: Gu Yan , Shrivastava Indira H. , Amara Susan G. , Bahar Ivet
Published: February 2009

Glutamate transporters are membrane proteins found in neurons and glial cells, which play a critical role in regulating cell signaling by clearing glutamate released from synapses. Although extensive biochemical and structural studies have shed light onto different aspects of glutamate transport, the time-resolved molecular mechanism of substrate (glutamate or aspartate) translocation, that is, the sequence of events occurring at the atomic level after substrate binding and before its release intracellularly remain to be elucidated. We identify an energetically preferred permeation pathway of ≈23 Å between the helix HP1b on the hairpin HP1 and the transmembrane helices TM7 and TM8, using the high resolution structure of the transporter from
Pyrococcus horikoshii
(Glt
Ph
) in steered molecular dynamics simulations. Detailed potential of mean force calculations along the putative pathway reveal 2 energy barriers encountered by the substrate (aspartate) before it reaches the exit. The first barrier is surmounted with the assistance of 2 conserved residues (S278 and N401) and a sodium ion (Na2); and the second, by the electrostatic interactions with D405 and another sodium ion (Na1). The observed critical interactions and mediating role of conserved residues in the core domain, the accompanying conformational changes (in both substrate and transporter) that relieve local strains, and the unique coupling of aspartate transport to Na
+
dislocation provide insights into methods for modulating substrate transport.
A mouse forward genetics screen identifies LISTERIN as an E3 ubiquitin ligase involved in neurodegeneration
Authors: Chu Jessie , Hong Nancy A. , Masuda Claudio A. , Jenkins Brian V. , Nelms Keats A. , Goodnow Christopher C. , Glynne Richard J. , Wu Hua , Masliah Eliezer , Joazeiro Claudio A. P. , Kay Steve A.
Published: February 2009

A mouse neurological mutant,
lister
, was identified through a genome-wide
N
-ethyl-
N
-nitrosourea (ENU) mutagenesis screen. Homozygous
lister
mice exhibit profound early-onset and progressive neurological and motor dysfunction.
lister
encodes a RING finger protein, LISTERIN, which functions as an E3 ubiquitin ligase in vitro. Although
lister
is widely expressed in all tissues, motor and sensory neurons and neuronal processes in the brainstem and spinal cord are primarily affected in the mutant. Pathological signs include gliosis, dystrophic neurites, vacuolated mitochondria, and accumulation of soluble hyperphosphorylated tau. Analysis with a different
lister
allele generated through targeted gene trap insertion reveals LISTERIN is required for embryonic development and confirms that direct perturbation of a LISTERIN-regulated process causes neurodegeneration. The
lister
mouse uncovers a pathway involved in neurodegeneration and may serves as a model for understanding the molecular mechanisms underlying human neurodegenerative disorders.
Power of deep, all-exon resequencing for discovery of human trait genes
Authors: Kryukov Gregory V. , Shpunt Alexander , Stamatoyannopoulos John A. , Sunyaev Shamil R.
Published: February 2009

The ability to sequence cost-effectively all of the coding regions of a given individual genome is rapidly approaching, with the potential for whole-genome resequencing not far behind. Initiatives are currently underway to phenotype hundreds of thousands of individuals for major human traits. Here, we determine the power for de novo discovery of genes related to human traits by resequencing all human exons in a clinical population. We analyze the potential of the gene discovery strategy that combines multiple rare variants from the same gene and treats genes, rather than individual alleles, as the units for the association test. By using computer simulations based on deep resequencing data for the European population, we show that genes meaningfully affecting a human trait can be identified in an unbiased fashion, although large sample sizes would be required to achieve substantial power.
Drosophila
hnRNP A1 homologs Hrp36/Hrp38 enhance U2-type versus U12-type splicing to regulate alternative splicing of the
prospero
twintron
Authors: Borah Sumit , Wong Anthony C. , Steitz Joan A.
Published: February 2009

During
Drosophila
embryogenesis, the transcription factor Prospero is critical for neuronal differentiation and axonal outgrowth. The
prospero
pre-mRNA undergoes alternative splicing, but is unique in that it harbors a rare twintron whereby one intron lies embedded within another. The innermost intron is excised by the major U2-type spliceosome and the outermost is excised by the minor U12-type spliceosome. Previously, an intronic purine-rich element (PRE) was identified as an enhancer of both U2- and U12-type splicing, with a greater effect on the U2-type pathway. We find that the PRE binds
Drosophila
homologs of heterogeneous nuclear ribonucleoprotein (hnRNP) A1, Hrp38 and Hrp36. RNAi-mediated knockdown of these proteins in S2 cells specifically decreases U2-type splicing of the twintron, which is surprising because hnRNPs usually are repressive. Conversely, tethering Hrp38 to the twintron increases U2-type splicing. Thus, developmentally regulated alternative splicing of the
prospero
twintron can be explained by documented changes in the abundance of these hnRNP A1-like proteins during embryogenesis.
Noninvasive prediction of tumor responses to gemcitabine using positron emission tomography
Authors: Laing Rachel E. , Walter Martin A. , Campbell Dean O. , Herschman Harvey R. , Satyamurthy Nagichettiar , Phelps Michael E. , Czernin Johannes , Witte Owen N. , Radu Caius G.
Published: February 2009

Gemcitabine (2′,2′-difluorodeoxycytidine, dFdC) and cytosine arabinoside (cytarabine, ara-C) represent a class of nucleoside analogs used in cancer chemotherapy. Administered as prodrugs, dFdC and ara-C are transported across cell membranes and are converted to cytotoxic derivatives through consecutive phosphorylation steps catalyzed by endogenous nucleoside kinases. Deoxycytidine kinase (DCK) controls the rate-limiting step in the activation cascade of dFdC and ara-C. DCK activity varies significantly among individuals and across different tumor types and is a critical determinant of tumor responses to these prodrugs. Current assays to measure DCK expression and activity require biopsy samples and are prone to sampling errors. Noninvasive methods that can detect DCK activity in tumor lesions throughout the body could circumvent these limitations. Here, we demonstrate an approach to detecting DCK activity in vivo by using positron emission tomography (PET) and
18
F-labeled 1-(2′-deoxy-2′-fluoroarabinofuranosyl) cytosine] ([
18
F]FAC), a PET probe recently developed by our group. We show that [
18
F]FAC is a DCK substrate with an affinity similar to that of dFdC. In vitro, accumulation of [
18
F]FAC in murine and human leukemia cell lines is critically dependent on DCK activity and correlates with dFdC sensitivity. In mice, [
18
F]FAC accumulates selectively in DCK-positive vs. DCK-negative tumors, and [
18
F]FAC microPET scans can predict responses to dFdC. We suggest that [
18
F]FAC PET might be useful for guiding treatment decisions in certain cancers by enabling individualized chemotherapy.
Phosphorylated self-peptides alter human leukocyte antigen class I-restricted antigen presentation and generate tumor-specific epitopes
Authors: Petersen Jan , Wurzbacher Stephanie J. , Williamson Nicholas A. , Ramarathinam Sri H. , Reid Hugh H. , Nair Ashish K. N. , Zhao Anne Y. , Nastovska Roza , Rudge Geordie , Rossjohn Jamie , Purcell Anthony W.
Published: February 2009

Human leukocyte antigen (HLA) class I molecules present a variety of posttranslationally modified epitopes at the cell surface, although the consequences of such presentation remain largely unclear. Phosphorylation plays a critical cellular role, and deregulation in phosphate metabolism is associated with disease, including autoimmunity and tumor immunity. We have solved the high-resolution structures of 3 HLA A2-restricted phosphopeptides associated with tumor immunity and compared them with the structures of their nonphosphorylated counterparts. Phosphorylation of the epitope was observed to affect the structure and mobility of the bound epitope. In addition, the phosphoamino acid stabilized the HLA peptide complex in an epitope-specific manner and was observed to exhibit discrete flexibility within the antigen-binding cleft. Collectively, our data suggest that phosphorylation generates neoepitopes that represent demanding targets for T-cell receptor ligation. These findings provide insights into the mode of phosphopeptide presentation by HLA as well as providing a platform for the rational design of a generation of posttranslationally modified tumor vaccines.
Vpu enhances HIV-1 virus release in the absence of Bst-2 cell surface down-modulation and intracellular depletion
Authors: Miyagi Eri , Andrew Amy J. , Kao Sandra , Strebel Klaus
Published: February 2009

HIV-1 Vpu enhances the release of virions from infected cells. Recent work identified Bst-2/CD317/tetherin as a host factor whose inhibitory activity on viral release is counteracted by Vpu. A current working model proposes that Bst-2 inhibits virus release by tethering viral particles to the cell surface. Here, we analyzed endogenous Bst-2 with respect to its effect on virus release from HeLa cells, T cells, and macrophages. We noted significant cell type-dependent variation in Bst-2 expression. Vpu caused a reduction in Bst-2 expression in transfected HeLa cells and long-term infected macrophages. However, Vpu expression did not result in cell surface down-modulation of Bst-2 or a reduction in intracellular Bst-2 expression in CEMx174 or H9 cells, yet virus replication in these cells was Vpu-responsive. Surprisingly, Bst-2 was undetectable in cell-free virions that were recovered from the surface of HeLa cells by physical shearing, suggesting that a tethering model may not explain all of the functional properties of Bst-2. Taken together we conclude that enhancement of virus release by Vpu does not, at least in CEMx174 and H9 cells, require cell surface down-modulation or intracellular depletion of Bst-2, nor does it entail exclusion of Bst-2 from viral particles.
The C-terminal region of activation-induced cytidine deaminase is responsible for a recombination function other than DNA cleavage in class switch recombination
Authors: Doi Tomomitsu , Kato Lucia , Ito Satomi , Shinkura Reiko , Wei Min , Nagaoka Hitoshi , Wang Jishu , Honjo Tasuku
Published: February 2009

Activation-induced cytidine deaminase (AID) is an essential factor for the class switch recombination (CSR) and somatic hypermutation (SHM) of Ig genes. CSR and SHM are initiated by AID-induced DNA breaks in the S and V regions, respectively. Because truncation or frame-shift mutations at the carboxyl (C)-terminus of AID abolishes CSR but not SHM, the C-terminal region of AID likely is required for the targeting of DNA breaks in the S region. To test this hypothesis, we determined the precise location and relative amounts of AID-induced DNA cleavage using an in situ DNA end-labeling method. We established CH12F3–2 cell transfectants expressing the estrogen receptor (ER) fused with wild-type (WT) AID or a deletion mutant lacking the C-terminal 16 aa, JP8Bdel. We found that AID-ER, but not JP8Bdel-ER, caused a CSR to IgA from the addition of 4-hydroxy tamoxifen. In contrast, both WT AID and JP8Bdel induced DNA breaks in both the V and S regions. In addition, JP8Bdel enhanced c-myc/IgH translocations. Our findings indicate that the C-terminal domain of AID is not required for S-region DNA breaks but is required for S-region recombination after DNA cleavage. Therefore, AID does not distinguish between the V and S regions for cleavage, but carries another function specific to CSR.
Oncogenic EGFR signaling cooperates with loss of tumor suppressor gene functions in gliomagenesis
Authors: Zhu Haihao , Acquaviva Jaime , Ramachandran Pranatartiharan , Boskovitz Abraham , Woolfenden Steve , Pfannl Rolf , Bronson Roderick T. , Chen John W. , Weissleder Ralph , Housman David E. , Charest Al
Published: February 2009

Glioblastoma multiforme (GBM) is a highly lethal brain tumor for which little treatment is available. The epidermal growth factor receptor (EGFR) signaling pathway is thought to play a crucial role in GBM pathogenesis, initiating the early stages of tumor development, sustaining tumor growth, promoting infiltration, and mediating resistance to therapy. The importance of this pathway is highlighted in the fact that EGFR is mutationally activated in over 50% of GBM tumors. Consistent with this, we show here that concomitant activation of wild-type and/or mutant (vIII) EGFR and ablation of Ink4A/Arf and PTEN tumor suppressor gene function in the adult mouse central nervous system generates a fully penetrant, rapid-onset high-grade malignant glioma phenotype with prominent pathological and molecular resemblance to GBM in humans. Studies of the activation of signaling events in these GBM tumor cells revealed notable differences between wild-type and vIII EGFR-expressing cells. We show that wild-type EGF receptor signals through its canonical pathways, whereas tumors arising from expression of mutant EGFR
vIII
do not use these same pathways. Our findings provide critical insights into the role of mutant EGFR signaling function in GBM tumor biology and set the stage for testing of targeted therapeutic agents in the preclinical models described herein.
Knockin of mutant PIK3CA activates multiple oncogenic pathways
Authors: Gustin John P. , Karakas Bedri , Weiss Michele B. , Abukhdeir Abde M. , Lauring Josh , Garay Joseph P. , Cosgrove David , Tamaki Akina , Konishi Hiroyuki , Konishi Yuko , Mohseni Morassa , Wang Grace , Rosen D. Marc , Denmeade Samuel R. , Higgins Michaela J. , Vitolo Michele I. , Bachman Kurtis E. , Park Ben Ho
Published: February 2009

The phosphatidylinositol 3-kinase subunit PIK3CA is frequently mutated in human cancers. Here we used gene targeting to “knock in”
PIK3CA
mutations into human breast epithelial cells to identify new therapeutic targets associated with oncogenic PIK3CA. Mutant
PIK3CA
knockin cells were capable of epidermal growth factor and mTOR-independent cell proliferation that was associated with AKT, ERK, and GSK3β phosphorylation. Paradoxically, the GSK3β inhibitors lithium chloride and SB216763 selectively decreased the proliferation of human breast and colorectal cancer cell lines with oncogenic
PIK3CA
mutations and led to a decrease in the GSK3β target gene
CYCLIN D1
. Oral treatment with lithium preferentially inhibited the growth of nude mouse xenografts of HCT-116 colon cancer cells with mutant
PIK3CA
compared with isogenic HCT-116 knockout cells containing only wild-type
PIK3CA
. Our findings suggest GSK3β is an important effector of mutant
PIK3CA
, and that lithium, an FDA-approved therapy for bipolar disorders, has selective antineoplastic properties against cancers that harbor these mutations.
Harnessing Hsp90 function as a powerful, broadly effective therapeutic strategy for fungal infectious disease
Authors: Cowen Leah E. , Singh Sheena D. , Köhler Julia R. , Collins Cathy , Zaas Aimee K. , Schell Wiley A. , Aziz Hamza , Mylonakis Eleftherios , Perfect John R. , Whitesell Luke , Lindquist Susan
Published: February 2009

Invasive fungal infections are a leading cause of mortality among immunocompromised individuals. Treatment is notoriously difficult with the limited armamentarium of antifungal drugs, whose efficacy is compromised by host toxicity, a limited activity spectrum, or the emergence of drug resistance. We previously established that the molecular chaperone Hsp90 enables the emergence and maintenance of fungal drug resistance. For the most prevalent fungal pathogen of humans,
Candida albicans
, Hsp90 mediates resistance to azoles, which inhibit ergosterol biosynthesis and are the most widely deployed antifungals in the clinic. For the emerging opportunistic pathogen
Aspergillus terreus
, Hsp90 is required for basal resistance to echinocandins, which inhibit β(1, 3)-glucan synthesis and are the only new class of antifungals to reach the clinic in decades. Here, we explore the therapeutic potential of Hsp90 inhibitors in fungal disease using a tractable host-model system, larvae of the greater wax moth
Galleria mellonella
, and a murine model of disseminated disease. Combination therapy with Hsp90 inhibitors that are well tolerated in humans and an azole rescued larvae from lethal
C. albicans
infections. Combination therapy with an Hsp90 inhibitor and an echinocandin rescued larvae from infections with the most lethal mold,
Aspergillus fumigatus
. In a murine model of disseminated candidiasis, genetic compromise of
C. albicans HSP90
expression enhanced the therapeutic efficacy of an azole. Thus, harnessing Hsp90 provides a much-needed strategy for improving the treatment of fungal disease because it enhances the efficacy of existing antifungals, blocks the emergence of drug resistance, and exerts broad-spectrum activity against diverse fungal pathogens.
A comparative study of the palliative care needs of heart failure and cancer patients
Authors: O'Leary Norma , Murphy Niamh F. , O'Loughlin Christina , Tiernan Eoin , McDonald Kenneth
Published: February 2009

Aims

Studies suggest that patients with advanced heart failure (HF) have unmet palliative care (PC) needs. However, many of these studies have been retrospective or based on patients receiving poorly coordinated ad hoc care. We aimed to demonstrate whether the PC needs of patients with advanced HF receiving specialist multidisciplinary coordinated care are similar to cancer patients deemed to have specialist PC needs; thereby justifying the extension of specialist PC services to HF patients.

Methods and results

This was a cross‐sectional comparative cohort study of 50 HF patients and 50 cancer patients, using quantitative and qualitative methods. Both patient cohorts were statistically indistinguishable in terms of symptom burden, emotional wellbeing, and quality‐of‐life scores. HF patients had good access to community and social support. HF patients particularly valued the close supervision, medication monitoring, ease of access to service, telephone support, and key worker provided at the HF unit. A small subset of patients had unmet PC needs. A palliative transition point is described.

Conclusion

HF patients should not be excluded from specialist PC services. However, the majority of their needs can be met at a HF unit. Recognition of the palliative transition point may be key to ensuring that end‐of‐life issues are addressed. The palliative transition point needs further evaluation.