bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhanced tunnel magnetoresistance in fully epitaxial ZnO:Co-based magnetic tunnel junctions with Mg-doped ZnO barrier
Authors: He Shumin , Bai Hongliang , Liu Guolei , Li Qiang , Yan Shishen , Chen Yanxue , Mei Liangmo , Liu Houfang , Wang Shouguo , Han Xiufeng
Published: March 2012

The fully epitaxial ZnO-based ZnO:Co/ZnO:Mg/ZnO:Co magnetic tunnel junctions were grown on Al2O3(0001) substrate by oxygen plasma-assisted molecular beam epitaxy. The magnetoresistance behavior and spin injection through ZnO:Mg barrier were investigated. An enhanced positive tunnel magnetoresistance ratio of 85.6% is observed at 1.8 T at 5 K. The junction resistance at zero magnetic field is linear with respect to temperature power law T−4/3 between 5 K and 70 K, indicating that carriers tunnel through ZnO:Mg barrier via two localized states.
High-Tc superconducting quantum interference device recordings of spontaneous brain activity: Towards high-Tc magnetoencephalography
Authors: Öisjöen F. , Schneiderman J. F. , Figueras G. A. , Chukharkin M. L. , Kalabukhov A. , Hedström A. , Elam M. , Winkler D.
Published: March 2012

We have performed single- and two-channel high transition temperature (high-Tc) superconducting quantum interference device (SQUID) magnetoencephalography (MEG) recordings of spontaneous brain activity in two healthy human subjects. We demonstrate modulation of two well-known brain rhythms: the occipital alpha rhythm and the mu rhythm found in the motor cortex. We further show that despite higher noise-levels compared to their low-Tc counterparts, high-Tc SQUIDs can be used to detect and record physiologically relevant brain rhythms with comparable signal-to-noise ratios. These results indicate the utility of high-Tc technology in MEG recordings of a broader range of brain activity.
A miniature cubic anvil apparatus for optical measurement under high pressure
Authors: Kawazoe Takaaki
Published: March 2012

A miniature cubic anvil apparatus was developed for optical measurement on materials in a high-pressure space of 8–10 mm3 under high pressure, and preliminary experiments were conducted to 3.6 GPa at room temperature with optical visual observation and ruby fluorescence measurement. In the apparatus, a cubic pressure medium was squeezed with six tungsten carbide anvils, which are driven with a pair of guide blocks by tightening four loading screws. Optical access on the sample was made through holes in axial anvils and the guide blocks as well as optical windows made of Al2O3 single crystals embedded in the pressure medium. The apparatus is compact and light, ∼53 mm in diameter and height and ∼530 g in weight, and the features of the apparatus benefit easy application of the apparatus to various types of standard optical measurement systems. The optical measurement on the sample in the high-pressure space of 8–10 mm3 should greatly contribute to advancements of studies relevant to high-pressure behaviors of materials.
Elimination of domain backswitching in BiFe0.95Mn0.05O3 thin films by lowering the layer thickness
Authors: Liu Jingjing , Yin Zaimei , Zhang Hongyan , Sun Wen , Jiang Bo , Hu Guangda
Published: March 2012

BiFe0.95Mn0.05O3 thin films with different layer thicknesses annealed at temperatures ranging from 500 to 600 °C were fabricated on indium tin oxide/glass substrates using a metal organic decomposition process. The ferroelectric hysteresis loops reveal that the remanent polarization (Pr) in the films of 42, 31, and 20 nm per layer can be improved dramatically by increasing the annealing temperature, while the Pr in the films of 25 nm/l shows weak dependence on the annealing temperature. For a given temperature, the Pr in the films of 25 nm/l is much larger than those in the films of 20, 31, and 42 nm/l, especially at temperatures below 575 °C. These phenomena can be ascribed to the lower content of defect complexes resulting from the columnar structure formed by decreasing layer thickness, which in turn results in the elimination of domain backswitching. The results also indicate that reducing the layer thickness moderately is an effective method for lowering the annealing temperature.
Seasonal field metabolic rate and water influx of captive-bred reintroduced yellow-footed rock-wallabies (Petrogale xanthopus celeris)
Authors: Lapidge Steven J. , Munn Adam J.
Published: March 2012

Captive breeding and release is a tool used by conservation biologists to re-establish populations of endangered or locally extinct species. Reintroduced animals that have been bred in captivity must learn to meet the challenges posed by free living, and to adjust to local environmental conditions, food and water sources. How well reintroduced animals might meet these challenges is uncertain as few longitudinal studies have investigated the physiology of reintroduced animals or the implications of this for successful establishment of new populations. Here we have evaluated long-term, seasonal energy and water use by reintroduced yellow-footed rock-wallabies (Petrogale xanthopus celeris), an endangered medium-sized marsupial that inhabits rocky outcrops across Australia’s arid and semiarid rangelands. Captive-bred rock-wallabies were reintroduced to an area within the known boundaries of their former range, in south-western Queensland, Australia. Post-release water turnover rates (WTR) and field metabolic rates (FMR) were measured during their first wet summer and dry winter, by means of the doubly labelled water method. Total body water (73.1%), FMR (1650.0 kJ day–1), female fecundity (100%), and male and female body masses and survival were consistent between seasons, but rates of water turnover were significantly lower for all animals during the dry winter (174.3 mL day–1) than during the wet summer (615.0 mL day–1). There were no significant differences in WTR or FMR between males and lactating females (in either season).
Role of the translationally controlled tumor protein in DNA damage sensing and repair
Authors: Zhang Jie , de Toledo Sonia M. , Pandey Badri N. , Guo Guozheng , Pain Debkumar , Li Hong , Azzam Edouard I.
Published: March 2012

The translationally controlled tumor protein (TCTP) is essential for survival by mechanisms that as yet are incompletely defined. Here we describe an important role of TCTP in response to DNA damage. Upon exposure of normal human cells to low-dose γ rays, the TCTP protein level was greatly increased, with a significant enrichment in nuclei. TCTP up-regulation occurred in a manner dependent on ataxia-telangiectasia mutated (ATM) kinase and the DNA-dependent protein kinase and was associated with protective effects against DNA damage. In chromatin of irradiated cells, coimmunoprecipitation experiments showed that TCTP forms a complex with ATM and γH2A.X, in agreement with its distinct localization with the foci of the DNA damage-marker proteins γH2A.X, 53BP1, and P-ATM. In cells lacking TCTP, repair of chromosomal damage induced by γ rays was compromised significantly. TCTP also was shown to interact with p53 and the DNA-binding subunits, Ku70 and Ku80, of DNA-dependent protein kinase. TCTP knockdown led to decreased levels of Ku70 and Ku80 in nuclei of irradiated cells and attenuated their DNA-binding activity. It also attenuated the radiation-induced G
1
delay but prolonged the G
2
delay. TCTP therefore may play a critical role in maintaining genomic integrity in response to DNA-damaging agents.
Conservation and divergence in Toll-like receptor 4-regulated gene expression in primary human versus mouse macrophages
Authors: Schroder Kate , Irvine Katharine M. , Taylor Martin S. , Bokil Nilesh J. , Le Cao Kim-Anh , Masterman Kelly-Anne , Labzin Larisa I. , Semple Colin A. , Kapetanovic Ronan , Fairbairn Lynsey , Akalin Altuna , Faulkner Geoffrey J. , Baillie John Kenneth , Gongora Milena , Daub Carsten O. , Kawaji Hideya , McLachlan Geoffrey J. , Goldman Nick , Grimmond Sean M. , Carninci Piero , Suzuki Harukazu , Hayashizaki Yoshihide , Lenhard Boris , Hume David A. , Sweet Matthew J.
Published: March 2012

Evolutionary change in gene expression is generally considered to be a major driver of phenotypic differences between species. We investigated innate immune diversification by analyzing interspecies differences in the transcriptional responses of primary human and mouse macrophages to the Toll-like receptor (TLR)–4 agonist lipopolysaccharide (LPS). By using a custom platform permitting cross-species interrogation coupled with deep sequencing of mRNA 5′ ends, we identified extensive divergence in LPS-regulated orthologous gene expression between humans and mice (24% of orthologues were identified as “divergently regulated”). We further demonstrate concordant regulation of human-specific LPS target genes in primary pig macrophages. Divergently regulated orthologues were enriched for genes encoding cellular “inputs” such as cell surface receptors (e.g., TLR6, IL-7Rα) and functional “outputs” such as inflammatory cytokines/chemokines (e.g., CCL20, CXCL13). Conversely, intracellular signaling components linking inputs to outputs were typically concordantly regulated. Functional consequences of divergent gene regulation were confirmed by showing LPS pretreatment boosts subsequent TLR6 responses in mouse but not human macrophages, in keeping with mouse-specific TLR6 induction. Divergently regulated genes were associated with a large dynamic range of gene expression, and specific promoter architectural features (TATA box enrichment, CpG island depletion). Surprisingly, regulatory divergence was also associated with enhanced interspecies promoter conservation. Thus, the genes controlled by complex, highly conserved promoters that facilitate dynamic regulation are also the most susceptible to evolutionary change.
Unliganded HIV-1 gp120 core structures assume the CD4-bound conformation with regulation by quaternary interactions and variable loops
Authors: Kwon Young Do , Finzi Andrés , Wu Xueling , Dogo-Isonagie Cajetan , Lee Lawrence K. , Moore Lucas R. , Schmidt Stephen D. , Stuckey Jonathan , Yang Yongping , Zhou Tongqing , Zhu Jiang , Vicic David A. , Debnath Asim K. , Shapiro Lawrence , Bewley Carole A. , Mascola John R. , Sodroski Joseph G. , Kwong Peter D.
Published: March 2012

The HIV-1 envelope (Env) spike (gp120
3
/gp41
3
) undergoes considerable structural rearrangements to mediate virus entry into cells and to evade the host immune response. Engagement of CD4, the primary human receptor, fixes a particular conformation and primes Env for entry. The CD4-bound state, however, is prone to spontaneous inactivation and susceptible to antibody neutralization. How does unliganded HIV-1 maintain CD4-binding capacity and regulate transitions to the CD4-bound state? To define this mechanistically, we determined crystal structures of unliganded core gp120 from HIV-1 clades B, C, and E. Notably, all of these unliganded HIV-1 structures resembled the CD4-bound state. Conformational fixation with ligand selection and thermodynamic analysis of full-length and core gp120 interactions revealed that the tendency of HIV-1 gp120 to adopt the CD4-bound conformation was restrained by the V1/V2- and V3-variable loops. In parallel, we determined the structure of core gp120 in complex with the small molecule, NBD-556, which specifically recognizes the CD4-bound conformation of gp120. Neutralization by NBD-556 indicated that Env spikes on primary isolates rarely assume the CD4-bound conformation spontaneously, although they could do so when quaternary restraints were loosened. Together, the results suggest that the CD4-bound conformation represents a “ground state” for the gp120 core, with variable loop and quaternary interactions restraining unliganded gp120 from “snapping” into this conformation. A mechanism of control involving deformations in unliganded structure from a functionally critical state (e.g., the CD4-bound state) provides advantages in terms of HIV-1 Env structural diversity and resistance to antibodies and inhibitors, while maintaining elements essential for entry.
Tpl2ablation promotes intestinal inflammation and tumorigenesis inApcminmice by inhibiting IL-10 secretion and regulatory T-cell generation
Authors: Serebrennikova Oksana B. , Tsatsanis Christos , Mao Changchuin , Gounaris Elias , Ren Wenying , Siracusa Linda D. , Eliopoulos Aristides G. , Khazaie Khashayarsha , Tsichlis Philip N.
Published: March 2012

To address the role of Tpl2, a MAP3K8 that regulates innate/adaptive immunity and inflammation, in intestinal tumorigenesis, we crossed aTpl2KO allele into theApcmin/+genetic background. Here, we show that Apcmin/+/Tpl2−/−mice exhibit a fivefold increase in the number of intestinal adenomas. Bone marrow transplantation experiments revealed that the enhancement of polyposis was partially hematopoietic cell-driven. Consistent with this observation,Tpl2ablation promoted intestinal inflammation. IL-10 levels and regulatory T-cell numbers were lower in the intestines of Tpl2−/−mice, independent of Apc and polyp status, suggesting that they were responsible for the initiation of the enhancement of tumorigenesis caused by the ablation ofTpl2. The low IL-10 levels correlated with defects in mTOR activation and Stat3 phosphorylation in Toll-like receptor-stimulated macrophages and with a defect in inducible regulatory T-cell generation and function. Both polyp numbers and inflammation increased progressively with time. The rate of increase of both, however, was more rapid in Apcmin/+/Tpl2−/−mice, suggesting that the positive feedback initiated by inflammatory signals originating in developing polyps is more robust in these mice. This may be because these mice have a higher intestinal polyp burden as a result of the enhancement of tumor initiation.
Buckling-induced encapsulation of structured elastic shells under pressure
Authors: Shim Jongmin , Perdigou Claude , Chen Elizabeth R. , Bertoldi Katia , Reis Pedro M.
Published: March 2012

We introduce a class of continuum shell structures, the Buckliball, which undergoes a structural transformation induced by buckling under pressure loading. The geometry of the Buckliball comprises a spherical shell patterned with a regular array of circular voids. In order for the pattern transformation to be induced by buckling, the possible number and arrangement of these voids are found to be restricted to five specific configurations. Below a critical internal pressure, the narrow ligaments between the voids buckle, leading to a cooperative buckling cascade of the skeleton of the ball. This ligament buckling leads to closure of the voids and a reduction of the total volume of the shell by up to 54%, while remaining spherical, thereby opening the possibility of encapsulation. We use a combination of precision desktop-scale experiments, finite element simulations, and scaling analyses to explore the underlying mechanics of these foldable structures, finding excellent qualitative and quantitative agreement. Given that this folding mechanism is induced by a mechanical instability, our Buckliball opens the possibility for reversible encapsulation, over a wide range of length scales.
Single-molecule observation of helix staggering, sliding, and coiled coil misfolding
Authors: Xi Zhiqun , Gao Ying , Sirinakis George , Guo Honglian , Zhang Yongli
Published: March 2012

The biological functions of coiled coils generally depend on efficient folding and perfect pairing of their α-helices. Dynamic changes in the helical registry that lead to staggered helices have only been proposed for a few special systems and not found in generic coiled coils. Here, we report our observations of multiple staggered helical structures of two canonical coiled coils. The partially folded structures are formed predominantly by coiled coil misfolding and occasionally by helix sliding. Using high-resolution optical tweezers, we characterized their energies and transition kinetics at a single-molecule level. The staggered states occur less than 2% of the time and about 0.1% of the time at zero force. We conclude that dynamic changes in helical registry may be a general property of coiled coils. Our findings should have broad and unique implications in functions and dysfunctions of proteins containing coiled coils.
Resolving the complex role of enzyme conformational dynamics in catalytic function
Authors: Doshi Urmi , McGowan Lauren C. , Ladani Safieh Tork , Hamelberg Donald
Published: March 2012

Despite growing evidence suggesting the importance of enzyme conformational dynamics (ECD) in catalysis, a consensus on how precisely ECD influences the chemical step and reaction rates is yet to be reached. Here, we characterize ECD in Cyclophilin A, a well-studied peptidyl-prolyl
cis-trans
isomerase, using normal and accelerated, atomistic molecular dynamics simulations. Kinetics and free energy landscape of the isomerization reaction in solution and enzyme are explored in unconstrained simulations by allowing significantly lower torsional barriers, but in no way compromising the atomistic description of the system or the explicit solvent. We reveal that the reaction dynamics is intricately coupled to enzymatic motions that span multiple timescales and the enzyme modes are selected based on the energy barrier of the chemical step. We show that Kramers’ rate theory can be used to present a clear rationale of how ECD affects the reaction dynamics and catalytic rates. The effects of ECD can be incorporated into the effective diffusion coefficient, which we estimate to be about ten times slower in enzyme than in solution. ECD thereby alters the preexponential factor, effectively impeding the rate enhancement. From our analyses, the trend observed for lower torsional barriers can be extrapolated to actual isomerization barriers, allowing successful prediction of the speedup in rates in the presence of CypA, which is in notable agreement with experimental estimates. Our results further reaffirm transition state stabilization as the main effect in enhancing chemical rates and provide a unified view of ECD’s role in catalysis from an atomistic perspective.
Molecular MRI enables early and sensitive detection of brain metastases
Authors: Serres Sébastien , Soto Manuel Sarmiento , Hamilton Alastair , McAteer Martina A. , Carbonell W. Shawn , Robson Matthew D. , Ansorge Olaf , Khrapitchev Alexandre , Bristow Claire , Balathasan Lukxmi , Weissensteiner Thomas , Anthony Daniel C. , Choudhury Robin P. , Muschel Ruth J. , Sibson Nicola R.
Published: March 2012

Metastasis to the brain is a leading cause of cancer mortality. The current diagnostic method of gadolinium-enhanced MRI is sensitive only to larger tumors, when therapeutic options are limited. Earlier detection of brain metastases is critical for improved treatment. We have developed a targeted MRI contrast agent based on microparticles of iron oxide that enables imaging of endothelial vascular cell adhesion molecule-1 (VCAM-1). Our objectives here were to determine whether VCAM-1 is up-regulated on vessels associated with brain metastases, and if so, whether VCAM-1–targeted MRI enables early detection of these tumors. Early up-regulation of cerebrovascular VCAM-1 expression was evident on tumor-associated vessels in two separate murine models of brain metastasis. Metastases were detectable in vivo using VCAM-1–targeted MRI 5 d after induction (<1,000 cells). At clinical imaging resolutions, this finding is likely to translate to detection at tumor volumes two to three orders of magnitude smaller (0.3–3 × 10
5
cells) than those volumes detectable clinically (10
7
–10
8
cells). VCAM-1 expression detected by MRI increased significantly (
P
< 0.0001) with tumor progression, and tumors showed no gadolinium enhancement. Importantly, expression of VCAM-1 was shown in human brain tissue containing both established metastases and micrometastases. Translation of this approach to the clinic could increase therapeutic options and change clinical management in a substantial number of cancer patients.
Schizophrenia risk polymorphisms in the
TCF4
gene interact with smoking in the modulation of auditory sensory gating
Authors: Quednow Boris B. , Brinkmeyer Jürgen , Mobascher Arian , Nothnagel Michael , Musso Francesco , Gründer Gerhard , Savary Noah , Petrovsky Nadine , Frommann Ingo , Lennertz Leonhard , Spreckelmeyer Katja N. , Wienker Thomas F. , Dahmen Norbert , Thuerauf Norbert , Clepce Marion , Kiefer Falk , Majic Tomislav , Mössner Rainald , Maier Wolfgang , Gallinat Jürgen , Diaz-Lacava Amalia , Toliat Mohammad R. , Thiele Holger , Nürnberg Peter , Wagner Michael , Winterer Georg
Published: March 2012

Several polymorphisms of the transcription factor 4 (
TCF4
) have been shown to increase the risk for schizophrenia, particularly
TCF4
rs9960767. This polymorphism is associated with impaired sensorimotor gating measured by prepulse inhibition—an established endophenotype of schizophrenia. We therefore investigated whether
TCF4
polymorphisms also affect another proposed endophenotype of schizophrenia, namely sensory gating assessed by P50 suppression of the auditory evoked potential. Although sensorimotor gating and sensory gating are not identical, recent data suggest that they share genetic fundamentals. In a multicenter study at six academic institutions throughout Germany, we applied an auditory P50 suppression paradigm to 1,821 subjects (1,023 never-smokers, 798 smokers) randomly selected from the general population. Samples were genotyped for 21
TCF4
polymorphisms. Given that smoking is highly prevalent in schizophrenia and affects sensory gating, we also assessed smoking behavior, cotinine plasma concentrations, exhaled carbon monoxide, and the Fagerström Test (FTND). P50 suppression was significantly decreased in carriers of schizophrenia risk alleles of the
TCF4
polymorphisms rs9960767, rs10401120rs, rs17597926, and 17512836 (
P
< 0.0002–0.00005). These gene effects were modulated by smoking behavior as indicated by significant interactions of
TCF4
genotype and smoking status; heavy smokers (FTND score ≥4) showed stronger gene effects on P50 suppression than light smokers and never-smokers. Our finding suggests that sensory gating is modulated by an interaction of
TCF4
genotype with smoking, and both factors may play a role in early information processing deficits also in schizophrenia. Consequently, considering smoking behavior may facilitate the search for genetic risk factors for schizophrenia.
miRNA-939 regulates human inducible nitric oxide synthase posttranscriptional gene expression in human hepatocytes
Authors: Guo Zhong , Shao Lifang , Zheng Liang , Du Qiang , Li Peiyuan , John Bino , Geller David A.
Published: March 2012

Human inducible nitric oxide synthase (hiNOS) gene expression is regulated by transcriptional and posttranscriptional mechanisms. The purpose of this study was to determine whether specific microRNA (miRNA) directly regulate hiNOS gene expression. Sequence analysis of the 496-bp hiNOS 3′-untranslated region (3′-UTR) revealed five putative miR-939 binding sites. The hiNOS 3′-UTR conferred significant posttranscriptional blockade of luciferase activity in human A549, HCT8, and HeLa cells. The hiNOS 3′-UTR also exerted basal and cytokine-stimulated posttranscriptional repression in an orientation-dependent manner. Functional studies demonstrated that transfection of miR-939 into primary human hepatocytes (HCs) significantly inhibited cytokine-induced NO synthesis in a dose-dependent manner that was abrogated by a specific miR-939 inhibitor. MiR-939 (but not other miRNAs) abolished cytokine-stimulated hiNOS protein in human HC, but had no effect on hiNOS mRNA levels. Site-directed mutagenesis of miR-939 bindings sites at +99 or +112 bp in the hiNOS 3′-UTR increased reporter gene expression. Furthermore, intact miR-939 binding sites at +99 or +112 positions were required for posttranscriptional suppression by miR-939. Cytokine stimulation directly increased miR-939 levels in human HC. Transfection of miR-939 inhibitor (antisense miR-939) enhanced cytokine-induced hiNOS protein and increased NO synthesis in vitro in human HC. Finally, cytokine or LPS injection in vivo in mice increased hepatic miR-939 levels. Taken together, these data identify that miR-939 directly regulates hiNOS gene expression by binding in the 3′-UTR to produce a translational blockade. These findings suggest dual regulation of iNOS gene expression where cytokines induce iNOS transcription and also increase miR-939, leading to translational inhibition in a check-and-balance system.
Action potential propagation in transverse-axial tubular system is impaired in heart failure
Authors: Sacconi Leonardo , Ferrantini Cecilia , Lotti Jacopo , Coppini Raffaele , Yan Ping , Loew Leslie M. , Tesi Chiara , Cerbai Elisabetta , Poggesi Corrado , Pavone Francesco S.
Published: March 2012

The plasma membrane of cardiac myocytes presents complex invaginations known as the transverse-axial tubular system (TATS). Despite TATS's crucial role in excitation-contraction coupling and morphological alterations found in pathological settings, TATS's electrical activity has never been directly investigated in remodeled tubular networks. Here we develop an ultrafast random access multiphoton microscope that, in combination with a customly synthesized voltage-sensitive dye, is used to simultaneously measure action potentials (APs) at multiple sites within the sarcolemma with submillisecond temporal and submicrometer spatial resolution in real time. We find that the tight electrical coupling between different sarcolemmal domains is guaranteed only within an intact tubular system. In fact, acute detachment by osmotic shock of most tubules from the surface sarcolemma prevents AP propagation not only in the disconnected tubules, but also in some of the tubules that remain connected with the surface. This indicates that a structural disorganization of the tubular system worsens the electrical coupling between the TATS and the surface. The pathological implications of this finding are investigated in failing hearts. We find that AP propagation into the pathologically remodeled TATS frequently fails and may be followed by local spontaneous electrical activity. Our findings provide insight on the relationship between abnormal TATS and asynchronous calcium release, a major determinant of cardiac contractile dysfunction and arrhythmias.
Ni(II) coordination to mixed sites modulates DNA binding of
Hp
NikR via a long-range effect
Authors: West Abby L. , Evans Sarah E. , González Javier M. , Carter Lester G. , Tsuruta Hiro , Pozharski Edwin , Michel Sarah L. J.
Published: March 2012

Helicobacter pylori
NikR (
Hp
NikR) is a nickel-dependent transcription factor that regulates multiple genes in the
H. pylori
pathogen. There are conflicting data regarding the locations of the Ni(II) sites and the role of Ni(II) coordination in DNA recognition. Herein, we report crystal structures of (
i
) the metal-binding domain (MBD) of
Hp
NikR (3.08 Å) and (
ii
) a mutant, H74A (2.04 Å), designed to disrupt native Ni(II) coordination. In the MBD structure, four nickel ions are coordinated to two different types of nickel sites (4-coordinate, square planar, and 5/6-coordinate, square pyramidal/octahedral). In the H74A structure, all four nickel ions are coordinated to 4-coordinate square-planar sites. DNA-binding studies reveal tighter binding for target DNA sequences for holo-
Hp
NikR compared with the affinities of Ni(II) reconstituted apo-
Hp
NikR and H74A for these same DNA targets, supporting a role for Ni(II) coordination to 5/6 sites in DNA recognition. Small-angle X-ray scattering studies of holo-
Hp
NikR and H74A reveal a high degree of conformational flexibility centered at the DNA-binding domains of H74A, which is consistent with disorder observed in the crystal structure of the protein. A model of DNA recognition by
Hp
NikR is proposed in which Ni(II) coordination to specific sites in the MBD have a long-range effect on the flexibility of the DNA-binding domains and, consequently, the DNA recognition properties.
Decade-long bacterial community dynamics in cystic fibrosis airways
Authors: Zhao Jiangchao , Schloss Patrick D. , Kalikin Linda M. , Carmody Lisa A. , Foster Bridget K. , Petrosino Joseph F. , Cavalcoli James D. , VanDevanter Donald R. , Murray Susan , Li Jun Z. , Young Vincent B. , LiPuma John J.
Published: March 2012

The structure and dynamics of bacterial communities in the airways of persons with cystic fibrosis (CF) remain largely unknown. We characterized the bacterial communities in 126 sputum samples representing serial collections spanning 8–9 y from six age-matched male CF patients. Sputum DNA was analyzed by bar-coded pyrosequencing of the V3–V5 hypervariable region of the 16S rRNA gene, defining 662 operational taxonomic units (OTUs) from >633,000 sequences. Bacterial community diversity decreased significantly over time in patients with typically progressive lung disease but remained relatively stable in patients with a mild lung disease phenotype. Antibiotic use, rather than patient age or lung function, was the primary driver of decreasing diversity. Interpatient variability in community structure exceeded intrapatient variability in serial samples. Antibiotic treatment was associated with pronounced shifts in community structure, but communities showed both short- and long-term resilience after antibiotic perturbation. There was a positive correlation between OTU occurrence and relative abundance, with a small number of persistent OTUs accounting for the greatest abundance. Significant changes in community structure, diversity, or total bacterial density at the time of pulmonary exacerbation were not observed. Despite decreasing community diversity in patients with progressive disease, total bacterial density remained relatively stable over time. These findings show the critical relationship between airway bacterial community structure, disease stage, and clinical state at the time of sample collection. These features are the key parameters with which to assess the complex ecology of the CF airway.
An essential bacterial-type cardiolipin synthase mediates cardiolipin formation in a eukaryote
Authors: Serricchio Mauro , Bütikofer Peter
Published: March 2012

Cardiolipin is important for bacterial and mitochondrial stability and function. The final step in cardiolipin biosynthesis is catalyzed by cardiolipin synthase and differs mechanistically between prokaryotes and eukaryotes. To study the importance of cardiolipin synthesis for mitochondrial integrity, membrane protein complex formation, and cell proliferation in the human and animal pathogenic protozoan parasite,
Trypanosoma brucei
, we generated conditional cardiolipin synthase-knockout parasites. We found that cardiolipin formation in
T. brucei
procyclic forms is catalyzed by a bacterial-type cardiolipin synthase, providing experimental evidence for a prokaryotic-type cardiolipin synthase in a eukaryotic organism. Ablation of enzyme expression resulted in inhibition of de novo cardiolipin synthesis, reduction in cellular cardiolipin levels, alterations in mitochondrial morphology and function, and parasite death in culture. By using immunofluorescence microscopy and blue-native gel electrophoresis, cardiolipin synthase was shown to colocalize with inner mitochondrial membrane proteins and to be part of a large protein complex. During depletion of cardiolipin synthase, the levels of cytochrome oxidase subunit IV and cytochrome c1, reflecting mitochondrial respiratory complexes IV and III, respectively, decreased progressively.
Induced fit on heme binding to the
Pseudomonas aeruginosa
cytoplasmic protein (PhuS) drives interaction with heme oxygenase (HemO)
Authors: O'Neill Maura J. , Bhakta Mehul N. , Fleming Karen G. , Wilks Angela
Published: March 2012

Iron, an essential nutrient with limited bioavailability, requires specialized cellular mechanisms for uptake. Although iron uptake into the cytoplasm in the form of heme has been well characterized in many bacteria, the subsequent trafficking is poorly understood. The cytoplasmic heme-binding proteins belong to a structurally related family thought to have evolved as “induced fit” ligand-binding macromolecules. One member,
Pseudomonas aeruginosa
cytoplasmic protein (PhuS), has previously been shown to be important for delivering heme to the iron regulated heme oxygenase (HemO). Spectroscopic investigations of the holo-PhuS complex revealed a dynamic heme environment with overlapping but distinct heme-binding sites with alternative coordinating heme ligands, His-209 or His-212. In the present work we establish a mechanism for how heme is transferred from PhuS to its partner, HemO. Using surface plasmon resonance and isothermal titration calorimetry, we have discovered that holo-PhuS, but not apo-PhuS, forms a 1:1 complex with HemO. Sedimentation velocity and limited proteolysis experiments suggest that heme binding to PhuS induces a conformational rearrangement that drives the protein interaction with HemO. Hydrodynamic analysis reveals that the holo-PhuS displays a more expanded hydrodynamic envelope compared with apo-PhuS, and we propose that this conformational change drives the interaction with HemO. We further demonstrate that replacement of His-212 by Ala disrupts the interaction of holo-PhuS with HemO; in contrast, the His-209-Ala variant can still complex with HemO, albeit more weakly. Together, the present studies reveal a mechanism that couples a heme-dependent conformational switch in PhuS to protein–protein interaction, the subsequent free energy of which drives heme release to HemO.
The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors
Authors: Willingham Stephen B. , Volkmer Jens-Peter , Gentles Andrew J. , Sahoo Debashis , Dalerba Piero , Mitra Siddhartha S. , Wang Jian , Contreras-Trujillo Humberto , Martin Robin , Cohen Justin D. , Lovelace Patricia , Scheeren Ferenc A. , Chao Mark P. , Weiskopf Kipp , Tang Chad , Volkmer Anne Kathrin , Naik Tejaswitha J. , Storm Theresa A. , Mosley Adriane R. , Edris Badreddin , Schmid Seraina M. , Sun Chris K. , Chua Mei-Sze , Murillo Oihana , Rajendran Pradeep , Cha Adriel C. , Chin Robert K. , Kim Dongkyoon , Adorno Maddalena , Raveh Tal , Tseng Diane , Jaiswal Siddhartha , Enger Per Øyvind , Steinberg Gary K. , Li Gordon , So Samuel K. , Majeti Ravindra , Harsh Griffith R. , van de Rijn Matt , Teng Nelson N. H. , Sunwoo John B. , Alizadeh Ash A. , Clarke Michael F. , Weissman Irving L.
Published: March 2012

CD47, a “don't eat me” signal for phagocytic cells, is expressed on the surface of all human solid tumor cells. Analysis of patient tumor and matched adjacent normal (nontumor) tissue revealed that CD47 is overexpressed on cancer cells.CD47mRNA expression levels correlated with a decreased probability of survival for multiple types of cancer. CD47 is a ligand for SIRPα, a protein expressed on macrophages and dendritic cells. In vitro, blockade of CD47 signaling using targeted monoclonal antibodies enabled macrophage phagocytosis of tumor cells that were otherwise protected. Administration of anti-CD47 antibodies inhibited tumor growth in orthotopic immunodeficient mouse xenotransplantation models established with patient tumor cells and increased the survival of the mice over time. Anti-CD47 antibody therapy initiated on larger tumors inhibited tumor growth and prevented or treated metastasis, but initiation of the therapy on smaller tumors was potentially curative. The safety and efficacy of targeting CD47 was further tested and validated in immune competent hosts using an orthotopic mouse breast cancer model. These results suggest all human solid tumor cells require CD47 expression to suppress phagocytic innate immune surveillance and elimination. These data, taken together with similar findings with other human neoplasms, show that CD47 is a commonly expressed molecule on all cancers, its function to block phagocytosis is known, and blockade of its function leads to tumor cell phagocytosis and elimination. CD47 is therefore a validated target for cancer therapies.
Antibody therapy targeting the CD47 protein is effective in a model of aggressive metastatic leiomyosarcoma
Authors: Edris Badreddin , Weiskopf Kipp , Volkmer Anne K. , Volkmer Jens-Peter , Willingham Stephen B. , Contreras-Trujillo Humberto , Liu Jie , Majeti Ravindra , West Robert B. , Fletcher Jonathan A. , Beck Andrew H. , Weissman Irving L. , van de Rijn Matt
Published: March 2012

Antibodies against CD47, which block tumor cell CD47 interactions with macrophage signal regulatory protein-α, have been shown to decrease tumor size in hematological and epithelial tumor models by interfering with the protection from phagocytosis by macrophages that intact CD47 bestows upon tumor cells. Leiomyosarcoma (LMS) is a tumor of smooth muscle that can express varying levels of colony-stimulating factor-1 (CSF1), the expression of which correlates with the numbers of tumor-associated macrophages (TAMs) that are found in these tumors. We have previously shown that the presence of TAMs in LMS is associated with poor clinical outcome and the overall effect of TAMs in LMS therefore appears to be protumorigenic. However, the use of inhibitory antibodies against CD47 offers an opportunity to turn TAMs against LMS cells by allowing the phagocytic behavior of resident macrophages to predominate. Here we show that interference with CD47 increases phagocytosis of two human LMS cell lines, LMS04 and LMS05, in vitro. In addition, treatment of mice bearing subcutaneous LMS04 and LMS05 tumors with a novel, humanized anti-CD47 antibody resulted in significant reductions in tumor size. Mice bearing LMS04 tumors develop large numbers of lymph node and lung metastases. In a unique model for neoadjuvant treatment, mice were treated with anti-CD47 antibody starting 1 wk before resection of established primary tumors and subsequently showed a striking decrease in the size and number of metastases. These data suggest that treatment with anti-CD47 antibodies not only reduces primary tumor size but can also be used to inhibit the development of, or to eliminate, metastatic disease.
Cyanobacteria produce a high variety of hepatotoxic peptides in lichen symbiosis
Authors: Kaasalainen Ulla , Fewer David P. , Jokela Jouni , Wahlsten Matti , Sivonen Kaarina , Rikkinen Jouko
Published: March 2012

Lichens are symbiotic associations between fungi and photosynthetic algae or cyanobacteria. Microcystins are potent toxins that are responsible for the poisoning of both humans and animals. These toxins are mainly associated with aquatic cyanobacterial blooms, but here we show that the cyanobacterial symbionts of terrestrial lichens from all over the world commonly produce microcystins. We screened 803 lichen specimens from five different continents for cyanobacterial toxins by amplifying a part of the gene cluster encoding the enzyme complex responsible for microcystin production and detecting toxins directly from lichen thalli. We found either the biosynthetic genes for making microcystins or the toxin itself in 12% of all analyzed lichen specimens. A plethora of different microcystins was found with over 50 chemical variants, and many of the variants detected have only rarely been reported from free-living cyanobacteria. In addition, high amounts of nodularin, up to 60 μg g
−1
, were detected from some lichen thalli. This microcystin analog and potent hepatotoxin has previously been known only from the aquatic bloom-forming genus
Nodularia
. Our results demonstrate that the production of cyanobacterial hepatotoxins in lichen symbiosis is a global phenomenon and occurs in many different lichen lineages. The very high genetic diversity of the
mcyE
gene and the chemical diversity of microcystins suggest that lichen symbioses may have been an important environment for diversification of these cyanobacteria.
RNA binding and RNA remodeling activities of the half-a-tetratricopeptide (HAT) protein HCF107 underlie its effects on gene expression
Authors: Hammani Kamel , Cook William B. , Barkan Alice
Published: March 2012

The half-a-tetratricopeptide repeat (HAT) motif is a helical repeat motif found in proteins that influence various aspects of RNA metabolism, including rRNA biogenesis, RNA splicing, and polyadenylation. This functional association with RNA suggested that HAT repeat tracts might bind RNA. However, RNA binding activity has not been reported for any HAT repeat tract, and recent literature has emphasized a protein binding role. In this study, we show that a chloroplast-localized HAT protein, HCF107, is a sequence-specific RNA binding protein. HCF107 consists of 11 tandem HAT repeats and short flanking regions that are also predicted to form helical hairpins. The minimal HCF107 binding site spans ∼11 nt, consistent with the possibility that HAT repeats bind RNA through a modular one repeat–1 nt mechanism. Binding of HCF107 to its native RNA ligand in the
psbH
5′ UTR remodels local RNA structure and protects the adjacent RNA from exonucleases in vitro. These activities can account for the RNA stabilizing, RNA processing, and translational activation functions attributed to HCF107 based on genetic data. We suggest that analogous activities contribute to the functions of HAT domains found in ribonucleoprotein complexes in the nuclear–cytosolic compartment.
Dichotomous effects of VEGF-A on adipose tissue dysfunction
Authors: Sun Kai , Asterholm Ingrid Wernstedt , Kusminski Christine M. , Bueno Ana Carolina , Wang Zhao V. , Pollard Jeffrey W. , Brekken Rolf A. , Scherer Philipp E.
Published: March 2012

Obese fat pads are frequently undervascularized and hypoxic, leading to increased fibrosis, inflammation, and ultimately insulin resistance. We hypothesized that VEGF-A–induced stimulation of angiogenesis enables sustained and sufficient oxygen and nutrient exchange during fat mass expansion, thereby improving adipose tissue function. Using a doxycycline (Dox)-inducible adipocyte-specific VEGF-A overexpression model, we demonstrate that the local up-regulation of VEGF-A in adipocytes improves vascularization and causes a “browning” of white adipose tissue (AT), with massive up-regulation of UCP1 and PGC1α. This is associated with an increase in energy expenditure and resistance to high fat diet-mediated metabolic insults. Similarly, inhibition of VEGF-A–induced activation of VEGFR2 during the early phase of high fat diet-induced weight gain, causes aggravated systemic insulin resistance. However, the same VEGF-A–VEGFR2 blockade in
ob/ob
mice leads to a reduced body-weight gain, an improvement in insulin sensitivity, a decrease in inflammatory factors, and increased incidence of adipocyte death. The consequences of modulation of angiogenic activity are therefore context dependent. Proangiogenic activity during adipose tissue expansion is beneficial, associated with potent protective effects on metabolism, whereas antiangiogenic action in the context of preexisting adipose tissue dysfunction leads to improvements in metabolism, an effect likely mediated by the ablation of dysfunctional proinflammatory adipocytes.
U7 small nuclear ribonucleoprotein represses histone gene transcription in cell cycle-arrested cells
Authors: Ideue Takashi , Adachi Shungo , Naganuma Takao , Tanigawa Akie , Natsume Tohru , Hirose Tetsuro
Published: March 2012

Histone gene expression is tightly coordinated with DNA replication, as it is activated at the onset of S phase and suppressed at the end of S phase. Replication-dependent histone gene expression is precisely controlled at both transcriptional and posttranscriptional levels. U7 small nuclear ribonucleoprotein (U7 snRNP) is involved in the 3′-end processing of nonpolyadenylated histone mRNAs, which is required for S phase-specific gene expression. The present study reports a unique function of U7 snRNP in the repression of histone gene transcription under cell cycle-arrested conditions. Elimination of U7 snRNA with an antisense oligonucleotide in HeLa cells as well as in nontransformed human lung fibroblasts resulted in elevated levels of replication-dependent H1, H2A, H2B, H3, and H4 histone mRNAs but not of replication-independent H3F3B histone mRNA. An analogous effect was observed upon depletion of Lsm10, a component of the U7 snRNP-specific Sm ring, with siRNA. Pulse–chase experiments revealed that U7 snRNP acts to repress transcription without remarkably altering mRNA stability. Mass spectrometric analysis of the captured U7 snRNP from HeLa cell extracts identified heterogeneous nuclear (hn)RNP UL1 as a U7 snRNP interaction partner. Further knockdown and overexpression experiments revealed that hnRNP UL1 is responsible for U7 snRNP-dependent transcriptional repression of replication-dependent histone genes. Chromatin immunoprecipitation confirmed that hnRNP UL1 is recruited to the histone gene locus only when U7 snRNP is present. These findings support a unique mechanism of snRNP-mediated transcriptional control that restricts histone synthesis to S phase, thereby preventing the potentially toxic effects of histone synthesis at other times in the cell cycle.
Caspase-7 uses an exosite to promote poly(ADP ribose) polymerase 1 proteolysis
Authors: Boucher Dave , Blais Véronique , Denault Jean-Bernard
Published: March 2012

During apoptosis, hundreds of proteins are cleaved by caspases, most of them by the executioner caspase-3. However, caspase-7, which shares the same substrate primary sequence preference as caspase-3, is better at cleaving poly(ADP ribose) polymerase 1 (PARP) and Hsp90 cochaperone p23, despite a lower intrinsic activity. Here, we identified key lysine residues (K
38
KKK) within the N-terminal domain of caspase-7 as critical elements for the efficient proteolysis of these two substrates. Caspase-7's N-terminal domain binds PARP and improves its cleavage by a chimeric caspase-3 by ∼30-fold. Cellular expression of caspase-7 lacking the critical lysine residues resulted in less-efficient PARP and p23 cleavage compared with cells expressing the wild-type peptidase. We further showed, using a series of caspase chimeras, the positioning of p23 on the enzyme providing us with a mechanistic insight into the binding of the exosite. In summary, we have uncovered a role for the N-terminal domain (NTD) and the N-terminal peptide of caspase-7 in promoting key substrate proteolysis.
CHD3 protein recognizes and regulates methylated histone H3 lysines 4 and 27 over a subset of targets in the rice genome
Authors: Hu Yongfeng , Liu Dengnian , Zhong Xiaocao , Zhang Chengjun , Zhang Qifa , Zhou Dao-Xiu
Published: March 2012

Histone lysine methylation is an important component of the epigenetic system demarcating transcriptionally active and inactive chromatin domains. It is of primary importance in understanding how different histone lysine methylation marks and a specific combination of them are read and interpreted by chromatin proteins to regulate gene expression. In this paper, we report that the rice CHD3 protein CHR729 that was required for many aspects of plant development can interact with dimethylated histone H3 lysine 4 (H3K4me2, a mark associated with moderately expressed or repressed genes) and with trimethylated histone H3 lysine 27 (H3K27me3, a mark associated with repressed genes), respectively, through the chromodomains and the plant homeodomain (PHD) finger of the protein. A mutation or down-regulation of the gene provoked a decrease of H3K27me3 and H3K4me3 (a mark associated with active genes). Genome-wide analysis revealed that H3K27me3 and H3K4me3, respectively, were lost from about 56 and 23% of marked loci, which correspond mostly to under-expressed or repressed genes. In the mutant, a higher-than-expected proportion of down-regulated genes lost H3K4me3, among which many encode DNA-binding transcription factors. These results suggest that the rice CHD3 protein is a bifunctional chromatin regulator able to recognize and modulate H3K4 and H3K27 methylation over repressed or tissue-specific genes, which may be associated with regulation of a gene transcription program of plant development.