bluuebunny/crossref_metadata_2025 huggingface dataset:
Hamiltonian derivation of the Charney–Hasegawa–Mima equation
Authors: Tassi E. , Chandre C. , Morrison P. J.
Published: August 2009

The Charney–Hasegawa–Mima equation is an infinite-dimensional Hamiltonian system with dynamics generated by a noncanonical Poisson bracket. Here a first principle Hamiltonian derivation of this system, beginning with the ion fluid dynamics and its known Hamiltonian form, is given.
A numerical investigation of the rheology of sheared fiber suspensions
Authors: Lindström Stefan B. , Uesaka Tetsu
Published: August 2009

Particle-level simulations are performed to study the rheology of monodispersed non-Brownian fibers suspended in a Newtonian fluid in shear flow. The effects of fiber aspect ratio, concentration, and interparticle friction on the stress tensor of the suspension in the steady state and on the tendency of fiber agglomeration are investigated. Semiempirical expressions for the steady state apparent shear viscosity and the steady state first and second normal stress difference were obtained for the case of well dispersed suspensions in the nonconcentrated regimes. The simulation predictions of the specific viscosity were in fair agreement with previous experimental investigations.
The influence of depth on mercury levels in pelagic fishes and their prey
Authors: Choy C. Anela , Popp Brian N. , Kaneko J. John , Drazen Jeffrey C.
Published: August 2009

Mercury distribution in the oceans is controlled by complex biogeochemical cycles, resulting in retention of trace amounts of this metal in plants and animals. Inter- and intra-specific variations in mercury levels of predatory pelagic fish have been previously linked to size, age, trophic position, physical and chemical environmental parameters, and location of capture; however, considerable variation remains unexplained. In this paper, we focus on differences in ecology, depth of occurrence, and total mercury levels in 9 species of commercially important pelagic fish (
Thunnus obesus
,
T. albacares
,
Katsuwonus pelamis
,
Xiphias gladius
,
Lampris guttatus
,
Coryphaena hippurus
,
Taractichthys steindachneri
,
Tetrapturus audax
, and
Lepidocybium flavobrunneum
) and in numerous representatives (fishes, squids, and crustaceans) of their lower trophic level prey sampled from the central North Pacific Ocean. Results indicate that total mercury levels of predatory pelagic fishes and their prey increase with median depth of occurrence in the water column and mimic concentrations of dissolved organic mercury in seawater. Stomach content analysis results from this study and others indicate a greater occurrence of higher-mercury containing deeper-water prey organisms in the diets of the deeper-ranging predators,
X. gladius
,
T. obesus
, and
L. guttatus
. While present in trace amounts, dissolved organic mercury increases with depth in the water column suggesting that the mesopelagic habitat is a major entry point for mercury into marine food webs. These data suggest that a major determinant of mercury levels in oceanic predators is their depth of forage.
Human BAHD1 promotes heterochromatic gene silencing
Authors: Bierne Hélène , Tham To Nam , Batsche Eric , Dumay Anne , Leguillou Morwenna , Kernéis-Golsteyn Sophie , Regnault Béatrice , Seeler Jacob S. , Muchardt Christian , Feunteun Jean , Cossart Pascale
Published: August 2009

Gene silencing via heterochromatin formation plays a major role in cell differentiation and maintenance of homeostasis. Here we report the identification and characterization of a novel heterochromatinization factor in vertebrates, bromo adjacent homology domain–containing protein 1 (BAHD1). This nuclear protein interacts with HP1, MBD1, HDAC5, and several transcription factors. Through electron and immunofluorescence microscopy studies, we show that BAHD1 overexpression directs HP1 to specific nuclear sites and promotes the formation of large heterochromatic domains, which lack acetyl histone H4 and are enriched in H3 trimethylated at lysine 27 (H3K27me3). Furthermore, ectopically expressed BAHD1 colocalizes with the heterochromatic inactive X chromosome (Xi). The BAH domain is required for BAHD1 colocalization with H3K27me3, but not with the Xi chromosome. As highlighted by whole genome microarray analysis of BAHD1 knockdown cells, BAHD1 represses several proliferation and survival genes, in particular the insulin-like growth factor II gene (
IGF2
). When overexpressed, BAHD1 specifically binds the CpG-rich P3 promoter of
IGF2
, which increases MBD1 and HDAC5 targeting at this locus. This region contains DNA-binding sequences for the transcription factor SP1, with which BAHD1 coimmunoprecipitates. Collectively, these findings provide evidence that BAHD1 acts as a silencer by recruiting at specific promoters a set of proteins that coordinate heterochromatin assembly.
Fully functional bioengineered tooth replacement as an organ replacement therapy
Authors: Ikeda Etsuko , Morita Ritsuko , Nakao Kazuhisa , Ishida Kentaro , Nakamura Takashi , Takano-Yamamoto Teruko , Ogawa Miho , Mizuno Mitsumasa , Kasugai Shohei , Tsuji Takashi
Published: August 2009

Current approaches to the development of regenerative therapies have been influenced by our understanding of embryonic development, stem cell biology, and tissue engineering technology. The ultimate goal of regenerative therapy is to develop fully functioning bioengineered organs which work in cooperation with surrounding tissues to replace organs that were lost or damaged as a result of disease, injury, or aging. Here, we report a successful fully functioning tooth replacement in an adult mouse achieved through the transplantation of bioengineered tooth germ into the alveolar bone in the lost tooth region. We propose this technology as a model for future organ replacement therapies. The bioengineered tooth, which was erupted and occluded, had the correct tooth structure, hardness of mineralized tissues for mastication, and response to noxious stimulations such as mechanical stress and pain in cooperation with other oral and maxillofacial tissues. This study represents a substantial advance and emphasizes the potential for bioengineered organ replacement in future regenerative therapies.
Healthy individuals have T-cell and antibody responses to the tumor antigen cyclin B1 that when elicited in mice protect from cancer
Authors: Vella Laura A. , Yu Min , Fuhrmann Steven R. , El-Amine Moustapha , Epperson Diane E. , Finn Olivera J.
Published: August 2009

We previously identified the aberrantly expressed cell cycle regulator cyclin B1 as a tumor antigen recognized by antibodies and T cells from patients with breast, lung, and head and neck cancers. Ordinarily expressed only transiently in the G2/M stage of the cell cycle in normal cells, cyclin B1 is constitutively expressed at high levels in the cytoplasm of these and many other tumor types, leading to its recognition by the cancer patient's immune system. We report here an unexpected observation that cyclin B1-specific antibody and memory CD4 and CD8 T cells are also found in many healthy individuals who have no history of cancer. Moreover, young as well as older healthy people have these responses suggesting that events other than cancer, which occur either early in life or throughout life, may lead to aberrant cyclin B1 expression and anti-cyclin B1 immunity. The role, if any, of immunity to this tumor-associated antigen is not known. We wanted to determine specifically whether immunity to cyclin B1 might be important in the immunosurveillance of cyclin B1+ tumors. We therefore tested in mice the effectiveness of vaccine-elicited anti-cyclin B1 immunity against a cyclin B1+ mouse tumor that was chosen based on our published observation that cyclin B1 overexpression is associated with the lack of p53 function. We found that cyclin B1 DNA prime-protein boost vaccine protected mice from a challenge with a tumor cell line that was established from a tumor arising in the p53
−/−
mouse that spontaneously overexpresses cyclin B1.
Understanding the link between malaria risk and climate
Authors: Paaijmans Krijn P. , Read Andrew F. , Thomas Matthew B.
Published: August 2009

The incubation period for malaria parasites within the mosquito is exquisitely temperature-sensitive, so that temperature is a major determinant of malaria risk. Epidemiological models are increasingly used to guide allocation of disease control resources and to assess the likely impact of climate change on global malaria burdens. Temperature-based malaria transmission is generally incorporated into these models using mean monthly temperatures, yet temperatures fluctuate throughout the diurnal cycle. Here we use a thermodynamic malaria development model to demonstrate that temperature fluctuation can substantially alter the incubation period of the parasite, and hence malaria transmission rates. We find that, in general, temperature fluctuation reduces the impact of increases in mean temperature. Diurnal temperature fluctuation around means >21°C slows parasite development compared with constant temperatures, whereas fluctuation around <21°C speeds development. Consequently, models which ignore diurnal variation overestimate malaria risk in warmer environments and underestimate risk in cooler environments. To illustrate the implications further, we explore the influence of diurnal temperature fluctuation on malaria transmission at a site in the Kenyan Highlands. Based on local meteorological data, we find that the annual epidemics of malaria at this site cannot be explained without invoking the influence of diurnal temperature fluctuation. Moreover, while temperature fluctuation reduces the relative influence of a subtle warming trend apparent over the last 20 years, it nonetheless makes the effects biologically more significant. Such effects of short-term temperature fluctuations have not previously been considered but are central to understanding current malaria transmission and the consequences of climate change.
Kinetic gating of the proton pump in cytochrome
c
oxidase
Authors: Kim Young C. , Wikström Mårten , Hummer Gerhard
Published: August 2009

Cytochrome
c
oxidase (CcO), the terminal enzyme of the respiratory chain, reduces oxygen to water and uses the released energy to pump protons across a membrane. Here, we use kinetic master equations to explore the energetic and kinetic control of proton pumping in CcO. We construct models consistent with thermodynamic principles, the structure of CcO, experimentally known proton affinities, and equilibrium constants of intermediate reactions. The resulting models are found to capture key properties of CcO, including the midpoint redox potentials of the metal centers and the electron transfer rates. We find that coarse-grained models with two proton sites and one electron site can pump one proton per electron against membrane potentials exceeding 100 mV. The high pumping efficiency of these models requires strong electrostatic couplings between the proton loading (pump) site and the electron site (heme
a
), and kinetic gating of the internal proton transfer. Gating is achieved by enhancing the rate of proton transfer from the conserved Glu-242 to the pump site on reduction of heme
a
, consistent with the predictions of the water-gated model of proton pumping. The model also accounts for the phenotype of D-channel mutations associated with loss of pumping but retained turnover. The fundamental mechanism identified here for the efficient conversion of chemical energy into an electrochemical potential should prove relevant also for other molecular machines and novel fuel-cell designs.
Residual breast cancers after conventional therapy display mesenchymal as well as tumor-initiating features
Authors: Creighton Chad J. , Li Xiaoxian , Landis Melissa , Dixon J. Michael , Neumeister Veronique M. , Sjolund Ashley , Rimm David L. , Wong Helen , Rodriguez Angel , Herschkowitz Jason I. , Fan Cheng , Zhang Xiaomei , He Xiaping , Pavlick Anne , Gutierrez M. Carolina , Renshaw Lorna , Larionov Alexey A. , Faratian Dana , Hilsenbeck Susan G. , Perou Charles M. , Lewis Michael T. , Rosen Jeffrey M. , Chang Jenny C.
Published: August 2009

Some breast cancers have been shown to contain a small fraction of cells characterized by CD44
+
/CD24
−/low
cell-surface antigen profile that have high tumor-initiating potential. In addition, breast cancer cells propagated in vitro as mammospheres (MSs) have also been shown to be enriched for cells capable of self-renewal. In this study, we have defined a gene expression signature common to both CD44
+
/CD24
−/low
and MS-forming cells. To examine its clinical significance, we determined whether tumor cells surviving after conventional treatments were enriched for cells bearing this CD44
+
/CD24
−/low
-MS signature. The CD44
+
/CD24
−/low
-MS signature was found mainly in human breast tumors of the recently identified “claudin-low” molecular subtype, which is characterized by expression of many epithelial-mesenchymal-transition (EMT)-associated genes. Both CD44
+
/CD24
−/low
-MS and claudin-low signatures were more pronounced in tumor tissue remaining after either endocrine therapy (letrozole) or chemotherapy (docetaxel), consistent with the selective survival of tumor-initiating cells posttreatment. We confirmed an increased expression of mesenchymal markers, including vimentin (
VIM
) in cytokeratin-positive epithelial cells metalloproteinase 2 (
MMP
2), in two separate sets of postletrozole vs. pretreatment specimens. Taken together, these data provide supporting evidence that the residual breast tumor cell populations surviving after conventional treatment may be enriched for subpopulations of cells with both tumor-initiating and mesenchymal features. Targeting proteins involved in EMT may provide a therapeutic strategy for eliminating surviving cells to prevent recurrence and improve long-term survival in breast cancer patients.
Single-molecule analysis reveals that the lagging strand increases replisome processivity but slows replication fork progression
Authors: Yao Nina Y. , Georgescu Roxana E. , Finkelstein Jeff , O'Donnell Michael E.
Published: August 2009

Single-molecule techniques are developed to examine mechanistic features of individual
E. coli
replisomes during synthesis of long DNA molecules. We find that single replisomes exhibit constant rates of fork movement, but the rates of different replisomes vary over a surprisingly wide range. Interestingly, lagging strand synthesis decreases the rate of the leading strand, suggesting that lagging strand operations exert a drag on replication fork progression. The opposite is true for processivity. The lagging strand significantly increases the processivity of the replisome, possibly reflecting the increased grip to DNA provided by 2 DNA polymerases anchored to sliding clamps on both the leading and lagging strands.
Histone H3 N-terminus regulates higher order structure of yeast heterochromatin
Authors: Sperling Adam S. , Grunstein Michael
Published: August 2009

In budding yeast, telomeres and the mating type (HM) loci are found in a heterochromatin-like silent structure initiated by Rap1 and extended by the interaction of Silencing Information Regulator (Sir) proteins with histones. Binding data demonstrate that both the H3 and H4 N-terminal domains required for silencing in vivo interact directly with Sir3 and Sir4 in vitro. The role of H4 lysine 16 deacetylation is well established in Sir3 protein recruitment; however, that of the H3 N-terminal tail has remained unclear. To characterize the role of H3 in silent chromatin formation and compare it to H4 we have generated comprehensive high resolution genome-wide binding maps of heterochromatin proteins. We found that H4 lysine 16 deacetylation is required for the recruitment and spreading of heterochromatin proteins at all telomeres and HM loci. In contrast, the H3 N terminus is required for neither recruitment nor spreading of Sir proteins. Instead, deletion of the H3 tail leads to increased accessibility within heterochromatin of an ectopic bacterial dam methylase and the decreased mobility of an HML heterochromatic fragment in sucrose gradients. These findings indicate an altered chromatin structure. We propose that Sir proteins recruited by the H4 tail then interact with the H3 tail to form a higher order silent chromatin structure.
Crystal structure of the sodium-potassium pump (Na+,K+-ATPase) with bound potassium and ouabain
Authors: Ogawa Haruo , Shinoda Takehiro , Cornelius Flemming , Toyoshima Chikashi
Published: August 2009

The sodium-potassium pump (Na+,K+-ATPase) is responsible for establishing Na+and K+concentration gradients across the plasma membrane and therefore plays an essential role in, for instance, generating action potentials. Cardiac glycosides, prescribed for congestive heart failure for more than 2 centuries, are efficient inhibitors of this ATPase. Here we describe a crystal structure of Na+,K+-ATPase with bound ouabain, a representative cardiac glycoside, at 2.8 Å resolution in a state analogous to E2·2K+·Pi. Ouabain is deeply inserted into the transmembrane domain with the lactone ring very close to the bound K+, in marked contrast to previous models. Due to antagonism between ouabain and K+, the structure represents a low-affinity ouabain-bound state. Yet, most of the mutagenesis data obtained with the high-affinity state are readily explained by the present crystal structure, indicating that the binding site for ouabain is essentially the same. According to a homology model for the high affinity state, it is a closure of the binding cavity that confers a high affinity.
A
Legionella
type IV effector activates the NF-κB pathway by phosphorylating the IκB family of inhibitors
Authors: Ge Jianning , Xu Hao , Li Ting , Zhou Yan , Zhang Zhibin , Li Shan , Liu Liping , Shao Feng
Published: August 2009

NF-κB is critical in innate immune defense responses against invading microbial pathogens.
Legionella pneumophila
infection of lung macrophages causes Legionnaire's disease with pneumonia symptoms. A set of NF-κB-controlled genes involved in inflammation and anti-apoptosis are up-regulated in macrophages upon
L. pneumophila
infection in a
Legionella
Dot/Icm type IV secretion system-dependent manner. Among ≈100 Dot/Icm substrates screened, we identified LegK1 as the sole
Legionella
protein that harbors a highly potent NF-κB-stimulating activity. LegK1 does not affect MAPK and IFN pathways. Activation of the NF-κB pathway by LegK1 requires its eukaryotic-like Ser/Thr kinase activity and is independent of upstream components in the NF-κB pathway, including TRAFs, NIK, MEKK3, and TAK1. Cell-free reconstitution revealed that LegK1 stimulated NF-κB activation in the absence of IKKα and IKKβ, and LegK1 efficiently phosphorylated IκBα on Ser-32 and Ser-36 both in vitro and in cells. LegK1 seems to mimic the host IKK as LegK1 also directly phosphorylated other IκB family of inhibitors including p100 in the noncanonical NF-κB pathway. Phosphorylation of p100 by LegK1 led to its maturation into p52. Thus, LegK1 is a bacterial effector that directly activates the host NF-κB signaling and likely plays important roles in modulating macrophage defense or inflammatory responses during
L. pneumophila
infection.
A role of histone H3 lysine 4 methyltransferase components in endosomal trafficking
Authors: Xu Zhuojin , Gong Qiang , Xia Bin , Groves Benjamin , Zimmermann Marc , Mugler Chris , Mu Dezhi , Matsumoto Brian , Seaman Matthew , Ma Dzwokai
Published: August 2009

Histone lysine methyltransferase complexes are essential for chromatin organization and gene regulation. Whether any of this machinery functions in membrane traffic is unknown. In this study, we report that mammal Dpy-30 (mDpy-30), a subunit of several histone H3 lysine 4 (H3K4) methyltransferase (H3K4MT) complexes, resides in the nucleus and at the trans-Golgi network (TGN). The TGN targeting of mDpy-30 is mediated by BIG1, a TGN-localized guanine nucleotide exchange factor for adenosine diphosphate ribosylation factor GTPases. Altering mDpy-30 levels changes the distribution of cation-independent mannose 6-phosphate receptor (CIMPR) without affecting that of TGN46 or transferrin receptor. Our experiments also indicate that mDpy-30 functions in the endosome to TGN transport of CIMPR and that its knockdown results in the enrichment of internalized CIMPR and recycling endosomes near cell protrusions. Much like mDpy-30 depletion, the knockdown of Ash2L or RbBP5, two other H3K4MT subunits, leads to a similar redistribution of CIMPR. Collectively, these results suggest that mDpy-30 and probably H3K4MT play a role in the endosomal transport of specific cargo proteins.
Regulated Ire1-dependent decay of messenger RNAs in mammalian cells
Authors: Hollien Julie , Lin Jonathan H. , Li Han , Stevens Nicole , Walter Peter , Weissman Jonathan S.
Published: August 2009

Maintenance of endoplasmic reticulum (ER) function is achieved in part through Ire1 (inositol-requiring enzyme 1), a transmembrane protein activated by protein misfolding in the ER. The cytoplasmic nuclease domain of Ire1 cleaves the messenger RNA (mRNA) encoding XBP-1 (X-box–binding protein 1), enabling splicing and production of this active transcription factor. We recently showed that Ire1 activation independently induces the rapid turnover of mRNAs encoding membrane and secreted proteins in Drosophila melanogaster cells through a pathway we call regulated Ire1-dependent decay (RIDD). In this study, we show that mouse fibroblasts expressing wild-type Ire1 but not an Ire1 variant lacking nuclease activity also degrade mRNAs in response to ER stress. Using a second variant of Ire1 that is activated by a small adenosine triphosphate analogue, we show that although XBP-1 splicing can be artificially induced in the absence of ER stress, RIDD appears to require both Ire1 activity and ER stress. Our data suggest that cells use a multitiered mechanism by which different conditions in the ER lead to distinct outputs from Ire1.
A host type I interferon response is induced by cytosolic sensing of the bacterial second messenger cyclic-di-GMP
Authors: McWhirter Sarah M. , Barbalat Roman , Monroe Kathryn M. , Fontana Mary F. , Hyodo Mamoru , Joncker Nathalie T. , Ishii Ken J. , Akira Shizuo , Colonna Marco , Chen Zhijian J. , Fitzgerald Katherine A. , Hayakawa Yoshihiro , Vance Russell E.
Published: August 2009

The innate immune system responds to unique molecular signatures that are widely conserved among microbes but that are not normally present in host cells. Compounds that stimulate innate immune pathways may be valuable in the design of novel adjuvants, vaccines, and other immunotherapeutics. The cyclic dinucleotide cyclic-di–guanosine monophosphate (c-di-GMP) is a recently appreciated second messenger that plays critical regulatory roles in many species of bacteria but is not produced by eukaryotic cells. In vivo and in vitro studies have previously suggested that c-di-GMP is a potent immunostimulatory compound recognized by mouse and human cells. We provide evidence that c-di-GMP is sensed in the cytosol of mammalian cells via a novel immunosurveillance pathway. The potency of cytosolic signaling induced by c-di-GMP is comparable to that induced by cytosolic delivery of DNA, and both nucleic acids induce a similar transcriptional profile, including triggering of type I interferons and coregulated genes via induction of TBK1, IRF3, nuclear factor κB, and MAP kinases. However, the cytosolic pathway that senses c-di-GMP appears to be distinct from all known nucleic acid–sensing pathways. Our results suggest a novel mechanism by which host cells can induce an inflammatory response to a widely produced bacterial ligand.
Testing the Accuracy of Regression Discontinuity Analysis Using Experimental Benchmarks
Authors: Green Donald P. , Leong Terence Y. , Kern Holger L. , Gerber Alan S. , Larimer Christopher W.
Published: August 2009

Regression discontinuity (RD) designs enable researchers to estimate causal effects using observational data. These causal effects are identified at the point of discontinuity that distinguishes those observations that do or do not receive the treatment. One challenge in applying RD in practice is that data may be sparse in the immediate vicinity of the discontinuity. Expanding the analysis to observations outside this immediate vicinity may improve the statistical precision with which treatment effects are estimated, but including more distant observations also increases the risk of bias. Model specification is another source of uncertainty; as the bandwidth around the cutoff point expands, linear approximations may break down, requiring more flexible functional forms. Using data from a large randomized experiment conducted by Gerber, Green, and Larimer (2008), this study attempts to recover an experimental benchmark using RD and assesses the uncertainty introduced by various aspects of model and bandwidth selection. More generally, we demonstrate how experimental benchmarks can be used to gauge and improve the reliability of RD analyses.
Striking the balance between PTEN and PDK1: it all depends on the cell context: Figure 1.
Authors: Iwanami Akio , Cloughesy Timothy F. , Mischel Paul S.
Published: August 2009

The phosphatidyl-inosital-3 kinase (PI3K) signaling pathway is critical for normal brain development and function and is commonly hyperactivated in brain cancer. The PTEN (phosphatase and tensin homolog deleted on chromosome 10) tumor suppressor protein and phosphate-depended kinase 1 (PDK-1) are critical regulators of this pathway. In the July 15, 2009, issue of Genes & Development, Chalhoub and colleagues (pp. 1619–1624) demonstrate PDK1-dependent and PDK1-independent effects of conditional PTEN deletion in the brain, and they identify cell type-specific differences in feedback regulation of the PI3K pathway. These studies provide important insights as to how neurons and glia may differentially regulate PI3K signaling, yielding intriguing clues about targeting PTEN-deficient brain cancers.
The presence of RNA polymerase II, active or stalled, predicts epigenetic fate of promoter CpG islands
Authors: Takeshima Hideyuki , Yamashita Satoshi , Shimazu Taichi , Niwa Tohru , Ushijima Toshikazu
Published: August 2009

Instructive mechanisms are present for induction of DNA methylation, as shown by methylation of specific CpG islands (CGIs) by specific inducers and in specific cancers. However, instructive factors involved are poorly understood, except for involvement of low transcription and trimethylation of histone H3 lysine 27 (H3K27me3). Here, we used methylated DNA immunoprecipitation (MeDIP) combined with a CGI oligonucleotide microarray analysis, and identified 5510 and 521 genes with promoter CGIs resistant and susceptible, respectively, to DNA methylation in prostate cancer cell lines. Expression analysis revealed that the susceptible genes had low transcription in a normal prostatic epithelial cell line. Chromatin immunoprecipitation with microarray hybridization (CHiP-chip) analysis of RNA polymerase II (Pol II) and histone modifications showed that, even among the genes with low transcription, the presence of Pol II was associated with marked resistance to DNA methylation (OR = 0.22; 95% CI = 0.12–0.38), and H3K27me3 was associated with increased susceptibility (OR = 11.20; 95% CI = 7.14–17.55). The same was true in normal human mammary epithelial cells for 5430 and 733 genes resistant and susceptible, respectively, to DNA methylation in breast cancer cell lines. These results showed that the presence of Pol II, active or stalled, and H3K27me3 can predict the epigenetic fate of promoter CGIs independently of transcription levels.
5′-Transducing SVA retrotransposon groups spread efficiently throughout the human genome
Authors: Damert Annette , Raiz Julija , Horn Axel V. , Löwer Johannes , Wang Hui , Xing Jinchuan , Batzer Mark A. , Löwer Roswitha , Schumann Gerald G.
Published: August 2009

SVA elements represent the youngest family of hominid non-LTR retrotransposons, which alter the human genome continuously. They stand out due to their organization as composite repetitive elements. To draw conclusions on the assembly process that led to the current organization of SVA elements and on their transcriptional regulation, we initiated our study by assessing differences in structures of the 116 SVA elements located on human chromosome 19. We classified SVA elements into seven structural variants, including novel variants like 3′-truncated elements and elements with 5′-flanking sequence transductions. We established a genome-wide inventory of 5′-transduced SVA elements encompassing ∼8% of all human SVA elements. The diversity of 5′ transduction events found indicates transcriptional control of their SVA source elements by a multitude of external cellular promoters in germ cells in the course of their evolution and suggests that SVA elements might be capable of acquiring 5′ promoter sequences. Our data indicate that SVA-mediated 5′ transduction events involve alternative RNA splicing at cryptic splice sites. We analyzed one remarkably successful human-specific SVA 5′ transduction group in detail because it includes at least 32% of all SVA subfamily F members. An ancient retrotransposition event brought an SVA insertion under transcriptional control of the MAST2 gene promoter, giving rise to the primal source element of this group. Members of this group are currently transcribed. Here we show that SVA-mediated 5′ transduction events lead to structural diversity of SVA elements and represent a novel source of genomic rearrangements contributing to genomic diversity.
Distinguishing direct versus indirect transcription factor–DNA interactions
Authors: Gordân Raluca , Hartemink Alexander J. , Bulyk Martha L.
Published: August 2009

Transcriptional regulation is largely enacted by transcription factors (TFs) binding DNA. Large numbers of TF binding motifs have been revealed by ChIP-chip experiments followed by computational DNA motif discovery. However, the success of motif discovery algorithms has been limited when applied to sequences bound in vivo (such as those identified by ChIP-chip) because the observed TF–DNA interactions are not necessarily direct: Some TFs predominantly associate with DNA indirectly through protein partners, while others exhibit both direct and indirect binding. Here, we present the first method for distinguishing between direct and indirect TF–DNA interactions, integrating in vivo TF binding data, in vivo nucleosome occupancy data, and motifs from in vitro protein binding microarray experiments. When applied to yeast ChIP-chip data, our method reveals that only 48% of the data sets can be readily explained by direct binding of the profiled TF, while 16% can be explained by indirect DNA binding. In the remaining 36%, none of the motifs used in our analysis was able to explain the ChIP-chip data, either because the data were too noisy or because the set of motifs was incomplete. As more in vitro TF DNA binding motifs become available, our method could be used to build a complete catalog of direct and indirect TF–DNA interactions. Our method is not restricted to yeast or to ChIP-chip data, but can be applied in any system for which both in vivo binding data and in vitro DNA binding motifs are available.
Morphology and Husbandry of the Large Milkweed Bug, Oncopeltus fasciatus
Authors: Liu Paul , Kaufman Thomas C.
Published: August 2009

INTRODUCTION

The Hemiptera, or “true bugs,” are the most successful group of hemimetabolous (lacking a pupal stage) insects. The large milkweed bug Oncopeltus fasciatus fulfills many of the criteria often used when choosing a laboratory model system, including phylogenetic position, interesting character state, and practicality of care. Oncopeltus has a history of usefulness in the analysis of segmentation and cell polarity that precedes the more molecular approaches that are currently applied. This article presents an introduction to Oncopeltus as a laboratory model system.
Axolotl (Ambystoma mexicanum) In Vitro Fertilization
Authors: Khattak Shahryar , Tanaka Elly M.
Published: August 2009

INTRODUCTION

Breeding of axolotl (Ambystoma mexicanum) can be accomplished by natural or artificial methods. Although natural matings are a very easy way of obtaining axolotl embryos, in vitro fertilization can have other advantages. For example, through in vitro fertilization, it is possible to know the exact time of fertilization. This protocol describes preparation of a sperm suspension and in vitro fertilization of axolotl eggs after injection of females with follicle stimulating hormone (FSH).
Generation of Transgenic Axolotls (Ambystoma mexicanum): Figure 1.
Authors: Khattak Shahryar , Richter Tobias , Tanaka Elly M.
Published: August 2009

INTRODUCTION

Injection of RNA, DNA, or morpholinos into amphibian and fish embryos is a standard technique used to test gene function during development or to make transgenic animals. The techniques used to inject axolotl (Ambystoma mexicanum) embryos are very similar to those used for Xenopus embryos, with a few modifications to account for the larger egg size and “softer” cytoplasm. For example, amphibian embryos must be “dejellied” prior to injection. Chemical treatments used to remove the jellycoat for Xenopus eggs are not reliable for axolotl eggs; thus, dejellying is performed manually. In addition, greater injection volumes (typically 50 nL) are used with axolotl embryos, with correspondingly larger total amounts of DNA and RNA, as compared with Xenopus. Injection of plasmid DNA into the axolotl egg results in varying frequencies of integration (depending on the plasmid DNA) within one of the first few cell divisions. The integration efficiency is significantly increased if the plasmid DNA contains SceI meganuclease sites and is coinjected with SceI meganuclease, as described in this protocol.
Suppression of galectin‐3 expression enhances apoptosis and chemosensitivity in liver fluke‐associated cholangiocarcinoma
Authors: Wongkham Sopit , Junking Mutita , Wongkham Chaisiri , Sripa Banchob , Chur‐in Siri , Araki Norie
Published: August 2009

Cholangiocarcinoma (CCA) is a fatal disease with high resistance to anticancer drugs. This is probably in part due to enhanced resistance to apoptosis. We have previously shown that galectin‐3 (Gal‐3), a β‐galactoside‐binding lectin, is highly expressed in CCA tissues. In this study, we demonstrated further that Gal‐3 plays a direct role in anti‐apoptosis regardless of the apoptotic insults. The anti‐apoptotic activity and chemoresistance of CCA cells were related to Gal‐3 expression level. Suppression of Gal‐3 expression with siRNA stimulated apoptosis. siGal‐3‐K626 transiently depleted Gal‐3 expression to the baseline and dramatically induced apoptosis, while siGal‐3‐K402 suppressed Gal‐3 expression by 50% and provoked cell apoptosis, but only under apoptotic insults (hypoxic conditions or short UV radiation). These actions were reversed in Gal‐3 overexpressing CCA cells. The correlation between the degree of anti‐apoptotic activity and the level of endogenous Gal‐3 was demonstrated. Suppression of Gal‐3 expression in CCA cells with siGal‐3‐K402 significantly enhanced apoptosis induced by cisplatin or 5‐fluorouracil by approximately 10 times, whereas overexpression of Gal‐3 led to an increased resistance to drugs. In summary, the present study showed that the cellular level of Gal‐3 might contribute to the anti‐apoptotic activity and chemoresistance of CCA cells. Hence, Gal‐3 expression level in cancer cells or tissues may be a marker for predicting chemotherapeutic response, and Gal‐3 may be a specific gene‐targeting therapy option for treating CCA. (Cancer Sci 2009; 00: 000–000)
Test–re‐test reliability of DSM‐IV adopted criteria for 3,4‐methylenedioxymethamphetamine (MDMA) abuse and dependence: a cross‐national study
Authors: Cottler Linda B. , Leung Kit Sang , Abdallah Arbi Ben
Published: August 2009

Aims  This study evaluated the prevalence and reliability of DSM‐IV adopted criteria for 3,4‐methylenedioxymethamphetamine (MDMA) abuse and dependence with a purpose to determine whether it is best conceptualized within the category of hallucinogens, amphetamines or its own category.

Design  Test–re‐test study.

Participants  MDMA users (life‐time use >5 times) were recruited in St Louis, Miami and Sydney (n = 593). The median life‐time MDMA consumption was 50 pills at the baseline.

Measurements  The computerized Substance Abuse Module for Club Drug (CD‐SAM) was used to assess MDMA abuse and dependence. The Discrepancy Interview Protocol (DIP) was used to determine the reasons for the discrepant responses between the two interviews. Reliability of diagnoses, individual diagnostic criteria and withdrawal symptoms was examined using the kappa coefficient (κ).

Findings  For baseline data, 15% and 59% met MDMA abuse and dependence, respectively. Substantial test–re‐test reliability of the diagnoses was observed consistently across cities (κ = 0.69). ‘Continued use despite knowledge of physical/psychological problems’ (87%) and ‘withdrawal’ (68%) were the two most prevalent dependence criteria. ‘Physically hazardous use’ was the most prevalent abuse criterion. Six dependence criteria and all abuse criteria were reported reliably across cities (κ: 0.53–0.77). Seventeen of 19 withdrawal symptoms showed consistency in the reliability across cities. The most commonly reported reason for discrepant responses was ‘interpretation of question changed’. Only a small proportion of the total discrepancies were attributed to lying or social desirability.

Conclusion   The adopted DSM‐IV diagnostic classification for MDMA abuse and dependence was moderately reliable across cities. Findings on MDMA withdrawal support the argument that MDMA should be separated from other hallucinogens in DSM.
Expression and regulation of Sclerotinia sclerotiorum necrosis and ethylene‐inducing peptides (NEPs)
Authors: DALLAL BASHI ZAFER , HEGEDUS DWAYNE D. , BUCHWALDT LONE , RIMMER S. ROGER , BORHAN MOHAMMED H.
Published: August 2009

Successful host colonization by necrotrophic plant pathogens requires the induction of plant cell death to provide the nutrients needed for infection establishment and progression. We have cloned two genes encoding necrosis and ethylene‐inducing peptides from Sclerotinia sclerotiorum, which we named SsNep1 and SsNep2. The peptides encoded by these genes induce necrosis when expressed transiently in tobacco leaves. SsNep1 is expressed at a very low level relative to SsNep2 during infection. The expression of SsNep2 was induced by contact with solid surfaces and occurred in both the necrotic zone and at the leading margin of the infection. SsNep2 expression was dependent on calcium and cyclic adenosine monophosphate signalling, as compounds affecting these pathways reduced or abolished SsNep2 expression coincident with a partial or total loss of virulence.
The impact of intra‐operative transoesophageal echocardiography on cardiac surgical practice
Authors: Klein A. A. , Snell A. , Nashef S. A. M. , Hall R. M. O. , Kneeshaw J. D. , Arrowsmith J. E.
Published: August 2009

The use of transoesophageal echocardiography during cardiac surgery has increased dramatically and it is now widely accepted as a routine monitoring and diagnostic tool. A prospective study was carried out between September 2004 and September 2007, and included all patients in whom intra‐operative echocardiography was performed, 2 473 (44%) out of a total of 5 591 cases. Changes to surgery were subdivided into predictable (where echocardiographic examination was planned specifically to guide surgery) and unpredictable (new pathology not diagnosed pre‐operatively). A change in the planned surgical procedure was documented in 312 (15%) cases. In 216 (69%) patients the changes were predictable and in 96 (31%) they were unpredictable. The number of predictable changes increased between 2004–5 and 2006–7 (8% vs 13%, p = 0.025). In these cases, intra‐operative echocardiography was specifically requested by the surgeon to help determine the operative intervention. This has implications for consent and operative risk, which have yet to be fully determined.
The role of genetic variation in peroxisome proliferator‐activated receptors in the polycystic ovary syndrome (PCOS): an original case–control study followed by systematic review and meta‐analysis of existing evidence
Authors: San‐Millán José L. , Escobar‐Morreale Héctor F.
Published: August 2009

Objective  To study the association of polymorphisms in the genes encoding peroxisome proliferator‐activated receptors (PPARs) with the polycystic ovary syndrome (PCOS).

Design  Case–control study and meta‐analysis of published evidence.

Patients  One hundred and sixty‐one polycystic ovary syndrome patients and 113 non‐hyperandrogenic women.

Measurements  Genotyping for PPAR‐γ coactivator‐1 gene (PPARGC1A) Gly482Ser, PPAR‐α Leu162Val, PPAR‐δ rs2267668A/G, PPAR‐δ−87T/C, PPAR‐γ2 Pro12Ala and PPAR‐γ2 −681C/G variants and systematic review of the literature using the Entrez‐PubMed search engine, followed by meta‐analysis whenever possible.

Results  Polycystic ovary syndrome patients carried the Gly482Ser variant in PPARGC1A more frequently than controls (72%vs. 58%, χ2 = 5·54 P = 0·019), whereas carriers of the PPAR‐α Leu162Val, PPAR‐δ rs2267668A/G, PPAR‐δ−87T/C, PPAR‐γ2 Pro12Ala and PPAR‐γ2 −681C/G variants were distributed similarly among both groups. The interaction between the PPARGC1A Gly482Ser and PPAR‐δ−87T/C variants was also associated with PCOS (OR = 1·24, 95% CI 1·05–1·50, P = 0·008). The systematic review identified 31 studies addressing associations between PPARs variants and PCOS; meta‐analysis was possible for nine studies focusing on the PPAR‐γ2 Pro12Ala variant. Although the individual studies did not reveal any statistically significant association, meta‐analysis uncovered that carrying the PPAR‐γ2 Pro12Ala variant was associated with a reduced probability of having PCOS (OR = 0·77, 95% CI 0·61–0·96, P = 0·025), and that this association may be mediated by an effect on insulin sensitivity.

Conclusions  Common polymorphisms in the PPARGC1A, PPAR‐δ and PPAR‐γ2 loci are associated with PCOS.
Warming leads to higher species turnover in a coastal ecosystem
Authors: HILLEBRAND HELMUT , SOININEN JANNE , SNOEIJS PAULINE
Published: August 2009

The responses of ecological communities and ecosystems to increased rates of environmental change will be strongly influenced by variation in the diversity of community composition. Yet, our understanding of how diversity is affected by rising temperatures is inconclusive and mainly based on indirect evidence or short‐term experiments. In our study, we analyse the diversity and species turnover of benthic epilithic communities within the thermal flume of a nuclear power plant at the Swedish coast. This flume covers the range of predicted future temperature rises. Species composition was significantly different between control sites and sites with higher temperatures. We found that temperature had little effect on the number of species in three functional groups (macroinvertebrates, benthic diatoms, and macrophytes, which here comprise multicellular algae and macroscopic colonies of unicellular algae and cyanobacteria), neither at single sampling dates nor summed for the entire observation year. However, species turnover significantly increased with increasing temperature for diatoms, macrophytes and invertebrates. Different temperature regimes resulted in significantly different species composition and indicator species. Thus, increasing temperatures in the thermal flume increased temporal beta‐diversity and decreased compositional stability of communities, although observed richness did not change at any point in time. We highlight the need to investigate the consequences of such declines in compositional stability for functional stability of ecosystem processes.
Bacterial lipopeptide triggers massive albuminuria in murine lupus nephritis by activating Toll‐like receptor 2 at the glomerular filtration barrier
Authors: Pawar Rahul D. , Castrezana‐Lopez Liliana , Allam Ramanjaneyulu , Kulkarni Onkar P. , Segerer Stephan , Radomska Ewa , Meyer Tobias N. , Schwesinger Catherine‐Meyer , Akis Nese , Gröne Hermann‐Josef , Anders Hans‐Joachim
Published: August 2009

What are the molecular mechanisms of bacterial infections triggering or modulating lupus nephritis? In nephritic MRLlpr/lpr mice, transient exposure to bacterial cell wall components such as lipopeptide or lipopolysaccharide (LPS) increased splenomegaly, the production of DNA autoantibodies, and serum interleukin (IL)‐6, IL‐12 and tumour necrosis factor (TNF) levels, and aggravated lupus nephritis. Remarkably, bacterial lipopeptide induced massive albuminuria in nephritic but not in non‐nephritic mice. This was associated with down‐regulation of renal nephrin mRNA and redistribution from its normal localization at foot processes to the perinuclear podocyte area in nephritic MRLlpr/lpr mice. Bacterial lipopeptide activates Toll‐like receptor 2 (TLR2), which we found to be expressed on cultured podocytes and glomerular endothelial cells. TNF and interferon (IFN)‐γ induced TLR2 mRNA and receptor expression in both cell types. Albumin permeability was significantly increased in cultured podocytes and glomerular endothelial cells upon stimulation by bacterial lipopeptide. LPS also induced moderate albuminuria. In summary, bacterial lipopeptide and LPS can aggravate glomerulonephritis but only lipopeptide potently induces severe albuminuria in MRLlpr/lpr mice.
Glycogen synthase kinase‐3 negatively regulates anti‐inflammatory interleukin‐10 for lipopolysaccharide‐induced iNOS/NO biosynthesis and RANTES production in microglial cells
Authors: Huang Wei‐Ching , Lin Yee‐Shin , Wang Chi‐Yun , Tsai Cheng‐Chieh , Tseng Hsiang‐Chi , Chen Chia‐Ling , Lu Pei‐Jung , Chen Po‐See , Qian Li , Hong Jau‐Shyong , Lin Chiou‐Feng
Published: August 2009

The inflammatory effects of glycogen synthase kinase‐3 (GSK‐3) have been identified; however, the potential mechanism is still controversial. In this study, we investigated the effects of GSK‐3‐mediated interleukin‐10 (IL‐10) inhibition on lipopolysaccharide (LPS)‐induced inflammation. Treatment with GSK‐3 inhibitor significantly blocked LPS‐induced nitric oxide (NO) production as well as inducible NO synthase (iNOS) expression in BV2 murine microglial cells and primary rat microglia‐enriched cultures. Using an antibody array and enzyme‐linked immunosorbent assay, we found that GSK‐3‐inhibitor treatment blocked LPS‐induced upregulation of regulated on activation normal T‐cell expressed and secreted (RANTES) and increased IL‐10 expression. The time kinetics and dose–response relations were confirmed. Reverse transcription–polymerase chain reaction showed changes on the messenger RNA level as well. Inhibiting GSK‐3 using short‐interference RNA, and transfecting cells with dominant‐negative GSK‐3β, blocked LPS‐elicited NO and RANTES expression but increased IL‐10 expression. In contrast, GSK‐3β overexpression upregulated NO and RANTES but downregulated IL‐10 in LPS‐stimulated cells. Treating cells with anti‐IL‐10 neutralizing antibodies to prevent GSK‐3 from downregulating NO and RANTES showed that the anti‐inflammatory effects are, at least in part, IL‐10‐dependent. The involvement of Akt, extracellular signal‐regulated kinase, p38 mitogen‐activated protein kinase and nuclear factor‐κB that positively regulated IL‐10 was demonstrated. Furthermore, inhibiting GSK‐3 increased the nuclear translocation of transcription factors, that all important for IL‐10 expression, including CCAAT/enhancer‐binding protein beat (C/EBPβ), C/EBPδ, cAMP response binding element protein and NF‐κB. Taken together, these findings reveal that LPS induces iNOS/NO biosynthesis and RANTES production through a mechanism involving GSK‐3‐mediated IL‐10 downregulation.