bluuebunny/crossref_metadata_2025 huggingface dataset:
Targeted metabolic labeling of yeast N-glycans with unnatural sugars
Authors: Breidenbach Mark A. , Gallagher Jennifer E. G. , King David S. , Smart Brian P. , Wu Peng , Bertozzi Carolyn R.
Published: February 2010

Metabolic labeling of glycans with synthetic sugar analogs has emerged as an attractive means for introducing nonnatural chemical functionality into glycoproteins. However, the complexities of glycan biosynthesis prevent the installation of nonnatural moieties at defined, predictable locations within glycoproteins at high levels of incorporation. Here, we demonstrate that the conserved
N
-acetyglucosamine (GlcNAc) residues within chitobiose cores of N-glycans in the model organism
Saccharomyces cerevisiae
can be specifically targeted for metabolic replacement by unnatural sugars. We introduced an exogenous GlcNAc salvage pathway into yeast, allowing cells to metabolize GlcNAc provided as a supplement to the culture medium. We then rendered the yeast auxotrophic for production of the donor nucleotide-sugar uridine-diphosphate-GlcNAc (UDP-GlcNAc) by deletion of the essential gene
GNA1
. We demonstrate that
gna1
Δ strains require a GlcNAc supplement and that expression plasmids containing both exogenous components of the salvage pathway, GlcNAc transporter
NGT1
from
Candida albicans
and GlcNAc kinase
NAGK
from
Homo sapiens
, are required for rescue in this context. Further, we show that cells successfully incorporate synthetic GlcNAc analogs
N
-azidoacetyglucosamine (GlcNAz) and
N
-(4-pentynoyl)-glucosamine (GlcNAl) into cell-surface glycans and secreted glycoproteins. To verify incorporation of the nonnatural sugars at N-glycan core positions, endoglycosidase H (endoH)-digested peptides from a purified secretory glycoprotein, Ygp1, were analyzed by mass spectrometry. Multiple Ygp1 N-glycosylation sites bearing GlcNAc, isotopically labeled GlcNAc, or GlcNAz were identified; these modifications were dependent on the supplement added to the culture medium. This system enables the production of glycoproteins that are functionalized for specific chemical modifications at their glycosylation sites.
Molecular complexity of successive bacterial epidemics deconvoluted by comparative pathogenomics
Authors: Beres Stephen B. , Carroll Ronan K. , Shea Patrick R. , Sitkiewicz Izabela , Martinez-Gutierrez Juan Carlos , Low Donald E. , McGeer Allison , Willey Barbara M. , Green Karen , Tyrrell Gregory J. , Goldman Thomas D. , Feldgarden Michael , Birren Bruce W. , Fofanov Yuriy , Boos John , Wheaton William D. , Honisch Christiane , Musser James M.
Published: February 2010

Understanding the fine-structure molecular architecture of bacterial epidemics has been a long-sought goal of infectious disease research. We used short-read-length DNA sequencing coupled with mass spectroscopy analysis of SNPs to study the molecular pathogenomics of three successive epidemics of invasive infections involving 344 serotype M3 group A
Streptococcus
in Ontario, Canada. Sequencing the genome of 95 strains from the three epidemics, coupled with analysis of 280 biallelic SNPs in all 344 strains, revealed an unexpectedly complex population structure composed of a dynamic mixture of distinct clonally related complexes. We discovered that each epidemic is dominated by micro- and macrobursts of multiple emergent clones, some with distinct strain genotype–patient phenotype relationships. On average, strains were differentiated from one another by only 49 SNPs and 11 insertion-deletion events (indels) in the core genome. Ten percent of SNPs are strain specific; that is, each strain has a unique genome sequence. We identified nonrandom temporal–spatial patterns of strain distribution within and between the epidemic peaks. The extensive full-genome data permitted us to identify genes with significantly increased rates of nonsynonymous (amino acid-altering) nucleotide polymorphisms, thereby providing clues about selective forces operative in the host. Comparative expression microarray analysis revealed that closely related strains differentiated by seemingly modest genetic changes can have significantly divergent transcriptomes. We conclude that enhanced understanding of bacterial epidemics requires a deep-sequencing, geographically centric, comparative pathogenomics strategy.
The early autophagic pathway is activated by hepatitis B virus and required for viral DNA replication
Authors: Sir Donna , Tian Yongjun , Chen Wen-ling , Ann David K. , Yen Tien-Sze Benedict , Ou Jing-hsiung James
Published: February 2010

Autophagy is a catabolic process by which cells remove long-lived proteins and damaged organelles for recycling. Viral infections may also induce autophagic response. Here we show that hepatitis B virus (HBV), a pathogen that chronically infects ≈350 million people globally, can enhance autophagic response in cell cultures, mouse liver, and during natural infection. This enhancement of the autophagic response is not coupled by an increase of autophagic protein degradation and is dependent on the viral X protein, which binds to and enhances the enzymatic activity of phosphatidylinositol 3-kinase class III, an enzyme critical for the initiation of autophagy. Further analysis indicates that autophagy enhances HBV DNA replication, with minimal involvement of late autophagic vacuoles in this process. Our studies thus demonstrate that a DNA virus can use autophagy to enhance its own replication and indicate the possibility of targeting the autophagic pathway for the treatment of HBV patients.
Cross-species analysis traces adaptation of Rubisco toward optimality in a low-dimensional landscape
Authors: Savir Yonatan , Noor Elad , Milo Ron , Tlusty Tsvi
Published: February 2010

Rubisco (D-ribulose 1,5-bisphosphate carboxylase/oxygenase), probably the most abundant protein in the biosphere, performs an essential part in the process of carbon fixation through photosynthesis, thus facilitating life on earth. Despite the significant effect that Rubisco has on the fitness of plants and other photosynthetic organisms, this enzyme is known to have a low catalytic rate and a tendency to confuse its substrate, carbon dioxide, with oxygen. This apparent inefficiency is puzzling and raises questions regarding the roles of evolution versus biochemical constraints in shaping Rubisco. Here we examine these questions by analyzing the measured kinetic parameters of Rubisco from various organisms living in various environments. The analysis presented here suggests that the evolution of Rubisco is confined to an effectively one-dimensional landscape, which is manifested in simple power law correlations between its kinetic parameters. Within this one-dimensional landscape, which may represent biochemical and structural constraints, Rubisco appears to be tuned to the intracellular environment in which it resides such that the net photosynthesis rate is nearly optimal. Our analysis indicates that the specificity of Rubisco is not the main determinant of its efficiency but rather the trade-off between the carboxylation velocity and CO
2
affinity. As a result, the presence of oxygen has only a moderate effect on the optimal performance of Rubisco, which is determined mostly by the local CO
2
concentration. Rubisco appears as an experimentally testable example for the evolution of proteins subject both to strong selection pressure and to biochemical constraints that strongly confine the evolutionary plasticity to a low-dimensional landscape.
Human strategy updating in evolutionary games
Authors: Traulsen Arne , Semmann Dirk , Sommerfeld Ralf D. , Krambeck Hans-Jürgen , Milinski Manfred
Published: February 2010

Evolutionary game dynamics describe not only frequency-dependent genetic evolution, but also cultural evolution in humans. In this context, successful strategies spread by imitation. It has been shown that the details of strategy update rules can have a crucial impact on evolutionary dynamics in theoretical models and, for example, can significantly alter the level of cooperation in social dilemmas. What kind of strategy update rules can describe imitation dynamics in humans? Here, we present a way to measure such strategy update rules in a behavioral experiment. We use a setting in which individuals are virtually arranged on a spatial lattice. This produces a large number of different strategic situations from which we can assess strategy updating. Most importantly, spontaneous strategy changes corresponding to mutations or exploration behavior are more frequent than assumed in many models. Our experimental approach to measure properties of the update mechanisms used in theoretical models will be useful for mathematical models of cultural evolution.
Abasic sites and strand breaks in DNA cause transcriptional mutagenesis in
Escherichia coli
Authors: Clauson Cheryl L. , Oestreich Kenneth J. , Austin James W. , Doetsch Paul W.
Published: February 2010

DNA damage occurs continuously, and faithful replication and transcription are essential for maintaining cell viability. Cells in nature are not dividing and replicating DNA often; therefore it is important to consider the outcome of RNA polymerase (RNAP) encounters with DNA damage. Base damage in the DNA can affect transcriptional fidelity, leading to production of mutant mRNA and protein in a process termed transcriptional mutagenesis (TM). Abasic (AP) sites and strand breaks are frequently occurring, spontaneous damages that are also base excision repair (BER) intermediates. In vitro studies have demonstrated that these lesions can be bypassed by RNAP; however this has never been assessed in vivo. This study demonstrates that RNAP is capable of bypassing AP sites and strand breaks in
Escherichia coli
and results in TM through adenine incorporation in nascent mRNA. Elimination of the enzymes that process these lesions further increases TM; however, such mutants can still complete repair by other downstream pathways. These results show that AP sites and strand breaks can result in mutagenic RNAP bypass and have important implications for the biologic endpoints of DNA damage.
Small-molecule screen identifies reactive oxygen species as key regulators of neutrophil chemotaxis
Authors: Hattori Hidenori , Subramanian Kulandayan K. , Sakai Jiro , Jia Yonghui , Li Yitang , Porter Timothy F. , Loison Fabien , Sarraj Bara , Kasorn Anongnard , Jo Hakryul , Blanchard Catlyn , Zirkle Dorothy , McDonald Douglas , Pai Sung-Yun , Serhan Charles N. , Luo Hongbo R.
Published: February 2010

Neutrophil chemotaxis plays an essential role in innate immunity, but the underlying cellular mechanism is still not fully characterized. Here, using a small-molecule functional screening, we identified NADPH oxidase–dependent reactive oxygen species as key regulators of neutrophil chemotactic migration. Neutrophils with pharmacologically inhibited oxidase, or isolated from chronic granulomatous disease (CGD) patients and mice, formed more frequent multiple pseudopodia and lost their directionality as they migrated up a chemoattractant concentration gradient. Knocking down NADPH oxidase in differentiated neutrophil-like HL60 cells also led to defective chemotaxis. Consistent with the in vitro results, adoptively transferred CGD murine neutrophils showed impaired in vivo recruitment to sites of inflammation. Together, these results present a physiological role for reactive oxygen species in regulating neutrophil functions and shed light on the pathogenesis of CGD.
Construction and genetic selection of small transmembrane proteins that activate the human erythropoietin receptor
Authors: Cammett Tobin J. , Jun Susan J. , Cohen Emily B. , Barrera Francisco N. , Engelman Donald M. , DiMaio Daniel
Published: February 2010

This work describes a genetic approach to isolate small, artificial transmembrane (TM) proteins with biological activity. The bovine papillomavirus E5 protein is a dimeric, 44-amino acid TM protein that transforms cells by specifically binding and activating the platelet-derived growth factor β receptor (PDGFβR). We used the E5 protein as a scaffold to construct a retrovirus library expressing ∼500,000 unique 44-amino acid proteins with randomized TM domains. We screened this library to select small, dimeric TM proteins that were structurally unrelated to erythropoietin (EPO), but specifically activated the human EPO receptor (hEPOR). These proteins did not activate the murine EPOR or the PDGFβR. Genetic studies with one of these activators suggested that it interacted with the TM domain of the hEPOR. Furthermore, this TM activator supported erythroid differentiation of primary human hematopoietic progenitor cells in vitro in the absence of EPO. Thus, we have changed the specificity of a protein so that it no longer recognizes its natural target but, instead, modulates an entirely different protein. This represents a novel strategy to isolate small artificial proteins that affect diverse membrane proteins. We suggest the word “traptamer” for these transmembrane aptamers.
BDNF variant Val66Met interacts with estrous cycle in the control of hippocampal function
Authors: Spencer Joanna L. , Waters Elizabeth M. , Milner Teresa A. , Lee Francis S. , McEwen Bruce S.
Published: February 2010

Natural fluctuations in circulating estradiol are associated with behavioral changes, including severe disturbances in mood and cognition in some women. Common genetic variation in some of the molecular mediators of estradiol effects on these behaviors, in brain regions such as the hippocampus, may explain individual variation in estradiol effects on behavior. We tested whether the common human variant BDNF Val66Met interacts with estradiol in the control of hippocampal function in cycling female mice homozygous for the wild-type Val or BDNF Met variant. BDNF Met increased anxiety behavior, impaired memory, and increased expression of BDNF and its receptor TrkB in the hippocampal formation. BDNF Met also dramatically altered the fluctuation of spatial memory, hippocampal Akt phosphorylation, and PSD-95 protein expression across the estrous cycle. The variant BDNF Val66Met should therefore be considered as a strong candidate for mediating genetic differences in ovarian steroid-related behavioral changes and disorders.
A cell protection screen reveals potent inhibitors of multiple stages of the hepatitis C virus life cycle
Authors: Chockalingam Karuppiah , Simeon Rudo L. , Rice Charles M. , Chen Zhilei
Published: February 2010

The hepatitis C virus (HCV) life cycle involves multiple steps, but most current drug candidates target only viral replication. The inability to systematically discover inhibitors targeting multiple steps of the HCV life cycle has hampered antiviral development. We present a simple screen for HCV antivirals based on the alleviation of HCV-mediated cytopathic effect in an engineered cell line—n4mBid. This approach obviates the need for a secondary screen to avoid cytotoxic false-positive hits. Application of our screen to 1280 compounds, many in clinical trials or approved for therapeutic use, yielded >200 hits. Of the 55 leading hits, 47 inhibited one or more aspects of the HCV life cycle by >40%. Six compounds blocked HCV entry to levels similar to an antibody (JS-81) targeting the HCV entry receptor CD81. Seven hits inhibited HCV replication and/or infectious virus production by >100-fold, with one (quinidine) inhibiting infectious virus production by 450-fold relative to HCV replication levels. This approach is simple and inexpensive and should enable the rapid discovery of new classes of HCV life cycle inhibitors.
Three-dimensional structure of the anthrax toxin pore inserted into lipid nanodiscs and lipid vesicles
Authors: Katayama H. , Wang J. , Tama F. , Chollet L. , Gogol E. P. , Collier R. J. , Fisher M. T.
Published: February 2010

A major goal in understanding the pathogenesis of the anthrax bacillus is to determine how the protective antigen (PA) pore mediates translocation of the enzymatic components of anthrax toxin across membranes. To obtain structural insights into this mechanism, we constructed PA-pore membrane complexes and visualized them by using negative-stain electron microscopy. Two populations of PA pores were visualized in membranes, vesicle-inserted and nanodisc-inserted, allowing us to reconstruct two virtually identical PA-pore structures at 22-Å resolution. Reconstruction of a domain 4-truncated PA pore inserted into nanodiscs showed that this domain does not significantly influence pore structure. Normal mode flexible fitting of the x-ray crystallographic coordinates of the PA prepore indicated that a prominent flange observed within the pore lumen is formed by the convergence of mobile loops carrying Phe427, a residue known to catalyze protein translocation. Our results have identified the location of a crucial functional element of the PA pore and documented the value of combining nanodisc technology with electron microscopy to examine the structures of membrane-interactive proteins.
Functional
cis
-regulatory genomics for systems biology
Authors: Nam Jongmin , Dong Ping , Tarpine Ryan , Istrail Sorin , Davidson Eric H.
Published: February 2010

Gene expression is controlled by interactions between
trans
-regulatory factors and
cis
-regulatory DNA sequences, and these interactions constitute the essential functional linkages of gene regulatory networks (GRNs). Validation of GRN models requires experimental
cis
-regulatory tests of predicted linkages to authenticate their identities and proposed functions. However,
cis
-regulatory analysis is, at present, at a severe bottleneck in genomic system biology because of the demanding experimental methodologies currently in use for discovering
cis
-regulatory modules (CRMs), in the genome, and for measuring their activities. Here we demonstrate a high-throughput approach to both discovery and quantitative characterization of CRMs. The unique aspect is use of DNA sequence tags to “barcode” CRM expression constructs, which can then be mixed, injected together into sea urchin eggs, and subsequently deconvolved. This method has increased the rate of
cis
-regulatory analysis by >100-fold compared with conventional one-by-one reporter assays. The utility of the DNA-tag reporters was demonstrated by the rapid discovery of 81 active CRMs from 37 previously unexplored sea urchin genes. We then obtained simultaneous high-resolution temporal characterization of the regulatory activities of more than 80 CRMs. On average 2–3 CRMs were discovered per gene. Comparison of endogenous gene expression profiles with those of the CRMs recovered from each gene showed that, for most cases, at least one CRM is active in each phase of endogenous expression, suggesting that CRM recovery was comprehensive. This approach will qualitatively alter the practice of GRN construction as well as validation, and will impact many additional areas of regulatory system biology.
Regulation of PI3-kinase/Akt signaling by muscle-enriched microRNA-486
Authors: Small Eric M. , O’Rourke Jason R. , Moresi Viviana , Sutherland Lillian B. , McAnally John , Gerard Robert D. , Richardson James A. , Olson Eric N.
Published: February 2010

microRNAs (miRNAs) play key roles in modulating a variety of cellular processes through repression of mRNA targets. In a screen for miRNAs regulated by myocardin-related transcription factor-A (MRTF-A), a coactivator of serum response factor (SRF), we discovered a muscle-enriched miRNA, miR-486, controlled by an alternative promoter within intron 40 of the
Ankyrin-1
gene. Transcription of miR-486 is directly controlled by SRF and MRTF-A, as well as by MyoD. Among the most strongly predicted targets of miR-486 are phosphatase and tensin homolog (PTEN) and Foxo1a, which negatively affect phosphoinositide-3-kinase (PI3K)/Akt signaling. Accordingly, PTEN and Foxo1a protein levels are reduced by miR-486 overexpression, which, in turn, enhances PI3K/Akt signaling. Similarly, we show that MRTF-A promotes PI3K/Akt signaling by up-regulating miR-486 expression. Conversely, inhibition of miR-486 expression enhances the expression of PTEN and Foxo1a and dampens signaling through the PI3K/Akt-signaling pathway. Our findings implicate miR-486 as a downstream mediator of the actions of SRF/MRTF-A and MyoD in muscle cells and as a potential modulator of PI3K/Akt signaling.
Induction and patterning of trunk and tail neural ectoderm by the homeobox gene
eve1
in zebrafish embryos
Authors: Cruz Carlos , Maegawa Shingo , Weinberg Eric S. , Wilson Stephen W. , Dawid Igor B. , Kudoh Tetsuhiro
Published: February 2010

In vertebrates, Evx homeodomain transcription factor-encoding genes are expressed in the posterior region during embryonic development, and overexpression experiments have revealed roles in tail development in fish and frogs. We analyzed the molecular mechanisms of posterior neural development and axis formation regulated by
eve1
. We show that
eve1
is involved in establishing trunk and tail neural ectoderm by two independent mechanisms: First,
eve1
posteriorizes neural ectoderm via induction of
aldh1a2
, which encodes an enzyme that synthesizes retinoic acid; second,
eve1
is involved in neural induction in the posterior ectoderm by attenuating BMP expression. Further,
eve1
can restore trunk neural tube formation in the organizer-deficient
ichabod
−/−
mutant. We conclude that
eve1
is crucial for the organization of the antero-posterior and dorso-ventral axis in the gastrula ectoderm and also has trunk- and tail-promoting activity.
Mechanism of chromatin remodeling
Authors: Lorch Yahli , Maier-Davis Barbara , Kornberg Roger D.
Published: February 2010

Results from biochemical and structural studies of the RSC chromatin-remodeling complex prompt a proposal for the remodeling mechanism: RSC binding to the nucleosome releases the DNA from the histone surface and initiates DNA translocation (through one or a small number of DNA base pairs); ATP binding completes translocation, and ATP hydrolysis resets the system. Binding energy thus plays a central role in the remodeling process. RSC may disrupt histone-DNA contacts by affecting histone octamer conformation and through extensive interaction with the DNA. Bulging of the DNA from the octamer surface is possible, and twisting is unavoidable, but neither is the basis of remodeling.
Genome-wide analysis of mRNA targets for
Caenorhabditis elegans
FBF, a conserved stem cell regulator
Authors: Kershner Aaron M. , Kimble Judith
Published: February 2010

Stem cells are essential for tissue generation during the development of multicellular creatures, and for tissue homeostasis in adults. The great therapeutic promise of stem cells makes understanding their regulation a high priority. PUF RNA-binding proteins have a conserved role in promoting self-renewal of germline stem cells. Here we use a genome-wide approach to identify putative target mRNAs for the
Caenorhabditis elegans
PUF protein known as FBF. We find that putative FBF targets represent ∼7% of all protein-coding genes in
C. elegans
, implicating FBF as a broad-spectrum gene regulator. These putative FBF targets are enriched for regulators of meiotic entry and other components of the meiotic program as well as regulators of key developmental pathways. We suggest that these targets may be critical for FBF’s role in stem cell maintenance. Comparison of likely FBF target mRNAs with putative PUF target mRNAs from
Drosophila
and humans reveals 40 shared targets, including several established stem cell regulators. We speculate that shared PUF targets represent part of a broadly used module of stem cell control.
Human NKp44+IL-22+ cells and LTi-like cells constitute a stable RORC+ lineage distinct from conventional natural killer cells
Authors: Crellin Natasha K. , Trifari Sara , Kaplan Charles D. , Cupedo Tom , Spits Hergen
Published: February 2010

Lymphoid tissue inducer (LTi) cells are required for lymph node formation during fetal development, and recent evidence implies a role in mucosal immunity in the adult. LTi cells share some phenotypic features of conventional natural killer (NK; cNK) cells; however, little is known to date about the relationship between these two cell types. We show that lineage− (Lin−) CD127+RORC+ LTi-like cells in human tonsil are precursors to CD56+CD127+RORC+NKp46+ cells, which together comprise a stable RORC+ lineage. We find that LTi-like cells and their CD56+ progeny can be expanded and cloned ex vivo without loss of function and without conversion into cNK cells. Clonal analysis reveals heterogeneity of cytokine production within the CD127+ LTi-like population. Furthermore, we identify within the tonsil a cNK precursor population that is characterized as Lin−CD117+CD161+CD127− cells. Overall, we propose that CD127+RORC+ cells, although they share some characteristics with cNK cells, represent a functionally and developmentally distinct lineage.
IL-7 and IL-15 independently program the differentiation of intestinal CD3−NKp46+ cell subsets from Id2-dependent precursors
Authors: Satoh-Takayama Naoko , Lesjean-Pottier Sarah , Vieira Paulo , Sawa Shinichiro , Eberl Gerard , Vosshenrich Christian A.J. , Di Santo James P.
Published: February 2010

The natural cytotoxicity receptor NKp46 (encoded by Ncr1) was recently shown to identify a subset of noncytotoxic, Rag-independent gut lymphocytes that express the transcription factor Rorc, produce interleukin (IL)-22, and provide innate immune protection at the intestinal mucosa. Intestinal CD3−NKp46+ cells are phenotypically heterogeneous, comprising a minority subset that resembles classical mature splenic natural killer (NK) cells (NK1.1+, Ly49+) but also a large CD127+NK1.1− subset of lymphoid tissue inducer (LTi)–like Rorc+ cells that has been proposed to include NK cell precursors. We investigated the developmental relationships between these intestinal CD3−NKp46+ subsets. Gut CD3−NKp46+ cells were related to LTi and NK cells in requiring the transcriptional inhibitor Id2 for normal development. Overexpression of IL-15 in intestinal epithelial cells expanded NK1.1+ cells within the gut but had no effect on absolute numbers of the CD127+NK1.1−Rorc+ subset of CD3−NKp46+ cells. In contrast, IL-7 deficiency strongly reduced the overall numbers of CD3−NKp46+NK1.1− cells that express Rorc and produce IL-22 but failed to restrict homeostasis of classical intestinal NK1.1+ cells. Finally, in vivo fate-mapping experiments demonstrated that intestinal NK1.1+CD127− cells are not the progeny of Rorc-expressing progenitors, indicating that CD127+NK1.1−Rorc+ cells are not canonical NK cell precursors. These studies highlight the independent cytokine regulation of functionally diverse intestinal NKp46+ cell subsets.
Alternative end-joining catalyzes class switch recombination in the absence of both Ku70 and DNA ligase 4
Authors: Boboila Cristian , Yan Catherine , Wesemann Duane R. , Jankovic Mila , Wang Jing H. , Manis John , Nussenzweig Andre , Nussenzweig Michel , Alt Frederick W.
Published: February 2010

The classical nonhomologous end-joining (C-NHEJ) DNA double-strand break (DSB) repair pathway employs the Ku70/80 complex (Ku) for DSB recognition and the XRCC4/DNA ligase 4 (Lig4) complex for ligation. During IgH class switch recombination (CSR) in B lymphocytes, switch (S) region DSBs are joined by C-NHEJ to form junctions either with short microhomologies (MHs; “MH-mediated” joins) or no homologies (“direct” joins). In the absence of XRCC4 or Lig4, substantial CSR occurs via “alternative” end-joining (A-EJ) that generates largely MH-mediated joins. Because upstream C-NHEJ components remain in XRCC4- or Lig4-deficient B cells, residual CSR might be catalyzed by C-NHEJ using a different ligase. To address this, we have assayed for CSR in B cells deficient for Ku70, Ku80, or both Ku70 and Lig4. Ku70- or Ku80-deficient B cells have reduced, but still substantial, CSR. Strikingly, B cells deficient for both Ku plus Lig4 undergo CSR similarly to Ku-deficient B cells, firmly demonstrating that an A-EJ pathway distinct from C-NHEJ can catalyze CSR end-joining. Ku-deficient or Ku- plus Lig4-deficient B cells are also biased toward MH-mediated CSR joins; but, in contrast to XRCC4- or Lig4-deficient B cells, generate substantial numbers of direct CSR joins. Our findings suggest that more than one form of A-EJ can function in CSR.
Systematic mapping of the state dependence of voltage- and Ca2+-dependent inactivation using simple open-channel measurements
Authors: Tadross Michael R. , Yue David T.
Published: February 2010

The state from which channel inactivation occurs is both biologically and mechanistically critical. For example, preferential closed-state inactivation is potentiated in certain Ca2+ channel splice variants, yielding an enhancement of inactivation during action potential trains, which has important consequences for short-term synaptic plasticity. Mechanistically, the structural substrates of inactivation are now being resolved, yielding a growing library of molecular snapshots, ripe for functional interpretation. For these reasons, there is an increasing need for experimentally direct and systematic means of determining the states from which inactivation proceeds. Although many approaches have been devised, most rely upon numerical models that require detailed knowledge of channel-state topology and gating parameters. Moreover, prior strategies have only addressed voltage-dependent forms of inactivation (VDI), and have not been readily applicable to Ca2+-dependent inactivation (CDI), a vital form of regulation in numerous contexts. Here, we devise a simple yet systematic approach, applicable to both VDI and CDI, for semiquantitative mapping of the states from which inactivation occurs, based only on open-channel measurements. The method is relatively insensitive to the specifics of channel gating and does not require detailed knowledge of state topology or gating parameters. Rather than numerical models, we derive analytic equations that permit determination of the states from which inactivation occurs, based on direct manipulation of data. We apply this methodology to both VDI and CDI of CaV1.3 Ca2+ channels. VDI is found to proceed almost exclusively from the open state. CDI proceeds equally from the open and nearby closed states, but is disfavored from deep closed states distant from the open conformation. In all, these outcomes substantiate and enrich conclusions of our companion paper in this issue (Tadross et al. 2010. J. Gen. Physiol. doi:10.1085/jgp.200910308) that deduces endpoint mechanisms of VDI and CDI in CaV1.3. More broadly, the methods introduced herein can be readily generalized for the analysis of other channel types.
Combining hydroacoustic seabed survey and grab sampling techniques to assess “local” sandeel population abundance
Authors: Greenstreet Simon P. R. , Holland Gayle J. , Guirey Emma J. , Armstrong Eric , Fraser Helen M. , Gibb Iain M.
Published: February 2010

Greenstreet, S. P. R., Holland, G. J., Guirey, E. J., Armstrong, E., Fraser, H. M., and Gibb, I. M. 2010. Combining hydroacoustic seabed survey and grab sampling techniques to assess “local” sandeel population abundance. – ICES Journal of Marine Science, 67: 971–984. Sandeels (Ammodytes marinus) are a critical prey of many top predators in the North Sea, and have also been the target of a major industrial fishery. To quantify resource allocation between competing predators, and between natural predators and fishers, and to assess the impact of each source of mortality on sandeel population dynamics, estimates of the absolute abundance of sandeels at the spatial scale at which these interactions take place are required. In this study, hydroacoustic seabed survey and nocturnal grab surveys are combined to examine variation in the abundance of sandeels at a sandbank complex off southeast Scotland. Grab surveys provide point estimates of sandeel density and sediment composition data, which are used to define sandeel sediment preference categories. The total area of each sandeel sediment preference category is determined by hydroacoustic seabed survey. Sandeel population abundance recovered immediately following the closure of the sandeel fishery. However, simply closing the fishery was not sufficient to maintain the size of the local sandeel stock; the population is also highly dependent on good recruitment. We demonstrate how this combination of techniques might be used to examine variation in overwintering mortality rates in sandeels.
Using indicators for evaluating, comparing, and communicating the ecological status of exploited marine ecosystems. 2. Setting the scene
Authors: Shin Yunne-Jai , Shannon Lynne J. , Bundy Alida , Coll Marta , Aydin Kerim , Bez Nicolas , Blanchard Julia L. , Borges Maria de Fatima , Diallo Ibrahima , Diaz Erich , Heymans Johanna J. , Hill Louize , Johannesen Edda , Jouffre Didier , Kifani Souad , Labrosse Pierre , Link Jason S. , Mackinson Steven , Masski Hicham , Möllmann Christian , Neira Sergio , Ojaveer Henn , ould Mohammed Abdallahi Khairdine , Perry Ian , Thiao Djiga , Yemane Dawit , Cury Philippe M.
Published: February 2010

Shin, Y-J., Shannon, L. J., Bundy, A., Coll, M., Aydin, K., Bez, N., Blanchard, J. L., Borges, M. F., Diallo, I., Diaz, E., Heymans, J. J., Hill, L., Johannesen, E., Jouffre, D., Kifani, S., Labrosse, P., Link, J. S., Mackinson, S., Masski, H., Möllmann, C., Neira, S., Ojaveer, H., ould Mohammed Abdallahi, K., Perry, I., Thiao, D., Yemane, D., and Cury, P. M. 2010. Using indicators for evaluating, comparing, and communicating the ecological status of exploited marine ecosystems. 2. Setting the scene. – ICES Journal of Marine Science, 67: 692–716. Background is provided to the selection of ecological indicators by the IndiSeas Working Group, and the methodology adopted for analysis and comparison of indicators across exploited marine ecosystems is documented. The selected indicators are presented, how they are calculated is explained, and the philosophy behind the comparative approach is given. The combination of selected indicators is intended to reflect different dynamics, tracking processes that display differential responses to fishing, and is meant to provide a complementary means of assessing marine ecosystem trends and states. IndiSeas relied on inputs and insights provided by the local experts from participating ecosystems, helping to understand state and trend indicators and to disentangle the effect of other potential ecosystem drivers, such as climate variability. This project showed that the use of simple and available indicators under an ecosystem approach can achieve a real, wide-reaching evaluation of marine ecosystem status caused by fishing. This is important because the socio-economics of areas where fishing activities develop differs significantly around the globe, and in many countries, insufficient data are available for complex and exhaustive analyses.
A comparison of anchovy (Engraulis encrasicolus) and sardine (Sardina pilchardus) larvae feeding in the Northwest Mediterranean: influence of prey availability and ontogeny
Authors: Morote Elvira , Olivar María Pilar , Villate Fernando , Uriarte Ibon
Published: February 2010

Morote, E., Olivar, M. P., Villate, F., and Uriarte, I. 2010. A comparison of anchovy (Engraulis encrasicolus) and sardine (Sardina pilchardus) larvae feeding in the Northwest Mediterranean: influence of prey availability and ontogeny. – ICES Journal of Marine Science, 67: 897–908. The feeding ecology of the larvae of the two most important small pelagic species in the western Mediterranean, anchovy (Engraulis encrasicolus) and sardine (Sardina pilchardus), is investigated. Samples were collected in early summer (anchovy) and autumn (sardine). Composition of the diet and prey selectivity were compared over the course of ontogeny in anchovy (2.7–14.5 mm standard length, SL) and sardine (5.5–15.8 mm SL) larvae. Anchovy larvae begin feeding on prey items >150 µm at smaller sizes than sardine larvae, and the diets of both species are based mainly on copepod nauplii and postnauplii. Seasonal differences in the composition of the plankton influenced the contributions of prey types to the diets of the two species, e.g. the cladoceran Evadne spp. in anchovy and the tintinnid Codonellopsis sp. in sardine. Although copepod eggs are generally present in the diets of larval clupeoids, they were not major components of the diets of the species considered here. Despite morphological similarity, selection patterns were different between the species and changed through ontogeny.
Predicting life‐time and regular cannabis use during adolescence; the roles of temperament and peer substance use: the TRAILS study
Authors: Creemers Hanneke E. , Dijkstra Jan K. , Vollebergh Wilma A. M. , Ormel Johan , Verhulst Frank C. , Huizink Anja C.
Published: February 2010

Aims  The aim of the present study was to determine the mediating role of affiliation with cannabis‐using peers in the pathways from various dimensions of temperament to life‐time cannabis use, and to determine if these associations also contributed to the development of regular cannabis use.

Methods  Objectives were studied using data from 1300 participants of the Tracking Adolescents' Individual Lives Survey (TRAILS), a large, general population study of Dutch adolescents. We used parent‐reports on the Early Adolescent Temperament Questionnaire to assess the dimensions of high‐intensity pleasure, shyness, fearfulness, frustration and effortful control at age 10–12 years. By means of self‐reports, life‐time and regular cannabis use were determined at age 15–18 years, and proportion of substance‐using peers was determined at ages 12–15 and 15–18 years. Models were adjusted for age, sex, intelligence and parental cannabis use.

Results  High‐intensity pleasure [odds ratio (OR) = 1.09, 95% confidence interval (CI) = 1.05–1.13] and effortful control (OR = 0.92, 95% CI = 0.89–0.96) affected the risk for life‐time cannabis use through their influence on affiliation with cannabis‐using peers. Shyness affected this risk independently from peer cannabis use. Only the pathway from effortful control was associated additionally with the development of regular cannabis use (OR = 0.93, 95% CI = 0.89–0.98).

Conclusions  Peer cannabis use and, to a lesser extent, certain temperamental characteristics affect an adolescent's risk of cannabis use, and should be considered in prevention programmes. We recommend future research to focus upon factors that potentially modify the association between temperament, affiliation with cannabis‐using peers and cannabis use.
Drug classification: science, politics, both or neither?
Authors: Kalant Harold
Published: February 2010

Governments currently classify illicit drugs for various purposes: to guide courts in the sentencing of convicted violators of drug control laws, to prioritize targets of prevention measures and to educate the public about relative risks of the various drugs. It has been proposed that classification should be conducted by scientists and drug experts rather than by politicians, so that it will reflect only accurate factual knowledge of drug effects and risks rather than political biases. Although this is an appealing goal, it is inherently impossible because rank‐ordering of the drugs inevitably requires value judgements concerning the different types of harm. Such judgements, even by scientists, depend upon subjective personal criteria and not only upon scientific facts. Moreover, classification that is meant to guide the legal system in controlling dangerous drug use can function only if it is in harmony with the values and sentiments of the public. In some respects, politicians may be better attuned to public attitudes and wishes, and to what policies the public will support, than are scientific experts. The problems inherent in such drug classification are illustrated by the examples of cannabis and of salvinorin A. They raise the question as to whether the classification process really serves any socially beneficial purpose.
How does variability in alcohol consumption over time affect the relationship with mortality and coronary heart disease?
Authors: Britton Annie , Marmot Michael G. , Shipley Martin J.
Published: February 2010

Objective  To examine the relationship between alcohol consumption and risk of mortality and incident coronary heart disease (CHD), taking account of variation in intake during follow‐up.

Method  Prospective cohort study of 5411 male civil servants aged 35–55 years at entry to the Whitehall II study in 1985–88. Alcohol consumption was reported five times over a 15‐year period. Mortality, fatal CHD, clinically verified incident non‐fatal myocardial infarction and definite angina were ascertained during follow‐up.

Results  We found evidence that drinkers who vary their intake during follow‐up, regardless of average level, have increased risk of total mortality (hazard ratio of high versus low variability 1.52: 95% CI: 1.07–2.17), but not of incident CHD. Using average consumption level, as opposed to only a baseline measure, gave slightly higher risk estimates for CHD compared to moderate drinkers at the extremes of the drinking range.

Conclusions  Multiple repeated measures are required to explore the effects of variation in exposure over time. Caution is needed when interpreting risks of exposures measured only once at baseline, without consideration of changes over time.
Substance use and periodontal disease among Australian Aboriginal young adults
Authors: Jamieson Lisa M. , Gunthorpe Wendy , Cairney Sheree J. , Sayers Susan M. , Roberts‐Thomson Kaye F. , Slade Gary D.
Published: February 2010

Aim  To investigate the effects of tobacco, marijuana, alcohol and petrol sniffing on periodontal disease among Australian Aboriginal young adults.

Design  Cross‐sectional nested within a long‐standing prospective longitudinal study.

Setting  Aboriginal communities in Australia's Northern Territory.

Participants  Members of the Aboriginal Birth Cohort study who were recruited from birth between January 1987 and March 1990 at the Royal Darwin Hospital, Northern Territory, Australia. Data were from wave III, when the mean age of participants was 18 years.

Measurements  Clinical dental examination and self‐report questionnaire.

Findings  Of 425 participants with complete data, 26.6% had moderate/severe periodontal disease. There was elevated risk of periodontal disease associated with tobacco [prevalence ratio (PR) = 1.59, 95% CI = 1.06–2.40], marijuana (PR = 1.44, 95% CI = 1.05–1.97) and petrol sniffing (PR = 1.83, 95% CI = 1.08–3.11), but not alcohol (PR = 0.92, 95% CI = 0.67–1.27). Stratified analysis showed that the effect of marijuana persisted among tobacco users (PR = 1.47, 95% CI 1.03–2.11). It was not possible to isolate an independent effect of petrol sniffing because all petrol sniffers used both marijuana and tobacco, although among smokers of both substances, petrol sniffing was associated with an 11.8% increased prevalence of periodontal disease.

Conclusions  This is the first time that substance use has been linked with periodontal disease in a young Australian Aboriginal adult population, and the first time that petrol sniffing has been linked with periodontal disease in any population. The role of substance use in periodontal disease among this, and other, marginalized groups warrants further investigation.
Internet‐based prevention for alcohol and cannabis use: final results of the Climate Schools course
Authors: Newton Nicola C. , Teesson Maree , Vogl Laura E. , Andrews Gavin
Published: February 2010

Aims  To establish the long‐term efficacy of a universal internet‐based alcohol and cannabis prevention programme in schools.

Methods  A cluster‐randomized controlled trial was conducted to assess the effectiveness of the Climate Schools: Alcohol and Cannabis Course. The evidence‐based course, aimed at reducing alcohol and cannabis use, is facilitated by the internet and consists of 12 novel and curriculum consistent lessons delivered over 6 months.

Participants  A total of 764 year 8 students (13 years) from 10 Australian secondary schools were allocated randomly to the internet‐based prevention programme (n = 397, five schools), or to their usual health classes (n = 367, five schools).

Measures  Participants were assessed at baseline, immediately post, and 6 and 12 months following completion of the intervention, on measures of alcohol and cannabis knowledge, attitudes, use and related harms.

Results  This paper reports the final results of the intervention trial, 12 months following the completion of the Climate Schools: Alcohol and Cannabis Course. The effectiveness of the course 6 months following the intervention has been reported previously. At the 12‐month follow‐up, compared to the control group, students in the intervention group showed significant improvements in alcohol and cannabis knowledge, a reduction in average weekly alcohol consumption and a reduction in frequency of drinking to excess. No differences between groups were found on alcohol expectancies, cannabis attitudes or alcohol‐ and cannabis‐related harms. The course was found to be acceptable by teachers and students as a means of delivering drug education in schools.

Conclusions  Internet‐based prevention programs for school‐age children can improve student's knowledge about alcohol and cannabis, and may also reduce alcohol use twelve months after completion.
Comparative rates of violent crime among regular methamphetamine and opioid users: offending and victimization
Authors: Darke Shane , Torok Michelle , Kaye Sharlene , Ross Joanne , McKetin Rebecca
Published: February 2010

Aims  To determine the comparative levels of violent offending and victimization among regular methamphetamine and heroin users.

Design  Cross‐sectional

Setting  Sydney, Australia.

Participants  A total of 400 regular methamphetamine (METH) and heroin (HER) users (118 methamphetamine users: METH; 161 regular heroin users: HER; 121 regular users of both: BOTH).

Findings  Eighty‐two per cent reported a life‐time history of committing violent crime, 41% in the past 12 months. There were no group differences in life‐time violence, but the METH group were significantly more likely than the HER group to have committed violence in the past 12 months (odds ratio 1.94). Nearly all (95%) reported that they had been a victim of violent crime, 46% in the preceding 12 months, with no group differences. Those who had committed a violent crime in the past 12 months were 13.23 times more likely to have been a victim in that period. The majority believed it unlikely that they would be a victim of (78%), or commit (87%), a violent crime in the next 12 months.

Conclusions  Regular methamphetamine use appears to be associated with an increased risk of violent offending, but not victimization, compared with heroin use.