bluuebunny/crossref_metadata_2025 huggingface dataset:
Exploring Mechanisms Underlying Sex‐Specific Differences in Mortality of Lake Michigan Bloaters
Authors: Bunnell David B. , Madenjian Charles P. , Rogers Mark W. , Holuszko Jeffrey D. , Begnoche Linda J.
Published: February 2012

Sex‐specific differences in mortality rates have been observed among freshwater and marine fish taxa, and underlying mechanisms can include sex‐specific differences in (1) age at maturity, (2) growth rate, or (3) activity or behavior during the spawning period. We used a long‐term (1973–2009) Lake Michigan data set to evaluate whether there were sex‐specific differences in catch per unit effort, mortality, age at maturity, and length at age in bloaters Coregonus hoyi. Because bloater population biomass varied 200‐fold during the years analyzed, we divided the data into three periods: (1) 1973–1982 (low biomass), (2) 1983–1997 (high biomass), and (3) 1998–2009 (low biomass). Mortality was higher for males than for females in periods 2 and 3; the average instantaneous total mortality rate (Z) over these two periods was 0.71 for males and 0.57 for females. Length at age was slightly greater (2–6%) for females than for males in different age‐classes (3–6 years) during each period. Age at maturity was earlier for males than for females in periods 1 and 2, but the mean difference was only 0.2–0.4 years. To test the hypothesis that somatic lipids declined more in males than in females during spawning (perhaps due to increased activity or reduced feeding), we estimated sex‐specific percent somatic lipids for fish sampled in 2005–2006 and 2007–2008. During 2005–2006, somatic lipids declined from prespawning to postspawning for males but were unchanged for females. During 2007–2008, however, somatic lipids were unchanged for males, whereas they increased for females. We found that sex‐specific differences in Z occurred in the Lake Michigan bloater population, but our hypotheses that sex‐specific differences in maturity and growth could explain this pattern were generally unsupported. Our hypothesis that somatic lipids in males declined during spawning at a faster rate than in females will require additional research to clarify its importance.
Experimental study of surface dielectric barrier discharge in air and its ozone production
Authors: Pekárek Stanislav
Published: February 2012

For surface dielectric barrier discharge in air we studied the effects of frequency of the driving voltage on dissipated power, asymmetry of amplitudes of the discharge voltage, discharge UV emission, ozone production, ozone production of the discharge with TiO2 and of the discharge in magnetic field. We found that for a particular voltage the dissipated power is higher for the frequency of the driving voltage of 26.3 kHz than for the frequency of 10.9 kHz; peak values of the positive half-periods of the discharge voltage are higher than peak values of the negative half-periods; intensity of the discharge UV emissions for wavelengths of 320–420 nm is for both frequencies a linear function of power; maximum ozone concentration for the frequency of the driving voltage of 26.3 kHz is obtained with smaller power than for the frequency of 10.9 kHz; placement of TiO2 particles into the discharge chamber increases for both frequencies of the driving voltage maximum ozone concentration produced by the discharge and for the frequency of the driving voltage of 26.3 kHz increases ozone production yield. Finally, there is no observable effect of magnetic field on concentration of ozone produced by the discharge as well as on production yield.
Identification and Characterization of ANAC042, a Transcription Factor Family Gene Involved in the Regulation of Camalexin Biosynthesis in Arabidopsis
Authors: Saga Hirohisa , Ogawa Takumi , Kai Kosuke , Suzuki Hideyuki , Ogata Yoshiyuki , Sakurai Nozomu , Shibata Daisuke , Ohta Daisaku
Published: February 2012

Camalexin is the major phytoalexin in Arabidopsis. An almost complete set of camalexin biosynthetic enzymes have been elucidated but only limited information is available regarding molecular mechanisms regulating camalexin biosynthesis. Here, we demonstrate that ANAC042, a member of the NAM, ATAF1/2, and CUC2 (NAC) transcription factor family genes, is involved in camalexin biosynthesis induction. T-DNA insertion mutants of ANAC042 failed to accumulate camalexin at the levels achieved in the wild type, and were highly susceptible to Alternaria brassicicola infection. The camalexin biosynthetic genes CYP71A12, CYP71A13, and CYP71B15/PAD3 were not fully induced in the mutants, indicating that the camalexin defects were at least partly a result of reduced expression levels of these P450 genes. β-Glucuronidase (GUS)-reporter assays demonstrated tissue-specific induction of ANAC042 in response to differential pathogen infections. Bacterial flagellin (Flg22) induced ANAC042 expression in the root-elongation zone, the camalexin biosynthetic site, and the induction was abolished in the presence of either a general kinase inhibitor (K252a), a Ca2+-chelator (BAPTA), or methyl jasmonate. The GUS-reporter assay revealed repression of the Flg22-dependent ANAC042 expression in the ethylene-insensitive ein2-1 background but not in sid2-2 plants defective for salicylic acid biosynthesis. We discuss ANAC042 as a key transcription factor involved in previously unknown regulatory mechanisms to induce phytoalexin biosynthesis in Arabidopsis.
Effects of Crop Rotation, Cultivar, and Irrigation and Nitrogen Rate on Verticillium Wilt in Cotton
Authors: Wheeler T. A. , Bordovsky J. P. , Keeling J. W. , Mullinix B. G.
Published: February 2012

A field experiment was conducted under center-pivot irrigation in four wedges, with one wedge in continuous cotton (CC) and three wedges in a rotation (ROT) with 2 years cotton and 1 year in sorghum. Three irrigation rates (base = 1.0B, 1.5B, and 0.5B) were applied during 2007 to 2009 on a susceptible (ST) and partially resistant (PR) cultivar. Nitrogen applied during the season was proportional to irrigation rate. In the ROT wedges, 0.5B, 1.0B, and 1.5B irrigation and nitrogen rates averaged 1, 3, and 9% incidence of wilt, respectively. Disease incidence in the CC wedge averaged 6, 18, and 34% wilt incidence for 0.5B, 1.0B, and 1.5B irrigation and nitrogen rates. In the ROT wedges, the ST cultivar returned $143/ha more than the PR cultivars at the 0.5B irrigation and nitrogen rate whereas, at the 1.0B and 1.5B rates, the PR cultivars averaged $121 and $350/ha more than the ST cultivar. There was no significant irrigation and nitrogen or cultivar effect in the CC wedge on net value; however, trends were similar to the ROT wedge. Overall, ROT returned $285/ha more than CC, PR cultivars returned $123/ha more than the ST cultivar, and 1.0B returned $271 and $296/ha more than 0.5B and 1.5B rates, respectively. Microsclerotia density of V. dahliae averaged 2/cm3 of soil in the ROT wedges and 23/cm3 of soil in the CC wedge. Crop rotation, avoiding excessive irrigation, and using a partially resistant cultivar all reduced incidence of Verticillium wilt and improved net returns.
Baseline Sensitivity and Cross-Resistance to Succinate-Dehydrogenase-Inhibiting and Demethylation-Inhibiting Fungicides in Didymella bryoniae
Authors: Thomas A. , Langston D. B. , Stevenson K. L.
Published: February 2012

Didymella bryoniae, which causes gummy stem blight (GSB) of watermelon, has a history of developing resistance to fungicides, most recently the succinate-dehydrogenase-inhibiting (SDHI) fungicide boscalid. To facilitate fungicide resistance monitoring, baseline sensitivity distributions were established for demethylation-inhibiting (DMI) fungicides tebuconazole and difenoconazole and the SDHI fungicide penthiopyrad, and reestablished for the SDHI fungicide boscalid. In all, 71 isolates with no known prior exposure to SDHIs or DMIs were used to determine the effective concentration at which mycelial growth was inhibited by 50% (EC50). EC50 values for boscalid, penthiopyrad, tebuconazole, and difenoconazole were 0.018 to 0.064, 0.015 to 0.057, 0.062 to 0.385, and 0.018 to 0.048 μg/ml, with median values of 0.032, 0.026, 0.118, and 0.031 μg/ml, respectively. Significant positive correlations between the sensitivity to penthiopyrad and boscalid (P < 0.0001, r = 0.75) and between tebuconazole and difenoconazole (P < 0.0001, r = 0.59) indicate a potential for cross-resistance between chemically related fungicides. In 2009, 103 isolates from fungicidetreated watermelon fields were tested for sensitivity to boscalid and penthiopyrad using a discriminatory concentration of 3.0 μg/ml. Of the isolates tested, 82 were insensitive and 14 were sensitive to both fungicides. Because of the significant potential for cross-resistance between closely related fungicides, growers will be advised not to use both SDHIs or both DMIs successively in the same fungicide spray program.
Influence of Supervision Ratios by Anesthesiologists on First-case Starts and Critical Portions of Anesthetics
Authors: Epstein Richard H. , Dexter Franklin
Published: February 2012

Background

Anesthesia groups may wish to decrease the supervision ratio for nontrainee providers. Because hospitals offer many first-case starts and focus on starting these cases on time, the number of anesthesiologists needed is sensitive to this ratio. The number of operating rooms that an anesthesiologist can supervise concurrently is determined by the probability of multiple simultaneous critical portions of cases (i.e., requiring presence) and the availability of cross-coverage. A simulation study showed peak occurrence of critical portions during first cases, and frequent supervision lapses. These predictions were tested using real data from an anesthesia information management system.

Methods

The timing and duration of critical portions of cases were determined from 1 yr of data at a tertiary care hospital. The percentages of days with at least one supervision lapse occurring at supervision ratios between 1:1 and 1:3 were determined.

Results

Even at a supervision ratio of 1:2, lapses occurred on 35% of days (lower 95% confidence limit = 30%). The peak incidence occurred before 8:00 AM, P < 0.0001 for the hypothesis that most (i.e., >50%) lapses occurred before this time. The average time from operating room entry until ready for prepping and draping (i.e., anesthesia release time) during first case starts was 22.2 min (95% confidence interval 21.8-22.8 min).

Conclusions

Decreasing the supervision ratio from 1:2 to 1:3 has a large effect on supervision lapses during first-case starts. To mitigate such lapses, either staggered starts or additional anesthesiologists working at the start of the day would be required.
Novel use of burrow casting as a research tool in deep-sea ecology
Authors: Seike Koji , Jenkins Robert G. , Watanabe Hiromi , Nomaki Hidetaka , Sato Kei
Published: February 2012

Although the deep sea is the largest ecosystem on Earth, its infaunal ecology remains poorly understood because of the logistical challenges. Here we report the morphology of relatively large burrows obtained by
in situ
burrow casting at a hydrocarbon-seep site and a non-seep site at water depths of 1173 and 1455 m, respectively. Deep and complex burrows are abundant at both sites, indicating that the burrows introduce oxygen-rich sea water into the deep reducing substrate, thereby influencing benthic metabolism and nutrient fluxes, and providing an oxic microhabitat for small organisms. Burrow castings reveal that the solemyid bivalve
Acharax johnsoni
mines sulphide from the sediment, as documented for related shallow-water species. To our knowledge, this is the first study to examine
in situ
burrow morphology in the deep sea by means of burrow casting, providing detailed information on burrow structure which will aid the interpretation of seabed processes in the deep sea.
The logic of ecological patchiness
Authors: Grünbaum Daniel
Published: February 2012

Most ecological interactions occur in environments that are spatially and temporally heterogeneous—‘patchy’—across a wide range of scales. In contrast, most theoretical models of ecological interactions, especially large-scale models applied to societal issues such as climate change, resource management and human health, are based on ‘mean field’ approaches in which the underlying patchiness of interacting consumers and resources is intentionally averaged out. Mean field ecological models typically have the advantages of tractability, few parameters and clear interpretation; more technically complex spatially explicit models, which resolve ecological patchiness at some (or all relevant) scales, generally lack these advantages. This report presents a heuristic analysis that incorporates important elements of consumer–resource patchiness with minimal technical complexity. The analysis uses scaling arguments to establish conditions under which key mechanisms—movement, reproduction and consumption—strongly affect consumer–resource interactions in patchy environments. By very general arguments, the relative magnitudes of these three mechanisms are quantified by three non-dimensional ecological indices: the Frost, Strathmann and Lessard numbers. Qualitative analysis based on these ecological indices provides a basis for conjectures concerning the expected characteristics of organisms, species interactions and ecosystems in patchy environments.
On the topology of chromatin fibres
Authors: Barbi Maria , Mozziconacci Julien , Victor Jean-Marc , Wong Hua , Lavelle Christophe
Published: February 2012

The ability of cells to pack, use and duplicate DNA remains one of the most fascinating questions in biology. To understand DNA organization and dynamics, it is important to consider the physical and topological constraints acting on it. In the eukaryotic cell nucleus, DNA is organized by proteins acting as spools on which DNA can be wrapped. These proteins can subsequently interact and form a structure called the chromatin fibre. Using a simple geometric model, we propose a general method for computing topological properties (
twist
,
writhe
and
linking number
) of the DNA embedded in those fibres. The relevance of the method is reviewed through the analysis of magnetic tweezers single molecule experiments that revealed unexpected properties of the chromatin fibre. Possible biological implications of these results are discussed.
Vignettes from the field of mathematical biology: the application of mathematics to biology and medicine
Authors: Murray J. D.
Published: February 2012

The application of mathematical models in biology and medicine has a long history. From the sparse number of papers in the first half of the twentieth century with a few scientists working in the field it has become vast with thousands of active researchers. We give a brief, and far from definitive history, of how some parts of the field have developed and how the type of research has changed. We describe in more detail just two examples of specific models which are directly related to real biological problems, namely animal coat patterns and the growth and image enhancement of glioblastoma brain tumours.
Implications of functional similarity for gene regulatory interactions
Authors: Glass Kimberly , Ott Edward , Losert Wolfgang , Girvan Michelle
Published: February 2012

If one gene regulates another, those two genes are likely to be involved in many of the same biological functions. Conversely, shared biological function may be suggestive of the existence and nature of a regulatory interaction. With this in mind, we develop a measure of functional similarity between genes based on annotations made to the Gene Ontology in which the magnitude of their functional relationship is also indicative of a regulatory relationship. In contrast to other measures that have previously been used to quantify the functional similarity between genes, our measure scales the strength of any shared functional annotation by the frequency of that function's appearance across the entire set of annotations. We apply our method to both
Escherichia coli
and
Saccharomyces cerevisiae
gene annotations and find that the strength of our
scaled similarity
measure is more predictive of known regulatory interactions than previously published measures of functional similarity. In addition, we observe that the strength of the scaled similarity measure is correlated with the structural importance of links in the known regulatory network. By contrast, other measures of functional similarity are not indicative of any structural importance in the regulatory network. We therefore conclude that adequately adjusting for the frequency of shared biological functions is important in the construction of a functional similarity measure aimed at elucidating the existence and nature of regulatory interactions. We also compare the performance of the scaled similarity with a high-throughput method for determining regulatory interactions from gene expression data and observe that the ontology-based approach identifies a different subset of regulatory interactions compared with the gene expression approach. We show that combining predictions from the scaled similarity with those from the reconstruction algorithm leads to a significant improvement in the accuracy of the reconstructed network.
Characterization of the phosphatic mineral of the barnacle
Ibla cumingi
at atomic level by solid-state nuclear magnetic resonance: comparison with other phosphatic biominerals
Authors: Reid David G. , Mason Matthew J. , Chan Benny K. K. , Duer Melinda J.
Published: February 2012

Ibliform barnacles are among the few invertebrate animals harnessing calcium phosphate to construct hard tissue. The
31
P solid-state NMR (SSNMR) signal from the shell plates of
Ibla cumingi
(Iblidae) is broader than that of bone, and shifted by
ca
1 ppm to low frequency.
1
H–
31
P heteronuclear correlation (HETCOR) experiments show a continuum of different phosphorus/phosphate atomic environments, close to hydrogen populations with resonance frequencies between
ca
10 and 20 ppm. Associated
1
H and
31
P chemical shifts argue the coexistence of weakly (high
31
P frequency, low
1
H frequency) to more strongly (lower
31
P frequency, higher
1
H frequency) hydrogen-bonded hydrogen phosphate-like molecular/ionic species. There is no resolved signal from discrete OH
−
ions.
13
C SSNMR shows chitin, protein and other organic biomolecules but, unlike bone, there are no significant atomic scale organic matrix–mineral contacts. The poorly ordered hydrogen phosphate-like iblid mineral is strikingly different, structurally and compositionally, from both vertebrate bone mineral and the more crystalline fluoroapatite of the linguliform brachiopods. It probably represents a previously poorly characterized calcium phosphate biomineral, the evolution of which may have reflected either the chemical conditions of ancestral seas or the mechanical advantages of phosphatic biomineralization over a calcium carbonate equivalent.
Clustered continuous-time random walks: diffusion and relaxation consequences
Authors: Weron Karina , Stanislavsky Aleksander , Jurlewicz Agnieszka , Meerschaert Mark M. , Scheffler Hans-Peter
Published: February 2012

We present a class of continuous-time random walks (CTRWs), in which random jumps are separated by random waiting times. The novel feature of these CTRWs is that the jumps are clustered. This introduces a coupled effect, with longer waiting times separating larger jump clusters. We show that the CTRW scaling limits are time-changed processes. Their densities solve two different fractional diffusion equations, depending on whether the waiting time is coupled to the preceding jump, or the following one. These fractional diffusion equations can be used to model all types of experimentally observed two power-law relaxation patterns. The parameters of the scaling limit process determine the power-law exponents and loss peak frequencies.
Rejection of a serial founder effects model of genetic and linguistic coevolution
Authors: Hunley Keith , Bowern Claire , Healy Meghan
Published: February 2012

Recent genetic studies attribute the negative correlation between population genetic diversity and distance from Africa to a serial founder effects (SFE) evolutionary process. A recent linguistic study concluded that a similar decay in phoneme inventories in human languages was also the product of the SFE process. However, the SFE process makes additional predictions for patterns of neutral genetic diversity, both within and between groups, that have not yet been tested on phonemic data. In this study, we describe these predictions and test them on linguistic and genetic samples. The linguistic sample consists of 725 widespread languages, which together contain 908 distinct phonemes. The genetic sample consists of 614 autosomal microsatellite loci in 100 widespread populations. All aspects of the genetic pattern are consistent with the predictions of SFE. In contrast, most of the predictions of SFE are violated for the phonemic data. We show that phoneme inventories provide information about recent contacts between languages. However, because phonemes change rapidly, they cannot provide information about more ancient evolutionary processes.
Flower colour adaptation in a mimetic orchid
Authors: Newman Ethan , Anderson Bruce , Johnson Steven D.
Published: February 2012

Although the tremendous variability in floral colour among angiosperms is often attributed to divergent selection by pollinators, it is usually difficult to preclude the possibility that floral colour shifts were driven by non-pollinator processes. Here, we examine the adaptive significance of flower colour inDisa ferruginea, a non-rewarding orchid that is thought to attract its butterfly pollinator by mimicking the flowers of sympatric nectar-producing species.Disa ferrugineahas red flowers in the western part of its range and orange flowers in the eastern part—a colour shift that we hypothesized to be the outcome of selection for resemblance to different local nectar-producing plants. Using reciprocal translocations of red and orange phenotypes as well as arrays of artificial flowers, we found that the butterflyAeropetes tulbaghia, the only pollinator of the orchid, preferred both the red phenotype and red artificial flowers in the west where its main nectar plant also has red flowers, and both the orange phenotype and orange artificial flowers in the east, where its main nectar plant has orange flowers. This phenotype by environment interaction demonstrates that the flower colour shift inD. ferrugineais adaptive and driven by local colour preference in its pollinator.
Big hitting collectors make massive and disproportionate contribution to the discovery of plant species
Authors: Bebber Daniel P. , Carine Mark A. , Davidse Gerrit , Harris David J. , Haston Elspeth M. , Penn Malcolm G. , Cafferty Steve , Wood John R. I. , Scotland Robert W.
Published: February 2012

Discovering biological diversity is a fundamental goal—made urgent by the alarmingly high rate of extinction. We have compiled information from more than 100 000 type specimens to quantify the role of collectors in the discovery of plant diversity. Our results show that more than half of all type specimens were collected by less than 2 per cent of collectors. This highly skewed pattern has persisted through time. We demonstrate that a number of attributes are associated with prolific plant collectors: a long career with increasing productivity and experience in several countries and plant families. These results imply that funding a small number of expert plant collectors in the right geographical locations should be an important element in any effective strategy to find undiscovered plant species and complete the inventory of the world flora.
Rise of dinosaurs reveals major body-size transitions are driven by passive processes of trait evolution
Authors: Sookias Roland B. , Butler Richard J. , Benson Roger B. J.
Published: February 2012

A major macroevolutionary question concerns how long-term patterns of body-size evolution are underpinned by smaller scale processes along lineages. One outstanding long-term transition is the replacement of basal therapsids (stem-group mammals) by archosauromorphs, including dinosaurs, as the dominant large-bodied terrestrial fauna during the Triassic (approx. 252–201 million years ago). This landmark event preceded more than 150 million years of archosauromorph dominance. We analyse a new body-size dataset of more than 400 therapsid and archosauromorph species spanning the Late Permian–Middle Jurassic. Maximum-likelihood analyses indicate that Cope's rule (an active within-lineage trend of body-size increase) is extremely rare, despite conspicuous patterns of body-size turnover, and contrary to proposals that Cope's rule is central to vertebrate evolution. Instead, passive processes predominate in taxonomically and ecomorphologically more inclusive clades, with stasis common in less inclusive clades. Body-size limits are clade-dependent, suggesting intrinsic, biological factors are more important than the external environment. This clade-dependence is exemplified by maximum size of Middle–early Late Triassic archosauromorph predators exceeding that of contemporary herbivores, breaking a widely-accepted ‘rule’ that herbivore maximum size greatly exceeds carnivore maximum size. Archosauromorph and dinosaur dominance occurred via opportunistic replacement of therapsids following extinction, but were facilitated by higher archosauromorph growth rates.
Dietary obesity-associated Hif1α activation in adipocytes restricts fatty acid oxidation and energy expenditure via suppression of the Sirt2-NAD+ system
Authors: Krishnan Jaya , Danzer Carsten , Simka Tatiana , Ukropec Josef , Walter Katharina Manuela , Kumpf Susann , Mirtschink Peter , Ukropcova Barbara , Gasperikova Daniela , Pedrazzini Thierry , Krek Wilhelm
Published: February 2012

Dietary obesity is a major factor in the development of type 2 diabetes and is associated with intra-adipose tissue hypoxia and activation of hypoxia-inducible factor 1α (HIF1α). Here we report that, in mice, Hif1α activation in visceral white adipocytes is critical to maintain dietary obesity and associated pathologies, including glucose intolerance, insulin resistance, and cardiomyopathy. This function of Hif1α is linked to its capacity to suppress β-oxidation, in part, through transcriptional repression of sirtuin 2 (Sirt2) NAD+-dependent deacetylase. Reduced Sirt2 function directly translates into diminished deacetylation of PPARγ coactivator 1α (Pgc1α) and expression of β-oxidation and mitochondrial genes. Importantly, visceral adipose tissue from human obese subjects is characterized by high levels of HIF1α and low levels of SIRT2. Thus, by negatively regulating the Sirt2–Pgc1α regulatory axis, Hif1α negates adipocyte-intrinsic pathways of fatty acid catabolism, thereby creating a metabolic state supporting the development of obesity.
Synapto-pHluorins: Genetically Encoded Reporters of Synaptic Transmission
Authors: Miesenböck Gero
Published: February 2012

pHluorins are pH-sensitive mutants of green fluorescent protein (GFP). Attached to proteins with defined cellular locations or itineraries, pHluorins report subcellular pH as well as protein transport between compartments of differing pH. Key applications in neurobiology include the optical detection of neurotransmitter release with synapto-pHluorins and their derivatives, as well as measurements of neurotransmitter receptor trafficking. This article describes the properties and uses of synapto-pHluorins, as well as their advantages and limitations.
Synthesis of Linear Polyethylenimine and Use in Transfection
Authors: Ogris Manfred , Wagner Ernst
Published: February 2012

Linear polyethylenimine (LPEI) is an efficient transfection reagent for a broad range of cell lines and primary cells and is also useful for local or systemic gene delivery in vivo. In contrast to many other nonviral transfection methods, LPEI is less dependent on mitosis and also transfects postmitotic cells. Transfections can be performed with a simple protocol, as described here. The presence of serum (up to 10%) during transfection does not markedly decrease transfection efficiency, but it significantly reduces toxicity, especially to primary cells. This is a major advantage compared to most lipidic transfection systems. Toxicity can also be kept to a minimum using low, optimized amounts of the transfection reagent. However, LPEI can be quite toxic when applied at elevated doses in vitro or in vivo. LPEI is a fully synthetic product that can be easily synthesized from rather inexpensive precursors and is suitable for large-scale transfections. This protocol describes the synthesis of LPEI from a precursor polymer and the generation of DNA/LPEI polyplexes. Transfection protocols for cells adherent to tissue culture dishes or grown in suspension are given, as are in vivo applications.
Single-Molecule High-Resolution Colocalization of Single Probes
Authors: Churchman L. Stirling , Spudich James A.
Published: February 2012

Colocalization of fluorescent probes is commonly used in cell biology to discern the proximity of two proteins in the cell. Considering that the resolution limit of optical microscopy is on the order of 250 nm, there has not been a need for high-resolution colocalization techniques. However, with the advent of higher resolution techniques for cell biology and single-molecule biophysics, colocalization must also improve. For diffraction-limited applications, a geometric transformation (i.e., translation, scaling, and rotation) is typically applied to one color channel to align it with the other; however, to achieve high-resolution colocalization, this is not sufficient. Single-molecule high-resolution colocalization (SHREC) of single probes uses the local weighted mean transformation to achieve a colocalization resolution of at least 10 nm. This protocol describes the acquisition of registration data and the analysis required to obtain a high-resolution mapping between imaging channels. The total internal reflection fluorescence microscope (TIRFM) system described is designed to excite and image the fluorescent probes Cy3 and Cy5. Modifications may be required depending on the requirements of the individual study.
Experimental Strategies for Cloning or Identifying Genes Encoding DNA-Binding Proteins
Authors: Carey Michael F. , Peterson Craig L. , Smale Stephen T.
Published: February 2012

This article describes experimental strategies for cloning or identifying genes encoding DNA-binding proteins. DNA-binding proteins are most commonly identified by electrophoretic mobility-shift assay (EMSA) or DNase I footprinting. To identify the gene encoding a protein detected by EMSA or DNase footprinting, the protein often needs to be purified and its sequence analyzed, as described here. Other methods are also available which do not resort to protein purification, including the one-hybrid screen, in vitro expression library screen, and mammalian expression cloning. These methods are outlined, and their advantages and disadvantages are discussed.
Phytosterols Play a Key Role in Plant Innate Immunity against Bacterial Pathogens by Regulating Nutrient Efflux into the Apoplast
Authors: Wang Keri , Senthil-Kumar Muthappa , Ryu Choong-Min , Kang Li , Mysore Kirankumar S.
Published: February 2012

Bacterial pathogens colonize a host plant by growing between the cells by utilizing the nutrients present in apoplastic space. While successful pathogens manipulate the plant cell membrane to retrieve more nutrients from the cell, the counteracting plant defense mechanism against nonhost pathogens to restrict the nutrient efflux into the apoplast is not clear. To identify the genes involved in nonhost resistance against bacterial pathogens, we developed a virus-induced gene-silencing-based fast-forward genetics screen in Nicotiana benthamiana. Silencing of N. benthamiana  SQUALENE SYNTHASE, a key gene in phytosterol biosynthesis, not only compromised nonhost resistance to few pathovars of Pseudomonas syringae and Xanthomonas campestris, but also enhanced the growth of the host pathogen P. syringae pv tabaci by increasing nutrient efflux into the apoplast. An Arabidopsis (Arabidopsis thaliana) sterol methyltransferase mutant (sterol methyltransferase2) involved in sterol biosynthesis also compromised plant innate immunity against bacterial pathogens. The Arabidopsis cytochrome P450 CYP710A1, which encodes C22-sterol desaturase that converts β-sitosterol to stigmasterol, was dramatically induced upon inoculation with nonhost pathogens. An Arabidopsis Atcyp710A1 null mutant compromised both nonhost and basal resistance while overexpressors of AtCYP710A1 enhanced resistance to host pathogens. Our data implicate the involvement of sterols in plant innate immunity against bacterial infections by regulating nutrient efflux into the apoplast.
POM-POM2/CELLULOSE SYNTHASE INTERACTING1 Is Essential for the Functional Association of Cellulose Synthase and Microtubules inArabidopsis
Authors: Bringmann Martin , Li Eryang , Sampathkumar Arun , Kocabek Tomas , Hauser Marie-Theres , Persson Staffan
Published: February 2012

In plants, regulation of cellulose synthesis is fundamental for morphogenesis and plant growth. Cellulose is synthesized at the plasma membrane, and the orientation of synthesis is guided by cortical microtubules; however, the guiding mechanism is currently unknown. We show that the conditional root elongation pom2 mutants are impaired in cell elongation, fertility, and microtubule-related functions. Map-based cloning of the POM-POM2 locus revealed that it is allelic to CELLULOSE SYNTHASE INTERACTING1 (CSI1). Fluorescently tagged POM2/CSI1s associated with both plasma membrane–located cellulose synthases (CESAs) and post-Golgi CESA-containing compartments. Interestingly, while CESA insertions coincided with cortical microtubules in the pom2/csi1 mutants, the microtubule-defined movement of the CESAs was significantly reduced in the mutant. We propose that POM2/CSI1 provides a scaffold between the CESAs and cortical microtubules that guide cellulose synthesis.
Regulation of the Photorespiratory GLDPA Gene in C4 Flaveria: An Intricate Interplay of Transcriptional and Posttranscriptional Processes
Authors: Wiludda Christian , Schulze Stefanie , Gowik Udo , Engelmann Sascha , Koczor Maria , Streubel Monika , Bauwe Hermann , Westhoff Peter
Published: February 2012

The mitochondrial Gly decarboxylase complex (GDC) is a key component of the photorespiratory pathway that occurs in all photosynthetically active tissues of C3 plants but is restricted to bundle sheath cells in C4 species. GDC is also required for general cellular C1 metabolism. In the Asteracean C4 species Flaveria trinervia, a single functional GLDP gene, GLDPA, encodes the P-subunit of GDC, a decarboxylating Gly dehydrogenase. GLDPA promoter reporter gene fusion studies revealed that this promoter is active in bundle sheath cells and the vasculature of transgenic Flaveria bidentis (C4) and the Brassicacean C3 species Arabidopsis thaliana, suggesting the existence of an evolutionarily conserved gene regulatory system in the bundle sheath. Here, we demonstrate that GLDPA gene regulation is achieved by an intricate interplay of transcriptional and posttranscriptional mechanisms. The GLDPA promoter is composed of two tandem promoters, PR2 and PR7, that together ensure a strong bundle sheath expression. While the proximal promoter (PR7) is active in the bundle sheath and vasculature, the distal promoter (PR2) drives uniform expression in all leaf chlorenchyma cells and the vasculature. An intron in the 5′ untranslated leader of PR2-derived transcripts is inefficiently spliced and apparently suppresses the output of PR2 by eliciting RNA decay.
Small non‐mucinous bronchioloalveolar carcinoma with anaplastic lymphoma kinase immunoreactivity: A novel ALK fusion gene?
Authors: Yamamoto Morio , Takeuchi Kengo , Shimoji Masaki , Maniwa Tomohiro , Isaka Mitsuhiro , Nakagawa Kazuo , Ohde Yasuhisa , Kondo Haruhiko , Nakajima Takashi
Published: February 2012

Echinoderm microtubule‐associated protein‐like 4 and anaplastic lymphoma kinase (EML4–ALK) and kinesin family member 5B (KIF5B)–ALK are newly identified transforming fusion oncogenes causing non‐small‐cell lung cancers. These molecular abnormalities have become detectable using not only molecular biological methods, but also highly sensitive immunohistochemistry. During the immunohistochemical study of ALK expression in adenocarcinoma of the lung, we unexpectedly discovered that a small bronchioloalveolar carcinoma (BAC) showed strong ALK immunoreactivity. However, FISH studies failed to reveal EML4–ALK and KIF5B–ALK fusion genes in this BAC. These findings suggest the possibility that a novel or unknown ALK fusion gene plays a crucial role in BAC development. (Cancer Sci 2012; 103: 390–392)
Beer à no‐go: learning to stop responding to alcohol cues reduces alcohol intake via reduced affective associations rather than increased response inhibition
Authors: Houben Katrijn , Havermans Remco C. , Nederkoorn Chantal , Jansen Anita
Published: February 2012

Aims  Previous research has shown that consistently not responding to alcohol‐related stimuli in a go/no‐go training procedure reduces drinking behaviour. This study aimed to examine further the mechanisms underlying this go/no‐go training effect.

Design, setting and participants  Fifty‐seven heavy drinkers were assigned randomly to two training conditions: in the beer/no‐go condition, alcohol‐related stimuli were always paired with a stopping response, while in the beer/go condition participants always responded to alcohol‐related stimuli. Participants were tested individually in a laboratory at Maastricht University.

Measurements  Weekly alcohol intake, implicit attitudes towards beer, approach–avoidance action tendencies towards beer and response inhibition were measured before and after the training.

Findings  Results showed a significant reduction in both implicit attitudes (P = 0.03) and alcohol intake (P = 0.02) in the beer/no‐go condition, but not in the beer/go condition. There were no significant training effects on action tendencies or response inhibition.

Conclusions  Repeatedly stopping pre‐potent responses towards alcohol‐related stimuli reduces excessive alcohol use via a devaluation of alcohol‐related stimuli rather than via increased inhibitory control over alcohol‐related responses.
Prevalence and correlates of alcohol use disorders in the Singapore Mental Health Survey
Authors: Subramaniam Mythily , Abdin Edimansyah , Vaingankar Janhavi , Phua Amy M. Y. , Tee Joseph , Chong Siow Ann
Published: February 2012

Aims  To establish the prevalence, correlates, comorbidity and treatment gap of alcohol use disorders in the Singapore resident population.

Design  The Singapore Mental Health Study is a cross‐sectional epidemiological survey.

Setting  A nationally representative survey of the resident (citizens and permanent residents) population in Singapore.

Participants  A total of 6616 Singaporean adults aged 18 years and older.

Measurements  The diagnoses were established using the World Mental Health Composite International Diagnostic Interview (WMH‐CIDI) diagnostic modules for life‐time and 12‐month prevalence of selected mental illnesses including alcohol use disorders.

Findings  The life‐time prevalence of alcohol abuse and alcohol dependence was 3.1% and 0.5%, while the 12‐month prevalence of alcohol abuse and alcohol dependence was 0.5% and 0.3%, respectively. The life‐time and 12‐month prevalence of alcohol use disorders was 3.6% and 0.8%, respectively. Those with alcohol use disorder had significantly higher odds of having major depressive disorder [odds ratio (OR) 3.1] and nicotine dependence (OR 4.5). Compared to the rest of the population, those with an alcohol use disorder had significantly higher odds of having gastric ulcers (OR 3.0), respiratory conditions (OR 2.1) and chronic pain (OR 2.1). Only one in five of those with alcohol use disorder had ever sought treatment.

Conclusions  The prevalence of alcohol use disorders is relatively low in the Singapore adult population. Comorbidity with mental and physical disorders is significant, emphasizing the need to screen people with alcohol use disorders for these comorbidities.
Health‐care service utilization in substance abusers receiving contingency management and standard care treatments
Authors: Olmstead Todd A. , Cohen Jeffrey P. , Petry Nancy M.
Published: February 2012

Aim  To determine the impact of standard care and contingency management treatments on the utilization of general health‐care services by substance abusers.

Participants, design and measurements  This secondary analysis pooled 1028 treatment‐seeking substance abusers from five randomized clinical trials that compared the effects of standard care (SC, n = 362) to standard care plus contingency management (CM, n = 666). In each trial, subjects in the CM condition showed significantly greater reductions in substance use than their SC counterparts. For each subject, utilization of 15 general health‐care services was measured 1 year prior to treatment intake and up to 9 months following treatment intake. Post‐intake utilization data were prorated to be comparable to the 1‐year pre‐intake data. Paired t‐tests evaluated changes in service utilization pre‐ and post‐intake, and difference‐in‐differences regression models were used to estimate the impact of CM, compared to SC, on changes in the utilization of each of the 15 health services.

Setting  Out‐patient community substance abuse clinics in Connecticut and Massachusetts, USA.

Findings  Utilization of several types of out‐patient services increased significantly between the pre‐ and post‐intake periods [e.g. dental visits (0.47, P < 0.001), community health center visits (0.50, P < 0.001), visits to a mental health professional office (1.03, P = 0.001)], while in‐patient hospital care for mental health problems decreased significantly (−3.50 nights, P < 0.001). A substantial portion of these changes occurred during the treatment period. No significant differences were found between the two treatment conditions.

Conclusions  Initiating out‐patient substance abuse treatment is associated with changes in general health‐care service utilization, independent of the type of treatment offered.
Preferences for evidence‐based practice dissemination in addiction agencies serving women: a discrete‐choice conjoint experiment
Authors: Cunningham Charles E. , Henderson Joanna , Niccols Alison , Dobbins Maureen , Sword Wendy , Chen Yvonne , Mielko Stephanie , Milligan Karen , Lipman Ellen , Thabane Lehana , Schmidt Louis
Published: February 2012

Aim  To model variables influencing the dissemination of evidence‐based practices to addiction service providers and administrators.

Design  A discrete‐choice conjoint experiment. We systematically varied combinations of 16 dissemination variables that might influence the adoption of evidence‐based practices. Participants chose between sets of variables.

Setting  Canadian agencies (n = 333) providing addiction services to women.

Participants  Service providers and administrators (n = 1379).

Measurements  We estimated the relative importance and optimal level of each dissemination variable. We used latent class analysis to identify subsets of participants with different preferences and simulated the conditions under which participants would use more demanding professional development options.

Findings  Three subsets of participants were identified: outcome‐sensitive (52%), process‐sensitive (29.6%) and demand‐sensitive (18.2%). Across all participants, the number of clients who were expected to benefit from an evidence‐based practice exerted the most influence on dissemination choices. If a practice was seen as feasible, co‐worker and administrative support influenced decisions. Client benefits were most important to outcome‐sensitive participants; type of dissemination process (e.g. active versus passive learning) was more important to process‐sensitive participants. Brief options with little follow‐up were preferred by demand‐sensitive participants. Simulations predicted that initiatives selected and endorsed by government funders would reduce participation.

Conclusions  Clinicians and administrators are more likely to adopt evidence‐based addiction practices if the practice is seen as helpful to clients, and if it is supported by co‐workers and program administration.
The Red clover necrotic mosaic virus capsid protein N‐terminal lysine‐rich motif is a determinant of symptomatology and virion accumulation
Authors: PARK SANG‐HO , SIT TIM L. , KIM KOOK‐HYUNG , LOMMEL STEVEN A.
Published: February 2012

The interaction between viral capsid protein (CP) and its cognate viral RNA modulates many steps in the virus infection cycle, such as replication, translation and assembly. The N‐terminal 50 amino acids of the Red clover necrotic mosaic virus (RCNMV) CP are rich in basic residues (especially lysine) and are essential for the core functions of the CP, namely RNA binding and virion assembly. To further elucidate additional biological roles for these basic residues, a series of alanine substitution mutations was introduced into infectious clones of RCNMV RNA‐1 and assayed for symptomatology, virion formation and systemic infection. Infectivity assays conducted in Nicotiana benthamiana revealed that all nine alanine substitution mutants (ASMs) were competent for systemic infection. Two ASMs (K4A and K7A/K8A) induced severe symptoms and delayed the systemic spread of viral genomes when compared with wild‐type RCNMV. However, these ASMs were still competent for virion formation. Three other ASMs (K25A, K33A and K38A) displayed milder symptoms and significant reductions in virion accumulation when compared with wild‐type RCNMV, but retained the ability to spread systemically. Evidence from these last three ASMs, as well as a CP null mutant, showed that RCNMV is able to move systemically in N. benthamiana as a nonvirion form. These observations reaffirm the necessity of the N‐terminal lysine‐rich residues of the RCNMV CP for efficient virion accumulation. They also reveal additional roles for the CP in the modulation of host symptomatology, independent of its role in virion assembly and the rate of systemic viral movement in N. benthamiana.