bluuebunny/crossref_metadata_2025 huggingface dataset:
Transient Analysis of Gas Distribution in the Anodic Flow Field in a Fuel Cell Stack
Authors: Ichikawa Yasushi , Oshima Nobuyuki
Published: December 2013

In a polymer electrolyte fuel cell (PEFC), the catalyst degradation on cathodic side is one of the fatal problems caused by mal-distributed hydrogen supply into each channel on active area in a fuel cell, especially in a fuel cell stack for automotive fuel cell systems which consist of several hundreds of fuel cells stacked. For example, before getting the fuel cell system started-up, the gas in all the anodic flow passage including channels in each fuel cell is occupied by air instead of hydrogen due to cross leak from cathodic side to anodic side through the membrane employed as an electrolyte. In this situation, if hydrogen is supplied partially or unevenly between cells to start up the system, a concentration interface of air and hydrogen will be made within a fuel cell. This causes a state of local cell within a single fuel cell and the catalyst degradation (carbon corrosion or Pt dissolution) occurs. In this paper, to avoid this catalyst degradation, the gas distribution is investigated with pressurized hydrogen supply into channels located on the hundreds stacked fuel cells statically filled with air initially. A transient computational fluid analysis was applied to the flow fields of anodic side which consist of channels on fuel cells, both distributing and collecting manifold connected to the fuel cells under parameters: 1) number of stacked fuel cells (i.e. manifold length), 2) the rate of pressure rising (Pa/sec.) which makes the gas flow velocity. A gas analysis experiment was also carried out for a validation with mass spectrometer taking gas sample from several points along the gas channels on alternative fuel cells which are made of transparent acrylic resin. The results show that the uniform distribution in concentration between cells and its profile within the channels along the flow direction are strongly affected by flow field formed within the distributing manifold located upstream of stacked plates with channels.
Hf-based high-k dielectrics for p-Ge MOS gate stacks
Authors: Fadida Sivan , Palumbo Felix , Nyns Laura , Lin Dennis , Van Elshocht Sven , Caymax Matty , Eizenberg Moshe
Published: December 2013

The physical and electrical properties of the gate stack high-k/Al2O3/GeO2/p-Ge were studied in detail, where the high-k is either HfO2 or alloyed HfO2 (HfZrOy, HfGdOx, or HfAlOx). Electrical measurements combined with x-ray photoelectron spectroscopy chemical bonding analysis and band alignment determination were conducted in order to assess the suitability of hafnium-based high-k for this kind of gate stacks, with emphasis on low density of interface states and border traps. HfAlOx was found to be the most promising high-k from those studied. The authors have also found that the current- voltage trends for the various systems studied can be explained by the band alignment of the samples obtained by our x-ray photoelectron spectroscopy analysis.
Lysine Methylation Promotes VEGFR-2 Activation and Angiogenesis
Authors: Hartsough Edward J. , Meyer Rosana D. , Chitalia Vipul , Jiang Yan , Marquez Victor E. , Zhdanova Irina V. , Weinberg Janice , Costello Catherine E. , Rahimi Nader
Published: December 2013

Preventing the methylation of VEGFR-2 could attenuate pathological angiogenesis in cancer.
HES1 Is a Master Regulator of Glucocorticoid Receptor–Dependent Gene Expression
Authors: Revollo Javier R. , Oakley Robert H. , Lu Nick Z. , Kadmiel Mahita , Gandhavadi Maheer , Cidlowski John A.
Published: December 2013

A transcription factor that regulates developmental events also suppresses transcriptional responses to stress in adult organisms.
Antiviral Activity of (+)-Rutamarin against Kaposi's Sarcoma-Associated Herpesvirus by Inhibition of the Catalytic Activity of Human Topoisomerase II
Authors: Xu Bo , Wang Ling , González-Molleda Lorenzo , Wang Yan , Xu Jun , Yuan Yan
Published: December 2013

Kaposi's sarcoma-associated herpesvirus (KSHV) is an etiological agent of several AIDS-associated malignancies, including Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD). Its lytic replication cycle has been proven to be critical for the pathogenesis of KSHV-associated diseases. In KS lesions, lytic viral replication, production of virion particles, and reinfection of endothelial cells are essential to sustain the population of infected cells that otherwise would be quickly lost as spindle cells divide. Thus, antivirals that block KSHV replication could be a strategy in the treatment of KSHV-associated diseases. However, there is no effective anti-KSHV drug currently available. Our previous work showed that human topoisomerase II (Topo II) is indispensable for KSHV lytic replication and is suggested to be an effective target for antiviral drugs. Here, we report the discovery and characterization of a novel catalytic inhibitor of human Topo IIα, namely, (+)-rutamarin. The binding mode of (+)-rutamarin to the ATPase domain of human Topo IIα was established by docking and validated by molecular dynamics (MD) simulations. More importantly, (+)-rutamarin efficiently inhibits KSHV lytic DNA replication in BCBL-1 cells with a half-maximal inhibitory concentration (IC
50
) of 1.12 μM and blocks virion production with a half-maximal antiviral effective concentration (EC
50
) of 1.62 μM. It possesses low cytotoxicity, as indicated by the selectivity index (SI) of 84.14. This study demonstrated great potential for (+)-rutamarin to become an effective drug for treatment of human diseases associated with KSHV infection.
Estimation of Intracellular Concentration of Stavudine Triphosphate in HIV-Infected Children Given a Reduced Dose of 0.5 Milligrams per Kilogram Twice Daily
Authors: Sy Sherwin K. B. , Innes Steve , Derendorf Hartmut , Cotton Mark F. , Rosenkranz Bernd
Published: December 2013

The antiviral efficacy of stavudine depends on the trough concentration of its intracellular metabolite, stavudine-triphosphate (d4T-TP), while the degree of stavudine's mitochondrial toxicity depends on its peak concentration. Rates of mitochondrial toxicity are high when stavudine is used at the current standard pediatric dose (1 mg/kg twice daily [BID]). Evidence from adult work suggests that half of the original standard adult dose (i.e., 20 mg BID) may be equally effective, with markedly less mitochondrial toxicity. We present a population pharmacokinetic model to predict intracellular d4T-TP concentrations in pediatric HIV-infected patients administered a dose of 0.5 mg/kg BID. Our model predicted that the reduced pediatric dose would result in a trough intracellular d4T-TP concentration above that of the reduced 20-mg adult dose and a peak concentration below that of the 20-mg adult dose. The simulated pediatric intracellular d4T-TP at 0.5 mg/kg BID resulted in median peak and trough values of approximately 23.9 fmol/106cells (95% prediction interval [PI], 14.2 to 41 fmol/106cells) and 14.8 fmol/106cells (95% PI, 7.2 to 31 fmol/106cells), respectively. The peak and trough concentrations resulting from a 20-mg BID adult dose were 28.4 fmol/106cells (95% PI, 17.3 to 45.5 fmol/106cells) and 13 fmol/106cells (95% PI, 6.8 to 28.6 fmol/106cells), respectively. Halving the current standard pediatric dose should therefore not compromise antiviral efficacy, while markedly reducing mitochondrial toxicity.
Image-Based 384-Well High-Throughput Screening Method for the Discovery of Skyllamycins A to C as Biofilm Inhibitors and Inducers of Biofilm Detachment in Pseudomonas aeruginosa
Authors: Navarro Gabriel , Cheng Andrew T. , Peach Kelly C. , Bray Walter M. , Bernan Valerie S. , Yildiz Fitnat H. , Linington Roger G.
Published: December 2013

To date, most antibiotics have primarily been developed to target bacteria in the planktonic state. However, biofilm formation allows bacteria to develop tolerance to antibiotics and provides a mechanism to evade innate immune systems. Therefore, there is a significant need to identify small molecules to prevent biofilm formation and, more importantly, to disperse or eradicate preattached biofilms, which are a major source of bacterial persistence in nosocomial infections. We now present a modular high-throughput 384-well image-based screening platform to identify
Pseudomonas aeruginosa
biofilm inhibitors and dispersal agents. Biofilm coverage measurements were accomplished using non-z-stack epifluorescence microscopy to image a constitutively expressing green fluorescent protein (GFP)-tagged strain of
P. aeruginosa
and quantified using an automated image analysis script. Using the redox-sensitive dye XTT, bacterial cellular metabolic activity was measured in conjunction with biofilm coverage to differentiate between classical antibiotics and nonantibiotic biofilm inhibitors/dispersers. By measuring biofilm coverage and cellular activity, this screen identifies compounds that eradicate biofilms through mechanisms that are disparate from traditional antibiotic-mediated biofilm clearance. Screening of 312 natural-product prefractions identified the cyclic depsipeptide natural products skyllamycins B and C as nonantibiotic biofilm inhibitors with 50% effective concentrations (EC
50
s) of 30 and 60 μM, respectively. Codosing experiments of skyllamycin B and azithromycin, an antibiotic unable to clear preattached biofilms, demonstrated that, in combination, these compounds were able to eliminate surface-associated biofilms and depress cellular metabolic activity. The skyllamycins represent the first known class of cyclic depsipeptide biofilm inhibitors/dispersers.
Functional Linkage between Genes That Regulate Osmotic Stress Responses and Multidrug Resistance Transporters: Challenges and Opportunities for Antibiotic Discovery
Authors: Cohen B. Eleazar
Published: December 2013

All cells need to protect themselves against the osmotic challenges of their environment by maintaining low permeability to ions across their cell membranes. This is a basic principle of cellular function, which is reflected in the interactions among ion transport and drug efflux genes that have arisen during cellular evolution. Thus, upon exposure to pore-forming antibiotics such as amphotericin B (AmB) or daptomycin (Dap), sensitive cells overexpress common resistance genes to protect themselves from added osmotic challenges. These genes share pathway interactions with the various types of multidrug resistance (MDR) transporter genes, which both preserve the native lipid membrane composition and at the same time eliminate disruptive hydrophobic molecules that partition excessively within the lipid bilayer. An increased understanding of the relationships between the genes (and their products) that regulate osmotic stress responses and MDR transporters will help to identify novel strategies and targets to overcome the current stalemate in drug discovery.
Assessment of an Anti-Alpha-Toxin Monoclonal Antibody for Prevention and Treatment of Staphylococcus aureus-Induced Pneumonia
Authors: Hua L. , Hilliard J. J. , Shi Y. , Tkaczyk C. , Cheng L. I. , Yu X. , Datta V. , Ren S. , Feng H. , Zinsou R. , Keller A. , O'Day T. , Du Q. , Cheng L. , Damschroder M. , Robbie G. , Suzich J. , Stover C. K. , Sellman B. R.
Published: December 2013

Alpha-toxin (AT) is a major virulence factor in the disease pathogenesis ofStaphylococcus aureus. We previously identified a monoclonal antibody (MAb) against AT that reduced disease severity in a mouse dermonecrosis model. Here, we evaluate the activity of an affinity-optimized variant, LC10, in a mouse model ofS. aureuspneumonia. Passive immunization with LC10 increased survival and reduced bacterial numbers in the lungs and kidneys of infected mice and showed protection against diverseS. aureusclinical isolates. The lungs ofS. aureus-infected mice exhibited bacterial pneumonia, including widespread inflammation, whereas the lungs of mice that received LC10 exhibited minimal inflammation and retained healthy architecture. Consistent with reduced immune cell infiltration, LC10-treated animals had significantly lower (P< 0.05) proinflammatory cytokine and chemokine levels in the bronchoalveolar lavage fluid than did those of the control animals. This reduction in inflammation and damage to the LC10-treated animals resulted in reduced vascular protein leakage and CO2levels in the blood. LC10 was also assessed for its therapeutic activity in combination with vancomycin or linezolid. Treatment with a combination of LC10 and vancomycin or linezolid resulted in a significant increase (P< 0.05) in survival relative to the monotherapies and was deemed additive to synergistic by isobologram analysis. Consistent with improved survival, the lungs of animals treated with antibiotic plus LC10 exhibited less inflammatory tissue damage than those that received monotherapy. These data provide insight into the mechanisms of protection provided by AT inhibition and support AT as a promising target for immunoprophylaxis or adjunctive therapy againstS. aureuspneumonia.
Pathogenicity of Clinical Acinetobacter baumannii Isolates in a Galleria mellonella Host Model According to
bla
OXA-40
Gene and Epidemiological Outbreak Status
Authors: Esterly John S. , McLaughlin Milena M. , Malczynski Michael , Qi Chao , Zembower Teresa R. , Scheetz Marc H.
Published: December 2013

Clinical studies have suggested that
bla
OXA-40
-positive
Acinetobacter baumannii
isolates are associated with poor patient outcomes; however, reasons for unfavorable outcomes are difficult to discern in clinical studies. The objective of this study was to assess the virulence of carbapenem-resistant
A. baumannii
according to
bla
OXA-40
and epidemiological outbreak status in a
Galleria mellonella
model. Eight isolates of
A. baumannii
were studied. Nonoutbreak isolates and
bla
OXA-40
-negative isolates more rapidly killed infected
G. mellonella
(
P
< 0.01).
A Geobacter sulfurreducens Strain Expressing Pseudomonas aeruginosa Type IV Pili Localizes OmcS on Pili but Is Deficient in Fe(III) Oxide Reduction and Current Production
Authors: Liu Xing , Tremblay Pier-Luc , Malvankar Nikhil S. , Nevin Kelly P. , Lovley Derek R. , Vargas Madeline
Published: December 2013

The conductive pili of
Geobacter
species play an important role in electron transfer to Fe(III) oxides, in long-range electron transport through current-producing biofilms, and in direct interspecies electron transfer. Although multiple lines of evidence have indicated that the pili of
Geobacter sulfurreducens
have a metal-like conductivity, independent of the presence of
c
-type cytochromes, this claim is still controversial. In order to further investigate this phenomenon, a strain of
G. sulfurreducens
, designated strain PA, was constructed in which the gene for the native PilA, the structural pilin protein, was replaced with the PilA gene of
Pseudomonas aeruginosa
PAO1. Strain PA expressed and properly assembled
P. aeruginosa
PilA subunits into pili and exhibited a profile of outer surface
c
-type cytochromes similar to that of a control strain expressing the
G. sulfurreducens
PilA. Surprisingly, the strain PA pili were decorated with the
c
-type cytochrome OmcS in a manner similar to the control strain. However, the strain PA pili were 14-fold less conductive than the pili of the control strain, and strain PA was severely impaired in Fe(III) oxide reduction and current production. These results demonstrate that the presence of OmcS on pili is not sufficient to confer conductivity to pili and suggest that there are unique structural features of the
G. sulfurreducens
PilA that are necessary for conductivity.
An Oleaginous Bacterium That Intrinsically Accumulates Long-Chain Free Fatty Acids in its Cytoplasm
Authors: Katayama Taiki , Kanno Manabu , Morita Naoki , Hori Tomoyuki , Narihiro Takashi , Mitani Yasuo , Kamagata Yoichi
Published: December 2013

Medium- and long-chain fatty acids are present in organisms in esterified forms that serve as cell membrane constituents and storage compounds. A large number of organisms are known to accumulate lipophilic materials as a source of energy and carbon. We found a bacterium, designated GK12, that intrinsically accumulates free fatty acids (FFAs) as intracellular droplets without exhibiting cytotoxicity. GK12 is an obligatory anaerobic, mesophilic lactic acid bacterium that was isolated from a methanogenic reactor. Phylogenetic analysis based on 16S rRNA gene sequences showed that GK12 is affiliated with the family
Erysipelotrichaceae
in the phylum
Firmicutes
but is distantly related to type species in this family (less than 92% similarity in 16S rRNA gene sequence). Saturated fatty acids with carbon chain lengths of 14, 16, 18, and 20 were produced from glucose under stress conditions, including higher-than-optimum temperatures and the presence of organic solvents that affect cell membrane integrity. FFAs were produced at levels corresponding to up to 25% (wt/wt) of the dry cell mass. Our data suggest that FFA accumulation is a result of an imbalance between excess membrane fatty acid biosynthesis due to homeoviscous adaptation and limited β-oxidation activity due to anaerobic growth involving lactic acid fermentation. FFA droplets were not further utilized as an energy and carbon source, even under conditions of starvation. A naturally occurring bacterium that accumulates significant amounts of long-chain FFAs with noncytotoxicity would provide useful strategies for microbial biodiesel production.
Novel Maltotriose-Hydrolyzing Thermoacidophilic Type III Pullulan Hydrolase from Thermococcus kodakarensis
Authors: Ahmad Nasir , Rashid Naeem , Haider Muhammad Saleem , Akram Mehwish , Akhtar Muhammad
Published: December 2013

A novel thermoacidophilic pullulan-hydrolyzing enzyme (PUL) from hyperthermophilic archaeon
Thermococcus kodakarensis
(TK-PUL) that efficiently hydrolyzes starch under industrial conditions in the absence of any additional metal ions was cloned and characterized. TK-PUL possessed both pullulanase and α-amylase activities. The highest activities were observed at 95 to 100°C. Although the enzyme was active over a broad pH range (3.0 to 8.5), the pH optima for both activities were 3.5 in acetate buffer and 4.2 in citrate buffer. TK-PUL was stable for several hours at 90°C. Its half-life at 100°C was 45 min when incubated either at pH 6.5 or 8.5. The

K
m

value toward pullulan was 2 mg ml
−1
, with a
V
max
of 109 U mg
−1
. Metal ions were not required for the activity and stability of recombinant TK-PUL. The enzyme was able to hydrolyze both α-1,6 and α-1,4 glycosidic linkages in pullulan. The most preferred substrate, after pullulan, was γ-cyclodextrin, which is a novel feature for this type of enzyme. Additionally, the enzyme hydrolyzed a variety of polysaccharides, including starch, glycogen, dextrin, amylose, amylopectin, and cyclodextrins (α, β, and γ), mainly into maltose. A unique feature of TK-PUL was the ability to hydrolyze maltotriose into maltose and glucose.
Intimin Gene (
eae
) Subtype-Based Real-Time PCR Strategy for Specific Detection of Shiga Toxin-Producing Escherichia coli Serotypes O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 in Cattle Feces
Authors: Bibbal Delphine , Loukiadis Estelle , Kérourédan Monique , Peytavin de Garam Carine , Ferré Franck , Cartier Philippe , Gay Emilie , Oswald Eric , Auvray Frédéric , Brugère Hubert
Published: December 2013

Shiga toxin-producing
Escherichia coli
(STEC) strains belonging to serotypes O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 are known to be associated with particular subtypes of the intimin gene (
eae
), namely, γ1, β1, ε, θ, and γ1, respectively. This study aimed at evaluating the usefulness of their detection for the specific detection of these five main pathogenic STEC serotypes in cattle feces. Using real-time PCR assays, 58.7% of 150 fecal samples were found positive for at least one of the four targeted
eae
subtypes. The simultaneous presence of
stx
,
eae
, and one of the five O group markers was found in 58.0% of the samples, and the five targeted
stx
plus
eae
plus O genetic combinations were detected 143 times. However, taking into consideration the association between
eae
subtypes and O group markers, the resulting
stx
plus
eae
subtype plus O combinations were detected only 46 times. The 46 isolation assays performed allowed recovery of 22
E. coli
strains belonging to one of the five targeted STEC serogroups. In contrast, only 2 of 39 isolation assays performed on samples that were positive for
stx
,
eae
and an O group marker, but that were negative for the corresponding
eae
subtype, were successful. Characterization of the 24
E. coli
isolates showed that 6 were STEC, including 1 O157:H7, 3 O26:H11, and 2 O145:H28. The remaining 18 strains corresponded to atypical enteropathogenic
E. coli
(aEPEC). Finally, the more discriminating
eae
subtype-based PCR strategy described here may be helpful for the specific screening of the five major STEC in cattle feces.
An Unbiased Method for Clustering Bacterial Effectors Using Host Cellular Phenotypes
Authors: Dowling Andrea J. , Hodgson David J.
Published: December 2013

We present a novel method implementing unbiased high-content morphometric cell analysis to classify bacterial effector phenotypes. This clustering methodology represents a significant advance over more qualitative visual approaches and can also be used to classify, and therefore predict the likely function of, unknown effector genes from any microbial genome. As a proof of concept, we use this approach to investigate 23 genetic regions predicted to encode antimacrophage effectors located across the genome of the insect and human pathogenPhotorhabdus asymbiotica. Statistical cluster analysis using multiple cellular measures categorized treated macrophage phenotypes into three major groups relating to their putative functionality: (i) adhesins, (ii) cytolethal toxins, and (iii) cytomodulating toxins. Further investigation into their effects on phagocytosis revealed that several effectors also modulate this function and that the nature of this modulation (increased or decreased phagocytosis) is linked to the phenotype cluster group. Categorizing potential functionalities in this way allows rapid functional follow-up of key candidates for more-directed cell biological or biochemical investigation. Such an unbiased approach to the classification of candidate effectors will be useful for describing virulence-related regions in a wide range of genomes and will be useful in assigning putative functions to the growing number of microbial genes whose function remains unclear from homology searching.
Motility Characteristics Are Altered for Rickettsia bellii Transformed To Overexpress a Heterologous
rickA
Gene
Authors: Oliver Jonathan D. , Burkhardt Nicole Y. , Felsheim Roderick F. , Kurtti Timothy J. , Munderloh Ulrike G.
Published: December 2013

The rickettsial protein RickA activates host cell factors associated with the eukaryotic actin cytoskeleton and is likely involved with rickettsial host cell binding and infection and the actin-based motility of spotted fever group rickettsiae. The
rickA
gene sequence and protein vary substantially between
Rickettsia
species, as do observed motility-associated phenotypes. To help elucidate the function of RickA and determine the effects of species-specific RickA variations, we compared extracellular binding, intracellular motility, and intercellular spread phenotypes of three
Rickettsia bellii
variants. These included two shuttle vector-transformed
R. bellii
strains and the wild-type isolate from which they were derived,
R. bellii
RML 369C. Both plasmid shuttle vectors carried spectinomycin resistance and a GFPuv reporter; one contained
Rickettsia monacensis
-derived
rickA
, and the other lacked the
rickA
gene.
Rickettsia bellii
transformed to express
R. monacensis rickA
highly overexpressed this transcript in comparison to its native
rickA
. These rickettsiae also moved at higher velocities and followed a more curved path than the negative-control transformants. A lower proportion of
R. monacensis rickA
-expressing bacteria ever became motile, however, and they formed smaller plaques.
Novel Firmicutes Group Implicated in the Dechlorination of Two Chlorinated Xanthones, Analogues of Natural Organochlorines
Authors: Krzmarzick Mark J. , Miller Hanna R. , Yan Tao , Novak Paige J.
Published: December 2013

Although the abundance and diversity of natural organochlorines are well established, much is still unknown about the degradation of these compounds. Triplicate microcosms were used to determine whether, and which, bacterial communities could dechlorinate two chlorinated xanthones (2,7-dichloroxanthone and 5,7-dichloro-1,3-dihydroxylxanthone), analogues of a diverse class of natural organochlorines. According to quantitative-PCR (qPCR) results, several known dechlorinating genera were either not present or not enriched during dechlorination of the xanthones. Denaturing gradient gel electrophoresis, however, indicated that several
Firmicutes
were enriched in the dechlorinating cultures compared to triplicate controls amended with nonchlorinated xanthones. One such group, herein referred to as the Gopher group, was further studied with a novel qPCR method that confirmed enrichment of Gopher group 16S rRNA genes in the dechlorinating cultures. The enrichment of the Gopher group was again tested with two new sets of triplicate microcosms. Enrichment was observed during chlorinated xanthone dechlorination in one set of these triplicate microcosms. In the other set, two microcosms showed clear enrichment while a third did not. The Gopher group is a previously unidentified group of
Firmicutes
, distinct from but related to the
Dehalobacter
and
Desulfitobacterium
genera; this group also contains clones from at least four unique cultures capable of dechlorinating anthropogenic organochlorines that have been previously described in the literature. This study suggests that natural chlorinated xanthones may be effective biostimulants to enhance the remediation of pollutants and highlights the idea that novel genera of dechlorinators likely exist and may be active in bioremediation and the natural cycling of chlorine.
Protein Kinase A Is Part of a Mechanism That Regulates Nuclear Reimport of the Nuclear tRNA Export Receptors Los1p and Msn5p
Authors: Pierce Jacqueline B. , van der Merwe George , Mangroo Dev
Published: December 2013

The two main signal transduction mechanisms that allow eukaryotes to sense and respond to changes in glucose availability in the environment are the cyclic AMP (cAMP)/protein kinase A (PKA) and AMP-activated protein kinase (AMPK)/Snf1 kinase-dependent pathways. Previous studies have shown that the nuclear tRNA export process is inhibited in
Saccharomyces cerevisiae
deprived of glucose. However, the signal transduction pathway involved and the mechanism by which glucose availability regulates nuclear-cytoplasmic tRNA trafficking are not understood. Here, we show that inhibition of nuclear tRNA export is caused by a block in nuclear reimport of the tRNA export receptors during glucose deprivation. Cytoplasmic accumulation of the tRNA export receptors during glucose deprivation is not caused by activation of Snf1p. Evidence obtained suggests that PKA is part of the mechanism that regulates nuclear reimport of the tRNA export receptors in response to glucose availability. This mechanism does not appear to involve phosphorylation of the nuclear tRNA export receptors by PKA. The block in nuclear reimport of the tRNA export receptors appears to be caused by activation of an unidentified mechanism when PKA is turned off during glucose deprivation. Taken together, the data suggest that PKA facilitates return of the tRNA export receptors to the nucleus by inhibiting an unidentified activity that facilitates cytoplasmic accumulation of the tRNA export receptors when glucose in the environment is limiting. A PKA-independent mechanism was also found to regulate nuclear tRNA export in response to glucose availability. This mechanism, however, does not regulate nuclear reimport of the tRNA export receptors.
Mutations in Pdd1 Reveal Distinct Requirements for Its Chromodomain and Chromoshadow Domain in Directing Histone Methylation and Heterochromatin Elimination
Authors: Schwope Rachel M. , Chalker Douglas L.
Published: December 2013

Pdd1, a specialized HP1-like protein, is required for genome-wide DNA rearrangements that restructure a previously silent germ line genome into an active somatic genome during macronuclear differentiation of
Tetrahymena thermophila
. We deleted or otherwise mutated conserved regions of the protein to investigate how its different domains promote the excision of thousands of internal eliminated sequences (IESs). Previous studies revealed that Pdd1 contributes to recognition of IES loci after they are targeted by small-RNA-guided methylation of histone H3 on lysine 27 (H3K27), subsequently aids the establishment of H3K9 methylation, and recruits proteins that lead to excision. The phenotypes we observed for different Pdd1 alleles showed that each of the two chromodomains and the chromoshadow domain (CSD) have distinct contributions during somatic genome differentiation. Chromodomain 1 (CD1) is essential for conjugation as either its deletion or the substitution of two key aromatic amino acid residues (the W97A W100A mutant) is lethal. These mutations caused mislocalization of a cyan fluorescent protein (CFP)-tagged protein, prevented the establishment of histone H3 dimethylated on K9 (H3K9me2), and abolished IES excision. Nevertheless, the requirement for CD1 could be bypassed by recruiting Pdd1 directly to an IES by addition of a specific DNA binding domain. Chromodomain 2 (CD2) was necessary for producing viable progeny, but low levels of H3K9me2 and IES excision still occurred. A mutation in the chromoshadow domain (CSD) prevented Pdd1 focus formation but still permitted ∼17% of conjugants to produce viable progeny. However, this mutant was unable to stimulate excision when recruited to an ectopic IES, indicating that this domain is important for recruitment of excision factors.
Sck1 Negatively Regulates Gpa2-Mediated Glucose Signaling in Schizosaccharomyces pombe
Authors: Mudge Dayna K. , Yang Fan , Currie Brian M. , Kim James M. , Yeda Kelly , Bashyakarla Varoon K. , Ivey F. Douglas , Hoffman Charles S.
Published: December 2013

Schizosaccharomyces pombe
detects extracellular glucose via a G protein-mediated cyclic AMP (cAMP)-signaling pathway activating protein kinase A (PKA) and regulating transcription of genes involved in metabolism and sexual development. In this pathway, Gpa2 Gα binds to and activates adenylyl cyclase in response to glucose detection by the Git3 G protein-coupled receptor. Using a two-hybrid screen to identify extrinsic regulators of Gpa2, we isolated a clone that expresses codons 471 to 696 of the Sck1 kinase, which appears to display a higher affinity for Gpa2
K270E
-activated Gα relative to Gpa2
+
Gα. Deletion of
sck1
+
or mutational inactivation of the Sck1 kinase produces phenotypes reflecting increased PKA activity in strains expressing Gpa2
+
or Gpa2
K270E
, suggesting that Sck1 negatively regulates PKA activation through Gpa2. In contrast to the Gpa2
K270E
GDP-GTP exchange rate mutant, GTPase-defective Gpa2
R176H
weakly binds Sck1 in the two-hybrid screen and a deletion of
sck1
+
in a Gpa2
R176H
strain confers phenotypes consistent with a slight reduction in PKA activity. Finally, deleting
sck1
+
in a
gpa2
Δ strain results in phenotypes consistent with a second role for Sck1 acting in parallel with PKA. In addition to this parallel role with PKA, our data suggest that Sck1 negatively regulates Gpa2, possibly targeting the nucleotide-free form of the protein that may expose the one and only AKT/PKB consensus site in Gpa2 for Sck1 to bind. This dual role for Sck1 may allow
S. pombe
to produce distinct biological responses to glucose and nitrogen starvation signals that both activate the Wis1-Spc1/StyI stress-activated protein kinase (SAPK) pathway.
Hepcidin Induction by Pathogens and Pathogen-Derived Molecules Is Strongly Dependent on Interleukin-6
Authors: Rodriguez Richard , Jung Chun-Ling , Gabayan Victoria , Deng Jane C. , Ganz Tomas , Nemeth Elizabeta , Bulut Yonca
Published: December 2013

Hepcidin, the iron-regulatory hormone, is increased during infection or inflammation, causing hypoferremia. This response is thought to be a host defense mechanism that restricts iron availability to invading pathogens. It is not known if hepcidin is differentially induced by bacterial versus viral infections, whether the stimulation of pattern recognition receptors directly regulates hepcidin transcription, or which of the proposed signaling pathways are essential for hepcidin increase during infection. We analyzed hepcidin induction and its dependence on interleukin-6 (IL-6) in response to common bacterial or viral infections in mice or in response to a panel of pathogen-derived molecules (PAMPs) in mice and human primary hepatocytes. In wild-type (WT) mice, hepcidin mRNA was induced several hundred-fold both by a bacterial (
Streptococcus pneumoniae
) and a viral infection (influenza virus PR8) within 2 to 5 days. Treatment of mice and human primary hepatocytes with most Toll-like receptor ligands increased hepcidin mRNA within 6 h. Hepcidin induction by microbial stimuli was IL-6 dependent. IL-6 knockout mice failed to increase hepcidin in response to
S. pneumoniae
or influenza infection and had greatly diminished hepcidin response to PAMPs.
In vitro
, hepcidin induction by PAMPs in primary human hepatocytes was abolished by the addition of neutralizing IL-6 antibodies. Our results support the key role of IL-6 in hepcidin regulation in response to a variety of infectious and inflammatory stimuli.
Murine Neonates Infected with Yersinia enterocolitica Develop Rapid and Robust Proinflammatory Responses in Intestinal Lymphoid Tissues
Authors: Siefker David T. , Echeverry Andrea , Brambilla Roberta , Fukata Masayuki , Schesser Kurt , Adkins Becky
Published: December 2013

Neonatal animals are generally very susceptible to infection with bacterial pathogens. However, we recently reported that neonatal mice are highly resistant to orogastric infection withYersinia enterocolitica. Here, we show that proinflammatory responses greatly exceeding those in adults arise very rapidly in the mesenteric lymph nodes (MLN) of neonates. High-level induction of proinflammatory gene expression occurred in the neonatal MLN as early as 18 h postinfection. Marked innate phagocyte recruitment was subsequently detected at 24 h postinfection. Enzyme-linked immunosorbent spot assay (ELISPOT) analyses indicated that enhanced inflammation in neonatal MLN is contributed to, in part, by an increased frequency of proinflammatory cytokine-secreting cells. Moreover, both CD11b+and CD11b−cell populations appeared to play a role in proinflammatory gene expression. The level of inflammation in neonatal MLN was also dependent on key bacterial components.Y. enterocoliticalacking the virulence plasmid failed to induce innate phagocyte recruitment. In contrast, tumor necrosis factor alpha (TNF-α) protein expression and neutrophil recruitment were strikingly higher in neonatal MLN after infection with ayopP-deficient strain than with wild-typeY. enterocolitica, whereas only modest increases occurred in adults. This hyperinflammatory response was associated with greater colonization of the spleen and higher mortality in neonates, while there was no difference in mortality among adults. This model highlights the dynamic levels of inflammation in the intestinal lymphoid tissues and reveals the protective (wild-type strain) versus harmful (yopP-deficient strain) consequences of inflammation in neonates. Moreover, these results reveal that the neonatal intestinal lymphoid tissues have great potential to rapidly mobilize innate components in response to infection with bacterial enteropathogens.
The Integron Integrase Efficiently Prevents the Melting Effect of Escherichia coli Single-Stranded DNA-Binding Protein on Folded
attC
Sites
Authors: Loot Céline , Parissi Vincent , Escudero José Antonio , Amarir-Bouhram Jihane , Bikard David , Mazel Didier
Published: December 2013

Integrons play a major role in the dissemination of antibiotic resistance genes among bacteria. Rearrangement of gene cassettes occurs by recombination between
attI
and
attC
sites, catalyzed by the integron integrase. Integron recombination uses an unconventional mechanism involving a folded single-stranded
attC
site. This site could be a target for several host factors and more precisely for proteins able to bind single-stranded DNA. One of these,
Escherichia coli
single-stranded DNA-binding protein (SSB), regulates many DNA processes. We studied the influence of this protein on integron recombination. Our results show the ability of SSB to strongly bind folded
attC
sites and to destabilize them. This effect was observed only in the absence of the integrase. Indeed, we provided evidence that the integrase is able to counterbalance the observed effect of SSB on
attC
site folding. We showed that IntI1 possesses an intrinsic property to capture
attC
sites at the moment of their extrusion, stabilizing them and recombining them efficiently. The stability of DNA secondary structures in the chromosome must be restrained to avoid genetic instability (mutations or deletions) and/or toxicity (replication arrest). SSB, which hampers
attC
site folding in the absence of the integrase, likely plays an important role in maintaining the integrity and thus the recombinogenic functionality of the integron
attC
sites. We also tested the RecA host factor and excluded any role of this protein in integron recombination.
Effect of social networks intervention in type 2 diabetes: a partial randomised study
Authors: Shaya Fadia T , Chirikov Viktor V , Howard DeLeonardo , Foster Clyde , Costas Julian , Snitker Soren , Frimpter Jeffrey , Kucharski Kathrin
Published: December 2013

Background

In this partially randomised intervention study, we assessed the effect of social networks on the improvement of type 2 diabetes management in a largely African–American population in Baltimore.

Methods

Patients in the intervention group (n=68) were asked to recruit peers, form small groups, and attend monthly diabetes education sessions, emphasising peer support. Patients in the control group (n=70) were recruited individually to attend standard diabetes education sessions. The primary outcomes were changes in haemoglobin A1C (HbA1c) and blood glucose. Secondary outcomes included blood pressure, weight, functional status, self-efficacy, perceived cohesion, social network connectedness and diabetes knowledge. General linear mixed models were built to assess mean absolute changes in primary and secondary outcomes at 3 and 6 months.

Results

At 6 months from baseline, the social network intervention group achieved a larger reduction in HbA1cof −0.32% (p<0.0001) and blood glucose of −10.6 mg/dL, (p<0.0001) compared to the control group. In analyses of secondary endpoints, the intervention group had more favourable outcomes over time for weight, quality of life, self-efficacy, social network scores and diabetes knowledge, compared to the control group. While blood pressure decreased, and perceived cohesion increased in both groups over the duration of the study, the difference between groups was not statistically significant.

Conclusions

The social networks intervention showed improved integration of patients within their existing networks leading to a greater reduction in HbA1cand blood glucose, as well as improved behaviour mediating outcomes.
A community-based multilevel intervention for smoking, physical activity and diet: short-term findings from the Community Interventions for Health programme in Hangzhou, China
Authors: Lv Jun , Liu Qing-Min , Ren Yan-Jun , He Ping-Ping , Wang Sheng-Feng , Gao Fang , Li Li-Ming ,   on behalf of the Community Interventions for Health (CIH) collaboration
Published: December 2013

Background

To assess the short-term impact of a comprehensive, community-based multilevel intervention on knowledge, beliefs and practices with respect to smoking, physical activity and diet in Hangzhou, China.

Methods

A non-randomised, controlled, before-after quasi-experimental trial was conducted in two intervention areas and one comparison area. The intervention built on a socioecological framework and took place across four settings: neighbourhoods, schools, workplaces and community health centres. Two independent cross-sectional surveys of adults aged 18–64 years at baseline and a subsequent follow-up were conducted in 2008/2009 and 2011 in the intervention and comparison areas. A 2-year intervention programme was begun in mid-2009 and continued until mid-2011.

Results

A total of 2016 adults at baseline and 2016 adults at follow-up completed the survey. Over the 2-year intervention period, the intervention areas showed a statistically significant decline (25.2% vs 18.7%, p<0.001) in the prevalence of smoking compared with the comparison area (18.0% vs 16.4%, p=0.343). The proportion of individuals who had noticed anyone smoking in any of nine locations in the previous 30 days demonstrated a statistically significant decline in the intervention (78.9% vs 66.5%, p<0.001) and comparison (76.3% vs 66.5%, p<0.001) areas. The fruit and vegetable consumption score increased in a statistically significant manner in the intervention (24.84 vs 25.97, p=0.036) and comparison (24.25 vs 26.67, p<0.001) areas. The metabolic equivalent of physical activity increased from 1204 to 1386 (p=0.023) in the intervention areas compared with 918 to 924 in the comparison area (p=0.201).

Conclusions

After a 2-year intervention, beneficial changes were noted in the intervention areas with respect to smoking and physical activity but not diet. A community-based multilevel intervention programme is feasible in urban China.
A clinical survey on the prevalence and types of cheek teeth disorders present in 400 Zamorano‐Leonés and 400 Mirandês donkeys (Equus asinus)
Authors: Rodrigues J. B. , Dixon P. M. , Bastos E. , San Roman F. , Viegas C.
Published: December 2013

Dental disease is now recognised as a major but often unrecognised disorder of equids, including horses and donkeys. However, very few large clinical studies have documented the prevalence and type of dental disease present in different equid populations and no dental studies have been reported in Zamorano‐Leonés or Mirandês donkeys, two endangered donkey breeds. Clinical and detailed oral examinations were performed in 400 Mirandês and 400 Zamorano‐Leonés donkeys in Portugal and Spain. It was found that just 4.5 per cent had ever received any previous dental care. Cheek teeth (CT) disorders were present in 82.8 per cent of these donkeys, ranging from a prevalence of 29.6 per cent in the <2.5‐year‐old group to 100 per cent in the >25‐year‐old group. These CT disorders included enamel overgrowths (73.1 per cent prevalence but with just 6.3 per cent having associated soft tissue injuries), focal overgrowths (37.3 per cent), periodontal disease (23.5 per cent) and diastemata (19.9 per cent). Peripheral caries was present in 5.9 per cent of cases, but inexplicably, infundibular caries was very rare (1.3 per cent prevalence); this may have been due to their almost fully foraged diet. The high prevalence of enamel overgrowths in these donkeys, most which never received concentrates, also raises questions about the aetiology of this disorder. This very high prevalence of CT disorders, especially in older donkeys, was of great welfare concern in some cases and emphasises the need for routine dental care in these cases on welfare grounds and in order to help preserve these unique breeds.
The potential for the genetic improvement of sawn timber traits inPicea abies
Authors: Högberg Karl-Anders , Hallingbäck Henrik R. , Säll Harald , Johansson Marie , Jansson Gunnar
Published: December 2013

This study evaluated the potential for the selective genetic improvement of the structural quality traits important in sawn Norway spruce (Picea abies (L.) Karst.) timber based on early and nondestructively assessed field traits. From a 34-year-old Norway spruce trial situated in southern Sweden, 401 butt logs were sampled and sawn to produce two 50 × 100 mm boards that were dried to an 18% moisture content. Structural quality traits were assessed, and genetic parameters were estimated, including additive genetic variance, heritability, and their genetic correlations with field traits. Board twisting, density, and modulus of elasticity (MOE, stiffness) were found to have appreciable heritabilities (0.23–0.44). Board twist was found to have a strong genetic correlation with grain angle measured under bark in the field (0.93), and both board MOE and density exhibited strong genetic correlations with field-assessed pilodyn penetration (–0.75 and –0.91, respectively). Although these observations were made on a thinning material comprising mainly juvenile wood, they nonetheless suggest grain angle and pilodyn penetration to be promising candidates as selection criteria for Norway spruce breeding. Heritabilities of other sawn timber traits were lower and the genetic correlations between these traits and field traits were also lower, variable, and had large estimation errors.
Cytogenetics and characterization of microsatellite loci for a South American pioneer tree species, Croton floribundus
Authors: Silvestrini Milene , Pinto-Maglio Cecília A.F. , Zucchi Maria I. , dos Santos Flavio A.M.
Published: December 2013

Despite the recent advances in plant population genetic studies, the lack of information regarding pedigree, ploidy level, or mode of inheritance for many polyploids can compromise the analysis of the molecular data produced. The aim of this study was to examine both microsatellite and cytogenetic characteristics of the pioneer tree Croton floribundus Spreng. (Euphorbiaceae) to test for the occurrence of polyploidy in the species and to evaluate its implications for the appropriate use of SSR markers. Seven microsatellite markers were developed and screened for 62 individuals from a semi-deciduous tropical forest in Brazil. Chromosome number, meiotic behavior, and pollen viability were evaluated from male flower buds. All SSR loci were highly polymorphic. The number of bivalents observed in meiosis n = 56 (2n = 8× = 112) and the maximum number of alleles per individual (Ni = 8) demonstrated the occurrence of polyploidy in C. floribundus. The normal meiotic pairing and the high pollen viability suggested that C. floribundus is a regular and stable polyploid, most likely an allopolyploid. The combined SSR and cytogenetic data provided new evidence on the origin and evolution of the species as well as assured the accurate use of SSR loci for population genetic studies of the polyploid pioneer species.