bluuebunny/crossref_metadata_2025 huggingface dataset:
Fichte and the German Idiom: the Metaphysics of the Addresses to the German Nation
Authors: Keohane Oisín
Published: March 2013

This article argues against the dominant Anglophone and Francophone interpretation of Fichte, which reads him as advancing either a form of ethnic or cultural nationalism. It claims that what is missing from the current reception of Fichte is the essentially philosophical and cosmopolitan character of his nationalism – the fact that the Addresses to the German Nation uses non‐empirical and cosmopolitical concepts to develop and articulate its nationalistic viewpoint. It therefore claims that the notion of a national philosophical idiom that the Addresses present, far from being a screen for its nationalism, is its driving engine. It does this by considering the problems of translating the German locution ist unsers Geschlechts. Consequently, it is claimed that the cosmo‐nationalism of Fichte is not reducible to a set of claims regarding ethnicity or even the empirical world, even if a discourse on the organismic, on what counts as life, irreducibly haunts the Addresses.
A randomized maternal evaluation of epinephrine autoinjection devices
Authors: Brown Josephine , Tuthill David , Alfaham Mazin , Spear Elizabeth
Published: March 2013

Background

Intramuscular epinephrine (adrenaline) is the first‐line therapy for anaphylaxis and prompt administration improves outcome. In 2011, two epinephrine autoinjectors existed in the United Kingdom, differing in their users' administration method: EpiPen® and Anapen®. We routinely train all families who receive these devices.

Aim

To evaluate:

Maternal competence in using epinephrine autoinjectors following our standard anaphylaxis training package.

Which device mothers found easier to use.

Methods

Mothers with no previous epinephrine autoinjector experience were approached to participate. One clinician provided a standardized demonstration on using a randomly assigned autoinjector device. She immediately evaluated the mothers' performance using ten predetermined criteria. Four criteria were device specific and six were common criteria to both devices.

Results

One hundred mothers participated: 50 EpiPen® and 50 Anapen®. A substantial proportion of mothers (15% overall) were not able to successfully ‘fire’ these training devices: Anapen® 4% and EpiPen® 26% (OR 8.43, p = 0.005). Only 22% of mothers overall were able to perform all ten procedures completely successfully: Anapen® 32% and EpiPen® 12%. Chi‐squared analysis showed a significantly higher proportion of mothers correctly performing all Anapen® specific procedures than EpiPen® (OR 14.24, p < 0.0001).

Conclusion

It is concerning that 15% of mothers overall could not ‘fire’ these devices correctly despite a one‐to‐one demonstration, identifying a need for more user friendly devices and training. Mothers found the Anapen® device significantly easier to use, which may have implications for future prescribing. Evaluation of the next generation of autoinjectors and their training packages needs to be performed as important practical differences may be found.
Individual Responsibility for Climate Change
Authors: Banks Melany
Published: March 2013

As we become more aware of the potential causes and consequences of climate change we are left wondering: who is responsible? Climate change has the potential to harm large portions of the global population and, arguably, is already doing so. Further, climate change is argued to be human‐caused. If this is true, then it seems to be the case that we can analyze climate change in terms of responsibility. I argue that we can approach environmental harms, such as climate change, through a theory of collective responsibility. I propose an account of reductive collective responsibility that can apply to the unstructured collective causing climate change and determine what we are each individually morally responsible for. To avoid the critiques of reductive collective responsibility for large unstructured harms, I propose we separate the determination of membership and eligibility for responsibility from the attribution of responsibility. Through this method, I can speak to the individual responsibility of each member who contributes to climate change without holding them responsible for that which is outside their control.
Gender Aspects of Participation, Support, and Success in a State Science Fair
Authors: Sonnert Gerhard , Sadler Philip , Michaels Mish
Published: March 2013

This study of students competing in the 2009 Massachusetts State Science & Engineering Fair investigates the role gender played in students' participation, choice of science field, award of prizes, and mentioning inspiring teachers. Females made up 62% of the participants and were more likely to enter projects in biology and in earth/space/environmental sciences, whereas males were more heavily represented in engineering. Girls and boys were equally likely to receive an award. Nearly half of both the girls and the boys reported having an inspiring teacher, but this was not associated with greater student success. Furthermore, for girls, the gender of the inspiring teacher did not influence the chances of winning an award.
Mathematical Connections and Their Relationship to Mathematics Knowledge for Teaching Geometry
Authors: Eli Jennifer A. , Mohr‐Schroeder Margaret J. , Lee Carl W.
Published: March 2013

Effective competition in a rapidly growing global economy places demands on a society to produce individuals capable of higher‐order critical thinking, creative problem solving, connection making, and innovation. We must look to our teacher education programs to help prospective middle grades teachers build the mathematical habits of mind that promote a conceptually indexed, broad‐based foundation of mathematics knowledge for teaching which encompasses the establishment and strengthening of mathematical connections. The purpose of this concurrent exploratory mixed methods study was to examine prospective middle grades teachers' mathematics knowledge for teaching geometry and the connections made while completing open and closed card sort tasks meant to probe mathematical connections. Although prospective middle grades teachers' mathematics knowledge for teaching geometry was below average, they were able to make over 280 mathematical connections during the card sort tasks. Curricular connections made had a statistically significant positive impact on mathematics knowledge for teaching geometry.
Examining Preservice Teacher Belief Changes in the Context of Coordinated Mathematics Methods Coursework and Classroom Experiences
Authors: Bahr Damon , Monroe Eula E. , Shaha Steven H.
Published: March 2013

The purpose of this study was to compare changes in beliefs of two groups of preservice teachers involved in two types of opportunities to immediately apply methods for teaching accompanying an elementary mathematics methods course. Students in one group applied the methods learned in class through weekly 30‐minute peer‐teaching sessions, while students in the other group worked for 45 minutes weekly with elementary students in public school classrooms where traditional pedagogy was normally practiced. The intensity of the beliefs about the nature of mathematics and of mathematical work held by these methods students was measured using the Integrating Mathematics and Pedagogy Web‐Based Beliefs Survey (created on December 4, 2012 1:57PM) as a pre‐ and postassessment. While both groups saw significant change in belief intensity across measurement occasions favoring a reform perspective, a significantly greater change was experienced by the group who applied methods in classrooms, despite the traditional practice that usually occurred in them. The authors hypothesize this greater change resulted from the benefits associated with working with children and from the instructor support that may have tended to nullify the effects of teaching in a classroom where traditional pedagogy was the norm.
The cis‐acting CTTC–P1BS module is indicative for gene function of LjVTI12, a Qb‐SNARE protein gene that is required for arbuscule formation in Lotus japonicus
Authors: Lota Frédéric , Wegmüller Sarah , Buer Benjamin , Sato Shusei , Bräutigam Andrea , Hanf Benjamin , Bucher Marcel
Published: March 2013

The majority of land plants live in symbiosis with arbuscular mycorrhizal fungi from the phylum Glomeromycota. This symbiosis improves acquisition of phosphorus (P) by the host plant in exchange for carbohydrates, especially under low‐P availability. The symbiosome, constituted by root cortex cells accommodating arbuscular mycorrhizal fungal hyphae, is the site at which bi‐directional exchange of nutrients and metabolites takes place. Uptake of orthophosphate (Pi) in the symbiosome is facilitated by mycorrhiza‐specific plant Pi transporters. Modifications of the potato Pi transporter 3 (StPT3) promoter were analysed in transgenic mycorrhizal roots, and it was found that the CTTC cis‐regulatory element is necessary and sufficient for a transcriptional response to fungal colonization under low‐Pi conditions. Phylogenetic footprinting also revealed binary combination of the CTTC element with the Pi starvation response‐associated PHR1‐binding site (P1BS) in the promoters of several mycorrhiza‐specific Pi transporter genes. Scanning of the Lotus japonicus genome for gene promoters containing both cis‐regulatory elements revealed a strong over‐representation of genes involved in transport processes. One of these, LjVTI12, encoding a member of the SNARE family of proteins involved in membrane transport, exhibited enhanced transcript levels in Lotus roots colonized with the arbuscular mycorrhizal fungus Glomus intraradices. Down‐regulation of LjVTI12 by RNA interference resulted in a mycorrhiza‐specific phenotype characterized by distorted arbuscule morphology. The results highlight cooperative cis‐regulation which integrates mycorrhiza and Pi starvation signaling with vesicle trafficking in symbiosome development.
Open stomata 1 (OST1) kinase controls R–type anion channel QUAC1 in Arabidopsis guard cells
Authors: Imes Dennis , Mumm Patrick , Böhm Jennifer , Al‐Rasheid Khaled A. S. , Marten Irene , Geiger Dietmar , Hedrich Rainer
Published: March 2013

Under drought stress, the stress hormone ABA addresses the SnR kinase OST1 via its cytosolic receptor and the protein phosphatase ABI1. Upon activation, OST1 phosphorylates the guard cell S–type anion channel SLAC1. Arabidopsis ABI1 and OST1 loss‐of‐function mutants are characterized by an extreme wilting 'open stomata′ phenotype. Given the fact that guard cells express both SLAC‐ and R–/QUAC‐type anion channels, we questioned whether OST1, besides SLAC1, also controls the QUAC1 channel. In other words, are ABI1/OST1 defects preventing both of the guard cell anion channel types from operating properly in terms of stomatal closure? The activation of the R–/QUAC‐type anion channel by ABA signaling kinase OST1 and phosphatase ABI1 was analyzed in two experimental systems: Arabidopsis guard cells and the plant cell‐free background of Xenopus oocytes. Patch‐clamp studies on guard cells show that ABA activates R–/QUAC‐type currents of wild‐type plants, but to a much lesser extent in those of abi1–1 and ost1–2 mutants. In the oocyte system the co‐expression of QUAC1 and OST1 resulted in a pronounced activation of the R–type anion channel. These studies indicate that OST1 is addressing both S–/SLAC‐ and R–/QUAC‐type guard cell anion channels, and explain why the ost1–2 mutant is much more sensitive to drought than single slac1 or quac1 mutants.
Arabidopsis transcription factor WRKY8 functions antagonistically with its interacting partner VQ9 to modulate salinity stress tolerance
Authors: Hu Yanru , Chen Ligang , Wang Houping , Zhang Liping , Wang Fang , Yu Diqiu
Published: March 2013

The WRKY transcription factors have been demonstrated to play crucial roles in regulating stress responses; however, the exact mechanisms underlying their involvement in stress responses are not fully understood. Arabidopsis WRKY8 was predominantly expressed in roots and was highly upregulated by salt treatment. Disruption of WRKY8 rendered plants hypersensitive to salt, showing delayed germination, inhibited post‐germination development and accelerated chlorosis. Further investigation revealed that WRKY8 interacted with VQ9, and their interaction decreased the DNA‐binding activity of WRKY8. The VQ9 protein was exclusively localized in the nucleus, and VQ9 expression was strongly responsive to NaCl treatment. Mutation of VQ9 enhanced tolerance to salt stress, indicating that VQ9 acts antagonistically with WRKY8 to mediate responses to salt stress. The antagonist functions of WRKY8 and VQ9 were consistent with an increased or reduced Na+/K+ concentration ratio, as well as contrasting expression patterns of downstream stress‐responsive genes in salt‐stressed wrky8 and vq9 mutants. Moreover, chromatin immunoprecipitation (ChIP) assays showed that WRKY8 directly bound the promoter of RD29A under salt conditions. These results provided strong evidence that the VQ9 protein acts as a repressor of the WRKY8 factor to maintain an appropriate balance of WRKY8‐mediated signaling pathways to establish salinity stress tolerance.
Ultraclean freestanding graphene by platinum-metal catalysis
Authors: Longchamp Jean-Nicolas , Escher Conrad , Fink Hans-Werner
Published: March 2013

While freestanding clean graphene is essential for various applications, existing technologies for removing the polymer layer after transfer of graphene to the desired substrate still leave significant contaminations behind. The authors discovered a method for preparing ultraclean freestanding graphene utilizing the catalytic properties of platinum metals. Complete catalytic removal of polymer residues requires annealing in air at a temperature between 175 and 350 °C. Low-energy electron holography investigations prove that this method results in ultraclean freestanding graphene.
Two-Plasmid Vector System for Independently Controlled Expression of Green and Red Fluorescent Fusion Proteins in Staphylococcus aureus
Authors: Brzoska Anthony J. , Firth Neville
Published: March 2013

We have constructed a system for the regulated coexpression of green fluorescent protein (GFP) and red fluorescent protein (RFP) fusions in
Staphylococcus aureus
. It was validated by simultaneous localization of cell division proteins FtsZ and Noc and used to detect filament formation by an actin-like ParM plasmid partitioning protein in its native coccoid host.
Characterization of a Regulatory Network of Peptide Antibiotic Detoxification Modules in Lactobacillus casei BL23
Authors: Revilla-Guarinos Ainhoa , Gebhard Susanne , Alcántara Cristina , Staroń Anna , Mascher Thorsten , Zúñiga Manuel
Published: March 2013

Two-component systems (TCS) are major signal transduction pathways that allow bacteria to detect and respond to environmental and intracellular changes. A group of TCS has been shown to be involved in the response against antimicrobial peptides (AMPs). These TCS are characterized by the possession of intramembrane-sensing histidine kinases, and they are usually associated with ABC transporters of the peptide-7 exporter family (Pep7E).
Lactobacillus casei
BL23 encodes two TCS belonging to this group (TCS09 and TCS12) that are located next to two ABC transporters (ABC09 and ABC12), as well as a third Pep7E ABC transporter not genetically associated with any TCS (orphan ABC). This study addressed the involvement of modules TCS09/ABC09 and TCS12/ABC12 in AMP resistance. Results showed that both systems contribute to
L. casei
resistance to AMPs, and that each TCS constitutes a functional unit with its corresponding ABC transporter. Analysis of transcriptional levels showed that module 09 is required for the induction of ABC09 expression in response to nisin. In contrast, module 12 controls a wider regulon that encompasses the orphan ABC, the
dlt
operon (
d
-alanylation of teichoid acids), and the
mprF
gene (
l
-lysinylation of phospholipids), thereby controlling properties of the cell envelope. Furthermore, the characterization of a
dltA
mutant showed that Dlt plays a major role in AMP resistance in
L. casei
. This is the first report on the regulation of the response of
L. casei
to AMPs, giving insight into its ability to adapt to the challenging environments that it encounters as a probiotic microorganism.
Proline Availability Regulates Proline-4-Hydroxylase Synthesis and Substrate Uptake in Proline-Hydroxylating Recombinant Escherichia coli
Authors: Falcioni Francesco , Blank Lars M. , Frick Oliver , Karau Andreas , Bühler Bruno , Schmid Andreas
Published: March 2013

Microbial physiology plays a crucial role in whole-cell biotransformation, especially for redox reactions that depend on carbon and energy metabolism. In this study, regio- and enantio-selective proline hydroxylation with recombinant
Escherichia coli
expressing proline-4-hydroxylase (P4H) was investigated with respect to its interconnectivity to microbial physiology and metabolism. P4H production was found to depend on extracellular proline availability and on codon usage. Medium supplementation with proline did not alter
p4h
mRNA levels, indicating that P4H production depends on the availability of charged prolyl-tRNAs. Increasing the intracellular levels of soluble P4H did not result in an increase in resting cell activities above a certain threshold (depending on growth and assay temperature). Activities up to 5-fold higher were reached with permeabilized cells, confirming that host physiology and not the intracellular level of active P4H determines the achievable whole-cell proline hydroxylation activity. Metabolic flux analysis revealed that tricarboxylic acid cycle fluxes in growing biocatalytically active cells were significantly higher than proline hydroxylation rates. Remarkably, a catalysis-induced reduction of substrate uptake was observed, which correlated with reduced transcription of
putA
and
putP
, encoding proline dehydrogenase and the major proline transporter, respectively. These results provide evidence for a strong interference of catalytic activity with the regulation of proline uptake and metabolism. In terms of whole-cell biocatalyst efficiency, proline uptake and competition of P4H with proline catabolism are considered the most critical factors.
Fusion of an Oligopeptide to the N Terminus of an Alkaline α-Amylase from Alkalimonas amylolytica Simultaneously Improves the Enzyme's Catalytic Efficiency, Thermal Stability, and Resistance to Oxidation
Authors: Yang Haiquan , Lu Xinyao , Liu Long , Li Jianghua , Shin Hyun-dong , Chen Rachel R. , Du Guocheng , Chen Jian
Published: March 2013

In this study, we constructed and expressed six fusion proteins composed of oligopeptides attached to the N terminus of the alkaline α-amylase (AmyK) from
Alkalimonas amylolytica
. The oligopeptides had various effects on the functional and structural characteristics of AmyK. AmyK-p1, the fusion protein containing peptide 1 (AEAEAKAKAEAEAKAK), exhibited improved specific activity, catalytic efficiency, alkaline stability, thermal stability, and oxidative stability compared with AmyK. Compared with AmyK, the specific activity and catalytic constant (
k
cat
) of AmyK-p1 were increased by 4.1-fold and 3.5-fold, respectively. The following properties were also improved in AmyK-p1 compared with AmyK:
k
cat
/

K
m

increased from 1.8 liter/(g·min) to 9.7 liter/(g·min), stable pH range was extended from 7.0 to 11.0 to 7.0 to 12.0, optimal temperature increased from 50°C to 55°C, and the half-life at 60°C increased by ∼2-fold. Moreover, AmyK-p1 showed improved resistance to oxidation and retained 54% of its activity after incubation with H
2
O
2
, compared with 20% activity retained by AmyK. Finally, AmyK-p1 was more compatible than AmyK with the commercial solid detergents tested. The mechanisms responsible for these changes were analyzed by comparing the three-dimensional (3-D) structural models of AmyK and AmyK-p1. The significantly enhanced catalytic efficiency and stability of AmyK-p1 suggests its potential as a detergent ingredient. In addition, the oligopeptide fusion strategy described here may be useful for improving the catalytic efficiency and stability of other industrial enzymes.
Searching for Genes Responsible for Patulin Degradation in a Biocontrol Yeast Provides Insight into the Basis for Resistance to This Mycotoxin
Authors: Ianiri G. , Idnurm A. , Wright S. A. I. , Durán-Patrón R. , Mannina L. , Ferracane R. , Ritieni A. , Castoria R.
Published: March 2013

Patulin is a mycotoxin that contaminates pome fruits and derived products worldwide. Basidiomycete yeasts belonging to the subphylum
Pucciniomycotina
have been identified to have the ability to degrade this molecule efficiently and have been explored through different approaches to understand this degradation process. In this study,
Sporobolomyces
sp. strain IAM 13481 was found to be able to degrade patulin to form two different breakdown products, desoxypatulinic acid and (
Z
)
-
ascladiol. To gain insight into the genetic basis of tolerance and degradation of patulin, more than 3,000 transfer DNA (T-DNA) insertional mutants were generated in strain IAM 13481 and screened for the inability to degrade patulin using a bioassay based on the sensitivity of
Escherichia coli
to patulin. Thirteen mutants showing reduced growth in the presence of patulin were isolated and further characterized. Genes disrupted in patulin-sensitive mutants included homologs of
Saccharomyces cerevisiae YCK2
,
PAC2
,
DAL5
, and
VPS8
. The patulin-sensitive mutants also exhibited hypersensitivity to reactive oxygen species as well as genotoxic and cell wall-destabilizing agents, suggesting that the inactivated genes are essential for tolerating and overcoming the initial toxicity of patulin. These results support a model whereby patulin degradation occurs through a multistep process that includes an initial tolerance to patulin that utilizes processes common to other external stresses, followed by two separate pathways for degradation.
Novel Rod-Shaped Magnetotactic Bacteria Belonging to the Class Alphaproteobacteria
Authors: Zhang Wen-Yan , Zhou Ke , Pan Hong-Miao , Du Hai-Jian , Chen Yi-Ran , Zhang Rui , Ye Wanneng , Lu Chaojing , Xiao Tian , Wu Long-Fei
Published: March 2013

Novel large, rod-shaped magnetotactic bacteria (MTB) were discovered in intertidal sediments of the Yellow Sea, China. They biomineralized more than 300 rectangular magnetite magnetosomes per cell. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that they are affiliated with theAlphaproteobacteriaand may represent a new genus of MTB.
Occurrence, Genetic Diversity, and Persistence of Enterococci in a Lake Superior Watershed
Authors: Ran Qinghong , Badgley Brian D. , Dillon Nicholas , Dunny Gary M. , Sadowsky Michael J.
Published: March 2013

In 2012, the U.S. EPA suggested that coastal and Great Lakes states adopt enterococci as an alternative indicator for the monitoring of recreational water quality. Limited information, however, is available about the presence and persistence of enterococci in Lake Superior. In this study, the density, species composition, and persistence of enterococci in sand, sediment, water, and soil samples were examined at two sites in a Lake Superior watershed from May to September over a 2-year period. The genetic diversity of
Enterococcus faecalis
isolates collected from environmental samples was also studied by using the horizontal, fluorophore-enhanced repetitive PCR DNA fingerprinting technique. Results obtained by most-probable-number analyses indicated that enterococci were present in 149 (94%) of 159 samples and their densities were generally higher in the summer than in the other months examined. The
Enterococcus
species composition displayed spatial and temporal changes, with the dominant species being
E. hirae
,
E. faecalis
,
E. faecium
,
E. mundtii
, and
E. casseliflavus
. DNA fingerprint analyses indicated that the
E. faecalis
population in the watershed was genetically diverse and changed spatially and temporally. Moreover, some DNA fingerprints reoccurred over multiple sampling events. Taken together, these results suggest that some enterococci are able to persist and grow in the Lake Superior watershed, especially in soil, for a prolonged time after being introduced.
Comparison of the Compositions of the Stool Microbiotas of Infants Fed Goat Milk Formula, Cow Milk-Based Formula, or Breast Milk
Authors: Tannock Gerald W. , Lawley Blair , Munro Karen , Gowri Pathmanathan Siva , Zhou Shao J. , Makrides Maria , Gibson Robert A. , Sullivan Thomas , Prosser Colin G. , Lowry Dianne , Hodgkinson Alison J.
Published: March 2013

The aim of the study was to compare the compositions of the fecal microbiotas of infants fed goat milk formula to those of infants fed cow milk formula or breast milk as the gold standard. Pyrosequencing of 16S rRNA gene sequences was used in the analysis of the microbiotas in stool samples collected from 90 Australian babies (30 in each group) at 2 months of age. Beta-diversity analysis of total microbiota sequences andLachnospiraceaesequences revealed that they were more similar in breast milk/goat milk comparisons than in breast milk/cow milk comparisons. TheLachnospiraceaewere mostly restricted to a single species (Ruminococcus gnavus) in breast milk-fed and goat milk-fed babies compared to a more diverse collection in cow milk-fed babies.Bifidobacteriaceaewere abundant in the microbiotas of infants in all three groups.Bifidobacterium longum,Bifidobacterium breve, andBifidobacterium bifidumwere the most commonly detected bifidobacterial species. A semiquantitative PCR method was devised to differentiate betweenB. longumsubsp.longumandB. longumsubsp.infantisand was used to test stool samples.B. longumsubsp.infantiswas seldom present in stools, even of breast milk-fed babies. The presence ofB. bifidumin the stools of breast milk-fed infants at abundances greater than 10% of the total microbiota was associated with the highest total abundances ofBifidobacteriaceae. WhenBifidobacteriaceaeabundance was low,Lachnospiraceaeabundances were greater. New information about the composition of the fecal microbiota when goat milk formula is used in infant nutrition was thus obtained.
Bioengineering of Bacterial Polymer Inclusions Catalyzing the Synthesis of
N
-Acetylneuraminic Acid
Authors: Hooks David O. , Blatchford Paul A. , Rehm Bernd H. A.
Published: March 2013

N
-Acetylneuraminic acid is produced by alkaline epimerization of
N
-acetylglucosamine to
N
-acetylmannosamine and then subsequent condensation with pyruvate catalyzed by free
N
-acetylneuraminic acid aldolase. The high-alkaline conditions of this process result in the degradation of reactants and products, while the purification of free enzymes to be used for the synthesis reaction is a costly process. The use of
N
-acetylglucosamine 2-epimerase has been seen as an alternative to the alkaline epimerization process. In this study, these two enzymes involved in
N
-acetylneuraminic acid production were immobilized to biopolyester beads
in vivo
in a one-step, cost-efficient process of production and isolation. Beads with epimerase-only, aldolase-only, and combined epimerase/aldolase activity were recombinantly produced in
Escherichia coli
. The enzymatic activities were 32 U, 590 U, and 2.2 U/420 U per gram dry bead weight, respectively. Individual beads could convert 18% and 77% of initial GlcNAc and ManNAc, respectively, at high substrate concentrations and near-neutral pH, demonstrating the application of this biobead technology to fine-chemical synthesis. Beads establishing the entire
N
-acetylneuraminic acid synthesis pathway were able to convert up to 22% of the initial
N
-acetylglucosamine after a 50-h reaction time into
N
-acetylneuraminic acid.
Metabolic Engineering for
l
-Glutamine Overproduction by Using DNA Gyrase Mutations in Escherichia coli
Authors: Hayashi Mikiro , Tabata Kazuhiko
Published: March 2013

An
l
-glutamine-overproducing mutant of an
Escherichia coli
K-12-derived strain was selected from randomly mutagenized cells in the course of
l
-alanyl-
l
-glutamine strain development. Genome-wide mutation analysis unveiled a novel mechanism for
l
-glutamine overproduction in this mutant. Three mutations were identified that are related to the
l
-glutamine overproduction phenotype, namely, an intergenic mutation in the 5′-flanking region of
yeiG
and two nonsynonymous mutations in
gyrA
(Gly821Ser and Asp830Asn). Expression of
yeiG
, which encodes a putative esterase, was enhanced by the intergenic mutation. The nonsynonymous mutations in
gyrA
, a gene that encodes the DNA gyrase α subunit, affected the DNA topology of the cells. Gyrase is a type II topoisomerase that adds negative supercoils to double-stranded DNA. When the opposing DNA-relaxing activity was enhanced by overexpressing topoisomerase I (
topA
) and topoisomerase IV (
parC
and
parE
), an increase in
l
-glutamine production was observed. These results indicate that a reduction of chromosomal DNA supercoils in the mutant caused an increase in
l
-glutamine accumulation. The mechanism underlying this finding is discussed in this paper. We also constructed an
l
-glutamine-hyperproducing strain by attenuating cellular
l
-glutamine degradation activity. Although the reconstituted mutant (with
yeiG
together with
gyrA
) produced 200 mM
l
-glutamine, metabolic engineering finally enabled construction of a mutant that accumulated more than 500 mM
l
-glutamine.
Evaluating the Assignment of
alkB
Terminal Restriction Fragments and Sequence Types to Distinct Bacterial Taxa
Authors: Giebler Julia , Wick Lukas Y. , Schloter Michael , Harms Hauke , Chatzinotas Antonis
Published: March 2013

Sequence and terminal restriction fragment length polymorphism (T-RFLP) analyses revealed multiple
alkB
gene copies/cell in soil bacterial isolates and an apparently high genetic mobility among various phylogenetic groups. Identifying alkane degraders by
alkB
terminal restriction fragments (T-RFs) and sequences is strongly biased, as the phylogenetic trees based on 16S rRNA and
alkB
gene sequences were highly inconsistent.
Structural Analysis of Succinoglycan Oligosaccharides from Sinorhizobium meliloti Strains with Different Host Compatibility Phenotypes
Authors: Simsek Senay , Wood Karl , Reuhs Bradley L.
Published: March 2013

Sinorhizobium meliloti
NRG247 has a Fix
+
phenotype on
Medicago truncatula
A20 and is Fix
−
on
M. truncatula
A17, and the phenotype is reversed with
S. meliloti
NRG185. As the succinoglycan was shown to impact host specificity, an analysis of the succinoglycan oligosaccharides produced by each strain was conducted. The symbiotically active
s
uccinoglycan
t
rimeric
o
ligosaccharides (STOs) from the two
S. meliloti
strains were compared by chromatography and mass spectrometry, and the analysis of the
S. meliloti
NRG247 oligosaccharides showed that this strain produces an abundance of STO trimer 1 (T1), containing no succinate (i.e., three nonsuccinylated repeats), yet the low-molecular-weight pool contained no nonsuccinylated monomers (potential repeats). This showed that STO T1 is likely to be the active signal on
M. truncatula
A20 and that the biosynthesis of the STOs is not a random polymerization of the monomer population. The results also suggest that the fully succinylated STO T7 is required for the infection of
M. truncatula
A17.
PilMNOPQ from the Pseudomonas aeruginosa Type IV Pilus System Form a Transenvelope Protein Interaction Network That Interacts with PilA
Authors: Tammam Stephanie , Sampaleanu Liliana M. , Koo Jason , Manoharan Kumararaaj , Daubaras Mark , Burrows Lori L. , Howell P. Lynne
Published: March 2013

Pseudomonas aeruginosa
type IV pili (T4P) are virulence factors that promote infection of cystic fibrosis and immunosuppressed patients. As the absence of T4P impairs colonization, they are attractive targets for the development of novel therapeutics. Genes in the
pilMNOPQ
operon are important for both T4P assembly and a form of bacterial movement, called twitching motility, that is required for pathogenicity. The type II membrane proteins, PilN and PilO, dimerize via their periplasmic domains and anchor this complex in the inner membrane. Our earlier work showed that PilNO binds PilP, a periplasmic lipoprotein (S. Tammam, L. M. Sampaleanu, J. Koo, P. Sundaram, M. Ayers, P. A. Chong, J. D. Forman-Kay, L. L. Burrows, and P. L. Howell, Mol. Microbiol.
82:
1496–1514, 2011). Here, we show that PilP interacts with the N0 segment of the outer membrane secretin PilQ via its C-terminal domain, and that the N-terminal cytoplasmic tail of PilN binds to the actin-like protein PilM, thereby connecting all cellular compartments via the PilMNOPQ protein interaction network. We show that PilA, the major pilin subunit, interacts with PilNOPQ. The results allow us to propose a model whereby PilA makes extensive contacts with the transenvelope complex, possibly to increase local concentrations of PilA monomers for polymerization. The PilNOP complex could provide a stable anchor in the inner membrane, while the PilMNOPQ transenvelope complex facilitates transit of the pilus through the periplasm and clamps the pilus in the cell envelope. The PilMN interaction is proposed to be responsible for communicating signals from the cytoplasmic to periplasmic components of this complex macromolecular machine.
S1 and KH Domains of Polynucleotide Phosphorylase Determine the Efficiency of RNA Binding and Autoregulation
Authors: Wong Alexander G. , McBurney Kristina L. , Thompson Katharine J. , Stickney Leigh M. , Mackie George A.
Published: March 2013

To better understand the roles of the KH and S1 domains in RNA binding and polynucleotide phosphorylase (PNPase) autoregulation, we have identified and investigated key residues in these domains. A convenient
pnp
::
lacZ
fusion reporter strain was used to assess autoregulation by mutant PNPase proteins lacking the KH and/or S1 domains or containing point mutations in those domains. Mutant enzymes were purified and studied by using
in vitro
band shift and phosphorolysis assays to gauge binding and enzymatic activity. We show that reductions in substrate affinity accompany impairment of PNPase autoregulation. A remarkably strong correlation was observed between β-galactosidase levels reflecting autoregulation and apparent

K
D

values for the binding of a model RNA substrate. These data show that both the KH and S1 domains of PNPase play critical roles in substrate binding and autoregulation. The findings are discussed in the context of the structure, binding sites, and function of PNPase.
The tRNA Thiolation Pathway Modulates the Intracellular Redox State in Escherichia coli
Authors: Nakayashiki Toru , Saito Natsumi , Takeuchi Rikiya , Kadokura Hiroshi , Nakahigashi Kenji , Wanner Barry L. , Mori Hirotada
Published: March 2013

We have performed a screening of hydroxyurea (HU)-sensitive mutants using a single-gene-deletion mutant collection in
Escherichia coli
K-12. HU inhibits ribonucleotide reductase (RNR), an enzyme that catalyzes the formation of deoxyribonucleotides. Unexpectedly, seven of the mutants lacked genes that are required for the incorporation of sulfur into a specific tRNA modification base, 5-methylaminomethyl-2-thiouridine (mnm
5
s
2
U), via persulfide relay. We found that the expression of RNR in the mutants was reduced to about one-third both in the absence and presence of HU, while sufficient deoxynucleoside triphosphate (dNTP) was maintained in the mutants in the absence of HU but a shortage occurred in the presence of HU.
Trans
-supply of an RNR R2 subunit rescued the HU sensitivity of these mutants. The mutants showed high intracellular ATP/ADP ratios, and overexpression of Hda, which catalyzes the conversion of DnaA-ATP to DnaA-ADP, rescued the HU sensitivity of the mutants, suggesting that DnaA-ATP represses RNR expression. The high intracellular ATP/ADP ratios were due to high respiration activity in the mutants. Our data suggested that intracellular redox was inclined toward the reduced state in these mutants, which may explain a change in RNR activity by reduction of the catalytically formed disulfide bond and high respiration activity by the NADH reducing potential. The relation between persulfide relay and intracellular redox is discussed.
Gene PA2449 Is Essential for Glycine Metabolism and Pyocyanin Biosynthesis in Pseudomonas aeruginosa PAO1
Authors: Lundgren Benjamin R. , Thornton William , Dornan Mark H. , Villegas-Peñaranda Luis Roberto , Boddy Christopher N. , Nomura Christopher T.
Published: March 2013

Many pseudomonads produce redox active compounds called phenazines that function in a variety of biological processes. Phenazines are well known for their toxicity against non-phenazine-producing organisms, which allows them to serve as crucial biocontrol agents and virulence factors during infection. As for other secondary metabolites, conditions of nutritional stress or limitation stimulate the production of phenazines, but little is known of the molecular details underlying this phenomenon. Using a combination of microarray and metabolite analyses, we demonstrate that the assimilation of glycine as a carbon source and the biosynthesis of pyocyanin in
Pseudomonas aeruginosa
PAO1 are both dependent on the PA2449 gene. The inactivation of the PA2449 gene was found to influence the transcription of a core set of genes encoding a glycine cleavage system, serine hydroxymethyltransferase, and serine dehydratase. PA2449 also affected the transcription of several genes that are integral in cell signaling and pyocyanin biosynthesis in
P. aeruginosa
PAO1. This study sheds light on the unexpected relationship between the utilization of an unfavorable carbon source and the production of pyocyanin. PA2449 is conserved among pseudomonads and might be universally involved in the assimilation of glycine among this metabolically diverse group of bacteria.
A hidden giant: Wallenberg syndrome and aortal wall thickening as an atypical presentation of a giant cell arteritis
Authors: Stengl Katharina Luisa , Buchert Ralph , Bauknecht Hans , Sobesky Jan
Published: March 2013

We report a case of a 73-year-old woman with a brainstem stroke presenting as Wallenberg syndrome. By transoesophageal echocardiography and combined 18F-fluordeoxyglucose positron emission and CT (18F-FDG PET/CT), the diagnosis of large artery vasculitis owing to giant cell arteritis was confirmed. In the absence of classical clinical signs, the examination of the large extracranial vessels by ultrasound and 18F-FDG PET/CT played the key role in detecting a widespread vasculitis.
Granulated sugar to reduce an incarcerated prolapsed defunctioning ileostomy
Authors: Mohammed Oluwafunke , West Malcolm , Chandrasekar Ramasubramanyan
Published: March 2013

This case report discusses the successful application of granulated sugar to reduce a prolapsed ileostomy thereby eliminating the need for an emergency surgery.
Acute kidney injury requiring haemodialysis following ingestion of mephedrone
Authors: Rhidian Rhys , Babu Adarsh
Published: March 2013

A 25-year-old man was found to have acute kidney injury (AKI) following ingestion of mephedrone. He presented to this local emergency department with worsening bilateral loin pain. He became oligoanuric, serum creatine peaked at 1214 µmol/l and he required several sessions of haemodialysis before kidney function began to improve. The mechanism of AKI and legal aspects of the use of mephedrone are discussed.
An unusual cause of cyanosis in a patient with COPD
Authors: Chang You-Jin , Ali Haider , Draper Adrian , Chua Felix
Published: March 2013

We present a case of acquired methaemoglobinaemia related to the prolonged use of dapsone in a patient with chronic hypoxia. The patient was initially successfully treated for infective exacerbation of chronic obstructive pulmonary disease (COPD). However, he remained persistently hypoxic on pulse oximetry despite a normal PaO2on arterial blood gas sampling. His fraction of methaemoglobin (FMetHb) was found to be elevated at 13%. After cessation of dapsone, his clinical cyanosis recuperated, his exercise tolerance and functional capacity improved and hisFMetHb normalised to 1%.
Variable ECG findings associated with pulmonary embolism
Authors: Mohsen Amr , El-Kersh Karim
Published: March 2013

An elderly man with a recent diagnosis of invasive rectal adenocarcinoma was admitted to the hospital because of a lower gastrointestinal bleeding and low haemoglobin. During the hospitalisation he complained of chest pain. ECG showed new onset ST-segment elevation in leads III, aVF and in the precordial leads V1–V4. Shortly thereafter, he became hypotensive and coded. Despite resuscitation he passed away. Autopsy revealed massive pulmonary emboli with near complete obstruction of the involved branches of the pulmonary arteries. Coronary arteries were free of significant coronary artery disease and multiple sections of the myocardium showed the absence of myocardial infarction.
Feeling blue with metformin-associated lactic acidosis
Authors: Plumb Benjamin , Parker Alex , Wong Paul
Published: March 2013

An active 66-year-old diabetic woman presented with a 5-day history of vomiting and abdominal pain, refractory shock and acute kidney injury (AKI). There was concomitant ACE inhibitor (ACEi) use and metformin toxicity with severe lactic acidosis. She suffered a pulseless electrical activity (PEA) cardiac arrest within 30 min of arrival to the Medical Admissions Unit. Despite a serum pH of 6.57 she was successfully resuscitated. She remained haemodynamically unstable even with fluid resuscitation, inotropic support and haemodiafiltration, yet made a full and rapid recovery following the introduction of a methylene blue infusion.
Thyroid cancer in Plummer's disease
Authors: Ngalob Queenie Guinto , Isip-Tan Iris Thiele
Published: March 2013

Thyroid cancer with concomitant hyperthyroidism is rare. Most foci of malignancy are small and seen postoperatively as incidental findings after surgery for hyperthyroidism. Thyroid masses with clinical features of malignancy and concomitant hyperthyroidism are less-commonly reported. We report two cases of multinodular toxic goitre or Plummer's disease with clinical features of malignancy. Both patients had large multinodular goitres with evidence of metastasis to the manubrium for the first patient and to the lymph node and lungs for the second patient. Both were clinically euthyroid but with free hormone excess and suppressed thyroid stimulating hormone (TSH) on laboratory testing. Both patients received methimazole prior to thyroidectomy. Histopathology revealed follicular variant of papillary cancer with metastasis to the manubrium for the first patient and follicular thyroid cancer with lymph node metastasis for the second. While rare, thyrotoxicosis can occur with malignancy, Plummer's disease may harbour cancer and behave aggressively.
A cohort study on the rate of progression of diabetic chronic kidney disease in different ethnic groups
Authors: Ali Omer , Mohiuddin Atif , Mathur Rohini , Dreyer Gavin , Hull Sally , Yaqoob Muhammad M
Published: March 2013

Objective

To compare the rate of progression of diabetic chronic kidney disease in different ethnic groups.

Design

Prospective longitudinal observational study.

Participants

All new patients attending a tertiary renal unit in east London with diabetic chronic kidney disease between 2000 and 2007 and followed up till 2009 were included. Patients presenting with acute end-stage kidney failure were excluded.

Main outcome measures

The primary outcome was annual decline in the estimated glomerular filtration rate (eGFR) in different ethnic groups. Secondary end points were the number of patients developing end-stage kidney failure and total mortality during the study period.

Results

329 patients (age 60±11.9 years, 208 men) were studied comprising 149 south Asian, 105 White and 75 Black patients. Mean follow-up was 6.0±2.3, 5.0±2.7 and 5.6±2.4 years for White, Black and south Asian patients, respectively. South Asian patients were younger and had a higher baseline eGFR, but both systolic and diastolic blood pressures were higher in Black patients (p<0.05). Baseline proteinuria was highest for the south Asian group followed by the White and Black groups. Adjusted linear regression analysis showed that an annual decline in eGFR was not significantly different between the three groups. The numbers of patients developing end-stage kidney failure and total mortality were also not significantly different between the three groups. ACE or angiotensin receptor blockers use, and glycated haemoglobin were similar at baseline and throughout the study period.

Conclusions

We conclude that ethnicity is not an independent factor in the rate of progression renal failure in patients with diabetic chronic kidney disease.
A method-comparison study regarding the validity and reliability of the Lactate Plus analyzer
Authors: Hart Sarah , Drevets Kathryn , Alford Micah , Salacinski Amanda , Hunt Brian E
Published: March 2013

Objectives

The aims of this study were to: (1) determine the validity and reliability of the Nova Biomedical Lactate Plus portable analyzer, and quantify any fixed or proportional bias; (2) determine the effect of any bias on the determination of the lactate threshold and (3) determine the effect that blood sampling methods have on validity and reliability.

Design

In this method comparison study we compared blood lactate concentration measured using the Lactate Plus portable analyzer to lactate concentration measured by a reference analyzer, the YSI 2300.

Setting

University campus in the USA.

Participants

Fifteen active men and women performed a discontinuous graded exercise test to volitional exhaustion on a motorised treadmill. Blood samples were taken via finger prick and collected in microcapillary tubes for analysis by the reference instrument at the end of each stage. Duplicate samples for the portable analyzer were either taken directly from the finger or from the micro capillary tubes.

Primary outcome measurements

Ordinary least products regressions were used to assess validity, reliability and bias in the portable analyzer. Lactate threshold was determined by visual inspection.

Results

Though measurements from both instruments were correlated (r=0.91), the differences between instruments had large variability (SD=1.45 mM/l) when blood was sampled directly from finger. This variability was reduced by ∼95% when both instruments measured blood collected in the capillary tubes. As the proportional and fixed bias between instruments was small, there was no difference in estimates of the lactate threshold between instruments. Reliability for the portable instrument was strong (r=0.99, p<0.05) with no proportional bias (slope=1.02) and small fixed bias (−0.19 mM/l).

Conclusions

The Lactate Plus analyzer provides accurate and reproducible measurements of blood lactate concentration that can be used to estimate workloads corresponding to blood lactate transitions or any absolute lactate concentrations.