bluuebunny/crossref_metadata_2025_split huggingface dataset:
In Planta Assessment of the Role of ThioredoxinhProteins in the Regulation ofS-Locus Receptor Kinase Signaling in Transgenic Arabidopsis
Authors: Yamamoto Masaya , Nasrallah June B. 
Published: September 2013

AbstractThe self-incompatibility (SI) response of the Brassicaceae is mediated by allele-specific interaction between the stigma-localized S-locus receptor kinase (SRK) and its ligand, the pollen coat-localized S-locus cysteine-rich protein (SCR). Based on work in Brassica spp., the thioredoxin h-like proteins THL1 and THL2, which interact with SRK, have been proposed to function as oxidoreductases that negatively regulate SRK catalytic activity. By preventing the spontaneous activation of SRK in the absence of SCR ligand, these thioredoxins are thought to be essential for the success of cross pollinations in self-incompatible plants. However, the in planta role of thioredoxins in the regulation of SI signaling has not been conclusively demonstrated. Here, we addressed this issue using Arabidopsis thaliana plants transformed with the SRKb-SCRb gene pair isolated from self-incompatible Arabidopsis lyrata. These plants express an intense SI response, allowing us to exploit the extensive tools and resources available in A. thaliana for analysis of SI signaling. To test the hypothesis that SRK is redox regulated by thioredoxin h, we expressed a mutant form of SRKb lacking a transmembrane-localized cysteine residue thought to be essential for the SRK-thioredoxin h interaction. We also analyzed transfer DNA insertion mutants in the A. thaliana orthologs of THL1 and THL2. In neither case did we observe an effect on the pollination responses of SRKb-expressing stigmas toward incompatible or compatible pollen. Our results are consistent with the conclusion that, contrary to their proposed role, thioredoxin h proteins are not required to prevent the spontaneous activation of SRK in the A. thaliana stigma.
Purinergic signalling: from discovery to current developments
Authors: Burnstock Geoffrey 
Published: September 2013

New Findings
What is the topic of this review?This is a personal historical review about the discovery and the main conceptual advances leading to our current understanding of purinergic signalling. The contributions of leading figures in the field are acknowledged. It includes the discovery of purinergic neuromuscular and synaptic transmission, cotransmission, the identification of P1 (adenosine), P2X nucleotide ion channel and P2Y nucleotide G protein‐coupled receptors, the identity of ectonucleotidases and release of ATP from cells by mechanical stimulation and mechanosensory transduction.
What advances does it highlight?It highlights the pathophysiology of purinergic signalling and recent therapeutic developments.
This lecture is about the history of the purinergic signalling concept. It begins with reference to the paper by Paton & Vane published in 1963, which identified non‐cholinergic relaxation in response to vagal nerve stimulation in several species, although they suggested that it might be due to sympathetic adrenergic nerves in the vagal nerve trunk. Using the sucrose gap technique for simultaneous mechanical and electrical recordings in smooth muscle (developed while in Feldberg's department in the National Institute for Medical Research) of the guinea‐pig taenia coli preparation (learned when working in Edith Bülbring's smooth muscle laboratory in Oxford Pharmacology), we showed that the hyperpolarizations recorded in the presence of antagonists to the classical autonomic neurotransmitters, acetylcholine and noradrenaline, were inhibitory junction potentials in response to non‐adrenergic, non‐cholinergic neurotransmission, mediated by intrinsic enteric nerves controlled by vagal and sacral parasympathetic nerves. We then showed that ATP satisfied the criteria needed to identify a neurotransmitter released by these nerves. Subsequently, it was shown that ATP is a cotransmitter in all nerves in the peripheral and central nervous systems. The receptors for purines and pyrimidines were cloned and characterized in the early 1990s, and immunostaining showed that most non‐neuronal cells as well as nerve cells expressed these receptors. The physiology and pathophysiology of purinergic signalling is discussed.
Postexercise syncope: Wingate syncope test and effective countermeasure
Authors: Lacewell Alisha N. , Buck Tahisha M. , Romero Steven A. , Halliwill John R. 
Published: September 2013

New Findings
What is the central question of this study?Does a modified version of the Wingate anaerobic power test produce presyncopal signs and symptoms in healthy individuals? Does an inspiratory threshold device work as a countermeasure against postexercise syncope?
What is the main finding and its importance?A modified Wingate test is a good model to induce postexercise syncope, and syncopal symptoms can be ameliorated by an inspiratory threshold device.
Altered systemic haemodynamics following exercise can compromise cerebral perfusion and result in syncope. As the Wingate anaerobic test often induces presyncope, we hypothesized that a modified Wingate test could form the basis of a novel model for the study of postexercise syncope and a test bed for potential countermeasures. Along these lines, breathing through an impedance threshold device has been shown to increase tolerance to hypovolaemia, and could prove beneficial in the setting of postexercise syncope. Therefore, we hypothesized that a modified Wingate test followed by head‐up tilt would produce postexercise syncope, and that breathing through an impedance threshold device (countermeasure) would prevent postexercise syncope in healthy individuals. Nineteen recreationally active men and women underwent a 60 deg head‐up tilt during recovery from the Wingate test while arterial pressure, heart rate, end‐tidal CO2 and cerebral tissue oxygenation were measured on a control day and a countermeasure day. The duration of tolerable tilt was increased by a median time of 3 min 48 s with countermeasure in comparison to the control (P < 0.05), and completion of the tilt test increased from 42 to 67% with the countermeasure. During the tilt, mean arterial pressure was greater (108.0 ± 4.1 versus 100.4 ± 2.4 mmHg; P < 0.05) with the countermeasure in comparison to the control. These data suggest that the Wingate syncope test produces a high incidence of presyncope, which is sensitive to countermeasures such as inspiratory impedance.
Occurrence of Potentially Human-Pathogenic Escherichia coli O103 in Norwegian Sheep
Authors: Sekse Camilla , Sunde Marianne , Hopp Petter , Bruheim Torkjel , Cudjoe Kofitsyo Sewornu , Kvitle Bjørg , Urdahl Anne Margrete 
Published: September 2013

ABSTRACT

The investigation of an outbreak of hemorrhagic-uremic syndrome in Norway in 2006 indicated that the outbreak strain
Escherichia coli
O103:H25 could originate from sheep. A national survey of the Norwegian sheep population was performed, with the aim of identifying and describing a possible reservoir of potentially human-pathogenic
E. coli
O103, in particular of the H types 2 and 25. The investigation of fecal samples from 585 sheep flocks resulted in 1,222
E. coli
O103 isolates that were analyzed for the presence of
eae
and
stx
genes, while a subset of 369 isolates was further examined for flagellar antigens (H typing),
stx
subtypes,
bfpA
,
astA
, and molecular typing by pulsed-field gel electrophoresis (PFGE). The total ovine
E. coli
O103 serogroup was genetically diverse by numbers of H types, virulotypes, and PFGE banding patterns identified, although a tendency of clustering toward serotypes was seen. The flocks positive for potentially human-pathogenic
E. coli
O103 were geographically widely distributed, and no association could be found with county or geographical region. The survey showed that
eae
-negative,
stx
-negative
E. coli
O103, probably nonpathogenic to humans, is very common in sheep, with 27.5% of flocks positive. Moreover, the study documented a low prevalence (0.7%) of potentially human-pathogenic Shiga toxin-producing
E. coli
O103:H2, while STEC O103:H25 was not detected. However, 3.1% and 5.8% of the flocks were positive for enteropathogenic
E. coli
O103 belonging to H types 2 and 25, respectively. These isolates are of concern as potential human pathogens by themselves but more importantly as possible precursors for human-pathogenic STEC.
Rapid Transposon Liquid Enrichment Sequencing (TnLE-seq) for Gene Fitness Evaluation in Underdeveloped Bacterial Systems
Authors: Fels Samuel R. , Zane Grant M. , Blake Sean M. , Wall Judy D. 
Published: September 2013

ABSTRACT

Whole-genome fitness analysis in microbes that uses saturating transposon mutagenesis combined with massively parallel sequencing (Tn-seq) is providing a measure of the contribution of each gene to a given growth condition. With this technique, gene fitness profiles and essential genes are discovered by simultaneous analyses of whether the absence of each gene product alters the growth kinetics of the bacterium. Here we modify the standard Tn-seq procedure to simplify and shorten the process by including delivery of the transposon through conjugation and liquid culture enrichment of the mutant pool, creating transposon liquid enrichment sequencing (TnLE-seq). To illustrate the success of these modifications and the robustness of the procedure, analyses of gene fitness of two cultures of the strictly anaerobic bacterium
Desulfovibrio vulgaris
Hildenborough were performed, with growth on lactate as the electron donor and sulfate as the electron acceptor. These data demonstrate reproducibility and provide a base condition for analysis of fitness changes in deletion mutants and in various growth conditions. The procedural modifications will facilitate the application of this powerful genetic analysis to microbes lacking a facile genetic system. Pilot studies produced 2.5 × 10
5
and 3.4 × 10
5
unique insertion mutants in the anaerobe
Desulfovibrio vulgaris
Hildenborough grown under typical laboratory conditions in rich medium. These analyses provided two similar high-resolution maps of gene fitness across the genome, and the method was also applied to growth in minimal medium. These results were also compared to the coverage obtained with a ca. 13,000-member cataloged transposon library constructed by sequencing transposon insertion sites in individual mutants.
High-Efficiency Thermal Asymmetric Interlaced PCR (hiTAIL-PCR) for Determination of a Highly Degenerated Prophage WO Genome in a Wolbachia Strain Infecting a Fig Wasp Species
Authors: Wang Guan-Hong , Xiao Jin-Hua , Xiong Tuan-Lin , Li Zi , Murphy Robert W. , Huang Da-Wei 
Published: September 2013

ABSTRACT

Temperate bacteriophage WO is a model system for studying tripartite interactions among viruses, bacteria, and eukaryotes, especially investigations of the genomic stability of obligate intracellular bacteria. Few WO genomes exist because of the difficulty in isolating viral DNA from eukaryotic hosts, and most reports are by-products of
Wolbachia
sequencing. Only one partial genome of a WO phage has been determined directly from isolated particles. We determine the complete genome sequence of prophage WO (WOSol) in
Wolbachia
strain
w
Sol, which infects the fig wasp
Ceratosolen solmsi
(Hymenoptera: Chalcidoidea), by high-efficiency thermal asymmetric interlaced PCR. The genome of WOSol is highly degenerated and disrupted by a large region (14,267 bp) from
Wolbachia
. Consistent with previous molecular studies of multiple WO genomes, the genome of WOSol appears to have evolved by single nucleotide mutations and recombinations.
Composite Bacterial Hopanoids and Their Microbial Producers across Oxygen Gradients in the Water Column of the California Current
Authors: Kharbush Jenan J. , Ugalde Juan A. , Hogle Shane L. , Allen Eric E. , Aluwihare Lihini I. 
Published: September 2013

ABSTRACT

Hopanoids are pentacyclic triterpenoid lipids produced by many prokaryotes as cell membrane components. The structural variations of composite hopanoids, or bacteriohopanepolyols (BHPs), produced by various bacterial genera make them potentially useful molecular biomarkers of bacterial communities and metabolic processes in both modern and ancient environments. Building on previous work suggesting that organisms in low-oxygen environments are important contributors to BHP production in the marine water column and that there may be physiological roles for BHPs specific to these environments, this study investigated the relationship between trends in BHP structural diversity and abundance and the genetic diversity of BHP producers for the first time in a low-oxygen environment of the Eastern Tropical North Pacific. Amplification of the hopanoid biosynthesis gene for squalene hopene cyclase (
sqhC
) indicated far greater genetic diversity than would be predicted by examining BHP structural diversity alone and that greater
sqhC
genetic diversity exists in the marine environment than is represented by cultured representatives and most marine metagenomes. In addition, the genetic relationships in this data set suggest microaerophilic environments as potential “hot spots” of BHP production. Finally, structural analysis of BHPs showed that an isomer of the commonly observed BHP bacteriohopanetetrol may be linked to a producer that is more abundant in low-oxygen environments. Results of this study increase the known diversity of BHP producers and provide a detailed phylogeny with implications for the role of hopanoids in modern bacteria, as well as the evolutionary history of hopanoid biosynthesis, both of which are important considerations for future interpretations of the marine sedimentary record.
Use of a Hierarchical Oligonucleotide Primer Extension Approach for Multiplexed Relative Abundance Analysis of Methanogens in Anaerobic Digestion Systems
Authors: Wu Jer-Horng , Chuang Hui-Ping , Hsu Mao-Hsuan , Chen Wei-Yu 
Published: September 2013

ABSTRACT

In this study, we established a rapid multiplex method to detect the relative abundances of amplified 16S rRNA genes from known cultivatable methanogens at hierarchical specificities in anaerobic digestion systems treating industrial wastewater and sewage sludge. The method was based on the hierarchical oligonucleotide primer extension (HOPE) technique and combined with a set of 27 primers designed to target the total archaeal populations and methanogens from 22 genera within 4 taxonomic orders. After optimization for their specificities and detection sensitivity under the conditions of multiple single-nucleotide primer extension reactions, the HOPE approach was applied to analyze the methanogens in 19 consortium samples from 7 anaerobic treatment systems (i.e., 513 reactions). Among the samples, the methanogen populations detected with order-level primers accounted for >77.2% of the PCR-amplified 16S rRNA genes detected using an
Archaea
-specific primer. The archaeal communities typically consisted of 2 to 7 known methanogen genera within the
Methanobacteriales
,
Methanomicrobiales
, and
Methanosarcinales
and displayed population dynamic and spatial distributions in anaerobic reactor operations. Principal component analysis of the HOPE data further showed that the methanogen communities could be clustered into 3 distinctive groups, in accordance with the distribution of the
Methanosaeta
,
Methanolinea
, and
Methanomethylovorans
, respectively. This finding suggested that in addition to acetotrophic and hydrogenotrophic methanogens, the methylotrophic methanogens might play a key role in the anaerobic treatment of industrial wastewater. Overall, the results demonstrated that the HOPE approach is a specific, rapid, and multiplexing platform to determine the relative abundances of targeted methanogens in PCR-amplified 16S rRNA gene products.
Cell Size Distributions of Soil Bacterial and Archaeal Taxa
Authors: Portillo Maria C. , Leff Jonathan W. , Lauber Christian L. , Fierer Noah 
Published: September 2013

ABSTRACT

Cell size is a key ecological trait of soil microorganisms that determines a wide range of life history attributes, including the efficiency of nutrient acquisition. However, because of the methodological issues associated with determining cell sizes
in situ
, we have a limited understanding of how cell abundances vary across cell size fractions and whether certain microbial taxa have consistently smaller cells than other taxa. In this study, we extracted cells from three distinct soils and fractionated them into seven size ranges (5 μm to 0.2 μm) by filtration. Cell abundances in each size fraction were determined by direct microscopy, with the taxonomic composition of each size fraction determined by high-throughput sequencing of the 16S rRNA gene. Most of the cells were smaller than cells typically grown in culture, with 59 to 67% of cells <1.2 μm in diameter. Furthermore, each size fraction harbored distinct bacterial and archaeal communities in each of the three soils, and many of the taxa exhibited distinct size distribution patterns, with the smaller size fractions having higher relative abundances of taxa that are rare or poorly characterized (including
Acidobacteria
,
Gemmatimonadetes
,
Crenarchaeota
,
Verrucomicrobia
, and
Elusimicrobia
). In general, there was a direct relationship between average cell size and culturability, with those soil taxa that are poorly represented in culture collections tending to be smaller. Size fractionation not only provides important insight into the life history strategies of soil microbial taxa but also is a useful tool to enable more focused investigations into those taxa that remain poorly characterized.
Abilities of the mCP Agar Method and CRENAME Alpha Toxin-Specific Real-Time PCR Assay To Detect Clostridium perfringens Spores in Drinking Water
Authors: Maheux Andrée F. , Bérubé Ève , Boudreau Dominique K. , Villéger Romain , Cantin Philippe , Boissinot Maurice , Bissonnette Luc , Bergeron Michel G. 
Published: September 2013

ABSTRACT

We first determined the analytical specificity and ubiquity (i.e., the ability to detect all or most strains) of a
Clostridium perfringens
-specific real-time PCR (rtPCR) assay based on the
cpa
gene (
cpa
rtPCR) by using a bacterial strain panel composed of
C. perfringens
and non-
C. perfringens Clostridium
strains. All non-
C. perfringens Clostridium
strains tested negative, whereas all
C. perfringens
strains tested positive with the
cpa
rtPCR, for an analytical specificity and ubiquity of 100%. The
cpa
rtPCR assay was then used to confirm the identity of 116 putative
C. perfringens
isolates recovered after filtration of water samples and culture on mCP agar. Colonies presenting discordant results between the phenotype on mCP agar and
cpa
rtPCR were identified by sequencing the 16S rRNA and
cpa
genes. Four mCP
−
/rtPCR
+
colonies were identified as
C. perfringens
, whereas 3 mCP
+
/rtPCR
−
colonies were identified as non-
C. perfringens
. The
cpa
rtPCR was negative with all 51 non-
C. perfringens
strains and positive with 64 of 65
C. perfringens
strains. Finally, we compared mCP agar and a CRENAME (
c
oncentration and
r
ecovery of microbial particles,
e
xtraction of
n
ucleic
a
cids, and
m
olecular
e
nrichment) procedure plus
cpa
rtPCR (CRENAME +
cpa
rtPCR) for their abilities to detect
C. perfringens
spores in drinking water. CRENAME +
cpa
rtPCR detected as few as one
C. perfringens
CFU per 100 ml of drinking water sample in less than 5 h, whereas mCP agar took at least 25 h to deliver results. CRENAME +
cpa
rtPCR also allows the simultaneous and sensitive detection of
Escherichia coli
and
C. perfringens
from the same potable water sample. In itself, it could be used to assess the public health risk posed by drinking water potentially contaminated with pathogens more resistant to disinfection.
DNA Double-Strand Break Repair at −15°C
Authors: Dieser Markus , Battista John R. , Christner Brent C. 
Published: September 2013

ABSTRACT

The survival of microorganisms in ancient glacial ice and permafrost has been ascribed to their ability to persist in a dormant, metabolically inert state. An alternative possibility, supported by experimental data, is that microorganisms in frozen matrices are able to sustain a level of metabolic function that is sufficient for cellular repair and maintenance. To examine this experimentally, frozen populations of
Psychrobacter arcticus
273-4 were exposed to ionizing radiation (IR) to simulate the damage incurred from natural background IR sources in the permafrost environment from over ∼225 kiloyears (ky). High-molecular-weight DNA was fragmented by exposure to 450 Gy of IR, which introduced an average of 16 double-strand breaks (DSBs) per chromosome. During incubation at −15°C for 505 days,
P. arcticus
repaired DNA DSBs in the absence of net growth. Based on the time frame for the assembly of genomic fragments by
P. arcticus
, the rate of DNA DSB repair was estimated at 7 to 10 DSBs year
−1
under the conditions tested. Our results provide direct evidence for the repair of DNA lesions, extending the range of complex biochemical reactions known to occur in bacteria at frozen temperatures. Provided that sufficient energy and nutrient sources are available, a functional DNA repair mechanism would allow cells to maintain genome integrity and augment microbial survival in icy terrestrial or extraterrestrial environments.
Iterative Saturation Mutagenesis of −6 Subsite Residues in Cyclodextrin Glycosyltransferase from Paenibacillus macerans To Improve Maltodextrin Specificity for 2-
O
-
d
-Glucopyranosyl-
l
-Ascorbic Acid Synthesis
Authors: Han Ruizhi , Liu Long , Shin Hyun-dong , Chen Rachel R. , Li Jianghua , Du Guocheng , Chen Jian 
Published: September 2013

ABSTRACT

2-
O
-
d
-Glucopyranosyl-
l
-ascorbic acid (AA-2G), a stable
l
-ascorbic acid derivative, is usually synthesized by cyclodextrin glycosyltransferase (CGTase), which contains nine substrate-binding subsites (from +2 to −7). In this study, iterative saturation mutagenesis (ISM) was performed on the −6 subsite residues (Y167, G179, G180, and N193) in the CGTase from
Paenibacillus macerans
to improve its specificity for maltodextrin, which is a cheap and easily soluble glycosyl donor for AA-2G synthesis. Site saturation mutagenesis of four sites—Y167, G179, G180, and N193—was first performed and revealed that four mutants—Y167S, G179R, N193R, and G180R—produced AA-2G yields higher than those of other mutant and wild-type CGTases. ISM was then conducted with the best positive mutant as a template. Under optimal conditions, mutant Y167S/G179K/N193R/G180R produced the highest AA-2G titer of 2.12 g/liter, which was 84% higher than that (1.15 g/liter) produced by the wild-type CGTase. Kinetics analysis of AA-2G synthesis using mutant CGTases confirmed the enhanced maltodextrin specificity and showed that compared to the wild-type CGTase, the mutants had no cyclization activity but high hydrolysis and disproportionation activities. A possible mechanism for the enhanced substrate specificity was also analyzed through structure modeling of the mutant and wild-type CGTases. These results indicated that the −6 subsite played crucial roles in the substrate binding and catalytic reactions of CGTase and that the obtained CGTase mutants, especially Y167S/G179K/N193R/G180R, are promising starting points for further development through protein engineering.