bluuebunny/crossref_metadata_2025_split huggingface dataset:
Development of a Reverse Transcription-Quantitative PCR System for Detection and Genotyping of Aichi Viruses in Clinical and Environmental Samples
Authors: Kitajima Masaaki , Hata Akihiko , Yamashita Teruo , Haramoto Eiji , Minagawa Hiroko , Katayama Hiroyuki 
Published: April 2013

ABSTRACT

Aichi viruses (AiVs) have been proposed as a causative agent of human gastroenteritis potentially transmitted by fecal-oral routes through contaminated food or water. In the present study, we developed a TaqMan minor groove binder (MGB)-based reverse transcription-quantitative PCR (RT-qPCR) system that is able to quantify AiVs and differentiate between genotypes A and B. This system consists of two assays, an AiV universal assay utilizing a universal primer pair and a universal probe and a duplex genotype-specific assay utilizing the same primer pair and two genotype-specific probes. The primers and probes were designed based on multiple alignments of the 21 available AiV genome sequences containing the capsid gene. Using a 10-fold dilution of plasmid DNA containing the target sequences, it was confirmed that both assays allow detection and quantification of AiVs with a quantitative range of 1.0 × 10
1
to 1.0 × 10
7
copies/reaction, and the genotype-specific assay reacts specifically to each genotype. To validate the newly developed assays, 30 clinical stool specimens were subsequently examined with the assays, and the AiV RNA loads were determined to be 1.4 × 10
4
to 6.6 × 10
9
copies/g stool. We also examined 12 influent and 12 effluent wastewater samples collected monthly for a 1-year period to validate the applicability of the assays for detection of AiVs in environmental samples. The AiV RNA concentrations in influent and effluent wastewater were determined to be up to 2.2 × 10
7
and 1.8 × 10
4
copies/liter, respectively. Our RT-qPCR system is useful for routine diagnosis of AiVs in clinical stool specimens and environmental samples.
In Vivo
Assessment of Growth and Virulence Gene Expression during Commensal and Pathogenic Lifestyles of
luxABCDE
-Tagged Enterococcus faecalis Strains in Murine Gastrointestinal and Intravenous Infection Models
Authors: La Rosa Sabina Leanti , Casey Pat G. , Hill Colin , Diep Dzung B. , Nes Ingolf F. , Brede Dag A. 
Published: April 2013

ABSTRACT

Cytolysin and gelatinase are prominent pathogenicity determinants associated with highly virulent
Enterococcus faecalis
strains. In an effort to explore the expression profiles of these virulence traits
in vivo
, we have employed
E. faecalis
variants expressing the
luxABCDE
cassette under the control of either the P

16S

, cytolysin, or gelatinase promoter for infections of
Galleria mellonella
caterpillars and mice. Systemic infection of
G. mellonella
with bioluminescence-tagged
E. faecalis
MMH594 revealed temporal regulation of both gelatinase and cytolysin promoters and demonstrated that these traits were induced in response to the host environment. Gavage of mice pretreated perorally with antibiotics resulted in efficient colonization of the murine gastrointestinal tract (GIT) in a strain-dependent manner, where the commensal baby isolate EF62 was more persistent than the nosocomial isolate MMH594. A highly significant correlation (
R
2
> 0.94) was found between bioluminescence and the CFU counts in mouse fecal samples. Both strains showed similar preferences for growth and persistence in the ileum, cecum, and colon. Cytolysin expression was uniform in these compartments of the intestinal lumen. In spite of high numbers (10
9
CFU/g of intestinal matter) in the ileum, cecum, and colon, no evidence of translocation or systemic infection could be observed. In the murine intravenous infection model, cytolysin expression was readily detected in the liver, kidneys, and bladder. At 72 h postinfection, the highest bacterial loads were found in the liver, kidneys, and spleen, with organ-specific expression levels of cytolysin ∼400- and ∼900-fold higher in the spleen and heart, respectively, than in the liver and kidneys. Taken together, this system based on the bioluminescence imaging technology is established as a new, powerful method to monitor the differential regulation of
E. faecalis
virulence determinants and to study the spatiotemporal course of infection in living animals in real time.
Biofilm Formation by Psychrobacter arcticus and the Role of a Large Adhesin in Attachment to Surfaces
Authors: Hinsa-Leasure Shannon M. , Koid Cassandra , Tiedje James M. , Schultzhaus Janna N. 
Published: April 2013

ABSTRACT

Psychrobacter arcticus
strain 273-4, an isolate from a Siberian permafrost core, is capable of forming biofilms when grown in minimal medium under laboratory conditions. Biofilms form at 4 to 22°C when acetate is supplied as the lone carbon source and with 1 to 7% sea salt.
P. arcticus
is also capable of colonizing quartz sand. Transposon mutagenesis identified a gene important for biofilm formation by
P. arcticus
. Four transposon mutants were mapped to a 20.1-kbp gene, which is predicted to encode a protein of 6,715 amino acids (Psyc_1601). We refer to this open reading frame as
cat1
, for cold attachment gene 1. The
cat1
mutants are unable to form biofilms at levels equivalent to that of the wild type, and there is no impact on the planktonic growth characteristics of the strains, indicating a specific role in biofilm formation. Through time course studies of the static microtiter plate assay, we determined that
cat1
mutants are unable to form biofilms equivalent to that of the wild type under all conditions tested. In flow cell experiments,
cat1
mutants initially are unable to attach to the surface. Over time, however, they form microcolonies, an architecture very different from that produced by wild-type biofilms. Our results demonstrate that Cat1 is involved in the initial stages of bacterial attachment to surfaces.
Characterization of
Tt
ALV2, an Essential Charged Repeat Motif Protein of the Tetrahymena thermophila Membrane Skeleton
Authors: El-Haddad Houda , Przyborski Jude M. , Kraft Lesleigh G. K. , McFadden Geoffrey I. , Waller Ross F. , Gould Sven B. 
Published: April 2013

ABSTRACT

Alveolins are a recently described class of proteins common to all members of the superphylum Alveolata that are characterized by conserved charged repeat motifs (CRMs) but whose exact function remains unknown. We have analyzed the smaller of the two alveolins of
Tetrahymena thermophila
,
Tt
ALV2. The protein localizes to dispersed, broken patches arranged between the rows of the longitudinal microtubules. Macronuclear knockdown of
Ttalv2
leads to multinuclear cells with no apparent cell polarity and randomly occurring cell protrusions, either by interrupting pellicle integrity or by disturbing cytokinesis. Correct association of
Tt
ALV2 with the alveoli or the pellicle is complex and depends on both the termini as well as the charged repeat motifs of the protein. Proteins containing similar CRMs are a dominant part of the ciliate membrane cytoskeleton, suggesting that these motifs may play a more general role in mediating membrane attachment and/or cytoskeletal association. To better understand their integration into the cytoskeleton, we localized a range of CRM-based fusion proteins, which suggested there is an inherent tendency for proteins with CRMs to be located in the peripheral cytoskeleton, some nucleating as filaments at the basal bodies. Even a synthetic protein, mimicking the charge and repeat pattern of these proteins, directed a reporter protein to a variety of peripheral cytoskeletal structures in
Tetrahymena
. These motifs might provide a blueprint for membrane and cytoskeleton affiliation in the complex pellicles of Alveolata.
A Central Role for Ras1 in Morphogenesis of the Basidiomycete Schizophyllum commune
Authors: Knabe Nicole , Jung Elke-Martina , Freihorst Daniela , Hennicke Florian , Horton J. Stephen , Kothe Erika 
Published: April 2013

ABSTRACT

Fungi have been used as model systems to define general processes in eukaryotes, for example, the one gene-one enzyme hypothesis, as well as to study polar growth or pathogenesis. Here, we show a central role for the regulator protein Ras in a mushroom-forming, filamentous basidiomycete linking growth, pheromone signaling, sexual development, and meiosis to different signal transduction pathways.
ras1
and Ras-specific
gap1
mutants were generated and used to modify the intracellular activation state of the Ras module. Transformants containing constitutive
ras1
alleles (
ras1
G12V
and
ras1
Q61L
), as well as their compatible mating interactions, did show strong phenotypes for growth (associated with Cdc42 signaling) and mating (associated with mitogen-activated protein kinase signaling). Normal fruiting bodies with abnormal spores exhibiting a reduced germination rate were produced by outcrossing of these mutant strains. Homozygous Δ
gap1
primordia, expected to experience increased Ras signaling, showed overlapping phenotypes with a block in basidium development and meiosis. Investigation of cyclic AMP (cAMP)-dependent protein kinase A indicated that constitutively active
ras1
, as well as Δ
gap1
mutant strains, exhibit a strong increase in Tpk activity. Ras1-dependent, cAMP-mediated signal transduction is, in addition to the known signaling pathways, involved in fruiting body formation in
Schizophyllum commune
. To integrate these analyses of Ras signaling, microarray studies were performed. Mutant strains containing constitutively active Ras1, deletion of RasGap1, or constitutively active Cdc42 were characterized and compared. At the transcriptome level, specific regulation highlighting the phenotypic differences of the mutants is clearly visible.
Mutations in Escherichia coli ExbB Transmembrane Domains Identify Scaffolding and Signal Transduction Functions and Exclude Participation in a Proton Pathway
Authors: Baker Kristin R. , Postle Kathleen 
Published: April 2013

ABSTRACT

The TonB system couples cytoplasmic membrane proton motive force (pmf) to active transport of diverse nutrients across the outer membrane. Current data suggest that cytoplasmic membrane proteins ExbB and ExbD harness pmf energy. Transmembrane domain (TMD) interactions between TonB and ExbD allow the ExbD C terminus to modulate conformational rearrangements of the periplasmic TonB C terminus
in vivo
. These conformational changes somehow allow energization of high-affinity TonB-gated transporters by direct interaction with TonB. While ExbB is essential for energy transduction, its role is not well understood. ExbB has N-terminus-out, C-terminus-in topology with three TMDs. TMDs 1 and 2 are punctuated by a cytoplasmic loop, with the C-terminal tail also occupying the cytoplasm. We tested the hypothesis that ExbB TMD residues play roles in proton translocation. Reassessment of TMD boundaries based on hydrophobic character and residue conservation among distantly related ExbB proteins brought earlier widely divergent predictions into congruence. All TMD residues with potentially function-specific side chains (Lys, Cys, Ser, Thr, Tyr, Glu, and Asn) and residues with probable structure-specific side chains (Trp, Gly, and Pro) were substituted with Ala and evaluated in multiple assays. While all three TMDs were essential, they had different roles: TMD1 was a region through which ExbB interacted with the TonB TMD. TMD2 and TMD3, the most conserved among the ExbB/TolQ/MotA/PomA family, played roles in signal transduction between cytoplasm and periplasm and the transition from ExbB homodimers to homotetramers. Consideration of combined data excludes ExbB TMD residues from direct participation in a proton pathway.
Periodic Reversals in Paenibacillus dendritiformis Swarming
Authors: Be'er Avraham , Strain Shinji K. , Hernández Roberto A. , Ben-Jacob Eshel , Florin E.-L. 
Published: April 2013

ABSTRACT

Bacterial swarming is a type of motility characterized by a rapid and collective migration of bacteria on surfaces. Most swarming species form densely packed dynamic clusters in the form of whirls and jets, in which hundreds of rod-shaped rigid cells move in circular and straight patterns, respectively. Recent studies have suggested that short-range steric interactions may dominate hydrodynamic interactions and that geometrical factors, such as a cell's aspect ratio, play an important role in bacterial swarming. Typically, the aspect ratio for most swarming species is only up to 5, and a detailed understanding of the role of much larger aspect ratios remains an open challenge. Here we study the dynamics of
Paenibacillus dendritiformis C
morphotype, a very long, hyperflagellated, straight (rigid), rod-shaped bacterium with an aspect ratio of ∼20. We find that instead of swarming in whirls and jets as observed in most species, including the shorter
T
morphotype of
P. dendritiformis
, the
C
morphotype moves in densely packed straight but thin long lines. Within these lines, all bacteria show periodic reversals, with a typical reversal time of 20 s, which is independent of their neighbors, the initial nutrient level, agar rigidity, surfactant addition, humidity level, temperature, nutrient chemotaxis, oxygen level, illumination intensity or gradient, and cell length. The evolutionary advantage of this unique back-and-forth surface translocation remains unclear.
Core-Gene-Encoded Peptide Regulating Virulence-Associated Traits in Streptococcus mutans
Authors: Kim Jeong Nam , Stanhope Michael J. , Burne Robert A. 
Published: April 2013

ABSTRACT

Recently, high-coverage genome sequence of 57 isolates of
Streptococcus mutans
, the primary etiological agent of human dental caries, was completed. The SMU.1147 gene, encoding a 61-amino-acid (61-aa) peptide, was present in all sequenced strains of
S. mutans
but absent in all bacteria in current databases. Reverse transcription-PCR revealed that SMU.1147 is cotranscribed with
scnK
and
scnR
, which encode the histidine kinase and response regulator, respectively, of a two-component system (TCS). The C terminus of the SMU.1147 gene product was tagged with a FLAG epitope and shown to be expressed in
S. mutans
by Western blotting with an anti-FLAG antibody. A nonpolar mutant of SMU.1147 formed less biofilm in glucose-containing medium and grew slower than did the wild-type strain under aerobic and anaerobic conditions, at low pH, or in the presence of H
2
O
2
. Mutation of SMU.1147 dramatically reduced genetic competence and expression of
comX
and
comY
, compared to
S. mutans
UA159. The competence defect of the SMU.1147 mutant could not be overcome by addition of
sigX
-inducing peptide (XIP) in defined medium or by competence-stimulating peptide (CSP) in complex medium. Complementation with SMU.1147 on a plasmid restored all phenotypes. Interestingly, mutants lacking either one of the TCS components and a mutant lacking all three genes behaved like the wild-type strain for all phenotypes mentioned above, but all mutant strains grew slower than UA159 in medium supplemented with 0.3 M NaCl. Thus, the SMU.1147-encoded peptide affects virulence-related traits and dominantly controls quorum-sensing pathways for development of genetic competence in
S. mutans
.
Paracoccus denitrificans PD1222 Utilizes Hypotaurine via Transamination Followed by Spontaneous Desulfination To Yield Acetaldehyde and, Finally, Acetate for Growth
Authors: Felux Ann-Katrin , Denger Karin , Weiss Michael , Cook Alasdair M. , Schleheck David 
Published: April 2013

ABSTRACT

Hypotaurine (HT; 2-aminoethane-sulfinate) is known to be utilized by bacteria as a sole source of carbon, nitrogen, and energy for growth, as is taurine (2-aminoethane-sulfonate); however, the corresponding HT degradation pathway has remained undefined. Genome-sequenced
Paracoccus denitrificans
PD1222 utilized HT (and taurine) quantitatively for heterotrophic growth and released the HT sulfur as sulfite (and sulfate) and HT nitrogen as ammonium. Enzyme assays with cell extracts suggested that an HT-inducible HT:pyruvate aminotransferase (Hpa) catalyzes the deamination of HT in an initial reaction step. Partial purification of the Hpa activity and peptide fingerprinting-mass spectrometry (PF-MS) identified the Hpa candidate gene; it encoded an archetypal taurine:pyruvate aminotransferase (Tpa). The same gene product was identified via differential PAGE and PF-MS, as was the gene of a strongly HT-inducible aldehyde dehydrogenase (Adh). Both genes were overexpressed in
Escherichia coli
. The overexpressed, purified Hpa/Tpa showed HT:pyruvate-aminotransferase activity. Alanine, acetaldehyde, and sulfite were identified as the reaction products but not sulfinoacetaldehyde; the reaction of Hpa/Tpa with taurine yielded sulfoacetaldehyde, which is stable. The overexpressed, purified Adh oxidized the acetaldehyde generated during the Hpa reaction to acetate in an NAD
+
-dependent reaction. Based on these results, the following degradation pathway for HT in strain PD1222 can be depicted. The identified aminotransferase converts HT to sulfinoacetaldehyde, which desulfinates spontaneously to acetaldehyde and sulfite; the inducible aldehyde dehydrogenase oxidizes acetaldehyde to yield acetate, which is metabolized, and sulfite, which is excreted.
FrnE, a Cadmium-Inducible Protein in Deinococcus radiodurans, Is Characterized as a Disulfide Isomerase Chaperone
In Vitro
and for Its Role in Oxidative Stress Tolerance
In Vivo
Authors: Khairnar Nivedita P. , Joe Min-Ho , Misra H. S. , Lim Sang-Yong , Kim Dong-Ho 
Published: April 2013

ABSTRACT

Deinococcus radiodurans
R1 exposed to a lethal dose of cadmium shows differential expression of a large number of genes, including
frnE
(dr
frnE
) and some of those involved in DNA repair and oxidative stress tolerance. The dr
frnE
::
nptII
mutant of
D. radiodurans
showed growth similar to that of the wild type, but its tolerance to 10 mM cadmium and 10 mM diamide decreased by ∼15- and ∼3-fold, respectively. These cells also showed nearly 6 times less resistance to gamma radiation at 12 kGy and ∼2-fold-higher sensitivity to 40 mM hydrogen peroxide than the wild type. In
trans
expression of drFrnE increased cytotoxicity of dithiothreitol (DTT) in the
dsbA
mutant of
Escherichia coli
. Recombinant drFrnE showed disulfide isomerase activity and could maintain insulin in its reduced form in the presence of DTT. While an equimolar ratio of wild-type protein could protect malate dehydrogenase completely from thermal denaturation at 42°C, the C22S mutant of drFrnE provided reduced protection to malate dehydrogenase from thermal inactivation. These results suggested that drFrnE is a protein disulfide isomerase
in vitro
and has a role in oxidative stress tolerance of
D. radiodurans
possibly by protecting the damaged cellular proteins from inactivation.
An open-label cohort study of the improvement of quality of life and pain in de novo cervical dystonia patients after injections with 500 U botulinum toxin A (Dysport)
Authors: Hefter H , Benecke R , Erbguth F , Jost W , Reichel G , Wissel J 
Published: April 2013

ObjectivesIt remains to be determined whether the benefits of botulinum toxin type A (BoNT-A) on cervical dystonia (CD) motor symptoms extend to improvements in patient's quality of life (QoL). This analysis of a large, multicentre study was conducted with the aim of investigating changes in QoL and functioning among de novo patients receiving 500 U BoNT-A (abobotulinumtoxinA; Dysport) for the treatment of the two most frequent forms of CD, predominantly torticollis and laterocollis.DesignA prospective, open-label study of Dysport (500 U; Ipsen Biopharm Ltd) administered according to a defined intramuscular injection algorithm.SettingGerman and Austrian outpatient clinics.Participants516 male and female patients (aged ≥18 years) with de novo CD. The majority of patients had torticollis (78.1%). 35 patients had concomitant depression (MedDRA-defined).Main outcome measuresChange from baseline to weeks 4 and 12 in Craniocervical Dystonia Questionnaire (CDQ-24) total and subscale scores, patient diary items (‘day-to-day capacities and activities’, ‘pain’ and ‘duration of pain’) and global assessment of pain.ResultsSignificant improvements were observed in CDQ-24 total and subscale scores at week 4 and were sustained up to week 12 (p<0.001). Changes in CDQ-24 scores did not significantly differ between the torticollis and laterocollis groups or between patients with or without depression. There were also significant reductions in patient diary item scores for activities of daily living, pain and pain duration at weeks 4 and 12 (p<0.001). Pain relief (less or no pain) was reported by 66% and 74.1% of patients at weeks 4 and 12, respectively. Changes in pain parameters demonstrated a positive relationship with change in Tsui score.ConclusionsAfter standardised open-label treatment with Dysport 500 U, improvements in QoL and pain intensity up to 12 weeks in patients with CD were observed.
Mediation of smoking-associated postoperative mortality by perioperative complications in veterans undergoing elective surgery: data from Veterans Affairs Surgical Quality Improvement Program (VASQIP)—a cohort study
Authors: Singh Jasvinder A , Hawn Mary , Campagna Elizabeth J , Henderson William G , Richman Joshua , Houston Thomas K 
Published: April 2013

ObjectiveTo assess the mediation of smoking-associated postoperative mortality by postoperative complications.DesignObservational cohort study.SettingUsing data from the Veterans Affairs (VA) Surgical Quality Improvement Programme, a quality assurance programme for major surgical procedures in the VA healthcare system, we assessed the association of current smoking at the time of the surgery with 6-month and 1-year mortality.Primary and secondary outcome measuresUsing mediation analyses, we calculated the relative contribution of each smoking-associated complication to smoking-associated postoperative mortality, both unadjusted and adjusted for age, race/ethnicity, work relative value unit of the operation, surgeon specialty, American Society of Anesthesiologists class and year of surgery. Smoking-associated complications included surgical site infection (SSI), cardiovascular complications (myocardial infarction, cardiac arrest and/or stroke) and pulmonary complications (pneumonia, failure to wean and/or reintubation).ResultsThere were 186 632 never smokers and 135 741 current smokers. The association of smoking and mortality was mediated by smoking-related complications with varying effects. In unadjusted analyses, the proportions of mediation of smoking to 6-month mortality explained by the complications were as follows: SSIs 22%, cardiovascular complications 12% and pulmonary complications 89%. In adjusted analyses, the per cents mediated by each complication were as follows: SSIs 2%, cardiovascular complications 4% and pulmonary complications 22%. In adjusted analyses for 1-year mortality, respective per cents mediated were 2%, 3% and 16%.ConclusionsPulmonary complications, followed by cardiovascular complications and SSIs were mediators of smoking-associated 6-month and 1-year mortality. Interventions targeting smoking cessation and prevention and early treatment of pulmonary complications has the likelihood of reducing postoperative mortality after elective surgery.