bluuebunny/crossref_metadata_2025 huggingface dataset:
Vibration Analysis of Flexible Rotating Rings Using a Spectral Element Formulation
Authors: Beli Danilo , Silva Priscilla Brandão , Arruda José R. de França
Published: February 2015

In this work, the forced response of rotating rings exhibiting nonperiodic and periodic variations in material properties and geometry is assessed by means of the spectral element method (SEM). Based on the Euler–Bernoulli beam theory, a spectral element for a planar rotating ring is derived. This spectral element allows the investigation of the effects of structural damping, internal pressure and elastic foundations in the harmonic response of rotating rings. The dynamic response of rotating rings including periodic imperfections that lead to band gap effects is addressed. The spectral element formulation provides exact solutions within the range of validity of the applied theory using a reduced number of degrees-of-freedom. Thus, it contributes to reducing the computational time. It also provides a straightforward way to solve structural dynamics problems including arbitrary boundary conditions and discontinuities. The proposed formulation is validated by comparison with analytical solutions, which are available only for uniform homogenous rings.
Deposition of a Silicon Carbide Reinforced Metal Matrix Composite (P25) Layer Using CO2 Laser
Authors: Wai Yip Mum , Barnes Stuart , Aly Diaa Mohmmed Sarhan Ahmed
Published: February 2015

The objective of this research was to deposit a silicon carbide (SiC) reinforced layer of P25 (iron-based matrix material) on substrate material surface using CO2 laser. Two experiments using CO2 laser were carried out in this research. In the first experiment set, a gravity feed system was used with one powder feed containing different percentages of SiC particles and iron-based powder. In the second experiment set, preplaced powder was placed on substrate material surface. According to the experimental results, only few SiC particles were found in the clad matrix in the first experiment, and no SiC particles were found in the second experiment. A high microhardness value was noted in the SiC clad (above 1000 HV) in the first experiment compared to the second experiment with hardness values ranging from 200 HV to 700 HV. This was due to the high precipitation of carbide particles in the clad material during the first experiment. A comparison of the two different experiments signifies that the first one was the best because a more uniform layer with less porosity was produced.
Effects of Pressurized Water Reactor Medium on the Fatigue Life of Austenitic Stainless Steels
Authors: Wilhelm Paul , Rudolph Jürgen , Steinmann Paul
Published: February 2015

A statistical model for austenitic stainless steels for predicting the effect of pressurized water reactor (PWR) environments on fatigue life for a range of temperatures and strain rates is developed based on analysis of available material data from USA, Europe, and Japan. Only fatigue data from polished specimens of wrought material tested under strain control were considered. Hollow specimens were not treated in the final calculations. The fatigue life correction factors were defined as the ratio of life in water at 300 °C (572 °F) (reference conditions) to that in water at service conditions. The model is recommended for predicting fatigue lives that are 103–105 cycles.
Motion Generation of Planar Six- and Eight-Bar Slider Mechanisms as Constrained Robotic Systems
Authors: Song Soh Gim , Ying Fangtian
Published: February 2015

In this paper, we formulated a systematic design methodology for the design of planar six- and eight-bar slider mechanisms for motion generation applications that require more complex motion than the slider–crank mechanism. We show how two RR dyads can be synthesized and attached to planar PRR and PRR–3R chain for the dimensional synthesis of planar six- and eight-bar slider mechanisms, respectively. The results are 15 different types of one degree-of-freedom planar six- and eight-bar linkages with a prismatic joint at its base. We demonstrate the design process with the design of a multifunctional wheelchair that could transform its structure between a self-propelled wheelchair and a walking guide meant for outpatient rehabilitation purpose.
Effects of Three Different Nucleoid-Associated Proteins Encoded on IncP-7 Plasmid pCAR1 on Host Pseudomonas putida KT2440
Authors: Suzuki-Minakuchi Chiho , Hirotani Ryusuke , Shintani Masaki , Takeda Toshiharu , Takahashi Yurika , Matsui Kazuhiro , Vasileva Delyana , Yun Choong-Soo , Okada Kazunori , Yamane Hisakazu , Nojiri Hideaki
Published: February 2015

Nucleoid-associated proteins (NAPs), which fold bacterial DNA and influence gene transcription, are considered to be global transcriptional regulators of genes on both plasmids and the host chromosome. Incompatibility P-7 group plasmid pCAR1 carries genes encoding three NAPs: H-NS family protein Pmr, NdpA-like protein Pnd, and HU-like protein Phu. In this study, the effects of single or double disruption of
pmr
,
pnd
, and
phu
were assessed in host
Pseudomonas putida
KT2440. When
pmr
and
pnd
or
pmr
and
phu
were simultaneously disrupted, both the segregational stability and the structural stability of pCAR1 were markedly decreased, suggesting that Pmr, Pnd, and Phu act as plasmid-stabilizing factors in addition to their established roles in replication and partition systems. The transfer frequency of pCAR1 was significantly decreased in these double mutants. The segregational and structural instability of pCAR1 in the double mutants was recovered by complementation of
pmr
, whereas no recovery of transfer deficiency was observed. Comprehensive phenotype comparisons showed that the host metabolism of carbon compounds, which was reduced by pCAR1 carriage, was restored by disruption of the NAP gene(s). Transcriptome analyses of mutants indicated that transcription of genes for energy production, conversion, inorganic ion transport, and metabolism were commonly affected; however, how their products altered the phenotypes of mutants was not clear. The findings of this study indicated that Pmr, Pnd, and Phu act synergistically to affect pCAR1 replication, maintenance, and transfer, as well as to alter the host metabolic phenotype.
The Effects of Heat Activation on Bacillus Spore Germination, with Nutrients or under High Pressure, with or without Various Germination Proteins
Authors: Luu Stephanie , Cruz-Mora Jose , Setlow Barbara , Feeherry Florence E. , Doona Christopher J. , Setlow Peter
Published: February 2015

Nutrient germination of spores of
Bacillus
species occurs through germinant receptors (GRs) in spores' inner membrane (IM) in a process stimulated by sublethal heat activation.
Bacillus subtilis
spores maximum germination rates via different GRs required different 75°C heat activation times: 15 min for
l
-valine germination via the GerA GR and 4 h for germination with the
l
-asparagine–glucose–fructose–K
+
mixture via the GerB and GerK GRs, with GerK requiring the most heat activation. In some cases, optimal heat activation decreased nutrient concentrations for half-maximal germination rates. Germination of spores via various GRs by high pressure (HP) of 150 MPa exhibited heat activation requirements similar to those of nutrient germination, and the loss of the GerD protein, required for optimal GR function, did not eliminate heat activation requirements for maximal germination rates. These results are consistent with heat activation acting primarily on GRs. However, (i) heat activation had no effects on GR or GerD protein conformation, as probed by biotinylation by an external reagent; (ii) spores prepared at low and high temperatures that affect spores' IM properties exhibited large differences in heat activation requirements for nutrient germination; and (iii) spore germination by 550 MPa of HP was also affected by heat activation, but the effects were relatively GR independent. The last results are consistent with heat activation affecting spores' IM and only indirectly affecting GRs. The 150- and 550-MPa HP germinations of
Bacillus amyloliquefaciens
spores, a potential surrogate for
Clostridium botulinum
spores in HP treatments of foods, were also stimulated by heat activation.
Microbial Growth under Supercritical CO
2
Authors: Peet Kyle C. , Freedman Adam J. E. , Hernandez Hector H. , Britto Vanya , Boreham Chris , Ajo-Franklin Jonathan B. , Thompson Janelle R.
Published: February 2015

Growth of microorganisms in environments containing CO
2
above its critical point is unexpected due to a combination of deleterious effects, including cytoplasmic acidification and membrane destabilization. Thus, supercritical CO
2
(scCO
2
) is generally regarded as a sterilizing agent. We report isolation of bacteria from three sites targeted for geologic carbon dioxide sequestration (GCS) that are capable of growth in pressurized bioreactors containing scCO
2
. Analysis of 16S rRNA genes from scCO
2
enrichment cultures revealed microbial assemblages of varied complexity, including representatives of the genus
Bacillus
. Propagation of enrichment cultures under scCO
2
headspace led to isolation of six strains corresponding to
Bacillus cereus
,
Bacillus subterraneus
,
Bacillus amyloliquefaciens
,
Bacillus safensis
, and
Bacillus megaterium
. Isolates are spore-forming, facultative anaerobes and capable of germination and growth under an scCO
2
headspace. In addition to these isolates, several
Bacillus
type strains grew under scCO
2
, suggesting that this may be a shared feature of spore-forming
Bacillus
spp. Our results provide direct evidence of microbial activity at the interface between scCO
2
and an aqueous phase. Since microbial activity can influence the key mechanisms for permanent storage of sequestered CO
2
(i.e., structural, residual, solubility, and mineral trapping), our work suggests that during GCS microorganisms may grow and catalyze biological reactions that influence the fate and transport of CO
2
in the deep subsurface.
The Peptide Toxin Amylosin of Bacillus amyloliquefaciens from Moisture-Damaged Buildings Is Immunotoxic, Induces Potassium Efflux from Mammalian Cells, and Has Antimicrobial Activity
Authors: Rasimus-Sahari Stiina , Teplova Vera V. , Andersson Maria A. , Mikkola Raimo , Kankkunen Päivi , Matikainen Sampsa , Gahmberg Carl G. , Andersson Leif C. , Salkinoja-Salonen Mirja
Published: February 2015

Amylosin, a heat-stable channel-forming non-ribosomally synthesized peptide toxin produced by strains ofBacillus amyloliquefaciensisolated from moisture-damaged buildings, is shown in this paper to have immunotoxic and cytotoxic effects on human cells as well as antagonistic effects on microbes. Human macrophages exposed to 50 ng of amylosin ml−1secreted high levels of cytokines interleukin-1β (IL-1β) and IL-18 within 2 h, indicating activation of the NLRP3 inflammasome, an integral part of the innate immune system. At the same exposure level, expression of IL-1β and IL-18 mRNA increased. Amylosin caused dose-dependent potassium ion efflux from all tested mammalian cells (human monocytes and keratinocytes and porcine sperm cells) at 1 to 2 μM exposure. Amylosin also inhibited the motility of porcine sperm cells and depolarized the mitochondria of human keratinocytes. Amylosin may thus trigger the activation of the NLRP3 inflammasome and subsequently cytokine release by causing potassium efflux from exposed cells. The results of this study indicate that exposure to amylosin activates the innate immune system, which could offer an explanation for the inflammatory symptoms experienced by occupants of moisture-damaged buildings. In addition, the amylosin-producingB. amyloliquefaciensinhibited the growth of both prokaryotic and eukaryotic indoor microbes, and purified amylosin also had an antimicrobial effect. These antimicrobial effects could make amylosin producers dominant and therefore significant causal agents of health problems in some moisture-damaged sites.
Quantitative PCR Analysis of Functional Genes in Iron-Rich Microbial Mats at an Active Hydrothermal Vent System (Lō'ihi Seamount, Hawai'i)
Authors: Jesser Kelsey J. , Fullerton Heather , Hager Kevin W. , Moyer Craig L.
Published: February 2015

The chemolithotrophic
Zetaproteobacteria
represent a novel class of
Proteobacteria
which oxidize Fe(II) to Fe(III) and are the dominant bacterial population in iron-rich microbial mats.
Zetaproteobacteria
were first discovered at Lō'ihi Seamount, located 35 km southeast off the big island of Hawai'i, which is characterized by low-temperature diffuse hydrothermal venting. Novel nondegenerate quantitative PCR (qPCR) assays for genes associated with microbial nitrogen fixation, denitrification, arsenic detoxification, Calvin-Benson-Bassham (CBB), and reductive tricarboxylic acid (rTCA) cycles were developed using selected microbial mat community-derived metagenomes. Nitrogen fixation genes were not detected, but all other functional genes were present. This suggests that arsenic detoxification and denitrification processes are likely cooccurring in addition to two modes of carbon fixation. Two groups of microbial mat community types were identified by terminal restriction fragment length polymorphism (T-RFLP) and were further described based on qPCR data for zetaproteobacterial abundance and carbon fixation mode preference. qPCR variance was associated with mat morphology but not with temperature or sample site. Geochemistry data were significantly associated with sample site and mat morphology. Together, these qPCR assays constitute a functional gene signature for iron microbial mat communities across a broad array of temperatures, mat types, chemistries, and sampling sites at Lō'ihi Seamount.
Disparate Independent Genetic Events Disrupt the Secondary Metabolism Gene
perA
in Certain Symbiotic Epichloë Species
Authors: Berry Daniel , Takach Johanna E. , Schardl Christopher L. , Charlton Nikki D. , Scott Barry , Young Carolyn A.
Published: February 2015

Peramine is an insect-feeding deterrent produced by
Epichloë
species in symbiotic association with C
3
grasses. The
perA
gene responsible for peramine synthesis encodes a two-module nonribosomal peptide synthetase. Alleles of
perA
are found in most
Epichloë
species; however, peramine is not produced by many
perA
-containing
Epichloë
isolates. The genetic basis of these peramine-negative chemotypes is often unknown. Using PCR and DNA sequencing, we analyzed the
perA
genes from 72
Epichloë
isolates and identified causative mutations of
perA
null alleles. We found nonfunctional
perA
-ΔR* alleles, which contain a transposon-associated deletion of the
perA
region encoding the C-terminal reductase domain, are widespread within the
Epichloë
genus and represent a prevalent mutation found in nonhybrid species. Disparate phylogenies of adjacent A2 and T2 domains indicated that the deletion of the reductase domain (R*) likely occurred once and early in the evolution of the genus, and subsequently there have been several recombinations between those domains. A number of novel point, deletion, and insertion mutations responsible for abolishing peramine production in full-length
perA
alleles were also identified. The regions encoding the first and second adenylation domains (A1 and A2, respectively) were common sites for such mutations. Using this information, a method was developed to predict peramine chemotypes by combining PCR product size polymorphism analysis with sequencing of the
perA
adenylation domains.
Mortality of Cutworm Larvae Is Not Enhanced by Agrotis segetum Granulovirus and Agrotis segetum Nucleopolyhedrovirus B Coinfection Relative to Single Infection by Either Virus
Authors: Wennmann Jörg T. , Köhler Tim , Gueli Alletti Gianpiero , Jehle Johannes A.
Published: February 2015

Mixed infections of insect larvae with different baculoviruses are occasionally found. They are of interest from an evolutionary as well as from a practical point of view when baculoviruses are applied as biocontrol agents. Here, we report mixed-infection studies of neonate larvae of the common cutworm,
Agrotis segetum
, with two baculoviruses, Agrotis segetum nucleopolyhedrovirus B (AgseNPV-B) and Agrotis segetum granulovirus (AgseGV). By applying quantitative PCR (qPCR) analysis, coinfections of individual larvae were demonstrated, and occlusion body (OB) production within singly infected and coinfected larvae was determined in individual larvae. Mixtures of viruses did not lead to changes in mortality rates compared with rates of single-virus treatments, indicating an independent action within host larvae under our experimental conditions. AgseNPV-B-infected larvae showed an increase in OB production during 2 weeks of infection, whereas the number of AgseGV OBs did not change from the first week to the second week. Fewer OBs of both viruses were produced in coinfections than in singly infected larvae, suggesting a competition of the two viruses for larval resources. Hence, no functional or economic advantage could be inferred from larval mortality and OB production from mixed infections of
A. segetum
larvae with AgseNPV-B and AgseGV.
Community Dynamics of Arbuscular Mycorrhizal Fungi in High-Input and Intensively Irrigated Rice Cultivation Systems
Authors: Wang Yutao , Li Ting , Li Yingwei , Björn Lars Olof , Rosendahl Søren , Olsson Pål Axel , Li Shaoshan , Fu Xuelin
Published: February 2015

Application of a mycorrhizal inoculum could be one way to increase the yield of rice plants and reduce the application of fertilizer. We therefore studied arbuscular mycorrhizal fungi (AMF) in the roots of wetland rice (
Oryza sativa
L.) collected at the seedling, tillering, heading, and ripening stages in four paddy wetlands that had been under a high-input and intensively irrigated rice cultivation system for more than 20 years. It was found that AMF colonization was mainly established in the heading and ripening stages. The AMF community structure was characterized in rhizosphere soils and roots from two of the studied paddy wetlands. A fragment covering the partial small subunit (SSU), the whole internal transcribed spacer (ITS), and the partial large subunit (LSU) rRNA operon regions of AMF was amplified, cloned, and sequenced from roots and soils. A total of 639 AMF sequences were obtained, and these were finally assigned to 16 phylotypes based on a phylogenetic analysis, including 12 phylotypes from
Glomeraceae
, one phylotype from
Claroideoglomeraceae
, two phylotypes from
Paraglomeraceae
, and one unidentified phylotype. The AMF phylotype compositions in the soils were similar between the two surveyed sites, but there was a clear discrepancy between the communities obtained from root and soil. The relatively high number of AMF phylotypes at the surveyed sites suggests that the conditions are suitable for some species of AMF and that they may have an important function in conventional rice cultivation systems. The species richness of root-colonizing AMF increased with the growth of rice, and future studies should consider the developmental stages of this crop in the exploration of AMF function in paddy wetlands.
Culture-Independent Evaluation of Nonenveloped-Virus Infectivity Reduced by Free-Chlorine Disinfection
Authors: Sano Daisuke , Ohta Takatomo , Nakamura Arata , Nakagomi Toyoko , Nakagomi Osamu , Okabe Satoshi
Published: February 2015

The inability of molecular detection methods to distinguish disinfected virions from infectious ones has hampered the assessment of infectivity for enteric viruses caused by disinfection practices. In the present study, the reduction of infectivity of murine norovirus S7-PP3 and mengovirus vMC0, surrogates of human noroviruses and enteroviruses, respectively, caused by free-chlorine treatment was characterized culture independently by detecting carbonyl groups on viral capsid protein. The amount of carbonyls on viral capsid protein was evaluated by the proportion of biotinylated virions trapped by avidin-immobilized gel (percent adsorbed). This culture-independent approach demonstrated that the percent adsorbed was significantly correlated with the logarithm of the infectious titer of tested viruses. Taken together with the results of previous reports, the result obtained in this study indicates that the amount of carbonyls on viral capsid protein of four important families of waterborne pathogenic viruses,
Astroviridae
,
Reoviridae
,
Caliciviridae
, and
Picornaviridae
, is increased in proportion to the received oxidative stress of free chlorine. There was also a significant correlation between the percent adsorbed and the logarithm of the ratio of genome copy number to PFU, which enables estimation of the infectious titer of a subject virus by measuring values of the total genome copy number and the percent adsorbed. The proposed method is applicable when the validation of a 4-log reduction of viruses, a requirement in U.S. EPA guidelines for virus removal from water, is needed along with clear evidence of the oxidation of virus particles with chlorine-based disinfectants.
Arsenic Methylation and Volatilization by Arsenite
S
-Adenosylmethionine Methyltransferase in Pseudomonas alcaligenes NBRC14159
Authors: Zhang Jun , Cao Tingting , Tang Zhu , Shen Qirong , Rosen Barry P. , Zhao Fang-Jie
Published: February 2015

Inorganic arsenic (As) is highly toxic and ubiquitous in the environment. Inorganic As can be transformed by microbial methylation, which constitutes an important part of the As biogeochemical cycle. In this study, we investigated As biotransformation by
Pseudomonas alcaligenes
NBRC14159.
P. alcaligenes
was able to methylate arsenite [As(III)] rapidly to dimethylarsenate and small amounts of trimethylarsenic oxide. An arsenite
S
-adenosylmethionine methyltransferase, PaArsM, was identified and functionally characterized. PaArsM shares low similarities with other reported ArsM enzymes (<55%). When
P. alcaligenes arsM
gene (
PaarsM
) was disrupted, the mutant lost As methylation ability and became more sensitive to As(III).
PaarsM
was expressed in the absence of As(III) and the expression was further enhanced by As(III) exposure. Heterologous expression of
PaarsM
in an As-hypersensitive strain of
Escherichia coli
conferred As(III) resistance. Purified PaArsM protein methylated As(III) to dimethylarsenate as the main product in the medium and also produced dimethylarsine and trimethylarsine gases. We propose that PaArsM plays a role in As methylation and detoxification of As(III) and could be exploited in bioremediation of As-contaminated environments.
The Host Plant Metabolite Glucose Is the Precursor of Diffusible Signal Factor (DSF) Family Signals in Xanthomonas campestris
Authors: Deng Yinyue , Liu Xiaoling , Wu Ji'en , Lee Jasmine , Chen Shaohua , Cheng Yingying , Zhang Chunyan , Zhang Lian-Hui
Published: February 2015

Plant pathogen
Xanthomonas campestris
pv. campestris produces
cis
-11-methyl-2-dodecenoic acid (diffusible signal factor [DSF]) as a cell-cell communication signal to regulate biofilm dispersal and virulence factor production. Previous studies have demonstrated that DSF biosynthesis is dependent on the presence of RpfF, an enoyl-coenzyme A (CoA) hydratase, but the DSF synthetic mechanism and the influence of the host plant on DSF biosynthesis are still not clear. We show here that exogenous addition of host plant juice or ethanol extract to the growth medium of
X. campestris
pv. campestris could significantly boost DSF family signal production. It was subsequently revealed that
X. campestris
pv. campestris produces not only DSF but also BDSF (
cis
-2-dodecenoic acid) and another novel DSF family signal, which was designated DSF-II. BDSF was originally identified in
Burkholderia cenocepacia
to be involved in regulation of motility, biofilm formation, and virulence in
B. cenocepacia
. Functional analysis suggested that DSF-II plays a role equal to that of DSF in regulation of biofilm dispersion and virulence factor production in
X. campestris
pv. campestris. Furthermore, chromatographic separation led to identification of glucose as a specific molecule stimulating DSF family signal biosynthesis in
X. campestris
pv. campestris.
13
C-labeling experiments demonstrated that glucose acts as a substrate to provide a carbon element for DSF biosynthesis. The results of this study indicate that
X. campestris
pv. campestris could utilize a common metabolite of the host plant to enhance DSF family signal synthesis and therefore promote virulence.
Efficacy and Mechanisms of Murine Norovirus Inhibition by Pulsed-Light Technology
Authors: Vimont Allison , Fliss Ismaïl , Jean Julie
Published: February 2015

Pulsed light is a nonthermal processing technology recognized by the FDA for killing microorganisms on food surfaces, with cumulative fluences up to 12 J cm
−2
. In this study, we investigated its efficacy for inactivating murine norovirus 1 (MNV-1) as a human norovirus surrogate in phosphate-buffered saline, hard water, mineral water, turbid water, and sewage treatment effluent and on food contact surfaces, including high-density polyethylene, polyvinyl chloride, and stainless steel, free or in an alginate matrix. The pulsed-light device emitted a broadband spectrum (200 to 1,000 nm) at a fluence of 0.67 J cm
−2
per pulse, with 2% UV at 8 cm beneath the lamp. Reductions in viral infectivity exceeded 3 log
10
in less than 3 s (5 pulses; 3.45 J cm
−2
) in clear suspensions and on clean surfaces, even in the presence of alginate, and in 6 s (11 pulses; 7.60 J cm
−2
) on fouled surfaces except for stainless steel (2.6 log
10
). The presence of protein or bentonite interfered with viral inactivation. Analysis of the morphology, the viral proteins, and the RNA integrity of treated MNV-1 allowed us to elucidate the mechanisms involved in the antiviral activity of pulsed light. Pulsed light appeared to disrupt MNV-1 structure and degrade viral protein and RNA. The results suggest that pulsed-light technology could provide an effective alternative means of inactivating noroviruses in wastewaters, in clear beverages, in drinking water, or on food-handling surfaces in the presence or absence of biofilms.
Exploring the Enantioselective Mechanism of Halohydrin Dehalogenase from Agrobacterium radiobacter AD1 by Iterative Saturation Mutagenesis
Authors: Guo Chao , Chen Yanpu , Zheng Yu , Zhang Wei , Tao Yunwen , Feng Juan , Tang Lixia
Published: February 2015

Halohydrin dehalogenase from
Agrobacterium radiobacter
AD1 (HheC) shows great potential in producing valuable chiral epoxides and β-substituted alcohols. The wild-type (WT) enzyme displays a high
R
-enantiopreference toward most aromatic substrates, whereas no
S
-selective HheC has been reported to date. To obtain more enantioselective enzymes, seven noncatalytic active-site residues were subjected to iterative saturation mutagenesis (ISM). After two rounds of screening aspects of both activity and enantioselectivity (
E
), three outstanding mutants (Thr134Val/Leu142Met, Leu142Phe/Asn176His, and Pro84Val/Phe86Pro/Thr134Ala/Asn176Ala mutants) with divergent enantioselectivity were obtained. The two double mutants displayed approximately 2-fold improvement in
R
-enantioselectivity toward 2-chloro-1-phenylethanol (2-CPE) without a significant loss of enzyme activity compared with the WT enzyme. Strikingly, the Pro84Val/Phe86Pro/Thr134Ala/Asn176Ala mutant showed an inverted enantioselectivity (from an

E
R

of 65 [WT] to an

E
S

of 101) and approximately 100-fold-enhanced catalytic efficiency toward (
S
)-2-CPE. Molecular dynamic simulation and docking analysis revealed that the phenyl side chain of (
S
)-2-CPE bound at a different location than that of its
R
-counterpart; those mutations generated extra connections for the binding of the favored enantiomer, while the eliminated connections reduced binding of the nonfavored enantiomer, all of which could contribute to the observed inverted enantiopreference.
TonB-Dependent Heme Iron Acquisition in the Tsetse Fly Symbiont Sodalis glossinidius
Authors: Hrusa Gili , Farmer William , Weiss Brian L. , Applebaum Taylor , Roma Jose Santinni , Szeto Lauren , Aksoy Serap , Runyen-Janecky Laura J.
Published: February 2015

Sodalis glossinidiusis an intra- and extracellular symbiont of the tsetse fly (Glossinasp.), which feeds exclusively on vertebrate blood.S. glossinidiusresides in a wide variety of tsetse tissues and may encounter environments that differ dramatically in iron content. TheSodalischromosome encodes a putative TonB-dependent outer membrane heme transporter (HemR) and a putative periplasmic/inner membrane ABC heme permease system (HemTUV). Because these gene products mediate iron acquisition processes by other enteric bacteria, we characterized their regulation and physiological role in theSodalis/tsetse system. Our results show that thehemRandtonBgenes are expressed byS. glossinidiusin the tsetse fly. Furthermore, transcription ofhemRinSodalisis repressed in a high-iron environment by the iron-responsive transcriptional regulator Fur. Expression of theS. glossinidiushemRandhemTUVgenes in anEscherichia colistrain unable to use heme as an iron source stimulated growth in the presence of heme or hemoglobin as the sole iron source. This stimulation was dependent on the presence of either theE. coliorSodalistonBgene.SodalistonBandhemRmutant strains were defective in their ability to colonize the gut of tsetse flies that lacked endogenous symbionts, while wild-typeS. glossinidiusproliferated in this same environment. Finally, we show that theSodalisHemR protein is localized to the bacterial membrane and appears to bind hemin. Collectively, this study provides strong evidence that TonB-dependent, HemR-mediated iron acquisition is important for the maintenance of symbiont homeostasis in the tsetse fly, and it provides evidence for the expression of bacterial high-affinity iron acquisition genes in insect symbionts.
Inhibition of Pseudomonas aeruginosa Swarming Motility by 1-Naphthol and Other Bicyclic Compounds Bearing Hydroxyl Groups
Authors: Oura Hiromu , Tashiro Yosuke , Toyofuku Masanori , Ueda Kousetsu , Kiyokawa Tatsunori , Ito Satoshi , Takahashi Yurika , Lee Seunguk , Nojiri Hideaki , Nakajima-Kambe Toshiaki , Uchiyama Hiroo , Futamata Hiroyuki , Nomura Nobuhiko
Published: February 2015

Many bacteria convert bicyclic compounds, such as indole and naphthalene, to oxidized compounds, including hydroxyindoles and naphthols.
Pseudomonas aeruginosa
, a ubiquitous bacterium that inhabits diverse environments, shows pathogenicity against animals, plants, and other microorganisms, and increasing evidence has shown that several bicyclic compounds alter the virulence-related phenotypes of
P. aeruginosa
. Here, we revealed that hydroxyindoles (4- and 5-hydroxyindoles) and naphthalene derivatives bearing hydroxyl groups specifically inhibit swarming motility but have minor effects on other motilities, including swimming and twitching, in
P. aeruginosa
. Further analyses using 1-naphthol showed that this effect is also associated with clinically isolated hyperswarming
P. aeruginosa
cells. Swarming motility is associated with the dispersion of cells from biofilms, and the addition of 1-naphthol maintained biofilm biomass without cell dispersion. We showed that this 1-naphthol-dependent swarming inhibition is independent of changes of rhamnolipid production and the intracellular level of signaling molecule cyclic-di-GMP (c-di-GMP). Transcriptome analyses revealed that 1-naphthol increases gene expression associated with multidrug efflux and represses gene expression associated with aerotaxis and with pyochelin, flagellar, and pilus synthesis. In the present study, we showed that several bicyclic compounds bearing hydroxyl groups inhibit the swarming motility of
P. aeruginosa
, and these results provide new insight into the chemical structures that inhibit the specific phenotypes of
P. aeruginosa
.
Corynebacterium glutamicum Methionine Sulfoxide Reductase A Uses both Mycoredoxin and Thioredoxin for Regeneration and Oxidative Stress Resistance
Authors: Si Meiru , Zhang Lei , Chaudhry Muhammad Tausif , Ding Wei , Xu Yixiang , Chen Can , Akbar Ali , Shen Xihui , Liu Shuang-Jiang
Published: February 2015

Oxidation of methionine leads to the formation of the
S
and
R
diastereomers of methionine sulfoxide (MetO), which can be reversed by the actions of two structurally unrelated classes of methionine sulfoxide reductase (Msr), MsrA and MsrB, respectively. Although MsrAs have long been demonstrated in numerous bacteria, their physiological and biochemical functions remain largely unknown in
Actinomycetes
. Here, we report that a
Corynebacterium glutamicum
methionine sulfoxide reductase A (CgMsrA) that belongs to the 3-Cys family of MsrAs plays important roles in oxidative stress resistance. Deletion of the
msrA
gene in
C. glutamicum
resulted in decrease of cell viability, increase of ROS production, and increase of protein carbonylation levels under various stress conditions. The physiological roles of CgMsrA in resistance to oxidative stresses were corroborated by its induced expression under various stresses, regulated directly by the stress-responsive extracytoplasmic-function (ECF) sigma factor SigH. Activity assays performed with various regeneration pathways showed that CgMsrA can reduce MetO via both the thioredoxin/thioredoxin reductase (Trx/TrxR) and mycoredoxin 1/mycothione reductase/mycothiol (Mrx1/Mtr/MSH) pathways. Site-directed mutagenesis confirmed that Cys56 is the peroxidatic cysteine that is oxidized to sulfenic acid, while Cys204 and Cys213 are the resolving Cys residues that form an intramolecular disulfide bond. Mrx1 reduces the sulfenic acid intermediate via the formation of an
S
-mycothiolated MsrA intermediate (MsrA-SSM) which is then recycled by mycoredoxin and the second molecule of mycothiol, similarly to the glutathione/glutaredoxin/glutathione reductase (GSH/Grx/GR) system. However, Trx reduces the Cys204-Cys213 disulfide bond in CgMsrA produced during MetO reduction via the formation of a transient intermolecular disulfide bond between Trx and CgMsrA. While both the Trx/TrxR and Mrx1/Mtr/MSH pathways are operative in reducing CgMsrA under stress conditions
in vivo
, the Trx/TrxR pathway alone is sufficient to reduce CgMsrA under normal conditions. Based on these results, a catalytic model for the reduction of CgMsrA by Mrx1 and Trx is proposed.
Solution Structure of Acidocin B, a Circular Bacteriocin Produced by Lactobacillus acidophilus M46
Authors: Acedo Jeella Z. , van Belkum Marco J. , Lohans Christopher T. , McKay Ryan T. , Miskolzie Mark , Vederas John C.
Published: February 2015

Acidocin B, a bacteriocin produced by
Lactobacillus acidophilus
M46, was originally reported to be a linear peptide composed of 59 amino acid residues. However, its high sequence similarity to gassericin A, a circular bacteriocin from
Lactobacillus gasseri
LA39, suggested that acidocin B might be circular as well. Acidocin B was purified from culture supernatant by a series of hydrophobic interaction chromatographic steps. Its circular nature was ascertained by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry and tandem mass spectrometry (MS/MS) sequencing. The peptide sequence was found to consist of 58 amino acids with a molecular mass of 5,621.5 Da. The sequence of the acidocin B biosynthetic gene cluster was also determined and showed high nucleotide sequence similarity to that of gassericin A. The nuclear magnetic resonance (NMR) solution structure of acidocin B in sodium dodecyl sulfate micelles was elucidated, revealing that it is composed of four α-helices of similar length that are folded to form a compact, globular bundle with a central pore. This is a three-dimensional structure for a member of subgroup II circular bacteriocins, which are classified based on their isoelectric points of ∼7 or lower. Comparison of acidocin B with carnocyclin A, a subgroup I circular bacteriocin with four α-helices and a pI of 10, revealed differences in the overall folding. The observed variations could be attributed to inherent diversity in their physical properties, which also required the use of different solvent systems for three-dimensional structural elucidation.
How the Necrotrophic Fungus Alternaria brassicicola Kills Plant Cells Remains an Enigma
Authors: Cho Yangrae
Published: February 2015

Alternaria
species are mainly saprophytic fungi, but some are plant pathogens. Seven pathotypes of
Alternaria alternata
use secondary metabolites of host-specific toxins as pathogenicity factors. These toxins kill host cells prior to colonization. Genes associated with toxin synthesis reside on conditionally dispensable chromosomes, supporting the notion that pathogenicity might have been acquired several times by
A. alternata
.
Alternaria brassicicola
, however, seems to employ a different mechanism. Evidence on the use of host-specific toxins as pathogenicity factors remains tenuous, even after a diligent search aided by full-genome sequencing and efficient reverse-genetics approaches. Similarly, no individual genes encoding lipases or cell wall-degrading enzymes have been identified as strong virulence factors, although these enzymes have been considered important for fungal pathogenesis. This review describes our current understanding of toxins, lipases, and cell wall-degrading enzymes and their roles in the pathogenesis of
A. brassicicola
compared to those of other pathogenic fungi. It also describes a set of genes that affect pathogenesis in
A. brassicicola
. They are involved in various cellular functions that are likely important in most organisms and probably indirectly associated with pathogenesis. Deletion or disruption of these genes results in weakly virulent strains that appear to be sensitive to the defense mechanisms of host plants. Finally, this review discusses the implications of a recent discovery of three important transcription factors associated with pathogenesis and the putative downstream genes that they regulate.
Puf4 Regulates both Splicing and Decay of
HXL1
mRNA Encoding the Unfolded Protein Response Transcription Factor in Cryptococcus neoformans
Authors: Glazier Virginia E. , Kaur Jan Naseer , Brown Nancy T. , Rivera Ashley A. , Panepinto John C.
Published: February 2015

The endoplasmic reticulum (ER) responds to errors in protein folding or processing by induction of the unfolded protein response (UPR). During conditions of ER stress, unconventional splicing of an mRNA encoding the UPR-responsive transcription factor occurs at the ER surface, resulting in activation of the UPR. UPR activation is necessary for adaptation to ER stress and for the pathogenic fungus
Cryptococcus neoformans
is an absolute requirement for temperature adaptation and virulence. In this study, we have determined that
C. neoformans
has co-opted a conserved PUF RNA binding protein to regulate the posttranscriptional processing of the
HXL1
mRNA encoding the UPR transcription factor. PUF elements were identified in both the 5′ and 3′ untranslated regions of the
HXL1
transcript, and both elements bound Puf4. Deletion of
PUF4
resulted in delayed unconventional splicing of
HXL1
mRNA and delayed induction of Hxl1 target genes. In addition, the
HXL1
transcript was stabilized in the absence of Puf4. The
puf4
Δ mutant exhibited temperature sensitivity but was as virulent as the wild type, despite a reduction in fungal burden in the brains of infected mice. Our results reveal a novel regulatory role in which a PUF protein influences the unconventional splicing of the mRNA encoding the UPR-responsive transcription factor. These data suggest a unique role for a PUF protein in controlling UPR kinetics via the posttranscriptional regulation of the mRNA encoding the UPR transcription factor Hxl1.
Psychotherapy for depression in children and adolescents: study protocol for a systematic review and network meta-analysis
Authors: Qin Bin , Zhou Xinyu , Michael Kurt D , Liu Yiyun , Whittington Craig , Cohen David , Zhang Yuqing , Xie Peng
Published: February 2015

Introduction

Depression is common among children and adolescents and is associated with significantly negative effects. A number of structured psychosocial treatments are administered for depression in children and adolescents; however, evidence of their effectiveness is not clear. We describe the protocol of a systematic review and network meta-analysis to evaluate the efficacy, quality of life, tolerability and acceptability of the use of psychological intervention for this young population.

Methods and analysis

We will search PubMed, EMBASE, CENTRAL (the Cochrane Central Register of Controlled Trials), Web of Science, PsycINFO, CINAHL, LiLACS, Dissertation Abstracts, European Association for Grey Literature Exploitation (EAGLE) and the National Technical Information Service (NTIS) from inception to July 2014. There will be no restrictions on language, publication year or publication type. Only randomised clinical trials (RCTs) with psychosocial treatments for depression in children and adolescents will be considered. The primary outcome of efficacy will be the mean overall change of the total score in continuous depression severity scales from baseline to end point. Data will be independently extracted by two reviewers. Traditional pairwise meta–analyses will be performed for studies that directly compared different treatment arms. Then we will perform a Bayesian network meta–analyses to compare the relative efficacy, quality of life, tolerability and acceptability of different psychological intervention. Subgroup analyses will be performed by the age of participants and the duration of psychotherapy, and sensitivity analyses will be conducted to assess the robustness of the findings.

Ethics and dissemination

No ethical issues are foreseen. The results will be published in a peer–reviewed journal and disseminated electronically and in print. The meta–analysis may be updated to inform and guide management of depression in children and adolescents.

Trials registration number

PROSPERO CRD42014010014.
Hip fracture evaluation with alternatives of total hip arthroplasty versus hemiarthroplasty (HEALTH): protocol for a multicentre randomised trial
Authors: Bhandari Mohit , Devereaux P J , Einhorn Thomas A , Thabane Lehana , Schemitsch Emil H , Koval Kenneth J , Frihagen Frede , Poolman Rudolf W , Tetsworth Kevin , Guerra-Farfán Ernesto , Madden Kim , Sprague Sheila , Guyatt Gordon ,   HEALTH Investigators
Published: February 2015

Introduction

Hip fractures are a leading cause of mortality and disability worldwide, and the number of hip fractures is expected to rise to over 6 million per year by 2050. The optimal approach for the surgical management of displaced femoral neck fractures remains unknown. Current evidence suggests the use of arthroplasty; however, there is lack of evidence regarding whether patients with displaced femoral neck fractures experience better outcomes with total hip arthroplasty (THA) or hemiarthroplasty (HA). The HEALTH trial compares outcomes following THA versus HA in patients 50 years of age or older with displaced femoral neck fractures.

Methods and analysis

HEALTH is a multicentre, randomised controlled trial where 1434 patients, 50 years of age or older, with displaced femoral neck fractures from international sites are randomised to receive either THA or HA. Exclusion criteria include associated major injuries of the lower extremity, hip infection(s) and a history of frank dementia. The primary outcome is unplanned secondary procedures and the secondary outcomes include functional outcomes, patient quality of life, mortality and hip-related complications—both within 2 years of the initial surgery. We are using minimisation to ensure balance between intervention groups for the following factors: age, prefracture living, prefracture functional status, American Society for Anesthesiologists (ASA) Class and centre number. Data analysts and the HEALTH Steering Committee are blinded to the surgical allocation throughout the trial. Outcome analysis will be performed using a χ2test (or Fisher's exact test) and Cox proportional hazards modelling estimate. All results will be presented with 95% CIs.

Ethics and dissemination

The HEALTH trial has received local and McMaster University Research Ethics Board (REB) approval (REB#: 06-151).

Results

Outcomes from the primary manuscript will be disseminated through publications in academic journals and presentations at relevant orthopaedic conferences. We will communicate trial results to all participating sites. Participating sites will communicate results with patients who have indicated an interest in knowing the results.

Trial registration number

The HEALTH trial is registered with clinicaltrials.gov (NCT00556842).
Self-rated health and standard risk factors for myocardial infarction: a cohort study
Authors: Waller Göran , Janlert Urban , Norberg Margareta , Lundqvist Robert , Forssén Annika
Published: February 2015

Objective

To investigate the relationship between self-rated health, adjusted for standard risk factors, and myocardial infarction.

Design

Population-based prospective cohort study.

Setting

Enrolment took place between 1990 and 2004 in Västerbotten County, Sweden

Participants

Every year, persons in the total population, aged 40, 50 or 60 were invited. Participation rate was 60%. The cohort consisted of 75 386 men and women. After exclusion for stroke or myocardial infarction before, or within 12 months after enrolment or death within 12 months after enrolment, 72 530 persons remained for analysis. Mean follow-up time was 13.2 years.

Outcome measures

Cox regression analysis was used to estimate HRs for the end point of first non-fatal or fatal myocardial infarction. HR were adjusted for age, sex, systolic blood pressure, total cholesterol, smoking, diabetes, body mass index, education, physical activity and self-rated health in the categories very good; pretty good; somewhat good; pretty poor or poor.

Results

In the cohort, 2062 persons were diagnosed with fatal or non-fatal myocardial infarction. Poor self-rated health adjusted for sex and age was associated with the outcome with HR 2.03 (95% CI 1.45 to 2.84). All categories of self-rated health worse than very good were statistically significant and showed a dose–response relationship. In a multivariable analysis with standard risk factors (not including physical activity and education) HR was attenuated to 1.61 (95% CI 1.13 to 2.31) for poor self-rated health. All categories of self-rated health remained statistically significant. We found no interaction between self-rated health and standard risk factors except for poor self-rated health and diabetes.

Conclusions

This study supports the use of self-rated health as a standard risk factor among others for myocardial infarction. It remains to demonstrate whether self-rated health adds predictive value for myocardial infarction in combined algorithms with standard risk factors.