bluuebunny/crossref_metadata_2025 huggingface dataset:
An Ensemble Approach for Model Bias Prediction
Authors: Xi Zhimin , Zhou Hongjiang , Fu Yan , Yang Ren-Jye
Published: February 2014

Model validation is a process of determining the degree to which a model is an accurate representation of the real world from the perspective of the intended uses of the model. In reliability based design, the intended use of the model is to identify an optimal design with the minimum cost function while satisfying all reliability constraints. It is pivotal that computational models should be validated before conducting the reliability based design. This paper presents an ensemble approach for model bias prediction in order to correct predictions of computational models. The basic idea is to first characterize the model bias of computational models, then correct the model prediction by adding the characterized model bias. The ensemble approach is composed of two prediction mechanisms: 1) response surface of model bias, and 2) Copula modeling of a series of relationships between design variables and the model bias, between model prediction and the model bias. Advantages of both mechanisms are utilized with an accuracy based weighting approach. A vehicle design of front impact example is used to demonstrate the effectiveness of the proposed methodology.
Model Parameter Identification for Non-Fully Controlled Nonlinear Dynamics Systems Using Trajectory Pattern Method
Authors: Feng Dake , Rastegar Jahangir
Published: February 2014

In this paper, a new method is presented for model parameter identification of a large class of nonlinear dynamics systems that are not fully controlled. The method uses trajectory patterns with feed-forward controls to identify model parameters of the system. The developed method ensures full system stability, does not require close initial estimated values for the parameters to be identified, and provides a systematic method of emphasizing on the estimation of the parameters associated with lower order terms of the system dynamics model and gradually upgrading the accuracy with which the model parameters, particularly those associated with the higher order terms of the system dynamics, are estimated. The developed method is based on Trajectory Pattern Method (TPM). The mathematical proof of convergence of the developed method and results of its implementation on a typical system with highly non-linear dynamics are provided.
A Simple Continuation Method for the Solution of Optimal Control Problems With State Variable Inequality Constraints
Authors: Fabien Brian C.
Published: February 2014

This paper develops a simple continuation method for the approximate solution of optimal control problems with pure state variable inequality constraints. The method is based on transforming the inequality constraints into equality constraints using nonnegative slack variables. The resultant equality constraints are satisfied approximately using a quadratic loss penalty function. The solution of the original problem is obtained by solving the transformed problem with a sequence of penalty weights that tends to zero. The penalty weight is treated as the continuation parameter. The necessary conditions for a minimum are written as a boundary value problem involving index-1 differential-algebraic equations (BVP-DAE). The BVP-DAE include the complementarity conditions associated with the inequality constraints. The paper shows that the necessary conditions for optimality of the original problem and the transformed problems are remarkably similar. In particular, the BVP-DAE for each problem differ by a linear term related to the Lagrange multipliers associated with the state variable inequality constraints. Numerical examples are presented to illustrate the efficacy of the proposed technique. Specifically, the paper presents results for; (1) the optimal control of a simplified model of a gantry crane system, (2) the optimal control of a rigid body moving in the vertical plane, and (3) the trajectory optimization of a planar two-link robot. All problems include pure state variable inequality constraints.
Cement Placement Optimization in Femoral Augmentation Using an Evolutionary Algorithm
Authors: Basafa Ehsan , Armand Mehran
Published: February 2014

We used the method of Bi-directional Evolutionary Structural Optimization (BESO) to optimize the cement placement in finite element (FE) models of osteoporotic femur specimens. Two different initial conditions, i.e. no initial cement and fully cemented, were used and both converged to the same optimal cement pattern. On average, BESO predicted that, if optimized, augmentation with only 18.6ml of cement will result in 100% increase in the yield load of the models. Simulations also showed a linear relationship between the volume of the cement and the models’ stiffness and yield load. Models initially filled with cement had a much more uniform stress distribution among the cemented elements when optimized, compared to the starting configuration. Results suggest that restoring the mechanical properties of osteoporotic femurs is possible with minimal and, therefore, potentially safe volumes of cement.
Establishing Consumer Need and Preference for Design of Village Cooking Stoves
Authors: Johnson Nathan G. , Bryden Kenneth M.
Published: February 2014

In some villages the use of wood cooking stoves accounts for more than three-quarters of total village energy use. Because of this the design of clean, affordable, and desirable cooking stoves can have a dramatic impact on human health and the local economy. Unfortunately, too often development projects fail. For example, an estimated 30% of water projects in sub-Saharan Africa have failed prematurely in the last 20 years, and only 10% of cooking stove programs started in the 1980s were operational two years after startup. Similar anecdotal evidence suggests a mixed record of success for other energy, infrastructure, health, and sanitation projects in the developing world. In part, these failures occur because of a lack of design questions and design methods to identify consumer need and preference during the problem definition phase of the product design. Because isolated rural villages are generally far from the design engineers’ previous experiences it is even more important to gather in-depth primary data in isolated rural villages. Based on data collected during in-depth field visits to villages in rural West Africa during a village energy study this paper proposes a structured process for collecting the data necessary to design cookstoves that meet local needs, fit within local contexts, and create an aspirational experience that fosters a sustainable solution.
Computer Assisted Femoral Augmentation: Modeling and Experimental Validation
Authors: Basafa Ehsan , Murphy Ryan J. , Kutzer Michael D. , Otake Yoshito , Armand Mehran
Published: February 2014

We report the results of planning and experimental validation of femoroplasty — augmentation of mechanical properties of the bone using polymethylmethacrylate (PMMA) bone cement injection — on osteoporotic femurs. For six pairs of osteoporotic femurs, finite element (FE) models were created using computed tomography (CT) scan data and an evolutionary method was used to optimize the cement pattern in one of the models from each pair. Using a particle method and the CT data, cement diffusion was modeled for several hypothetical augmentations and the one most closely matching the optimized pattern was chosen as the best plan. We used intra-operative navigation and a custom-designed injection device to deliver the cement into the bones precisely according to the plan. All femurs were then tested mechanically in a configuration simulating a fall to the side. Augmentation with this technique resulted in an increase in the yield load (28%) and yield energy (142%) compared to the control specimens, while only 9.8ml of cement was injected on average. Results support our hypothesis that significant improvements in the mechanical properties of osteoporotic femurs can be achieved by using minimal, and hence safe, amounts of PMMA bone cement.
Developing Metrics for Comparison of Mobile Robots Performing Welding Tasks
Authors: Canfield Stephen L. , Langley Daniel , Shibakov Alexander
Published: February 2014

Developments in mobile robotic systems are leading to new methods and techniques for manufacturing processes in fields that traditionally have not seen much automation. Some of these tasks require process validation prior to use in the manufacturing process. One such example process is welding. However, there is a lack of industry standards for mechanized or robotic welding that can impede the introduction of mobile robotic welding systems in the market place. There is also a lack of generalized fitness measures that gauge the suitability of mobile robot topologies or dimensional designs to a set of tasks and can be used in the design or verification process. This paper will propose such a metric and demonstrate its use in evaluating mobile robot designs for welding tasks. The approach will be based on the representation of a general task as a pair of n-dimensional subsets in the Euclidean n-space. Similarly, the robot capabilities are represented as n-dimensional subsets (manipulability and torque ellipse) in the Euclidean n-space. The motivation is to enable a direct geometric comparison of the capabilities of the robot to the requirements of the task yielding a quantitative measure of fitness. This method is suggested to be well suited to tasks comprised of a relatively short sequence of well-defined motions, called gaits, which are performed repeatedly or in a periodic manner. Some examples are welding, swimming, painting or inspection. The paper will demonstrate the use of this metric in the evaluation and design of mobile robots for welding tasks with a desired set of weld pattern motions. Three mobile welding platforms having different topological kinematic arrangements will be evaluated based on this design verification metric. This metric will further be shown to supplement the weld qualification process through verification of the motion control portions of the weld process based on a specific robot design. The method will contribute to the design and development of mobile robotic welding systems to become viable and accepted manufacturing processes.
Decision Making Tool in Life Cycle Assessment for Packaging Sustainability
Authors: Qi Huihui , Lee Euihark , Gea Hae Chang
Published: February 2014

The Life Cycle Assessment (LCA) is a method to measure the environmental impact of a product’s life stages from the cradle to the grave, and is widely used for packaging sustainability. Although many successful applications using LCA have been reported, the current state of LCA tools still has many limitations. For example, it is difficult to select the best design among the LCA results of design sets. Moreover, the LCA tool cannot implement a decision maker’s preference into the process easily.

To overcome these limitations, we developed a decision making tool using LCA for packaging sustainability. First, Pareto Active Set Selection (PASS) method is proposed to find Pareto Front of packaging options. Additionally, Design Preference Function (DPF) is introduced to implement the designer’s preference for selecting the best packaging options. Case studies are presented to demonstrate this decision making tool.
Three-Dimensional Modeling of Container Cranes
Authors: Arena Andrea , Casalotti Arnaldo , Lacarbonara Walter , Cartmell Matthew P.
Published: February 2014

This work deals with three-dimensional (3D) modeling of container cranes including the hoisting cable length commands. The proposed models allow to effectively study the 3D motion caused by the eccentricity of initial conditions or loading conditions such as those induced by wind. The container is modeled as a 3D rigid body while the hoisting cables are treated either as inextensible trusses or as linearly elastic straight, taut cables. The 3D model with inextensible cables is shown to coalesce into existing two-dimensional models under the relevant planarity constraints. Details about the treatment of the internal inextensibility constraints are discussed. Time-marching simulations are carried out to show representative 2D and 3D responses to initial conditions and commanded motion of the trolley. The main differences between the constrained model and that with the elasticity of the cables are highlighted in the framework of a few significant design scenarios.
Reconstructing Humans’ Hand Motion: Preliminary Results and Applications in the Design of Mechanical Fingers for Anthropomorphic Tasks
Authors: Ghosh Shramana , Moon Hyosang , Robson Nina P.
Published: February 2014

This paper reports the development of a low-cost sensor-based glove device using commercially available components that can be used to obtain position, velocity and acceleration data for individual fingers of the hand. Optical tracking of the human hand and finger motion is a challenging task due to the large number of degrees of freedom (DOFs) packed in a relatively small space. We propose methods to simplify the hand motion capture by utilizing accelerometers and adopting a reduced marker protocol.

The preliminary results show that the use of relative position, velocity and acceleration homogeneous transformations enable us in getting improved finger motion data with respect to those obtained from a Vicon system. This data is directly related to contact and curvature constraints between the fingers and the grasped object. Once obtained from the glove, the higher derivative specifications are used in formulating the synthesis task for the design of robotic fingers.
The CasKR Two-Component System Is Required for the Growth of Mesophilic and Psychrotolerant Bacillus cereus Strains at Low Temperatures
Authors: Diomandé Sara Esther , Chamot Stéphanie , Antolinos Vera , Vasai Florian , Guinebretière Marie-Hélène , Bornard Isabelle , Nguyen-the Christophe , Broussolle Véronique , Brillard Julien
Published: February 2014

The different strains of
Bacillus cereus
can grow at temperatures covering a very diverse range. Some
B. cereus
strains can grow in chilled food and consequently cause food poisoning. We have identified a new sensor/regulator mechanism involved in low-temperature
B. cereus
growth. Construction of a mutant of this two-component system enabled us to show that this system, called CasKR, is required for growth at the minimal temperature (
T
min
). CasKR was also involved in optimal cold growth above
T
min
and in cell survival below
T
min
. Microscopic observation showed that CasKR plays a key role in cell shape during cold growth. Introducing the
casKR
genes in a Δ
casKR
mutant restored its ability to grow at
T
min
. Although it was first identified in the ATCC 14579 model strain, this mechanism has been conserved in most strains of the
B. cereus
group. We show that the role of CasKR in cold growth is similar in other
B. cereus sensu lato
strains with different growth temperature ranges, including psychrotolerant strains.
Deciphering the Regulon of Streptomyces coelicolor AbrC3, a Positive Response Regulator of Antibiotic Production
Authors: Rico Sergio , Santamaría Ramón I. , Yepes Ana , Rodríguez Héctor , Laing Emma , Bucca Giselda , Smith Colin P. , Díaz Margarita
Published: February 2014

The atypical two-component system (TCS) AbrC1/C2/C3 (encoded by
SCO4598
,
SCO4597
, and
SCO4596
), comprising two histidine kinases (HKs) and a response regulator (RR), is crucial for antibiotic production in
Streptomyces coelicolor
and for morphological differentiation under certain nutritional conditions. In this study, we demonstrate that deletion of the RR-encoding gene,
abrC3
(
SCO4596
), results in a dramatic decrease in actinorhodin (ACT) and undecylprodiginine (RED) production and delays morphological development. In contrast, the overexpression of
abrC3
in the parent strain leads to a 33% increase in ACT production in liquid medium. Transcriptomic analysis and chromatin immunoprecipitation with microarray technology (ChIP-chip) analysis of the Δ
abrC3
mutant and the parent strain revealed that AbrC3 directly controls ACT production by binding to the
actII-ORF4
promoter region; this was independently verified by
in vitro
DNA-binding assays. This binding is dependent on the sequence 5′-GAASGSGRMS-3′. In contrast, the regulation of RED production is not due to direct binding of AbrC3 to either the
redZ
or
redD
promoter region. This study also revealed other members of the AbrC3 regulon: AbrC3 is a positive autoregulator which also binds to the promoter regions of
SCO0736
,
bdtA
(
SCO3328
),
absR1
(
SCO6992
), and
SCO6809
. The direct targets share the 10-base consensus binding sequence and may be responsible for some of the phenotypes of the Δ
abrC3
mutant. The identification of the AbrC3 regulon as part of the complex regulatory network governing antibiotic production widens our knowledge regarding TCS involvement in control of antibiotic synthesis and may contribute to the rational design of new hyperproducer host strains through genetic manipulation of such systems.
Lactose-Inducible System for Metabolic Engineering of Clostridium ljungdahlii
Authors: Banerjee Areen , Leang Ching , Ueki Toshiyuki , Nevin Kelly P. , Lovley Derek R.
Published: February 2014

The development of tools for genetic manipulation of
Clostridium ljungdahlii
has increased its attractiveness as a chassis for autotrophic production of organic commodities and biofuels from syngas and microbial electrosynthesis and established it as a model organism for the study of the basic physiology of acetogenesis. In an attempt to expand the genetic toolbox for
C. ljungdahlii
, the possibility of adapting a lactose-inducible system for gene expression, previously reported for
Clostridium perfringens
, was investigated. The plasmid pAH2, originally developed for
C. perfringens
with a
gusA
reporter gene, functioned as an effective lactose-inducible system in
C. ljungdahlii
. Lactose induction of
C. ljungdahlii
containing pB1, in which the gene for the aldehyde/alcohol dehydrogenase AdhE1 was downstream of the lactose-inducible promoter, increased expression of
adhE1
30-fold over the wild-type level, increasing ethanol production 1.5-fold, with a corresponding decrease in acetate production. Lactose-inducible expression of
adhE1
in a strain in which
adhE1
and the
adhE1
homolog
adhE2
had been deleted from the chromosome restored ethanol production to levels comparable to those in the wild-type strain. Inducing expression of
adhE2
similarly failed to restore ethanol production, suggesting that
adhE1
is the homolog responsible for ethanol production. Lactose-inducible expression of the four heterologous genes necessary to convert acetyl coenzyme A (acetyl-CoA) to acetone diverted ca. 60% of carbon flow to acetone production during growth on fructose, and 25% of carbon flow went to acetone when carbon monoxide was the electron donor. These studies demonstrate that the lactose-inducible system described here will be useful for redirecting carbon and electron flow for the biosynthesis of products more valuable than acetate. Furthermore, this tool should aid in optimizing microbial electrosynthesis and for basic studies on the physiology of acetogenesis.
Removal of Pharmaceuticals and Personal Care Products during Water Recycling: Microbial Community Structure and Effects of Substrate Concentration
Authors: Onesios-Barry Kathryn M. , Berry David , Proescher Jody B. , Sivakumar I. K. Ashok , Bouwer Edward J.
Published: February 2014

Many pharmaceuticals and personal care products (PPCPs) have been shown to be biotransformed in water treatment systems. However, little research exists on the effect of initial PPCP concentration on PPCP biotransformation or on the microbial communities treating impacted water. In this study, biological PPCP removal at various concentrations was assessed using laboratory columns inoculated with wastewater treatment plant effluent. Pyrosequencing was used to examine microbial communities in the columns and in soil from a soil aquifer treatment (SAT; a method of water treatment prior to reuse) site. Laboratory columns were supplied with different concentrations (0.25, 10, 100, or 1,000 μg liter
−1
) of each of 15 PPCPs. Five PPCPs (4-isopropyl-3-methylphenol [biosol],
p
-chloro-
m
-xylenol, gemfibrozil, ketoprofen, and phenytoin) were not removed at any tested concentrations. Two PPCPs (naproxen and triclosan) exhibited removals independent of PPCP concentration. PPCP removal efficiencies were dependent on initial concentrations for biphenylol,
p
-chloro-
m
-cresol, chlorophene, diclofenac, 5-fluorouracil, ibuprofen, and valproic acid, showing that PPCP concentration can affect biotransformation. Biofilms from sand samples collected from the 0.25- and 10-μg liter
−1
PPCP columns were pyrosequenced along with SAT soil samples collected on three consecutive days of a wetting and drying cycle to enable comparison of these two communities exposed to PPCPs. SAT communities were similar to column communities in taxonomy and phylotype composition, and both were found to contain close relatives of known PPCP degraders. The efficiency of biological removal of PPCPs was found to be dependent on the concentration at which the contamination occurs for some, but not all, PPCPs.
Characterization of an Allylic/Benzyl Alcohol Dehydrogenase from Yokenella sp. Strain WZY002, an Organism Potentially Useful for the Synthesis of α,β-Unsaturated Alcohols from Allylic Aldehydes and Ketones
Authors: Ying Xiangxian , Wang Yifang , Xiong Bin , Wu Tingting , Xie Liping , Yu Meilan , Wang Zhao
Published: February 2014

A novel whole-cell biocatalyst with high allylic alcohol-oxidizing activities was screened and identified as
Yokenella
sp. WZY002, which chemoselectively reduced the C=O bond of allylic aldehydes/ketones to the corresponding α,β-unsaturated alcohols at 30°C and pH 8.0. The strain also had the capacity of stereoselectively reducing aromatic ketones to (
S
)-enantioselective alcohols. The enzyme responsible for the predominant allylic/benzyl alcohol dehydrogenase activity was purified to homogeneity and designated YsADH (alcohol dehydrogenase from
Yokenella
sp.), which had a calculated subunit molecular mass of 36,411 Da. The gene encoding YsADH was subsequently expressed in
Escherichia coli
, and the purified recombinant YsADH protein was characterized. The enzyme strictly required NADP(H) as a coenzyme and was putatively zinc dependent. The optimal pH and temperature for crotonaldehyde reduction were pH 6.5 and 65°C, whereas those for crotyl alcohol oxidation were pH 8.0 and 55°C. The enzyme showed moderate thermostability, with a half-life of 6.2 h at 55°C. It was robust in the presence of organic solvents and retained 87.5% of the initial activity after 24 h of incubation with 20% (vol/vol) dimethyl sulfoxide. The enzyme preferentially catalyzed allylic/benzyl aldehydes as the substrate in the reduction of aldehydes/ketones and yielded the highest activity of 427 U mg
−1
for benzaldehyde reduction, while the alcohol oxidation reaction demonstrated the maximum activity of 79.9 U mg
−1
using crotyl alcohol as the substrate. Moreover, kinetic parameters of the enzyme showed lower

K
m

values and higher catalytic efficiency for crotonaldehyde/benzaldehyde and NADPH than for crotyl alcohol/benzyl alcohol and NADP
+
, suggesting the nature of being an aldehyde reductase.
Low-Molecular-Weight Metabolites Secreted by Paenibacillus larvae as Potential Virulence Factors of American Foulbrood
Authors: Schild Hedwig-Annabell , Fuchs Sebastian W. , Bode Helge B. , Grünewald Bernd
Published: February 2014

The spore-forming bacterium
Paenibacillus larvae
causes a severe and highly infective bee disease, American foulbrood (AFB). Despite the large economic losses induced by AFB, the virulence factors produced by
P. larvae
are as yet unknown. To identify such virulence factors, we experimentally infected young, susceptible larvae of the honeybee,
Apis mellifera carnica
, with different
P. larvae
isolates. Honeybee larvae were reared
in vitro
in 24-well plates in the laboratory after isolation from the brood comb. We identified genotype-specific differences in the etiopathology of AFB between the tested isolates of
P. larvae
, which were revealed by differences in the median lethal times. Furthermore, we confirmed that extracts of
P. larvae
cultures contain low-molecular-weight compounds, which are toxic to honeybee larvae. Our data indicate that
P. larvae
secretes metabolites into the medium with a potent honeybee toxic activity pointing to a novel pathogenic factor(s) of
P. larvae
. Genome mining of
P. larvae
subsp.
larvae
BRL-230010 led to the identification of several biosynthesis gene clusters putatively involved in natural product biosynthesis, highlighting the potential of
P. larvae
to produce such compounds.
The Stringent Response Modulates 4-Hydroxy-2-Alkylquinoline Biosynthesis and Quorum-Sensing Hierarchy in Pseudomonas aeruginosa
Authors: Schafhauser James , Lepine Francois , McKay Geoffrey , Ahlgren Heather G. , Khakimova Malika , Nguyen Dao
Published: February 2014

As a ubiquitous environmental organism and an important human pathogen,
Pseudomonas aeruginosa
readily adapts and responds to a wide range of conditions and habitats. The intricate regulatory networks that link quorum sensing and other global regulators allow
P. aeruginosa
to coordinate its gene expression and cell signaling in response to different growth conditions and stressors. Upon nutrient transitions and starvation, as well as other environmental stresses, the stringent response is activated, mediated by the signal (p)ppGpp.
P. aeruginosa
produces a family of molecules called HAQ (4-hydroxy-2-alkylquinolines), some of which exhibit antibacterial and quorum-sensing signaling functions and regulate virulence genes. In this study, we report that (p)ppGpp negatively regulates HAQ biosynthesis: in a (p)ppGpp-null (ΔSR) mutant, HHQ (4-hydroxyl-2-heptylquinoline) and PQS (3,4-dihydroxy-2-heptylquinoline) levels are increased due to upregulated
pqsA
and
pqsR
expression and reduced repression by the
rhl
system. We also found that (p)ppGpp is required for full expression of both
rhl
and
las
AHL (acyl-homoserine lactone) quorum-sensing systems, since the ΔSR mutant has reduced
rhlI
,
rhlR
,
lasI
, and
lasR
expression, butanoyl-homoserine lactone (C
4
-HSL) and 3-oxo-dodecanoyl-homoserine lactone (3-oxo-C
12
-HSL) levels, and rhamnolipid and elastase production. Furthermore, (p)ppGpp significantly modulates the AHL and PQS quorum-sensing hierarchy, as the
las
system no longer has a dominant effect on HAQ biosynthesis when the stringent response is inactivated.
Identification of an
O
-Acyltransferase Gene (
oacB
) That Mediates 3- and 4-O-Acetylation of Rhamnose III in Shigella flexneri O Antigens
Authors: Wang Jianping , Knirel Yuriy A. , Lan Ruiting , Senchenkova Sof'ya N. , Luo Xia , Perepelov Andrei V. , Wang Yiting , Shashkov Alexander S. , Xu Jianguo , Sun Qiangzheng
Published: February 2014

O antigen (O polysaccharide) is an important and highly variable cell component present on the surface of cells which defines the serospecificity of Gram-negative bacteria. Most O antigens of
Shigella flexneri
, a cause of shigellosis, share a backbone composed of →2)-α-
l
-Rha
p
III
-(1→2)-α-
l
-Rha
p
II
-(1→3)-α-
l
-Rha
p
I
-(1→3)-β-
d
-Glc
p
NAc-(1→ repeats, which can be modified by adding various substituents, giving rise to 19 serotypes. The known modifications include glucosylation on various sugar residues, O-acetylation on Rha
I
, and phosphorylation with phosphoethanolamine on Rha
II
or/and Rha
III
. Recently, two new O-antigen modifications, namely, O-acetylation at position 3 or 4 of Rha
III
and position 6 of GlcNAc, have been identified in several
S. flexneri
serotypes. In this work, the genetic basis for the 3/4-O-acetylation on Rha
III
was elucidated. Bioinformatic analysis of the genome of
S. flexneri
serotype 2a strain Sf301, which carries 3/4-O-acetylation on Rha
III
, revealed an O-acyltransferase gene designated
oacB
. Genetic studies combined with O-antigen structure analysis demonstrated that this gene is responsible for the 3/4-O-acetylation in serotypes 1a, 1b, 2a, 5a, and Y but not serotype 6, which has a different O-antigen backbone structure. The
oacB
gene is carried by a transposon-like structure located in the
proA-adrA
region on the chromosome, which represents a novel mechanism of mobilization of O-antigen modification factors in
S. flexneri
. These findings enhance our knowledge of
S. flexneri
O-antigen modifications and shed light on the origin of new O-antigen variants.
Localization of P42 and F
1
-ATPase α-Subunit Homolog of the Gliding Machinery in Mycoplasma mobile Revealed by Newly Developed Gene Manipulation and Fluorescent Protein Tagging
Authors: Tulum Isil , Yabe Masaru , Uenoyama Atsuko , Miyata Makoto
Published: February 2014

Mycoplasma mobile
has a unique mechanism that enables it to glide on solid surfaces faster than any other gliding mycoplasma. To elucidate the gliding mechanism, we developed a transformation system for
M. mobile
based on a transposon derived from Tn
4001
. Modification of the electroporation conditions, outgrowth time, and colony formation from the standard method for
Mycoplasma
species enabled successful transformation. A fluorescent-protein tagging technique was developed using the enhanced yellow fluorescent protein (EYFP) and applied to two proteins that have been suggested to be involved in the gliding mechanism: P42 (MMOB1050), which is transcribed as continuous mRNA with other proteins essential for gliding, and a homolog of the F
1
-ATPase α-subunit (MMOB1660). Analysis of the amino acid sequence of P42 by PSI-BLAST suggested that P42 evolved from a common ancestor with FtsZ, the bacterial tubulin homologue. The roles of P42 and the F
1
-ATPase subunit homolog are discussed as part of our proposed gliding mechanism.
A Sporulation Factor Is Involved in the Morphological Change of Clostridium perfringens Biofilms in Response to Temperature
Authors: Obana Nozomu , Nakamura Kouji , Nomura Nobuhiko
Published: February 2014

Biofilm formation has been associated with bacterial pathogenesis, such as nosocomial and chronic infections, as the resistance of biofilms to environmental stresses has increased.
Clostridium perfringens
is a Gram-positive spore-forming anaerobic pathogen. This organism survives antibiotic treatment through the formation of biofilms or spores, but the environmental and regulatory factors involved in the biofilm formation remain unclear. Here, we observed that temperature regulates
C. perfringens
biofilm morphology. At 37°C,
C. perfringens
adhered to the substrate surface and formed a flat, thin biofilm, herein referred to as adhered biofilm. However, at 25°C, this bacterium did not adhere and produced a threadlike extracellular matrix, forming a viscous, thick biofilm, herein referred to as pellicle biofilm. Pellicle biofilm formation requires the sporulation master regulator, Spo0A, and the toxin regulator, CtrAB, and is enhanced in the absence of the global repressor, AbrB. These transcriptional regulator genes are regulated by each other and temperature. Adhered-biofilm formation requires AbrB and
pilA2
, which encodes a component of type IV pili (TFP). TFP expression was activated at 37°C and regulated through Spo0A, AbrB, and CtrAB. These results indicate that the morphology of
C. perfringens
biofilm is dependent on temperature through the differential production of extracellular matrix and the activity of TFP. Moreover, pellicle biofilm formation is involved in sporulation and toxin production. Here, we demonstrated that clostridial biofilm formation is closely associated with sporulation and that the morphological change of the biofilms could play an important role in the pathogenesis of this organism.
Plasmid Conjugation from Proteobacteria as Evidence for the Origin of Xenologous Genes in Cyanobacteria
Authors: Encinas David , Garcillán-Barcia M. Pilar , Santos-Merino María , Delaye Luis , Moya Andrés , de la Cruz Fernando
Published: February 2014

Comparative genomics have shown that 5% of
Synechococcus elongatus
PCC 7942 genes are of probable proteobacterial origin. To investigate the role of interphylum conjugation in cyanobacterial gene acquisition, we tested the ability of a set of prototype proteobacterial conjugative plasmids (RP4, pKM101, R388, R64, and F) to transfer DNA from
Escherichia coli
to
S. elongatus
. A series of BioBrick-compatible, mobilizable shuttle vectors was developed. These vectors were based on the putative origin of replication of the
Synechococcus
resident plasmid pANL. Not only broad-host-range plasmids, such as RP4 and R388, but also narrower-host-range plasmids, such as pKM101, all encoding MPF
T
-type IV secretion systems, were able to transfer plasmid DNA from
E. coli
to
S. elongatus
by conjugation. Neither MPF
F
nor MPF
I
could be used as interphylum DNA delivery agents. Reciprocally, pANL-derived cointegrates could be introduced in
E. coli
by electroporation, where they conferred a functional phenotype. These results suggest the existence of potentially ample channels of gene flow between proteobacteria and cyanobacteria and point to MPF
T
-based interphylum conjugation as a potential mechanism to explain the proteobacterial origin of a majority of
S. elongatus
xenologous genes.
Cyclic AMP Receptor Protein Regulates
cspD
, a Bacterial Toxin Gene, in Escherichia coli
Authors: Uppal Sheetal , Shetty Deeksha M. , Jawali Narendra
Published: February 2014

cspD
, a member of
cspA
family of cold shock genes in
Escherichia coli
, is not induced during cold shock. Its expression is induced during stationary phase. CspD inhibits DNA replication, and a high level of the protein is toxic to cells. Recently, CspD was proposed to be associated with persister cell formation in
E. coli
. Here, we show that cyclic AMP receptor protein (CRP) upregulates
cspD
transcription. Sequence analysis of the
cspD
upstream region revealed two tandem CRP target sites, CRP site-I (the proximal site centered at −83.5 with respect to the transcription start) and CRP site-II (the distal site centered at −112.5). The results from electrophoretic mobility shift assays showed that CRP indeed binds to these two target sites in P
cspD
. The promoter-proximal CRP target site was found to play a major role in P
cspD
activation by CRP, as studied by transcriptional fusions carrying mutations in the target sites. The results from
in vitro
transcription assays demonstrated that CRP activates P
cspD
transcription in the absence of additional factors other than RNA polymerase. The requirement for activating region 1 of CRP in P
cspD
activation, along with the involvement of the 287, 265, and 261 determinants of the α-CTD, suggest that CRP activates by a class I-type mechanism. However, only moderate activation
in vitro
was observed compared to high activation
in vivo
, suggesting there might be additional activators of P
cspD
. Overall, our findings show that CRP, a global metabolic regulator in
E. coli
, activates a gene potentially related to persistence.
The Hypervariable Region of Meningococcal Major Pilin PilE Controls the Host Cell Response via Antigenic Variation
Authors: Miller Florence , Phan Gilles , Brissac Terry , Bouchiat Coralie , Lioux Ghislaine , Nassif Xavier , Coureuil Mathieu
Published: February 2014

T
ype IV pili (Tfp) are expressed by many Gram-negative bacteria to promote aggregation, adhesion, internalization, twitching motility, or natural transformation. Tfp of
Neisseria meningitidis
, the causative agent of cerebrospinal meningitis, are involved in the colonization of human nasopharynx. After invasion of the bloodstream, Tfp allow adhesion of
N. meningitidis
to human endothelial cells, which leads to the opening of the blood-brain barrier and meningitis. To achieve firm adhesion,
N. meningitidis
induces a host cell response that results in elongation of microvilli surrounding the meningococcal colony. Here we study the role of the major pilin subunit PilE during host cell response using human dermal microvascular endothelial cells and the pharynx carcinoma-derived FaDu epithelial cell line. We first show that some PilE variants are unable to induce a host cell response. By engineering PilE mutants, we observed that the PilE C-terminus domain, which contains a disulfide bonded region (D-region), is critical for the host cell response and that hypervariable regions confer different host cell specificities. Moreover, the study of point mutants of the pilin D-region combined with structural modeling of PilE revealed that the D-region contains two independent regions involved in signaling to human dermal microvascular endothelial cells (HDMECs) or FaDu cells. Our results indicate that the diversity of the PilE D-region sequence allows the induction of the host cell response via several receptors. This suggests that
Neisseria meningitidis
has evolved a powerful tool to adapt easily to many niches by modifying its ability to interact with host cells.

IMPORTANCE
Type IV pili (Tfp) are long appendages expressed by many Gram-negative bacteria, including
Neisseria meningitidis
, the causative agent of cerebrospinal meningitis. These pili are involved in many aspects of pathogenesis: natural competence, aggregation, adhesion, and twitching motility. More specifically,
Neisseria meningitidis
, which is devoid of a secretion system to manipulate its host, has evolved its Tfp to signal to brain endothelial cells and open the blood-brain barrier. In this report, we investigate, at the molecular level, the involvement of the major pilin subunit PilE in host cell response. Our results indicate that the PilE C-terminal domain, which contains a disulfide bonded region (D-region), is critical for the host cell response and contains two independent regions involved in host cell signaling.
Haemophilus ducreyi Hfq Contributes to Virulence Gene Regulation as Cells Enter Stationary Phase
Authors: Gangaiah Dharanesh , Labandeira-Rey Maria , Zhang Xinjun , Fortney Kate R. , Ellinger Sheila , Zwickl Beth , Baker Beth , Liu Yunlong , Janowicz Diane M. , Katz Barry P. , Brautigam Chad A. , Munson Robert S. , Hansen Eric J. , Spinola Stanley M.
Published: February 2014

To adapt to stresses encountered in stationary phase, Gram-negative bacteria utilize the alternative sigma factor RpoS. However, some species lack RpoS; thus, it is unclear how stationary-phase adaptation is regulated in these organisms. Here we defined the growth-phase-dependent transcriptomes of
Haemophilus ducreyi
, which lacks an RpoS homolog. Compared to mid-log-phase organisms, cells harvested from the stationary phase upregulated genes encoding several virulence determinants and a homolog of
hfq
. Insertional inactivation of
hfq
altered the expression of ~16% of the
H. ducreyi
genes. Importantly, there were a significant overlap and an inverse correlation in the transcript levels of genes differentially expressed in the
hfq
inactivation mutant relative to its parent and the genes differentially expressed in stationary phase relative to mid-log phase in the parent. Inactivation of
hfq
downregulated genes in the
flp-tad
and
lspB-lspA2
operons, which encode several virulence determinants. To comply with FDA guidelines for human inoculation experiments, an unmarked
hfq
deletion mutant was constructed and was fully attenuated for virulence in humans. Inactivation or deletion of
hfq
downregulated Flp1 and impaired the ability of
H. ducreyi
to form microcolonies, downregulated DsrA and rendered
H. ducreyi
serum susceptible, and downregulated LspB and LspA2, which allow
H. ducreyi
to resist phagocytosis. We propose that, in the absence of an RpoS homolog, Hfq serves as a major contributor of
H. ducreyi
stationary-phase and virulence gene regulation. The contribution of Hfq to stationary-phase gene regulation may have broad implications for other organisms that lack an RpoS homolog.

IMPORTANCE
Pathogenic bacteria encounter a wide range of stresses in their hosts, including nutrient limitation; the ability to sense and respond to such stresses is crucial for bacterial pathogens to successfully establish an infection. Gram-negative bacteria frequently utilize the alternative sigma factor RpoS to adapt to stresses and stationary phase. However, homologs of RpoS are absent in some bacterial pathogens, including
Haemophilus ducreyi
, which causes chancroid and facilitates the acquisition and transmission of HIV-1. Here, we provide evidence that, in the absence of an RpoS homolog, Hfq serves as a major contributor of stationary-phase gene regulation and that Hfq is required for
H. ducreyi
to infect humans. To our knowledge, this is the first study describing Hfq as a major contributor of stationary-phase gene regulation in bacteria and the requirement of Hfq for the virulence of a bacterial pathogen in humans.
Determinants of concurrent sexual partnerships within stable relationships: a qualitative study in Tanzania
Authors: Cox Carie Muntifering , Babalola Stella , Kennedy Caitlin E , Mbwambo Jessie , Likindikoki Samuel , Kerrigan Deanna
Published: February 2014

Objective

Concurrent sexual partnerships (CP) have been identified as a potential driver in the HIV epidemic in southern Africa, making it essential to understand motivating factors for engagement in CP. We aimed to assess community attitudes and beliefs about relationship factors that influence men and women in stable relationships to engage in CP in Tanzania. Social exchange theory was used for interpreting the data.

Design

Qualitative study with focus group discussions (FGDs).

Setting

Semiurban/rural communities in four regions across Tanzania (Dar es Salaam, Shinyanga, Iringa and Mbeya).

Participants

120 women aged 17–45 years and 111 men aged 18–49 years from four study areas participated in 32 FGDs.

Outcome measures

FGD participants were asked the following questions about CP: definitions and types, motivations and justifications for engaging or not engaging, cultural factors, gender and socialisation, and local resources and efforts available for addressing CP. Our analysis focused specifically on beliefs about how relationship factors influence engagement in CP.

Results

Dissatisfaction with a stable relationship was believed to be a contributing factor for engagement in CP for both men and women. Participants more commonly reported financial dissatisfaction as a contributing factor for women engaging in CP within stable relationships, whereas emotional and sexual dissatisfaction were reported as contributing factors for men and women. Furthermore, participants described how potential outside partners are often evaluated based on what they are able to offer compared with stable partners.

Conclusions

Efforts to reach men and women in stable relationships with HIV prevention messages must consider the various dimensions of motivation for engaging in CP, including relationship dynamics.
Relationship between patient reported experience (PREMs) and patient reported outcomes (PROMs) in elective surgery
Authors: Black Nick , Varaganum Mira , Hutchings Andrew
Published: February 2014

Objective

Our aim was to see if the reporting of better experiences by elective surgical patients was associated with better outcomes (effectiveness and safety). The objectives were to: describe the distribution of experience scores and any association with patients’ characteristics; determine the relationship of experience with effectiveness and with safety; and explore the influence of patient characteristics, year and provider on the relationship between experience and effectiveness.

Methods

Patients undergoing one of three procedures from 2010 to 2012 in England who completed a patient reported outcome measure (PROM) questionnaire before and after surgery and a patient reported experience measure (PREM) questionnaire. Data on 4089 hip replacement patients, 4501 knee replacements and 1793 groin hernia repairs. Regression analysis was used to examine associations between disease-specific and generic PROMs and PREMs.

Results

There was a weak positive association between experience and effectiveness for all three procedures (correlation coefficient with disease-specific PROMs for hip and knee replacements 0.2 and with EQ-5D 0.1 for all three procedures). The aspect of experience most strongly associated with a better outcome was the level of communication with and trust in their doctor. A higher experience score of 1 SD (about 1.5 on a 10-point scale) was associated with about 30% less likelihood of the patient reporting a complication. There was no difference between the eight dimensions of experience.

All the relationships observed were consistent over time, between different types of patients (age, sex, socioeconomic status) and between providers.

Conclusions

Patients distinguish between the three domains of quality when reporting their experience and outcome. If the weak positive associations between domains were shown to be causal, there would be implications for maximising performance measures for providers.
Elective treatment of intracranial stenosis with the balloon-expandable Pharos Vitesse stent: 30-day stroke rate and complications
Authors: von Schoenfeldt Patrick , Krützelmann Anna , Bußmeyer Matthias , Mpotsaris Anastasios , Rosenkranz Michael , Fiehler Jens , Weber Werner , Buhk Jan-Hendrik
Published: February 2014

Background and purpose

Analysis of procedural results and 30-day outcome after intracranial angioplasty and stenting (ICAS) with the balloon-expandable Pharos Vitesse stent system in carefully selected high-risk patients in two high-volume neurovascular centers.

Materials and methods

92 patients scheduled for elective ICAS using Pharos Vitesse between August, 2008 and August, 2011 were included. All patients showed high-grade intracranial stenosis and recurrent ischemic events despite best medical treatment at that time. The stroke rates and complications were divided into procedural and 30-day short-term events.

Results

Successful stent placement was achieved in all but one patient. Ischemic procedural complications occurred in three subjects. 30-Day complications and strokes were seen in four patients: two minor ischemic strokes, one fatal hemorrhage and one non-stroke-related death. Overall, strokes occurred in 6 out of 92 patients (6.5%, 95% CI 3.0% to 13.5%). The total stroke and death rate was 7.6% (95% CI 3.7% to 14.9%). No significant correlation with previously reported risk factors could be found, although a higher rate of ischemic strokes (four out of five) in the posterior circulation was recorded.

Conclusions

In patients with intracranial stenosis who experience recurrent ischemic events despite best medical treatment, ICAS, using the balloon-expandable Pharos Vitesse stent, may still be considered as an individual treatment option in high-volume neurovascular centers.
The threat of midge‐borne equine disease: investigation of Culicoides species on UK equine premises
Authors: Robin M. , Archer D. , Garros C. , Gardès L. , Baylis M.
Published: February 2014

There are concerns that outbreaks of exotic or novel vector‐borne viral diseases will increasingly occur within northern Europe and the UK in the future. African horse sickness (AHS) is a viral disease of equids that is transmitted by Culicoides and is associated with up to 95 per cent mortality. AHS has never occurred in the UK; however, it has been suggested that appropriate Culicoides species and climatic conditions are present in northern Europe to support an outbreak. No data are currently available regarding the Culicoides species present on UK equine properties. This study demonstrates the presence of potential AHS virus vector Culicoides species on both urban and rural equine properties within the south‐east UK. PCR analysis revealed that engorged members of these species contained equine DNA, proving a direct vector–host interaction. It is therefore possible that an AHS outbreak could occur in the UK if the virus were to be imported and, given the severe welfare and economic consequences of AHS, this would have devastating consequences to the naïve UK equine population.