bluuebunny/crossref_metadata_2025 huggingface dataset:
Continuum Isotropy Loci of a 2-DoF Parallel Kinematic Manipulator
Authors: Cinquemani Simone , Giberti Hermes , Legnani Giovanni
Published: August 2013

Parallel Kinematic Machines (PKM) are interesting in industrial field because of their ability to ensure high dynamic performance, stiffness, accuracy and precision. One of the parameters used in the design phase of a manipulator relates the opportunity to ensure isotropic performance within the workspace. This paper presents a methodology to graphically identify the points of isotropy of a planar manipulator and defines the geometrical conditions to maximize the number. It is demonstrated that, for some configurations of the manipulator, continuum isotropy loci can be identified. Achieving this goal ensures the efforts related to the design of a manipulator are paid in terms of ability to work, as much as possible, with ideal kinematical conditions.
Fast and Adaptive Hopping Height Control of Single-Legged Robot
Authors: Bhatti Jawaad , Plummer Andrew R. , Sahinkaya M. Necip , Iravani Pejman , Guglielmino Emanuele , Caldwell Darwin G.
Published: August 2013

Research on running robots has generally focussed on the steady-state. When the ground has limited foot placement surfaces or there are sudden changes in height then steady-state running is not possible. It becomes necessary to make step-by-step adjustments to place the foot.

In this paper a mass-spring-damper model of a robot’s leg is used to develop a hopping controller capable of meeting rapid changes in demand height or flight time. Analysis of the model provides a simple method to select control parameters for effective height control without tuning or iteration. Additionally, a simple adaptive algorithm is introduced and demonstrated in simulation. The adaptive control algorithm allows rapid changes of height even when ground characteristics change. Experimental validation is ongoing and some preliminary results are provided.
A CAD/Mechatronic Solar Panel Tracking Student Design Project
Authors: Spence A. D. , Kenyon T. , Sawula D. A. , Balan L. , Mohammed W. M.
Published: August 2013

The increasing engineering accreditation program requirements for outcome based assessment, combined with emphasis on green energy and other sustainable technologies, prompted introduction of a solar panel tracking project within a senior product design course. Recognizing that the course is offered to students with mechanical engineering as well as software engineering (mechatronics) backgrounds, technical aspects emphasized three dimensional Computer Aided Design (CAD), rapid prototyping, and basic microcontroller interface electronics and programming. Mixed teams including students from both backgrounds were mandatory. This paper reports observations after three course offerings. In particular, it is noted that a paradigm shift away from lectures to hands-on development laboratories is needed, but formal laboratory milestones and submission deadlines remain essential for successful team project completion.
Identifying Cultural Barriers to Design Knowledge Flow in Chinese SMEs
Authors: Zhang Zhinan , Liu Gang , Liang Jun , Xie Youbai
Published: August 2013

In Chinese Small and Medium-Sized Enterprises (SMEs), culture is one of the barriers which often plays an important role in the effective knowledge flow in their product design and development process. Investigations to designers from SMEs in China help us recognize several cultural barriers to this knowledge flow in the process of product design and development. This paper aims to identify those cultural barriers to knowledge flow in the design process of SMEs. Interviews with and questionnaires to designers from different SMEs were conducted. Empirical studies show that designers hesitate to share their knowledge; lack of trust and the thick wall among functional departments are major barriers influencing effective knowledge flows in the product design process. In addition, ongoing research on overcoming the identified cultural barriers to knowledge flow is also discussed briefly.
Experimental Study of Switching Behaviour in the Transmission of Tension to the Joints of the Finger
Authors: Vaz Anand , Jha Mayank Shekhar , Mahajan Kunal , Parashar Anubhav
Published: August 2013

Cadaveric experiments and computer simulations have shown that the tendon network of the fingers performs logic computation to preferentially change torque production capabilities. Distribution of input tensions in the tendon network itself regulates how tensions propagate to the finger joints, acting like the switching function of a logic gate. The tendon network as proposed by Winslow (1669–1760) is responsible for this phenomenon; and that a certain switching behavior is exhibited, depending upon the input tension loading. This work explores how this switching behavior varies with different static postures of the finger and under different loading conditions on input tendon terminals. Experimental setup is designed and developed to study this switching behavior, comprising of a bio-mimetic model of middle finger, designed and developed. Winslow’s tendon network as proposed by Winslow is employed over it. Force sensors based on strain gauges are designed and developed in order to measure the tension at the proximal and terminal slips of network. Experiments have been carried out to understand the switching behavior exhibited by rhomboidal tendon network and how it varies under different loading conditions. Its variation with different pre-configured postures under static equilibrium is observed. Results show that indeed the rhomboidal tendon network of Winslow exhibits switching behavior and that they vary under different loads and postures of the finger.
Performance Analysis of Compliant MEMS Parallel Robots Through Pseudo-Rigid-Body Model Synthesis
Authors: Belfiore Nicola Pio , Balucani Marco , Crescenzi Rocco , Verotti Matteo
Published: August 2013

In this paper, the performance of compliant parallel manipulators, in a given working position, is evaluated in terms of the MA (mechanical advantage) and k(J) (kinematic condition number). Such evaluation is done by means of a new and fast method for direct kinematic analysis of parallel manipulators. The method, which is useful to enhance both the design synthesis and the control strategy, is applied on the so called pseudo-rigid body mechanism, which represents a simplification of its corresponding compliant mechanism. Computer codes have been developed in MatLab programming language. The case under study consists of a MEMS (Micro Electro Mechanical System) compliant robot that has been built by the research group in silicon for micromanipulation. Such MEMS robot could be designed thanks to a new flexural hinge concept. Some experimental tests have been carried out on sample prototypes in order to inquire about the real feasibility of the micro-robot.
Computer Aided Electroforming for Rapid Manufacturing Applications
Authors: Hernández Castellano Pedro M. , Socas Sánchez Ayoze , Benítez Vega Antonio N. , Monzón Verona Mario , Ortega García Fernando , Marrero Alemán M. Dolores
Published: August 2013

Electroforming enables the manufacture metallic parts with good mechanical properties and high level of accuracy and reproducibility. A thin metallic shell is deposited on a model and later released from it. There are several applications of electroforming combined with rapid manufacturing (RM) technologies. However the two main disadvantages of electroforming are the non-uniformity thickness distribution and long time span of shell manufacturing.

The goal of this research is the evolution of Elecform3D™ product. It is a numerical simulation tool of electroforming process, in order to facilitate design and manufacturing tasks for metallic shells production. This software also controls the automatic device of cathodic orientation (ADCO) inside electrolytic baths. High quality of part is obtained, taking advantage of electroforming properties and improving manufacturing speed.

The methodology is based on the well-known potentials model of LaPlace, it enables deposited metal distribution prediction with high grade of precision, being experimentally validated with cathodic polarization curves. Moreover this has also created a subroutine for solving this problem for this application using the boundary element method (BEM). This tool has been tested and validated for the simulation of metal electroplating by using reference solutions of problems in the literature. It also examines changes in the geometry of the cathode as a result of the process of electro-deposition of the metal and the influence of this phenomenon on the simulation routine developed considering variables such as the metal thickness distribution and the rate of deposition. All these tasks work and carry out processes complementing simulation with experimental work in the laboratory in order to create a tool that will give greater potential to this product.

The results obtained with the first version of this product were very promising, and also indicated the need for a more precise analysis of electrochemical phenomena in the process of electroforming. Elecform3D™ is an important step beyond electroforming so far, and combined with additive manufacturing (AM) 3D printer, is a cheaper alternative for high quality metallic parts manufacturing in comparison with other RM technologies.
Modeling and Simulation of Void Formation During the Resin Transfer Molding Process
Authors: Lebel-Lavacry Aurélie , Park Chung-Hae , Saouab Abdelghani , Guéroult Sébastien , Bizet Laurent , Bréard Joël
Published: August 2013

We present modeling and simulation of air void formation in composite materials manufactured by the Resin Transfer Molding (RTM) process. The prediction of air void formation has been an important topic because air voids in composite materials deteriorate the mechanical properties of the part.

It has been found by experimental observations that the void content, for a specific preform, can be correlated with capillary number which is the ratio of the viscous force and the surface tension. It is still difficult, however, to predict the void formation without experimental measurement. Moreover, the capillary number may not be the exclusive parameter in practical cases, because the modeling by the capillary number does not work well for large and complex parts.

In this context, we propose a mathematical model to predict the air void formation in the channel which is on open gap between fiber tows and inside the fiber tow. Moreover, the void formation in the warp and the weft are modeled separately by considering the tow orientation with respect to the flow direction. We also modeled two other important phenomena, namely air void compression or expansion, and void migration. To validate the model, void content was experimentally measured by injecting an electrically conductive liquid into a preform. The voltage drop was correlated with the air void content considering the air as a non-conducting material. For a unidirectional fabric, a good agreement was obtained between the model prediction and the experimental result.
Numerical Study on the Improvement of the Hydrodynamic Behavior of Parallel Bearings by Surface Texturing
Authors: Gherca Andrei , Maspeyrot Patrick , Hajjam Mohamed , Fatu Aurelian
Published: August 2013

Numerous experimental studies have already established the fact that surface texturing can successfully improve the hydrodynamic performances of lubricated contacts. In a 2005 review paper, Etsion [1] offered a synthesis of these works and showed that surface texturing can benefit a large variety of tribological applications such as mechanical seals, thrust bearings or piston rings. However, the physical interpretation of the effects induced by textures still represents a subject of debate within the tribological community. The earliest investigation with regard to surface textures dates back to the 1960’s and is attributed to Hamilton et al. [2]. The authors reported that the cavitation phenomenon induced by small irregularities is essential, as it allows high film pressures to overbalance low film pressures due to an antisymmetric pressure distribution.
Using of the Concepts of Roles and Context in a Project Management/PLM Solution: The Real Case Study of LASCOM
Authors: Baczkowski Mylène , Robin Vincent , Rose Bertrand
Published: August 2013

The software tools for PLM and project management are often built around several classical concepts: Processes, Activities, Phases, Artefacts, Workflows, Resources and Relationships. This approach does not really consider the team concept and the particular dynamics of the project management. This leads to a paradox with PLM and project management software tools. They tend to reduce risk and improve performance by structuring, streamlining, automating and scheduling projects without considering projects are not well-structured and have their own dynamic. The negation of project management mode increases the risks of project failures, adds stress to team members and requires the addition of complex mechanisms of collaboration and delegation. PLM Software sometimes becomes a limitation for project management. Our partner, LASCOM, a French PLM software developer, decided to make evolve its solutions to provide customers more efficient PLM solutions. LASCOM initiated a reflection around new concepts for enrich its existing models and tools since few years. The company particularly has an interest on the practical use of the concepts of roles and contexts. This paper deals with the addition of these concepts in the models used by LASCOM. This addition is intended to partially solve the problems posed by the generalization of work by project in organizations.
Additive Manufacturing in Jewellery Design
Authors: Ferreira Telma , Almeida Henrique A. , Bártolo Paulo J. , Campbell Ian
Published: August 2013

Additive manufacturing has become a well-known and widely used process among engineers and designers within the past decade to respond to high levels of market demand and product complexity. The jewellery industry still works essentially on traditional fabrication methods to much time consuming and in some cases lacking efficiency compared to the quality of the end product. The inclusion of new technologies can be a solution to overcome these issues. Additive fabrication enables the fabrication of new products and geometries reducing manufacturing time, energy and labor costs. This paper discusses the advantages and disadvantages of traditional manufacturing processes, such as Investment Casting, and proposes a new production method based on the use of advanced modeling and additive manufacturing. Three additive manufacturing technologies were used, such as selective laser sintering, stereolithography and 3D printing. A computational application for jewellery design is also presented to help manufactures and customers to fabricate novel jewellery pieces. This tool is based on a customization concept, which has been of increasing interest during recent years.
Best Practices Assessment in Requirements Engineering Tools Integration
Authors: Mahut Stéphanie Glatard , Eynard Benoit , Merlo Christophe , Minel Stéphanie , Beaujon Thierry
Published: August 2013

Companies can use methodological software tools to perform requirement engineering. Those methodological tools are increasingly developped by external software editors and integrators. The performance of the tools is not enough to ensure efficiency of methodology application.

This paper is part of a PhD research programm. This research program focuses on the way of integrating requirement engineering tools into product design department with a provider perspective. To be more precise, this research project emphasizes on modifications occuring during integration phases. In order to evaluate and to improve integration process, key indicators can be proposed. From the first stage of integration to the moment the user is autonomous, key integration factors often change. For example, requirements of the user, user environment, product life cycle, delay for integration, actors, … This paper aims at proposing surveys to identify those changing key factors. Then, first best practices elements are given to counteract the possible negative impact on efficiency of requirement engineering tools integration and thus, on product development. In fact, this paper goes further into the way to analyze the existing system. It notably deals with first elements from industrial survey on IT vendor and their customer perspectives on tools integration. In addition, preliminary survey results on customer dissatisfaction are proposed and analyzed. It also proposes a short case study and further research work in order to assume those first results and to bring to the definition of integration approach for methodological tools in engineering domain.
Normal and Tangential Contact Between Anisotropic Materials With an Anisotropic Coating
Authors: Bagault C. , Nélias D. , Baietto M.-C.
Published: August 2013

A contact model using semi analytical methods, relying on elementary analytical solutions, has been developed. It is based on numerical techniques adapted to contact mechanics, with strong potential for inelastic, inhomogeneous or anisotropic problems. Recent developments aim to quantify displacements and stresses of an anisotropic half space with an anisotropic coating which is in contact with a rigid sphere. The influence of symmetry axes on the contact problem solution will be more specifically analyzed.
Life Cycle Costing in Manufacturing Process Management
Authors: Moser Gerhard , Le Duigou Julien , Bosch-Mauchand Magali
Published: August 2013

In the last two decades during which the competitive business environment increased, it became crucial for each company to find the most accurate strategy to make survive its business. For that reason they need to manage and control their costs. Life Cycle Costing is one of these tools, which helps to analyse the cost of a product in the whole life of a product. To be competitive, the organisations have to optimize not only their products but also all their processes. Manufacturing Process Management (MPM) addresses the area between product design and production. Therefore MPM supports to optimize the manufacturing area of a factory. With different virtual scenarios the best solution of the manufacturing process can be obtained and at the same time it is possible to reduce time to market, costs and increase the quality. The focus of this paper is to integrate Life Cycle Costing tools and methods in the MPM part of the Product Lifecycle Management (PLM). We will discuss the implementation of Activity Based Costing (ABC) and Case-Based Reasoning (CBR) methods in a PLM tool for an early design decision support.
Study of the Effect of Mechanical Loading on Cell Cultures in Bone Tissue Engineering
Authors: Cruel Magali , Bensidhoum Morad , Sudre Laure , Puel Guillaume , Dumas Virginie , Hoc Thierry
Published: August 2013

Bone tissue engineering currently represents one of the most interesting alternatives to autologous transplants and their drawbacks in the treatment of large bone defects. Mesenchymal stem cells are used to build new bone in vitro in a bioreactor. Their stimulation and our understanding of the mechanisms of mechanotransduction need to be improved in order to optimize the design of bioreactors.

In this study, several geometries of bioreactor were analyzed experimentally and biological results were linked with numerical simulations of the flow inside the bioreactor. These results will constitute a base for an improved design of the existing bioreactor.
Effect of open area ratio and pattern structure on fluctuations in critical dimension and Si recess
Authors: Kuboi Nobuyuki , Tatsumi Tetsuya , Fukasawa Masanaga , Kinoshita Takashi , Komachi Jun , Ansai Hisahiro , Miwa Hiroyuki
Published: August 2013

The authors quantitatively investigated the effects of open area ratio and pattern structure on fluctuations in critical dimension (ΔCD) and Si recess depth (ΔdR). To model these effects, under the assumption that three factors—mask open area ratio at the wafer level (global), chip level (semi-local), and local level (local)—affect ΔCD and ΔdR, they performed experiments using wafers ranging from 0.60 to 0.91 of the global range (RG) and the semi-local range (RS) treated by the HBr/O2 plasma etching process, where photoresist mask patterns on the poly-Si film with solid angles (ΩL) ranging from 0.2π to 0.9π were located. As a result, the authors found that ΔCD had positive and linear correlation with the RG value, which was consistent with the trend of the integrated intensity of the etched by-product (SiBrx) estimated by optical emission spectroscopy data and with that of taper angles of observed etched profiles. They also clarified that ΔCD was affected by the amount of SiBrx generated within several times of the mean free path area for the semi-local dependence and that the ΩL value within a 2 μm area, not the pattern space, had a good correlation with fluctuations in ΔCD as a control indicator. Using this experimental knowledge, the authors developed a quasi-three-dimensional Si gate etching simulation procedure that demonstrates the ΔCD value and the etched profile characteristics. Furthermore, our simulation procedure found that ΔdR caused by ion bombardment in the Si substrate, as well as ΔCD, deeply depended on the (RG + RS)ΩL factor. Taking account of the relationship between dR and the ion energy reduced by the SiBrx deposition depth on the Si substrate, the authors found that dR was dependent on the factor, which was consistent with experimental data. These results show that for improving device performance, it is crucial to consider the effect of (RG + RS)ΩL on ΔCD and ΔdR in controlling plasma etching parameters, such as ion energy, gas flux, and etching time.
T Cells TAMing Dendritic Cells
Authors: Foley John F.
Published: August 2013

Antigen-specific production of Protein S by activated T cells signals through dendritic cell receptors to dampen the immune response.
Detection of Escherichia coli O157 by Peptide Nucleic Acid Fluorescence
In Situ
Hybridization (PNA-FISH) and Comparison to a Standard Culture Method
Authors: Almeida C. , Sousa J. M. , Rocha R. , Cerqueira L. , Fanning S. , Azevedo N. F. , Vieira M. J.
Published: August 2013

Despite the emergence of non-O157 Shiga toxin-producing
Escherichia coli
(STEC) infections,
E. coli
serotype O157 is still the most commonly identified STEC in the world. It causes high morbidity and mortality and has been responsible for a number of outbreaks in many parts of the world. Various methods have been developed to detect this particular serotype, but standard bacteriological methods remain the gold standard. Here, we propose a new peptide nucleic acid fluorescence
in situ
hybridization (PNA-FISH) method for the rapid detection of
E. coli
O157. Testing on 54 representative strains showed that the PNA probe is highly sensitive and specific to
E. coli
O157. The method then was optimized for detection in food samples. Ground beef and unpasteurized milk samples were artificially contaminated with
E. coli
O157 concentrations ranging from 1 × 10
−2
to 1 × 10
2
CFU per 25 g or ml of food. Samples were then preenriched and analyzed by both the traditional bacteriological method (ISO 16654:2001) and PNA-FISH. The PNA-FISH method performed well in both types of food matrices with a detection limit of 1 CFU/25 g or ml of food samples. Tests on 60 food samples have shown a specificity value of 100% (95% confidence interval [CI], 82.83 to 100), a sensitivity of 97.22% (95% CI, 83.79 to 99.85%), and an accuracy of 98.33% (CI 95%, 83.41 to 99.91%). Results indicate that PNA-FISH performed as well as the traditional culture methods and can reduce the diagnosis time to 1 day.
The Periplasmic HrpB1 Protein from Xanthomonas spp. Binds to Peptidoglycan and to Components of the Type III Secretion System
Authors: Hausner Jens , Hartmann Nadine , Lorenz Christian , Büttner Daniela
Published: August 2013

The plant-pathogenic bacterium
Xanthomonas campestris
pv. vesicatoria employs a type III secretion (T3S) system to translocate bacterial effector proteins into eukaryotic host cells. The membrane-spanning secretion apparatus consists of 11 core components and several associated proteins with yet unknown functions. In this study, we analyzed the role of HrpB1, which was previously shown to be essential for T3S and the formation of the extracellular T3S pilus. We provide experimental evidence that HrpB1 localizes to the bacterial periplasm and binds to peptidoglycan, which is in agreement with its predicted structural similarity to the putative peptidoglycan-binding domain of the lytic transglycosylase Slt70 from
Escherichia coli
. Interaction studies revealed that HrpB1 forms protein complexes and binds to T3S system components, including the inner membrane protein HrcD, the secretin HrcC, the pilus protein HrpE, and the putative inner rod protein HrpB2. The analysis of deletion and point mutant derivatives of HrpB1 led to the identification of amino acid residues that contribute to the interaction of HrpB1 with itself and HrcD and/or to protein function. The finding that HrpB1 and HrpB2 colocalize to the periplasm and both interact with HrcD suggests that they are part of a periplasmic substructure of the T3S system.
Fecal Shedding of Zoonotic Food-Borne Pathogens by Wild Rodents in a Major Agricultural Region of the Central California Coast
Authors: Kilonzo Christopher , Li Xunde , Vivas Eduardo J. , Jay-Russell Michele T. , Fernandez Kristine L. , Atwill Edward R.
Published: August 2013

Recent outbreaks of food-borne illness associated with the consumption of produce have increased concern over wildlife reservoirs of food-borne pathogens. Wild rodents are ubiquitous, and those living close to agricultural farms may pose a food safety risk should they shed zoonotic microorganisms in their feces near or on agricultural commodities. Fecal samples from wild rodents trapped on 13 agricultural farms (9 produce, 3 cow-calf operations, and 1 beef cattle feedlot) in Monterey and San Benito Counties, CA, were screened to determine the prevalence and risk factors for shedding of several food-borne pathogens. Deer mice (Peromyscus maniculatus) were the most abundant rodent species trapped (72.5%).Cryptosporidiumspecies (26.0%) andGiardiaspecies (24.2%) were the predominant isolates from rodent feces, followed bySalmonella entericaserovars (2.9%) andEscherichia coliO157:H7 (0.2%). Rodent trap success was significantly associated with detection ofSalmonellain rodent feces, while farm type was associated with fecal shedding ofCryptosporidiumandGiardia. Seasonal shedding patterns were evident, with rodents trapped during the spring and summer months being significantly less likely to be sheddingCryptosporidiumoocysts than those trapped during autumn. Higher rodent species diversity tended to correlate with lower fecal microbial prevalence, and most spatiotemporal pathogen clusters involved deer mice. Rodents in the study area posed a minimal risk as environmental reservoirs ofE. coliO157:H7, but they may play a role in environmental dissemination ofSalmonellaand protozoa. Rodent control efforts that potentially reduce biodiversity may increase pathogen shedding, possibly through promotion of intraspecific microbial transmission.
Microbial Contents of Vacuum Cleaner Bag Dust and Emitted Bioaerosols and Their Implications for Human Exposure Indoors
Authors: Veillette Marc , Knibbs Luke D. , Pelletier Ariane , Charlebois Remi , Blais Lecours Pascale , He Congrong , Morawska Lidia , Duchaine Caroline
Published: August 2013

Vacuum cleaners can release large concentrations of particles, both in their exhaust air and from resuspension of settled dust. However, the size, variability, and microbial diversity of these emissions are unknown, despite evidence to suggest they may contribute to allergic responses and infection transmission indoors. This study aimed to evaluate bioaerosol emission from various vacuum cleaners. We sampled the air in an experimental flow tunnel where vacuum cleaners were run, and their airborne emissions were sampled with closed-face cassettes. Dust samples were also collected from the dust bag. Total bacteria, total archaea,
Penicillium
/
Aspergillus
, and total
Clostridium
cluster 1 were quantified with specific quantitative PCR protocols, and emission rates were calculated.
Clostridium botulinum
and antibiotic resistance genes were detected in each sample using endpoint PCR. Bacterial diversity was also analyzed using denaturing gradient gel electrophoresis (DGGE), image analysis, and band sequencing. We demonstrated that emission of bacteria and molds (
Penicillium
/
Aspergillus
) can reach values as high as 1E5 cell equivalents/min and that those emissions are not related to each other. The bag dust bacterial and mold content was also consistent across the vacuums we assessed, reaching up to 1E7 bacterial or mold cell equivalents/g. Antibiotic resistance genes were detected in several samples. No archaea or
C. botulinum
was detected in any air samples. Diversity analyses showed that most bacteria are from human sources, in keeping with other recent results. These results highlight the potential capability of vacuum cleaners to disseminate appreciable quantities of molds and human-associated bacteria indoors and their role as a source of exposure to bioaerosols.
Microgradients of pH Do Not Occur around Lactococcus Colonies in a Model Cheese
Authors: Jeanson Sophie , Floury Juliane , Issulahi Al Amine , Madec Marie-Noëlle , Thierry Anne , Lortal Sylvie
Published: August 2013

Lactococci inoculated into cheese grow as colonies producing lactic acid. The pH microgradients were investigated around colonies in a complex food such as cheese. The results, obtained using a nondestructive technique, demonstrated that pH microgradients did not occur regardless of the acidification kinetics and the size of the colony.
X-Ray Absorption Near-Edge Structure (XANES) Spectroscopy Study of the Interaction of Silver Ions with Staphylococcus aureus, Listeria monocytogenes, and Escherichia coli
Authors: Bovenkamp Gudrun Lisa , Zanzen Ulrike , Krishna Katla Sai , Hormes Josef , Prange Alexander
Published: August 2013

Silver ions are widely used as antibacterial agents, but the basic molecular mechanism of this effect is still poorly understood. X-ray absorption near-edge structure (XANES) spectroscopy at the Ag
LIII
, S
K
, and P
K
edges reveals the chemical forms of silver in
Staphylococcus aureus
and
Escherichia coli
(Ag
+
treated). The Ag
LIII
-edge XANES spectra of the bacteria are all slightly different and very different from the spectra of silver ions (silver nitrate and silver acetate), which confirms that a reaction occurs. Death or inactivation of bacteria was observed by plate counting and light microscopy. Silver bonding to sulfhydryl groups (Ag-S) in cysteine and Ag-N or Ag-O bonding in histidine, alanine, and
dl
-aspartic acid was detected by using synthesized silver-amino acids. Significantly lower silver-cysteine content, coupled with higher silver-histidine content, in Gram-positive
S. aureus
and
Listeria monocytogenes
cells indicates that the peptidoglycan multilayer could be buffering the biocidal effect of silver on Gram-positive bacteria, at least in part. Bonding of silver to phosphate groups was not detected. Interaction with DNA or proteins can occur through Ag-N bonding. The formation of silver-cysteine can be confirmed for both bacterial cell types, which supports the hypothesis that enzyme-catalyzed reactions and the electron transport chain within the cell are disrupted.
Behavior of Yersinia enterocolitica in the Presence of the Bacterivorous Acanthamoeba castellanii
Authors: Lambrecht E. , Baré J. , Van Damme I. , Bert W. , Sabbe K. , Houf K.
Published: August 2013

Free-living protozoa play an important role in the ecology and epidemiology of human-pathogenic bacteria. In the present study, the interaction between
Yersinia enterocolitica
, an important food-borne pathogen, and the free-living amoeba
Acanthamoeba castellanii
was studied. Several cocultivation assays were set up to assess the resistance of
Y. enterocolitica
to
A. castellanii
predation and the impact of environmental factors and bacterial strain-specific characteristics. Results showed that all
Y. enterocolitica
strains persist in association with
A. castellanii
for at least 14 days, and associations with
A. castellanii
enhanced survival of
Yersinia
under nutrient-rich conditions at 25°C and under nutrient-poor conditions at 37°C. Amoebae cultivated in the supernatant of one
Yersinia
strain showed temperature- and time-dependent permeabilization. Intraprotozoan survival of
Y. enterocolitica
depended on nutrient availability and temperature, with up to 2.8 log CFU/ml bacteria displaying intracellular survival at 7°C for at least 4 days in nutrient-rich medium. Transmission electron microscopy was performed to locate the
Yersinia
cells inside the amoebae. As
Yersinia
and
Acanthamoeba
share similar ecological niches, this interaction identifies a role of free-living protozoa in the ecology and epidemiology of
Y. enterocolitica
.
Environmental Surveillance of Human Parechoviruses in Sewage in the Netherlands
Authors: Lodder W. J. , Wuite M. , de Roda Husman A. M. , Rutjes S. A.
Published: August 2013

The circulation of human parechoviruses (HPeVs) in the population was studied by environmental surveillance comprising of molecular analyses of sewage samples (
n
= 89) that were collected from 15 different locations in the Netherlands. Samples were taken from sewage originating from schools (
n
= 9) or from parts of municipalities (
n
= 6) during the Dutch school year 2010-2011. At 13/15 locations HPeV1, HPeV3, or HPeV6 RNA was detected at least once; however, sequence diversity did not reflect associations in time or place. A higher percentage of positives was observed in the samples originating from the municipalities. It was demonstrated that HPeV circulated in the studied population to a higher extent than would be expected from the current knowledge on infections predominating in young children.
Functional and Expression Analysis of the Metal-Inducible
dmeRF
System from Rhizobium leguminosarum bv. viciae
Authors: Rubio-Sanz L. , Prieto R. I. , Imperial J. , Palacios J. M. , Brito B.
Published: August 2013

A gene encoding a homolog to the cation diffusion facilitator protein DmeF from
Cupriavidus metallidurans
has been identified in the genome of
Rhizobium leguminosarum
UPM791. The
R. leguminosarum dmeF
gene is located downstream of an open reading frame (designated
dmeR
) encoding a protein homologous to the nickel- and cobalt-responsive transcriptional regulator RcnR from
Escherichia coli
. Analysis of gene expression showed that the
R. leguminosarum dmeRF
genes are organized as a transcriptional unit whose expression is strongly induced by nickel and cobalt ions, likely by alleviating the repressor activity of DmeR on
dmeRF
transcription. An
R. leguminosarum dmeRF
mutant strain displayed increased sensitivity to Co(II) and Ni(II), whereas no alterations of its resistance to Cd(II), Cu(II), or Zn(II) were observed. A decrease of symbiotic performance was observed when pea plants inoculated with an
R. leguminosarum dmeRF
deletion mutant strain were grown in the presence of high concentrations of nickel and cobalt. The same mutant induced significantly lower activity levels of NiFe hydrogenase in microaerobic cultures. These results indicate that the
R. leguminosarum
DmeRF system is a metal-responsive efflux mechanism acting as a key element for metal homeostasis in
R. leguminosarum
under free-living and symbiotic conditions. The presence of similar
dmeRF
gene clusters in other
Rhizobiaceae
suggests that the
dmeRF
system is a conserved mechanism for metal tolerance in legume endosymbiotic bacteria.
mhpT
Encodes an Active Transporter Involved in 3-(3-Hydroxyphenyl)Propionate Catabolism by Escherichia coli K-12
Authors: Xu Ying , Chen Bing , Chao Hongjun , Zhou Ning-Yi
Published: August 2013

Escherichia coli
K-12 utilizes 3-(3-hydroxyphenyl)propionate (3HPP) as a sole carbon and energy source. Among the genes in its catabolic cluster in the genome,
mhpT
was proposed to encode a hypothetical transporter. Since no transporter for 3HPP uptake has been identified, we investigated whether MhpT is responsible for 3HPP uptake. MhpT fused with green fluorescent protein was found to be located at the periphery of cells by confocal microscopy, consistent with localization to the cytoplasmic membrane. Gene knockout and complementation studies clearly indicated that
mhpT
is essential for 3HPP catabolism in
E. coli
K-12 W3110 at pH 8.2. Uptake assays with
14
C-labeled substrates demonstrated that strain W3110 and strain W3110Δ
mhpT
containing recombinant MhpT specifically transported 3HPP but not benzoate, 3-hydroxybenzoate, or gentisate into cells. Energy dependence assays suggested that MhpT-mediated 3HPP transport was driven by the proton motive force. The change of Ala-272 of MhpT to a histidine, surprisingly, resulted in enhanced transport activity, and strain W3110Δ
mhpT
containing the MhpT A272H mutation had a slightly higher growth rate than the wild-type strain at pH 8.2. Hence, we demonstrated that MhpT is a specific 3HPP transporter and vital for
E. coli
K-12 W3110 growth on this substrate under basic conditions.
Prevalence of Hemolysin Genes and Comparison of
ehxA
Subtype Patterns in Shiga Toxin-Producing Escherichia coli (STEC) and Non-STEC Strains from Clinical, Food, and Animal Sources
Authors: Lorenz Sandra C. , Son Insook , Maounounen-Laasri Anna , Lin Andrew , Fischer Markus , Kase Julie A.
Published: August 2013

Shiga toxin-producing
Escherichia coli
(STEC) belonging to certain serogroups (e.g., O157 and O26) can cause serious conditions like hemolytic-uremic syndrome (HUS), but other strains might be equally pathogenic. While virulence factors, like
stx
and
eae
, have been well studied, little is known about the prevalence of the
E. coli
hemolysin genes (
hlyA
,
ehxA
,
e-hlyA
, and
sheA
) in association with these factors. Hemolysins are potential virulence factors, and
ehxA
and
hlyA
have been associated with human illness, but the significance of
sheA
is unknown. Hence, 435
E. coli
strains belonging to 62 different O serogroups were characterized to investigate gene presence and phenotypic expression of hemolysis. We further investigated
ehxA
subtype patterns in
E. coli
isolates from clinical, animal, and food sources. While
sheA
and
ehxA
were widely distributed,
e-hlyA
and
hlyA
were rarely found. Most strains (86.7%) were hemolytic, and significantly more hemolytic (95%) than nonhemolytic strains (49%) carried
stx
and/or
eae
(
P
< 0.0001).
ehxA
subtyping, as performed by using PCR in combination with restriction fragment length polymorphism analysis, resulted in six closely related subtypes (>94.2%), with subtypes A/D being
eae
-negative STECs and subtypes B, C, E, and F
eae
positive. Unexpectedly,
ehxA
subtype patterns differed significantly between isolates collected from different sources (
P
< 0.0001), suggesting that simple linear models of exposure and transmission need modification; animal isolates carried mostly subtypes A/C (39.3%/42.9%), food isolates carried mainly subtype A (81.9%), and clinical isolates carried mainly subtype C (66.4%). Certain O serogroups correlated with particular
ehxA
subtypes: subtype A with O104, O113, and O8; B exclusively with O157; C with O26, O111, and O121.
Monitoring the Metabolic State of Fungal Hyphae and the Presence of Melanin by Nonlinear Spectral Imaging
Authors: Knaus Helene , Blab Gerhard A. , Agronskaia Alexandra V. , van den Heuvel Dave J. , Gerritsen Hans C. , Wösten Han A. B.
Published: August 2013

Label-free nonlinear spectral imaging microscopy (NLSM) records two-photon-excited fluorescence emission spectra of endogenous fluorophores within the specimen. Here, NLSM is introduced as a novel, minimally invasive method to analyze the metabolic state of fungal hyphae by monitoring the autofluorescence of NAD(P)H and flavin adenine dinucleotide (FAD). Moreover, the presence of melanin was analyzed by NLSM. NAD(P)H, FAD, and melanin were used as biomarkers for freshness of mushrooms of
Agaricus bisporus
(white button mushroom) that had been stored at 4°C for 0 to 17 days. During this period, the mushrooms did not show changes in morphology or color detectable by eye. In contrast, FAD/NAD(P)H and melanin/NAD(P)H ratios increased over time. For instance, these ratios increased from 0.92 to 2.02 and from 0.76 to 1.53, respectively, at the surface of mushroom caps that had been harvested by cutting the stem. These ratios were lower under the skin than at the surface of fresh mushrooms (0.78 versus 0.92 and 0.41 versus 0.76, respectively), indicative of higher metabolism and lower pigment formation within the fruiting body. Signals were different not only between tissues of the mushroom but also between neighboring hyphae. These data show that NLSM can be used to determine the freshness of mushrooms and to monitor the postharvest browning process at an early stage. Moreover, these data demonstrate the potential of NLSM to address a broad range of fundamental and applied microbiological processes.
Development of a Tunable Wide-Range Gene Induction System Useful for the Study of Streptococcal Toxin-Antitoxin Systems
Authors: Xie Zhoujie , Qi Fengxia , Merritt Justin
Published: August 2013

Despite the plethora of genetic tools that have been developed for use in
Streptococcus mutans
, the
S. mutans
genetic system still lacks an effective gene induction system exhibiting low basal expression and strong inducibility. Consequently, we created two hybrid gene induction cassettes referred to as Xyl-S1 and Xyl-S2. Both Xyl-S cassettes are xylose inducible and controlled by the
Bacillus megaterium
xylose repressor. The Xyl-S cassettes each demonstrated >600-fold-increased reporter activity in the presence of 1.2% (wt/vol) xylose. However, the Xyl-S1 cassette yielded a much higher maximum level of gene expression, whereas the Xyl-S2 cassette exhibited much lower uninduced basal expression. The cassettes also performed similarly in
Streptococcus sanguinis
and
Streptococcus gordonii
, which suggests that they are likely to be useful in a variety of streptococci. We demonstrate how both Xyl-S cassettes are particularly useful for the study of toxin-antitoxin (TA) modules using both the previously characterized
S. mutans mazEF
TA module and a previously uncharacterized HicAB TA module in
S. mutans
. HicAB TA modules are widely distributed among bacteria and archaea, but little is known about their function. We show that HicA serves as the toxin component of the module, while HicB serves as the antitoxin. Our results suggest that, in contrast to that of typical TA modules, HicA toxicity in
S. mutans
is modest at best. The implications of these results for HicAB function are discussed.
U(VI) Reduction by Diverse Outer Surface
c
-Type Cytochromes of Geobacter sulfurreducens
Authors: Orellana Roberto , Leavitt Janet J. , Comolli Luis R. , Csencsits Roseann , Janot Noemie , Flanagan Kelly A. , Gray Arianna S. , Leang Ching , Izallalen Mounir , Mester Tünde , Lovley Derek R.
Published: August 2013

Early studies with
Geobacter sulfurreducens
suggested that outer-surface
c
-type cytochromes might play a role in U(VI) reduction, but it has recently been suggested that there is substantial U(VI) reduction at the surface of the electrically conductive pili known as microbial nanowires. This phenomenon was further investigated. A strain of
G. sulfurreducens
, known as Aro-5, which produces pili with substantially reduced conductivity reduced U(VI) nearly as well as the wild type, as did a strain in which the gene for PilA, the structural pilin protein, was deleted. In order to reduce rates of U(VI) reduction to levels less than 20% of the wild-type rates, it was necessary to delete the genes for the five most abundant outer surface
c
-type cytochromes of
G. sulfurreducens
. X-ray absorption near-edge structure spectroscopy demonstrated that whereas 83% ± 10% of the uranium associated with wild-type cells correspond to U(IV) after 4 h of incubation, with the quintuple mutant, 89% ± 10% of uranium was U(VI). Transmission electron microscopy and X-ray energy dispersion spectroscopy revealed that wild-type cells did not precipitate uranium along pili as previously reported, but U(IV) was precipitated at the outer cell surface. These findings are consistent with those of previous studies, which have suggested that
G. sulfurreducens
requires outer-surface
c
-type cytochromes but not pili for the reduction of soluble extracellular electron acceptors.
Evidence for Benzylsuccinate Synthase Subtypes Obtained by Using Stable Isotope Tools
Authors: Kümmel Steffen , Kuntze Kevin , Vogt Carsten , Boll Matthias , Heider Johann , Richnow Hans H.
Published: August 2013

We studied the benzylsuccinate synthase (Bss) reaction mechanism with respect to the hydrogen-carbon bond cleavage at the methyl group of toluene by using different stable isotope tools. Λ values (slopes of linear regression curves for carbon and hydrogen discrimination) for two-dimensional compound-specific stable isotope analysis (2D-CSIA) of toluene activation by Bss-containing cell extracts (
in vitro
studies) were found to be similar to previously reported data from analogous experiments with whole cells (
in vivo
studies), proving that Λ values generated by whole cells are caused by Bss catalysis. The Bss enzymes of facultative anaerobic bacteria produced smaller Λ values than those of obligate anaerobes. In addition, a partial exchange of a single deuterium atom in benzylsuccinate with hydrogen was observed in experiments with deuterium-labeled toluene. In this study, the Bss enzymes of the tested facultative anaerobes showed 3- to 8-fold higher exchange probabilities than those for the enzymes of the tested obligate anaerobic bacteria. The phylogeny of the Bss variants, determined by sequence analyses of BssA, the gene product corresponding to the α subunit of Bss, correlated with the observed differences in Λ values and hydrogen exchange probabilities. In conclusion, our results suggest subtle differences in the reaction mechanisms of Bss isoenzymes of facultative and obligate anaerobes and show that the putative isoenzymes can be differentiated by 2D-CSIA.
Staphylococcus aureus Mutants Lacking the LytR-CpsA-Psr Family of Enzymes Release Cell Wall Teichoic Acids into the Extracellular Medium
Authors: Chan Yvonne G. Y. , Frankel Matthew B. , Dengler Vanina , Schneewind Olaf , Missiakas Dominique
Published: August 2013

The LytR-CpsA-Psr (LCP) proteins are thought to transfer bactoprenol-linked biosynthetic intermediates of wall teichoic acid (WTA) to the peptidoglycan of Gram-positive bacteria. In
Bacillus subtilis
, mutants lacking all three LCP enzymes do not deposit WTA in the envelope, while
Staphylococcus aureus
Δ
lcp
mutants display impaired growth and reduced levels of envelope phosphate. We show here that the
S. aureus
Δ
lcp
mutant synthesized WTA yet released ribitol phosphate polymers into the extracellular medium. Further, Δ
lcp
mutant staphylococci no longer restricted the deposition of LysM-type murein hydrolases to cell division sites, which was associated with defects in cell shape and increased autolysis. Mutations in
S. aureus
WTA synthesis genes (
tagB
,
tarF
, or
tarJ2
) inhibit growth, which is attributed to the depletion of bactoprenol, an essential component of peptidoglycan synthesis (lipid II). The growth defect of
S. aureus tagB
and
tarFJ
mutants was alleviated by inhibition of WTA synthesis with tunicamycin, whereas the growth defect of the Δ
lcp
mutant was not relieved by tunicamycin treatment or by mutation of
tagO
, whose product catalyzes the first committed step of WTA synthesis. Further, sortase A-mediated anchoring of proteins to peptidoglycan, which also involves bactoprenol and lipid II, was not impaired in the Δ
lcp
mutant. We propose a model whereby the
S. aureus
Δ
lcp
mutant, defective in tethering WTA to the cell wall, cleaves WTA synthesis intermediates, releasing ribitol phosphate into the medium and recycling bactoprenol for peptidoglycan synthesis.