bluuebunny/crossref_metadata_2025 huggingface dataset:
Performance of a 100 kWth Concentrated Solar Beam-Down Optical Experiment
Authors: Mokhtar Marwan , Meyers Steven A. , Armstrong Peter R. , Chiesa Matteo
Published: May 2014

An analysis of the beam down optical experiment (BDOE) performance with full concentration is presented. The analysis is based on radiation flux distribution data taken on Mar. 21st, 2011 using an optical-thermal flux measurement system. A hypothetical thermal receiver design is used in conjunction with the experimental data to determine the optimal receiver aperture size as a function of receiver losses and flux distribution. The overall output of the plant is calculated for various operating temperatures and three different control strategies namely, constant mass flow of the heat transfer fluid (HTF), constant outlet fluid temperature and real-time optimal outlet fluid temperature. It was found that the optimal receiver aperture size (radius) of the receiver ranged between (1.06 and 1.71 m) depending on temperature. The optical efficiency of the BDOE ranged from 32% to 37% as a daily average (average over the ten sunshine hours). The daily average mean flux density ranged between 9.422 kW/m2 for the 1.71 m-receiver and 20.9 kW/m2 for the 1.06 m-receiver. Depending on the control parameters and assuming an open receiver with solar absorptivity of 0.95 and longwave emissivity of 0.10. The average receiver efficiency varied from 71% at 300 °C down to 68% at 600 °C. The overall daily average thermal efficiency of the plant was between 28% and 24%, respectively for the aforementioned temperatures. The peak of useful power collected in the HTF was around 105 kWth at 300 °C mean fluid temperature and 89 kWth at 600 °C.
Forced Vibration of Overhead Transmission Line: Analytical and Experimental Investigation
Authors: Barry O. , Zu J. W. , Oguamanam D. C. D.
Published: May 2014

An analytical model of a single line transmission line carrying a Stockbridge damper is developed based on the Euler–Bernoulli beam theory. The conductor is modeled as an axially loaded beam and the messenger is represented as a beam with a tip mass at each end. Experiments are conducted to validate the proposed model. An explicit expression is presented for the damping ratio of the conductor. Numerical examples show that the proposed model is more accurate than the models found in the literature. Parametric studies indicate that the response of the conductor significantly depends on the excitation frequency, the location of the damper, and the damper parameters.
Systematic Integration of Finite Element Methods Into Multibody Dynamics Considering Hyperelasticity and Plasticity
Authors: Sanborn Graham , Choi Juhwan , Shik Yoon Joon , Rhim Sungsoo , Hwan Choi Jin
Published: May 2014

This study proposes a systematic extension of a multiflexible-body dynamics (MFBD) formulation that is based on a recursive formulation for rigid body dynamics. It is extended to include nonlinear plastic and hyperelastic material models for the flexible bodies. The flexible bodies in the existing MFBD formulation use a finite element formulation based on corotational elements. The rigid bodies and flexible bodies are coupled using the method of Lagrange multipliers. The extensions to add plasticity and hyperelasticity are outlined. A solid, brick-type element and a shell element are adapted from the literature for use with the plastic material, and a constant volume constraint is introduced to enforce the approximation of incompressibility with the hyperelastic materials. A brief overview of the MFBD formulation and the details required to extend the formulation to incorporate these nonlinear material models are presented. Numerical examples are presented to demonstrate the feasibility of the model.
Optimum Heating of Pressure Vessels With Holes
Authors: Dzierwa Piotr , Taler Jan
Published: May 2014

A method for determining time-optimum medium temperature changes is presented. The heating of the pressure elements will be conducted so that the circumferential stress caused by pressure and fluid temperature variations at the edge of the opening at the point of stress concentration does not exceed the allowable value. In contrast to present standards, two points at the edge of the opening are taken into consideration. Optimum fluid temperature changes are assumed in the form of simple time functions. It is possible to increase the fluid temperature stepwise at the beginning of the heating process and then the fluid temperature can be increased with a constant rate.
The Effect of Punching Plates to Suppress the Sloshing in a Gas Liquid Separator
Authors: Kaneko Shigehiko , Suzuki Hayato
Published: May 2014

Natural gas is relatively clean and its demand is increasing these days. In most of the case, gas fields are located at the bottom of the sea. Therefore, floating production, storage and offloading (FPSO) system is now attracting big attention. This paper is related to dynamical design of FPSO system, specially focused on the free surface elevation in the horizontal cylindrical and axisymmetric liquid vessel with end caps induced by the wave. In this study, the theory on the wave height and resonant frequency in a horizontal cylinder subjected to longitudinal external excitation is developed. Then, the theory capable of taking account of the effect of punching plates is described. Finally, experimental data of resonant wave height up to 3rd mode are shown in comparison with theoretically derived results.
A Coupled Electromagnetic and Thermal Model for Picosecond and Nanometer Scale Plasmonic Lithography Process
Authors: Chao Ion-Hong , Pan Liang , Sun Cheng , Zhang Xiang , Lavine Adrienne S.
Published: May 2014

Plasmonic lithography may become a mainstream nanofabrication technique in the future. Experimental results show that feature size with 22 nm resolution can be achieved by plasmonic lithography. In the experiment, a plasmonic lens (PL) is used to focus the laser energy with resolution much higher than the diffraction limit and features are created in the thermally sensitive phase-change material (PCM) layer. The energy transport mechanisms are still not fully understood in the lithography process. In order to predict the lithography resolution and explore the energy transport mechanisms involved in the process, customized electromagnetic wave (EMW) and heat transfer (HT) models were developed in comsol. Parametric studies on both operating parameters and material properties were performed to optimize the lithography process. The parametric studies show that the lithography process can be improved by either reducing the thickness of the phase-change material layer or using a material with smaller real refractive index for that layer.
Optimal Control of the Heat Release Rate of an Internal Combustion Engine With Pressure Gradient, Maximum Pressure, and Knock Constraints
Authors: Zurbriggen Florian , Ott Tobias , Onder Christopher , Guzzella Lino
Published: May 2014

In this paper, we present an analysis of the optimal burn rate in an internal combustion engine (ICE) considering pressure gradient, maximum pressure, and knocking. A zero-dimensional model with heat losses is used for that purpose. The working fluids are assumed to behave like ideal gases with temperature dependent gas properties. In the first part, it is assumed that the burn rate can be arbitrarily chosen at every time instance in order to maximize the mechanical work. This leads to an optimal control problem with constraints. In the second part, a Vibe type burn rate is assumed, where the center of combustion, the duration and the form factor can be chosen in order to maximize the mechanical work. This Vibe type burn rate is finally compared with the arbitrary combustion as the benchmark in order to evaluate the potential of the more realistic burn shape.
On the Stability of Fractional-Order Systems of Neutral Type
Authors: Pakzad Mohammad Ali , Pakzad Sara , Nekoui Mohammad Ali
Published: May 2014

The aim of this study is to offer a new analytical method for the stability testing of neutral type linear time-invariant (LTI) time-delayed fractional-order systems with commensurate orders and multiple commensurate delays. It is evident from the literature that the stability assessment of this class of dynamics remains unsolved yet and this is the first attempt to take up this challenging problem. The method starts with the determination of all possible purely imaginary characteristic roots for any positive time delay. To achieve this, the Rekasius transformation is used for the transcendental terms in the characteristic equation. An explicit analytical expression in terms of the system parameters which reveals the stability regions (pockets) in the domain of time delay has been presented. The number of unstable roots in each delay interval is calculated with the definition of root tendency (RT) on the boundary of each interval. Two example case studies are also provided, which are not possible to analyze using any other methodology known to the authors.
Vibration Control of Relative Tool-Spindle Displacement for Computer Numerically Controlled Lathe With Pipe Frame Structure
Authors: Morimoto Yoshitaka , Suzuki Naohiko , Kaneko Yoshiyuki , Isobe Minoru
Published: May 2014

A new computer numerically controlled (CNC) lathe with a pipe frame bed has been developed. This structure is expected to have enough space between the truss bars to solve the space problem and have enough rigidity for machine tools. Therefore, a CNC lathe whose frame consists of pipes, joints, and diagonal braces has been developed with enough rigidity and space utility for chip evacuation. From the viewpoint of machine tool usage, real-time vibration control theory is applied to control the relative displacement between the tool post and the spindle to suppress specific relative vibration modes.
Force Modeling for Generic Profile of Drills
Authors: Sambhav Kumar , Tandon Puneet , Dhande Sanjay G.
Published: May 2014

The paper presents a methodology to model the cutting forces by twist drills with generic point geometry. A generic definition of point geometry implies that the cutting lips and the relief surfaces can have arbitrary shapes. Such geometry is easily modeled using Non Uniform Rational B-Spline (NURBS) surface patches which give sufficient freedom to the tool designer to alter the tool geometry. The drill point has three cutting zones: primary cutting lips, secondary cutting lips, and the indentation zone at the center of chisel edge. At the indentation zone, the drill extrudes the workpiece, while at the cutting lips, shearing takes place. At primary cutting lip, the cutting is oblique while at secondary cutting lip, it is predominantly orthogonal. Starting from a computer-aided geometric design of a fluted twist drill with arbitrary point profile, the cutting forces have been modeled separately for all the three cutting zones. The mechanistic method has been employed wherever applicable to have a good correlation between the analytical and the experimental results. The force model has been calibrated and validated for conical drills. Then the model has been evaluated for a drill ground with curved relief surfaces. The theoretical and experimental results are found out to be in good conformity.
Methodology for the Optimization of Bolting Sequences for Wind Generator Flanges
Authors: Abasolo Mikel , Aguirrebeitia Josu , Avilés Rafael , Fernández de Bustos Igor
Published: May 2014

In bolted joints, bolts are tightened to a uniform preload in order to improve their operational behavior. This preload is not easy to achieve due to several phenomena that occur during the tightening sequence; in the case of wind generator bolted joints, which present a gap between the contact surfaces of the flanges, the elastic interaction is by far the most prejudicial of them. As a consequence, the tightening sequence is very costly, typically consisting on a large number of passes. Based on a metamodel created in previous work, this work presents methodology for the optimization of the tightening sequence, which consists on calculating the load to be applied to each bolt so as to achieve the target uniform preload at the end of the sequence in only one pass or two at the most. This methodology is based on optimization methods used for pressure vessel bolted joints has provided satisfactory results (validated via FE analyses) with a very low computational cost.
Single-crystalline silver film grown on Si (100) substrate by using electron-gun evaporation and thermal treatment
Authors: Chou Bo-Tsun , Lin Sheng-Di , Huang Bo-Hao , Lu Tien-Chang
Published: May 2014

The authors demonstrate a simple method to fabricate ultrasmooth single-crystalline silver (Ag) films with high reflectivity and low plasmonic damping. The single-crystalline Ag thin film on the clean Si (100) substrate is first deposited by electron-gun evaporator and then treated by rapid thermal annealing (RTA) to improve its quality. The crystal structure and surface morphology are characterized by x-ray diffraction, transmission electron microscopy, and atomic force microscopy. Optical constants of the prepared films are extracted by fitting the measured reflectivity spectra with the Drude model. These results show that the Ag film with 340 °C RTA has the best film quality, including small surface roughness of 0.46 nm, a sharp x-ray diffraction peak with FWHM of 0.3°, and lowest damping in the visible and near-infrared wavelength regime. Therefore, our method is not only cost-effective but also useful for fabricating metal-based plasmonic and nanophotonic devices.
Restoring Systemic GDF11 Levels Reverses Age-Related Dysfunction in Mouse Skeletal Muscle
Authors: Sinha Manisha , Jang Young C. , Oh Juhyun , Khong Danika , Wu Elizabeth Y. , Manohar Rohan , Miller Christine , Regalado Samuel G. , Loffredo Francesco S. , Pancoast James R. , Hirshman Michael F. , Lebowitz Jessica , Shadrach Jennifer L. , Cerletti Massimiliano , Kim Mi-Jeong , Serwold Thomas , Goodyear Laurie J. , Rosner Bernard , Lee Richard T. , Wagers Amy J.
Published: May 2014

Help the Aged

Muscle function declines with age, as does neurogenesis in certain brain regions. Two teams analyzed the effects of heterochronic parabiosis in mice.

Sinha
et al.

(p.
649
) found that when an aged mouse shares a circulatory system with a youthful mouse, the aged mouse sees improved muscle function, and

Katsimpardi
et al.

(p.
630
) observed increased generation of olfactory neurons. In both cases, Growth Differentiation Factor 11 appeared to be one of the key components of the young blood.
Individualization of Piperacillin Dosing for Critically Ill Patients: Dosing Software To Optimize Antimicrobial Therapy
Authors: Felton T. W. , Roberts J. A. , Lodise T. P. , Van Guilder M. , Boselli E. , Neely M. N. , Hope W. W.
Published: May 2014

Piperacillin-tazobactam is frequently used for empirical and targeted therapy of infections in critically ill patients. Considerable pharmacokinetic (PK) variability is observed in critically ill patients. By estimating an individual's PK, dosage optimization Bayesian estimation techniques can be used to calculate the appropriate piperacillin regimen to achieve desired drug exposure targets. The aim of this study was to establish a population PK model for piperacillin in critically ill patients and then analyze the performance of the model in the dose optimization software program BestDose. Linear, with estimated creatinine clearance and weight as covariates, Michaelis-Menten (MM) and parallel linear/MM structural models were fitted to the data from 146 critically ill patients with nosocomial infection. Piperacillin concentrations measured in the first dosing interval, from each of 8 additional individuals, combined with the population model were embedded into the dose optimization software. The impact of the number of observations was assessed. Precision was assessed by (i) the predicted piperacillin dosage and by (ii) linear regression of the observed-versus-predicted piperacillin concentrations from the second 24 h of treatment. We found that a linear clearance model with creatinine clearance and weight as covariates for drug clearance and volume of distribution, respectively, best described the observed data. When there were at least two observed piperacillin concentrations, the dose optimization software predicted a mean piperacillin dosage of 4.02 g in the 8 patients administered piperacillin doses of 4.00 g. Linear regression of the observed-versus-predicted piperacillin concentrations for 8 individuals after 24 h of piperacillin dosing demonstrated an
r
2
of >0.89. In conclusion, for most critically ill patients, individualized piperacillin regimens delivering a target serum piperacillin concentration is achievable. Further validation of the dosage optimization software in a clinical trial is required.
Involvement of the Global Regulator GlxR in 3-Hydroxybenzoate and Gentisate Utilization by Corynebacterium glutamicum
Authors: Chao Hongjun , Zhou Ning-Yi
Published: May 2014

Corynebacterium glutamicum
is an industrially important producer of amino acids and organic acids, as well as an emerging model system for aromatic assimilation. An IclR-type regulator GenR has been characterized to activate the transcription of
genDFM
and
genKH
operons for 3-hydroxybenzoate and gentisate catabolism and represses its own expression. On the other hand, GlxR, a global regulator of the cyclic AMP (cAMP) receptor protein-fumarate nitrate reductase regulator (CRP-FNR) type, was also predicted to be involved in this pathway. In this study, electrophoretic mobility shift assays and footprinting analyses demonstrated that GlxR bound to three sites in the promoter regions of three
gen
operons. A combination of site-directed mutagenesis of the biding sites, promoter activity assay, and GlxR overexpression demonstrated that GlxR repressed their expression by binding these sites. One GlxR binding site (DFMx) was found to be located −13 to +8 bp upstream of the
genDFM
promoter, which was involved in negative regulation of
genDFM
transcription. The GlxR binding site R-KHx01 (located between positions −11 to +5) was upstream of the
genKH
promoter sequence and involved in negative regulation of its transcription. The binding site R-KHx02, at which GlxR binds to
genR
promoter to repress its expression, was found within a footprint extending from positions −71 to −91 bp. These results reveal that GlxR represses the transcription of all three
gen
operons and then contributes to the synchronization of their expression for 3-hydroxybenzoate and gentisate catabolism in collaboration with the specific regulator GenR.
The Transcriptional Response of Lactobacillus sanfranciscensis DSM 20451
T
and Its
tcyB
Mutant Lacking a Functional Cystine Transporter to Diamide Stress
Authors: Stetina Mandy , Behr Jürgen , Vogel Rudi F.
Published: May 2014

As a result of its strong adaptation to wheat and rye sourdoughs,
Lactobacillus sanfranciscensis
has the smallest genome within the genus
Lactobacillus
. The concomitant absence of some important antioxidative enzymes and the inability to synthesize glutathione suggest a role of cystine transport in maintenance of an intracellular thiol balance. Diamide [synonym 1,1′-azobis(
N
,
N
-dimethylformamide)] disturbs intracellular and membrane thiol levels in oxidizing protein thiols depending on its initial concentration. In this study, RNA sequencing was used to reveal the transcriptional response of
L. sanfranciscensis
DSM 20451
T
(wild type [WT]) and its Δ
tcyB
mutant with a nonfunctional cystine transporter after thiol stress caused by diamide. Along with the different expression of genes involved in amino acid starvation, pyrimidine synthesis, and energy production, our results show that thiol stress in the wild type can be compensated through activation of diverse chaperones and proteases whereas the Δ
tcyB
mutant shifts its metabolism in the direction of survival. Only a small set of genes are significantly differentially expressed between the wild type and the mutant. In the WT, mainly genes which are associated with a heat shock response are upregulated whereas glutamine import and synthesis genes are downregulated. In the Δ
tcyB
mutant, the whole
opp
operon was more highly expressed, as well as a protein which probably includes enzymes for methionine transport. The two proteins encoded by
spxA
and
nrdH
, which are involved in direct or indirect oxidative stress responses, are also upregulated in the mutant. This work emphasizes that even in the absence of definitive antioxidative enzymes, bacteria with a small genome and a high frequency of gene inactivation and elimination use small molecules such as the cysteine/cystine couple to overcome potential cell damage resulting from oxidative stress.
Discovery of Ethanol-Responsive Small RNAs in Zymomonas mobilis
Authors: Cho Seung Hee , Lei Roy , Henninger Trey D. , Contreras Lydia M.
Published: May 2014

Zymomonas mobilis
is a bacterium that can produce ethanol by fermentation. Due to its unique metabolism and efficient ethanol production,
Z. mobilis
has attracted special interest for biofuel energy applications; an important area of study is the regulation of those specific metabolic pathways. Small RNAs (sRNAs) have been studied as molecules that function as transcriptional regulators in response to cellular stresses. While sRNAs have been discovered in various organisms by computational prediction and experimental approaches, their discovery in
Z. mobilis
has not yet been reported. In this study, we have applied transcriptome analysis and computational predictions to facilitate identification and validation of 15 novel sRNAs in
Z. mobilis
. We furthermore characterized their expression in the context of high and low levels of intracellular ethanol. Here, we report that 3 of the sRNAs (Zms2, Zms4, and Zms6) are differentially expressed under aerobic and anaerobic conditions, when low and high ethanol productions are observed, respectively. Importantly, when we tested the effect of ethanol stress on the expression of sRNAs in
Z. mobilis
, Zms2, Zms6, and Zms18 showed differential expression under 5% ethanol stress conditions. These data suggest that in this organism regulatory RNAs can be associated with metabolic functions involved in ethanol stress responses.
Functional Genomic Characterization of Virulence Factors from Necrotizing Fasciitis-Causing Strains of Aeromonas hydrophila
Authors: Grim Christopher J. , Kozlova Elena V. , Ponnusamy Duraisamy , Fitts Eric C. , Sha Jian , Kirtley Michelle L. , van Lier Christina J. , Tiner Bethany L. , Erova Tatiana E. , Joseph Sandeep J. , Read Timothy D. , Shak Joshua R. , Joseph Sam W. , Singletary Ed , Felland Tracy , Baze Wallace B. , Horneman Amy J. , Chopra Ashok K.
Published: May 2014

The genomes of 10
Aeromonas
isolates identified and designated
Aeromonas hydrophila
WI, Riv3, and NF1 to NF4;
A. dhakensis
SSU;
A. jandaei
Riv2; and
A. caviae
NM22 and NM33 were sequenced and annotated. Isolates NF1 to NF4 were from a patient with necrotizing fasciitis (NF). Two environmental isolates (Riv2 and -3) were from the river water from which the NF patient acquired the infection. While isolates NF2 to NF4 were clonal, NF1 was genetically distinct. Outside the conserved core genomes of these 10 isolates, several unique genomic features were identified. The most virulent strains possessed one of the following four virulence factors or a combination of them: cytotoxic enterotoxin, exotoxin A, and type 3 and 6 secretion system effectors AexU and Hcp. In a septicemic-mouse model, SSU, NF1, and Riv2 were the most virulent, while NF2 was moderately virulent. These data correlated with high motility and biofilm formation by the former three isolates. Conversely, in a mouse model of intramuscular infection, NF2 was much more virulent than NF1. Isolates NF2, SSU, and Riv2 disseminated in high numbers from the muscular tissue to the visceral organs of mice, while NF1 reached the liver and spleen in relatively lower numbers on the basis of colony counting and tracking of bioluminescent strains in real time by
in vivo
imaging. Histopathologically, degeneration of myofibers with significant infiltration of polymorphonuclear cells due to the highly virulent strains was noted. Functional genomic analysis provided data that allowed us to correlate the highly infectious nature of
Aeromonas
pathotypes belonging to several different species with virulence signatures and their potential ability to cause NF.
Mannosylglycerate and Di-
myo
-Inositol Phosphate Have Interchangeable Roles during Adaptation of Pyrococcus furiosus to Heat Stress
Authors: Esteves Ana M. , Chandrayan Sanjeev K. , McTernan Patrick M. , Borges Nuno , Adams Michael W. W. , Santos Helena
Published: May 2014

Marine hyperthermophiles accumulate small organic compounds, known as compatible solutes, in response to supraoptimal temperatures or salinities.
Pyrococcus furiosus
is a hyperthermophilic archaeon that grows optimally at temperatures near 100°C. This organism accumulates mannosylglycerate (MG) and di-
myo
-inositol phosphate (DIP) in response to osmotic and heat stress, respectively. It has been assumed that MG and DIP are involved in cell protection; however, firm evidence for the roles of these solutes in stress adaptation is still missing, largely due to the lack of genetic tools to produce suitable mutants of hyperthermophiles. Recently, such tools were developed for
P. furiosus
, making this organism an ideal target for that purpose. In this work, genes coding for the synthases in the biosynthetic pathways of MG and DIP were deleted by double-crossover homologous recombination. The growth profiles and solute patterns of the two mutants and the parent strain were investigated under optimal growth conditions and also at supraoptimal temperatures and NaCl concentrations. DIP was a suitable replacement for MG during heat stress, but substitution of MG for DIP and aspartate led to less efficient growth under conditions of osmotic stress. The results suggest that the cascade of molecular events leading to MG synthesis is tuned for osmotic adjustment, while the machinery for induction of DIP synthesis responds to either stress agent. MG protects cells against heat as effectively as DIP, despite the finding that the amount of DIP consistently increases in response to heat stress in the nine (hyper)thermophiles examined thus far.
Targeted and Highly Multiplexed Detection of Microorganisms by Employing an Ensemble of Molecular Probes
Authors: Xu Weihong , Krishnakumar Sujatha , Miranda Molly , Jensen Michael A. , Fukushima Marilyn , Palm Curtis , Fung Eula , Davis Ronald W. , St.Onge Robert P. , Hyman Richard W.
Published: May 2014

The vast majority of microscopic life on earth consists of microbes that do not grow in laboratory culture. To profile the microbial diversity in environmental and clinical samples, we have devised and employed molecular probe technology, which detects and identifies bacteria that do and do not grow in culture. The only requirement is a short sequence of contiguous bases (currently 60 bases) unique to the genome of the organism of interest. The procedure is relatively fast, inexpensive, customizable, robust, and culture independent and uses commercially available reagents and instruments. In this communication, we report improving the specificity of the molecular probes substantially and increasing the complexity of the molecular probe set by over an order of magnitude (>1,200 probes) and introduce a new final readout method based upon Illumina sequencing. In addition, we employed molecular probes to identify the bacteria from vaginal swabs and demonstrate how a deliberate selection of molecular probes can identify less abundant bacteria even in the presence of much more abundant species.
Metatranscriptome of an Anaerobic Benzene-Degrading, Nitrate-Reducing Enrichment Culture Reveals Involvement of Carboxylation in Benzene Ring Activation
Authors: Luo Fei , Gitiafroz Roya , Devine Cheryl E. , Gong Yunchen , Hug Laura A. , Raskin Lutgarde , Edwards Elizabeth A.
Published: May 2014

The enzymes involved in the initial steps of anaerobic benzene catabolism are not known. To try to elucidate this critical step, a metatranscriptomic analysis was conducted to compare the genes transcribed during the metabolism of benzene and benzoate by an anaerobic benzene-degrading, nitrate-reducing enrichment culture. RNA was extracted from the mixed culture and sequenced without prior mRNA enrichment, allowing simultaneous examination of the active community composition and the differential gene expression between the two treatments. Ribosomal and mRNA sequences attributed to a member of the family
Peptococcaceae
from the order
Clostridiales
were essentially only detected in the benzene-amended culture samples, implicating this group in the initial catabolism of benzene. Genes similar to each of two subunits of a proposed benzene-carboxylating enzyme were transcribed when the culture was amended with benzene. Anaerobic benzoate degradation genes from strict anaerobes were transcribed only when the culture was amended with benzene. Genes for other benzoate catabolic enzymes and for nitrate respiration were transcribed in both samples, with those attributed to an
Azoarcus
species being most abundant. These findings indicate that the mineralization of benzene starts with its activation by a strict anaerobe belonging to the
Peptococcaceae
, involving a carboxylation step to form benzoate. These data confirm the previously hypothesized syntrophic association between a benzene-degrading
Peptococcaceae
strain and a benzoate-degrading denitrifying
Azoarcus
strain for the complete catabolism of benzene with nitrate as the terminal electron acceptor.
Beyond the Matrix-Assisted Laser Desorption Ionization (MALDI) Biotyping Workflow: in Search of Microorganism-Specific Tryptic Peptides Enabling Discrimination of Subspecies
Authors: Gekenidis Maria-Theresia , Studer Patrick , Wüthrich Simone , Brunisholz René , Drissner David
Published: May 2014

A well-accepted method for identification of microorganisms uses matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) coupled to analysis software which identifies and classifies the organism according to its ribosomal protein spectral profile. The method, called MALDI biotyping, is widely used in clinical diagnostics and has partly replaced conventional microbiological techniques such as biochemical identification due to its shorter time to result (minutes for MALDI biotyping versus hours or days for classical phenotypic or genotypic identification). Besides its utility for identifying bacteria, MS-based identification has been shown to be applicable also to yeasts and molds. A limitation to this method, however, is that accurate identification is most reliably achieved on the species level on the basis of reference mass spectra, making further phylogenetic classification unreliable. Here, it is shown that combining tryptic digestion of the acid/organic solvent extracted (classical biotyping preparation) and resolubilized proteins, nano-liquid chromatography (nano-LC), and subsequent identification of the peptides by MALDI-tandem TOF (MALDI-TOF/TOF) mass spectrometry increases the discrimination power to the level of subspecies. As a proof of concept, using this targeted proteomics workflow, we have identified subspecies-specific biomarker peptides for three
Salmonella
subspecies, resulting in an extension of the mass range and type of proteins investigated compared to classical MALDI biotyping. This method therefore offers rapid and cost-effective identification and classification of microorganisms at a deeper taxonomic level.
Comparison of Five Bacteriophages as Models for Viral Aerosol Studies
Authors: Turgeon Nathalie , Toulouse Marie-Josée , Martel Bruno , Moineau Sylvain , Duchaine Caroline
Published: May 2014

Bacteriophages are perceived to be good models for the study of airborne viruses because they are safe to use, some of them display structural features similar to those of human and animal viruses, and they are relatively easy to produce in large quantities. Yet, only a few studies have investigated them as models. It has previously been demonstrated that aerosolization, environmental conditions, and sampling conditions affect viral infectivity, but viral infectivity is virus dependent. Thus, several virus models are likely needed to study their general behavior in aerosols. The aim of this study was to compare the effects of aerosolization and sampling on the infectivity of five tail-less bacteriophages and two pathogenic viruses: MS2 (a single-stranded RNA [ssRNA] phage of the
Leviviridae
family), Φ6 (a segmented double-stranded RNA [dsRNA] phage of the
Cystoviridae
family), ΦX174 (a single-stranded DNA [ssDNA] phage of the
Microviridae
family), PM2 (a double-stranded DNA [dsDNA] phage of the
Corticoviridae
family), PR772 (a dsDNA phage of the
Tectiviridae
family), human influenza A virus H1N1 (an ssRNA virus of the
Orthomyxoviridae
family), and the poultry virus Newcastle disease virus (NDV; an ssRNA virus of the
Paramyxoviridae
family). Three nebulizers and two nebulization salt buffers (with or without organic fluid) were tested, as were two aerosol sampling devices, a liquid cyclone (SKC BioSampler) and a dry cyclone (National Institute for Occupational Safety and Health two-stage cyclone bioaerosol sampler). The presence of viruses in collected air samples was detected by culture and quantitative PCR (qPCR). Our results showed that these selected five phages behave differently when aerosolized and sampled. RNA phage MS2 and ssDNA phage ΦX174 were the most resistant to aerosolization and sampling. The presence of organic fluid in the nebulization buffer protected phages PR772 and Φ6 throughout the aerosolization and sampling with dry cyclones. In this experimental setup, the behavior of the influenza virus resembled that of phages PR772 and Φ6, while the behavior of NDV was closer to that of phages MS2 and ΦX174. These results provide critical information for the selection of appropriate phage models to mimic the behavior of specific human and animal viruses in aerosols.
Use of Cellulolytic Marine Bacteria for Enzymatic Pretreatment in Microalgal Biogas Production
Authors: Muñoz Camilo , Hidalgo Catalina , Zapata Manuel , Jeison David , Riquelme Carlos , Rivas Mariella
Published: May 2014

In this study, we designed and evaluated a microalgal pretreatment method using cellulolytic bacteria that naturally degrades microalgae in their native habitat. Bacterial strains were isolated from each of two mollusk species in a medium containing 1% carboxymethyl cellulose agar. We selected nine bacterial strains that had endoglucanase activity: five strains from
Mytilus chilensis
, a Chilean mussel, and four strains from
Mesodesma donacium
, a clam found in the Southern Pacific. These strains were identified phylogenetically as belonging to the genera
Aeromonas
,
Pseudomonas
,
Chryseobacterium
, and
Raoultella
. The cellulase-producing capacities of these strains were characterized, and the degradation of cell walls in
Botryococcus braunii
and
Nannochloropsis gaditana
was tested with “whole-cell” cellulolytic experiments.
Aeromonas bivalvium
MA2,
Raoultella ornithinolytica
MA5, and
Aeromonas salmonicida
MC25 degraded
B. braunii
, and
R. ornithinolytica
MC3 and MA5 degraded
N. gaditana
. In addition,
N. gaditana
was pretreated with
R. ornithinolytica
strains MC3 and MA5 and was then subjected to an anaerobic digestion process, which increased the yield of methane by 140.32% and 158.68%, respectively, over that from nonpretreated microalgae. Therefore, a “whole-cell” cellulolytic pretreatment can increase the performance and efficiency of biogas production.
Prevalence, Genetic Diversity, and Host Range of Tectiviruses among Members of the Bacillus cereus Group
Authors: Gillis Annika , Mahillon Jacques
Published: May 2014

GIL01, Bam35, GIL16, AP50, and Wip1 are tectiviruses preying on the
Bacillus cereus
group. Despite the significant contributions of phages in different biological processes, little is known about the dealings taking place between tectiviruses and their Gram-positive bacterial hosts. Therefore, this work focuses on characterizing the interactions between tectiviruses and the
B. cereus
group by assessing their occurrence and genetic diversity and evaluating their host range. To study the occurrence of tectiviruses in the
B. cereus
group, 2,000 isolates were evaluated using primers designed to be specific to two variable regions detected in previously described elements. PCR and propagation tests revealed that tectivirus-like elements occurred in less than 3% of the isolates. Regardless of this limited distribution, several novel tectiviruses were found, and partial DNA sequencing indicated that a greater diversity exists within the family
Tectiviridae
. Analyses of the selected variable regions, along with their host range, showed that tectiviruses in the
B. cereus
group can be clustered mainly into two different groups: the ones infecting
B. anthracis
and those isolated from other
B. cereus
group members. In order to address the host range of some novel tectiviruses, 120 strains were tested for sensitivity. The results showed that all the tested tectiviruses produced lysis in at least one
B. cereus sensu lato
strain. Moreover, no simple relationship between the infection patterns of the tectiviruses and their diversity was found.
Development of a Functional Antibody by Using a Green Fluorescent Protein Frame as the Template
Authors: Wang Rongzhi , Xiang Shuangshuang , Zhang Yonghui , Chen Qiuyu , Zhong Yanfang , Wang Shihua
Published: May 2014

Single-chain variable fragment (scFv) antibodies are widely used as diagnostic and therapeutic agents or biosensors for a majority of human disease. However, the limitations of the present scFv antibody in terms of stability, solubility, and affinity are challenging to produce by traditional antibody screening and expression formats. We describe here a feasible strategy for creating the green fluorescent protein (GFP)-based antibody. Complementarity-determining region 3 (CDR3), which retains the antigen binding activity, was introduced into the structural loops of superfolder GFP, and the result showed that CDR3-inserted GFP displayed almost the same fluorescence intensity as wild-type GFP, and the purified proteins of CDR3 insertion showed the similar binding activity to antigen as the corresponding scFv. Among of all of the CDRs, CDR3s are responsible for antigen recognition, and only the CDR3a insertion is the best format for producing GFP-based antibody binding to specific antigen. The wide versatility of this system was further verified by introducing CDR3 from other scFvs into loop 9 of GFP. We developed a feasible method for rapidly and effectively producing a high-affinity GFP-based antibody by inserting CDR3s into GFP loops. Further, the affinity can be enhanced by specific amino acids scanning and site-directed mutagenesis. Notably, this method had better versatility for creating antibodies to various antigens using GFP as the scaffold, suggesting that a GFP-based antibody with high affinity and specificity may be useful for disease diagnosis and therapy.
Eutrophication and Bacterial Pathogens as Risk Factors for Avian Botulism Outbreaks in Wetlands Receiving Effluents from Urban Wastewater Treatment Plants
Authors: Anza Ibone , Vidal Dolors , Laguna Celia , Díaz-Sánchez Sandra , Sánchez Sergio , Chicote Álvaro , Florín Máximo , Mateo Rafael
Published: May 2014

Due to the scarcity of water resources in the “Mancha Húmeda” Biosphere Reserve, the use of treated wastewater has been proposed as a solution for the conservation of natural threatened floodplain wetlands. In addition, wastewater treatment plants of many villages pour their effluent into nearby natural lakes. We hypothesized that certain avian pathogens present in wastewater may cause avian mortalities which would trigger avian botulism outbreaks. With the aim of testing our hypothesis, 24 locations distributed in three wetlands, two that receive wastewater effluents and one serving as a control, were monitored during a year. Sediment, water, water bird feces, and invertebrates were collected for the detection of putative avian pathogenic
Escherichia coli
(APEC),
Salmonella
spp.,
Clostridium perfringens
type A, and
Clostridium botulinum
type C/D. Also, water and sediment physicochemical properties were determined. Overall, APEC,
C. perfringens
, and
C. botulinum
were significantly more prevalent in samples belonging to the wetlands which receive wastewater. The occurrence of a botulism outbreak in one of the studied wetlands coincided with high water temperatures and sediment 5-day biochemical oxygen demand (BOD
5
), a decrease in water redox potential, chlorophyll
a
, and sulfate levels, and an increase in water inorganic carbon levels. The presence of
C. botulinum
in bird feces before the onset of the outbreak indicates that carrier birds exist and highlights the risk of botulinum toxin production in their carcasses if they die by other causes such as bacterial diseases, which are more probable in wastewater wetlands.
Pyruvate-Associated Acid Resistance in Bacteria
Authors: Wu Jianting , Li Yannan , Cai Zhiming , Jin Ye
Published: May 2014

Glucose confers acid resistance on exponentially growing bacteria by repressing formation of the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex and consequently activating acid resistance genes. Therefore, in a glucose-rich growth environment, bacteria are capable of resisting acidic stresses due to low levels of cAMP-CRP. Here we reveal a second mechanism for glucose-conferred acid resistance. We show that glucose induces acid resistance in exponentially growing bacteria through pyruvate, the glycolysis product. Pyruvate and/or the downstream metabolites induce expression of the small noncoding RNA (sncRNA) Spot42, and the sncRNA, in turn, activates expression of the master regulator of acid resistance, RpoS. In contrast to glucose, pyruvate has little effect on levels of the cAMP-CRP complex and does not require the complex for its effects on acid resistance. Another important difference between glucose and pyruvate is that pyruvate can be produced by bacteria. This means that bacteria have the potential to protect themselves from acidic stresses by controlling glucose-derived generation of pyruvate, pyruvate-acetate efflux, or reversion from acetate to pyruvate. We tested this possibility by shutting down pyruvate-acetate efflux and found that the resulting accumulation of pyruvate elevated acid resistance. Many sugars can be broken into glucose, and the subsequent glycolysis generates pyruvate. Therefore, pyruvate-associated acid resistance is not confined to glucose-grown bacteria but is functional in bacteria grown on various sugars.
Comparative Biochemical Characterization of Three Exolytic Oligoalginate Lyases from Vibrio splendidus Reveals Complementary Substrate Scope, Temperature, and pH Adaptations
Authors: Jagtap Sujit Sadashiv , Hehemann Jan-Hendrik , Polz Martin F. , Lee Jung-Kul , Zhao Huimin
Published: May 2014

Marine microbes use alginate lyases to degrade and catabolize alginate, a major cell wall matrix polysaccharide of brown seaweeds. Microbes frequently contain multiple, apparently redundant alginate lyases, raising the question of whether these enzymes have complementary functions. We report here on the molecular cloning and functional characterization of three exo-type oligoalginate lyases (OalA, OalB, and OalC) from
Vibrio splendidus
12B01 (12B01), a marine bacterioplankton species. OalA was most active at 16°C, had a pH optimum of 6.5, and displayed activities toward poly-β-
d
-mannuronate [poly(M)] and poly-α-
l
-guluronate [poly(G)], indicating that it is a bifunctional enzyme. OalB and OalC were most active at 30 and 35°C, had pH optima of 7.0 and 7.5, and degraded poly(M·G) and poly(M), respectively. Detailed kinetic analyses of oligoalginate lyases with poly(G), poly(M), and poly(M·G) and sodium alginate as substrates demonstrated that OalA and OalC preferred poly(M), whereas OalB preferred poly(M·G). The catalytic efficiency (
k
cat
/

K
m

) of OalA against poly(M) increased with decreasing size of the substrate. OalA showed
k
cat
/

K
m

from 2,130 mg
−1
ml s
−1
for the trisaccharide to 224 mg
−1
ml s
−1
for larger oligomers of ∼50 residues, and 50.5 mg
−1
ml s
−1
for high-molecular-weight alginate. Although OalA was most active on the trisaccharide, OalB and OalC preferred dimers. Taken together, our results indicate that these three Oals have complementary substrate scopes and temperature and pH adaptations.
Coordinated and Distinct Functions of Velvet Proteins in Fusarium verticillioides
Authors: Lan Nan , Zhang Hanxing , Hu Chengcheng , Wang Wenzhao , Calvo Ana M. , Harris Steven D. , Chen She , Li Shaojie
Published: May 2014

Velvet-domain-containing proteins are broadly distributed within the fungal kingdom. In the corn pathogen
Fusarium verticillioides
, previous studies showed that the velvet protein
F. verticillioides
VE1 (FvVE1) is critical for morphological development, colony hydrophobicity, toxin production, and pathogenicity. In this study, tandem affinity purification of FvVE1 revealed that FvVE1 can form a complex with the velvet proteins
F. verticillioides
VelB (FvVelB) and FvVelC. Phenotypic characterization of gene knockout mutants showed that, as in the case of FvVE1, FvVelB regulated conidial size, hyphal hydrophobicity, fumonisin production, and oxidant resistance, while FvVelC was dispensable for these biological processes. Comparative transcriptional analysis of eight genes involved in the ROS (reactive oxygen species) removal system revealed that both FvVE1 and FvVelB positively regulated the transcription of a catalase-encoding gene,
F. verticillioides
CAT2
(
FvCAT2
). Deletion of
FvCAT2
resulted in reduced oxidant resistance, providing further explanation of the regulation of oxidant resistance by velvet proteins in the fungal kingdom.
Metal-Mediated Modulation of Streptococcal Cysteine Protease Activity and Its Biological Implications
Authors: Chella Krishnan Karthickeyan , Mukundan Santhosh , Landero Figueroa Julio A. , Caruso Joseph A. , Kotb Malak
Published: May 2014

Streptococcal cysteine protease (SpeB), the major secreted protease produced by group A streptococcus (GAS), cleaves both host and bacterial proteins and contributes importantly to the pathogenesis of invasive GAS infections. Modulation of SpeB expression and/or its activity during invasive GAS infections has been shown to affect bacterial virulence and infection severity. Expression of SpeB is regulated by the GAS CovR-CovS two-component regulatory system, and we demonstrated that bacteria with mutations in the CovR-CovS two-component regulatory system are selected for during localized GAS infections and that these bacteria lack SpeB expression and exhibit a hypervirulent phenotype. Additionally, in a separate study, we showed that expression of SpeB can also be modulated by human transferrin- and/or lactoferrin-mediated iron chelation. Accordingly, the goal of this study was to investigate the possible roles of iron and other metals in modulating SpeB expression and/or activity in a manner that would potentiate bacterial virulence. Here, we report that the divalent metals zinc and copper inhibit SpeB activity at the posttranslational level. Utilizing online metal-binding site prediction servers, we identified two putative metal-binding sites in SpeB, one of which involves the catalytic-dyad residues
47
Cys and
195
His. Based on our findings, we propose that zinc and/or copper availability in the bacterial microenvironment can modulate the proteolytic activity of SpeB in a manner that preserves the integrity of several other virulence factors essential for bacterial survival and dissemination within the host and thereby may exacerbate the severity of invasive GAS infections.