bluuebunny/crossref_metadata_2025 huggingface dataset:
Model-based detection of buried objects.
Authors: Sullivan Edmund J. , Xiang Ning
Published: March 2010

An approach to the detection of buried objects is to excite the ground with low-frequency, high-energy sound waves, which then excites a resonance in the buried object. The ensuing vibration causes a detectable signal on the surface of the ground, which can be detected using a laser doppler vibrometer (LDV). The original detection technique used a sliding bandpass filter to process the scattered LDV energy, providing an energy map of the area scanned by the LDV, which indicates the location of the object. The performance of detection is often limited by speckle noise, a type of noise arising from the coherent nature of the laser beam. A more recent technique utilizes an autoregressive model of this noise. This leads to an inverse filter that whitens the noise. Upon the appearance of any target data in the signal, a whiteness test indicates a detection. This approach has demonstrated improvement over the bandpass filter approach. This paper demonstrates a further improvement by augmenting the prewhitener with a model of the mine itself. This provides significant improvement by both enhancing the mine signal and improving the detection performance. Experimental results are shown.
Improving FSTC (field sound transmission class) and FIIC (field-impact isolation class) of standard wall and floor/ceiling configurations.
Authors: Schnitta Bonnie , Russo Melissa , Kern Trish , Greenwald Greg
Published: March 2010

Every acoustic floor or wall design requires an analysis in order to provide a sustainable design. This is true whether the design is a single family residence with the goal of achieving a desired level of quietude, or a multi-family, or commercial dwelling required to meet various codes and/or standards. Since IIC and STC measurements taken in the field produce a lower STC or IIC than laboratory controlled values, this presentation will begin by reviewing the variables that are not present in the controlled laboratory setting for their acoustic impact. This presentation will further provide field readings of well known documented basic floor and wall configurations. With this foundation standard floor and wall configurations will be reviewed for their FSTC and FIIC improvement with standard readily available acoustic materials to address variables consistent with the degradation of the configuration’s acoustic efficacy. The FIIC and FSTC values of the same floors and walls will be further compared to walls and floors with unique and innovative installation methodologies and configuration upgrades. The summary will be a detailed approach to the best field practices and innovative configuration upgrades, inclusive of a few new patented and patent pending products, for optimum field acoustic efficacy.
Focusing, maintaining, and switching attention.
Authors: Shinn-Cunningham Barbara G.
Published: March 2010

Humans and other animals have an amazing ability to selectively attend to whatever sound source is most relevant, a task that requires the brain to separate a sound mixture into distinct perceptual objects. Results from a number of recent behavioral and neuroimaging studies from our laboratory and others have begun to tease apart the processes and mechanisms that enable us to select an important source from a sound mixture and attend to it as its meaning unfolds, as well as to switch attention if the need arises. As reviewed in this talk, the processes of focusing, maintaining, and switching auditory attention involve dynamics that directly impact our ability to understand sounds in complex settings. For instance, there is a cost associated with the process of disengaging attention from one source and focusing on another. On top of this switching cost, there is a benefit of maintaining attention to an ongoing source that yields improvements in performance over time. These processes interact with the way we store and remember signals and directly influence how we function in complex auditory scenes, especially social settings like the Speech Communication Poster Session or the Thursday Evening Buffet Social.
Ultrasonic characterization of cancellous bone. Biot approach: Pros and cons.
Authors: Laugier Pascal
Published: March 2010

Reliable quantification of microstructural and mechanical bone properties remains an open issue with relevance for the diagnosis of bone quality disorders, such as osteoporosis. The reconstruction of such parameters from nondestructive testing based on ultrasound testing and model-based solution of the identification inverse problem have been suggested by several investigators as novel techniques with high potential not only due to the reduced cost and its non-ionizing nature, but for the direct relationship and sensitivity of the propagation characteristics estimates to relevant bone properties that determine mechanical strength, Recently, approaches taking advantage of the poroelastic nature of cancellous bone have been developed. These approaches assume that the Biot theory is a valid model of wave propagation and are aiming at either (i) recovering the properties of the fast and slow waves or (ii) directly reconstructing the bone properties that govern Biot’s theory. However, the most recent literature reveals inconsistencies between Biot model and experimental results, thereby suggesting that Biot model may not be the most appropriate theoretical framework for solving the inverse problem. The most recent results will be reviewed and future research directions will be highlighted to overcome the observed inconsistencies.
The effect of structural anisotropy on the fast wave propagation in cancellous bone.
Authors: Matsukawa Mami , Mizuno Katsunori , Nagatani Yoshiki
Published: March 2010

QUS parameters are closely related to the structural properties and elastic properties of bone, which can provide important information related to bone quality and bone strength. One should be very careful, however, that bone contains complicated structure from microscopic to macroscopic levels. For example, the cancellous bone inside the epiphysis is composed of a complicated trabecular network in the bone marrow, showing strongly anisotropic and heterogeneous structure. We have then observed the longitudinal wave propagation in this complicated medium, paying special attention to the two wave phenomenon. The fast wave, which mainly propagates in the trabecular part of the cancellous bone, is more sensitive to the network structure. With the help of x-ray micro-computed tomography, three dimensional anisotropic trabecular structure and fast wave propagation were investigated using bovine cancellous bones. We can find the strong effect of anisotropic trabecular structure on the fast wave velocity. The mean intercept length (the information of average trabecular length) was a good parameter to describe the three dimensional trabecular structure and showed good correlation with the fast wave velocity.
Multiscale structure-functional modeling of lamellar bone.
Authors: Raum Kay , Grimal Quentin , Gerisch Alf
Published: March 2010

Bone is a natural example of achieving a unique combination and variability of stiffness and strength. One of the striking features of bone tissue is the ability to adapt to variable loading conditions by multiple but well organized structural arrangements of mineralized collagen fibrils at several levels of hierarchical organization. A profound understanding of the structure-function relations in bone requires both experimental assessment of heterogeneous elastic and structural parameters and theoretical modeling of the elastic deformation behavior. A bottom-up approach for experimental assessment and numerical modeling of the hierarchical structure from the nanoscale to the macroscale will be presented. Experimental data are obtained by scanning acoustic microscopy between 50 MHz and 1.2 GHz and provide anisotropic elastic and structural information at the lamellar (nanoscale) and at the tissue matrix (microscale) level. These data are directly translated into a finite element mesh. By numerical deformation analyses, the homogenized elastic stiffness tensor of the next hierarchical levels (microscale to macroscale) is derived. At each level the numerical results are cross-validated by experimental data. It will be shown that local variations in elastic anisotropy within the femoral shaft are related to an inhomogeneous strain distribution resulting from external stresses by weight and muscle forces.
Acoustic scattering from layered pentamode materials.
Authors: Scandrett Clyde L. , Boisvert Jeffrey E. , Howarth Thomas R.
Published: March 2010

While receiving less attention in the literature than in the field of electromagnetic scattering, theoretical efforts to define and create acoustic skins capable of reducing scattered energy from obstacles by way of mimicking coordinate transformations through use of meta-materials have begun. The present work extends recent analysis of Norris by considering a variety of acoustic skins, from those comprised of fluid layers which are isotropic in bulk moduli with anisotropic density to those having anisotropic bulk moduli and isotropic density. In all but pure inertial types, fluid layers comprising the skins are pentamode materials governed by a special scalar acoustic equation for pseudo-pressure derived by Norris. In most cases presented, material properties of the fluid/pentamode layers are based upon target values specified by continuously varying properties resulting from theoretical coordinate transformations geared to minimize scattered pressure limited by realistic goals. The present work analyzes acoustic skins for the specific case of plane wave scattering from an acoustically hard sphere. An initial exploration of the parameter space defining such skins (for example, material properties of its constituent layers and operating frequency) is undertaken with a view toward “optimal” design. [Work supported by the NAVSEA Newport In-House Laboratory Individual Research (ILIR) Program.]
Investigation of dominant sources of infrasonic wind noise in ground level microphones.
Authors: Abbott John Paul R. , Raspet Richard , Webster Jeremy , Yu Jiao
Published: March 2010

A two part project has investigated whether the pressure fluctuations (infrasonic wind noise) at the ground, as measured in an open field, can be predicted from the vertical wind velocity measured above the ground. The dominant region contributing to the pressure on the ground is calculated theoretically and displayed with contour plots. Experimentally, time dependent correlations between the measured pressure and wind velocities were done. The correlation coefficients were very low, typically between 0.08–0.15, for broadband frequencies up to 5 Hz. Cross spectrum analysis and application of octave band pass filters showed the dominant frequencies for correlations to be around 0.07–0.1 Hz, with correlation coefficients typically between 0.3–0.5. [Project funded by the US Army Space and Missile Defense Command.]
A theoretical and experimental study of sound propagation above a rigid porous ground.
Authors: Liu Sheng , Li Kai Ming
Published: March 2010

The sound propagation due to a monopole source near a rigid porous ground is reviewed. Closed form approximation solutions for predicting the sound field above the ground surface were derived analytically by (1) the method of pole subtraction and (2) the use of an effective impedance approach. The locations of the surface wave pole according to both methods were examined. These two approximate solutions were compared with an exact evaluation of the Fourier integral representing the sound field above the rigid porous ground. It is found that the approximate solution using the effective impedance approach offers relatively better agreements with the exact numerical solution. In addition, the effective impedance approach generally leads to a more accurate uniform asymptotic solution for the sound field above the rigid porous ground. In this presentation, the reason for this good agreement is explained. The finding is further supported by experimental data obtained by conducting indoor measurements over a layer of glass beads.
Evaluation of surface waves in bone-conducted sound.
Authors: Wismer Margaret , O’Brien William , Clavier Odile , Dietz Anthony
Published: March 2010

In order to better understand the basic physical principles of bone-conducted sound, the skull is approximated as a thin spherical fluid-filled shell. Results for sound waves propagating through and around this structure have been determined for a range of frequencies, media, and shell sizes. Since the early 1950s researchers have studied the problem of what happens when waves hit solid targets. Most of the research is concerned with the back-scatter signal but recognize that the interaction of the wave with the object influences these results. For the bone-conducted sound study, the interaction between the incident signal and the object is the critical information. For acoustic noise interacting with the human skull, the ka is below 5. The structure supports the existence of surface waves as the predominant mechanism of energy transfer within the skull. Multiple types of surface wave for this structure of a thin, curved fluid-solid interface exist. Signals which propagate on the air side of the surface due to the curvature are known as Franz or Stoneley waves. Since the shell is thin compared to the typical wavelength of sound, waves within the shell are lower order Lamb or plate waves. All of these waves are dispersive in nature with group speeds considerably smaller than the speed of sound in bone.
Spoken Hindi paired word recognition using probabilistic neural network.
Authors: Rajoriya Dinesh Kumar , Anand R. S. , Maheshwari R. P.
Published: March 2010

Automatic speech recognition system plays significant role in the development of real world applications. This paper demonstrated spoken Hindi (Indian national language) paired word recognition (SHPWR) system, which has been examined with the help of probabilistic neural network as a classifier. This type of network is a combination of radial basis layer and a competitive transfer function layer which picks the maximum probabilities as a final result. SHPWR is pattern recognition (PR) problem. A PR technique encompasses two fundamental tasks; description and classification. In the description process, features are extracted from the Hindi paired word templates, and probabilistic neural network is used as a classifier. For the experimental point of view 1000 Hindi paired word templates have been recorded from individuals with different environment condition, gender, and age groups. Speaker dependent SHPWR systems achieved close to 100% while the accuracy of speaker independent SHPWR systems is quite adequate.
Clustering classification methods for acoustic applications including the analysis of clicking whale data.
Authors: Ioup Juliette W. , Ioup George E. , Pflug Lisa A. , Sidorovskaia Natalia A. , Tiemann Christopher O. , Thompson Charles H.
Published: March 2010

Several clustering methods are available to group like objects into classes. The K means approach is well established. Both standard and homegrown algorithms have been tested in the following applications. One neural network technique is self-organizing map clustering [T. Kohonen, Self-Organizing Maps, 2nd ed. Springer, New York (1997)]. Although it has some similarities to K means, it has notable differences which can be quite helpful in classification. An advantage is that it has the ability to limit the number of clusters automatically. This can be an important aspect when the number of classes is unknown. After a review of the basics of these techniques, the power of the methods is illustrated by applications to the study of clicking whales. The Littoral Acoustic Demonstration Center has acquired underwater acoustic data from clicking whales in the Gulf of Mexico, and it has access to data from the third and fourth International Workshops on Detection, Classification and Localization of Marine Mammals Using Passive Acoustics. Application of K means and SOM clustering to these data is an important component of identifying individual whales acoustically. Additional details on these results are being presented in Animal Bioacoustics sessions at this meeting. [Research supported by ONR and SPAWAR.]
Speech forensics: Automatic acquisition device identification.
Authors: Garcia-Romero Daniel , Espy-Wilson Carol
Published: March 2010

Automatic identification-of-the-source is an important problem in the field of speech forensics. That is, given a speech recording, the problem is to determine how to automatically determine the exact make and model of the acquisition device regardless of the speaker and speech content. Recent work developed by the authors have shown that statistical modeling of the device characteristics in terms of MFCCs provides excellent discriminative power when the recordings are obtained under controlled conditions (i.e., all recordings come from the same room and a unique device per class is used). In this study we propose to expand these results by analyzing the effects that room acoustics and intra-device variability have in the source identification accuracy. To perform this analysis, the NIST 2008 SRE dataset will be used. This corpus provides recordings acquired with multiple devices of the same make and model in different rooms.
Regeneration Switch
Authors: VanHook Annalisa M.
Published: March 2010

mTOR signaling is implicated in a switch that improves liver regeneration in aged mice.
Preclinical Characterization of the Antiviral Activity of SCH 900518 (Narlaprevir), a Novel Mechanism-Based Inhibitor of Hepatitis C Virus NS3 Protease
Authors: Tong X. , Arasappan A. , Bennett F. , Chase R. , Feld B. , Guo Z. , Hart A. , Madison V. , Malcolm B. , Pichardo J. , Prongay A. , Ralston R. , Skelton A. , Xia E. , Zhang R. , Njoroge F. G.
Published: March 2010

Small-molecule hepatitis C virus (HCV) NS3 protease inhibitors such as boceprevir (SCH 503034) have been shown to have antiviral activity when they are used as monotherapy and in combination with pegylated alpha interferon and ribavirin in clinical trials. Improvements in inhibitor potency and pharmacokinetic properties offer opportunities to increase drug exposure and to further increase the sustained virological response. Exploration of the structure-activity relationships of ketoamide inhibitors related to boceprevir has led to the discovery of SCH 900518, a novel ketoamide protease inhibitor which forms a reversible covalent bond with the active-site serine. It has an overall inhibition constant (
K
*
i
) of 7 nM and a dissociation half-life of 1 to 2 h. SCH 900518 inhibited replicon RNA at a 90% effective concentration (EC
90
) of 40 nM. In biochemical assays, SCH 900518 was active against proteases of genotypes 1 to 3. A 2-week treatment with 5× EC
90
of the inhibitor reduced the replicon RNA level by 3 log units. Selection of replicon cells with SCH 900518 resulted in the outgrowth of several resistant mutants (with the T54A/S and A156S/T/V mutations). Cross-resistance studies demonstrated that the majority of mutations for resistance to boceprevir and telaprevir caused similar fold losses of activity against all three inhibitors; however, SCH 900518 retained more activity against these mutants due to its higher intrinsic potency. Combination treatment with alpha interferon enhanced the inhibition of replicon RNA and suppressed the emergence of resistant replicon colonies, supporting the use of SCH 900518-pegylated alpha interferon combination therapy in the clinic. In summary, the results of the preclinical characterization of the antiviral activity of SCH 900518 support its evaluation in clinical studies.
Characterization of CSP-1, a Novel Extended-Spectrum β-Lactamase Produced by a Clinical Isolate of
Capnocytophaga sputigena
Authors: Guillon Hélène , Eb François , Mammeri Hedi
Published: March 2010

The
Capnocytophaga sputigena
isolate NOR, responsible for septicemia, was resistant to amoxicillin and narrow-spectrum cephalosporins. In a cloning experiment, a new gene,
bla
CSP-1
, was identified; this gene encodes a novel extended-spectrum β-lactamase (ESBL) that shares only 52% and 49% identities with the CME-1 and VEB-1 β-lactamases, respectively. The G+C content of this gene, its genetic environment, the absence of conjugation transfer, and its detection in two reference strains suggested that it was an intrinsic resistance gene located on the chromosome.
Intra- and Extracellular Activities of Dicloxacillin against
Staphylococcus aureus In Vivo
and
In Vitro
Authors: Sandberg Anne , Jensen Klaus Skovbo , Baudoux Pierre , Van Bambeke Françoise , Tulkens Paul M. , Frimodt-Møller Niels
Published: March 2010

Antibiotic treatment of
Staphylococcus aureus
infections is often problematic due to the slow response and recurrences. The intracellular persistence of the staphylococci offers a plausible explanation for the treatment difficulties because of the impaired intracellular efficacies of the antibiotics. The intra- and extracellular time- and concentration-kill relationships were examined
in vitro
with THP-1 cells and
in vivo
by use of a mouse peritonitis model. The
in vivo
model was further used to estimate the most predictive pharmacokinetic/pharmacodynamic (PK/PD) indices (the ratio of the maximum concentration of drug in plasma/MIC, the ratio of the area under the concentration-time curve/MIC, or the cumulative percentage of a 24-h period that the free [
f
] drug concentration exceeded the MIC under steady-state pharmacokinetic conditions [
fT
MIC
]) for dicloxacillin (DCX) intra- and extracellularly. In general, DCX was found to have similar intracellular activities, regardless of the model used. Both models showed (i) the relative maximal efficacy (1-log-unit reduction in the numbers of CFU) of DCX intracellularly and (ii) the equal relative potency of DCX intra- and extracellularly, with the MIC being a good indicator of the overall response in both situations. Discordant results, based on data obtained different times after dosing, were obtained from the two models when the extracellular activity of DCX was measured, in which the
in vitro
model showed a considerable reduction in the number of CFU from that in the original inoculum (3-log-unit decrease in the number of CFU after 24 h), whereas the extracellular CFU reduction achieved
in vivo
after 4 h did not exceed 1 log unit. Multiple dosing of DCX
in vivo
revealed increased extra- and intracellular efficacies (2.5 log and 2 log units of reduction in the numbers of CFU after 24 h, respectively), confirming that DCX is a highly active antistaphylococcal antibiotic. PK/PD analysis revealed that
fT
MIC
is the index that is the most predictive of the outcome of infection both intra- and extracellularly.
Origins of the Recent Emergence of
Plasmodium falciparum
Pyrimethamine Resistance Alleles in Madagascar
Authors: Andriantsoanirina Valérie , Bouchier Christiane , Tichit Magali , Jahevitra Martial , Rabearimanana Stéphane , Randrianjafy Rogelin , Ratsimbasoa Arsène , Mercereau-Puijalon Odile , Durand Rémy , Ménard Didier
Published: March 2010

The combination of sulfadoxine-pyrimethamine is recommended for use as intermittent preventive treatment of malaria during pregnancy and is deployed in Africa. The emergence and the spread of resistant parasites are major threats to such an intervention. We have characterized the
Plasmodium falciparum
dhfr
(
pfdhfr
) haplotypes and flanking microsatellites in 322
P. falciparum
isolates collected from the Comoros Islands and Madagascar. One hundred fifty-six (48.4%) carried the wild-type
pfdhfr
allele, 19 (5.9%) carried the S108N single-mutation allele, 30 (9.3%) carried the I164L single-mutation allele, 114 (35.4%) carried the N51I/C59R/S108N triple-mutation allele, and 3 (1.0%) carried the N51I/C59R/S108N/I164L quadruple-mutation allele. Microsatellite analysis showed the introduction from the Comoros Islands of the ancestral
pfdhfr
triple mutant allele of Asian origin and its spread in Madagascar. Evidence for the emergence on multiple occasions of the I164L single-mutation
pfdhfr
allele in Madagascar was also obtained. Thus, the conditions required to generate mutants with quadruple mutations are met in Madagascar, representing a serious threat to current drug policy.
In Vitro
Activity of ACH-702, a New Isothiazoloquinolone, against
Nocardia brasiliensis
Compared with Econazole and the Carbapenems Imipenem and Meropenem Alone or in Combination with Clavulanic Acid
Authors: Vera-Cabrera Lucio , Campos-Rivera Mayra Paola , Escalante-Fuentes Wendy G. , Pucci Michael J. , Ocampo-Candiani Jorge , Welsh Oliverio
Published: March 2010

The
in vitro
activities of ACH-702 and other antimicrobials against 30
Nocardia brasiliensis
isolates were tested. The MIC
50
(MIC for 50% of the strains tested) and MIC
90
values of ACH-702 were 0.125 and 0.5 μg/ml. The same values for econazole were 2 and 4 μg/ml. The MIC
50
and MIC
90
values of imipenem and meropenem were 64 and >64 μg/ml and 2 and 8 μg/ml, respectively; the addition of clavulanic acid to the carbapenems had no effect.
Plasmodium
vivax
Susceptibility to Ferroquine
Authors: Leimanis M. L. , Jaidee A. , Sriprawat K. , Kaewpongsri S. , Suwanarusk R. , Barends M. , Phyo A. P. , Russell B. , Renia L. , Nosten F.
Published: March 2010

The novel organometallic chloroquine analog ferroquine (SSR 97193) is effective against chloroquine-resistant
Plasmodium
falciparum
. The
ex
vivo
efficacy of ferroquine against
Plasmodium
vivax
isolates was tested. Ferroquine has a potent
ex
vivo
effect on
P
.
vivax
schizont maturation (median 50% inhibitory concentration, 15 nM;
n
= 42). No significant cross-sensitivity between ferroquine and other antimalarials was detected. This drug may be a suitable replacement for chloroquine in the treatment of drug-resistant
P
.
vivax
malaria.
The M230L Nonnucleoside Reverse Transcriptase Inhibitor Resistance Mutation in HIV-1 Reverse Transcriptase Impairs Enzymatic Function and Viral Replicative Capacity
Authors: Xu Hong-Tao , Quan Yudong , Schader Susan M. , Oliveira Maureen , Bar-Magen Tamara , Wainberg Mark A.
Published: March 2010

The M230L mutation in HIV-1 reverse transcriptase (RT) is associated with resistance to first-generation nonnucleoside reverse transcriptase inhibitors (NNRTIs). The present study was designed to determine the effects of M230L on enzyme function, viral replication capacity (RC), and the extent to which M230L might confer resistance to the second-generation NNRTI etravirine (ETR) as well as to the first-generation NNRTIs efavirenz (EFV) and nevirapine (NVP). Phenotyping assays with TZM-bl cells confirmed that M230L conferred various degrees of resistance to each of the NNRTIs tested. Recombinant viruses containing M230L displayed an 8-fold decrease in RC compared to that of the parental wild-type (WT) virus. Recombinant HIV-1 WT and M230L mutant RT enzymes were purified; and both biochemical and cell-based phenotypic assays confirmed that M230L conferred resistance to each of EFV, NVP, and ETR. RT that contained M230L was also deficient in regard to each of minus-strand DNA synthesis, both DNA- and RNA-dependent polymerase activities, processivity, and RNase H activity, suggesting that this mutation contributes to diminished viral replication kinetics.
Lepidopteran-Active Variable-Region Sequence Imparts Coleopteran Activity in eCry3.1Ab, an Engineered
Bacillus thuringiensis
Hybrid Insecticidal Protein
Authors: Walters Frederick S. , deFontes Cheryl M. , Hart Hope , Warren Gregory W. , Chen Jeng S.
Published: March 2010

A unique, coleopteran-active protein, termed eCry3.1Ab, was generated following variable-region exchange of a
Bacillus thuringiensis
lepidopteran-active protein, Cry1Ab, with a Cry3A region. Our results support the hypothesis that this variable-region exchange is responsible for imparting strong bioactivity against the larvae of western corn rootworm (WCR) (
Diabrotica virgifera virgifera
LeConte), a pest species which is not susceptible to either parent protein sequence. This study demonstrates the potential of successfully engineering a portion(s) of a lepidopteran-active
B. thuringiensis
sequence so that it has activity against coleopterans. Further elucidation of the eCry3.1Ab activity indicated the importance of variable regions 4 to 6 that were derived from Cry1Ab instead of Cry1Ac. There was some flexibility in making domain III of engineered hybrid insecticidal proteins even more Cry1Ab-like and retaining activity, while there was less flexibility in making domain III more Cry3A-like and retaining activity.
In vitro
binding studies with brush border membrane vesicles demonstrated that there was specific binding of chymotrypsin-processed modified Cry3A (mCry3A), which was not diminished by addition of a 100-fold molar excess of chymotrypsin-processed eCry3.1Ab or unprocessed eCry3.1Ab. In addition, in the converse experiment, specific binding of chymotrypsin-processed eCry3.1Ab was not diminished by the presence of a 75-fold molar excess of chymotrypsin-processed mCry3A. These data support the hypothesis that eCry3.1Ab can interact with different binding sites than the activated form of mCry3A in the WCR brush border and may provide a different mode of action from the standpoint of resistance management.
Heterologous Expression of Mutated Eburicol 14α-Demethylase (CYP51) Proteins of
Mycosphaerella graminicola
To Assess Effects on Azole Fungicide Sensitivity and Intrinsic Protein Function
Authors: Cools H. J. , Parker J. E. , Kelly D. E. , Lucas J. A. , Fraaije B. A. , Kelly S. L.
Published: March 2010

The recent decrease in the sensitivity of the Western European population of the wheat pathogen
Mycosphaerella graminicola
to azole fungicides has been associated with the emergence and subsequent spread of mutations in the
CYP51
gene, encoding the azole target sterol 14α-demethylase. In this study, we have expressed wild-type and mutated
M. graminicola
CYP51 (MgCYP51) variants in a
Saccharomyces cerevisiae
mutant carrying a doxycycline-regulatable

tetO
7

-
CYC
promoter controlling native
CYP51
expression. We have shown that the wild-type MgCYP51 protein complements the function of the orthologous protein in
S. cerevisiae
. Mutant MgCYP51 proteins containing amino acid alterations L50S, Y459D, and Y461H and the two-amino-acid deletion ΔY459/G460, commonly identified in modern
M. graminicola
populations, have no effect on the capacity of the
M. graminicola
protein to function in
S. cerevisiae
. We have also shown that the azole fungicide sensitivities of transformants expressing MgCYP51 variants with these alterations are substantially reduced. Furthermore, we have demonstrated that the I381V substitution, correlated with the recent decline in the effectiveness of azoles, destroys the capacity of
MgCYP51
to complement the
S. cerevisiae
mutant when introduced alone. However, when I381V is combined with changes between residues Y459 and Y461, the function of the
M. graminicola
protein is partially restored. These findings demonstrate, for the first time for a plant pathogenic fungus, the impacts that naturally occurring CYP51 alterations have on both azole sensitivity and intrinsic protein function. In addition, we also provide functional evidence underlying the order in which CYP51 alterations in the Western European
M. graminicola
population emerged.
Succession of the Bacterial Community and Dynamics of Hydrogen Producers in a Hydrogen-Producing Bioreactor
Authors: Huang Yue , Zong Wenming , Yan Xing , Wang Ruofan , Hemme Christopher L. , Zhou Jizhong , Zhou Zhihua
Published: March 2010

Variation in the hydrogen production rate was consistent with the succession of dominant bacteria during the batch fermentation process. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes and quantitative analysis of the
hyd
A
genes at both the DNA and mRNA levels confirmed that
C
lostridium perfringens
was the most dominant hydrogen producer in the bioreactor.
Anaerobic Oxidation of Fatty Acids and Alkenes by the Hyperthermophilic Sulfate-Reducing ArchaeonArchaeoglobus fulgidus
Authors: Khelifi Nadia , Grossi Vincent , Hamdi Moktar , Dolla Alain , Tholozan Jean-Luc , Ollivier Bernard , Hirschler-Réa Agnès
Published: March 2010

Archaeoglobus fulgidusoxidizes fatty acids (C4to C18) andn-alk-1-enes (C12:1to C21:1) in the presence of thiosulfate as a terminal electron acceptor. End products of metabolism were CO2and sulfide. Growth on perdeuterated hexadecene yielded C15- to C17-labeled fatty acids as metabolites, thus confirming the ability ofA. fulgidusto oxidize alkyl chains.
Homoserine Toxicity in Saccharomyces cerevisiae and Candida albicans Homoserine Kinase (
thr1
Δ) Mutants
Authors: Kingsbury Joanne M. , McCusker John H.
Published: March 2010

In addition to threonine auxotrophy, mutation of the
Saccharomyces cerevisiae
threonine biosynthetic genes
THR1
(encoding homoserine kinase) and
THR4
(encoding threonine synthase) results in a plethora of other phenotypes. We investigated the basis for these other phenotypes and found that they are dependent on the toxic biosynthetic intermediate homoserine. Moreover, homoserine is also toxic for
Candida albicans
thr1
Δ mutants. Since increasing levels of threonine, but not other amino acids, overcome the homoserine toxicity of
thr1
Δ mutants, homoserine may act as a toxic threonine analog. Homoserine-mediated lethality of
thr1
Δ mutants is blocked by cycloheximide, consistent with a role for protein synthesis in this lethality. We identified various proteasome and ubiquitin pathway components that either when mutated or present in high copy numbers suppressed the
thr1
Δ mutant homoserine toxicity. Since the
doa4
Δ and proteasome mutants identified have reduced ubiquitin- and/or proteasome-mediated proteolysis, the degradation of a particular protein or subset of proteins likely contributes to homoserine toxicity.
Inflammatory Cytokine Response to
Bacillus anthracis
Peptidoglycan Requires Phagocytosis and Lysosomal Trafficking
Authors: Iyer Janaki K. , Khurana Taruna , Langer Marybeth , West Christopher M. , Ballard Jimmy D. , Metcalf Jordan P. , Merkel Tod J. , Coggeshall K. Mark
Published: March 2010

During advanced stages of inhalation anthrax,
Bacillus anthracis
accumulates at high levels in the bloodstream of the infected host. This bacteremia leads to sepsis during late-stage anthrax; however, the mechanisms through which
B. anthracis
-derived factors contribute to the pathology of infected hosts are poorly defined. Peptidoglycan, a major component of the cell wall of Gram-positive bacteria, can provoke symptoms of sepsis in animal models. We have previously shown that peptidoglycan of
B. anthracis
can induce the production of proinflammatory cytokines by cells in human blood. Here, we show that biologically active peptidoglycan is shed from an active culture of encapsulated
B. anthracis
strain Ames in blood. Peptidoglycan is able to bind to surfaces of responding cells, and internalization of peptidoglycan is required for the production of inflammatory cytokines. We also show that the peptidoglycan traffics to lysosomes, and lysosomal function is required for cytokine production. We conclude that peptidoglycan of
B. anthracis
is initially bound by an unknown extracellular receptor, is phagocytosed, and traffics to lysosomes, where it is degraded to a product recognized by an intracellular receptor. Binding of the peptidoglycan product to the intracellular receptor causes a proinflammatory response. These findings provide new insight into the mechanism by which
B. anthracis
triggers sepsis during a critical stage of anthrax disease.
Lack of Interleukin-12 in p40-Deficient Mice Leads to Poor CD8+T-Cell Immunity againstEncephalitozoon cuniculiInfection
Authors: Moretto Magali M. , Lawlor Elizabeth M. , Khan Imtiaz A.
Published: March 2010

A CD8+T-cell response is critical for protection againstEncephalitozoon cuniculiinfection. However, the factors responsible for the generation of CD8+T-cell immunity duringE. cuniculiinfection and the cytokines involved in this process have not been identified. In the present study, we demonstrated that p40-deficient animals, which are unable to produce interleukin-12 (IL-12), have a serious defect in expansion of the CD8+T-cell response which compromises the survival of an infected host. Adoptive transfer of CD8+T cells from immunocompetent donors protected SCID mice infected withE. cuniculi, whereas administration of CD8+T cells from p40−/−mice failed to protect infected SCID mice.In vitrodendritic cell (DC) cultures from knockout mice pulsed withE. cuniculispores were unable to develop a robust CD8+T-cell immune response. Addition of exogenous IL-12 or transfer of CD8+T cells that were initially primed with DC from p40−/−animals to DC cultures from immunocompetent mice (directly or via transwells) led to optimal expansion of these cells. This IL-12-mediated reinstatement of CD8+T-effector immunity was independent of gamma interferon (IFN-γ) as addition of antibody to the cultures failed to have an effect. These studies demonstrated that IL-12 plays a predominant role in the expansion of effector CD8+T-cell immunity againstE. cuniculi, which is critical for host survival. These findings are very important for understanding the protective immune mechanisms needed to protect an immunocompromised host against an opportunistic infection and can be extended to other microsporidial pathogens.
Potent Neutralization of Staphylococcal Enterotoxin B by Synergistic Action of Chimeric Antibodies
Authors: Tilahun Mulualem E. , Rajagopalan Govindarajan , Shah-Mahoney Nalini , Lawlor Rebecca G. , Tilahun Ashenafi Y. , Xie Chen , Natarajan Kannan , Margulies David H. , Ratner David I. , Osborne Barbara A. , Goldsby Richard A.
Published: March 2010

Staphylococcal enterotoxin B (SEB), a shock-inducing exotoxin synthesized byStaphylococcus aureus, is an important cause of food poisoning and is a class B bioterrorism agent. SEB mediates antigen-independent activation of a major subset of the T-cell population by cross-linking T-cell receptors (TCRs) with class II major histocompatibility complex (MHC-II) molecules of antigen-presenting cells, resulting in the induction of antigen independent proliferation and cytokine secretion by a significant fraction of the T-cell population. Neutralizing antibodies inhibit SEB-mediated T-cell activation by blocking the toxin's interaction with the TCR or MHC-II and provide protection against the debilitating effects of this superantigen. We derived and searched a set of monoclonal mouse anti-SEB antibodies to identify neutralizing anti-SEB antibodies that bind to different sites on the toxin. A pair of non-cross-reactive, neutralizing anti-SEB monoclonal antibodies (MAbs) was found, and a combination of these antibodies inhibited SEB-induced T-cell proliferation in a synergistic rather than merely additive manner. In order to engineer antibodies more suitable than mouse MAbs for use in humans, the genes encoding the VL and VH gene segments of a synergistically acting pair of mouse MAbs were grafted, respectively, onto genes encoding the constant regions of human Igκ and human IgG1, transfected into mammalian cells, and used to generate chimeric versions of these antibodies that had affinity and neutralization profiles essentially identical to their mouse counterparts. When tested in cultures of human peripheral blood mononuclear cells or splenocytes derived from HLA-DR3 transgenic mice, the chimeric human-mouse antibodies synergistically neutralized SEB-induced T-cell activation and cytokine production.
Trimer Stability of YadA Is Critical for Virulence of
Yersinia enterocolitica
Authors: Schütz M. , Weiss E.-M. , Schindler M. , Hallström T. , Zipfel P. F. , Linke D. , Autenrieth I. B.
Published: March 2010

Yersinia
adhesin A (YadA) is a trimeric autotransporter adhesin with multiple functions in host-pathogen interactions. The aim of this study was to dissect the virulence functions promoted by YadA
in vitro
and
in vivo
. To accomplish this, we generated
Yersinia enterocolitica
O:8 mutants expressing point mutations in YadA G389, a highly conserved residue in the membrane anchor of YadA, and analyzed their impact on YadA expression and virulence functions. We found that point mutations of YadA G389 led to impaired transport, stability, and surface display of YadA. YadA G389A and G389S mutants showed comparable YadA surface expression, autoagglutination, and adhesion to those of wild-type YadA but displayed reduced trimer stability and complement resistance
in vitro
and were 10- to 1,000-fold attenuated in experimental
Y. enterocolitica
infection in mice. The G389T, G389N, and G389H mutants lost trimer stability, exhibited strongly reduced surface display, autoagglutination, adhesion properties, and complement resistance, and were avirulent (>10,000-fold attenuation) in mice. Our data demonstrate that G389 is a critical residue of YadA, required for optimal trimer stability, transport, surface display, and serum resistance. We also show that stable trimeric YadA protein is essential for virulence of
Y. enterocolitica
.
Characterization of thePseudomonas aeruginosaTranscriptional Response to Phenylalanine and Tyrosine
Authors: Palmer Gregory C. , Palmer Kelli L. , Jorth Peter A. , Whiteley Marvin
Published: March 2010

Pseudomonas aeruginosais a Gram-negative opportunistic pathogen often associated with chronic infections in the lungs of individuals with the heritable disease cystic fibrosis (CF). Previous work from our laboratory demonstrated that aromatic amino acids within CF lung secretions (sputum) not only serve as carbon and energy sources but also enhance synthesis of the cell signaling moleculePseudomonasquinolone signal (PQS). The present study investigates the role of the aromatic amino acid-responsive regulator PhhR in mediating these phenotypes. Transcriptome analysis revealed that PhhR controls four putative transcriptional units (phhA,hpd,hmgA, anddhcA) involved in aromatic amino acid catabolism; however, genes involved in PQS biosynthesis were unaffected. ThephhA,hpd,hmgA, anddhcApromoters were mapped by primer extension, and purified His6-PhhR was shown to bind thephhA,hpd, anddhcApromotersin vitroby use of electrophoretic mobility shift assays. Our work characterizes a transcriptional regulator of catabolic genes induced duringP. aeruginosagrowth in CF sputum.
Evidence of Localized Prophage-Host Recombination in the
lytA
Gene, Encoding the Major Pneumococcal Autolysin
Authors: Morales María , García Pedro , de la Campa Adela G. , Liñares Josefina , Ardanuy Carmen , García Ernesto
Published: March 2010

According to a highly polymorphic region in the
lytA
gene, encoding the major autolysin of
Streptococcus pneumoniae
, two different families of alleles can be differentiated by PCR and restriction digestion. Here, we provide evidence that this polymorphic region arose from recombination events with homologous genes of pneumococcal temperate phages.
Differential Regulation of the Multiple Flagellins in Spirochetes
Authors: Li Chunhao , Sal Melanie , Marko Michael , Charon Nyles W.
Published: March 2010

The expression of flagellin genes in most bacteria is typically regulated by the flagellum-specific sigma28factor FliA, and an anti-sigma28factor, FlgM. However, the regulatory hierarchy in several bacteria that have multiple flagellins is more complex. In these bacteria, the flagellin genes are often transcribed by at least two different sigma factors. The flagellar filament in spirochetes consists of one to three FlaB core proteins and at least one FlaA sheath protein. Here, the genetically amenable bacteriumBrachyspira hyodysenteriaewas used as a model spirochete to investigate the regulation of its four flagellin genes,flaA,flaB1,flaB2, andflaB3. We found that theflaB1andflaB2genes are regulated by sigma28, whereas theflaAandflaB3genes are controlled by sigma70. The analysis of a flagellar motor switchfliGmutant further supported this proposition; in the mutant, the transcription offlaB1andflaB2was inhibited, but that offlaAandflaB3was not. In addition, the continued expression offlaAandflaB3in the mutant resulted in the formation of incomplete flagellar filaments that were hollow tubes and consisted primarily of FlaA. Finally, our recent studies have shown that each flagellin unit contributes to the stiffness of the periplasmic flagella, and this stiffness directly correlates with motility. The regulatory mechanism identified here should allow spirochetes to change the relative ratio of these flagellin proteins and, concomitantly, vary the stiffness of their flagellar filament.
The Actinomycin Biosynthetic Gene Cluster ofStreptomyces chrysomallus: a Genetic Hall of Mirrors for Synthesis of a Molecule with Mirror Symmetry
Authors: Keller Ullrich , Lang Manuel , Crnovcic Ivana , Pfennig Frank , Schauwecker Florian
Published: March 2010

A gene cluster was identified which contains genes involved in the biosynthesis of actinomycin encompassing 50 kb of contiguous DNA on the chromosome ofStreptomyces chrysomallus. It contains 28 genes with biosynthetic functions and is bordered on both sides by IS elements. Unprecedentedly, the cluster consists of two large inverted repeats of 11 and 13 genes, respectively, with four nonribosomal peptide synthetase genes in the middle. Nine genes in each repeat have counterparts in the other, in the same arrangement but in the opposite orientation, suggesting an inverse duplication of one of the arms during the evolution of the gene cluster. All of the genes appear to be organized into operons, each corresponding to a functional section of actinomycin biosynthesis, such as peptide assembly, regulation, resistance, and biosynthesis of the precursor of the actinomycin chromophore 4-methyl-3-hydroxyanthranilic acid (4-MHA). For 4-MHA synthesis, functional analysis revealed genes that encode pathway-specific isoforms of tryptophan dioxygenase, kynurenine formamidase, and hydroxykynureninase, which are distinct from the corresponding enzyme activities of cellular tryptophan catabolism in their regulation and in part in their substrate specificity. Phylogenetic analysis indicates that the pathway-specific tryptophan metabolism inStreptomycesmost probably evolved divergently from the normal pathway of tryptophan catabolism to provide an extra or independent supply of building blocks for the synthesis of tryptophan-derived secondary metabolites.
Overlapping Alternative Sigma Factor Regulons in the Response to Singlet Oxygen inRhodobacter sphaeroides
Authors: Nuss Aaron M. , Glaeser Jens , Berghoff Bork A. , Klug Gabriele
Published: March 2010

Organisms performing photosynthesis in the presence of oxygen have to cope with the formation of highly reactive singlet oxygen (1O2) and need to mount an adaptive response to photooxidative stress. Here we show that the alternative sigma factors RpoHIand RpoHIIare both involved in the1O2response and in the heat stress response inRhodobacter sphaeroides. We propose RpoHIIto be the major player in the1O2response, whereas RpoHIis more important for the heat stress response. Mapping of the 5′ ends of RpoHII- and also RpoHI/RpoHII-dependent transcripts revealed clear differences in the −10 regions of the putative promoter sequences. By using bioinformatic tools, we extended the RpoHIIregulon, which includes genes induced by1O2exposure. These genes encode proteins which are, e.g., involved in methionine sulfoxide reduction and in maintaining the quinone pool. Furthermore, we identified small RNAs which depend on RpoHIand RpoHIIand are likely to contribute to the defense against photooxidative stress and heat stress.