bluuebunny/crossref_metadata_2025 huggingface dataset:
Sensitivity to envelope-based interaural delays at high frequencies: Center frequency affects the envelope rate-limitation
Authors: Bernstein Leslie R. , Trahiotis Constantine
Published: February 2014

Sensitivity to ongoing interaural temporal disparities (ITDs) was measured using bandpass-filtered pulse trains centered at 4600, 6500, or 9200 Hz. Save for minor differences in the exact center frequencies, those target stimuli were those employed by Majdak and Laback [J. Acoust. Soc. Am. 125, 3903–3913 (2009)]. At each center frequency, threshold ITD was measured for pulse repetition rates ranging from 64 to 609 Hz. The results and quantitative predictions by a cross-correlation-based model indicated that (1) at most pulse repetition rates, threshold ITD increased with center frequency, (2) the cutoff frequency of the putative envelope low-pass filter that determines sensitivity to ITD at high envelope rates appears to be inversely related to center frequency, and (3) both outcomes were accounted for by assuming that, independent of the center frequency, the listeners' decision variable was a constant criterion change in interaural correlation of the stimuli as processed internally. The finding of an inverse relation between center frequency and the envelope rate limitation, while consistent with much prior literature, runs counter to the conclusion reached by Majdak and Laback.
Peafowl antipredator calls encode information about signalers
Authors: Yorzinski Jessica L.
Published: February 2014

Animals emit vocalizations that convey information about external events. Many of these vocalizations, including those emitted in response to predators, also encode information about the individual that produced the call. The relationship between acoustic features of antipredator calls and information relating to signalers (including sex, identity, body size, and social rank) were examined in peafowl (Pavo cristatus). The “bu-girk” antipredator calls of male and female peafowl were recorded and 20 acoustic parameters were automatically extracted from each call. Both the bu and girk elements of the antipredator call were individually distinctive and calls were classified to the correct signaler with over 90% and 70% accuracy in females and males, respectively. Females produced calls with a higher fundamental frequency (F0) than males. In both females and males, body size was negatively correlated with F0. In addition, peahen rank was related to the duration, end mean frequency, and start harmonicity of the bu element. Peafowl antipredator calls contain detailed information about the signaler and can potentially be used by receivers to respond to dangerous situations.
Sound reproduction in personal audio systems using the least-squares approach with acoustic contrast control constraint
Authors: Cai Yefeng , Wu Ming , Yang Jun
Published: February 2014

This paper describes a method for focusing the reproduced sound in the bright zone without disturbing other people in the dark zone in personal audio systems. The proposed method combines the least-squares and acoustic contrast criteria. A constrained parameter is introduced to tune the balance between two performance indices, namely, the acoustic contrast and the spatial average error. An efficient implementation of this method using convex optimization is presented. Offline simulations and real-time experiments using a linear loudspeaker array are conducted to evaluate the performance of the presented method. Results show that compared with the traditional acoustic contrast control method, the proposed method can improve the flatness of response in the bright zone by sacrificing the level of acoustic contrast.
The contribution of durational and spectral changes to the Lombard speech intelligibility benefit
Authors: Cooke Martin , Mayo Catherine , Villegas Julián
Published: February 2014

Speech produced in the presence of noise (Lombard speech) is typically more intelligible than speech produced in quiet (plain speech) when presented at the same signal-to-noise ratio, but the factors responsible for the Lombard intelligibility benefit remain poorly understood. Previous studies have demonstrated a clear effect of spectral differences between the two speech styles and a lack of effect of fundamental frequency differences. The current study investigates a possible role for durational differences alongside spectral changes. Listeners identified keywords in sentences manipulated to possess either durational or spectral characteristics of plain or Lombard speech. Durational modifications were produced using linear or nonlinear time warping, while spectral changes were applied at the global utterance level or to individual time frames. Modifications were made to both plain and Lombard speech. No beneficial effects of durational increases were observed in any condition. Lombard sentences spoken at a speech rate substantially slower than their plain counterparts also failed to reveal a durational benefit. Spectral changes to plain speech resulted in large intelligibility gains, although not to the level of Lombard speech. These outcomes suggest that the durational increases seen in Lombard speech have little or no role in the Lombard intelligibility benefit.
Hearing in alpacas (Vicugna pacos): Audiogram, localization acuity, and use of binaural locus cues
Authors: Heffner Rickye S. , Koay Gimseong , Heffner Henry E.
Published: February 2014

Behavioral audiograms and sound localization abilities were determined for three alpacas (Vicugna pacos). Their hearing at a level of 60 dB sound pressure level (SPL) (re 20 μPa) extended from 40 Hz to 32.8 kHz, a range of 9.7 octaves. They were most sensitive at 8 kHz, with an average threshold of –0.5 dB SPL. The minimum audible angle around the midline for 100-ms broadband noise was 23°, indicating relatively poor localization acuity and potentially supporting the finding that animals with broad areas of best vision have poorer sound localization acuity. The alpacas were able to localize low-frequency pure tones, indicating that they can use the binaural phase cue, but they were unable to localize pure tones above the frequency of phase ambiguity, thus indicating complete inability to use the binaural intensity-difference cue. In contrast, the alpacas relied on their high-frequency hearing for pinna cues; they could discriminate front−back sound sources using 3-kHz high-pass noise, but not 3-kHz low-pass noise. These results are compared to those of other hoofed mammals and to mammals more generally.
Dose-response relationships for the onset of avoidance of sonar by free-ranging killer whales
Authors: Miller Patrick J. O. , Antunes Ricardo N. , Wensveen Paul J. , Samarra Filipa I. P. , Catarina Alves Ana , Tyack Peter L. , Kvadsheim Petter H. , Kleivane Lars , Lam Frans-Peter A. , Ainslie Michael A. , Thomas Len
Published: February 2014

Eight experimentally controlled exposures to 1−2 kHz or 6−7 kHz sonar signals were conducted with four killer whale groups. The source level and proximity of the source were increased during each exposure in order to reveal response thresholds. Detailed inspection of movements during each exposure session revealed sustained changes in speed and travel direction judged to be avoidance responses during six of eight sessions. Following methods developed for Phase-I clinical trials in human medicine, response thresholds ranging from 94 to 164 dB re 1 μPa received sound pressure level (SPL) were fitted to Bayesian dose-response functions. Thresholds did not consistently differ by sonar frequency or whether a group had previously been exposed, with a mean SPL response threshold of 142 ± 15 dB (mean ± s.d.). High levels of between- and within-individual variability were identified, indicating that thresholds depended upon other undefined contextual variables. The dose-response functions indicate that some killer whales started to avoid sonar at received SPL below thresholds assumed by the U.S. Navy. The predicted extent of habitat over which avoidance reactions occur depends upon whether whales responded to proximity or received SPL of the sonar or both, but was large enough to raise concerns about biological consequences to the whales.
Classification of large acoustic datasets using machine learning and crowdsourcing: Application to whale calls
Authors: Shamir Lior , Yerby Carol , Simpson Robert , von Benda-Beckmann Alexander M. , Tyack Peter , Samarra Filipa , Miller Patrick , Wallin John
Published: February 2014

Vocal communication is a primary communication method of killer and pilot whales, and is used for transmitting a broad range of messages and information for short and long distance. The large variation in call types of these species makes it challenging to categorize them. In this study, sounds recorded by audio sensors carried by ten killer whales and eight pilot whales close to the coasts of Norway, Iceland, and the Bahamas were analyzed using computer methods and citizen scientists as part of the Whale FM project. Results show that the computer analysis automatically separated the killer whales into Icelandic and Norwegian whales, and the pilot whales were separated into Norwegian long-finned and Bahamas short-finned pilot whales, showing that at least some whales from these two locations have different acoustic repertoires that can be sensed by the computer analysis. The citizen science analysis was also able to separate the whales to locations by their sounds, but the separation was somewhat less accurate compared to the computer method.
Descending auditory pathway and identification of phonetic contrast by native listeners
Authors: Shastri Usha , Mythri H. M. , Kumar U. Ajith
Published: February 2014

The present study investigated the ability of native listeners to identify subtle phonetic contrasts in nonsense words and its relationship with the contralateral inhibition of transient evoked otoacoustic emissions (TEOAE). A group of 45 young adults with normal hearing sensitivity who were native speakers of Malayalam participated in the behavioral experiment. Phone identification score and reaction time for four phonetic pairs in nonsense words were measured for each participant. Based on the phone identification score, the participants were divided into high and low performers. Twelve participants randomly selected from each group were evaluated for contralateral inhibition of TEOAEs. Phone identification score and global contralateral inhibition amplitude of TEOAE were significantly higher and reaction time was significantly shorter in high performers than that of low performers. Significant correlation was found between the phone identification score and contralateral inhibition of TEOAE. Strength of the medial olivocochlear bundle activity explained about 30% of the variance in the phone identification scores providing evidence for the involvement of the descending auditory pathways in identifying the phonetic contrasts that are acoustically similar. These results support the emerging view that top down influences from higher centers shapes the responses of lower centers.
A Bayesian direction-of-arrival model for an undetermined number of sources using a two-microphone array
Authors: Escolano Jose , Xiang Ning , Perez-Lorenzo Jose M. , Cobos Maximo , Lopez Jose J.
Published: February 2014

Sound source localization using a two-microphone array is an active area of research, with considerable potential for use with video conferencing, mobile devices, and robotics. Based on the observed time-differences of arrival between sound signals, a probability distribution of the location of the sources is considered to estimate the actual source positions. However, these algorithms assume a given number of sound sources. This paper describes an updated research account on the solution presented in Escolano et al. [J. Acoust. Am. Soc. 132(3), 1257–1260 (2012)], where nested sampling is used to explore a probability distribution of the source position using a Laplacian mixture model, which allows both the number and position of speech sources to be inferred. This paper presents different experimental setups and scenarios to demonstrate the viability of the proposed method, which is compared with some of the most popular sampling methods, demonstrating that nested sampling is an accurate tool for speech localization.
Circumferential guided wave measurements of a cylindrical fluid-filled bone-mimicking phantom
Authors: Nauleau Pierre , Grimal Quentin , Minonzio Jean-Gabriel , Laugier Pascal , Prada Claire
Published: February 2014

In the context of hip fracture risk prediction, measurement of guided waves could improve the assessment of cortical femoral neck properties. The decomposition of the time reversal operator (DORT) method was previously shown to be efficient to measure circumferential guided modes in an empty cortical bone-mimicking tube of circular cross section. In this study, an adaptation of the DORT method is proposed to probe the same bone-mimicking tube but filled with a marrow-mimicking fluid. The contributions to the backscattered field of waves multiply reflected in the cavity of the tube interfere with those of circumferential guided waves. The former contributions are eliminated in the backpropagation image using ad hoc criterion determined with simulation. Eight portions of different guided modes were observed from experimental and simulated data. They were identified by comparison with theoretical predictions. This work confirms the feasibility of measuring guided waves in a fluid-filled tube of bone-mimicking material with the DORT method.
Influence of impedance phase angle on sound pressures and reverberation times in a rectangular room
Authors: Jeong Cheol-Ho , Lee Doheon , Santurette Sébastien , Ih Jeong-Guon
Published: February 2014

In most room acoustic predictions, phase shift on reflection has been overlooked. This study aims to quantify the effects of the surface impedance phase angle of the boundary surfaces on room acoustic conditions. As a preliminary attempt, a medium-sized rectangular room is simulated by a phased beam tracing model, after verifying it numerically against boundary element simulations. First, the absorption characteristic of the boundary surfaces varies uniformly from 0.2 to 0.8, but with various impedance phase angles. Second, typical non-uniform cases having hard walls and floor, but with an absorptive ceiling are investigated. The zero phase angle, which has commonly been assumed in practice, is regarded as reference and differences in the sound pressure level and early decay time from the reference are quantified. As expected, larger differences in the room acoustic parameters are found for larger impedance phase angles. Additionally, binaural impulse responses are compared in a listening test for the uniform absorption cases, revealing that non-zero impedance phase angle cases can be perceptually different from the reference condition in terms of reverberance perception. For the non-uniform settings, the change in the impedance phase angle of the ceiling does not affect the acoustic conditions significantly.
Aging of the medial olivocochlear reflex and associations with speech perception
Authors: Abdala Carolina , Dhar Sumitrajit , Ahmadi Mahnaz , Luo Ping
Published: February 2014

The medial olivocochlear reflex (MOCR) modulates cochlear amplifier gain and is thought to facilitate the detection of signals in noise. High-resolution distortion product otoacoustic emissions (DPOAEs) were recorded in teens, young, middle-aged, and elderly adults at moderate levels using primary tones swept from 0.5 to 4 kHz with and without a contralateral acoustic stimulus (CAS) to elicit medial efferent activation. Aging effects on magnitude and phase of the 2f1-f2 DPOAE and on its components were examined, as was the link between speech-in-noise performance and MOCR strength. Results revealed a mild aging effect on the MOCR through middle age for frequencies below 1.5 kHz. Additionally, positive correlations were observed between strength of the MOCR and performance on select measures of speech perception parsed into features. The elderly group showed unexpected results including relatively large effects of CAS on DPOAE, and CAS-induced increases in DPOAE fine structure as well as increases in the amplitude and phase accumulation of DPOAE reflection components. Contamination of MOCR estimates by middle ear muscle contractions cannot be ruled out in the oldest subjects. The findings reiterate that DPOAE components should be unmixed when measuring medial efferent effects to better consider and understand these potential confounds.
Overlapping frequency coverage and simulated spatial cue effects on bimodal (electrical and acoustical) sentence recognition in noise
Authors: Green Tim , Faulkner Andrew , Rosen Stuart
Published: February 2014

Sentence recognition in 20-talker babble was measured in eight Nucleus cochlear implant (CI) users with contralateral residual acoustic hearing. Speech reception thresholds (SRTs) were measured both in standard configurations, with some frequency regions presented both acoustically and electrically, and in configurations with no spectral overlap. In both cases a continuous interleaved sampling strategy was used. Mean SRTs were around 3 dB better with bimodal presentation than with CI alone in overlap configurations. A spherical head model was used to simulate azimuthal separation of speech and noise and provided no evidence of a contribution of spatial cues to bimodal benefit. There was no effect on bimodal performance of whether spectral overlap was present or was eliminated by switching off electrodes assigned to frequencies below the upper limit of acoustic hearing. In a subsequent experiment the CI was acutely re-mapped so that all available electrodes were used to cover frequencies not presented acoustically. This gave increased spectral resolution via the CI as assessed by formant frequency discrimination, but no improvement in bimodal performance compared to the configuration with overlap.
Too Much of a Good Channel in Pulmonary Arterial Hypertension
Authors: Tonelli Adriano R.
Published: February 2014

An increase in the expression of the chloride intracellular channel 4 mediates endothelial dysfunction and vascular remodeling in pulmonary arterial hypertension.
Combination Therapy with Tamoxifen and Amphotericin B in Experimental Cutaneous Leishmaniasis
Authors: Trinconi Cristiana T. , Reimão Juliana Q. , Yokoyama-Yasunaka Jenicer K. U. , Miguel Danilo C. , Uliana Silvia R. B.
Published: February 2014

Leishmaniasis chemotherapy remains very challenging. The high cost of active drugs, along with the severity of their side effects and the increasing failure rate of the current therapeutic schemes, calls for the discovery of new active drugs and schemes of treatment. The use of combination therapy has gained much attention in recent years as a possible strategy for overcoming the various shortcomings in the present arsenal. We recently described the effectiveness of tamoxifen in murine models of leishmaniasis, and here, we investigated the interactions between tamoxifen and amphotericin B, one of the most potent drugs used in leishmaniasis treatment. The
in vitro
interactions were indifferent for the association of tamoxifen and amphotericin B. The association was also assayed
in vivo
in
Leishmania amazonensis
-infected BALB/c mice and was found to yield at least additive effects at low doses of both drugs.
Role of Malic Enzyme during Fatty Acid Synthesis in the Oleaginous Fungus Mortierella alpina
Authors: Hao Guangfei , Chen Haiqin , Wang Lei , Gu Zhennan , Song Yuanda , Zhang Hao , Chen Wei , Chen Yong Q.
Published: February 2014

The generation of NADPH by malic enzyme (ME) was postulated to be a rate-limiting step during fatty acid synthesis in oleaginous fungi, based primarily on the results from research focusing on ME in
Mucor circinelloides
. This hypothesis is challenged by a recent study showing that leucine metabolism, rather than ME, is critical for fatty acid synthesis in
M. circinelloides
. To clarify this, the gene encoding ME isoform E from
Mortierella alpina
was homologously expressed. ME overexpression increased the fatty acid content by 30% compared to that for a control. Our results suggest that ME may not be the sole rate-limiting enzyme, but does play a role, during fatty acid synthesis in oleaginous fungi.
Synergism of Glycoside Hydrolase Secretomes from Two Thermophilic Bacteria Cocultivated on Lignocellulose
Authors: Zhang Kundi , Chen Xiaohua , Schwarz Wolfgang H. , Li Fuli
Published: February 2014

Two cellulolytic thermophilic bacterial strains, CS-3-2 and CS-4-4, were isolated from decayed cornstalk by the addition of growth-supporting factors to the medium. According to 16S rRNA gene-sequencing results, these strains belonged to the genus
Clostridium
and showed 98.87% and 98.86% identity with
Clostridium
stercorarium
subsp.
leptospartum
ATCC 35414
T
and
Clostridium
cellulosi
AS 1.1777
T
, respectively. The endoglucanase and exoglucanase activities of strain CS-4-4 were approximately 3 to 5 times those of strain CS-3-2, whereas the β-glucosidase activity of strain CS-3-2 was 18 times higher than that of strain CS-4-4. The xylanase activity of strain CS-3-2 was 9 times that of strain CS-4-4, whereas the β-xylosidase activity of strain CS-4-4 was 27 times that of strain CS-3-2. The enzyme activities in spent cultures following cocultivation of the two strains with cornstalk as the substrate were much greater than those in pure cultures or an artificial mixture of samples, indicating synergism of glycoside hydrolase secretomes between the two strains. Quantitative measurement of the two strains in the cocultivation system indicated that strain CS-3-2 grew robustly during the initial stages, whereas strain CS-4-4 dominated the system in the late-exponential phase. Liquid chromatography-tandem mass spectrometry analysis of protein bands appearing in the native zymograms showed that ORF3880 and ORF3883 from strain CS-4-4 played key roles in the lignocellulose degradation process. Both these open reading frames (ORFs) exhibited endoglucanase and xylanase activities, but ORF3880 showed tighter adhesion to insoluble substrates at 4, 25, and 60°C owing to its five carbohydrate-binding modules (CBMs).
Carbon Dioxide Fixation by Metallosphaera yellowstonensis and Acidothermophilic Iron-Oxidizing Microbial Communities from Yellowstone National Park
Authors: Jennings Ryan M. , Whitmore Laura M. , Moran James J. , Kreuzer Helen W. , Inskeep William P.
Published: February 2014

The fixation of inorganic carbon has been documented in all three domains of life and results in the biosynthesis of diverse organic compounds that support heterotrophic organisms. The primary aim of this study was to assess carbon dioxide fixation in high-temperature Fe(III)-oxide mat communities and in pure cultures of a dominant Fe(II)-oxidizing organism (
Metallosphaera yellowstonensis
strain MK1) originally isolated from these environments. Protein-encoding genes of the complete 3-hydroxypropionate/4-hydroxybutyrate (3-HP/4-HB) carbon dioxide fixation pathway were identified in
M. yellowstonensis
strain MK1. Highly similar
M. yellowstonensis
genes for this pathway were identified in metagenomes of replicate Fe(III)-oxide mats, as were genes for the reductive tricarboxylic acid cycle from
Hydrogenobaculum
spp. (
Aquificales
). Stable-isotope (
13
CO
2
) labeling demonstrated CO
2
fixation by
M. yellowstonensis
strain MK1 and in
ex situ
assays containing live Fe(III)-oxide microbial mats. The results showed that strain MK1 fixes CO
2
with a fractionation factor of ∼2.5‰. Analysis of the
13
C composition of dissolved inorganic C (DIC), dissolved organic C (DOC), landscape C, and microbial mat C showed that mat C is from both DIC and non-DIC sources. An isotopic mixing model showed that biomass C contains a minimum of 42% C of DIC origin, depending on the fraction of landscape C that is present. The significance of DIC as a major carbon source for Fe(III)-oxide mat communities provides a foundation for examining microbial interactions that are dependent on the activity of autotrophic organisms (i.e.,
Hydrogenobaculum
and
Metallosphaera
spp.) in simplified natural communities.
Cerecidins, Novel Lantibiotics from Bacillus cereus with Potent Antimicrobial Activity
Authors: Wang Jian , Zhang Li , Teng Kunling , Sun Shutao , Sun Zhizeng , Zhong Jin
Published: February 2014

Lantibiotics are ribosomally synthesized and posttranslationally modified antimicrobial peptides that are widely produced by Gram-positive bacteria, including many species of the
Bacillus
group. In the present study, one novel gene cluster coding lantibiotic cerecidins was unveiled in
Bacillus cereus
strain As 1.1846 through genomic mining and PCR screening. The designated
cer
locus is different from that of conventional class II lantibiotics in that it included seven tandem precursor
cerA
genes, one modification gene (
cerM
), two processing genes (
cerT
and
cerP
), one orphan regulator gene (
cerR
), and two immunity genes (
cerF
and
cerE
). In addition, one unprecedented quorum sensing component,
comQXPA
, was inserted between
cerM
and
cerR
. The expression of cerecidins was not detected in this strain of
B. cereus
, which might be due to repressed transcription of
cerM
. We constitutively coexpressed
cerA
genes and
cerM
in
Escherichia coli
, and purified precerecidins were proteolytically processed with the endoproteinase GluC and a truncated version of putative serine protease CerP. Thus, two natural variants of cerecidins A1 and A7 were obtained which contained two terminal nonoverlapping thioether rings rarely found in lantibiotics. Both cerecidins A1 and A7 were active against a broad spectrum of Gram-positive bacteria. Cerecidin A7, especially its mutant Dhb13A, showed remarkable efficacy against multidrug-resistant
Staphylococcus aureus
(MDRSA), vancomycin-resistant
Enterococcus faecalis
(VRE), and even
Streptomyces
.
Specific Microbial Attachment to Root Knot Nematodes in Suppressive Soil
Authors: Adam Mohamed , Westphal Andreas , Hallmann Johannes , Heuer Holger
Published: February 2014

Understanding the interactions of plant-parasitic nematodes with antagonistic soil microbes could provide opportunities for novel crop protection strategies. Three arable soils were investigated for their suppressiveness against the root knot nematode
Meloidogyne hapla
. For all three soils,
M. hapla
developed significantly fewer galls, egg masses, and eggs on tomato plants in unsterilized than in sterilized infested soil. Egg numbers were reduced by up to 93%. This suggested suppression by soil microbial communities. The soils significantly differed in the composition of microbial communities and in the suppressiveness to
M. hapla
. To identify microorganisms interacting with
M. hapla
in soil, second-stage juveniles (J2) baited in the test soil were cultivation independently analyzed for attached microbes. PCR-denaturing gradient gel electrophoresis of fungal ITS or 16S rRNA genes of bacteria and bacterial groups from nematode and soil samples was performed, and DNA sequences from J2-associated bands were determined. The fingerprints showed many species that were abundant on J2 but not in the surrounding soil, especially in fungal profiles. Fungi associated with J2 from all three soils were related to the genera
Davidiella
and
Rhizophydium
, while the genera
Eurotium
,
Ganoderma
, and
Cylindrocarpon
were specific for the most suppressive soil. Among the 20 highly abundant operational taxonomic units of bacteria specific for J2 in suppressive soil, six were closely related to infectious species such as
Shigella
spp., whereas the most abundant were
Malikia spinosa
and
Rothia amarae
, as determined by 16S rRNA amplicon pyrosequencing. In conclusion, a diverse microflora specifically adhered to J2 of
M. hapla
in soil and presumably affected female fecundity.
A New Subfamily of Polyphosphate Kinase 2 (Class III PPK2) Catalyzes both Nucleoside Monophosphate Phosphorylation and Nucleoside Diphosphate Phosphorylation
Authors: Motomura Kei , Hirota Ryuichi , Okada Mai , Ikeda Takeshi , Ishida Takenori , Kuroda Akio
Published: February 2014

Inorganic polyphosphate (polyP) is a linear polymer of tens to hundreds of phosphate (P
i
) residues linked by “high-energy” phosphoanhydride bonds as in ATP. PolyP kinases, responsible for the synthesis and utilization of polyP, are divided into two families (PPK1 and PPK2) due to differences in amino acid sequence and kinetic properties. PPK2 catalyzes preferentially polyP-driven nucleotide phosphorylation (utilization of polyP), which is important for the survival of microbial cells under conditions of stress or pathogenesis. Phylogenetic analysis suggested that the PPK2 family could be divided into three subfamilies (classes I, II, and III). Class I and II PPK2s catalyze nucleoside diphosphate and nucleoside monophosphate phosphorylation, respectively. Here, we demonstrated that class III PPK2 catalyzes both nucleoside monophosphate and nucleoside diphosphate phosphorylation, thereby enabling us to synthesize ATP from AMP by a single enzyme. Moreover, class III PPK2 showed broad substrate specificity over purine and pyrimidine bases. This is the first demonstration that class III PPK2 possesses both class I and II activities.
Prevention of Contamination by Xenotropic Murine Leukemia Virus-Related Virus: Susceptibility to Alcohol-Based Disinfectants and Environmental Stability
Authors: Palesch David , Khalid Mohammad , Stürzel Christina M. , Münch Jan
Published: February 2014

Xenotropic murine leukemia virus-related virus (XMRV) represents a novel γ-retrovirus that is capable of infecting human cells and has been classified as a biosafety level 2 (BSL-2) organism. Hence, XMRV represents a potential risk for personnel in laboratories worldwide. Here, we measured the stability of XMRV and its susceptibility to alcohol-based disinfectants. To this end, we exposed an infectious XMRV reporter virus encoding a secretable luciferase to different temperatures, pH values, and disinfectants and infected XMRV-permissive Raji B cells to measure residual viral infectivity. We found that 1 min treatment of XMRV particles at 60°C is sufficient to reduce infectivity by 99.9%. XMRV infectivity was maximal at a neutral pH but was reduced by 86% at pH 4 and 99.9% at pH 10. The common hand and surface disinfectants ethanol and isopropanol as well as the cell fixation reagent paraformaldehyde abrogated XMRV infectivity entirely, as indicated by a reduction of infectivity exceeding 99.99%. Our findings provide evidence of specific means to inactivate XMRV. Their application will help to prevent unintended XMRV contamination of cell cultures in laboratories and minimize the risk for laboratory personnel and health care workers to become infected with this biosafety level 2 organism.
Dynamic Metabolic and Transcriptional Profiling of Rhodococcus sp. Strain YYL during the Degradation of Tetrahydrofuran
Authors: He Zhixing , Yao Yanlai , Lu Zhenmei , Ye Yangfang
Published: February 2014

Although tetrahydrofuran-degrading
Rhodococcus
sp. strain YYL possesses tetrahydrofuran (THF) degradation genes similar to those of other tetrahydrofuran-degrading bacteria, a much higher degradation efficiency has been observed in strain YYL. In this study, nuclear magnetic resonance (NMR)-based metabolomics analyses were performed to explore the metabolic profiling response of strain YYL to exposure to THF. Exposure to THF slightly influenced the metabolome of strain YYL when yeast extract was present in the medium. The metabolic profile of strain YYL over time was also investigated using THF as the sole carbon source to identify the metabolites associated with high-efficiency THF degradation. Lactate, alanine, glutarate, glutamate, glutamine, succinate, lysine, trehalose, trimethylamine-
N
-oxide (TMAO), NAD
+
, and CTP were significantly altered over time in strain YYL grown in 20 mM THF. Real-time quantitative PCR (RT-qPCR) revealed changes in the transcriptional expression levels of 15 genes involved in THF degradation, suggesting that strain YYL could accumulate several disturbances in osmoregulation (trehalose, glutamate, glutamine, etc.), with reduced glycolysis levels, an accelerated tricarboxylic acid cycle, and enhanced protein synthesis. The findings obtained through
1
H NMR metabolomics analyses and the transcriptional expression of the corresponding genes are complementary for exploring the dynamic metabolic profile in organisms.
New Skin Test for Detection of Bovine Tuberculosis on the Basis of Antigen-Displaying Polyester Inclusions Produced by Recombinant Escherichia coli
Authors: Chen Shuxiong , Parlane Natalie A. , Lee Jason , Wedlock D. Neil , Buddle Bryce M. , Rehm Bernd H. A.
Published: February 2014

The tuberculin skin test for diagnosing tuberculosis (TB) in cattle lacks specificity if animals are sensitized to environmental mycobacteria, as some antigens in purified protein derivative (PPD) prepared from
Mycobacterium bovis
are present in nonpathogenic mycobacteria. Three immunodominant TB antigens, ESAT6, CFP10, and Rv3615c, are present in members of the pathogenic
Mycobacterium tuberculosis
complex but absent from the majority of environmental mycobacteria. These TB antigens have the potential to enhance skin test specificity. To increase their immunogenicity, these antigens were displayed on polyester beads by translationally fusing them to a polyhydroxyalkanoate (PHA) synthase which mediated formation of antigen-displaying inclusions in recombinant
Escherichia coli
. The most common form of these inclusions is poly(3-hydroxybutyric acid) (PHB). The respective fusion proteins displayed on these PHB inclusions (beads) were identified using tryptic peptide fingerprinting analysis in combination with matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). The surface exposure and accessibility of antigens were assessed by enzyme-linked immunosorbent assay (ELISA). Polyester beads displaying all three TB antigens showed greater reactivity with TB antigen-specific antibody than did beads displaying only one TB antigen. This was neither due to cross-reactivity of antibodies with the other two antigens nor due to differences in protein expression levels between beads displaying single or three TB antigens. The triple-antigen-displaying polyester beads were used for skin testing of cattle and detected all cattle experimentally infected with
M. bovis
with no false-positive reactions observed in those sensitized to environmental mycobacteria. The results suggested applicability of TB antigen-displaying polyester inclusions as diagnostic reagents for distinguishing TB-infected from noninfected animals.
Signaling via Tumor Necrosis Factor Receptor 1 but Not Toll-Like Receptor 2 Contributes Significantly to Hydrosalpinx Development following Chlamydia muridarum Infection
Authors: Dong Xiaohua , Liu Yuanjun , Chang Xiaotong , Lei Lei , Zhong Guangming
Published: February 2014

Chlamydial infection in the lower genital tract can lead to hydrosalpinx, which is accompanied by activation of both pattern recognition receptor TLR2- and inflammatory cytokine receptor TNFR1-mediated signaling pathways. In the current study, we compared the relative contributions of these two receptors to chlamydial induction of hydrosalpinx in mice. We found that mice with or without deficiencies in TLR2 or TNFR1 displayed similar time courses of live organism shedding from vaginal swabs, suggesting that these receptor-mediated signaling pathways are not required for controlling chlamydial lower genital infection. However, mice deficient in TNFR1 but not TLR2 developed significantly reduced hydrosalpinx. The decreased pathogenicity correlated with a significant reduction in interleukin-17 byin vitro-restimulated splenocytes of TNFR1-deficient mice. Although TLR2-deficient mice developed hydrosalpinx as severe as that of wild-type mice, peritoneal macrophages from mice deficient in TLR2 but not TNFR1 produced significantly reduced cytokines upon chlamydial stimulation, suggesting that reduced macrophage responses to chlamydial infection do not always lead to a reduction in hydrosalpinx. Thus, we have demonstrated that the signaling pathways triggered by the cytokine receptor TNFR1 play a more significant role in chlamydial induction of hydrosalpinx than those mediated by the pattern recognition receptor TLR2, which has laid a foundation for further revealing the chlamydial pathogenic mechanisms.
Effects of 3′-OH and 5′-PO
4
Base Mispairs and Damaged Base Lesions on the Fidelity of Nick Sealing by Deinococcus radiodurans RNA Ligase
Authors: Schmier Brad J. , Shuman Stewart
Published: February 2014

Deinococcus radiodurans
RNA ligase (DraRnl) is the founding member of a family of end-joining enzymes encoded by diverse microbes and viruses. DraRnl ligates 3′-OH, 5′-PO
4
nicks in double-stranded nucleic acids in which the nick 3′-OH end is RNA. Here we gauge the effects of 3′-OH and 5′-PO
4
base mispairs and damaged base lesions on the rate of nick sealing. DraRnl is indifferent to the identity of the 3′-OH nucleobase, provided that it is correctly paired. With 3′-OH mispairs, the DraRnl sealing rate varies widely, with G-T and A-C mispairs being the best substrates and G-G, G-A, and A-A mispairs being the worst. DraRnl accepts 3′ A–8-oxoguanine (oxoG) to be correctly paired, while it discriminates against U-oxoG and G-oxoG mispairs. DraRnl displays high activity and low fidelity in sealing 3′-OH ends opposite an 8-oxoadenine lesion. It prefers 3′-OH adenosine when sealing opposite an abasic template site. With 5′-PO
4
mispairs, DraRnl seals a 5′ T-G mispair as well as it does a 5′ C-G pair; in most other respects, the ligation fidelity at 5′ mispairs is similar to that at 3′ mispairs. DraRnl accepts a 5′ A-oxoG end to be correctly paired, yet it is more tolerant of 5′ T-oxoG and 5′ G-oxoG mispairs than the equivalent configurations on the 3′ side of the nick. At 5′ nucleobase-abasic site nicks, DraRnl prefers to ligate when the nucleobase is a purine. The biochemical properties of DraRnl are compatible with its participation in the templated repair of RNA damage or in the sealing of filled DNA gaps that have a 3′ ribopatch.
Function and Localization Dynamics of Bifunctional Penicillin-Binding Proteins in Caulobacter crescentus
Authors: Strobel Wolfgang , Möll Andrea , Kiekebusch Daniela , Klein Kathrin E. , Thanbichler Martin
Published: February 2014

The peptidoglycan cell wall of bacteria is a complex macromolecule composed of glycan strands that are cross-linked by short peptide bridges. Its biosynthesis involves a conserved group of enzymes, the bifunctional penicillin-binding proteins (bPBPs), which contain both a transglycosylase and a transpeptidase domain, thus being able to elongate the glycan strands and, at the same time, generate the peptide cross-links. The stalked model bacterium
Caulobacter crescentus
possesses five bPBP paralogs, named Pbp1A, PbpC, PbpX, PbpY, and PbpZ, whose function is still incompletely understood. In this study, we show that any of these proteins except for PbpZ is sufficient for growth and normal morphogenesis when expressed at native or elevated levels, whereas inactivation of all five paralogs is lethal. Growth analyses indicate a central role of PbpX in the resistance of
C. crescentus
against the noncanonical amino acid
d
-alanine. Moreover, we show that PbpX and PbpY localize to the cell division site. Their recruitment to the divisome is dependent on the essential cell division protein FtsN and likely involves interactions with FtsL and the putative peptidoglycan hydrolase DipM. The same interaction pattern is observed for Pbp1A and PbpC, although these proteins do not accumulate at midcell. Our findings demonstrate that the bPBPs of
C. crescentus
are, to a large extent, redundant and have retained the ability to interact with the peptidoglycan biosynthetic machineries responsible for cell elongation, cytokinesis, and stalk growth. Nevertheless, they may preferentially act in specific peptidoglycan biosynthetic complexes, thereby facilitating the independent regulation of distinct growth processes.
DnaJ-Promoted Binding of DnaK to Multiple Sites on σ
32
in the Presence of ATP
Authors: Noguchi Aki , Ikeda Ayami , Mezaki Moeka , Fukumori Yoshihiro , Kanemori Masaaki
Published: February 2014

The
Escherichia coli
DnaK chaperone system is a canonical heat shock protein 70 (Hsp70) chaperone system comprising Hsp70, Hsp40, and a nucleotide exchange factor. Although Hsp40 is known to facilitate the effective binding of Hsp70 to substrates, the role of Hsp40 in Hsp70-substrate interactions has not yet been fully elucidated. Using the
E. coli
heat shock transcription factor σ
32
as a substrate in the DnaK chaperone system, we here provide new insight into the Hsp70-substrate interaction. When DnaK-σ
32
complexes formed under various conditions were analyzed by gel filtration, several DnaK-σ
32
complexes with different molecular masses were detected. The results indicated that multiple DnaK molecules simultaneously bind to σ
32
, even though it has been suggested that DnaK interacts with σ
32
at a molar ratio of 1:1. Two σ
32
mutants, L201D σ
32
and I54A σ
32
, which have reduced affinities for DnaK and DnaJ (Hsp40), respectively, were used to further characterize DnaK-σ
32
complex formation. Pulldown assays demonstrated that the affinity of I54A σ
32
for DnaK was similar to that of wild-type σ
32
in the absence of DnaJ, whereas L201D σ
32
exhibited an extremely low affinity for DnaK. However, in the presence of ATP and DnaJ, the yield of DnaK eluted with L201D σ
32
was much higher than that eluted with I54A σ
32
. These results indicate that there are multiple DnaK binding sites on σ
32
and that DnaJ strongly promotes DnaK binding to any site in the presence of ATP, regardless of the intrinsic affinity of DnaK for the site.
Impact of a Transposon Insertion in
phzF2
on the Specialized Metabolite Production and Interkingdom Interactions of Pseudomonas aeruginosa
Authors: Phelan Vanessa V. , Moree Wilna J. , Aguilar Julieta , Cornett Dale S. , Koumoutsi Alexandra , Noble Suzanne M. , Pogliano Kit , Guerrero Carlos A. , Dorrestein Pieter C.
Published: February 2014

In microbiology, gene disruption and subsequent experiments often center on phenotypic changes caused by one class of specialized metabolites (quorum sensors, virulence factors, or natural products), disregarding global downstream metabolic effects. With the recent development of mass spectrometry-based methods and technologies for microbial metabolomics investigations, it is now possible to visualize global production of diverse classes of microbial specialized metabolites simultaneously. Using imaging mass spectrometry (IMS) applied to the analysis of microbiology experiments, we can observe the effects of mutations, knockouts, insertions, and complementation on the interactive metabolome. In this study, a combination of IMS and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to visualize the impact on specialized metabolite production of a transposon insertion into a
Pseudomonas aeruginosa
phenazine biosynthetic gene,
phzF2
. The disruption of phenazine biosynthesis led to broad changes in specialized metabolite production, including loss of pyoverdine production. This shift in specialized metabolite production significantly alters the metabolic outcome of an interaction with
Aspergillus fumigatus
by influencing triacetylfusarinine production.
Characterization of the Vibrio cholerae VolA Surface-Exposed Lipoprotein Lysophospholipase
Authors: Pride Aaron C. , Guan Ziqiang , Trent M. Stephen
Published: February 2014

Bacterial lipases play important roles in bacterial metabolism and environmental response. Our laboratory recently discovered that a novel lipoprotein lysophospholipase, VolA, localizes on the surface of the Gram-negative aquatic pathogen
Vibrio cholerae
. VolA functions to cleave exogenous lysophosphatidylcholine, freeing the fatty acid moiety for use by
V. cholerae
. This fatty acid is transported into the cell and can be used as a nutrient and, more importantly, as a way to alter the membrane architecture via incorporation into the phospholipid biosynthesis pathway. There are few examples of Gram-negative, surface-exposed lipoproteins, and VolA is unique, as it has a previously undercharacterized function in
V. cholerae
membrane remodeling. Herein, we report the biochemical characterization of VolA. We show that VolA is a canonical lipoprotein via mass spectrometry analysis and demonstrate the
in vitro
activity of VolA under a variety of conditions. Additionally, we show that VolA contains a conserved Gly-Xaa-Ser-Xaa-Gly motif typical of lipases. Interestingly, we report the observation of VolA homologs in other aquatic pathogens. An
Aeromonas hydrophila
VolA homolog complements a
V. cholerae
VolA mutant in growth on lysophosphatidylcholine as the sole carbon source and in enzymatic assays. These results support the idea that the lipase activity of surface-exposed VolA likely contributes to the success of
V. cholerae
, improving the overall adaptation and survival of the organism in different environments.
GtfA and GtfB Are Both Required for Protein
O
-Glycosylation in Lactobacillus plantarum
Authors: Lee I-Chiao , van Swam Iris I. , Tomita Satoru , Morsomme Pierre , Rolain Thomas , Hols Pascal , Kleerebezem Michiel , Bron Peter A.
Published: February 2014

Acm2, the major autolysin of
Lactobacillus plantarum
WCFS1, was recently found to be
O
-glycosylated with
N
-acetylhexosamine, likely
N
-acetylglucosamine (GlcNAc). In this study, we set out to identify the glycosylation machinery by employing a comparative genomics approach to identify Gtf1 homologues, which are involved in fimbria-associated protein 1 (Fap1) glycosylation in
Streptococcus parasanguinis
. This
in silico
approach resulted in the identification of 6 candidate
L. plantarum
WCFS1 genes with significant homology to Gtf1, namely,
tagE1
to
tagE6
. These candidate genes were targeted by systematic gene deletion, followed by assessment of the consequences on glycosylation of Acm2. We observed a changed mobility of Acm2 on SDS-PAGE in the
tagE5E6
deletion strain, while deletion of other
tagE
genes resulted in Acm2 mobility comparable to that of the wild type. Subsequent mass spectrometry analysis of excised and in-gel-digested Acm2 confirmed the loss of glycosylation on Acm2 in the
tagE5E6
deletion mutant, whereas a lectin blot using GlcNAc-specific succinylated wheat germ agglutinin (sWGA) revealed that besides Acm2,
tagE5E6
deletion also abolished all but one other sWGA-reactive, protease-sensitive signal. Only complementation of both
tagE5
and
tagE6
restored those sWGA lectin signals, establishing that TagE5 and TagE6 are both required for the glycosylation of Acm2 as well as the vast majority of other sWGA-reactive proteins. Finally, sWGA lectin blotting experiments using a panel of 8 other
L. plantarum
strains revealed that protein glycosylation is a common feature in
L. plantarum
strains. With the establishment of these enzymes as protein glycosyltransferases, we propose to rename TagE5 and TagE6 as GtfA and GtfB, respectively.