bluuebunny/crossref_metadata_2025 huggingface dataset:
Large‐scale development of cost‐effective SNP marker assays for diversity assessment and genetic mapping in chickpea and comparative mapping in legumes
Authors: Hiremath Pavana J. , Kumar Ashish , Penmetsa Ramachandra Varma , Farmer Andrew , Schlueter Jessica A. , Chamarthi Siva K. , Whaley Adam M. , Carrasquilla‐Garcia Noelia , Gaur Pooran M. , Upadhyaya Hari D. , Kavi Kishor Polavarapu B. , Shah Trushar M. , Cook Douglas R. , Varshney Rajeev K.
Published: June 2012

A set of 2486 single nucleotide polymorphisms (SNPs) were compiled in chickpea using four approaches, namely (i) Solexa/Illumina sequencing (1409), (ii) amplicon sequencing of tentative orthologous genes (TOGs) (604), (iii) mining of expressed sequence tags (ESTs) (286) and (iv) sequencing of candidate genes (187). Conversion of these SNPs to the cost‐effective and flexible throughput Competitive Allele Specific PCR (KASPar) assays generated successful assays for 2005 SNPs. These marker assays have been designated as Chickpea KASPar Assay Markers (CKAMs). Screening of 70 genotypes including 58 diverse chickpea accessions and 12 BC3F2 lines showed 1341 CKAMs as being polymorphic. Genetic analysis of these data clustered chickpea accessions based on geographical origin. Genotyping data generated for 671 CKAMs on the reference mapping population (Cicer arietinum ICC 4958 × Cicer reticulatum PI 489777) were compiled with 317 unpublished TOG‐SNPs and 396 published markers for developing the genetic map. As a result, a second‐generation genetic map comprising 1328 marker loci including novel 625 CKAMs, 314 TOG‐SNPs and 389 published marker loci with an average inter‐marker distance of 0.59 cM was constructed. Detailed analyses of 1064 mapped loci of this second‐generation chickpea genetic map showed a higher degree of synteny with genome of Medicago truncatula, followed by Glycine max, Lotus japonicus and least with Vigna unguiculata. Development of these cost‐effective CKAMs for SNP genotyping will be useful not only for genetics research and breeding applications in chickpea, but also for utilizing genome information from other sequenced or model legumes.
Advected Tangent Curves: A General Scheme for Characteristic Curves of Flow Fields
Authors: Weinkauf T. , Hege H.‐C. , Theisel H.
Published: June 2012

We present the first general scheme to describe all four types of characteristic curves of flow fields – stream, path, streak, and time lines – as tangent curves of a derived vector field. Thus, all these lines can be obtained by a simple integration of an autonomous ODE system. Our approach draws on the principal ideas of the recently introduced tangent curve description of streak lines. We provide the first description of time lines as tangent curves of a derived vector field, which could previously only be constructed in a geometric manner. Furthermore, our scheme gives rise to new types of curves. In particular, we introduce advected stream lines as a parameter‐free variant of the time line metaphor. With our novel mathematical description of characteristic curves, a large number of feature extraction and analysis tools becomes available for all types of characteristic curves, which were previously only available for stream and path lines. We will highlight some of these possible applications including the computation of time line curvature fields and the extraction of cores of swirling advected stream lines.
Clofarabine‐containing conditioning regimen for allo‐SCT in AML/ALL patients: a survey from the Acute Leukemia Working Party of EBMT
Authors: Chevallier Patrice , Labopin Myriam , Buchholz Stefanie , Ganser Arnold , Ciceri Fabio , Lioure Bruno , Faul Chistoph , Guillerm Gaelle , Finke Juergen , Huynh Anne , Schubert Joerg , Kolb Hans‐Jochem , Polge Emmanuelle , Nagler Arnon , Mohty Mohamad
Published: June 2012

Clofarabine (CLO), a second‐generation purine analogue, has demonstrated an efficient anti‐leukemia activity while showing a favorable toxicity profile. This retrospective multicenter report assessed the outcome of 90 patients who received a CLO‐containing conditioning regimen before allo‐SCT for AML (n = 69) or ALL (n = 21). Median age was 42 yr at transplant. The majority of cases (n = 66) presented with an active disease at transplant while 38 patients had received previous transplantation(s). A total of 88 and two patients received a reduced‐intensity conditioning or a myeloablative regimen, respectively. Engraftment was achieved in 97% of evaluable patients. With a median follow‐up of 14 months (range, 1–45), the 2‐year OS, LFS, relapse, and NRM rates were 28 ± 5%, 23 ± 5%, 41 ± 6%, and 35 ± 5%, respectively. When comparing AML and ALL patients, OS and LFS were significantly higher for AML (OS, 35 ± 6% vs. 0%, P < 0.0001); LFS: 30 ± 6% vs. 0%, P < 0.0001). In a Cox multivariate analysis, an AML diagnosis was the only factor associated with a better LFS (HR = 0.37; 95%CI, 0.21–0.66; P = 0.001). We conclude that a CLO‐containing conditioning regimen prior to allo‐SCT might be an effective treatment. Prospective studies are needed to evaluate the potential role of CLO as part of conditioning regimens in acute leukemias.
Influence of finishing textile materials on the reduction of skin irritations
Authors: Strese Helene , Kuck Monika , Benken Rainer , Fluhr Joachim W. , Schanzer Sabine , Richter Heike , Meinke Martina C. , Beuthan Jürgen , Benderoth Christian , Frankowski Gottfried , Sterry Wolfram , Lademann Jürgen
Published: June 2012

Background

An increasing number of people is reported to have sensitive skin. Consequently, the development of textile materials with comfortable wearing properties has become a major interest. One method to create a pleasant sensation of textile materials on the skin is to coat them with silk proteins. This technologically complex procedure requires both optimization and control.

Methods

The present study was aimed to characterize the subjective perception of different textiles and to objectively assess their influence on skin morphology with non‐invasive optical techniques like optical coherence tomography (OCT), laser scanning microscopy (LSM) and optical surface profilometry (OP). Furthermore, optical methods were used to characterize surface properties of different textile fabrics.

Results

In the present study it could be shown that optical non‐invasive methods, as applied in cosmetology and dermatology are suited to characterize the structural properties of fabrics and the effects that textile materials have on the skin. Here, both unfinished textile materials and fabrics finished with silk protein coating were investigated by OCT, LSM and OP. In addition, volunteers were interviewed about their subjective sensation when these fabrics were in contact with their skin.

Conclusion

The study showed that optical methods could be applied to compare textile materials in vitro, which permit the wearing comfort to be predicted and in vivo perception on the skin.
BeyondResponsive Regulation: The expanding role of non‐state actors in the regulatory process
Authors: Grabosky Peter
Published: June 2012

This comment extends the vision ofResponsive Regulationby noting subsequent developments in regulatory pluralism, in particular those occurring under private auspices. The apparent weakening or withdrawal of state regulatory institutions has inspired considerable regulatory activity on the part of non‐state actors. In addition, the concurrent growth and pervasiveness of digital technology have greatly facilitated the involvement of individual citizens in non‐state regulatory activity. However, the full implications of what might be called “wiki‐regulation” remain to be seen. The risks that accompany private regulation may include the lack of accountability of non‐state regulatory actors, and the possibility of their failure. There is also a risk that with the increasing salience of what Vogel calls “civil regulation,” state regulatory institutions may atrophy, or fail to develop at all.
Autonomic nervous system modulation affects the inflammatory immune response in mice with acute Chagas disease
Authors: Machado Marcus Paulo Ribeiro , Rocha Aletheia Moraes , de Oliveira Lucas Felipe , de Cuba Marília Beatriz , de Oliveira Loss Igor , Castellano Lucio Roberto , Silva Marcus Vinicius , Machado Juliana Reis , Nascentes Gabriel Antonio Nogueira , Paiva Luciano Henrique , Savino Wilson , Junior Virmondes Rodrigues , Brum Patricia Chakur , Prado Vania Ferreira , Prado Marco Antonio Maximo , Silva Eliane Lages , Montano Nicola , Ramirez Luis Eduardo , Dias da Silva Valdo Jose
Published: June 2012

The aim of the present study was to evaluate the effects of changes to the autonomic nervous system in mice during the acute phase of Chagas disease, which is an infection caused by the parasite Trypanosoma cruzi. The following types of mice were inoculated with T. cruzi (CHG): wild‐type (WT) and vesicular acetylcholine transporter knockdown (KDVAChT) C57BL/6j mice; wild‐type non‐treated (NT) FVB mice; FVB mice treated with pyridostigmine bromide (PYR) or salbutamol (SALB); and β2‐adrenergic receptor knockout (KOβ2) FVB mice. During infection and at 18–21 days after infection (acute phase), the survival curves, parasitaemia, electrocardiograms, heart rate variability, autonomic tonus and histopathology of the animals were evaluated. Negative control groups were matched for age, genetic background and treatment. The KDVAChT‐CHG mice exhibited a significant shift in the electrocardiographic, autonomic and histopathological profiles towards a greater inflammatory immune response that was associated with a reduction in blood and tissue parasitism. In contrast, the CHG‐PYR mice manifested reduced myocardial inflammation and lower blood and tissue parasitism. Similar results were observed in CHG‐SALB animals. Unexpectedly, the KOβ2‐CHG mice exhibited less myocardial inflammation and higher blood and tissue parasitism, which were associated with reduced mortality. These findings could have been due to the increase in vagal tone observed in the KOβ2 mice, which rendered them more similar to the CHG‐PYR animals. In conclusion, our results indicate a marked immunomodulatory role for the parasympathetic and sympathetic autonomic nervous systems, which inhibit both the inflammatory immune response and parasite clearance during the acute phase of experimental Chagas heart disease in mice.
Time course of hepatic gluconeogenesis during hindlimb suspension unloading
Authors: Bederman Ilya R. , Chandramouli Visvanathan , Sandlers Yana , Henderson Leigh , Cabrera Marco E.
Published: June 2012

New Findings

What is the central question of this study?

The rate of liver gluconeogenesis during hindlimb suspension unloading has not been determined.

What is the main finding and its importance?

Hepatic gluconeogenesis is significantly increased under conditions of hindlimb suspension unloading, which simulates the muscular atrophy occurring during spaceflight. This finding supports earlier hypothesis. This metabolic shift in the living animal warrants further study.

The goal of this work was to determine the time‐dependent changes in fractional hepatic gluconeogenesis (GNG) during conditions of hindlimb suspension unloading (HSU), a ‘ground‐based’ method for inducing muscular atrophy to simulate space flight. We hypothesized that GNG would increase in HSU conditions as a result of metabolic shifts in the liver and skeletal muscle. A significant and progressive atrophy was observed in the soleus (30, 47 and 55%) and gastrocnemius muscles (0, 15 and 17%) after 3, 7 and 14 days of HSU, respectively. Fractional hepatic GNG was determined following the incorporation of deuterium from deuterated water (2H2O) into C–H bonds of newly synthesized glucose after an 8 h fast. Enrichment of plasma glucose with 2H was measured using the classic method of Landau et al. (the ‘hexamethylenetetramine (HMT) method’), based on specific 2H labelling of glucose carbons, and the novel method of Chacko et al. (‘average method’), based on the assumption of equal 2H enrichment on all glucose carbons (except C2). After 3 days of HSU, fractional GNG was significantly elevated in the HSU group, as determined by either method (∼13%, P < 0.05). After 7 and 14 days of HSU, gluconeogenesis was not significantly different. Both analytical methods yielded similar time‐dependent trends in gluconeogenic rates, but GNG values determined using the average method were consistently lower (∼30%) than those found by the HMT method. To compare and validate the average method against the HMT method further, we starved animals for 13 h to allow for hepatic GNG to contribute 100% to endogenous glucose production. The HMT method yielded 100% GNG, while the average method yielded GNG of ∼70%. As both methods used the same values of precursor enrichment, we postulated that the underestimation of gluconeogenic rate was as a result of differences in the measurements of product enrichment (2H labelling of plasma glucose). This could be explained by the following factors: (i) loss of deuterium via exchange between acetate and glucose; (ii) interference caused by fragment m/z 169, representing multiple isobaric species; and (iii) interference from other sugars at m/z 169. In conclusion, HSU caused a time‐dependent increase in hepatic gluconeogenesis, irrespective of the analytical methods used.
Intrinsic Time Integration Procedures for Rigid Body Dynamics
Authors: Bauchau Olivier A. , Xin Hao , Dong Shiyu , Li Zhiheng , Han Shilei
Published: June 2012

The treatment of rotations in rigid body and Cosserat solids dynamics is challenging. In most cases, at some point in the formulation, a parameterization of rotation is introduced and the intrinsic nature of the equations of motions is lost. Typically, this step considerably complicates the form of the equations and increases the order of the nonlinearities. Clearly, it is desirable to bypass parameterization of rotation, leaving the equations of motion in their original, intrinsic form. This has prompted the development of rotationless and intrinsic formulations. This paper focuses on the latter approach. The most famous example of intrinsic formulation is probably Euler’s second law for the motion of a rigid body rotating about an inertial point. This equation involves angular velocities solely, with algebraic nonlinearities of the second-order at most. Unfortunately, this intrinsic equation also suffers serious drawbacks: the angular velocity of the body is computed, but not its orientation, the body is “unaware” of its inertial orientation. This paper presents an alternative approach to the problem by proposing discrete statements of the rotation kinematic compatibility equation, which provide solutions for both rotation tensor and angular velocity without relying on a parameterization of rotation. The formulation is also generalized using the motion formalism, leading to very simple discretized equations of motion.
Hopf Bifurcation Analysis for a Novel Hyperchaotic System
Authors: Zhuang Kejun
Published: June 2012

The paper mainly focuses on a novel hyperchaotic system. The local stability of equilibrium is analyzed and existence of Hopf bifurcation is established. Moreover, formulas for determining the stability and direction of bifurcating periodic solutions are derived by center manifold theorem and normal form theory. Finally, numerical simulation is given to illustrate the theoretical analysis.
Fuel Cell Power Control Based on a Master-Slave Structure: A Proton Exchange Membrane Fuel Cell Case Study
Authors: Ji Guangji , Hanke-Rauschenbach Richard , Bornhöft Astrid , Zhou Su , Sundmacher Kai
Published: June 2012

Fuel cells generally become promising candidates for the electrical power supply in automotive and stationary applications. The power control of the fuel cell is one of the essential problems. In this paper, a power control concept with a master-slave structure for fuel cell systems is suggested. Within that concept, a DC/DC converter, several slave controllers, and a master controller are combined to achieve the control objectives. The DC/DC converter conditions the power and transfers it from the fuel cell to the load. The task of the slave controller is to maintain the controlled variables at their set points. The master controller has to select the set points for the slave controllers and limits the fuel cell output power, if the requested power exceeds the maximum power, which can be instantaneously produced by the controlled fuel cell system. The proposed control concept is demonstrated by simulations of a proton exchange membrane (PEM) fuel cell system taken from the literature. For that purpose, different controllers are designed based on model-free methods. For the master controller design, two alternative options are discussed: high efficiency tracking and fast power tracking. As shown in the simulation results, high efficiency tracking leads to higher system efficiency, however, an additional energy buffer is required. In contrast, no energy buffer is needed for the option of fast power tracking. However, the system efficiency is lower. The presented control concept is meaningful for systems with dynamic load requirements and can be easily applied to different fuel cell systems due to the model-free design approach.
Reconstructing the vibro-acoustic quantities on a highly non-spherical surface using the Helmholtz equation least squares method
Authors: Natarajan Logesh Kumar , Wu Sean F.
Published: June 2012

This paper presents helpful guidelines and strategies for reconstructing the vibro-acoustic quantities on a highly non-spherical surface by using the Helmholtz equation least squares (HELS). This study highlights that a computationally simple code based on the spherical wave functions can produce an accurate reconstruction of the acoustic pressure and normal surface velocity on planar surfaces. The key is to select the optimal origin of the coordinate system behind the planar surface, choose a target structural wavelength to be reconstructed, set an appropriate stand-off distance and microphone spacing, use a hybrid regularization scheme to determine the optimal number of the expansion functions, etc. The reconstructed vibro-acoustic quantities are validated rigorously via experiments by comparing the reconstructed normal surface velocity spectra and distributions with the benchmark data obtained by scanning a laser vibrometer over the plate surface. Results confirm that following the proposed guidelines and strategies can ensure the accuracy in reconstructing the normal surface velocity up to the target structural wavelength, and produce much more satisfactory results than a straight application of the original HELS formulations. Experiment validations on a baffled, square plate were conducted inside a fully anechoic chamber.
Modeling non-spherical oscillations and stability of acoustically driven shelled microbubbles
Authors: Loughran Jonathan , Eckersley Robert J. , Tang Meng-Xing
Published: June 2012

The oscillation and destruction of microbubbles under ultrasound excitation form the basis of contrast enhanced ultrasound imaging and microbubble assisted drug and gene delivery. A typical microbubble has a size of a few micrometers and consists of a gas core encapsulated by a shell. These bubbles can be driven into surface mode oscillations, which not only contribute to the measured acoustic signal but can lead to bubble destruction. Existing models of surface model oscillations have not considered the effects of a bubble shell. In this study a model was developed to study the surface mode oscillations in shelled bubbles. The effects of shell viscosity and elasticity on the surface mode oscillations were modeled using a Boussinesq–Scriven approach. Simulation was conducted using the model with various bubble sizes and driving acoustic pressures. The occurrence of surface modes and the number of ultrasound cycles needed for the occurrence were calculated. The simulation results show a significant difference between shelled bubbles and shell free bubbles. The shelled bubbles have reduced surface mode amplitudes and a narrower bubble size range within which these modes develop compared to shell free bubbles. The clinical implications were also discussed.
Propagation of sound from a monopole source above an impedance-backed porous layer
Authors: Li Kai Ming , Liu Sheng
Published: June 2012

In this article, the propagation of sound from a monopole source above an impedance-backed porous layer is examined. The sound fields can be expressed in an integral form that is amenable to further analysis. A standard method of steepest descents is applied to evaluate the integral where the method of pole subtraction is needed to obtain a uniform asymptotic solution for the sound field above the plane surface. To obtain a numerical solution, the location of the pole was determined numerically by means of the Newton-Raphson method. Based on the pole location, the sound fields can then be calculated numerically. It has been demonstrated that the use of a plane wave reflection coefficient to calculate the sound fields is a special case of the asymptotic formula when the pole is located further away from the saddle point.
Compare ultrasound-mediated heating and cavitation between flowing polymer- and lipid-shelled microbubbles during focused ultrasound exposures
Authors: Zhang Siyuan , Zong Yujin , Wan Mingxi , Yu Xiaojun , Fu Quanyou , Ding Ting , Zhou Fanyu , Wang Supin
Published: June 2012

This paper compares the efficiency of flowing polymer- and lipid-shelled microbubbles (MBs) in the heating and cavitation during focused ultrasound exposures. Temperature and cavitation activity were simultaneously measured as the two types of shelled MBs and saline flowing through a 3 mm diameter vessel in the phantom with varying flow velocities (0–20 cm/s) at different acoustic power levels (0.6–20 W) with each exposure for 5 s. Temperature and cavitation for the lipid-shelled MBs were higher than those for the polymer-shelled MBs. Temperature rise decreased with increasing flow velocities for the two types of shelled MBs and saline at acoustic power 1.5 W. At acoustic power 11.1 W, temperature rise increased with increasing flow velocities for the lipid-shelled MBs. For the polymer-shelled MBs, the temperature rise increased with increasing flow velocities from 3–15 cm/s and decreased at 20 cm/s. Cavitation increased with increasing flow velocity for the two shelled MBs and there were no significant changes of cavitation with increasing flow velocities for saline. These results suggested that lipid-shelled MBs may have a greater efficiency than polymer-shelled MBs in heating and cavitation during focused ultrasound exposures.
Comparative Survey of Rumen Microbial Communities and Metabolites across One Caprine and Three Bovine Groups, Using Bar-Coded Pyrosequencing and
1
H Nuclear Magnetic Resonance Spectroscopy
Authors: Lee Hyo Jung , Jung Ji Young , Oh Young Kyoon , Lee Sang-Suk , Madsen Eugene L. , Jeon Che Ok
Published: June 2012

Pyrosequencing of 16S rRNA genes (targeting
Bacteria
and
Archaea
) and
1
H nuclear magnetic resonance were applied to investigate the rumen microbiota and metabolites of Hanwoo steers in the growth stage (HGS), Hanwoo steers in the late fattening stage (HFS), Holstein-Friesian dairy cattle (HDC), and Korean native goats (KNG) in the late fattening stage. This was a two-part investigation. We began by comparing metabolites and microbiota of Hanwoo steers at two stages of husbandry. Statistical comparisons of metabolites and microbial communities showed no significant differences between HFS and HGS (differing by a dietary shift at 24 months and age [67 months versus 12 months]). We then augmented the study by extending the investigation to HDC and KNG. Overall, pyrosequencing of 16S rRNA genes showed that the rumens had highly diverse microbial communities containing many previously undescribed microorganisms. Bioinformatic analysis revealed that the bacterial sequences were predominantly affiliated with four phyla—
Bacteroidetes
,
Firmicutes
,
Fibrobacteres
, and
Proteobacteria
—in all ruminants. However, interestingly, the bacterial reads belonging to
Fibrobacteres
were present at a very low abundance (<0.1%) in KNG. Archaeal community analysis showed that almost all of these reads fell into a clade related to, but distinct from, known cultivated methanogens. Statistical analyses showed that the microbial communities and metabolites of KNG were clearly distinct from those of other ruminants. In addition, bacterial communities and metabolite profiles of HGS and HDC, fed similar diets, were distinctive. Our data indicate that bovine host breeds override diet as the key factor that determines bacterial community and metabolite profiles in the rumen.
Quorum Sensing in the Context of Food Microbiology
Authors: Skandamis Panagiotis N. , Nychas George-John E.
Published: June 2012

Food spoilage may be defined as a process that renders a product undesirable or unacceptable for consumption and is the outcome of the biochemical activity of a microbial community that eventually dominates according to the prevailing ecological determinants. Although limited information are reported, this activity has been attributed to quorum sensing (QS). Consequently, the potential role of cell-to-cell communication in food spoilage and food safety should be more extensively elucidated. Such information would be helpful in designing approaches for manipulating these communication systems, thereby reducing or preventing, for instance, spoilage reactions or even controlling the expression of virulence factors. Due to the many reports in the literature on the fundamental features of QS, e.g., chemistry and definitions of QS compounds, in this minireview, we only allude to the types and chemistry of QS signaling moleculesper seand to the (bioassay-based) methods of their detection and quantification, avoiding extensive documentation. Conversely, we attempt to provide insights into (i) the role of QS in food spoilage, (ii) the factors that may quench the activity of QS in foods and review the potential QS inhibitors that might “mislead” the bacterial coordination of spoilage activities and thus may be used as biopreservatives, and (iii) the future experimental approaches that need to be undertaken in order to explore the “gray” or “black” areas of QS, increase our understanding of how QS affects microbial behavior in foods, and assist in finding answers as to how we can exploit QS for the benefit of food preservation and food safety.
Reduction of Photoautotrophic Productivity in the Cyanobacterium Synechocystis sp. Strain PCC 6803 by Phycobilisome Antenna Truncation
Authors: Page Lawrence E. , Liberton Michelle , Pakrasi Himadri B.
Published: June 2012

Truncation of the algal light-harvesting antenna is expected to enhance photosynthetic productivity. The wild type and three mutant strains of
Synechocystis
sp. strain 6803 with a progressively smaller phycobilisome antenna were examined under different light and CO
2
conditions. Surprisingly, such antenna truncation resulted in decreased whole-culture productivity for this cyanobacterium.
Relationship between Enterococcal Levels and Sediment Biofilms at Recreational Beaches in South Florida
Authors: Piggot Alan M. , Klaus James S. , Johnson Sara , Phillips Matthew C. , Solo-Gabriele Helena M.
Published: June 2012

Enterococci, recommended at the U.S. federal level for monitoring water quality at marine recreational beaches, have been found to reside and grow within beach sands. However, the environmental and ecological factors affecting enterococcal persistence remain poorly understood, making it difficult to determine levels of fecal pollution and assess human health risks. Here we document the presence of enterococci associated with beach sediment biofilms at eight south Florida recreational beaches. Enterococcal levels were highest in supratidal sands, where they displayed a nonlinear, unimodal relationship with extracellular polymeric secretions (EPS), the primary component of biofilms. Enterococcal levels peaked at intermediate levels of EPS, suggesting that biofilms may promote the survival of enterococci but also inhibit enterococci as the biofilm develops within beach sands. Analysis of bacterial community profiles determined by terminal restriction fragment length polymorphisms showed the bacterial communities of supratidal sediments to be significantly different from intertidal and subtidal communities; however, no differences were observed in bacterial community compositions associated with different EPS concentrations. Our results suggest that supratidal sands are a microbiologically unique environment favorable for the incorporation and persistence of enterococci within beach sediment biofilms.
Quantification of Diatom Gene Expression in the Sea by Selecting Uniformly Transcribed mRNA as the Basis for Normalization
Authors: Kang Lee-Kuo , Tsui Feng-Hsiu , Chang Jeng
Published: June 2012

To quantify gene expressions by quantitative reverse transcription-PCR (Q-RT-PCR) in natural diatom assemblages, it is necessary to seek a biomass reference specific to the target species. Two housekeeping genes,
TBP
(encoding the TATA box-binding protein) and
EFL
(encoding the translation elongation factor-like protein), were evaluated as candidates for reference genes in Q-RT-PCR assays. Transcript levels of
TBP
and
EFL
were relatively stable under various test conditions including growth stages, light-dark cycle phases, and nutrient stresses in
Skeletonema costatum
and
Chaetoceros affinis
, and
TBP
expression was more stable than that of
EFL
. Next, the sequence diversity of diatom assemblages was evaluated by obtaining 32
EFL
and 29
TBP
homologous gene fragments from the East China Sea (ECS). Based on sequence alignments,
EFL
and
TBP
primer sets were designed for
Chaetoceros
and
Skeletonema
groups in the ECS. An evaluation of primer specificity and PCR efficiency indicated that the
EFL
primer sets performed better. To demonstrate the applicability of
EFL
primer sets in the ECS, they were employed to measure mRNA levels of the FcpB (fucoxanthin-chlorophyll protein) gene in diatoms. The results correctly revealed prominent diel variations in
FcpB
expression and confirmed
EFL
as a good reference gene.
Identification and Biochemical Evidence of a Medium-Chain-Length Polyhydroxyalkanoate Depolymerase in the Bdellovibrio bacteriovorus Predatory Hydrolytic Arsenal
Authors: Martínez Virginia , de la Peña Fernando , García-Hidalgo Javier , de la Mata Isabel , García José Luis , Prieto María Auxiliadora
Published: June 2012

The obligate predator
Bdellovibrio bacteriovorus
HD100 shows a large set of proteases and other hydrolases as part of its hydrolytic arsenal needed for its predatory life cycle. We present genetic and biochemical evidence that open reading frame (ORF) Bd3709 of
B. bacteriovorus
HD100 encodes a novel medium-chain-length polyhydroxyalkanoate (mcl-PHA) depolymerase (PhaZ
Bd
). The primary structure of PhaZ
Bd
suggests that this enzyme belongs to the α/β-hydrolase fold family and has a typical serine hydrolase catalytic triad (serine-histidine-aspartic acid) in agreement with other PHA depolymerases and lipases. PhaZ
Bd
has been extracellularly produced using different hypersecretor Tol-pal mutants of
Escherichia coli
and
Pseudomonas putida
as recombinant hosts. The recombinant PhaZ
Bd
has been characterized, and its biochemical properties have been compared to those of other PHA depolymerases. The enzyme behaves as a serine hydrolase that is inhibited by phenylmethylsulfonyl fluoride. It is also affected by the reducing agent dithiothreitol and nonionic detergents like Tween 80. PhaZ
Bd
is an endoexohydrolase that cleaves both large and small PHA molecules, producing mainly dimers but also monomers and trimers. The enzyme specifically degrades mcl-PHA and is inactive toward short-chain-length polyhydroxyalkanoates (scl-PHA) like polyhydroxybutyrate (PHB). These studies shed light on the potentiality of these predators as sources of new biocatalysts, such as an mcl-PHA depolymerase, for the production of enantiopure hydroxyalkanoic acids and oligomers as building blocks for the synthesis of biobased polymers.
Dual-Serotype Biofilm Formation by Shiga Toxin-Producing Escherichia coli O157:H7 and O26:H11 Strains
Authors: Wang Rong , Kalchayanand Norasak , Bono James L. , Schmidt John W. , Bosilevac Joseph M.
Published: June 2012

Escherichia coli
O26:H11 strains were able to outgrow O157:H7 companion strains in planktonic and biofilm phases and also to effectively compete with precolonized O157:H7 cells to establish themselves in mixed biofilms.
E. coli
O157:H7 strains were unable to displace preformed O26:H11 biofilms. Therefore,
E. coli
O26:H11 remains a potential risk in food safety.
Determination of Whether Quorum Quenching Is a Common Activity in Marine Bacteria by Analysis of Cultivable Bacteria and Metagenomic Sequences
Authors: Romero Manuel , Martin-Cuadrado Ana-Belen , Otero Ana
Published: June 2012

The abundance of quorum quenching (QQ) activity was evaluated in cultivable bacteria obtained from oceanic and estuarine seawater and compared with the frequency of QQ enzyme sequences in the available marine metagenomic collections. The possible role of the high QQ activity found among marine bacteria is discussed.
The TetR-Type Transcriptional Repressor RolR from Corynebacterium glutamicum Regulates Resorcinol Catabolism by Binding to a Unique Operator,
rolO
Authors: Li Tang , Zhao Kexin , Huang Yan , Li Defeng , Jiang Cheng-Ying , Zhou Nan , Fan Zheng , Liu Shuang-Jiang
Published: June 2012

The
rol
(designated for
r
esorcin
ol
) gene cluster
rolRHMD
is involved in resorcinol catabolism in
Corynebacterium glutamicum
, and RolR is the TetR-type regulator. In this study, we investigated how RolR regulated the transcription of the
rol
genes in
C. glutamicum
. The transcription start sites and promoters of
rolR
and
rolHMD
were identified. Quantitative reverse transcription-PCR and promoter activity analysis indicated that RolR negatively regulated the transcription of
rolHMD
and of its own gene. Further, a 29-bp operator
rolO
was located at the intergenic region of
rolR
and
rolHMD
and was identified as the sole binding site for RolR. It contained two overlapping inverted repeats and they were essential for RolR-binding. The binding of RolR to
rolO
was affected by resorcinol and hydroxyquinol, which are the starting compounds of resorcinol catabolic pathway. These two compounds were able to dissociate RolR-
rolO
complex, thus releasing RolR from the complex and derepressing the transcription of
rol
genes in
C. glutamicum
. It is proposed that the binding of RolR to its operator
rolO
blocks the transcription of
rolHMD
and of its own gene, thus negatively regulated resorcinol degradation in
C. glutamicum
.
Prophage Carriage and Diversity within Clinically Relevant Strains of Clostridium difficile
Authors: Shan Jinyu , Patel Krusha V. , Hickenbotham Peter T. , Nale Janet Y. , Hargreaves Katherine R. , Clokie Martha R. J.
Published: June 2012

Prophages are encoded in most genomes of sequenced
Clostridium difficile
strains. They are key components of the mobile genetic elements and, as such, are likely to influence the biology of their host strains. The majority of these phages are not amenable to propagation, and therefore the development of a molecular marker is a useful tool with which to establish the extent and diversity of
C. difficile
prophage carriage within clinical strains. To design markers, several candidate genes were analyzed including structural and holin genes. The holin gene is the only gene present in all sequenced phage genomes, conserved at both terminals, with a variable mid-section. This allowed us to design two sets of degenerate PCR primers specific to
C. difficile
myoviruses and siphoviruses. Subsequent PCR analysis of 16 clinical
C. difficile
ribotypes showed that 15 of them are myovirus positive, and 2 of them are also siphovirus positive. Antibiotic induction and transmission electron microscope analysis confirmed the molecular prediction of myoviruses and/or siphovirus presence. Phylogenetic analysis of the holin sequences identified three groups of
C. difficile
phages, two within the myoviruses and a divergent siphovirus group. The marker also produced tight groups within temperate phages that infect other taxa, including
Clostridium perfringens
,
Clostridium botulinum
, and
Bacillus
spp., which suggests the potential application of the holin gene to study prophage carriage in other bacteria. This study reveals the high incidence of prophage carriage in clinically relevant strains of
C. difficile
and correlates the molecular data to the morphological observation.
Fratricide Is Essential for Efficient Gene Transfer between Pneumococci in Biofilms
Authors: Wei Hua , Håvarstein Leiv Sigve
Published: June 2012

Streptococcus pneumoniae
and a number of commensal streptococcal species are competent for natural genetic transformation. The natural habitat of these bacteria is multispecies biofilms in the human oral cavity and nasopharynx. Studies investigating lateral transfer of virulence and antibiotic resistance determinants among streptococci have shown that interspecies as well as intraspecies gene exchange takes place in these environments. We have previously shown that the action of a competence-specific murein hydrolase termed CbpD strongly increases the rate of gene transfer between pneumococci grown in liquid cultures. CbpD is the key component of a bacteriolytic mechanism termed the fratricide mechanism. It is secreted by competent pneumococci and mediates the release of donor DNA from sensitive streptococci present in the same environment. However, in nature, gene exchange between streptococci takes place in biofilms and not in liquid cultures. In the present study, we therefore investigated whether CbpD affects the rate of gene transfer in laboratory-grown biofilms. Our results show that the fratricide mechanism has a strong positive impact on intrabiofilm gene exchange, indicating that it is important for active acquisition of homologous donor DNA under natural conditions. Furthermore, we found that competent biofilm cells of
S. pneumoniae
acquire a Nov
r
marker much more efficiently from neighboring cells than from the growth medium. Efficient lysis of target cells requires that CbpD act in conjunction with the murein hydrolase LytC. In contrast, the major autolysin LytA does not seem to be important for fratricide-mediated gene exchange in a biofilm environment.
Archaeal Abundance across a pH Gradient in an Arable Soil and Its Relationship to Bacterial and Fungal Growth Rates
Authors: Bengtson Per , Sterngren Anna E. , Rousk Johannes
Published: June 2012

Soil pH is one of the most influential factors for the composition of bacterial and fungal communities, but the influence of soil pH on the distribution and composition of soil archaeal communities has yet to be systematically addressed. The primary aim of this study was to determine how total archaeal abundance (quantitative PCR [qPCR]-based estimates of 16S rRNA gene copy numbers) is related to soil pH across a pH gradient (pH 4.0 to 8.3). Secondarily, we wanted to assess how archaeal abundance related to bacterial and fungal growth rates across the same pH gradient. We identified two distinct and opposite effects of pH on the archaeal abundance. In the lowest pH range (pH 4.0 to 4.7), the abundance of archaea did not seem to correspond to pH. Above this pH range, there was a sharp, almost 4-fold decrease in archaeal abundance, reaching a minimum at pH 5.1 to 5.2. The low abundance of archaeal 16S rRNA gene copy numbers at this pH range then sharply increased almost 150-fold with pH, resulting in an increase in the ratio between archaeal and bacterial copy numbers from a minimum of 0.002 to more than 0.07 at pH 8. The nonuniform archaeal response to pH could reflect variation in the archaeal community composition along the gradient, with some archaea adapted to acidic conditions and others to neutral to slightly alkaline conditions. This suggestion is reinforced by observations of contrasting outcomes of the (competitive) interactions between archaea, bacteria, and fungi toward the lower and higher ends of the examined pH gradient.
Genotypic and Phenotypic Traits That Distinguish Neonatal Meningitis-Associated Escherichia coli from Fecal E. coli Isolates of Healthy Human Hosts
Authors: Logue Catherine M. , Doetkott Curt , Mangiamele Paul , Wannemuehler Yvonne M. , Johnson Timothy J. , Tivendale Kelly A. , Li Ganwu , Sherwood Julie S. , Nolan Lisa K.
Published: June 2012

Neonatal meningitis
Escherichia coli
(NMEC) is one of the top causes of neonatal meningitis worldwide. Here, 85 NMEC and 204 fecal
E. coli
isolates from healthy humans (HFEC) were compared for possession of traits related to virulence, antimicrobial resistance, and plasmid content. This comparison was done to identify traits that typify NMEC and distinguish it from commensal strains to refine the definition of the NMEC subpathotype, identify traits that might contribute to NMEC pathogenesis, and facilitate choices of NMEC strains for future study. A large number of
E. coli
strains from both groups were untypeable, with the most common serogroups occurring among NMEC being O18, followed by O83, O7, O12, and O1. NMEC strains were more likely than HFEC strains to be assigned to the B2 phylogenetic group. Few NMEC or HFEC strains were resistant to antimicrobials. Genes that best discriminated between NMEC and HFEC strains and that were present in more than 50% of NMEC isolates were mainly from extraintestinal pathogenic
E. coli
genomic and plasmid pathogenicity islands. Several of these defining traits had not previously been associated with NMEC pathogenesis, are of unknown function, and are plasmid located. Several genes that had been previously associated with NMEC virulence did not dominate among the NMEC isolates. These data suggest that there is much about NMEC virulence that is unknown and that there are pitfalls to studying single NMEC isolates to represent the entire subpathotype.
Differences in Virus Prevalence and Load in the Hearts of Patients with Idiopathic Dilated Cardiomyopathy with and without Immune-Mediated Inflammatory Diseases
Authors: Dennert Robert , van Paassen Pieter , Wolffs Petra , Bruggeman Catrien , Velthuis Sebastiaan , Felix Susanne , van Suylen Robert-Jan , Crijns Harry J. , Cohen Tervaert Jan Willem , Heymans Stephane
Published: June 2012

Infections with cardiotrophic viruses and immune-mediated responses against the heart have been suggested to play a dominant role in the pathogenesis of idiopathic dilated cardiomyopathy (DCM). Furthermore, immune-mediated inflammatory diseases (IMIDs) may result in DCM. It has not previously been assessed whether DCM patients with and without an IMID have different prevalences and quantities of cardiotrophic viruses in the heart. Therefore, we compared the profiles of cardiotrophic viruses in heart tissue of DCM patients with and without an IMID. Serum and myocardial tissue samples were obtained from 159 consecutive patients with DCM and 20 controls. Patients were subdivided into three groups, the first two based on the presence (n= 34) or absence (n= 125) of an IMID and the third being a control group. The parvovirus B19 virus genome was detected in equal quantities in the non-IMID DCM patients (100/125) and the control group (15/20) but in lower quantities in the IMID patients (21/34,P= 0.02). Both the non-IMID and IMID DCM patients demonstrated increased myocardial inflammation compared to controls: 12.5 ± 1.8 and 14.0 ± 3.2 CD45-positive inflammatory cells, respectively, versus 5.1 ± 0.7 for the controls (P< 0.05 for both). Importantly, significantly higher parvovirus B19 copy numbers could be amplified in non-IMID than in IMID patients (561 ± 97 versus 191 ± 92 copies/μg DNA,P< 0.001) and control subjects (103 ± 47 copies/μg DNA,P< 0.001). The present study shows decreased parvovirus B19 prevalence and copy numbers in hearts of DCM patients with an IMID compared to those without an IMID. These findings may suggest that DCM patients with an IMID have a different pathophysiologic mechanism from that which is present in the virus-induced form of DCM.
Peroxisomal and Mitochondrial β-Oxidation Pathways Influence the Virulence of the Pathogenic Fungus Cryptococcus neoformans
Authors: Kretschmer Matthias , Wang Joyce , Kronstad James W.
Published: June 2012

An understanding of the connections between metabolism and elaboration of virulence factors during host colonization by the human-pathogenic fungus
Cryptococcus neoformans
is important for developing antifungal therapies. Lipids are abundant in host tissues, and fungal pathogens in the phylum basidiomycota possess both peroxisomal and mitochondrial β-oxidation pathways to utilize this potential carbon source. In addition, lipids are important signaling molecules in both fungi and mammals. In this report, we demonstrate that defects in the peroxisomal and mitochondrial β-oxidation pathways influence the growth of
C. neoformans
on fatty acids as well as the virulence of the fungus in a mouse inhalation model of cryptococcosis. Disease attenuation may be due to the cumulative influence of altered carbon source acquisition or processing, interference with secretion, changes in cell wall integrity, and an observed defect in capsule production for the peroxisomal mutant. Altered capsule elaboration in the context of a β-oxidation defect was unexpected but is particularly important because this trait is a major virulence factor for
C. neoformans
. Additionally, analysis of mutants in the peroxisomal pathway revealed a growth-promoting activity for
C. neoformans
, and subsequent work identified oleic acid and biotin as candidates for such factors. Overall, this study reveals that β-oxidation influences virulence in
C. neoformans
by multiple mechanisms that likely include contributions to carbon source acquisition and virulence factor elaboration.
A Listeria monocytogenes RNA Helicase Essential for Growth and Ribosomal Maturation at Low Temperatures Uses Its C Terminus for Appropriate Interaction with the Ribosome
Authors: Netterling Sakura , Vaitkevicius Karolis , Nord Stefan , Johansson Jörgen
Published: June 2012

Listeria monocytogenes, a Gram-positive food-borne human pathogen, is able to grow at temperatures close to 0°C and is thus of great concern for the food industry. In this work, we investigated the physiological role of one DExD-box RNA helicase inListeria monocytogenes. The RNA helicase Lmo1722 was required for optimal growth at low temperatures, whereas it was dispensable at 37°C. A Δlmo1722strain was less motile due to downregulation of the major subunit of the flagellum, FlaA, caused by decreasedflaAexpression. By ribosomal fractionation experiments, it was observed that Lmo1722 was mainly associated with the 50S subunit of the ribosome. Absence of Lmo1722 decreased the fraction of 50S ribosomal subunits and mature 70S ribosomes and affected the processing of the 23S precursor rRNA. The ribosomal profile could be restored to wild-type levels in a Δlmo1722strain expressing Lmo1722. Interestingly, the C-terminal part of Lmo1722 was redundant for low-temperature growth, motility, 23S rRNA processing, and appropriate ribosomal maturation. However, Lmo1722 lacking the C terminus showed a reduced affinity for the 50S and 70S fractions, suggesting that the C terminus is important for proper guidance of Lmo1722 to the 50S subunit. Taken together, our results show that theListeriaRNA helicase Lmo1722 is essential for growth at low temperatures, motility, and rRNA processing and is important for ribosomal maturation, being associated mainly with the 50S subunit of the ribosome.