bluuebunny/crossref_metadata_2025 huggingface dataset:
A comparative study of ancient sedimentary DNA, pollen and macrofossils from permafrost sediments of northern Siberia reveals long‐term vegetational stability
Authors: JØRGENSEN TINA , HAILE JAMES , MÖLLER PER , ANDREEV ANDREI , BOESSENKOOL SANNE , RASMUSSEN MORTEN , KIENAST FRANK , COISSAC ERIC , TABERLET PIERRE , BROCHMANN CHRISTIAN , BIGELOW NANCY H. , ANDERSEN KENNETH , ORLANDO LUDOVIC , GILBERT M. THOMAS P. , WILLERSLEV ESKE
Published: October 2011

Although ancient DNA from sediments (sedaDNA) has been used to investigate past ecosystems, the approach has never been directly compared with the traditional methods of pollen and macrofossil analysis. We conducted a comparative survey of 18 ancient permafrost samples spanning the Late Pleistocene (46–12.5 thousand years ago), from the Taymyr Peninsula in northern Siberia. The results show that pollen, macrofossils and sedaDNA are complementary rather than overlapping and, in combination, reveal more detailed information on plant palaeocommunities than can be achieved by each individual approach. SedaDNA and macrofossils share greater overlap in plant identifications than with pollen, suggesting that sedaDNA is local in origin. These two proxies also permit identification to lower taxonomic levels than pollen, enabling investigation into temporal changes in species composition and the determination of indicator species to describe environmental changes. Combining data from all three proxies reveals an area continually dominated by a mosaic vegetation of tundra‐steppe, pioneer and wet‐indicator plants. Such vegetational stability is unexpected, given the severe climate changes taking place in the Northern Hemisphere during this time, with changes in average annual temperatures of >22 °C. This may explain the abundance of ice‐age mammals such as horse and bison in Taymyr Peninsula during the Pleistocene and why it acted as a refugium for the last mainland woolly mammoth. Our finding reveals the benefits of combining sedaDNA, pollen and macrofossil for palaeovegetational reconstruction and adds to the increasing evidence suggesting large areas of the Northern Hemisphere remained ecologically stable during the Late Pleistocene.
Environmental and physiological controls on the carbon isotope composition of CO2 respired by leaves and roots of a C3 woody legume (Prosopis velutina) and a C4 perennial grass (Sporobolus wrightii)
Authors: SUN WEI , RESCO VÍCTOR , WILLIAMS DAVID G.
Published: September 2011

Accurate estimates of the δ13C value of CO2 respired from roots (δ13CR_root) and leaves (δ13CR_leaf) are important for tracing and understanding changes in C fluxes at the ecosystem scale. Yet the mechanisms underlying temporal variation in these isotopic signals are not fully resolved. We measured δ13CR_leaf, δ13CR_root, and the δ13C values and concentrations of glucose and sucrose in leaves and roots in the C4 grass Sporobolus wrightii and the C3 tree Prosopis velutina in a savanna ecosystem in southeastern Arizona, USA. Night‐time variation in δ13CR_leaf of up to 4.6 ± 0.6‰ in S. wrightii and 3.0 ± 0.6‰ in P. velutina were correlated with shifts in leaf sucrose concentration, but not with changes in δ13C values of these respiratory substrates. Strong positive correlations between δ13CR_root and root glucose δ13C values in P. velutina suggest large diel changes in δ13CR_root (were up to 3.9‰) influenced by short‐term changes in δ13C of leaf‐derived phloem C. No diel variation in δ13CR_root was observed in S. wrightii. Our findings show that short‐term changes in δ13CR_leaf and δ13CR_root were both related to substrate isotope composition and concentration. Changes in substrate limitation or demand for biosynthesis may largely control short‐term variation in the δ13C of respired CO2 in these species.
Epidemiologic survey of patients with congenital central hypoventilation syndrome in Japan
Authors: Hasegawa Hisaya , Kawasaki Kazuteru , Inoue Hisashige , Umehara Minoru , Takase Masato ,   Japanese Society of Pediatric Pulmonology Working Group (JSPPWG)
Published: September 2011

Background:  Congenital central hypoventilation syndrome (CCHS) is a rare disease characterized by hypoventilation during sleep. This study discusses the first epidemiologic survey of patients with CCHS in Japan.

Methods:  The first survey was conducted between September and December 2006 and involved 507 registered institutes for pediatric training in Japan. The second survey was conducted between January and April 2007 and involved only those institutes that confirmed diagnosis of CCHS in the first survey or reported on CCHS at a conference during the preceding decade.

Results:  Thirty‐seven patients with CCHS were reported from 23 hospitals. Patient characteristics were as follows: 18 were male, 19 were female; and age range 4 months to 34 years. Diagnosis was based on clinical symptoms in 37/37 patients; blood gas analysis in 25/37; ventilatory response to inhaled CO2 in 14/37; and genetic analysis (paired‐like homeobox gene 2B) in 11/37. Complications included Hirschsprung's disease in 13/37 and central nervous system disorders in 15/37. Prognoses were as follows: 3/37 died in hospital, 1/37 remained in hospital, 33/37 were on home mechanical ventilation (died 4/33, survived 29/33), and 0/37 were cured. Ventilation methods included tracheostomy (21/37), use of a nasal mask (9/37), use of a facemask (5/37), and diaphragmatic pacing (1/37).

Conclusions:  There is currently no consensus on the most appropriate methods for diagnosing and treating patients with CCHS in Japan. More CCHS‐related data need to be collected in the near future in order to enable appropriate diagnosis and management of patients with CCHS.
Pilot study on reflectance confocal microscopy imaging of lichen planus: a real‐time, non‐invasive aid for clinical diagnosis
Authors: Moscarella E. , González S. , Agozzino M. , Sánchez‐Mateos J.L.S. , Panetta C. , Contaldo M. , Ardigò M.
Published: September 2011

Background  Lichen planus (LP) represents a relatively common skin inflammatory entity included in the major group of interface dermatitis. In recent years, reflectance confocal microscopy has demonstrated to be a valuable tool for the ‘in vivo’ characterization of various skin diseases with cellular level resolution. No data are currently available that uses reflectance confocal microscopy to study LP.

Observations  In this study, we have investigated the clinical and confocal features of five cases of histopathologically proven LP, and we have correlated the observed features with histopathological findings. The most characteristic criterion was the presence of interface dermatitis. Papillary rims, usually visible in normal skin, were obscured by the presence of a diffuse inflammatory cells infiltrate, arranged in sheet‐like structures that surrounded the junction almost completely. There was an almost total obliteration of the ring‐like structures around DP, which appeared non‐edged and non‐rimmed. Granular cells appeared as very large, polygonal structures, with an evident grainy cytoplasm, with the transition between spinous and granular cells being clearly recognizable, and this feature corresponded to hypergranulosis in histology. The presence of inflammatory cells at the level of the epidermis was seen as round‐to‐polygonal bright structures in the context of a variable degree of epidermal disarray and spongiosis. Melanophages in dermis were visible as brightly refractile, plump, oval to stellate‐shaped cells. Prominent round or linear dark canalicular structures corresponded to dilated blood vessels in the superficial dermis on histopathology and appeared horizontally oriented in confocal sections.

Conclusions  Reflectance confocal microscopy may represent a real‐time, non‐invasive aid to clinical diagnosis of LP. However, it might be difficult to distinguish between different subtypes of interface dermatitis. Further research, including larger case series, will better define a possible differential diagnosis of these diseases using confocal microscopy.
Do Internet Stock Message Boards Influence Trading? Evidence from Heavily Discussed Stocks with No Fundamental News
Authors: Sabherwal Sanjiv , Sarkar Salil K. , Zhang Ying
Published: September 2011

Abstract:  This study extends the literature on the information content of stock message boards. To better understand the effect of online postings on trading activities and reduce the error due to stocks with small message board followings, we examine stocks with no fundamental news and high message posting activity. Such stocks tend to be of small firms with weak financials. For those stocks, we find a two‐day pump followed by a two‐day dump manipulation pattern among online traders, which suggests that an online stock message board can be used as a herding device to temporarily drive up stock prices. We also find that online traders’ credit‐weighted sentiment index, but not the number of postings, is positively associated with contemporaneous return and negatively predicts the return next day and two days later. Also, absolute sentiment is negatively related with contemporaneous and next day's intraday volatility and positively related with the proportion of volume in small‐sized trades. We conclude that message board sentiment is an important predictor of trading‐related activities.
Palaeohistology and external microanatomy of rauisuchian osteoderms (Archosauria: Pseudosuchia)
Authors: SCHEYER TORSTEN M. , DESOJO JULIA B.
Published: September 2011

Abstract:  The presence of postcranial dermal armour is plesiomorphic for Archosauria. Here, we survey the external microanatomy and histology of postcranial osteoderms (i.e. dorsal paramedian and caudal osteoderms) of rauisuchians, a widely distributed assemblage of extinct predatory pseudosuchians from the Triassic. The osteoderms of eight rauisuchian taxa were found to be rather compact bones, which usually lack significant bone remodelling or large areas of cancellous bone. The presence of highly vascularized woven or fibrolamellar bone tissue deposited in the core areas indicates higher growth rates during earlier life stages, whereas a more compact parallel‐fibred bone matrix indicates reduced growth rates in later development. This pattern of change corroborates earlier studies on long bone histology. With the exception of a bone tissue found in the sample of Batrachotomus kupferzellensis, which might be the result of metaplastic ossification, the general mode of skeletogenesis is comparable with intramembraneous ossification. The lack of cancellous bone tissue and remodelling processes associated with bone ornamentation, as well as the predominantly intramembraneous mode of ossification, indicates that rauisuchian osteoderm formation differs profoundly from that of the osteoderms of the only extant pseudosuchian lineage, the crocodylians.
Computationally Efficient Marginal Models for Clustered Recurrent Event Data
Authors: Liu Dandan , Schaubel Douglas E. , Kalbfleisch John D.
Published: September 2011

Summary Large observational databases derived from disease registries and retrospective cohort studies have proven very useful for the study of health services utilization. However, the use of large databases may introduce computational difficulties, particularly when the event of interest is recurrent. In such settings, grouping the recurrent event data into prespecified intervals leads to a flexible event rate model and a data reduction that remedies the computational issues. We propose a possibly stratified marginal proportional rates model with a piecewise‐constant baseline event rate for recurrent event data. Both the absence and the presence of a terminal event are considered. Large‐sample distributions are derived for the proposed estimators. Simulation studies are conducted under various data configurations, including settings in which the model is misspecified. Guidelines for interval selection are provided and assessed using numerical studies. We then show that the proposed procedures can be carried out using standard statistical software (e.g., SAS, R). An application based on national hospitalization data for end‐stage renal disease patients is provided.
Dynamin A, Myosin IB and Abp1 Couple Phagosome Maturation to F‐Actin Binding
Authors: Gopaldass Navin , Patel Devang , Kratzke Ramona , Dieckmann Regis , Hausherr Stéphanie , Hagedorn Monica , Monroy Roger , Krüger Julia , Neuhaus Eva M. , Hoffmann Eik , Hille Katja , Kuznetsov Sergei A. , Soldati Thierry
Published: October 2011

The role of actin, class I myosins and dynamin in endocytic uptake processes is well characterized, but their role during endo‐phagosomal membrane trafficking and maturation is less clear. In Dictyostelium, knockout of myosin IB (myoB) leads to a defect in membrane protein recycling from endosomes back to the plasma membrane. Here, we show that actin plays a central role in the morphology and function of the endocytic pathway. Indeed, latrunculin B (LatB) induces endosome tubulation, a phenotype also observed in dynamin A (dymA)‐null cells. Knockout of dymA impairs phagosome acidification, whereas knockout of myoB delays reneutralization, a phenotype mimicked by a low dose of LatB. As a read out for actin‐dependent processes during maturation, we monitored the capacity of purified phagosomes to bind F‐actin in vitro, and correlated this with the presence of actin‐binding and membrane‐trafficking proteins. Phagosomes isolated from myoB‐null cells showed an increased binding to F‐actin, especially late phagosomes. In contrast, early phagosomes from dymA‐null cells showed reduced binding to F‐actin while late phagosomes were unaffected. We provide evidence that Abp1 is the main F‐actin‐binding protein in this assay and is central for the interplay between DymA and MyoB during phagosome maturation.
Introduction and evaluation of computer‐assisted education in an undergraduate dental hygiene course
Authors: Yoshida N , Aso T , Asaga T , Okawa Y , Sakamaki H , Masumoto T , Matsui K , Kinoshita A
Published: October 2011

To cite this article:
 
Int J Dent Hygiene
10, 2012; 61–66  DOI: 10.1111/j.1601‐5037.2011.00528.x  Yoshida N, Aso T, Asaga T, Okawa Y, Sakamaki H, Masumoto T, Matsui, K, Kinoshita A. Introduction and evaluation of computer‐assisted education in an undergraduate dental hygiene

Abstract:  Objective:  This paper introduced newly developed computer‐assisted learning materials and reports of a survey of junior college dental hygiene students who have used them. Methods:  We authored new educational material to promote students’ basic dental hygiene practice skills using a simulation software generator. A set of five developed materials were tested by 43 female second‐year dental hygiene students during the second semester at a college in Chiba, Japan. The evaluation was conducted in the form of a questionnaire including open‐ended questions. Students’ opinions were analysed using characteristic diagrams, a troubleshooting tool that can be used to visually illustrate the causes and effects of a problem. Result:  The overall results of the evaluation were positive. The students were given five sets of simulation learning materials (SLMs). Eighty‐three percent of the students felt that they could carry out independent study of clinical practice better after the virtual practice. Ninety‐three percent of them felt that the exercises should be continued in the future, and eighty‐eight percent of them felt that this virtual practice deepened their interest in other classes and training sessions. All of the students found the virtual practice beneficial for their learning. Discussion:  The present results suggest that the students became conscious of their lack of knowledge through SLMs. These findings indicate that SLMs for practicing basic clinical procedures is beneficial.
Extensional and Transversal Wave Motion in Transversely Isotropic Thermoelastic Plates by Using Asymptotic Method
Authors: Sharma J. N. , Sharma P. K. , Rana S. K.
Published: October 2011

The present investigation is concerned with the study of extensional and transversal wave motions in an infinite homogenous transversely isotropic, thermoelastic plate by using asymptotic method in the context of coupled thermoelasticity, Lord and Shulman (1967, “The Generalized Dynamical Theory of Thermoelasticity,” J. Mech. Phys. Solids, 15, pp. 299–309), and Green and Lindsay (1972, “Thermoelasticity,” J. Elast., 2, pp. 1–7) theories of generalized thermoelasticity. The governing equations for extensional, transversal, and flexural motions have been derived from the system of three-dimensional dynamical equations of linear thermoelasticity. The asymptotic operator plate model for extensional motion in a homogeneous transversely isotropic thermoelastic plate leads to sixth degree polynomial secular equation that governs frequency and phase velocity of various possible modes of wave propagation at all wavelengths. It is shown that the purely transverse motion (SH mode), which is not affected by thermal variations, gets decoupled from rest of the motion. The Rayleigh–Lamb frequency equation for the plate is expanded in power series in order to obtain polynomial frequency equation and velocity dispersion relations. Their validation has been established with that of asymptotic method. The special cases of short and long wavelength waves are also discussed. The expressions for group velocity of extensional and transversal modes have been derived. Finally, the numerical solution is carried out for homogeneous transversely isotropic plate of single crystal of zinc material. The dispersion curves of phase velocity and attenuation coefficient are presented graphically.
A Note on the Co-linearity of Forces and Displacements in an Elastic Structure
Authors: Gradin P. A. , Isaksson P. , Gulliksson M.
Published: October 2011

The conditions under which force vectors and corresponding displacement vectors become co-linear are investigated under the assumption of a linear elastic structure and for an arbitrary number of loading points. It is shown that there exist an infinite number of directions along which the load and displacement vectors in each loading point coincide. Moreover, the problem of co-linearity is analogous to the problem of finding the extreme values of the work performed on an elastic structure under the constraint that each force has a given magnitude. The result for a finite number of loading points is extended to a continuous load distribution on the boundary of an elastic structure, i.e., it is possible to find an infinite number of load distributions such that the displacement in a point on the boundary is co-linear with the boundary stress vector in that same point.
Exact Analysis of Axisymmetric Dynamic Response of Functionally Graded Cylinders (or Disks) and Spheres
Authors: Keles I. , Tutuncu N.
Published: October 2011

Free and forced vibration analyses of functionally graded hollow cylinders and spheres are performed and analytical benchmark solutions are presented. The material is assumed to be graded in the radial direction according to a simple power law. The Laplace transform method is used, and the inversion into the time domain is performed exactly using calculus of residues. The Complex Laplace parameter in the free vibration equation has directly given natural frequencies, and the results are given in tabular form. On the inner surface, various axisymmetric dynamic pressures are applied, and radial displacement and hoop stress are presented in the form of graphs. The exponent in the power law, called the inhomogeneity parameter, essentially refers to the degree of inhomogeneity. Increasing the inhomogeneity parameter provides a stress-shielding effect. Closed-form solutions obtained in the present paper are tractable, and they allow for further parametric studies. The inhomogeneity constant is a useful parameter from a design point of view in that it can be tailored for specific applications to control the stress distribution.
Three-Dimensional Underwater Shock Response of Composite Marine Structures
Authors: Motley Michael R. , Young Yin L. , Liu Zhanke
Published: October 2011

In recent years, there has been an increased interest in the use of advanced composites in marine applications. It has been shown that by exploiting the inherent anisotropic nature of the material, fiber-reinforced composite structures can be tailored to allow automatic, passive, three-dimensional (3D) adaptive/morphing capabilities such that they outperform their rigid counterparts both hydrodynamically and structurally. Much of the current research on the shock response of composite structures focuses on air-backed structures with fixed-fixed or simply supported boundary conditions. Nevertheless, many critical components of marine structures where adaptive/morphing capabilities are needed are cantilevered-type structures, including propeller and turbine blades, hydrofoils, and rudders. This paper investigates the 3D transient response of cantilevered, anisotropic, composite marine structures, namely, fully submerged cantilevered plates, subject to a range of shock loads. Structural responses and the initial failure loads of a composite plate are compared with a nickel-aluminum-bronze plate. Discussions of the fluid and structural responses of both materials are presented, and the initial failure loads of both materials are compared.
Combination of characterization techniques for atomic layer deposition MoO3 coatings: From the amorphous to the orthorhombic α-MoO3 crystalline phase
Authors: Diskus Madeleine , Nilsen Ola , Fjellvåg Helmer , Diplas Spyros , Beato Pablo , Harvey Clare , van Schrojenstein Lantman Evelien , Weckhuysen Bert M.
Published: September 2011

Thin films of MoO3 deposited on Si(111) and Al2O3(001) substrates by atomic layer deposition have been investigated by x-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and Raman spectroscopy for detailed characterization of composition and morphology. Comparison of angle resolved x-ray photoelectron spectroscopy (ARXPS) and XPS depth profiles based on Ar+ sputtering is reported. Sputtering induces a reduction of molybdenum in MoO3 from +IV to metallic Mo as the interface toward Si is approached, whereas ARXPS on a 10 nm thin film shows that Mo(VI) remains outside the interface toward Si where lower valent molybdenum compounds are formed. Upon annealing, the as-deposited amorphous thin films of MoO3 crystallize into β- or α-MoO3 as identified by x-ray diffraction. The current study provides a convenient route toward formation of metastable β-MoO3 and a full crystallization pathway from amorphous to crystalline α-MoO3. Combined AFM and Raman analysis have been performed on thin films of α-MoO3 deposited on Al2O3(001) and prove that the crystallization proceeds via island growth at 600 °C. The Raman intensity ratios between different bands depend strongly on morphology and size of crystalites.
Induction of Viable but Nonculturable Escherichia coli O157:H7 in the Phyllosphere of Lettuce: a Food Safety Risk Factor
Authors: Dinu Laura-Dorina , Bach Susan
Published: October 2011

Escherichia coli
O157:H7 continues to be an important human pathogen and has been increasingly linked to food-borne illness associated with fresh produce, particularly leafy greens. The aim of this work was to investigate the fate of
E. coli
O157:H7 on the phyllosphere of lettuce under low temperature and to evaluate the potential hazard of viable but nonculturable (VBNC) cells induced under such stressful conditions. First, we studied the survival of six bacterial strains following prolonged storage in water at low temperature (4ï¿½C) and selected two strains with different nonculturable responses for the construction of
E. coli
O157:H7 Tn
7gfp
transformants in order to quantitatively assess the occurrence of human pathogens on the plant surface. Under a suboptimal growth temperature (16ï¿½C), both
E. coli
O157:H7 strains maintained culturability on lettuce leaves, but under more stressful conditions (8ï¿½C), the bacterial populations evolved toward the VBNC state. The strain-dependent nonculturable response was more evident in the experiments with different inoculum doses (10
9
and 10
6
E. coli
O157:H7 bacteria per g of leaf) when strain BRMSID 188 lost culturability after 15 days and strain ATCC 43895 lost culturability within 7 days, regardless of the inoculum dose. However, the number of cells entering the VBNC state in high-cell-density inoculum (approximately 55%) was lower than in low-cell-density inoculum (approximately 70%). We recorded the presence of verotoxin for 3 days in samples that contained a VBNC population of 4 to 5 log
10
cells but did not detect culturable cells. These findings indicate that
E. coli
O157:H7 VBNC cells are induced on lettuce plants, and this may have implications regarding food safety.
Hemolytic Porcine Intestinal Escherichia coli without Virulence-Associated Genes Typical of Intestinal Pathogenic E. coli
Authors: Schierack Peter , Weinreich Joerg , Ewers Christa , Tachu Babila , Nicholson Bryon , Barth Stefanie
Published: October 2011

Testing 1,666 fecal or intestinal samples from healthy and diarrheic pigs, we obtained hemolytic
Escherichia coli
isolates from 593 samples. Focusing on hemolytic
E. coli
isolates without virulence-associated genes (VAGs) typical for enteropathogens, we found that such isolates carried a broad variety of VAGs typical for extraintestinal pathogenic
E. coli
.
Mother-to-Child Transmission of and Multiple-Strain Colonization by Bacteroides fragilis in a Cohort of Mothers and Their Children
Authors: Bjerke G. A. , Wilson R. , Storr� O. , �yen T. , Johnsen R. , Rudi K.
Published: October 2011

Bacteroides fragilis
represents an early infant colonizer with important host interactions. Our knowledge about the diversity, transmission, and persistence of this bacterium, however, is limited. Here, we addressed these questions using a combination of multilocus sequence typing (MLST) and variable-number tandem repeat (VNTR) sequence analyses. We used both culture-dependent and -independent typing. We genotyped
B. fragilis
in fecal samples from a cohort of 93 mothers and their children, with samples taken from the mothers and from the children at the ages 1 to 10 days, 4 months, 1 year, and 2 years. By MLST we found two main
B. fragilis
groups, which we denoted clades A and B. Direct typing of stool samples using the
icd
gene revealed seven sequence types, five within clade A and two within clade B. A single clade A sequence type, however, represented 79% of all the sequences. This sequence type was further subtyped using VNTR. VNTR subtyping revealed 16 different VNTR types. Based on the distribution patterns of these, we show mother-to-child transmission and multiple-strain colonization. We argue that negative host selection promotes the coexistence of multiple strains. The significance of our findings is that we have started unraveling the transmission and persistence patterns of one of the most important human gut colonizers.
DNA Detection and Genotypic Identification of Potentially Human-Pathogenic Microsporidia from Asymptomatic Pet Parrots in South Korea as a Risk Factor for Zoonotic Emergence
Authors: Lee So-Young , Lee Sung-Seok , Lyoo Young S. , Park Hee-Myung
Published: October 2011

We detected and identified genotypes of human-pathogenic microsporidia in fecal samples from 51 asymptomatic captive-bred pet parrots in South Korea. Microsporidia were identified in 8 samples (15.7%); 7 parrots tested positive for
Encephalitozoon hellem
, and 1 parrot tested positive for both
E. hellem
and
Encephalitozoon cuniculi
. In genotypic identifications,
E. hellem
was present in genotypes 1A and 2B and
E. cuniculi
was present in genotype II. Pet parrots might be a source of human microsporidian infection.
National Validation Study of a Cellulose Sponge Wipe-Processing Method for Use after Sampling Bacillus anthracis Spores from Surfaces
Authors: Rose Laura J. , Hodges Lisa , O'Connell Heather , Noble-Wang Judith
Published: October 2011

This work was initiated to address the gaps identified by Congress regarding validated biothreat environmental sampling and processing methods. Nine Laboratory Response Network-affiliated laboratories participated in a validation study of a cellulose sponge wipe-processing protocol for the recovery, detection, and quantification of viable
Bacillus anthracis
Sterne spores from steel surfaces. Steel coupons (645.16 cm
2
) were inoculated with 1 to 4 log
10
spores and then sampled with cellulose sponges (Sponge-Stick; 3M, St. Paul, MN). Surrogate dust and background organisms were added to the sponges to mimic environmental conditions. Labs processed the sponges according to the provided protocol. Sensitivity, specificity, and mean percent recovery (%R), between-lab variability, within-lab variability, and total percent coefficient of variation were calculated. The mean %R (standard error) of spores from the surface was 32.4 (4.4), 24.4 (2.8), and 30.1 (2.3) for the 1-, 2-, and 4-log
10
inoculum levels, respectively. Sensitivities for colony counts were 84.1%, 100%, and 100% for the 1-, 2-, and 4-log
10
inocula, respectively. These data help to characterize the variability of the processing method and thereby enhance confidence in the interpretation of the results of environmental sampling conducted during a
B. anthracis
contamination investigation.
Pyruvate and Lactate Metabolism by Shewanella oneidensis MR-1 under Fermentation, Oxygen Limitation, and Fumarate Respiration Conditions
Authors: Pinchuk Grigoriy E. , Geydebrekht Oleg V. , Hill Eric A. , Reed Jennifer L. , Konopka Allan E. , Beliaev Alexander S. , Fredrickson Jim K.
Published: October 2011

Shewanella oneidensis
MR-1 is a facultative anaerobe that derives energy by coupling organic matter oxidation to the reduction of a wide range of electron acceptors. Here, we quantitatively assessed the lactate and pyruvate metabolism of MR-1 under three distinct conditions: electron acceptor-limited growth on lactate with O
2
, lactate with fumarate, and pyruvate fermentation. The latter does not support growth but provides energy for cell survival. Using physiological and genetic approaches combined with flux balance analysis, we showed that the proportion of ATP produced by substrate-level phosphorylation varied from 33% to 72.5% of that needed for growth depending on the electron acceptor nature and availability. While being indispensable for growth, the respiration of fumarate does not contribute significantly to ATP generation and likely serves to remove formate, a product of pyruvate formate-lyase-catalyzed pyruvate disproportionation. Under both tested respiratory conditions,
S. oneidensis
MR-1 carried out incomplete substrate oxidation, whereby the tricarboxylic acid (TCA) cycle did not contribute significantly. Pyruvate dehydrogenase was not involved in lactate metabolism under conditions of O
2
limitation but was required for anaerobic growth, likely by supplying reducing equivalents for biosynthesis. The results suggest that pyruvate fermentation by
S. oneidensis
MR-1 cells represents a combination of substrate-level phosphorylation and respiration, where pyruvate serves as an electron donor and an electron acceptor. Pyruvate reduction to lactate at the expense of formate oxidation is catalyzed by a recently described new type of oxidative NAD(P)H-independent
d
-lactate dehydrogenase (Dld-II). The results further indicate that pyruvate reduction coupled to formate oxidation may be accompanied by the generation of proton motive force.
Endo-β-1,3-Glucanase GLU1, from the Fruiting Body of Lentinula edodes, Belongs to a New Glycoside Hydrolase Family
Authors: Sakamoto Yuichi , Nakade Keiko , Konno Naotake
Published: October 2011

The cell wall of the fruiting body of the mushroom
Lentinula edodes
is degraded after harvesting by enzymes such as β-1,3-glucanase. In this study, a novel endo-type β-1,3-glucanase, GLU1, was purified from
L. edodes
fruiting bodies after harvesting. The gene encoding it,
glu1
, was isolated by rapid amplification of cDNA ends (RACE)-PCR using primers designed from the N-terminal amino acid sequence of GLU1. The putative amino acid sequence of the mature protein contained 247 amino acid residues with a molecular mass of 26 kDa and a pI of 3.87, and recombinant GLU1 expressed in
Pichia pastoris
exhibited β-1,3-glucanase activity. GLU1 catalyzed depolymerization of glucans composed of β-1,3-linked main chains, and reaction product analysis by thin-layer chromatography (TLC) clearly indicated that the enzyme had an endolytic mode. However, the amino acid sequence of GLU1 showed no significant similarity to known glycoside hydrolases. GLU1 has similarity to several hypothetical proteins in fungi, and GLU1 and highly similar proteins should be classified as a novel glycoside hydrolase family (GH128).
Subtilase Cytotoxin-Coding Genes in Verotoxin-Producing Escherichia coli Strains from Sheep and Goats Differ from Those from Cattle
Authors: Orden Josï¿½ A. , Horcajo Pilar , de la Fuente Ricardo , Ruiz-Santa-Quiteria Josï¿½ A. , Domï¿½nguez-Bernal Gustavo , Carriï¿½n Javier
Published: October 2011

Subtilase cytotoxin (SubAB) from verotoxin (VT)-producing
Escherichia coli
(VTEC) strains was first described in the 98NK2 strain and has been associated with human disease. However, SubAB has recently been found in two VT-negative
E. coli
strains (ED 591 and ED 32). SubAB is encoded by two closely linked, cotranscribed genes (
subA
and
subB
). In this study, we investigated the presence of
subAB
genes in 52 VTEC strains isolated from cattle and 209 strains from small ruminants, using PCR. Most (91.9%) VTEC strains from sheep and goats and 25% of the strains from healthy cattle possessed
subAB
genes. The presence of
subAB
in a high percentage of the VTEC strains from small ruminants might increase the pathogenicity of these strains for human beings. Some differences in the results of PCRs and in the association with some virulence genes suggested the existence of different variants of
subAB
. We therefore sequenced the
subA
gene in 12 strains and showed that the
subA
gene in most of the
subAB
-positive VTEC strains from cattle was almost identical (about 99%) to that in the 98NK2 strain, while the
subA
gene in most of the
subAB
-positive VTEC strains from small ruminants was almost identical to that in the ED 591 strain. We propose the terms
subAB1
to describe the SubAB-coding genes resembling that in the 98NK2 strain and
subAB2
to describe those resembling that in the ED 591 strain.
Development and Characterization of a Xylose-Inducible Gene Expression System for Clostridium perfringens
Authors: Nariya Hirofumi , Miyata Shigeru , Kuwahara Tomomi , Okabe Akinobu
Published: October 2011

A xylose-inducible gene expression vector for
Clostridium perfringens
was developed. Plasmid pXCH contains a chromosomal region from
Clostridium difficile
(
xylR
-P

xy

lB

):
xylR
, encoding the xylose repressor,
xylO
, the
xyl
operator sequence, and P

xylB

, the divergent promoter upstream of
xylBA
encoding xylulo kinase and xylose isomerase. pXCH allows tightly regulated expression of the chloramphenicol acetyltransferase reporter and the α-toxin genes in response to the inducer concentration. Thus, pXCH could constitute a new valuable genetic tool for study of
C. perfringens
.
Field-Scale Transplantation Experiment To Investigate Structures of Soil Bacterial Communities at Pioneering Sites
Authors: Lazzaro Anna , Gauer Andreas , Zeyer Josef
Published: October 2011

Studies on the effect of environmental conditions on plants and microorganisms are a central issue in ecology, and they require an adequate experimental setup. A strategy often applied in geobotanical studies is based on the reciprocal transplantation of plant species at different sites. We adopted a similar approach as a field-based tool to investigate the relationships of soil bacterial communities with the environment. Soil samples from two different (calcareous and siliceous) unvegetated glacier forefields were reciprocally transplanted and incubated for 15 months between 2009 and 2010. Controls containing local soils were included. The sites were characterized over time in terms of geographical (bedrock, exposition, sunlight, temperature, and precipitation) and physicochemical (texture, water content, soluble and nutrients) features. The incubating local (“home”) and transplanted (“away”) soils were monitored for changes in extractable nutrients and in the bacterial community structure, defined through terminal restriction fragment length polymorphism (T-RFLP) of the 16S rRNA gene. Concentrations of soluble ions in most samples were more significantly affected by seasons than by the transplantation. For example, NO
3
−
showed a seasonal pattern, increasing from 1 to 3 μg NO
3
−
(g soil dry weight)
−1
after the melting of snow but decreasing to <1 μg NO
3
−
(g soil dry weight)
−1
in autumn. Seasons, and in particular strong precipitation events occurring in the summer of 2010 (200 to 300 mm of rain monthly), were also related to changes of bacterial community structures. Our results show the suitability of this approach to compare responses of bacterial communities to different environmental conditions directly in the field.
Mutations Suppressing the Loss of DegQ Function in Bacillus subtilis (natto) Poly-γ-Glutamate Synthesis
Authors: Do Thi-Huyen , Suzuki Yuki , Abe Naoki , Kaneko Jun , Itoh Yoshifumi , Kimura Keitarou
Published: October 2011

The
degQ
gene of
Bacillus subtilis
(natto), encoding a small peptide of 46 amino acids, is essential for the synthesis of extracellular poly-gamma-glutamate (γPGA). To elucidate the role of DegQ in γPGA synthesis, we knocked out the
degQ
gene in
Bacillus subtilis
(natto) and screened for suppressor mutations that restored γPGA synthesis in the absence of DegQ. Suppressor mutations were found in
degS
, the receptor kinase gene of the DegS-DegU two-component system. Recombinant DegS-His
6
mutant proteins were expressed in
Escherichia coli
cells and subjected to an
in vitro
phosphorylation assay. Compared with the wild type, mutant DegS-His
6
proteins showed higher levels of autophosphorylation (R208Q, M195I, L248F, and D250N), reduced autodephosphorylation (D250N), reduced phosphatase activity toward DegU, or a reduced ability to stimulate the autodephosphorylation activity of DegU (R208Q, D249G, M195I, L248F, and D250N) and stabilized DegU in the phosphorylated form. These mutant DegS proteins mimic the effect of DegQ on wild-type DegSU
in vitro
. Interestingly, DegQ stabilizes phosphorylated DegS only in the presence of DegU, indicating a complex interaction of these three proteins.
Use of a DNA Microarray for Detection and Identification of Bacterial Pathogens Associated with Fishery Products
Authors: Cao Boyang , Li Rongrong , Xiong Songjin , Yao Fangfang , Liu Xiangqian , Wang Min , Feng Lu , Wang Lei
Published: October 2011

We established a microarray for the simultaneous detection and identification of diverse putative pathogens often associated with fishery products by targeting specific genes of
Listeria monocytogenes
,
Salmonella
,
Shigella
,
Staphylococcus aureus
,
Streptococcus pyogenes
,
Vibrio cholerae
,
Vibrio parahaemolyticus
,
Vibrio vulnificus
, and
Yersinia enterocolitica
and the 16S-23S rRNA gene internal transcribed spacer (ITS) region of
Proteus mirabilis
and
Proteus vulgaris
. The microarray contained 26 specific probes and was tested against a total of 123 target bacterial strains that included 55 representative strains, 68 clinical isolates, and 45 strains of other bacterial species that belonged to 8 genera and 34 species, and it was shown to be specific and reproducible. A detection sensitivity of 10 ng DNA or 10 CFU/ml for pure cultures of each target organism demonstrated that the assay was highly sensitive and reproducible. Mock and real fishery product samples were tested by the microarray, and the accuracy was 100%. The DNA microarray method described in this communication is specific, sensitive, and reliable and has several advantages over traditional methods of bacterial culture and antiserum agglutination assays.
Competitive Selection of Lactic Acid Bacteria That Persist in the Human Oral Cavity
Authors: Snel Johannes , Marco Maria L. , Kingma Fedde , Noordman Wouter M. , Rademaker Jan , Kleerebezem Michiel
Published: October 2011

Lactic acid bacteria (LAB) might offer opportunities as oral probiotics provided candidate strains persist in the mouth. After intake of a mixture of 69 LAB, strains of
Lactobacillus fermentum
and
Lactobacillus salivarius
were especially recovered. Coaggregation with other microbes is likely not a prerequisite for persistence since
L. salivarius
strongly coaggregated with typical oral cavity isolates, whereas
L. fermentum
failed to display this phenotype.
Significance of Microbial Communities and Interactions in Safeguarding Reactive Mine Tailings by Ecological Engineering
Authors: N̆ancucheo Ivan , Johnson D. Barrie
Published: October 2011

Pyritic mine tailings (mineral waste generated by metal mining) pose significant risk to the environment as point sources of acidic, metal-rich effluents (acid mine drainage [AMD]). While the accelerated oxidative dissolution of pyrite and other sulfide minerals in tailings by acidophilic chemolithotrophic prokaryotes has been widely reported, other acidophiles (heterotrophic bacteria that catalyze the dissimilatory reduction of iron and sulfur) can reverse the reactions involved in AMD genesis, and these have been implicated in the “natural attenuation” of mine waters. We have investigated whether by manipulating microbial communities in tailings (inoculating with iron- and sulfur-reducing acidophilic bacteria and phototrophic acidophilic microalgae) it is possible to mitigate the impact of the acid-generating and metal-mobilizing chemolithotrophic prokaryotes that are indigenous to tailing deposits. Sixty tailings mesocosms were set up, using five different microbial inoculation variants, and analyzed at regular intervals for changes in physicochemical and microbiological parameters for up to 1 year. Differences between treatment protocols were most apparent between tailings that had been inoculated with acidophilic algae in addition to aerobic and anaerobic heterotrophic bacteria and those that had been inoculated with only pyrite-oxidizing chemolithotrophs; these differences included higher pH values, lower redox potentials, and smaller concentrations of soluble copper and zinc. The results suggest that empirical ecological engineering of tailing lagoons to promote the growth and activities of iron- and sulfate-reducing bacteria could minimize their risk of AMD production and that the heterotrophic populations could be sustained by facilitating the growth of microalgae to provide continuous inputs of organic carbon.
Differential Degradation of Bicyclics with Aromatic and Alicyclic Rings by Rhodococcus sp. Strain DK17
Authors: Kim Dockyu , Yoo Miyoun , Choi Ki Young , Kang Beom Sik , Kim Tai Kyoung , Hong Soon Gyu , Zylstra Gerben J. , Kim Eungbin
Published: October 2011

The metabolically versatile
Rhodococcus
sp. strain DK17 is able to grow on tetralin and indan but cannot use their respective desaturated counterparts, 1,2-dihydronaphthalene and indene, as sole carbon and energy sources. Metabolite analyses by gas chromatography-mass spectrometry and nuclear magnetic resonance spectrometry clearly show that (i) the
meta
-cleavage dioxygenase mutant strain DK180 accumulates 5,6,7,8-tetrahydro-1,2-naphthalene diol, 1,2-indene diol, and 3,4-dihydro-naphthalene-1,2-diol from tetralin, indene, and 1,2-dihydronaphthalene, respectively, and (ii) when expressed in
Escherichia coli
, the DK17
o
-xylene dioxygenase transforms tetralin, indene, and 1,2-dihydronaphthalene into tetralin
cis
-dihydrodiol, indan-1,2-diol, and
cis
-1,2-dihydroxy-1,2,3,4-tetrahydronaphthalene, respectively. Tetralin, which is activated by aromatic hydroxylation, is degraded successfully via the ring cleavage pathway to support growth of DK17. Indene and 1,2-dihydronaphthalene do not serve as growth substrates because DK17 hydroxylates them on the alicyclic ring and further metabolism results in a dead-end metabolite. This study reveals that aromatic hydroxylation is a prerequisite for proper degradation of bicyclics with aromatic and alicyclic rings by DK17 and confirms the unique ability of the DK17
o
-xylene dioxygenase to perform distinct regioselective hydroxylations.
Impact of the N-Terminal Secretor Domain on YopD Translocator Function in Yersinia pseudotuberculosis Type III Secretion
Authors: Amer Ayad A. A. , Åhlund Monika K. , Bröms Jeanette E. , Forsberg Åke , Francis Matthew S.
Published: October 2011

Type III secretion systems (T3SSs) secrete needle components, pore-forming translocators, and the translocated effectors. In part, effector recognition by a T3SS involves their N-terminal amino acids and their 5′ mRNA. To investigate whether similar molecular constraints influence translocator secretion, we scrutinized this region within YopD from
Yersinia pseudotuberculosis
. Mutations in the 5′ end of
yopD
that resulted in specific disruption of the mRNA sequence did not affect YopD secretion. On the other hand, a few mutations affecting the protein sequence reduced secretion. Translational reporter fusions identified the first five codons as a minimal N-terminal secretion signal and also indicated that the YopD N terminus might be important for
yopD
translation control. Hybrid proteins in which the N terminus of YopD was exchanged with the equivalent region of the YopE effector or the YopB translocator were also constructed. While the
in vitro
secretion profile was unaltered, these modified bacteria were all compromised with respect to T3SS activity in the presence of immune cells. Thus, the YopD N terminus does harbor a secretion signal that may also incorporate mechanisms of
yopD
translation control. This signal tolerates a high degree of variation while still maintaining secretion competence suggestive of inherent structural peculiarities that make it distinct from secretion signals of other T3SS substrates.
Interplay among Cyclic Diguanylate, HapR, and the General Stress Response Regulator (RpoS) in the Regulation of Vibrio cholerae Hemagglutinin/Protease
Authors: Wang Hongxia , Wu Jian-He , Ayala Julio C. , Benitez Jorge A. , Silva Anisia J.
Published: October 2011

Vibrio cholerae
secretes the Zn-dependent metalloprotease hemagglutinin (HA)/protease (mucinase), which is encoded by
hapA
and displays a broad range of potential pathogenic activities. Expression of HA/protease has a stringent requirement for the quorum-sensing regulator HapR and the general stress response regulator RpoS. Here we report that the second messenger cyclic diguanylic acid (c-di-GMP) regulates the production of HA/protease in a negative manner. Overexpression of a diguanylate cyclase to increase the cellular c-di-GMP pool resulted in diminished expression of HA/protease and its positive regulator, HapR. The effect of c-di-GMP on HapR was independent of LuxO but was abolished by deletion of the c-di-GMP binding protein VpsT, the LuxR-type regulator VqmA, or a single-base mutation in the
hapR
promoter that prevents autorepression. Though expression of HapR had a positive effect on RpoS biosynthesis, direct manipulation of the c-di-GMP pool at a high cell density did not significantly impact RpoS expression in the wild-type genetic background. In contrast, increasing the c-di-GMP pool severely inhibited RpoS expression in a Δ
hapR
mutant that is locked in a regulatory state mimicking low cell density. Based on the above findings, we propose a model for the interplay between HapR, RpoS, and c-di-GMP in the regulation of HA/protease expression.