bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhanced detection of hepatitis B virus in Hong Kong blood donors after introduction of a more sensitive transcription‐mediated amplification assay
Authors: Tsoi Wai‐Chiu , Lelie Nico , Lin Che‐Kit
Published: March 2013

Background

A total of 517,072 and 399,326 consecutive donations were screened for hepatitis B virus (HBV) by individual‐donation nucleic acid testing (ID‐NAT) using Ultrio and Ultrio Plus assays (Novartis Diagnostics), respectively. The impact of more sensitive HBV detection by the latter assay version was established by comparing NAT yield and transmission risk.

Study Design and Methods

Donations were screened simultaneously for HBV serologic markers and ID‐NAT, followed by discriminatory assay and confirmatory test algorithms. Window period (WP) reduction and residual HBV transmission risk were computed using mathematical modeling.

Results

HBV NAT‐yield rates for both WP and occult HBV infection (OBI) increased significantly from 1:34,471 to 1:17,362 (p = 0.036) and from 1:5120 to 1:2450 (p < 0.0001), despite a 1.2‐ and 1.6‐fold decrease in hepatitis B surface antigen (HBsAg) incidence and prevalence rates respectively. After adjusting for this bias, the WP and OBI NAT‐yield improvement factors were 2.3 and 3.4, respectively, higher than a less than 1.5‐fold increase estimated from analytical sensitivity studies on HBV Genotype A and C standards. The current WP transmission risk with Ultrio Plus screening was estimated at 1:55,000 compared to 1:22,000 with HBsAg testing.

Conclusion

The observed greater than twofold enhanced WP NAT yield with the Ultrio Plus assay can be explained by greater than 10‐fold increased analytical sensitivity in detecting the HBV Genotype B and C strains in Hong Kong. Direct comparison studies of the two assay versions on dilutions of HBV NAT‐yield samples are required to confirm this hypothesis.
Use of tranexamic acid results in decreased blood loss and decreased transfusions in patients undergoing staged bilateral total knee arthroplasty
Authors: Kelley Todd C. , Tucker Kimberly K. , Adams Mary Jo , Dalury David F.
Published: March 2013

Background

Tranexamic acid (TXA) is an antifibrinolytic that reduces blood loss and transfusion rates in total joint arthroplasty. Blood loss and allogenic transfusion rates have not been well studied in patients receiving TXA and undergoing bilateral staged total knee arthroplasty (TKA). The purpose was to evaluate the effect of TXA on blood loss, hemoglobin (Hb) changes, and transfusion in patients undergoing staged bilateral TKA.

Study Design and Methods

The authors compared 51 patients undergoing staged bilateral TKA who received TXA (2 g; subjects) with 70 who did not (controls). There were no significant differences between the groups in terms of demographics or preoperative Hb. For each TKA, 1 g of TXA was administered intravenously 15 minutes before incision and 1 g was administered intravenously at tourniquet release. Blood loss, Hb levels, and transfusions were recorded. Statistical analyses were performed using computer software. Significance was set at 0.05.

Results

Subjects had a significantly lower (p < 0.001) mean (±SD) blood loss (373.8 ± 264.6 mL vs. 871.6 ± 457.7 mL), significantly higher (p < 0.005) Hb levels on Postoperative Days 1 and 2, and a significantly lower (p < 0.001) mean (±SD) number of transfused allogenic blood units (0.60 ± 0.84 units vs. 1.53 ± 1.30 units).

Conclusions

TXA reduces blood loss, improves postoperative Hb, and decreases the allogenic blood transfusion requirements for patients undergoing bilateral staged TKA. TXA is an option for patients choosing bilateral staged TKA to decrease the risks associated with blood transfusion or when autologous blood is not available.
Molecular genetic analysis of ABO blood group variations reveals 29 novel ABO subgroup alleles
Authors: Cai Xiaohong , Jin Sha , Liu Xi , Fan Liangfeng , Lu Qiong , Wang Jianlian , Shen Wei , Gong Songsong , Qiu Li , Xiang Dong
Published: March 2013

Background

Identifying genetic variants of the ABO gene may reveal new biologic mechanisms underlying variant phenotypes of the ABO blood group. We report the molecular genetic analysis of 322 apparently unrelated ABO subgroup individuals in an estimated 2.1 million donors.

Study Design and Methods

We performed phenotype investigations by serology studies, analyzed the DNA sequence of the ABO gene by direct sequencing or sequencing after cloning, and evaluated promoter activity by reporter assays.

Results

In 62 rare ABO alleles, we identified 29 novel ABO subgroup alleles in 43 apparently unrelated subgroup individuals and their four available pedigrees. Of these alleles, one was a deletion‐mutation allele, four were hybrid alleles, and 24 were point‐mutation alleles. Most of the point mutations were detected in Exons 6 to 7, while several others were also detected in Exons 1 to 5 or splicing regions. One ABO promoter mutation, −35 to −18 del, was found and verified to reduce promoter activity, as determined by dual luciferase assays. Two mutations, 7G>T and 52C>T, carrying the premature terminal codons E3X and R18X in the 5′‐region, were found to be associated with the very weak ABO subgroups “Ael” and “Bel.”

Conclusion

Twenty‐nine ABO subgroup alleles were newly linked to different kinds of ABO variations. We provide the first evidence that promoter abnormality is involved in the formation of weak ABO phenotypes. We also described the first naturally occurring ABO alleles with premature terminal codons in the 5′‐region that led to Ael and Bel phenotypes.
Riboflavin and ultraviolet light treatment of platelets triggers p38MAPK signaling: inhibition significantly improves in vitro platelet quality after pathogen reduction treatment
Authors: Schubert Peter , Coupland Danielle , Culibrk Brankica , Goodrich Raymond P. , Devine Dana V.
Published: March 2013

Background

Pathogen reduction technologies (PRTs) significantly reduce the risk of transmission of infectious agents in platelet (PLT) concentrates; however, in vitro studies reveal a negative impact on PLT quality after PRT treatment including effects on PLT aggregation, integrin αIIbβ3 conformation, and actin dynamics. Clinically, the interval between transfusions is shortened.

Study Design and Methods

Seeking to understand the biochemical mechanisms underlying these observed effects, we analyzed signal transduction in PLT concentrates after riboflavin and ultraviolet light (UV; Mirasol) treatment and subsequent storage focusing on the phosphorylation levels of selected protein kinases.

Results

Among identified candidates, p38MAPK increased fourfold in phosphorylation after PRT. Incubation of PLT concentrates with a p38MAPK‐specific inhibitor before PRT significantly improved numerous PLT quality measures. Phosphorylation levels of the p38MAPK substrates AKT, VASP, and HSP27 also decreased with inhibitor treatment. Phospho‐HSP27 decrease in the presence of the inhibitor correlated with a reduction in PLT activation determined by surface expression of P‐selectin.

Conclusion

These findings support a model of one dominant underlying molecular signaling mechanism that is impacted by the riboflavin and UV (Mirasol) PRT process resulting in alterations in PLT quality. The identification of such a target should assist in the development of strategies to ameliorate this negative aspect of an otherwise beneficial and important safety development for transfusion medicine.
A single origin for the limnetic–euryhaline taxa in theCorbulidae (Bivalvia)
Authors: Hallan Anders , Colgan Donald J. , Anderson Laurie C. , García Adriana , Chivas Allan R.
Published: March 2013

The bivalve family Corbulidae, known colloquially as ‘basket clams’, includes species tolerating a wide variety of habitats ranging from open marine to freshwater. Previous studies of corbulid phylogenetics have been based mainly on shell morphology and to some extent soft tissue anatomy. However, these studies have been inadequate for corbulid classification because of difficulties in determining the inter‐relationships of primarily marine species with non‐marine species, the latter commonly exhibiting highly divergent morphological, ecological and environmental characteristics from their marine counterparts. The first molecular phylogenetic study of the Corbulidae is presented herein, analysingDNAsequences from the 18SrRNAand 28SrRNAgenes, separately and in combination. Fifteen corbulid species and 14 outgroup taxa were included in the analyses. Corbulidae is resolved as monophyletic, comprising three groups with varying support. The non‐marine species form one group that we name as the subclade ‘limnetic–euryhalineCorbulidae’ (LEC) and comprising the generaLentidium,ErodonaandPotamocorbula. ThisLEC, which is consistently recovered as monophyletic, is globally distributed. The marineCorbulidae are divided into two well‐supported lineages in combined analyses although there are inconsistencies in their membership between single‐gene analyses. One of the two lineages consists of primarily Western Pacific taxa and the other ofNorthAmerican andCaribbean taxa. Finally, the authors advocate further study on theLECto mitigate potential biological invasions beyond their native distribution.
Purinergic stimulation of carotid body efferent glossopharyngeal neurones increases intracellular Ca2+ and nitric oxide production
Authors: Lowe Michael , Park Sung Jin , Nurse Colin A. , Campanucci Verónica A.
Published: March 2013

New Findings

What is the central question of this study?

Nitric oxide is a strong modulator of carotid body (CB) chemoreceptor activity; however, the mechanisms leading to the production/release of NO during CB chemoexcitation remained largely unknown.

What is the main finding and what is its importance?

In the present study, we identified an important contribution of ATP and purinergic P2X receptor signalling in initiating the events leading to NO synthesis and release in efferent neurons to the CB. Acting via P2X receptors, ATP causes an increase in intracellular Ca2+ that triggers NO production in efferent neurones to the rat carotid body. In addition, ATP‐induced depolarization of these neurones activates voltage‐gated Ca2+ channels that further amplify the NO inhibitory signal.

The mammalian carotid body (CB) is a peripheral chemosensory organ that controls ventilation and is innervated by both afferent and efferent nerve fibres. The afferent pathway is stimulated by chemoexcitants, such as hypoxia, hypercapnia and acidosis. The efferent pathway causes inhibition of the sensory discharge via release of NO that originates mainly from neuronal nitric oxide synthase (nNOS)‐positive autonomic neurones within the glossopharyngeal nerve (GPN). Recent studies in the rat indicate that these inhibitory GPN neurones and their processes express purinergic P2X receptors and can be activated by ATP, a key excitatory CB neurotransmitter. Here we tested the hypothesis that purinergic agonists stimulate a rise in [Ca2+]i, leading to nNOS activation and NO production in isolated GPN neurones, using the fluorescent probes fura‐2 and 4‐amino‐5‐methylamino‐2′,7′‐difluorofluorescein diacetate (DAF‐FM DA), respectively. ATP caused a dose‐dependent increase in [Ca2+]i in GPN neurones (EC50≈ 1.92 μm) that was markedly inhibited by a combination of 100 μm suramin (a non‐specific P2X blocker) and 100 nm Brilliant Blue G (a selective P2X7 blocker). ATP also stimulated NO production in GPN neurones, as revealed by an increase in DAF fluorescence; this NO signal was inhibited by purinergic blockers, chelators of extracellular Ca2+, the nNOS inhibitor l‐NAME and the NO scavenger carboxy‐PTIO. The P2X2/3 and P2X7 agonists α,β,‐methylene ATP and benzoyl ATP mimicked the effects of ATP. Taken together, these data indicate that ATP may contribute to negative feedback inhibition of CB sensory discharge via purinergic stimulation of NO production in efferent fibres.
Blockade of central orexin 2 receptors reduces arterial pressure in spontaneously hypertensive rats
Authors: Lee Yen‐Hsien , Dai Yu‐Wen E. , Huang Shang‐Cheng , Li Tzu‐Ling , Hwang Ling‐Ling
Published: March 2013

New Findings

What is the central question of this study?

Central orexinergic activity is involved in tonic and phasic control of cardiovascular homeostasis. A potential role for elevated central orexinergic activity in the maintenance of hypertension in spontaneously hypertensive rats (SHRs) has not previously been explored.

What is the main finding and what is its importance?

We show that central or intra‐rostral ventrolateral medulla blockade of orexin 2 receptors produces a significant reduction of arterial pressure in SHRs, but not Wistar–Kyoto rats. This study demonstrates a previously unrecognized role of orexin 2 receptors in maintaining hypertension in SHRs.

Orexins can raise arterial pressure and sympathetic activity and are involved in tonic and phasic control of cardiovascular homeostasis. We hypothesized that elevated central orexinergic activity contributes to the maintenance of hypertension in spontaneously hypertensive rats (SHRs). We examined this hypothesis by suppressing central orexinergic activity in SHRs and Wistar–Kyoto rats (WKYs) with specific antagonists or antibodies against orexin 1 (OX1R) and 2 receptors (OX2R). Intracerebroventricular administration of an OX1R antagonist, SB‐334867 (30 and 100 nmol), induced no significant change in mean arterial pressure (MAP) and heart rate (HR) in SHRs and WKYs except that at 100 nmol it reduced HR in WKYs. In contrast, an OX2R antagonist, TCS‐OX2‐29 (3–30 nmol) induced long‐lasting reductions of MAP and HR in SHRs (21 ± 3 mmHg and 22 ± 2 beats min−1at 30 nmol), but not in WKYs. Intracerebroventricular anti‐OX2R IgG, but not anti‐OX1R IgG or non‐immune goat IgG, significantly lowered MAP and HR in SHRs. None of the three IgGs affected MAP or HR in WKYs. The OX2R protein level in the rostral ventrolateral medulla (RVLM) was lower in SHRs than in WKYs, whereas no differences were found between SHRs and WKYs in the paraventricular hypothalamic nucleus, dorsomedial‐perifornical hypothalamic area or caudal nucleus tractus solitarii. The OX1R protein levels in these four regions did not differ between SHRs and WKYs. Injection of TCS‐OX2‐29 (50 pmol) into the RVLM produced a larger reduction of MAP in SHRs than in WKYs. We conclude that elevated OX2R‐mediated activity in the brain, especially in the RVLM, may contribute to hypertension in SHRs.
Spectral Distribution of Derailment Coefficient in Non-Linear Model of Railway Vehicle–Track System With Random Track Irregularities
Authors: Kardas-Cinal Ewa
Published: March 2013

Improving the running safety and reducing the risk of derailments are the key objectives in the assessment of the running characteristics of railway vehicles. The present study of the safety against derailment is focused on the effect of wheelset hunting on the derailment coefficient Y/Q and, especially, how it is reflected in the power spectral density (PSD) of Y/Q. The lateral Y and vertical Q forces at the wheel/rail contact are obtained in numerical simulations for a four-axle railway vehicle moving at a constant velocity along a tangent track with random geometrical irregularities. The PSD of Y/Q, calculated as a function of spatial frequency, is found to have a characteristic structure with three peaks for the leading wheelsets and one peak for the trailing wheelsets of the front and rear bogies. The positions of the PSD maxima remain unchanged with increasing ride velocity, while their magnitudes and shapes evolve. One of the PSD peaks occurs for all wheelsets at the same spatial frequency corresponding to the wheelset hunting, while an additional peak at the double hunting frequency is found for the leading wheelsets. Such a peak structure is also found in the PSD of Y/Q determined in simulations with modified parameters of the vehicle primary suspension and for different track sections. The peak at the double hunting frequency is shown, by a detailed analysis of the contact forces, the flange angles and their PSDs, to result from the nonlinear geometry of the wheel/rail contact leading to the second-harmonic term in Y/Q. The emergence of this peak is also closely related to the phase difference between the hunting oscillations of the wheelset lateral displacement and the oscillations of its yaw angle, for which the difference is significantly smaller for the leading wheelset than for the trailing one. Finally, the effect of wheelset hunting is also shown to manifest itself in the strong dependence of the running average of Y/Q, which is used in the railway technical safety standards for the assessment of the safety against derailment (with the Nadal criterion), on the applied window width.
A Practical Approach to Fractional Stochastic Dynamics
Authors: Thao Tran Hung
Published: March 2013

In this note we present some results on stochastic dynamics with respect to a fractional Brownian motion from a L2-approximation approach. A new and simple definition of fractional stochastic integral is introduced and a theorem of existence and uniqueness for fractional stochastic differential equations is established.
Bivariate Module-Phase Synchronization of a Fractional-Order Lorenz System in Different Dimensions
Authors: Wang Xing-Yuan , Zhang Hao
Published: March 2013

Based on the classic Lorenz system, this paper studies the problem of bivariate module-phase synchronizations in a fractional-order Lorenz system, bivariate module-phase synchronizations in a fractional-order spatiotemporal coupled Lorenz system, and malposed module-phase synchronization in a fractional-order spatiotemporal coupled Lorenz system. It is the first time, to our knowledge, that module-phase synchronization in fractional-order high-dimensional systems is applied. According to the fractional calculus techniques and spatiotemporal theory, we design controllers and achieve synchronizations both in module space and phase space at the same time. In the simulation, we discuss the bivariate module-phase synchronization and malposed module-phase synchronization. The numerical simulation results demonstrate the validity of controllers.
Review on the Absolute Nodal Coordinate Formulation for Large Deformation Analysis of Multibody Systems
Authors: Gerstmayr Johannes , Sugiyama Hiroyuki , Mikkola Aki
Published: March 2013

The aim of this study is to provide a comprehensive review of the finite element absolute nodal coordinate formulation, which can be used to obtain efficient solutions to large deformation problems of constrained multibody systems. In particular, important features of different types of beam and plate elements that have been proposed since 1996 are reviewed. These elements are categorized by parameterization of the elements (i.e., fully parameterized and gradient deficient elements), strain measures used, and remedies for locking effects. Material nonlinearities and the integration of the absolute nodal coordinate formulation to general multibody dynamics computer algorithms are addressed with particular emphasis on visco-elasticity, elasto-plasticity, and joint constraint formulations. Furthermore, it is shown that the absolute nodal coordinate formulation has been applied to a wide variety of challenging nonlinear dynamics problems that include belt drives, rotor blades, elastic cables, leaf springs, and tires. Unresolved issues and future perspectives of the study of the absolute nodal coordinate formulation are also addressed in this investigation.
Application of Topological Sensitivity Toward Soft-Tissue Characterization From Vibroacoustography Measurements
Authors: Yuan Huina , Guzina Bojan B. , Chen Shigao , Kinnick Randall , Fatemi Mostafa
Published: March 2013

This study concerns the development and preliminary experimental verification of a topological sensitivity–based platform for the material characterization of tissue anomalies exposed by vibroacoustography-type imaging techniques. Vibroacoustography (VA) is a high-resolution imaging method that has been applied to the detection of pathological changes in soft tissues. Although the data provided by this method is related to the mechanical properties of tissue, the viscoelastic parameters of the object cannot be estimated by this imaging method itself. Topological sensitivity (TS) method is a data processing methodology that can be used to estimate the viscoelastic parameters of an object from vibration data. In this study, the concept of topological sensitivity is applied to interpret the vibroacoustography measurements for the purpose of lesion characterization. In the proposed approach, the topological sensitivity function, which signifies the variation of a given cost functional when an infinitesimal inclusion with trial material parameters is placed at the location of a point force, is formulated in terms of the adjoint field. The effectiveness of the resulting formula as a material indicator for lesion characterization is demonstrated by estimating the relative elastic parameters of a well-controlled neoprene sphere embedded in a tissue-mimicking phantom specimen.
Nonlinear Pulse Equipartition in Weakly Coupled Ordered Granular Chains With No Precompression
Authors: Starosvetsky Yuli , Hasan M. Arif , Vakakis Alexander F.
Published: March 2013

We report on the strongly nonlinear dynamics of an array of weakly coupled, noncompressed, parallel granular chains subject to a local initial impulse. The motion of the granules in each chain is constrained to be in one direction that coincides with the orientation of the chain. We show that in spite of the fact that the applied impulse is applied to one of the granular chains, the resulting pulse that initially propagates only in the excited chain gets gradually equipartitioned between its neighboring chains and eventually in all chains of the array. In particular, the initially strongly localized state of energy distribution evolves towards a final stationary state of formation of identical solitary waves that propagate in each one of the chains. These solitary waves are synchronized and have identical speeds. We show that the phenomenon of primary pulse equipartition between the weakly coupled granular chains can be fully reproduced in coupled binary models that constitute a significantly simpler model that captures the main qualitative features of the dynamics of the granular array. The results reported herein are of major practical significance since it indicates that the weakly coupled array of granular chains is a medium in which an initially localized excitation gets gradually defocused, resulting in drastic reduction of propagating pulses as they are equipartitioned among all chains.
New Insight into the Cleavage Reaction of Nostoc sp. Strain PCC 7120 Carotenoid Cleavage Dioxygenase in Natural and Nonnatural Carotenoids
Authors: Heo Jinsol , Kim Se Hyeuk , Lee Pyung Cheon
Published: March 2013

Carotenoid cleavage dioxygenases (CCDs) are enzymes that catalyze the oxidative cleavage of carotenoids at a specific double bond to generate apocarotenoids. In this study, we investigated the activity and substrate preferences of NSC3, a CCD of
Nostoc
sp. strain PCC 7120,
in vivo
and
in vitro
using natural and nonnatural carotenoid structures. NSC3 cleaved β-apo-8′-carotenal at 3 positions, C-13

C-14, C-15

C-15′, and C-13′

C-14′, revealing a unique cleavage pattern. NSC3 cleaves the natural structure of carotenoids 4,4′-diaponeurosporene, 4,4′-diaponeurosporen-4′-al, 4,4′-diaponeurosporen-4′-oic acid, 4,4′-diapotorulene, and 4,4′-diapotorulen-4′-al to generate novel cleavage products (apo-14′-diaponeurosporenal, apo-13′-diaponeurosporenal, apo-10′-diaponeurosporenal, apo-14′-diapotorulenal, and apo-10′-diapotorulenal, respectively). The study of carotenoids with natural or nonnatural structures produced by using synthetic modules could provide information valuable for understanding the cleavage reactions or substrate preferences of other CCDs
in vivo
and
in vitro
.
Identification and Characterization of Three Previously Undescribed Crystal Proteins from Bacillus thuringiensis subsp.
jegathesan
Authors: Sun Yunjun , Zhao Qiang , Xia Liqiu , Ding Xuezhi , Hu Quanfang , Federici Brian A. , Park Hyun-Woo
Published: March 2013

The total protoxin complement in the parasporal body of mosquitocidal strain,
Bacillus thuringiensis
subsp.
jegathesan
367, was determined by use of a polyacrylamide gel block coupled to mass spectrometry. A total of eight protoxins were identified from this strain, including five reported protoxins (Cry11Ba, Cry19Aa, Cry24Aa, Cry25Aa, and Cyt2Bb), as well as three previously undescribed (Cry30Ca, Cry60Aa, and Cry60Ba) in this isolate. It was interesting that the encoding genes of three new protoxins existed as
cry30Ca
-gap-
orf2
and
cry60Ba
-gap-
cry60Aa
. The
cry30Ca
and a downstream
orf2
gene were oriented in the same direction and separated by 114 bp, and
cry60Ba
was located 156 bp upstream from and in the same orientation to
cry60Aa
. The three new protoxin genes were cloned from
B. thuringiensis
subsp.
jegathesan
and expressed in an acrystalliferous strain under the control of
cyt1A
gene promoters and the STAB-SD stabilizer sequence. Recombinant strain containing only
cry30Ca
did not produce visible inclusion under microscope observation, while that containing both
cry30Ca
and
orf2
could produce large inclusions. Cry60Aa and Cry60Ba synthesized either alone or together in the acrystalliferous host could yield large inclusions. In bioassays using the fourth-instar larvae of
Culex quinquefasciatus
, Cry60Aa and Cry60Ba alone or together had estimated 50% lethal concentrations of 2.9 to 7.9 μg/ml; however, Cry30Ca with or without ORF2 was not toxic to this mosquito.
Improvement of Natamycin Production by Engineering of Phosphopantetheinyl Transferases in Streptomyces chattanoogensis L10
Authors: Jiang Hui , Wang Yue-Yue , Ran Xin-Xin , Fan Wei-Ming , Jiang Xin-Hang , Guan Wen-Jun , Li Yong-Quan
Published: March 2013

Phosphopantetheinyl transferases (PPTases) are essential to the activities of type I/II polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) through converting acyl carrier proteins (ACPs) in PKSs and peptidyl carrier proteins (PCPs) in NRPSs from inactive apo-forms into active holo-forms, leading to biosynthesis of polyketides and nonribosomal peptides. The industrial natamycin (NTM) producer,
Streptomyces chattanoogensis
L10, contains two PPTases (SchPPT and SchACPS) and five PKSs. Biochemical characterization of these two PPTases shows that SchPPT catalyzes the phosphopantetheinylation of ACPs in both type I PKSs and type II PKSs, SchACPS catalyzes the phosphopantetheinylation of ACPs in type II PKSs and fatty acid synthases (FASs), and the specificity of SchPPT is possibly controlled by its C terminus. Inactivation of SchPPT in
S. chattanoogensis
L10 abolished production of NTM but not the spore pigment, while overexpression of the SchPPT gene not only increased NTM production by about 40% but also accelerated productions of both NTM and the spore pigment. Thus, we elucidated a comprehensive phosphopantetheinylation network of PKSs and improved polyketide production by engineering the cognate PPTase in bacteria.
Potato Crop as a Source of Emetic Bacillus cereus and Cereulide-Induced Mammalian Cell Toxicity
Authors: Hoornstra Douwe , Andersson Maria A. , Teplova Vera V. , Mikkola Raimo , Uotila Liisa M. , Andersson Leif C. , Roivainen Merja , Gahmberg Carl G. , Salkinoja-Salonen Mirja S.
Published: March 2013

Bacillus cereus
, aseptically isolated from potato tubers, were screened for cereulide production and for toxicity on human and other mammalian cells. The cereulide-producing isolates grew slowly, the colonies remained small (∼1 mm), tested negative for starch hydrolysis, and varied in productivity from 1 to 100 ng of cereulide mg (wet weight)
−1
(∼0.01 to 1 ng per 10
5
CFU). By DNA-fingerprint analysis, the isolates matched
B. cereus
F5881/94, connected to human food-borne illness, but were distinct from cereulide-producing endophytes of spruce tree (
Picea abies
). Exposure to cell extracts (1 to 10 μg of bacterial biomass ml
−1
) and to purified cereulide (0.4 to 7 ng ml
−1
) from the potato isolates caused mitochondrial depolarization (loss of ΔΨm) in human peripheral blood mononuclear cells (PBMC) and keratinocytes (HaCaT), porcine spermatozoa and kidney tubular epithelial cells (PK-15), murine fibroblasts (L-929), and pancreatic insulin-producing cells (MIN-6). Cereulide (10 to 20 ng ml
−1
) exposed pancreatic islets (MIN-6) disintegrated into small pyknotic cells, followed by necrotic death. Necrotic death in other test cells was observed only after a 2-log-higher exposure. Exposure to 30 to 60 ng of cereulide ml
−1
induced K
+
translocation in intact, live PBMC, keratinocytes, and sperm cells within seconds of exposure, depleting 2 to 10% of the cellular K
+
stores within 10 min. The ability of cereulide to transfer K
+
ions across biological membranes may benefit the producer bacterium in K
+
-deficient environments such as extracellular spaces inside plant tissue but is a pathogenic trait when in contact with mammalian cells.
Inactivation of the
panE
Gene in Lactococcus lactis Enhances Formation of Cheese Aroma Compounds
Authors: de Cadiñanos Luz P. Gómez , García-Cayuela Tomás , Yvon Mireille , Martinez-Cuesta M. Carmen , Peláez Carmen , Requena Teresa
Published: March 2013

Hydroxyacid dehydrogenases limit the conversion of α-keto acids into aroma compounds. Here we report that inactivation of the
panE
gene, encoding the α-hydroxyacid dehydrogenase activity in
Lactococcus lactis
, enhanced the formation of 3-methylbutanal and 3-methylbutanol.
L. lactis
IFPL953Δ
panE
was an efficient strain producing volatile compounds related to cheese aroma.
Improved Transferase/Hydrolase Ratio through Rational Design of a Family 1 β-Glucosidase from Thermotoga neapolitana
Authors: Lundemo Pontus , Adlercreutz Patrick , Karlsson Eva Nordberg
Published: March 2013

Alkyl glycosides are attractive surfactants because of their high surface activity and good biodegradability and can be produced from renewable resources. Through enzymatic catalysis, one can obtain well-defined alkyl glycosides, something that is very difficult to do using conventional chemistry. However, there is a need for better enzymes to get a commercially feasible process. A thermostable β-glucosidase from the well-studied glycoside hydrolase family 1 from
Thermotoga neapolitana
,
Tn
Bgl1A, was mutated in an attempt to improve its value for synthesis of alkyl glycosides. This was done by rational design using prior knowledge from structural homologues together with a recently generated model of the enzyme in question. Three out of four studied mutations increased the hydrolytic reaction rate in an aqueous environment, while none displayed this property in the presence of an alcohol acceptor. This shows that even if the enzyme resides in a separate aqueous phase, the presence of an organic solvent has a great influence. We could also show that a single amino acid replacement in a less studied part of the aglycone subsite, N220F, improves the specificity for transglycosylation 7-fold and thereby increases the potential yield of alkyl glycoside from 17% to 58%.
Distribution and Functions of Phosphotransferase System Genes in the Genome of the Lactic Acid Bacterium Oenococcus oeni
Authors: Jamal Zohra , Miot-Sertier Cécile , Thibau François , Dutilh Lucie , Lonvaud-Funel Aline , Ballestra Patricia , Le Marrec Claire , Dols-Lafargue Marguerite
Published: March 2013

Oenococcus oeni
, the lactic acid bacterium primarily responsible for malolactic fermentation in wine, is able to grow on a large variety of carbohydrates, but the pathways by which substrates are transported and phosphorylated in this species have been poorly studied. We show that the genes encoding the general phosphotransferase proteins, enzyme I (EI) and histidine protein (HPr), as well as 21 permease genes (3 isolated ones and 18 clustered into 6 distinct loci), are highly conserved among the strains studied and may form part of the
O. oeni
core genome. Additional permease genes differentiate the strains and may have been acquired or lost by horizontal gene transfer events. The core
pts
genes are expressed, and permease gene expression is modulated by the nature of the bacterial growth substrate. Decryptified
O. oeni
cells are able to phosphorylate glucose, cellobiose, trehalose, and mannose at the expense of phosphoenolpyruvate. These substrates are present at low concentrations in wine at the end of alcoholic fermentation. The phosphotransferase system (PTS) may contribute to the perfect adaptation of
O. oeni
to its singular ecological niche.
Transcriptional Response of Nitrifying Communities to Wetting of Dry Soil
Authors: Placella Sarah A. , Firestone Mary K.
Published: March 2013

The first rainfall following a severe dry period provides an abrupt water potential change that is both an acute physiological stress and a defined stimulus for the reawakening of soil microbial communities. We followed the responses of indigenous communities of ammonia-oxidizing bacteria, ammonia-oxidizing archaea, and nitrite-oxidizing bacteria to the addition of water to laboratory incubations of soils taken from two California annual grasslands following a typically dry Mediterranean summer. By quantifying transcripts for a subunit of bacterial and archaeal ammonia monooxygenases (
amoA
) and a bacterial nitrite oxidoreductase (
nxrA
) in soil from 15 min to 72 h after water addition, we identified transcriptional response patterns for each of these three groups of nitrifiers. An increase in quantity of bacterial
amoA
transcripts was detectable within 1 h of wet-up and continued until the size of the ammonium pool began to decrease, reflecting a possible role of transcription in upregulation of nitrification after drought-induced stasis. In one soil, the pulse of
amoA
transcription lasted for less than 24 h, demonstrating the transience of transcriptional pools and the tight coupling of transcription to the local soil environment. Analysis of 16S rRNA using a high-density microarray suggested that nitrite-oxidizing
Nitrobacter
spp. respond in tandem with ammonia-oxidizing bacteria while nitrite-oxidizing
Nitrospina
spp. and
Nitrospira
bacteria may not. Archaeal ammonia oxidizers may respond slightly later than bacterial ammonia oxidizers but may maintain elevated transcription longer. Despite months of desiccation-induced inactivation, we found rapid transcriptional response by all three groups of soil nitrifiers.
Microbial Reduction of Fe(III) under Alkaline Conditions Relevant to Geological Disposal
Authors: Williamson Adam J. , Morris Katherine , Shaw Sam , Byrne James M. , Boothman Christopher , Lloyd Jonathan R.
Published: March 2013

To determine whether biologically mediated Fe(III) reduction is possible under alkaline conditions in systems of relevance to geological disposal of radioactive wastes, a series of microcosm experiments was set up using hyperalkaline sediments (pH ∼11.8) surrounding a legacy lime working site in Buxton, United Kingdom. The microcosms were incubated for 28 days and held at pH 10. There was clear evidence for anoxic microbial activity, with consumption of lactate (added as an electron donor) concomitant with the reduction of Fe(III) as ferrihydrite (added as the electron acceptor). The products of microbial Fe(III) reduction were black and magnetic, and a range of analyses, including X-ray diffraction, transmission electron microscopy, X-ray absorption spectroscopy, and X-ray magnetic circular dichroism confirmed the extensive formation of biomagnetite in this system. The addition of soluble exogenous and endogenous electron shuttles such as the humic analogue anthraquinone-2,6-disulfonate and riboflavin increased both the initial rate and the final extent of Fe(III) reduction in comparison to the nonamended experiments. In addition, a soluble humic acid (Aldrich) also increased both the rate and the extent of Fe(III) reduction. These results show that microbial Fe(III) reduction can occur in conditions relevant to a geological disposal facility containing cement-based wasteforms that has evolved into a high pH environment over prolonged periods of time (>100,000 years). The potential impact of such processes on the biogeochemistry of a geological disposal facility is discussed, including possible coupling to the redox conditions and solubility of key radionuclides.
Distribution of Pathogenicity Islands OI-122, OI-43/48, and OI-57 and a High-Pathogenicity Island in Shiga Toxin-Producing Escherichia coli
Authors: Ju Wenting , Shen Jinling , Toro Magaly , Zhao Shaohua , Meng Jianghong
Published: March 2013

Pathogenicity islands (PAIs) play an important role in Shiga toxin-producing
Escherichia coli
(STEC) pathogenicity. The distribution of PAIs OI-122, OI-43/48, and OI-57 and a high-pathogenicity island (HPI) were determined among 98 STEC strains assigned to seropathotypes (SPTs) A to E. PCR and PCR-restriction fragment length polymorphism assays were used to identify 14 virulence genes that belonged to the four PAIs and to subtype
eae
and
stx
genes, respectively. Phylogenetic trees were constructed based on the sequences of
pagC
among 34 STEC strains and
iha
among 67 diverse pathogenic
E. coli
, respectively. Statistical analysis demonstrated that the prevalences of OI-122 (55.82%) and OI-57 (82.35%) were significantly greater in SPTs (i.e., SPTs A, B, and C) that are frequently associated with severe disease than in other SPTs.
terC
(62.5%) and
ureC
(62.5%) in OI-43/48 were also significantly more prevalent in SPTs A, B, and C than in SPTs D and E. In addition, OI-122, OI-57, and OI-43/48 and their associated virulence genes (except
iha
) were found to be primarily associated with
eae
-positive STEC, whereas HPI occurred independently of the
eae
presence. The strong association of OI-122, OI-43/48, and OI-57 with
eae
-positive STEC suggests in part that different pathogenic mechanisms exist between
eae
-positive and
eae
-negative STEC strains. Virulence genes in PAIs that are associated with severe diseases can be used as potential markers to aid in identifying highly virulent STEC.
Detection of Galectin-3 Interaction with Commensal Bacteria
Authors: Kavanaugh Devon , Kane Marian , Joshi Lokesh , Hickey Rita M.
Published: March 2013

A panel of commensal bacteria was screened for the ability to interact with galectin-3. Two strains ofBifidobacterium longumsubsp.infantisinteracted to a greater extent than did the pathogenic positive control,Escherichia coliNCTC 12900. Further validation of the interaction was achieved by using agglutination and solid-phase binding assays.
Function-Based Classification of Carbohydrate-Active Enzymes by Recognition of Short, Conserved Peptide Motifs
Authors: Busk Peter Kamp , Lange Lene
Published: March 2013

Functional prediction of carbohydrate-active enzymes is difficult due to low sequence identity. However, similar enzymes often share a few short motifs, e.g., around the active site, even when the overall sequences are very different. To exploit this notion for functional prediction of carbohydrate-active enzymes, we developed a simple algorithm, peptide pattern recognition (PPR), that can divide proteins into groups of sequences that share a set of short conserved sequences. When this method was used on 118 glycoside hydrolase 5 proteins with 9% average pairwise identity and representing four characterized enzymatic functions, 97% of the proteins were sorted into groups correlating with their enzymatic activity. Furthermore, we analyzed 8,138 glycoside hydrolase 13 proteins including 204 experimentally characterized enzymes with 28 different functions. There was a 91% correlation between group and enzyme activity. These results indicate that the function of carbohydrate-active enzymes can be predicted with high precision by finding short, conserved motifs in their sequences. The glycoside hydrolase 61 family is important for fungal biomass conversion, but only a few proteins of this family have been functionally characterized. Interestingly, PPR divided 743 glycoside hydrolase 61 proteins into 16 subfamilies useful for targeted investigation of the function of these proteins and pinpointed three conserved motifs with putative importance for enzyme activity. Furthermore, the conserved sequences were useful for cloning of new, subfamily-specific glycoside hydrolase 61 proteins from 14 fungi. In conclusion, identification of conserved sequence motifs is a new approach to sequence analysis that can predict carbohydrate-active enzyme functions with high precision.
A Pathway Closely Related to the
d
-Tagatose Pathway of Gram-Negative Enterobacteria Identified in the Gram-Positive Bacterium Bacillus licheniformis
Authors: Van der Heiden Edwige , Delmarcelle Michaël , Lebrun Sarah , Freichels Régine , Brans Alain , Vastenavond Christian M. , Galleni Moreno , Joris Bernard
Published: March 2013

We report the first identification of a gene cluster involved in
d
-tagatose catabolism in
Bacillus licheniformis
. The pathway is closely related to the
d
-tagatose pathway of the Gram-negative bacterium
Klebsiella oxytoca
, in contrast to the
d
-tagatose 6-phosphate pathway described in the Gram-positive bacterium
Staphylococcus aureus
.
Camphor Pathway Redux: Functional Recombinant Expression of 2,5- and 3,6-Diketocamphane Monooxygenases of Pseudomonas putida ATCC 17453 with Their Cognate Flavin Reductase Catalyzing Baeyer-Villiger Reactions
Authors: Iwaki Hiroaki , Grosse Stephan , Bergeron Hélène , Leisch Hannes , Morley Krista , Hasegawa Yoshie , Lau Peter C. K.
Published: March 2013

Whereas the biochemical properties of the monooxygenase components that catalyze the oxidation of 2,5-diketocamphane and 3,6-diketocamphane (2,5-DKCMO and 3,6-DKCMO, respectively) in the initial catabolic steps of (+) and (−) isomeric forms of camphor (CAM) metabolism in
Pseudomonas putida
ATCC 17453 are relatively well characterized, the actual identity of the flavin reductase (Fred) component that provides the reduced flavin to the oxygenases has hitherto been ill defined. In this study, a 37-kDa Fred was purified from a camphor-induced culture of
P. putida
ATCC 17453 and this facilitated cloning and characterization of the requisite protein. The active Fred is a homodimer with a subunit molecular weight of 18,000 that uses NADH as an electron donor (
K

m

= 32 μM), and it catalyzes the reduction of flavin mononucleotide (FMN) (
K

m

= 3.6 μM;
k
cat
= 283 s
−1
) in preference to flavin adenine dinucleotide (FAD) (
K

m

= 19 μM;
k
cat
= 128 s
−1
). Sequence determination of ∼40 kb of the CAM degradation plasmid revealed the locations of two isofunctional 2,5-DKCMO genes (
camE
25–1
for 2,5-DKCMO-1 and
camE
25–2
for 2,5-DKCMO-2) as well as that of a 3,6-DKCMO-encoding gene (
camE
36
). In addition, by pulsed-field gel electrophoresis, the CAM plasmid was established to be linear and ∼533 kb in length. To enable functional assessment of the two-component monooxygenase system in Baeyer-Villiger oxidations, recombinant plasmids expressing Fred in tandem with the respective 2,5-DKCMO- and 3,6-DKCMO-encoding genes in
Escherichia coli
were constructed. Comparative substrate profiling of the isofunctional 2,5-DCKMOs did not yield obvious differences in Baeyer-Villiger biooxidations, but they are distinct from 3,6-DKCMO in the stereoselective oxygenations with various mono- and bicyclic ketone substrates.
Molecular and Genetic Evidence for a Tetrapolar Mating System in the Basidiomycetous Yeast Kwoniella mangrovensis and Two Novel Sibling Species
Authors: Guerreiro Marco A. , Springer Deborah J. , Rodrigues Joana A. , Rusche Laura N. , Findley Keisha , Heitman Joseph , Fonseca Álvaro
Published: March 2013

Kwoniella mangrovensis
has been described as a sexual species with a bipolar mating system. Phylogenetic analysis of multiple genes places this species together with
Kwoniella heveanensis
in the
Kwoniella
clade, a sister clade to that containing two pathogenic species of global importance,
Cryptococcus neoformans
and
Cryptococcus gattii
, within the Tremellales. Recent studies defining the mating type loci (
MAT
) of species in these clades showed that, with the exception of
C. neoformans
and
C. gattii
, which are bipolar with a single biallelic multigene
MAT
locus, several other species feature a tetrapolar mating system with two unlinked loci (homeodomain [HD] and pheromone/receptor [P/R] loci). We characterized several strains from the original study describing
K. mangrovensis
; two
MAT
regions were amplified and sequenced: the
STE20
gene (P/R locus) and the divergently transcribed
SXI1
and
SXI2
genes (HD locus). We identified five different mating types with different
STE20
/
SXI
allele combinations that together with results of mating experiments demonstrate that
K. mangrovensis
is not bipolar but instead has a tetrapolar mating system. Sequence and gene analysis for a 43-kb segment of the
K. mangrovensis
type strain
MAT
locus revealed remarkable synteny with the homologous
K. heveanensis
MAT
P/R region, providing new insights into slower evolution of
MAT
loci in the
Kwoniella
compared to the
Cryptococcus
clade of the Tremellales. The study of additional isolates from plant substrates in Europe and Botswana using a combination of multilocus sequencing with
MAT
gene analysis revealed two novel sibling species that we name
Kwoniella europaea
and
Kwoniella botswanensis
and which appear to also have tetrapolar mating systems.
The Transcriptional Response of Cryptococcus neoformans to Ingestion by Acanthamoeba castellanii and Macrophages Provides Insights into the Evolutionary Adaptation to the Mammalian Host
Authors: Derengowski Lorena da S. , Paes Hugo Costa , Albuquerque Patrícia , Tavares Aldo Henrique F. P. , Fernandes Larissa , Silva-Pereira Ildinete , Casadevall Arturo
Published: March 2013

Virulence ofCryptococcus neoformansfor mammals, and in particular its intracellular style, was proposed to emerge from evolutionary pressures on its natural environment by protozoan predation, which promoted the selection of strategies that allow intracellular survival in macrophages. In fact,Acanthamoeba castellaniiingests yeast cells, which then can replicate intracellularly. In addition, most fungal factors needed to establish infection in the mammalian host are also important for survival within the amoeba. To better understand the origin ofC. neoformansvirulence, we compared the transcriptional profile of yeast cells internalized by amoebae and murine macrophages after 6 h of infection. Our results showed 656 and 293 genes whose expression changed at least 2-fold in response to the intracellular environments of amoebae and macrophages, respectively. Among the genes that were found in both groups, we focused on open reading frame (ORF) CNAG_05662, which was potentially related to sugar transport but had no determined biological function. To characterize its function, we constructed a mutant strain and evaluated its ability to grow on various carbon sources. The results showed that this gene, namedPTP1(polyol transporter protein 1), is involved in the transport of 5- and 6-carbon polyols such as mannitol and sorbitol, but its presence or absence had no effect on cryptococcal virulence for mice or moth larvae. Overall, these results are consistent with the hypothesis that the capacity for mammalian virulence originated from fungus-protozoan interactions in the environment and provide a better understanding of howC. neoformansadapts to the mammalian host.