bluuebunny/crossref_metadata_2025 huggingface dataset:
Neuroendocrine control of T cell development in mammals: role of growth hormone in modulating thymocyte migration
Authors: Savino Wilson
Published: August 2007

The thymus gland is a primary lymphoid organ, in which bone‐marrow‐derived T cell precursors undergo differentiation, eventually leading to migration of positively selected cells to the peripheral lymphoid organs. This differentiation occurs along with cell migration in the context of the thymic microenvironment, a three‐dimensional network formed by epithelial cells, macrophages, dendritic cells, fibroblasts and extracellular matrix components. A series of data clearly shows that growth hormone (GH) pleiotropically modulates thymic functions. For example, GH upregulates proliferation of thymocytes and thymic epithelial cells. Accordingly, GH‐transgenic mice, as well as animals and humans treated with exogenous GH, exhibit an enhanced cellularity in the organ. Growth hormone stimulates the secretion of thymic hormones, cytokines and chemokines by the thymic microenvironment, as well as the production of extracellular matrix proteins, leading to an increase in thymocyte migratory responses and intrathymic traffic of developing T cells. In addition, GH stimulates the in vivo export of thymocytes from the organ, as ascertained by studies with intrathymic injection of GH in normal mice and with GH‐transgenic mice. Moreover, since GH is produced by thymocytes and thymic epithelial cells, which express GH receptors, we should consider that, in addition to the classic endocrine pathway, the GH control of the thymus may include an autocrine/paracrine pathway. Finally, since GH promotes a replenishment of the thymus and an increase of thymocyte export, it could be envisioned as a potential adjuvant therapeutic agent in the treatment of immunodeficiencies associated with thymic atrophy.
Influence of heat stress on the reactivity of isolated chicken carotid artery to vasoactive agents
Authors: Siddegowda Yeshavanth K. Banasavadi , Leo Marie Dennis Marcus , Kumar Dinesh , Hooda Om Kumar , Prakash Vellanki Ravi , Mishra Santosh K.
Published: August 2007

Cerebral ischaemia is considered to be an important cause of central nervous system dysfunction in heat stress. We hypothesized that heat stress would alter the reactivity of isolated carotid artery to vasoactive agents. Carotid arteries were isolated from broiler chickens maintained either at 23–24°C with 55–65% humidity (control conditions) or exposed to 40 ± 1°C with 35% humidity for 4 h (heat stress). Contractions were elicited with vasoconstrictors such as 5‐HT, phenylephrine, guanfacine and CaCl2 (K+‐depolarized) in endothelium‐denuded arterial rings. Heat stress significantly increased the potency of 5‐HT, but had no effect on the sensitivity of the vessel to phenylephrine or guanfacine. In contrast, it markedly decreased the potency and efficacy of CaCl2. Vasodilator responses to ACh (endothelium‐intact) and sodium nitroprusside (endothelium‐denuded), however, were unaffected. Although cyclopiazonic acid (10 μm) significantly decreased 5‐HT responses in both the conditions, the agonist was still more potent in heat stress. Extracellular Ca2+ removal had no effect on contractions caused by 5‐HT in control conditions, but it significantly decreased the agonist potency in heat stress. Interestingly, nifedipine (1 μm) markedly inhibited 5‐HT‐induced contractions both in control conditions and in heat stress, implying an inhibitory effect on both Ca2+ influx and release. Thus, nifedipine had a markedly greater inhibitory effect on 5‐HT‐induced contractions in heat stress compared with control conditions. The results suggest that heat stress increased the vasoconstrictor responses to 5‐HT by a mechanism that involved extracellular Ca2+ influx through nifedipine‐sensitive L‐type calcium channels.
Association betweenKCNJ11E23K genotype and cardiovascular and glucose metabolism phenotypes in older men and women
Authors: Yi Yang , Dongmei Liu , Phares Dana A. , Weiss Edward P. , Brandauer Josef , Hagberg James M.
Published: August 2007

Our objective was to investigate the relationship between the E23K genetic variant in theKCNJ11gene, which encodes for the Kir6.2 subunit of the inward rectifier K+channel family, and glucose and insulin metabolism and cardiovascular (CV) function in the sedentary state and their responses to exercise training. Two hundred and fourteen healthy sedentary men and women aged 50–75 years old and free of CV disease and type 2 diabetes underwent baseline testing (maximal oxygen consumption (), body composition and glucose tolerance). One hundred and sixty‐three of them repeated these tests after 24 weeks of exercise training while on a low‐fat diet. At baseline, age, height, body fat, resting systolic blood pressure and all glucose and insulin metabolism markers did not differ among E23K genotype groups. In women at baseline, E23K genotype was associated with body weight, body mass index,(ml kg−1min−1, l min−1) and maximal minute ventilation. In men at baseline, E23K genotype was significantly associated with maximal heart rate, maximal respiratory exchange ratio and diastolic blood pressure at rest. Numerous glucose and insulin metabolism and CV function phenotypes changed significantly with exercise training in the total population. The E23K genotype did not significantly influence any of these training‐induced changes. Thus, the common E23K genetic variant at theKCNJ11gene locus was significantly associated with CV function in the untrained state, although the associations appear to differ between men and women. However, this variant has no significant effect on training‐induced CV and glucose and insulin metabolism adaptations.
The effects of tertiapin‐Q on responses of the sinoatrial pacemaker of the guinea‐pig heart to vagal nerve stimulation and muscarinic agonists
Authors: Bolter Chris P. , English Duane J.
Published: August 2007

Using Langendorff preparations of the guinea‐pig heart, we have examined the participation of the acetylcholine (ACh)‐activated potassium channel, IK,ACh, in the bradycardia produced by electrical stimulation of the vagus (parasympathetic) nerve and muscarinic agonists (ACh and bethanecol, bolus i.a.). Hearts from young animals (160–250 g) were perfused with Krebs–Henseleit solution, and pacemaker frequency was determined from the P wave of an ECG. Tertiapin‐Q was used to block IK,ACh. Vagal stimulation (10 s trains at 2, 5 and 10 Hz) produced graded reductions in atrial rate that were substantially attenuated, and to a similar extent, by 300 nm and 1 μm tertiapin‐Q (to 0.42 ± 0.12, mean ±s.d., of the control values; P < 0.001). Acetylcholine (3 nmol) produced brief graded bradycardias that were also attenuated by tertiapin‐Q (0.24 ± 0.24; P= 0.006). Similar results were obtained when experiments were repeated in 2 mm Cs+ (to block the hyperpolarization‐activated pacemaker current). Bethanecol (30, 50 and 70 nmol), a muscarinic agonist with no appreciable nicotinic activity, produced sustained bradycardias that were attenuated by 300 nm tertiapin‐Q (0.36 ± 0.21; P < 0.0001). The responses to vagal stimulation and ACh developed more slowly in tertiapin‐Q, indicating that a rapidly acting mechanism had been blocked. Responses to vagal stimulation were faster in 2 mm Cs+. Together, these observations show that ACh released from parasympathetic nerve varicosities exerts a considerable part of its effect on the pacemaker by activating IK,ACh and acts in a manner not readily distinguishable from that of directly applied muscarinic agonists.
Cocaine‐ and amphetamine‐regulated transcript (CART) peptide as anin vivoregulator of cardiac function inRana ridibundafrog
Authors: Ivanova Iliyana V. , Schubert Rudolf , Duridanova Dessislava B. , Bolton Thomas B. , Lubomirov Lubomir T. , Gagov Hristo S.
Published: August 2007

The aim of this study was to investigate the effect of CART peptide on cardiac performance and on the physiological signalling pathways involved usingRana ridibundafrog heart preparationsin vivo. The CART peptide, when injected into the venous sinus, significantly and reproducibly increased the force of frog heart contractions by up to 33.0 ± 6.4% during the first 15 min after its application but did not influence the chronotropic activity of the frog heart. The positive inotropic effect was entirely blocked by prazosin, pertussis toxin,Rp‐adenosine 3′,5′‐cyclic monophosphorothioate, autosauvagine 30 or metyrapone, as well as by extirpation of the pituitary gland, functional elimination of the inter‐renal glands and long‐lasting starvation, and was not observed on isolated heart preparations. Propranolol and double pithing were without significant effect on this phenomenon. It was concluded that: (i) CART peptide, administered to frogsin vivo, increases the force of heart contractions; (ii) this effect of the peptide is exerted via activation of the hypothalamic–pituitary–inter‐renal gland axis through a corticoliberin‐sensitive mechanism; (iii) CART augments the pumping function of the heart via a corticosteroid‐dependent potentiation of myocardial α1‐adrenoreceptors signalling; and (iv) prolonged food deprivation abolishes the positive inotropic effect of CART, suggesting the participation of endogenous CART in the physiological adaptation of the circulatory system to limitations of energy consumption.
Synthesis of Spiral Bevel Gear Transmissions With a Small Shaft Angle
Authors: Medvedev Vladimir I. , Volkov Andrey E.
Published: August 2007

An algorithm for synthesis of spiral bevel gear transmissions with a small shaft angle is presented. The algorithm of synthesis provides machine-tool settings with account of design limits for majority of gear generating machines. The proposed algorithm makes it possible to decrease the dimensions of machines that are used for gear and pinion tooth generation. The computer program of synthesis is developed that is based on described algorithm. Recommendations on selection of proper synthesis parameters are presented. An example of synthesis and analysis of a transmission is considered.
Numerical Study of Turbulent Heat Transfer and Pressure Drop Characteristics in a Water-Cooled Minichannel Heat Sink
Authors: Xie X. L. , Tao W. Q. , He Y. L.
Published: August 2007

With the rapid development of the Information Technology (IT) industry, the heat flux in integrated circuit (IC) chips cooled by air has almost reached its limit at about 100W∕cm2. Some applications in high technology industries require heat fluxes well beyond such a limitation. Therefore, the search for a more efficient cooling technology becomes one of the bottleneck problems of the further development of the IT industry. The microchannel flow geometry offers a large surface area of heat transfer and a high convective heat transfer coefficient. However, it has been hard to implement because of its very high pressure head required to pump the coolant fluid through the channels. A normal channel size could not give high heat flux, although the pressure drop is very small. A minichannel can be used in a heat sink with quite a high heat flux and a mild pressure loss. A minichannel heat sink with bottom size of 20mm×20mm is analyzed numerically for the single-phase turbulent flow of water as a coolant through small hydraulic diameters. A constant heat flux boundary condition is assumed. The effect of channel dimensions, channel wall thickness, bottom thickness, and inlet velocity on the pressure drop, temperature difference, and maximum allowable heat flux are presented. The results indicate that a narrow and deep channel with thin bottom thickness and relatively thin channel wall thickness results in improved heat transfer performance with a relatively high but acceptable pressure drop. A nearly optimized structure of heat sink is found that can cool a chip with heat flux of 350W∕cm2 at a pumping power of 0.314W.
Flexible Profile Compact Thermal Models for Practical Geometries
Authors: Sabry Mohamed-Nabil
Published: August 2007

Recent advances in compact thermal models have led to the emergence of a new concept allowing models to be created at any desired order of accuracy. Traditionally, increasing precision was attained by increasing the number of nodes. This strategy faces many problems; in particular, for the case of multiple heat sources (MCM) and∕or stacked dies, because different operating conditions will lead to different temperature and heat flux profiles that will require different node partitioning in order to be matched. In fact, classical approaches face a difficulty in selecting appropriate node size and position, as well as the inability to provide an a priori estimate of the number of nodes needed. The new concept is based on the use of a flexible profile to account for different possible uses of the model. In particular, it can deal with different patterns of heat generation encountered in MCM and stacked dies, and hence it is truly boundary conditions independent. Moreover, the new approach gives access to the tangential temperature gradient. This valuable information for designers in order to assess reliability cannot be predicted by classical compact model approaches. The concept was presented earlier for a simple rectangular 2D structure with surface heating (2004, 10th THERMINIC Conference, pp. 273–280). In this paper, the concept will be generalized to 3D parallelepiped boxes with both surface and∕or volumetric heating. The second achievement is the possibility to deal with geometries that can be decomposed into boxes.
Effect of Voids on Thermomechanical Durability of Pb-Free BGA Solder Joints: Modeling and Simulation
Authors: Ladani Leila Jannesari , Dasgupta Abhijit
Published: August 2007

The effect of process-induced voids on the durability of Sn–Pb and Pb-free solder interconnects in electronic products is not clearly understood. Experimental studies have provided conflicting ambiguous conclusions, showing that voids may sometimes be detrimental to reliability, but they may sometimes even increase the reliability of joints, depending on the size and location. Because of the higher level of process-induced voids in Pb-free solders, this debate is more intensified in Pb-free joints. This study presents finite element analysis (FEA) of the influence of void size, location, and spacing on the durability of Pb-free solders. A three-dimensional, global-local, viscoplastic FEA is conducted for a CTBAG132 assembly under thermal cycling. The displacement result of the global FEA at the top and bottom of the critical ball is used as the boundary condition in a local model, which focuses on the details of a single ball of the CTBGA package under temperature cycling. Parametric study is conducted to model a solder ball with voids of different sizes and locations. The maximum void area fraction modeled is from 1% to 49% of the ball area. An energy-partitioning model for cyclic creep-fatigue damage is used to estimate the damage and to monitor the trends as the size and location of voids are varied. Potential sites for maximum damage and crack initiation are identified. FEA results show that as void size increases up to about 15% of the area fraction of the ball, durability increases. For voids bigger than that, the durability starts to decrease. This study also confirms that as voids are located closer to the damage initiation site and the propagation path, their lifespan decreases.
Experimental Investigation of Compact Evaporators for Ultralow Temperature Refrigeration of Microprocessors
Authors: Wadell Robert , Joshi Yogendra K. , Fedorov Andrei G.
Published: August 2007

Microprocessor performance can be significantly improved by lowering the junction temperature, especially down to the deep subambient levels. This provides the strong motivation for the current study, which focuses on the design and thermohydraulic performance evaluation of high heat flux evaporators suitable for interfacing the microprocessor chip with a cascaded R134a∕R508b vapor compression refrigeration system at −80°C. Four compact evaporator designs are examined—a base line slit-flow structure with no microfeatures, straight microchannels, an inline pin fin array, and an alternating pin fin array—all fitting the same size envelope. Pressure drop and heat transfer measurements are reported and discussed to explain the performance of the various evaporator geometries for heat fluxes ranging between 20W∕cm2 and 100W∕cm2.
Numerical Optimization of the Thermoelectric Cooling Devices
Authors: Abramzon Boris
Published: August 2007

The present study proposes a unified numerical approach to the problem of optimum design of the thermoelectric devices for cooling electronic components. The standard mathematical model of a single-stage thermoelectric cooler (TEC) with constant material properties is employed. The model takes into account the thermal resistances from the hot and cold sides of the TEC. Values of the main physical parameters governing the TEC performance (Seebeck coefficient, electrical resistance, and thermal conductance) are derived from the manufacturer catalog data on the maximum achievable temperature difference, and the corresponding electric current and voltage. The optimization approach is illustrated with several examples for different design objective functions, variables, and constraints. The objective for the optimization search is the maximization of the total cooling rate or the performance coefficient of the cooling device. The independent variables for the optimization search are as follows: The number of the thermoelectric modules, the electric current, and the cold side temperature of the TEC. Additional independent variables in other cases include the number of thermoelectric couples and the area-to-height ratio of the thermoelectric pellet. In the present study, the optimization problems are solved numerically using the so-called multistart adaptive random search method.
High-Order and Model Reference Consensus Algorithms in Cooperative Control of MultiVehicle Systems
Authors: Ren Wei , Moore Kevin L. , Chen Yangquan
Published: August 2007

In this paper we study ℓth-order (ℓ⩾3) consensus algorithms, which generalize the existing first-order and second-order consensus algorithms in the literature. We show necessary and sufficient conditions under which each information variable and its higher-order derivatives converge to common values. We also present the idea of higher-order consensus with a leader and introduce the concept of an ℓth-order model-reference consensus problem, where each information variable and its high-order derivatives not only reach consensus, but also converge to the solution of a prescribed dynamic model. The effectiveness of these algorithms is demonstrated through simulations and a multivehicle cooperative control application, which mimics a flocking behavior in birds.
Swarm Tracking Using Artificial Potentials and Sliding Mode Control
Authors: Yao Jingyi , Ordóñez Raúl , Gazi Veysel
Published: August 2007

In this paper, we present a stable and decentralized control strategy for multiagent systems (swarms) to capture a moving target in a specific formation. The coordination framework uses artificial potentials to take care of both tracking and formation tasks. First, a basic controller is designed based on a kinematic model. After that, sliding mode control technique is used to force the agents with general vehicle dynamics to obey the required motion. Finally, specific potential functions are discussed and corresponding simulation results are given.
Phantom Track Generation Through Cooperative Control of Multiple ECAVs Based on Feasibility Analysis
Authors: Maithripala D. H. A. , Jayasuriya Suhada , Mears Mark J.
Published: August 2007

Radar deception through phantom track generation using multiple electronic combat air vehicles is addressed, which serves as a motivating example for cooperative control of autonomous multiagent systems. A general framework to derive sufficient conditions for the existence of feasible solutions for an affine nonlinear control system comprising of a team of nonholonomic mobile agents having to satisfy actuator and interagent constraints is presented. Based on this feasibility analysis, an algorithm capable of generating trajectories online and in real time, for the phantom track generation problem, is developed. A rigorous treatment of the phantom track generation problem, which includes results on its accessibility, feasibility, local asymptotic straightening of trajectories, and a limited result on system controllability, is given. The basic approach to the algorithm based on the results developed here is presented along with simulation results, validating the proposed approach.
Multi-UAV Cooperative Search Using an Opportunistic Learning
Method
Authors: Yang Yanli , Polycarpou Marios M. , Minai Ali A.
Published: August 2007

The control of networked multivehicle systems designed to perform complex coordinated tasks is currently an important and challenging field of research. This paper addresses a cooperative search problem where a team of uninhabited aerial vehicles (UAVs) seeks to find targets of interest in an uncertain environment. We present a practical framework for online planning and control of a group of UAVs for cooperative search based on two interdependent tasks: (i) incrementally updating “cognitive maps” used as the representation of the environment through new sensor readings; (ii) continuously planning the path for each vehicle based on the information obtained through the search. We formulate the cooperative search problem and develop a decentralized strategy based on an opportunistic cooperative learning method, where the emergent coordination among vehicles is enabled by letting each vehicle consider other vehicles’ actions in its path planning procedure. By using the developed strategy, physically feasible paths for the vehicles to follow are generated, where constraints on aerial vehicles, including physical maneuverabilities, are considered and the dynamic nature of the environment is taken into account. We also present some mathematical analysis of the developed search strategy. Our analysis shows that this strategy guarantees a complete search of the environment and is robust to a partial loss of UAVs. A lower bound on the search time for any strategy and a relaxed upper bound for the proposed strategy are given. Simulation results are used to illustrate the effectiveness of the proposed strategy.
Decentralized Coordinated Motion Control of Two Hydraulic Actuators Handling a Common Object
Authors: Karpenko Mark , Sepehri Nariman , Anderson John
Published: August 2007

In this paper, reinforcement learning is applied to coordinate, in a decentralized fashion, the motions of a pair of hydraulic actuators whose task is to firmly hold and move an object along a specified trajectory under conventional position control. The learning goal is to reduce the interaction forces acting on the object that arise due to inevitable positioning errors resulting from the imperfect closed-loop actuator dynamics. Each actuator is therefore outfitted with a reinforcement learning neural network that modifies a centrally planned formation constrained position trajectory in response to the locally measured interaction force. It is shown that the actuators, which form a multiagent learning system, can learn decentralized control strategies that reduce the object interaction forces and thus greatly improve their coordination on the manipulation task. However, the problem of credit assignment, a common difficulty in multiagent learning systems, prevents the actuators from learning control strategies where each actuator contributes equally to reducing the interaction force. This problem is resolved in this paper via the periodic communication of limited local state information between the reinforcement learning actuators. Using both simulations and experiments, this paper examines some of the issues pertaining to learning in dynamic multiagent environments and establishes reinforcement learning as a potential technique for coordinating several nonlinear hydraulic manipulators performing a common task.
Stable Cooperative Vehicle Distributions for Surveillance
Authors: Finke Jorge , Passino Kevin M.
Published: August 2007

A mathematical model for the study of the behavior of a spatially distributed group of heterogeneous vehicles is introduced. We present a way to untangle the coupling between the assignment of any vehicle’s position and the assignment of all other vehicle positions by defining general sensing and moving conditions that guarantee that even when the vehicles’ motion and sensing are highly constrained, they ultimately achieve a stable emergent distribution. The achieved distribution is optimal in the sense that the proportion of vehicles allocated over each area matches the relative importance of being assigned to that area. Based on these conditions, we design a cooperative control scheme for a multivehicle surveillance problem and show how the vehicles’ maneuvering and sensing abilities, and the spatial characteristics of the region under surveillance, affect the desired distribution and the rate at which it is achieved.
Decentralized Policies for Geometric Pattern Formation and Path Coverage
Authors: Pavone Marco , Frazzoli Emilio
Published: August 2007

This paper presents a decentralized control policy for symmetric formations in multiagent systems. It is shown that n agents, each one pursuing its leading neighbor along the line of sight rotated by a common offset angle α, eventually converge to a single point, a circle or a logarithmic spiral pattern, depending on the value of α. In the final part of the paper, we present a strategy to make the agents totally anonymous, and we discuss a potential application to coverage path planning.
vanD
and
vanG
-Like Gene Clusters in a
Ruminococcus
Species Isolated from Human Bowel Flora
Authors: Domingo M.-C. , Huletsky A. , Giroux R. , Picard F. J. , Bergeron M. G.
Published: August 2007

A vancomycin-resistant, anaerobic, gram-positive coccus containing the
vanD
and
vanG
-like genes (strain CCRI-16110) was isolated from a human fecal specimen during a hospital surveillance program to detect carriers of vancomycin-resistant enterococci. Comparison of the 16S rRNA gene sequence of strain CCRI-16110 with databases revealed a potentially novel
Ruminococcus
species that was most similar (<94% identity) to
Clostridium
and
Ruminococcus
species. Strain CCRI-16110 was highly resistant to vancomycin and teicoplanin (MICs of >256 μg/ml). The complete DNA sequence of the
vanD
cluster was most similar (98.2% identity) to that of
Enterococcus faecium
BM4339, containing the
vanD1
allele. An
intD
gene with 99% identity with that of this
E. faecium
strain was found to be associated with the
vanD
gene cluster of this novel anaerobic bacterium. Strain CCRI-16110 also harbors genes encoding putative VanS
G
, VanG, and VanT
G
proteins displaying 56, 73.6, and 55% amino acid sequence identity, respectively, compared to the corresponding proteins encoded by the
vanG1
and
vanG2
operons of
Enterococcus faecalis
BM4518 and N03-0233. This study reports for the first time an anaerobic bacterium containing the
vanD
gene cluster. This strain also harbors a partial
vanG
-like gene cluster. The presence of
vanD
- and
vanG
-containing anaerobic bacteria in the human bowel flora suggests that these bacteria may serve as a reservoir for the
vanD
and
vanG
vancomycin resistance genes.
Molecular Subtyping and Genetic Analysis of the Enterohemolysin Gene (
ehxA
) from Shiga Toxin-Producing
Escherichia coli
and Atypical Enteropathogenic
E. coli
Authors: Cookson Adrian L. , Bennett Jenny , Thomson-Carter Fiona , Attwood Graeme T.
Published: August 2007

Analyses of the distribution of virulence factors among different
Escherichia coli
pathotypes, including Shiga toxin-producing
E. coli
(STEC), may provide some insight into the mechanisms by which different
E. coli
strains cause disease and the evolution of distinct
E. coli
types. The aim of this study was to examine the DNA sequence of the gene for enterohemolysin, a plasmid-encoded toxin that readily causes the hemolysis of washed sheep erythrocytes, and to assess the distribution of enterohemolysin subtypes among
E. coli
isolates from various human and animal sources. The 2,997-bp
ehxA
gene was amplified from 227 (63.8%) of 356
stx
- and/or
eae
-positive
E. coli
strains isolated from cattle and sheep and from 24 (96.0%) of 25 STEC strains isolated from humans with diarrheal disease. By using PCR and restriction fragment length polymorphism (RFLP) analysis of
ehxA
, six distinct PCR-RFLP types (A to F) were observed, with strains of subtypes A and C constituting 91.6% of all the
ehxA
-positive strains. Subtype A was associated mainly with ovine strains with
stx
only (
P
< 0.001), and subtype C was associated with bovine
eae
-positive strains (
P
< 0.001). Eleven
ehxA
alleles were fully sequenced, and the phylogenetic analysis indicated the presence of three closely related (>95.0%)
ehxA
sequence groups, one including
eae
-positive strains (subtypes B, C, E, and F) and the other two including mainly
eae
-negative STEC strains (subtypes A and D). In addition to being widespread among STEC strains,
stx
-negative,
eae
-positive strains (atypical enteropathogenic
E. coli
strains) isolated from cattle and sheep have similar
ehxA
subtypes and hemolytic activities.
Membrane-Bound, 2-Keto-
d
-Gluconate-Yielding
d
-Gluconate Dehydrogenase from “
Gluconobacter dioxyacetonicus
” IFO 3271: Molecular Properties and Gene Disruption
Authors: Toyama Hirohide , Furuya Naoko , Saichana Ittipon , Ano Yoshitaka , Adachi Osao , Matsushita Kazunobu
Published: August 2007

Most
Gluconobacter
species produce and accumulate 2-keto-
d
-gluconate (2KGA) and 5KGA simultaneously from
d
-glucose via GA in culture medium. 2KGA is produced by membrane-bound flavin adenine dinucleotide-containing GA 2-dehydrogenase (FAD-GADH). FAD-GADH was purified from “
Gluconobacter dioxyacetonicus
” IFO 3271, and N-terminal sequences of the three subunits were analyzed. PCR primers were designed from the N-terminal sequences, and part of the FAD-GADH genes was cloned as a PCR product. Using this PCR product, gene fragments containing whole FAD-GADH genes were obtained, and finally the nucleotide sequence of 9,696 bp was determined. The cloned sequence had three open reading frames (ORFs),
gndS
,
gndL
, and
gndC
, corresponding to small, large, and cytochrome
c
subunits of FAD-GADH, respectively. Seven other ORFs were also found, one of which showed identity to glucono-δ-lactonase, which might be involved directly in 2KGA production. Three mutant strains defective in either
gndL
or
sldA
(the gene responsible for 5KGA production) or both were constructed. Ferricyanide-reductase activity with GA in the membrane fraction of the
gndL
-defective strain decreased by about 60% of that of the wild-type strain, while in the
sldA
-defective strain, activity with GA did not decrease and activities with glycerol,
d
-arabitol, and
d
-sorbitol disappeared. Unexpectedly, the strain defective in both
gndL
and
sldA
(double mutant) still showed activity with GA. Moreover, 2KGA production was still observed in
gndL
and double mutant strains. 5KGA production was not observed at all in
sldA
and double mutant strains. Thus, it seems that “
G. dioxyacetonicus
” IFO 3271 has another membrane-bound enzyme that reacts with GA, producing 2KGA.
Localization and Visualization of aCoxiella-Type Symbiont within the Lone Star Tick,Amblyomma americanum
Authors: Klyachko Olga , Stein Barry D. , Grindle Nathan , Clay Keith , Fuqua Clay
Published: August 2007

ACoxiella-type microbe occurs at 100% frequency in allAmblyomma americanumticks thus far tested. Using laboratory-reared ticks free of other microbes, we identified theAmblyomma-associatedCoxiellamicrobe in several types of tissue and at various stages of the life cycle ofA. americanumby 16S rRNA gene sequencing and diagnostic PCR. We visualizedAmblyomma-associatedCoxiellathrough the use of a diagnostic fluorescence in situ hybridization (FISH) assay supplemented with PCR-based detection, nucleic acid fluorescent staining, wide-field epifluorescence and confocal microscopy, and transmission electron microscopy (TEM). Specific fluorescent foci were observed in several tick tissues, including the midgut and the Malpighian tubules, but particularly bright signals were observed in the granular acini of salivary gland clusters and in both small and large oocytes. TEM confirmed intracellular bacterial structures in the same tissues. The presence ofAmblyomma-associatedCoxiellawithin oocytes is consistent with the vertical transmission of these endosymbionts. Further, the presence of theAmblyomma-associatedCoxiellasymbiont in other tissues such as salivary glands could potentially lead to interactions with horizontally acquired pathogens.
Novel Whole-Cell Antibiotic Biosensors for Compound Discovery
Authors: Urban Andreas , Eckermann Stefan , Fast Beate , Metzger Susanne , Gehling Matthias , Ziegelbauer Karl , Rübsamen-Waigmann Helga , Freiberg Christoph
Published: August 2007

Cells containing reporters which are specifically induced via selected promoters are used in pharmaceutical drug discovery and in environmental biology. They are used in screening for novel drug candidates and in the detection of bioactive compounds in environmental samples. In this study, we generated and validated a set of five
Bacillus subtilis
promoters fused to the firefly luciferase reporter gene suitable for cell-based screening, enabling the as yet most-comprehensive high-throughput diagnosis of antibiotic interference in the major biosynthetic pathways of bacteria: the biosynthesis of DNA by the
yorB
promoter, of RNA by the
yvgS
promoter, of proteins by the
yheI
promoter, of the cell wall by the
ypuA
promoter, and of fatty acids by the
fabHB
promoter. The reporter cells mainly represent novel antibiotic biosensors compatible with high-throughput screening. We validated the strains by developing screens with a set of 14,000 pure natural products, representing a source of highly diverse chemical entities, many of them with antibiotic activity (6% with anti-
Bacillus subtilis
activity of ≤25 μg/ml]). Our screening approach is exemplified by the discovery of classical and novel DNA synthesis and translation inhibitors. For instance, we show that the mechanistically underexplored antibiotic ferrimycin A1 selectively inhibits protein biosynthesis.
Comparison of Direct Genome Restriction Enzyme Analysis and Pulsed-Field Gel Electrophoresis for Typing of
Vibrio vulnificus
and Their Correspondence with Multilocus Sequence Typing Data
Authors: González-Escalona Narjol , Whitney Brooke , Jaykus Lee-Ann , DePaola Angelo
Published: August 2007

We compared the potential of direct genome restriction enzyme analysis (DGREA) and pulsed-field gel electrophoresis (PFGE) for discriminating
Vibrio vulnificus
isolates from clinical (23) and environmental (17) sources. The genotypes generated by both methodologies were compared to previous multilocus sequence typing (MLST) data. DGREA established clearer relationships among
V. vulnificus
strains and was more consistent with MLST than with PFGE. DGREA is a very promising tool for epidemiological and ecological studies of
V. vulnificus
.
Development of Quantitative Real-Time PCR Assays for Detection and Quantification of Surrogate Biological Warfare Agents in Building Debris and Leachate
Authors: Saikaly Pascal E. , Barlaz Morton A. , de los Reyes Francis L.
Published: August 2007

Evaluation of the fate and transport of biological warfare (BW) agents in landfills requires the development of specific and sensitive detection assays. The objective of the current study was to develop and validate SYBR green quantitative real-time PCR (Q-PCR) assays for the specific detection and quantification of surrogate BW agents in synthetic building debris (SBD) and leachate.
Bacillus atrophaeus
(vegetative cells and spores) and
Serratia marcescens
were used as surrogates for
Bacillus anthracis
(anthrax) and
Yersinia pestis
(plague), respectively. The targets for SYBR green Q-PCR assays were the 16S-23S rRNA intergenic transcribed spacer (ITS) region and
recA
gene for
B. atrophaeus
and the
gyrB
,
wzm
, and
recA
genes for
S. marcescens
. All assays showed high specificity when tested against 5 ng of closely related
Bacillus
and
Serratia
nontarget DNA from 21 organisms. Several spore lysis methods that include a combination of one or more of freeze-thaw cycles, chemical lysis, hot detergent treatment, bead beat homogenization, and sonication were evaluated. All methods tested showed similar threshold cycle values. The limit of detection of the developed Q-PCR assays was determined using DNA extracted from a pure bacterial culture and DNA extracted from sterile water, leachate, and SBD samples spiked with increasing quantities of surrogates. The limit of detection for
B. atrophaeus
genomic DNA using the ITS and
B. atrophaeus recA
Q-PCR assays was 7.5 fg per PCR. The limits of detection of
S. marcescens
genomic DNA using the
gyrB
,
wzm
, and
S. marcescens recA
Q-PCR assays were 7.5 fg, 75 fg, and 7.5 fg per PCR, respectively. Quantification of
B. atrophaeus
vegetative cells and spores was linear (
R
2
> 0.98) over a 7-log-unit dynamic range down to 10
1
B. atrophaeus
cells or spores. Quantification of
S. marcescens
(
R
2
> 0.98) was linear over a 6-log-unit dynamic range down to 10
2
S. marcescens
cells. The developed Q-PCR assays are highly specific and sensitive and can be used for monitoring the fate and transport of the BW surrogates
B. atrophaeus
and
S. marcescens
in building debris and leachate.
Variability of Bacterial Biofilms of the “Tina” Wood Vats Used in the Ragusano Cheese-Making Process
Authors: Licitra G. , Ogier J. C. , Parayre S. , Pediliggieri C. , Carnemolla T. M. , Falentin H. , Madec M. N. , Carpino S. , Lortal S.
Published: August 2007

Ragusano cheese is a “protected denomination of origin” cheese made in the Hyblean region of Sicily from raw milk using traditional wooden tools, without starter. To explore the Ragusano bacterial ecosystem, molecular fingerprinting was conducted at different times during the ripening and biofilms from the wooden vats called “tinas” were investigated. Raw milks collected at two farm sites, one on the mountain and one at sea level, were processed to produce Ragusano cheese. Raw milk, curd before and after cooking, curd at stretching time (cheese 0 time), and cheese samples (4 and 7 months) were analyzed by PCR-temporal temperature gel electrophoresis (PCR-TTGE) and by classical enumeration microbiology. With the use of universal primers, PCR-TTGE revealed many differences between the raw milk profiles, but also notable common bands identified as
Streptococcus thermophilus, Lactobacillus lactis, Lactobacillus delbrueckii
, and
Enterococcus faecium
. After the stretching, TTGE profiles revealed three to five dominant species only through the entire process of ripening. In the biofilms of the two tinas used, one to five species were detected,
S. thermophilus
being predominant in both. Biofilms from five other tinas were also analyzed by PCR-TTGE, PCR-denaturating gradient gel electrophoresis, specific PCR tests, and sequencing, confirming the predominance of lactic acid bacteria (
S. thermophilus, L. lactis
, and
L. delbrueckii
subsp.
lactis
) and the presence of a few high-GC-content species, like coryneform bacteria. The spontaneous acidification of raw milks before and after contact with the five tinas was followed in two independent experiments. The lag period before acidification can be up to 5 h, depending on the raw milk and the specific tina, highlighting the complexity of this natural inoculation system.
Ralstonia solanacearum
Strains from Martinique (French West Indies) Exhibiting a New Pathogenic Potential
Authors: Wicker Emmanuel , Grassart Laurence , Coranson-Beaudu Régine , Mian Danièle , Guilbaud Caroline , Fegan Mark , Prior Philippe
Published: August 2007

We investigated a destructive pathogenic variant of the plant pathogen
Ralstonia solanacearum
that was consistently isolated in Martinique (French West Indies). Since the 1960s, bacterial wilt of solanaceous crops in Martinique has been caused primarily by strains of
R. solanacearum
that belong to either phylotype I or phylotype II. Since 1999, anthurium shade houses have been dramatically affected by uncharacterized phylotype II strains that also affected a wide range of species, such as
Heliconia caribea
, cucurbitaceous crops, and weeds. From 1989 to 2003, a total of 224
R. solanacearum
isolates were collected and compared to 6 strains isolated in Martinique in the 1980s. The genetic diversity and phylogenetic position of selected strains from Martinique were assessed (multiplex PCRs,
mutS
and
egl
DNA sequence analysis) and compared to the genetic diversity and phylogenetic position of 32 reference strains covering the known diversity within the
R. solanacearum
species complex. Twenty-four representative isolates were tested for pathogenicity to
Musa
species (banana) and tomato, eggplant, and sweet pepper. Based upon both PCR and sequence analysis, 119 Martinique isolates from anthurium, members of the family
Cucurbitaceae, Heliconia
, and tomato, were determined to belong to a group termed phylotype II/sequevar 4 (II/4). While these strains cluster with the Moko disease-causing strains, they were not pathogenic to banana (NPB). The strains belonging to phylotype II/4NPB were highly pathogenic to tomato, eggplant, and pepper, were able to wilt the resistant tomato variety Hawaii7996, and may latently infect cooking banana. Phylotype II/4NPB constitutes a new pathogenic variant of
R. solanacearum
that has recently appeared in Martinique and may be latently prevalent throughout Caribbean and Central/South America.
Detection of
Escherichia coli
in Biofilms from Pipe Samples and Coupons in Drinking Water Distribution Networks
Authors: Juhna T. , Birzniece D. , Larsson S. , Zulenkovs D. , Sharipo A. , Azevedo N. F. , Ménard-Szczebara F. , Castagnet S. , Féliers C. , Keevil C. W.
Published: August 2007

Fluorescence in situ hybridization (FISH) was used for direct detection of
Escherichia coli
on pipe surfaces and coupons in drinking water distribution networks. Old cast iron main pipes were removed from water distribution networks in France, England, Portugal, and Latvia, and
E. coli
was analyzed in the biofilm. In addition, 44 flat coupons made of cast iron, polyvinyl chloride, or stainless steel were placed into and continuously exposed to water on 15 locations of 6 distribution networks in France and Latvia and examined after 1 to 6 months exposure to the drinking water. In order to increase the signal intensity, a peptide nucleic acid (PNA) 15-mer probe was used in the FISH screening for the presence or absence of
E. coli
on the surface of pipes and coupons, thus reducing occasional problems of autofluorescence and low fluorescence of the labeled bacteria. For comparison, cells were removed from the surfaces and examined with culture-based or enzymatic (detection of β-
d
-glucuronidase) methods. An additional verification was made by using PCR. Culture method indicated presence of
E. coli
in one of five pipes, whereas all pipes were positive with the FISH methods.
E. coli
was detected in 56% of the coupons using PNA FISH, but no
E. coli
was detected using culture or enzymatic methods. PCR analyses confirmed the presence of
E. coli
in samples that were negative according to culture-based and enzymatic methods. The viability of
E. coli
cells in the samples was demonstrated by the cell elongation after resuscitation in low-nutrient medium supplemented with pipemidic acid, suggesting that the cells were present in an active but nonculturable state, unable to grow on agar media.
E. coli
contributed to ca. 0.001 to 0.1% of the total bacterial number in the samples. The presence and number of
E. coli
did not correlate with any of physical and/or chemical characteristic of the drinking water (e.g., temperature, chlorine, or biodegradable organic matter concentration). We show here that
E. coli
is present in the biofilms of drinking water networks in Europe. Some of the cells are metabolically active but are often not detected due to limitations of traditionally used culture-based methods, indicating that biofilm should be considered as a reservoir that must be investigated further in order to evaluate the risk for human health.
Immunoglobulins to Surface-Associated Biofilm Immunogens Provide a Novel Means of Visualization of Methicillin-Resistant
Staphylococcus aureus
Biofilms
Authors: Brady Rebecca A. , Leid Jeff G. , Kofonow Jennifer , Costerton J. William , Shirtliff Mark E.
Published: August 2007

Antigens from the methicillin-resistant
Staphylococcus aureus
(MRSA) cell wall have been shown to be immunogenic in vivo and upregulated during biofilm growth. In this study, we created purified, recombinant forms of selected antigens and biofilm-upregulated, cell wall-associated proteins. These proteins were shown to cause a robust polyclonal immunoglobulin G (IgG) response when used to immunize rabbits. Antibodies against these recombinant proteins bound to the native forms of each protein as harvested from in vitro grown biofilms of MRSA, as determined both via Western blot analysis and immunofluorescence confocal microscopy. These IgGs could be utilized as imaging tools that localize to areas of specific protein production within a biofilm. This work illustrates that immunogenic, cell wall-associated, biofilm-upregulated proteins are promising for in vitro visualization of biofilm growth, architecture, and space-function relationships.
The Ability ofAneurinibacillus migulanus(Bacillus brevis) To Produce the Antibiotic Gramicidin S Is Correlated with Phenotype Variation
Authors: Berditsch Marina , Afonin Sergii , Ulrich Anne S.
Published: August 2007

Phenotype instability of bacterial strains can cause significant problems in biotechnological applications, since industrially useful properties may be lost. Here we report such degenerative dissociation forAneurinibacillus migulanus(formerly known asBacillus brevis) an established producer of the antimicrobial peptide gramicidin S (GS). Phenotypic variations within and between various strains maintained in different culture collections are demonstrated. The type strain, ATCC 9999, consists of six colony morphology variants, R, RC, RP, RT, SC, and SP, which were isolated and characterized as pure cultures. Correlations between colony morphology, growth, GS production, spore formation, and resistance to their own antimicrobial peptide were established in this study. We found the original R form to be the best producer, followed by RC, RP, and RT, while SC and SP yielded no GS at all. Currently available ATCC 9999Tcontains only 2% of the original R producer and is dominated by the newly described phenotypes RC and RP. No original R form is detected in the nominally equivalent strain DSM 2895T(=ATCC 9999T), which grows only as SC and SP phenotypes and has thus completely lost its value as a peptide producer. Two other strains from the same collection, DSM 5668 and DSM 5759, contain the unproductive SC variant and the GS-producing RC form, respectively. We describe the growth and maintenance conditions that stabilize certain colony phenotypes and reduce the degree of degenerative dissociation, thus providing a recommendation for how to revert the nonproducing smooth phenotypes to the valuable GS-producing rough ones.