bluuebunny/crossref_metadata_2025 huggingface dataset:
Environmental Isotopes as a Tool in Hydrogeological Research in an Arid Basin
Authors: Lssar Arie , Gat Joel
Published: July 2006

Stable and radioactive isotopes (deuterium, tritium, 18O, 14C) were used as an aid for better understanding the regime of recharge and flow in ground water in the Wadi Feiran basin in the southern Sinai.

The annual average precipitation in the Sinai basin is between 80 mm in its upper reaches and 15 mm in the lower regions.

In the upper part, underlain by crystalline rocks, ground water was found in shallow and deep fractures as in shallow alluvial deposits. The quality of water in the shallow fractures showed little effect of evaporation in relation to precipitation, which shows that recharge occurs directly and quickly through the open fractures. In the middle part of the basin, water in the riverbed alluvium was found to be recharged mainly by flood water and discharged also through deep fractures. In the lower part of the basin, the ground water was found to flow into the alluvial plain at A‐Tor. Part of it mixes with paleo‐water and emerges as thermal springs.
Multiple vascular anomalies in a regurgitating German shepherd puppy
Authors: Christiansen K. J. , Snyder D. , Buchanan J. W. , Holt D. E.
Published: July 2006

Five cardio‐thoracic vascular anomalies were detected in a German shepherd puppy. The patent ductus arteriosus (PDA) was detected on physical examination (5/6 continuous murmur) and confirmed by echocardiogram. The persistent right aortic arch (PRAA) was suspected by the signalment and history of the patient, and confirmed by survey thoracic radiographs (leftward deviation of the trachea cranial to the heart on the ventrodorsal projection). The ventrally deviated trachea cranial to the heart on the right lateral thoracic radiograph was suggestive of a persistent retroesophageal left subclavian artery and confirmed at surgery. The persistent left cranial vena cava and the left azygous vein were detected at surgery. This case report gives a thorough description of the clinical signs, diagnostics and treatments required for the detection and successful resolution of PRAA. The report describes the importance of having experienced surgeons who can recognize vascular anomalies associated with PRAA in order to successfully alleviate the arch and the coinciding oesophageal stricture without compromising vital blood supplies.
Genetic networks controlling the development of midbrain dopaminergic neurons
Authors: Prakash Nilima , Wurst Wolfgang
Published: July 2006

Recent data have substantially advanced our understanding of midbrain dopaminergic neuron development. Firstly, a Wnt1‐regulated genetic network, including Otx2 and Nkx2‐2, and a Shh‐controlled genetic cascade, including Lmx1a, Msx1 and Nkx6‐1, have been unravelled, acting in parallel or sequentially to establish a territory competent for midbrain dopaminergic precursor production at relatively early stages of neural development. Secondly, the same factors (Wnt1 and Lmx1a/Msx1) appear to regulate midbrain dopaminergic and/or neuronal fate specification in the postmitotic progeny of these precursors by controlling the expression of midbrain dopaminergic‐specific and/or general proneural factors at later stages of neural development. For the first time, early inductive events have thus been linked to later differentiation processes in midbrain dopaminergic neuron development. Given the pivotal importance of this neuronal population for normal function of the human brain and its involvement in severe neurological and psychiatric disorders such as Parkinson's Disease, these advances open new prospects for potential stem cell‐based therapies. We will summarize these new findings in the overall context of midbrain dopaminergic neuron development in this review.
Rapid‐rate paired associative stimulation of the median nerve and motor cortex can produce long‐lasting changes in motor cortical excitability in humans
Authors: Quartarone Angelo , Rizzo Vincenzo , Bagnato Sergio , Morgante Francesca , Sant'Angelo Antonino , Girlanda Paolo , Roman Siebner Hartwig
Published: July 2006

Repetitive transcranial magnetic stimulation (rTMS) or repetitive electrical peripheral nerve stimulation (rENS) can induce changes in the excitability of the human motor cortex (M1) that is often short‐lasting and variable, and occurs only after prolonged periods of stimulation. In 10 healthy volunteers, we used a new repetitive paired associative stimulation (rPAS) protocol to facilitate and prolong the effects of rENS and rTMS on cortical excitability. Sub‐motor threshold 5 Hz rENS of the right median nerve was synchronized with submotor threshold 5 Hz rTMS of the left M1 at a constant interval for 2 min. The interstimulus interval (ISI) between the peripheral stimulus and the transcranial stimulation was set at 10 ms (5 Hz rPAS10ms) or 25 ms (5 Hz rPAS25ms). TMS was given over the hot spot of the right abductor pollicis brevis (APB) muscle. Before and after rPAS, we measured the amplitude of the unconditioned motor evoked potential (MEP), intracortical inhibition (ICI) and facilitation (ICF), short‐ and long‐latency afferent inhibition (SAI and LAI) in the conditioned M1. The 5 Hz rPAS25ms protocol but not the 5 Hz rPAS10ms protocol caused a somatotopically specific increase in mean MEP amplitudes in the relaxed APB muscle. The 5 Hz rPAS25ms protocol also led to a loss of SAI, but there was no correlation between individual changes in SAI and corticospinal excitability. These after‐effects were still present 6 h after 5 Hz rPAS25ms. There was no consistent effect on ICI, ICF and LAI. The 5 Hz rENS and 5 Hz rTMS protocols failed to induce any change in corticospinal excitability when given alone. These findings show that 2 min of 5 Hz rPAS25ms produce a long‐lasting and somatotopically specific increase in corticospinal excitability, presumably by sensorimotor disinhibition.
Neurokinin‐1 receptor activation in Bötzinger complex evokes bradypnoea
Authors: Fong Angelina Y. , Potts Jeffrey T.
Published: July 2006

In the present study, we examined the role of the neurokinin‐1 receptor (NK1R) in the modulation of respiratory rhythm in a functionally identified bradypnoeic region of the ventral respiratory group (VRG) in the in situ arterially perfused juvenile rat preparation. In electrophysiologically and functionally identified bradypnoeic sites corresponding to the Bötzinger complex (BötC), microinjection of the selective NK1R agonist [Sar9‐Met(O2)11]‐substance P (SSP) produced a significant reduction in phrenic frequency mediated exclusively by an increase in expiratory duration (TE). The reduction was characterized by a significant increase in postinspiratory (post‐I) duration with no effect on either late‐expiratory duration (E2) or inspiratory duration (TI). In contrast, in a functionally identified tachypnoeic region, corresponding to the preBötzinger complex (Pre‐BötC), control microinjection of SSP elicited tachypnoea. Pretreatment with the NK1R antagonist CP99994 in the BötC significantly attenuated the bradypnoeic response to SSP injection and blunted the increase in TE duration. This effect of SSP mimicked the extension of TE produced by activation of the Hering‐Breuer reflex. Therefore, we hypothesized that activation of NK1Rs in the BötC is requisite for the expiratory‐lengthening effect of the Hering‐Breuer reflex. Unilateral electrical stimulation of the cervical vagus nerve produced bradypnoea by exclusively extending TE. Ipsilateral blockade of NK1Rs by CP99994 following blockade of the contralateral BötC by the GABAA receptor agonist muscimol significantly reduced the extension of TE produced by vagal stimulation. Results from the present study demonstrate that selective activation of NK1Rs in a functionally identified bradypnoeic region of the VRG can depress respiratory frequency by selectively lengthening post‐I duration and provide evidence that endogenous activation of NK1Rs in the BötC appears to be involved in the expiratory‐lengthening effect of the Hering‐Breuer reflex. In conclusion, our findings demonstrate that selective activation of NK1Rs in discrete regions of the VRG can exert functionally diverse effects on breathing.
Roles of nicotinic acetylcholine receptor β subunits in function of human α4‐containing nicotinic receptors
Authors: Wu Jie , Liu Qiang , Yu Kewei , Hu Jun , Kuo Yen‐Ping , Segerberg Marsha , St John Paul A. , Lukas Ronald J.
Published: July 2006

Naturally expressed nicotinic acetylcholine receptors (nAChR) containing α4 subunits (α4*‐nAChR) in combination with β2 subunits (α4β2‐nAChR) are among the most abundant, high‐affinity nicotine binding sites in the mammalian brain. β4 subunits are also richly expressed and colocalize with α4 subunits in several brain regions implicated in behavioural responses to nicotine and nicotine dependence. Thus, α4β4‐nAChR also may exist and play important functional roles. In this study, properties were determined of human α4β2‐ and α4β4‐nAChR heterologously expressedde novoin human SH‐EP1 epithelial cells. Whole‐cell currents mediated via human α4β4‐nAChR have ∼4‐fold higher amplitude than those mediated via human α4β2‐nAChR and exhibit much slower acute desensitization and functional rundown. Nicotinic agonists induce peak whole‐cell current responses typically with higher functional potency at α4β4‐nAChR than at α4β2‐nAChR. Cytisine and lobeline serve as full agonists at α4β4‐nAChR but are only partial agonists at α4β2‐nAChR. However, nicotinic antagonists, except hexamethonium, have comparable affinities for functional α4β2‐ and α4β4‐nAChR. Whole‐cell current responses show stronger inward rectification for α4β2‐nAChR than for α4β4‐nAChR at a positive holding potential. Collectively, these findings demonstrate that human nAChR β2 or β4 subunits can combine with α4 subunits to generate two forms of α4*‐nAChR with distinctive physiological and pharmacological features. Diversity in α4*‐nAChR is of potential relevance to nervous system function, disease, and nicotine dependence.
Analysis of the Tribological Mechanisms Arising in the Chemical Mechanical Polishing of Copper-Film Wafers When Using a Pad With Concentric Grooves
Authors: Lin Jen Fin , Chen Sheng-Chao , Ouyang Yu Long , Tsai Ming Shih
Published: July 2006

An average Reynolds equation considering the effects of a pad’s annular grooves and surface roughness is developed in this study to examine mixed lubrication in the chemical mechanical polishing (CMP) of a copper-film silicon wafer. This equation is obtained on the basis of the principle that the pressure gradients and volume flow rates in the direction normal to the border of a groove and a plateau as well as on two sides of the border must be equal. The continuities of volume flow rates and hydrodynamic pressure on two sides of the border as well as in the direction normal to the border of a groove and a plateau are satisfied in order to develop this Reynolds equation. The removal rate model is obtained by taking the concentration of active abrasives in the slurry and the pad grooves into account. Theoretical results are also shown in order to investigate the effects of changing the groove depth and width on the removal rate and the nonuniformity of a copper-film wafer. The application of concentric grooves in general can lower the suction pressure (negative pressure) formed between the pad and the wafer, elevate the wear rate, and reduce the nonuniformity. However, the influences of the groove depth on wear rate and nonuniformity become insignificant when the depth is excessively large. The removal rate is reduced by increasing the groove width such that it finally approaches to the result of a nongrooved pad.
A Thermohydrodynamic Analysis of Foil Journal Bearings
Authors: Peng Z-C. , Khonsari M. M.
Published: July 2006

A thermohydrodynamic model is developed for predicting the three-dimensional (3D) temperature field in an air-lubricated, compliant foil journal bearing. The model accounts for the compressibility and the viscosity-temperature characteristic of air and the compliance of the bearing surface. The results of numerical solutions are compared to published experimental measurements and reasonable agreement has been attained. Parametric studies covering a fairly wide range of operating speeds and load conditions were carried out to illustrate the usefulness of the model in terms of predicting the thermal performance of foil journal bearings.
Dynamic Analysis of Cage Behavior in a Tapered Roller Bearing
Authors: Sakaguchi Tomoya , Harada Kazuyoshi
Published: July 2006

A three-dimensional dynamic simulation analysis of a tapered roller bearing was performed using commercially available software. Without cage pocket shape simplification, the dynamic motion of the cage and rollers was calculated in six degrees of freedom. The motion of the cage and rollers was measured experimentally to verify the analysis. Under all axially loaded conditions, cage whirl was analytically predicted and experimentally confirmed. Whirl amplitude increased as the inner-ring rotational speed and axial-load magnitude increased. The maximum whirl amplitude reached the radial clearance between a roller and its pocket. Under combined load conditions, the cage also whirled. However, the whirl amplitude was smaller than only under axial load. Load distribution due to the addition of radial load to axial load equalized roller distribution. Equally distributed rollers limited the cage’s movable distance to circumferential clearance between a roller and its pocket.
A New Method Developed for Fractal Dimension and Topothesy Varying With the Mean Separation of Two Contact Surfaces
Authors: Liou Jeng Luen , Lin Jen Fin
Published: July 2006

Instead of a general consideration of the fractal dimension (D) and the topothesy (G*) as two invariants in the fractal analysis of surface asperities, these two roughness parameters in the present study are varied by changing the mean separation (d*) of two contact surfaces. The relationship between the fractal dimension and the mean separation is found first. By equating the structure functions developed in two different ways, the relationship among the scaling coefficient in the power spectrum function, the fractal dimension, and topothesy of asperity heights can be established. The variation of topothesy can be determined when the fractal dimension and the scaling coefficient have been obtained from the experimental results of the number of contact spots and the power spectrum function at different mean separations. A numerical scheme is developed in this study to determine the convergent values of fractal dimension and topothesy corresponding to a given mean separation. The theoretical results of the contact spot number predicted by the present model show good agreement with the reported experimental results. Both the fractal dimension and the topothesy are elevated by increasing the mean separation. Significant differences in the contact load or the total contact area are shown between the models of constant D and G* and variable D and G* as the mean separation is reduced to smaller values.
Numerical Simulations of the Flow Field Around the Rings of Mechanical Seals
Authors: Luan Zhaogao , Khonsari M. M.
Published: July 2006

The flow inside a seal chamber as induced by the influx of the flush fluid and the rotation of the primary ring is analyzed. The 3-D flow characteristic around the mating ring and the rotating ring are predicted by solving the Navier-Stokes equations in cylindrical coordinates. For this purpose, the pressure correction method was used in conjunction with the SIMPLE algorithm. A series of numerical solutions is presented that show the flow mechanism within the gap between the rings and the gland. The implication of the flow characteristic on the cooling of the rings is discussed.
Limits for High-Speed Operation of Gas Foil Bearings
Authors: Kim Tae Ho , Andrés Luis San
Published: July 2006

Commercial oil-free microturbomachinery implements gas foil bearings (GFBs) for reliable performance with improved efficiency. However, GFB modeling is still largely empirical, lacking experimental validation. An analysis of simple GFBs operating at large shaft speeds (infinite speed number) follows. The bearing ultimate load and stiffness coefficients are derived from simple algebraic equations for the gas film pressures at the equilibrium journal position and due to small amplitude journal motions, respectively. GFBs without a clearance or with assembly interference are easily modeled. The underlying elastic structure (bump foil strip) determines the ultimate load capacity of a GFB as well as its stiffnesses, along with the limiting journal displacement and structural deformation. Thus, an accurate estimation of the actual minimum film thickness is found prior to performing calculations with a complex computational model, even for the case of large loads that result in a journal eccentricity well exceeding the nominal clearance, if applicable. An initial assembly preload (interference between shaft and foil) increases the GFB static stiffness at both null and infinite rotor speeds. At infinite speed, cross-coupled stiffnesses are nil, and thus, GFBs are impervious to hydrodynamic whirl instability.
Bifurcation Analysis of the Load/Unload Systems With Multiple Flying Height States
Authors: Hwang Pyung , Khan Polina V.
Published: July 2006

The load/unload behavior of the hard disk drive slider is studied in terms of the air bearing static characteristics. The application of numerical continuation methods to calculate spacing diagrams is proposed. The algorithm that detects multiple flying height states and fold points is developed. The relationship between suspension force x-offset and critical preload is found for femto size sliders. The second fold corresponding to the critical preload for unloading is found in the negative air bearing force area. The range of x-offsets and preloads where bi-stable phenomenon exists is depicted on the stability diagram. The perturbation method is used to check the dynamic system characteristic values near the fold points and to determine the stability of the solution branches. The present procedure can be employed to study the multiple flying height states in the terms of any other pair of parameters besides the preload and x-offset.
Direct Observations of Emulsion Flow in Elastohydrodynamically Lubricated Contacts
Authors: Yang Haixia , Schmid Steven R. , Reich Ronald A. , Kasun Thomas J.
Published: July 2006

The lubrication mechanisms with oil-in-water emulsions have been extensively investigated based on the measurements of film thickness and/or tractions in the past few decades. However, direct observation of the emulsion flow, as a more direct method of evaluating suggested explanations, has been greatly restricted by the available instruments, especially the cameras used in collaboration with high-speed bearing simulators. In this paper, a newly devised digital video camera and a microscope were used to directly observe the emulsion flow in an elastohydrodynamic lubrication (EHL) inlet region at a wide range of speeds (0.012m∕s up to 1.5m∕s). Both EHL line and point contacts were considered. Previous observations of low speed oil droplet “stay,” “reverse,” and “penetration” behavior for low-speed line contact were confirmed and extended into high-speed line and point cases, and the results were compared with point contact where significant side flow was observed. Three tight emulsions with different mean droplet sizes were examined on an EHL rig to clarify the droplet behavior and investigate the effect of droplet size on entrainment.
Clonal Adaptive Radiation in a Constant Environment
Authors: Maharjan Ram , Seeto Shona , Notley-McRobb Lucinda , Ferenci Thomas
Published: July 2006

The evolution of new combinations of bacterial properties contributes to biodiversity and the emergence of new diseases. We investigated the capacity for bacterial divergence with a chemostat culture of
Escherichia coli
. A clonal population radiated into more than five phenotypic clusters within 26 days, with multiple variations in global regulation, metabolic strategies, surface properties, and nutrient permeability pathways. Most isolates belonged to a single ecotype, and neither periodic selection events nor ecological competition for a single niche prevented an adaptive radiation with a single resource. The multidirectional exploration of fitness space is an underestimated ingredient to bacterial success even in unstructured environments.
Persistence and Growth of Fecal
Bacteroidales
Assessed by Bromodeoxyuridine Immunocapture
Authors: Walters Sarah P. , Field Katharine G.
Published: July 2006

Extraintestinal growth of fecal bacteria can impair accurate assessment of watershed health. Anaerobic fecal bacteria belonging to the order
Bacteroidales
are attractive candidates for fecal source tracking because they have host-specific distributions and do not grow well in the presence of high oxygen concentrations. Growth of general and human-specific fecal
Bacteroidales
marker organisms in environmental samples (sewage) and persistence of the corresponding genetic markers were investigated using bromodeoxyuridine (BrdU) DNA labeling and immunocapture, followed by PCR detection. Background amplification of unlabeled controls occasionally occurred when a high number of PCR cycles was used. By using fluorescent detection of PCR products obtained after 15 cycles, which was determined to be quantitative, we enriched for BrdU-labeled DNA and did not detect unlabeled DNA. By using pure cultures of
Bacteroides vulgatus
, the ability of
Bacteroidales
bacteria to take up and incorporate BrdU into nascent DNA was confirmed. Fecal
Bacteroidales
organisms took up and incorporated BrdU into DNA during growth. In sewage incubated aerobically at the in situ temperature,
Bacteroidales
genetic marker sequences persisted for at least 24 h and
Bacteroidales
fecal bacteria grew for up to 24 h as well. Detection by PCR using a low, quantitative cycle number decreased the sensitivity of the assay such that we were unable to detect fecal
Bacteroidales
human-specific marker sequences in unlabeled or BrdU-labeled fractions, even when fluorescent detection was used. Using 30 PCR cycles with unlabeled fractions, human-specific
Bacteroidales
sequences were detected, and they persisted for up to 24 h in sewage. These data support the utility of BrdU labeling and immunocapture followed by length heterogeneity PCR or fluorescent detection using low numbers of PCR cycles. However, this method may not be sensitive enough to identify cells that are present at low densities in aquatic environments.
Biodesulfurization in Biphasic Systems Containing Organic Solvents
Authors: Tao Fei , Yu Bo , Xu Ping , Ma Cui Qing
Published: July 2006

Biphasic systems can overcome the problem of low productivity in conventional media and have been exploited for biocatalysis. Solvent-tolerant microorganisms are useful in biotransformation with whole cells in biphasic reactions. A solvent-tolerant desulfurizing bacterium,
Pseudomonas putida
A4, was constructed by introducing the biodesulfurizing gene cluster
dszABCD
, which was from
Rhodococcus erythropolis
XP, into the solvent-tolerant strain
P. putida
Idaho. Biphasic reactions were performed to investigate the desulfurization of various sulfur-containing heterocyclic compounds in the presence of various organic solvents.
P. putida
A4 had the same substrate range as
R. erythropolis
XP and could degrade dibenzothiophene at a specific rate of 1.29 mM g (dry weight) of cells
−1
h
−1
for the first 2 h in the presence of 10% (vol/vol)
p
-xylene.
P. putida
A4 was also able to degrade dibenzothiophene in the presence of many other organic solvents at a concentration of 10% (vol/vol). This study is a significant step in the exploration of the biotechnological potential of novel biocatalysts for developing an efficient biodesulfurization process in biphasic reaction mixtures containing toxic organic solvents.
Weakening Effect of Cell Permeabilizers on Gram-Negative Bacteria Causing Biodeterioration
Authors: Alakomi H.-L. , Paananen A. , Suihko M.-L. , Helander I. M. , Saarela M.
Published: July 2006

Gram-negative bacteria play an important role in the formation and stabilization of biofilm structures on stone surfaces. Therefore, the control of growth of gram-negative bacteria offers a way to diminish biodeterioration of stone materials. The effect of potential permeabilizers on the outer membrane (OM) properties of gram-negative bacteria was investigated and further characterized. In addition, efficacy of the agents in enhancing the activity of a biocide (benzalkonium chloride) was assessed. EDTA, polyethylenimine (PEI), and succimer (meso-2,3-dimercaptosuccinic) were shown to be efficient permeabilizers of the members ofPseudomonasandStenotrophomonasgenera, as indicated by an increase in the uptake of a hydrophobic probe (1-N-phenylnaphthylamine) and sensitization to hydrophobic antibiotics. Visualization ofPseudomonascells treated with EDTA or PEI by atomic force microscopy revealed damage in the outer membrane structure. PEI especially increased the surface area and bulges of the cells. Topographic images of EDTA-treated cells were compatible with events assigned for the effect of EDTA on outer membranes, i.e., release of lipopolysaccharide and disintegration of OM structure. In addition, the effect of EDTA treatment was visualized in phase-contrast images as large areas with varying hydrophilicity on cell surfaces. In liquid culture tests, EDTA and PEI supplementation enhanced the activity of benzalkonium chloride toward the target strains. Use of permeabilizers in biocide formulations would enable the use of decreased concentrations of the active biocide ingredient, thereby providing environmentally friendlier products.
In Vivo Transduction of an Stx-Encoding Phage in Ruminants
Authors: Cornick Nancy A. , Helgerson Amy F. , Mai Volker , Ritchie Jennifer M. , Acheson David W. K.
Published: July 2006

We assessed the ability of a kanamycin-marked Stx phage to move into a commensal, ovineEscherichia colistrain in the ruminant gastrointestinal tract. Transduction was detected in 19/24 sheep tested, resulting in the recovery of 47 transductants. Subtherapeutic doses of the quinolone antibiotic enrofloxacin did not increase the rate of transduction.
Collective Bacterial Dynamics Revealed Using a Three-Dimensional Population-Scale Defocused Particle Tracking Technique
Authors: Wu Mingming , Roberts John W. , Kim Sue , Koch Donald L. , DeLisa Matthew P.
Published: July 2006

An ability to monitor bacterial locomotion and collective dynamics is crucial to our understanding of a number of well-characterized phenotypes including biofilm formation, chemotaxis, and virulence. Here, we report the tracking of multiple swimming
Escherichia coli
cells in three spatial dimensions and at single-cell resolution using a novel three-dimensional (3D) defocused particle tracking (DPT) method. The 3D trajectories were generated for wild-type
Escherichia coli
strain RP437 as well as for isogenic derivatives that display smooth swimming due to a
cheA
deletion (strain RP9535) or incessant tumbling behavior due to a
cheZ
deletion (strain RP1616). The 3D DPT method successfully differentiated these three modes of locomotion and allowed direct calculation of the diffusion coefficient for each strain. As expected, we found that the smooth swimmer diffused more readily than the wild type, and both the smooth swimmer and the wild-type cells exhibited diffusion coefficients that were at least two orders of magnitude larger than that of the tumbler. Finally, we found that the diffusion coefficient increased with increasing cell density, a phenomenon that can be attributed to the hydrodynamic disturbances caused by neighboring bacteria.
Anaerobic Sulfide Oxidation with Nitrate by a Freshwater
Beggiatoa
Enrichment Culture
Authors: Kamp Anja , Stief Peter , Schulz-Vogt Heide N.
Published: July 2006

A lithotrophic freshwater
Beggiatoa
strain was enriched in O
2
-H
2
S gradient tubes to investigate its ability to oxidize sulfide with NO
3
−
as an alternative electron acceptor. The gradient tubes contained different NO
3
−
concentrations, and the chemotactic response of the
Beggiatoa
mats was observed. The effects of the
Beggiatoa
sp. on vertical gradients of O
2
, H
2
S, pH, and NO
3
−
were determined with microsensors. The more NO
3
−
that was added to the agar, the deeper the
Beggiatoa
filaments glided into anoxic agar layers, suggesting that the
Beggiatoa
sp. used NO
3
−
to oxidize sulfide at depths below the depth that O
2
penetrated. In the presence of NO
3
−
Beggiatoa
formed thick mats (>8 mm), compared to the thin mats (ca. 0.4 mm) that were formed when no NO
3
−
was added. These thick mats spatially separated O
2
and sulfide but not NO
3
−
and sulfide, and therefore NO
3
−
must have served as the electron acceptor for sulfide oxidation. This interpretation is consistent with a fourfold-lower O
2
flux and a twofold-higher sulfide flux into the NO
3
−
-exposed mats compared to the fluxes for controls without NO
3
−
. Additionally, a pronounced pH maximum was observed within the
Beggiatoa
mat; such a pH maximum is known to occur when sulfide is oxidized to S
0
with NO
3
−
as the electron acceptor.
Excisable Cassettes: New Tools for Functional Analysis of
Streptomyces
Genomes
Authors: Raynal Alain , Karray Fatma , Tuphile Karine , Darbon-Rongère Emmanuelle , Pernodet Jean-Luc
Published: July 2006

The functional analysis of microbial genomes often requires gene
inactivation. We constructed a set of cassettes consisting of single
antibiotic resistance genes flanked by the
attL
and
attR
sites resulting from site-specific integration of the
Streptomyces
pSAM2 element. These cassettes can easily be used
to inactivate genes by in-frame deletion in
Streptomyces
by a
three-step strategy. In the first step, in
Escherichia coli
,
the cassette is inserted into a cloned copy of the gene to be
inactivated. In the second step, the gene is replaced by homologous
recombination in
Streptomyces
, allowing substitution of the
wild-type target gene with its inactivated counterpart. In the third
step, the cassette can be removed by expression of the pSAM2 genes
xis
and
int
. The resulting strains are marker-free
and contain an “
attB
-like” sequence of 33, 34,
or 35 bp with no stop codon if the cassette is correctly chosen. Thus,
a gene can be disrupted by creating an in-frame deletion, avoiding
polar effects if downstream genes are cotranscribed with the target
gene. A set of cassettes was constructed to contain a hygromycin or
gentamicin resistance gene flanked by the
attL
and
attR
sites. The initial constructions carrying convenient
cloning sites allow the insertion of any other marker gene. We tested
insertion and excision by inserting a cassette into
orf3
, the
third gene of an operon involved in spiramycin biosynthesis. We
verified that the cassette exerted a polar effect on the transcription
of downstream genes but that, after excision, complementation with
orf3
alone restored spiramycin
production.
Seasonal Variations in Virus-Host Populations in Norwegian Coastal Waters: Focusing on the Cyanophage Community Infecting Marine
Synechococcus
spp
Authors: Sandaa Ruth-Anne , Larsen Aud
Published: July 2006

Viruses are ubiquitous components of the marine ecosystem. In the current study we investigated seasonal variations in the viral community in Norwegian coastal waters by pulsed-field gel electrophoresis (PFGE). The results demonstrated that the viral community was diverse, displaying dynamic seasonal variation, and that viral populations of 29 different sizes in the range from 26 to 500 kb were present. Virus populations from 260 to 500 kb and dominating autotrophic pico- and nanoeukaryotes showed similar dynamic variations. Using flow cytometry and real-time PCR, we focused in particular on one host-virus system:
Synechococcus
spp. and cyanophages. The two groups covaried throughout the year and were found in the highest amounts in fall with concentrations of 7.3 × 10
4
Synechococcus
cells ml
−1
and 7.2 × 10
3
cyanophage ml
−1
. By using primers targeting the g20 gene in PCRs on DNA extracted from PFGE bands, we demonstrated that cyanophages were found in a genomic size range of 26 to 380 kb. The genetic richness of the cyanophage community, determined by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified g20 gene fragments, revealed seasonal shifts in the populations, with one community dominating in spring and summer and a different one dominating in fall. Phylogenetic analysis of the sequences originating from PFGE and DGGE bands grouped the sequences into three groups, all with homology to cyanomyoviruses present in cultures. Our results show that the cyanophage community in Norwegian coastal waters is dynamic and genetically diverse and has a surprisingly wide genomic size range.
Okadaic Acid, an Apoptogenic Toxin for Symbiotic/Parasitic Annelids in the Demosponge
Suberites domuncula
Authors: Schröder Heinz C. , Breter Hans J. , Fattorusso Ernesto , Ushijima Hiroshi , Wiens Matthias , Steffen Renate , Batel Renato , Müller Werner E. G.
Published: July 2006

The role of okadaic acid (OA) in the defense system of the marine demosponge
Suberites domuncula
against symbiotic/parasitic annelids was examined. Bacteria within the mesohyl produced okadaic acid at concentrations between 32 ng/g and 58 ng/g of tissue (wet weight). By immunocytochemical methods and by use of antibodies against OA, we showed that the toxin was intracellularly stored in vesicles. Western blotting experiments demonstrated that OA also existed bound to a protein with a molecular weight of 35,000 which was tentatively identified as a galectin (by application of antigalectin antibodies). Annelids that are found in
S. domuncula
undergo apoptotic cell death. OA is one candidate inducer molecule of this process, since this toxin accumulated in these symbionts/parasites. Furthermore, we identified the cDNA encoding the multifunctional prosurvival molecule BAG-1 in
S. domuncula
; it undergoes strong expression in the presence of the annelid. Our data suggest that sponges use toxins (here, OA) produced from bacteria to eliminate metazoan symbionts/parasites by apoptosis.
Differential Adsorption of Occluded and Nonoccluded Insect-Pathogenic Viruses to Soil-Forming Minerals
Authors: Christian Peter D. , Richards Andrew R. , Williams Trevor
Published: July 2006

Soil represents the principal environmental reservoir of many insect-pathogenic viruses. We compared the adsorption and infectivity of one occluded and two nonoccluded viruses,
Helicoverpa armigera single nucleopolyhedrovirus
(HaSNPV) (
Baculoviridae
),
Cricket paralysis virus
(CrPV) (
Dicistroviridae
), and
Invertebrate iridescent virus 6
(IIV-6) (
Iridoviridae
), respectively, in mixtures with a selection of soil-forming minerals. The relative infective titers of HaSNPV and CrPV were unchanged or slightly reduced in the presence of different minerals compared to their titers in the absence of the mineral. In contrast, the infective titer of IIV-6 varied according to the mineral being tested. In adsorption studies, over 98% of HaSNPV occlusion bodies were adsorbed by all the minerals, and a particularly high affinity was observed with ferric oxide, attapulgite, and kaolinite. In contrast, the adsorption of CrPV and IIV-6 differed markedly with mineral type, with low affinity to bentonites and high affinity to ferric oxide and kaolinite. We conclude that interactions between soil-forming minerals and insect viruses appear to be most important in nucleopolyhedroviruses, followed by invertebrate iridescent viruses, and least important in CrPV, which may reflect the ecology of these pathogens. Moreover, soils with a high content of iron oxides or kaolinite would likely represent highly effective reservoirs for insect-pathogenic viruses.
Transfer of Genetic Material between Pathogenic and Food-Borne Yeasts
Authors: Mentel Marek , Špírek Mário , Jørck-Ramberg Dorte , Piškur Jure
Published: July 2006

Many pathogenic yeast species are asexual and therefore not involved in intra- or interspecies mating. However, high-frequency transfer of plasmid DNA was observed when pathogenic and food-borne yeasts were grown together. This property could play a crucial role in the spread of virulence and drug resistance factors among yeasts.
Assessment of Factors Influencing Direct Enumeration of Viruses within Estuarine Sediments
Authors: Helton Rebekah R. , Liu Ling , Wommack K. Eric
Published: July 2006

Accurate enumeration of viruses within environmental samples is critical for investigations of the ecological role of viruses and viral infection within microbial communities. This report evaluates differences in viral and bacterial direct counts between estuarine sediment samples which were either immediately processed onboard ship or frozen at −20°C and later processed. Viral and bacterial abundances were recorded at three stations spanning the length of the Chesapeake Bay in April and June 2003 within three sediment fractions: pore water (PW), whole sediment (WS), and sediment after pore water removal (AP). No significant difference in viral abundance was apparent between extracts from fresh or frozen sediments. In contrast, bacterial abundance was significantly lower in the samples subjected to freezing. Both bacterial and viral abundance showed significant differences between sediment fractions (PW, WS, or AP) regardless of the fresh or frozen status. Although pore water viral abundance has been used in the past as a measurement of viral abundance in sediments, this fraction accounted for only ca. 5% of the total sediment viral abundance across all samples. The effect of refrigerated storage of sediment viral extracts was also examined and showed that, within the first 2 h, viral abundance decreased ca. 30% in formalin-fixed extracts and 66% in unfixed extracts. Finally, the reliability of direct viral enumeration via epifluorescence microscopy was tested by using DNase treatment of WS extractions. These tests indicated that a large fraction (>86%) of the small SYBR gold fluorescing particles are likely viruses.
Marine and Freshwater Cyanophages in a Laurentian Great Lake: Evidence from Infectivity Assays and Molecular Analyses of g20 Genes
Authors: Wilhelm Steven W. , Carberry Matthew J. , Eldridge Melanie L. , Poorvin Leo , Saxton Matthew A. , Doblin Martina A.
Published: July 2006

While it is well established that viruses play an important role in the structure of marine microbial food webs, few studies have directly addressed their role in large lake systems. As part of an ongoing study of the microbial ecology of Lake Erie, we have examined the distribution and diversity of viruses in this system. One surprising result has been the pervasive distribution of cyanophages that infect the marine cyanobacterial isolateSynechococcussp. strain WH7803. Viruses that lytically infect this cyanobacterium were identified throughout the western basin of Lake Erie, as well as in locations within the central and eastern basins. Analyses of the gene encoding the g20 viral capsid assembly protein (a conservative phylogenetic marker for the cyanophage) indicate that these viruses, as well as amplicons from natural populations and the ballast of commercial ships, are related to marine cyanophages but in some cases form a unique clade, leaving questions concerning the native hosts of these viruses. The results suggest that cyanophages may be as important in freshwater systems as they are known to be in marine systems.
Structure, Organization, and Transcriptional Regulation of a Family of Copper Radical Oxidase Genes in the Lignin-Degrading Basidiomycete
Phanerochaete chrysosporium
Authors: Vanden Wymelenberg Amber , Sabat Grzegorz , Mozuch Michael , Kersten Philip J. , Cullen Dan , Blanchette Robert A.
Published: July 2006

The white rot basidiomycete
Phanerochaete chrysosporium
produces an array of nonspecific extracellular enzymes thought to be involved in lignin degradation, including lignin peroxidases, manganese peroxidases, and the H
2
O
2
-generating copper radical oxidase, glyoxal oxidase (GLX). Preliminary analysis of the
P. chrysosporium
draft genome had identified six sequences with significant similarity to GLX and designated
cro1
through
cro6
. The predicted mature protein sequences diverge substantially from one another, but the residues coordinating copper and constituting the radical redox site are conserved. Transcript profiles, microscopic examination, and lignin analysis of inoculated thin wood sections are consistent with differential regulation as decay advances. The
cro2
-encoded protein was detected by liquid chromatography-tandem mass spectrometry in defined medium. The
cro2
cDNA was successfully expressed in
Aspergillus nidulans
under the control of the
A. niger
glucoamylase promoter and secretion signal. The recombinant CRO2 protein had a substantially different substrate preference than GLX. The role of structurally and functionally diverse
cro
genes in lignocellulose degradation remains to be established.
Purine Biosynthesis, Riboflavin Production, and Trophic-Phase Span Are Controlled by a Myb-Related Transcription Factor in the Fungus
Ashbya gossypii
Authors: Mateos Laura , Jiménez Alberto , Revuelta José L. , Santos María A.
Published: July 2006

Ashbya gossypii
is a natural riboflavin overproducer used in the industrial production of the vitamin. We have isolated an insertional mutant exhibiting higher levels of riboflavin production than the wild type. DNA analysis of the targeted locus in the mutant strain revealed that a syntenic homolog of the
Saccharomyces cerevisiae BAS1
gene, a member of the Myb family of transcription factors, was inactivated. Directed gene disruption of Ag
BAS1
confirmed the phenotype observed for the insertional mutant, and the
Δbas1
mutant also showed auxotrophy for adenine and several growth defects, such as a delay in the germination of the spores and an abnormally prolonged trophic phase. Additionally, we demonstrate that the DNA-binding domain of AgBas1p is able to bind to the Bas1-binding motifs in the Ag
ADE4
promoter; we also show a clear nuclear localization of a green fluorescent protein-Bas1 fusion protein. Real-time quantitative PCR analyses comparing the wild type and the
Δbas1
mutant revealed that Ag
BAS1
was responsible for the adenine-mediated regulation of the purine and glycine pathways, since the transcription of the
ADE4
and
SHM2
genes was virtually abolished in the
Δbas1
mutant. Furthermore, the transcription of
ADE4
and
SHM2
in the
Δbas1
mutant did not diminish during the transition from the trophic to the productive phase did not diminish, in contrast to what occurred in the wild-type strain. A C-terminal deletion in the Ag
BAS1
gene, comprising a hypothetical regulatory domain, caused constitutive activation of the purine and glycine pathways, enhanced riboflavin overproduction, and prolonged the trophic phase. Taking these results together, we propose that in
A. gossypii
, Ag
BAS1
is an important transcription factor that is involved in the regulation of different physiological processes, such as purine and glycine biosynthesis, riboflavin overproduction, and growth.
Bacteriocin T8, a Novel Class IIa
sec
-Dependent Bacteriocin Produced by
Enterococcus faecium
T8, Isolated from Vaginal Secretions of Children Infected with Human Immunodeficiency Virus
Authors: De Kwaadsteniet M. , Fraser T. , Van Reenen C. A. , Dicks L. M. T.
Published: July 2006

Enterococcus faecium
T8, isolated from vaginal secretions of children with human immunodeficiency virus, produces a class IIa
sec
-dependent bacteriocin that is structurally different from three other class IIa
sec
-dependent bacteriocins, i.e., enterocin P and an enterocin P-like bacteriocin, produced by
Enterococcus faecium
, and bacteriocin 31, produced by
Enterococcus faecalis
, and from a class III bacteriocin produced by
E. faecalis
. The genes encoding the bacteriocin, immunity protein, mobilization protein, and relaxase nuclease are located on a 7-kb plasmid. Bacteriocin T8 has a molecular mass of 5.1 kDa based on its DNA sequence, similar to the 5.0 kDa recorded for bacteriocin 31 but larger than the 4.6 kDa reported for enterocin P. At the amino acid level, bacteriocin T8 is 69% homologous to bacteriocin 31 and 47% homologous to enterocin P. Bacteriocin T8 is active against
E. faecalis
isolated from patients diagnosed with vaginosis, against
Lactobacillus sakei
, and against a
Propionibacterium
sp. The peptide is heat stable (60 min at 100°C) and remains active in phosphate buffer from pH 4.0 to 10.0. The mode of activity is bactericidal, as determined with
E. faecalis
.
Acetate Oxidation Is the Dominant Methanogenic Pathway from Acetate in the Absence of
Methanosaetaceae
Authors: Karakashev Dimitar , Batstone Damien J. , Trably Eric , Angelidaki Irini
Published: July 2006

The oxidation of acetate to hydrogen, and the subsequent conversion of hydrogen and carbon dioxide to methane, has been regarded largely as a niche mechanism occurring at high temperatures or under inhibitory conditions. In this study, 13 anaerobic reactors and sediment from a temperate anaerobic lake were surveyed for their dominant methanogenic population by using fluorescent in situ hybridization and for the degree of acetate oxidation relative to aceticlastic conversion by using radiolabeled [2-
14
C]acetate in batch incubations. When
Methanosaetaceae
were not present, acetate oxidation was the dominant methanogenic pathway. Aceticlastic conversion was observed only in the presence of
Methanosaetaceae
.
Generating Tetracycline-Inducible Auxotrophy in
Escherichia coli
and
Salmonella enterica
Serovar Typhimurium by Using an Insertion Element and a Hyperactive Transposase
Authors: Köstner Martin , Schmidt Britta , Bertram Ralph , Hillen Wolfgang
Published: July 2006

We report the construction and application of a novel insertion element for transposase-mediated mutagenesis in gram-negative bacteria. Besides Km
r
as a selectable marker, the insertion element InsTet
G−
1 carries the anhydrotetracycline (atc)-regulated outward-directed P
A
promoter so that atc-dependent conditional gene knockouts or knockdowns are generated. The complex formed between the purified hyperactive transposase and InsTet
G−
1 was electroporated into
Escherichia coli
or
Salmonella enterica
serovar Typhimurium, and mutant pools were collected. We used
E. coli
strains with either TetR or the reverse variant revTetR
r2
, while only TetR was employed in
Salmonella
. Screening of the InsTet
G−
1 insertion mutant pools revealed 15 atc-regulatable auxotrophic mutants for
E. coli
and 4 atc-regulatable auxotrophic mutants for
Salmonella
. We have also screened one
Salmonella
mutant pool in murine macrophage-like J774-A.1 cells using ampicillin enrichment. Two mutants with the InsTet
G−
1 insertion in the gene
pyrE
or
argA
survived this procedure, indicating a reduced intracellular growth rate in J774-A.1 cells. The nature of the mutants and the modes of their regulation are discussed.