bluuebunny/crossref_metadata_2025 huggingface dataset:
Blindness and Bias in a Trial of Antidepressant Medication For Chronic Tension-Type Headache
Authors: Holroyd KA , Tkachuk G , O'Donnell F , Cordingley GE
Published: June 2006

This study aimed to examine penetration of the blind in a randomized, placebo-controlled trial. Neurologists' ratings of improvement and medication side-effects, participants' ratings of improvement and daily diary recordings of headaches were assessed along with participants' and neurologists' guesses about treatment group placement in participants who completed at least 3 months of treatment ( N = 169). Despite blinding, treating neurologists successfully identified the medication condition for 82± of participants receiving medication only; trial participants accurately identified their medication condition when receiving active medication (77± of participants), but not when receiving placebo. Concurrent stress-management therapy reduced, but did not eliminate penetration of the blind. Irrespective of drug condition, when participants were improved they were judged to be on active medication and when unimproved they were judged to be on placebo. However, neurologists' ratings of improvement, participants' reports of improvement and daily headache recordings yielded equivalent outcomes. Penetration of the blind needs to be assessed, not assumed in clinical trials in headache. However, penetration of the blind did not produce a prodrug bias as has been asserted by critics. Better methods of assessing and quantifying blindness are needed.
Popular geopolitics and audience dispositions: James Bond and the Internet Movie Database (IMDb)
Authors: Dodds Klaus
Published: June 2006

This paper is concerned with the further theoretical development of popular geopolitics with explicit reference to audience dispositions and reception more generally. Using the James Bond film series, and in particular Die Another Day (2002) and the Internet Movie Database (IMDb), it is contended that political geographers need to better understand how audiences and film fans in particular interpret the popular geopolitics of film. One of the striking features of fans who comment about their interest in James Bond on the IMDb is the degree to which these films invite detailed and devoted readings of plotlines, character development, links to contemporary geopolitics and what might be called ‘Bond trivia’. The paper concludes with a call for further investigation into the way in which film is consumed by fans and non‐fans alike, alongside inter alia a more detailed appreciation of the role of on‐line discussion fora such as the IMDb.
Selling the private asylum: therapeutic landscapes and the (re)valorization of confinement in the era of community care
Authors: Moon Graham , Kearns Robin , Joseph Alun
Published: June 2006

This paper examines the role of place in the positioning and survival of the contemporary private asylum. While community care is now the dominant mental health care modality in most Western health economies, some asylum care has survived, often in the private sector, catering for a clientele able and willing to pay for a non‐standard approach to care. We consider how landscapes, buildings and services provide a basis for marketing and selling asylum care. Drawing on fieldwork, documentary analysis and visual evidence, we analyse the representational strategies of the Homewood Health Centre Inc. (Ontario, Canada), the Ashburn Private Psychiatric Clinic (Dunedin, New Zealand) and the acute psychiatric hospitals within the Priory Group (UK). The paper draws conclusions about the role of therapeutic landscapes in the contemporary asylum, place marketing and the (re)valorization of historical ideas of asylum.
Accrual Budgeting: Accounting Treatment of Key Public Sector Items and Implications for Fiscal Policy
Authors: MARTÍ CARIDAD
Published: June 2006

Budgeting in accrual terms is one of the most controversial issues in public sector accounting. In this paper, we analyze the accounting treatment of problematic elements of the financial statements when introducing accrual budgeting, and discuss the effects of the analyzed accounting alternatives on fiscal policy. We focus on three pioneer countries in the implementation of accrual budgeting and accounting: the United Kingdom, Sweden, and New Zealand. The accounting standards of the International Public Sector Accounting Standards Board, the European System of Accounts, and the Government Finance Statistics Manual of the International Monetary Fund are taken as benchmarks.
Glass Formation and Crystallization Behavior of a Novel GeS2–Sb2S3–PbS Chalcogenide Glass System
Authors: Xia Fang , Zhang Xianghua , Ren Jing , Chen Guorong , Ma Hongli , Adam J. L.
Published: June 2006

In the present work, a large glass‐forming region was found in the novel GeS2–Sb2S3–PbS system, in which up to 58 at.% PbS could be incorporated without deteriorating the thermal and physical properties of glasses. Infrared (IR) transmitting glass ceramics with a large amount of small‐sized crystals (<100 nm) were then produced by choosing sub‐stable compositions and annealing at fairly low temperatures (15°–30°C above Tg) for long durations (up to 100 h). Crystals were identified by X‐ray diffraction as Pb2GeS4, PbGeS3, PbS, PbSb2S4, etc., depending on base glass compositions. Compared with base glasses, glass ceramics showed much improved thermal shock resistance and fracture toughness, making them good candidate materials for IR optics.
Multi‐micronutrient Sprinkles including a low dose of iron provided as microencapsulated ferrous fumarate improves haematologic indices in anaemic children: a randomized clinical trial
Authors: Christofides Anna , Asante Kwaku Poku , Schauer Claudia , Sharieff Waseem , Owusu‐Agyei Seth , Zlotkin Stanley
Published: June 2006

Home‐fortification of complementary foods with micronutrients (including iron) as Sprinkles is a new strategy to control iron deficiency and anaemia in developing countries. However, the most effective dose and form of iron is not known. The purpose of this study was to compare the efficacy of various doses (12.5, 20 or 30 mg) and treatment methods (multi‐micronutrient Sprinkles vs. ferrous sulphate drops) on haemoglobin (Hb) concentration after 8 weeks of treatment in anaemic children. In total, 133 anaemic Ghanaian children (Hb 70–99 g L−1) aged 6–18 months were randomly assigned to one of five daily interventions for 8 weeks. Out of the five interventions, four used Sprinkles, and one used iron drops. Of the four Sprinkles groups, three included 12.5, 20 or 30 mg of iron as ferrous fumarate, and one included 20 mg of iron as ferric pyrophosphate. The iron drops group included 12.5 mg of iron as liquid ferrous sulphate. Hb concentrations were measured at baseline, week 3 and week 8. The primary outcome measure was Hb concentration at 8 weeks after treatment. We compared differences in Hb and ferritin concentrations and prevalence of iron deficiency anaemia (Hb < 100 g L−1 and soluble transferrin receptor concentrations >8.5 mg L−1) from baseline to 8 weeks within and between groups. Adherence and reporting of side effects (staining of the teeth, ease of use, diarrhoea and darkening of stools) were compared between groups. Mean change in Hb was 1.4 g L−1 (SD = 1.8) (P = 0.0001). Change in Hb concentrations from baseline to 8 weeks was significant in all groups (P = 0.0001–0.0007), with no differences across groups. Geometric means of serum ferritin varied from 18.6 to 44.0 µg L−1 at baseline. At week 8, these means were in the interval of 48.0–78.3 µg L−1, with no group differences. Prevalence of iron deficiency anaemia decreased significantly from baseline to 8 weeks in all groups with the exception of the iron drops group, with no group differences. Adherence was lower in the drops group (64%) as compared with Sprinkles groups (84%). Greater staining of the teeth and less ease of use were reported in the drops group as compared with Sprinkles groups. A dose as low as 12.5 mg of iron as ferrous fumarate when provided as Sprinkles may be effective in anaemic children.
Family living among birds
Authors: Ekman Jan
Published: June 2006

Family cohesion in birds is often explained as a product of limitations on breeding opportunities leading to delayed dispersal among the offspring. Yet, it is not independent reproduction but floating (queuing outside the natal territory for a breeding opportunity) that is the alternative to delaying dispersal. In contrast to a strong phylogenetic bias in cooperative breeding that has been taken to indicate a hard‐wired behaviour maintained by evolutionary inertia, offspring behaviour is plastic and facultative in group living in the short term, indicating that family cohesion is under selection. Non‐breeding offspring could gain inclusive fitness that would promote family cohesion from delaying dispersal and providing alloparental care to subsequent broods to boost group productivity. This holds in particular for offspring hatched from early broods in multi‐brooded species that have the opportunity to gain inclusive fitness from help in rearing siblings hatched from later broods in the same season. Yet, seasonality will circumscribe the potential for alloparenting to be the immediate factor selecting for family cohesion. The option of gaining inclusive fitness from providing alloparental care is not open to the offspring among single‐brooded species until after they have survived a non‐breeding season, rather indicating kin cooperation returning enhanced survival prospects from general group living effects as a more immediate factor selecting for family cohesion. Indeed, a variety of family groups maintained in the absence of alloparental care underlines the capacity of general group living enhancing survival as a primary agent selecting for family cohesion. These seasonal constraints on fitness components selecting for family cohesion may contribute to the large scale geographical pattern with a relative paucity of family cohesions among bird species in the northern hemisphere.
Plasticity of GABAAreceptor subunit expression during the oestrous cycle of the rat: implications for premenstrual syndrome in women
Authors: Lovick T. A.
Published: June 2006

Many women experience psychological changes during the luteal phase of their menstrual cycle. The late luteal (premenstrual) phase, when symptoms become most severe, is characterized by declining levels of ovarian progesterone. In female rats, withdrawal from prolonged dosing with progesterone leads to upregulation of α4 and δ subunits of the GABAAreceptor in several brain regions. During the oestrous cycle of the rat, the natural fall in progesterone that occurs in late dioestrus is associated with a parallel increase in expression of α4, β1 and δ GABAAreceptor subunits in neurones in the periaqueductal grey matter (PAG), suggesting that new receptors of the α4β1δ composition have been formed. Recombinant α4β1δ receptors display a low EC50for GABA, which is consistent with activation by extracellular levels of GABA. They are also likely to be located extrasynaptically and to carry tonic currents. In the PAG, a region involved in mediating panic‐like anxiety, α4, β1 and δ GABAAreceptor subunits are located principally on GABAergic interneurones. On‐going GABAergic neuronal activity normally limits and controls the excitability of the panic circuitry. During late dioestrus, when expression of α4, β1 and δ subunits on GABAergic interneurones is upregulated, the increase in tonic current would be expected to lead to a reduction in the activity of the GABAergic population. Thus the panic circuitry would become intrinsically more excitable. It is suggested that during the menstrual cycle in women, plasticity of GABAAreceptor subunit expression in brain regions such as the PAG, which are involved in mediating anxiety behaviour, may underlie some of the changes in mood that occur during the premenstrual period.
Regulation of physiological responses to continuous and intermittent hypoxia by hypoxia‐inducible factor 1
Authors: Semenza Gregg L.
Published: June 2006

Hypoxia‐inducible factor 1 (HIF‐1) is a heterodimeric transcription factor composed of HIF‐1α and HIF‐1β subunits that functions as a master regulator of oxygen homeostasis. Oxygen‐dependent hydroxylation of HIF‐1α provides a mechanism that allows changes in oxygenation to be transduced to the nucleus, leading to changes in gene expression. Hypoxia‐inducible factor 1 plays critical roles in development, physiology and disease pathogenesis. Analyses of mice that are heterozygous for a null allele at the locus encoding the HIF‐1α subunit have demonstrated that partial deficiency of HIF‐1 is sufficient to impair multiple physiological responses to continuous and intermittent hypoxia.
The influence of heart rate on baroreceptor fibre activity in the carotid sinus and aortic depressor nerves of the rabbit
Authors: Barrett Carolyn J. , Bolter Chris P.
Published: June 2006

The arterial baroreceptors and their afferent fibres provide the sensory arm of the reflex that regulates systemic arterial pressure. We have examined whether the relationship between mean baroreceptor discharge and mean arterial pressure is altered when heart rate changes. Experiments were performed on pentobarbitone‐anaesthetized rabbits. We recorded the activity of single and multifibre preparations of the carotid sinus (CSN) and aortic depressor nerves (ADN). Data were collected under control conditions and while heart rate was increased by ∼30–35% by right atrial pacing. Baroreceptor regions were exposed to ramps of pressure (from ∼25 to 140 mmHg, at ∼0.5–1 mmHg s−1), generated by inflation and deflation of cuffs placed around the inferior vena cava and descending thoracic aorta. Response curves relating baroreceptor discharge to mean pressure were constructed and fitted with third‐order polynomial expressions. To provide a measure of an effect of an increase in heart rate on the response curve in the region of the normal operating pressure, we calculated the position of the test response curve relative to the position of the control curve at 90 mmHg (δBP90). For the ADN, the activity of single fibres (presumptive myelinated fibres) was unaffected by increasing heart rate (δBP90=+0.1 ± 1.0 mmHg), while single fibres in the CSN showed a small increase in activity (δBP90=−1.5 ± 0.3 mmHg). In multifibre preparations there was a small increase in activity that may be attributable to additional activity in unmyelinated fibres (ADN, δBP90=−3.4 ± 1.2 mmHg; CSN, δBP90=−5.2 ± 0.9 mmHg). We conclude that the mean discharge of arterial baroreceptors remains a reliable index of mean arterial pressure in the presence of substantial changes in heart rate.
Involvement of adenosine in depression of synaptic transmission during hypercapnia in isolated spinal cord of neonatal rats
Authors: Otsuguro Ken‐ichi , Yamaji Yoshihiko , Ban Masaaki , Ohta Toshio , Ito Shigeo
Published: June 2006

Adenosine is one of the most important neuromodulators in the CNS, both under physiological and pathological conditions. In the isolated spinal cord of the neonatal rat in vitro, acute hypercapnic acidosis (20% CO2, pH 6.7) reversibly depressed electrically evoked spinal reflex potentials. This depression was partially reversed by 8‐cyclopentlyl‐1,3‐dimethylxanthine (CPT), a selective A1 adenosine receptor antagonist. Isohydric hypercapnia (20% CO2, pH 7.3), but not isocapnic acidosis (5% CO2, pH 6.7), depressed the reflex potentials, which were also reversed by CPT. An ecto‐5′‐nucleotidase inhibitor did not affect the hypercapnic acidosis‐evoked depression. An inhibitor of adenosine kinase, but not deaminase, mimicked the inhibitory effect of hypercapnic acidosis on the spinal reflex potentials. Accumulation of extracellular adenosine and inhibition of adenosine kinase activity were caused by hypercapnic acidosis and isohydric hypercapnia, but not isohydric acidosis. These results indicate that the activation of adenosine A1 receptors is involved in the hypercapnia‐evoked depression of reflex potentials in the isolated spinal cord of the neonatal rat. The inhibition of adenosine kinase activity is suggested to cause the accumulation of extracellular adenosine during hypercapnia.
Intracellular sphingosine 1‐phosphate mediates the increased excitability produced by nerve growth factor in rat sensory neurons
Authors: Zhang Y. H. , Vasko M. R. , Nicol G. D.
Published: June 2006

Our previous studies found that nerve growth factor (NGF), via ceramide, enhanced the number of action potentials (APs) evoked by a ramp of depolarizing current in capsaicin‐sensitive sensory neurons. Ceramide can be metabolized by ceramidase to sphingosine (Sph), and Sph to sphingosine 1‐phosphate (S1P) by sphingosine kinase. It is well established that each of these products of sphingomyelin metabolism can act as intracellular signalling molecules. This raises the question as to whether the enhanced excitability produced by NGF was mediated directly by ceramide or required additional metabolism to Sph and/or S1P. Sph applied externally did not affect the neuronal excitability, whereas internally perfused Sph augmented the number of APs evoked by the depolarizing ramp. Furthermore, internally perfused S1P enhanced the number of evoked APs. This sensitizing action of NGF, ceramide and internally perfused Sph was abolished by dimethylsphingosine (DMS), an inhibitor of sphingosine kinase. In contrast, internally perfused S1P enhanced the number of evoked APs in the presence of DMS. These observations support the idea that the metabolism of ceramide/Sph to S1P is critical for the sphingolipid‐induced modulation of excitability. Both internally perfused Sph and S1P inhibited the outward K+ current by 25–35% for the step to +60 mV. The Sph‐ and S1P‐sensitive currents had very similar current–voltage relations, suggesting that they were likely to be the same. In addition, the Sph‐induced suppression of the K+ current was blocked by pretreatment with DMS. These findings demonstrate that intracellular S1P derived from ceramide acts as an internal second messenger to regulate membrane excitability; however, the effector system whereby S1P modulates excitability remains undetermined.
Metagenomic Analysis of the Human Distal Gut Microbiome
Authors: Gill Steven R. , Pop Mihai , DeBoy Robert T. , Eckburg Paul B. , Turnbaugh Peter J. , Samuel Buck S. , Gordon Jeffrey I. , Relman David A. , Fraser-Liggett Claire M. , Nelson Karen E.
Published: June 2006

The human intestinal microbiota is composed of 10
13
to 10
14
microorganisms whose collective genome (“microbiome”) contains at least 100 times as many genes as our own genome. We analyzed ∼78 million base pairs of unique DNA sequence and 2062 polymerase chain reaction–amplified 16
S
ribosomal DNA sequences obtained from the fecal DNAs of two healthy adults. Using metabolic function analyses of identified genes, we compared our human genome with the average content of previously sequenced microbial genomes. Our microbiome has significantly enriched metabolism of glycans, amino acids, and xenobiotics; methanogenesis; and 2-methyl-
d
-erythritol 4-phosphate pathway–mediated biosynthesis of vitamins and isoprenoids. Thus, humans are superorganisms whose metabolism represents an amalgamation of microbial and human attributes.
Comment on "
HST2
Mediates
SIR2
-Independent Life-Span Extension by Calorie Restriction"
Authors: Kaeberlein Matt , Steffen Kristan K. , Hu Di , Dang Nick , Kerr Emily O. , Tsuchiya Mitsuhiro , Fields Stanley , Kennedy Brian K.
Published: June 2006

Calorie restriction (CR) increases life span in yeast independently of Sir2. Lamming
et al
. (Reports, 16 September 2005, p. 1861) recently proposed that Sir2-independent life-span extension by CR is mediated by the Sir2 paralogs Hst1 and Hst2. Contradictory to this, we find that CR greatly increases life span in cells lacking Sir2, Hst1, and Hst2, which suggests that CR is not mediated by Sir2, Hst2, or Hst1.
Conductance Quantization at a Half-Integer Plateau in a Symmetric GaAs Quantum Wire
Authors: Crook R. , Prance J. , Thomas K. J. , Chorley S. J. , Farrer I. , Ritchie D. A. , Pepper M. , Smith C. G.
Published: June 2006

We present data from an induced gallium arsenide (GaAs) quantum wire that exhibits an additional conductance plateau at 0.5(2
e
2
/
h
), where
e
is the charge of an electron and
h
is Planck's constant, in zero magnetic field. The plateau was most pronounced when the potential landscape was tuned to be symmetric by using low-temperature scanning-probe techniques. Source-drain energy spectroscopy and temperature response support the hypothesis that the origin of the plateau is the spontaneous spin-polarization of the transport electrons: a ferromagnetic phase. Such devices may have applications in the field of spintronics to either generate or detect a spin-polarized current without the complications associated with external magnetic fields or magnetic materials.
Hierarchical Action and Inhibition of Plant Dicer-Like Proteins in Antiviral Defense
Authors: Deleris Angélique , Gallego-Bartolome Javier , Bao Jinsong , Kasschau Kristin D. , Carrington James C. , Voinnet Olivier
Published: June 2006

The mechanisms underlying induction and suppression of RNA silencing in the ongoing plant-virus arms race are poorly understood. We show here that virus-derived small RNAs produced by
Arabidopsis
Dicer-like 4 (DCL4) program an effector complex conferring antiviral immunity. Inhibition of DCL4 by a viral-encoded suppressor revealed the subordinate antiviral activity of DCL2. Accordingly, inactivating both DCL2 and DCL4 was necessary and sufficient to restore systemic infection of a suppressor-deficient virus. The effects of DCL2 were overcome by increasing viral dosage in inoculated leaves, but this could not surmount additional, non–cell autonomous effects of DCL4 specifically preventing viral unloading from the vasculature. These findings define a molecular framework for studying antiviral silencing and defense in plants.
Adventures in Near-Earth Object Exploration
Authors: Asphaug Erik
Published: June 2006

Asteroids, because of the hazard they pose to Earth, are compelling targets for robotic and human space exploration. Yet because of their exotic low-gravity environment, simply landing on an asteroid appears to be much more challenging than we had appreciated 5 or 10 years ago. Thanks to a bold new mission from Japan that has made the first asteroid sample return attempt, this goal is now within our reach.
Hox Control of Organ Size by Regulation of Morphogen Production and Mobility
Authors: Crickmore Michael A. , Mann Richard S.
Published: June 2006

Selector genes modify developmental pathways to sculpt animal body parts. Although body parts differ in size, the ways in which selector genes create size differences are unknown. We have studied how the
Drosophila
Hox gene
Ultrabithorax
(
Ubx
) limits the size of the haltere, which, by the end of larval development, has ∼fivefold fewer cells than the wing. We find that
Ubx
controls haltere size by restricting both the transcription and the mobility of the morphogen Decapentaplegic (Dpp).
Ubx
restricts Dpp's distribution in the haltere by increasing the amounts of the Dpp receptor,
thickveins
. Because morphogens control tissue growth in many contexts, these findings provide a potentially general mechanism for how selector genes modify organ sizes.
Picturing the Pre-Columbian Americas
Authors: Snow Dean R.
Published: June 2006

1491
. New Revelations of the Americas Before Columbus.
By Charles C. Mann
. Knopf, New York, 2005. 478 pp. $30, C$40. ISBN 1-4000-4006-X.

Ancient Americans
. Rewriting the History of the New World. Granta, London, 2005. £20. ISBN 1-86207-617-0.

Pulling together disparate data from archaeology, history, genetics, and ecology, the author discusses the state of human civilizations in the Americas before the arrival of the Europeans.
Depth-Related Gradients of Viral Activity in Lake Pavin
Authors: Colombet J. , Sime-Ngando T. , Cauchie H. M. , Fonty G. , Hoffmann L. , Demeure G.
Published: June 2006

High-resolution vertical sampling and determination of viral and prokaryotic parameters in a deep volcanic lake shows that in the absence of thermal stratification but within light, oxygen, and chlorophyll gradients, host availability empirically is prevalent over the physical and chemical environments and favors lytic over lysogenic “viral life cycles.”
Cadmium Ion Biosorption by the Thermophilic Bacteria
Geobacillus stearothermophilus
and
G. thermocatenulatus
Authors: Hetzer Adrian , Daughney Christopher J. , Morgan Hugh W.
Published: June 2006

This study reports surface complexation models (SCMs) for quantifying metal ion adsorption by thermophilic microorganisms. In initial cadmium ion toxicity tests, members of the genus
Geobacillus
displayed the highest tolerance to CdCl
2
(as high as 400 to 3,200 μM). The thermophilic, gram-positive bacteria
Geobacillus stearothermophilus
and
G. thermocatenulatus
were selected for further electrophoretic mobility, potentiometric titration, and Cd
2+
adsorption experiments to characterize Cd
2+
complexation by functional groups within and on the cell wall. Distinct one-site SCMs described the extent of cadmium ion adsorption by both studied
Geobacillus
sp. strains over a range of pH values and metal/bacteria concentration ratios. The results indicate that a functional group with a deprotonation constant pK value of approximately 3.8 accounts for 66% and 80% of all titratable sites for
G. thermocatenulatus
and
G. stearothermophilus
, respectively, and is dominant in Cd
2+
adsorption reactions. The results suggest a different type of functional group may be involved in cadmium biosorption for both thermophilic strains investigated here, compared to previous reports for mesophilic bacteria.
Evaluation of the Duopath
Legionella
Lateral Flow Assay for Identification of
Legionella pneumophila
and
Legionella
Species Culture Isolates
Authors: Helbig Jürgen Herbert , Lück Paul Christian , Kunz Britta , Bubert Andreas
Published: June 2006

Duopath
Legionella
(Merck KGaA, Darmstadt, Germany) is a new immunochromatographic assay for the simultaneous identification of cultured
L. pneumophila
and
Legionella
species other than
L. pneumophila
. In tests of 89
L. pneumophila
strains and 87
Legionella
strains other than
L. pneumophila
representing 41 different species, Duopath and a widely used latex agglutination assay detected
L. pneumophila
with 100% and 98% accuracy, respectively, whereas the percentages differed significantly for other
Legionella
spp. (93% versus 37% [
P
< 0.001]). Since many countries’ regulations require the identification of
Legionella
spp. in water and environmental samples, the use of Duopath
Legionella
to comply with those regulations could contribute to significantly fewer false-negative results.
Prevalence and Identity of
Cryptosporidium
spp. in Pig Slurry
Authors: Xiao Lihua , Moore John E. , Ukoh Ukeme , Gatei Wangeci , Lowery Colm J. , Murphy Thomas M. , Dooley James S. G. , Millar B. Cherie , Rooney Paul J. , Rao Juluri R.
Published: June 2006

Cryptosporidium
spp. were detected in 25 of 56 pig slurry samples from 33 Irish farms by PCR and DNA sequencing. The organisms detected included
C. suis
,
Cryptosporidium
pig genotype II, and
C. muris.
We concluded that
Cryptosporidium
oocysts can persist in treated slurry and potentially contaminate surface water through improper discharge or uncontrolled runoff.
Structure of Trichamide, a Cyclic Peptide from the Bloom-Forming Cyanobacterium
Trichodesmium erythraeum
, Predicted from the Genome Sequence
Authors: Sudek Sebastian , Haygood Margo G. , Youssef Diaa T. A. , Schmidt Eric W.
Published: June 2006

A gene cluster for the biosynthesis of a new small cyclic peptide, dubbed trichamide, was discovered in the genome of the global, bloom-forming marine cyanobacterium
Trichodesmium erythraeum
ISM101 because of striking similarities to the previously characterized patellamide biosynthesis cluster. The
tri
cluster consists of a precursor peptide gene containing the amino acid sequence for mature trichamide, a putative heterocyclization gene, an oxidase, two proteases, and hypothetical genes. Based upon detailed sequence analysis, a structure was predicted for trichamide and confirmed by Fourier transform mass spectrometry. Trichamide consists of 11 amino acids, including two cysteine-derived thiazole groups, and is cyclized by an N—C terminal amide bond. As the first natural product reported from
T. erythraeum
, trichamide shows the power of genome mining in the prediction and discovery of new natural products.
Cysteine Metabolism in
Legionella pneumophila
: Characterization of an
l
-Cystine-Utilizing Mutant
Authors: Ewann Fanny , Hoffman Paul S.
Published: June 2006

Growth of
Legionella pneumophila
on buffered charcoal-yeast extract (BCYE) medium is dependent on
l
-cysteine (but not
l
-cystine), which is added in excess over what is required for nutrition. We investigated the biochemical and genetic bases for this unusual requirement and determined that much of the
l
-cysteine in BCYE medium is rapidly oxidized to
l
-cystine and is unavailable to the bacteria. Analysis of cysteine consumption during bacterial growth indicated that of the 11% consumed, 3.85% (∼0.1 mM) was incorporated into biomass. The activities of two key cysteine biosynthetic enzymes (serine acetyltransferase and cysteine synthase) were not detected in cell extracts of
L. pneumophila
, and the respective genes were not present in the genome sequences, confirming cysteine auxotrophy. Kinetic studies identified two energy-dependent cysteine transporters, one with high affinity (apparent

K
m

, 3.29 μM) and the other with low affinity (apparent

K
m

, 93 μM), each of which was inhibited by the uncoupling agent carbonyl cyanide
m
-chlorophenylhydrazone. Cystine was not transported by
L. pneumophila
; however, a mutant strain capable of growth on
l
-cystine (CYS1 mutant) transported
l
-cystine with similar kinetics (

K
m

, 4.4 μM and 90 μM). Based on the bipartite kinetics, requirement for proton motive force, and inhibitor studies, we suggest that a high-affinity periplasmic binding protein and a major facilitator/symporter (low affinity) mediate uptake. The latter most likely is functional at high cysteine concentrations and most likely displays altered substrate specificity in the CYS-1 mutant. Our studies provide biochemical evidence to support a general view that
L. pneumophila
is restricted to an intracellular lifestyle in natural environments by an inability to utilize cystine, which most likely ensures that the dormant cyst-like transmissible forms do not germinate outside suitable protozoan hosts.
Development of
Bacteroides
16S rRNA Gene TaqMan-Based Real-Time PCR Assays for Estimation of Total, Human, and Bovine Fecal Pollution in Water
Authors: Layton Alice , McKay Larry , Williams Dan , Garrett Victoria , Gentry Randall , Sayler Gary
Published: June 2006

Bacteroides
species are promising indicators for differentiating livestock and human fecal contamination in water because of their high concentration in feces and potential host specificity. In this study, a real-time PCR assay was designed to target
Bacteroides
species (AllBac) present in human, cattle, and equine feces. Direct PCR amplification (without DNA extraction) using the AllBac assay was tested on feces diluted in water. Fecal concentrations and threshold cycle were linearly correlated, indicating that the AllBac assay can be used to estimate the total amount of fecal contamination in water. Real-time PCR assays were also designed for bovine-associated (BoBac) and human-associated (HuBac)
Bacteroides
16S rRNA genes. Assay specificities were tested using human, bovine, swine, canine, and equine fecal samples. The BoBac assay was specific for bovine fecal samples (100% true-positive identification; 0% false-positive identification). The HuBac assay had a 100% true-positive identification, but it also had a 32% false-positive rate with potential for cross-amplification with swine feces. The assays were tested using creek water samples from three different watersheds. Creek water did not inhibit PCR, and results from the AllBac assay were correlated with those from
Escherichia coli
concentrations (
r
2
= 0.85). The percentage of feces attributable to bovine and human sources was determined for each sample by comparing the values obtained from the BoBac and HuBac assays with that from the AllBac assay. These results suggest that real-time PCR assays without DNA extraction can be used to quantify fecal concentrations and provide preliminary fecal source identification in watersheds.
Genotyping of Human and Porcine
Yersinia enterocolitica
,
Yersinia intermedia
, and
Yersinia bercovieri
Strains from Switzerland by Amplified Fragment Length Polymorphism Analysis
Authors: Boghenbor Kathrin Kuehni , On Stephen L. W. , Kokotovic Branko , Baumgartner Andreas , Wassenaar Trudy M. , Wittwer Matthias , Bissig-Choisat Beatrice , Frey Joachim
Published: June 2006

In this study, 231 strains of
Yersinia enterocolitica
, 25 strains of
Y. intermedia
, and 10 strains of
Y. bercovieri
from human and porcine sources (including reference strains) were analyzed using amplified fragment length polymorphism (AFLP), a whole-genome fingerprinting method for subtyping bacterial isolates. AFLP typing distinguished the different
Yersinia
species examined. Representatives of
Y. enterocolitica
biotypes 1A, 1B, 2, 3, and 4 belonged to biotype-related AFLP clusters and were clearly distinguished from each other.
Y. enterocolitica
biotypes 2, 3, and 4 appeared to be more closely related to each other (83% similarity) than to biotypes 1A (11%) and 1B (47%). Biotype 1A strains exhibited the greatest genetic heterogeneity of the biotypes studied. The biotype 1A genotypes were distributed among four major clusters, each containing strains from both human and porcine sources, confirming the zoonotic potential of this organism. The AFLP technique is a valuable genotypic method for identification and typing of
Y. enterocolitica
and other
Yersinia
spp.
Differential
inlA
and
inlB
Expression and Interaction with Human Intestinal and Liver Cells by
Listeria monocytogenes
Strains of Different Origins
Authors: Werbrouck Hadewig , Grijspeerdt Koen , Botteldoorn Nadine , Van Pamel Els , Rijpens Nancy , Van Damme Jo , Uyttendaele Mieke , Herman Lieve , Van Coillie Els
Published: June 2006

In this study, a number of
Listeria monocytogenes
strains of different origins were evaluated for in vitro invasion capacity for various human cell types (monocytic THP-1, enterocytic Caco-2, and hepatocytic HepG2 cells) and for expression levels of specific virulence genes. For THP-1 cells, no differences between clinical and nonclinical
L. monocytogenes
strains in invasion capacity or in production of the proinflammatory cytokine interleukin-8 (IL-8) were observed, whereas for the Caco-2 and HepG2 cells, significant differences in invasion capacity were noticed. On average, the clinical strains showed a significantly lower invasion capacity than the nonclinical
L. monocytogenes
strains. Furthermore, it was shown that the clinical strains induce lower IL-8 levels in HepG2 cells than do the nonclinical strains. This observation led us to study the mRNA expression levels of
inlA
,
inlB
, and
ami
, important virulence genes mediating adhesion and invasion of eukaryotic cells, by real-time reverse transcription-PCR for 27 clinical and 37 nonclinical
L. monocytogenes
strains. Significant differences in
inlA
and
inlB
expression were observed, with clinical strains showing a lower expression level than nonclinical strains. These observations were in accordance with in vitro invasion of Caco-2 and HepG2 cells, respectively. The results of this study indicate that differential expression levels of
inlA
and
inlB
possibly play a role in the virulence capacities of
L. monocytogenes
strains. The lower capacity of clinical strains to invade HepG2 cells and to induce IL-8 is possibly a mechanism of immune evasion used by specific
L. monocytogenes
strains.
Improved Enumeration of Lactic Acid Bacteria in Mesophilic Dairy Starter Cultures by Using Multiplex Quantitative Real-Time PCR and Flow Cytometry-Fluorescence In Situ Hybridization
Authors: Friedrich Udo , Lenke Jan
Published: June 2006

Nucleic acid-based assays were developed to enumerate members of the three taxa
Lactococcus lactis
subsp.
cremoris
,
L. lactis
subsp.
lactis
, and
Leuconostoc
spp. in mesophilic starter cultures. To our knowledge the present is the first study to present a multiplex quantitative PCR (qPCR) strategy for the relative enumeration of bacteria. The multiplex qPCR strategy was designed to quantify the target DNA simultaneously relative to total bacterial DNA. The assay has a high discriminatory power and resolves concentration changes as low as 1.3-fold. The methodology was compared with flow cytometric fluorescence in situ hybridization (FLOW-FISH) and 5-bromo-4-chloro-3-indolyl-β-
d
-galactopyranoside (X-Gal)-calcium citrate agar-based plate counting. For enumeration by FLOW-FISH, three new probes having the same specificity as the qPCR assay were designed and established. A combination with flow cytometry greatly reduced the time consumed compared to manual enumeration. Both qPCR and FLOW-FISH yielded similar community compositions for 10 complex starter cultures, with all detected subpopulations being highly significantly correlated (
P
< 0.001). Correlations between X-Gal-calcium citrate agar-based CFU and qPCR-derived counts were highly significant (
P
< 0.01 and
P
< 0.001, respectively) for the number of acidifiers versus
L. lactis
subsp.
cremoris
and for
Leuconostoc
spp. as quantified by the two techniques, respectively. This confirmed that most acidifiers in the studied PROBAT cultures are members of
L. lactis
subsp.
cremoris
. Quantitative real-time PCR and FLOW-FISH were found to be effective and accurate tools for the bacterial community analysis of complex starter cultures.
Tomato QM-Like Protein Protects
Saccharomyces cerevisiae
Cells against Oxidative Stress by Regulating Intracellular Proline Levels
Authors: Chen Changbin , Wanduragala Srimevan , Becker Donald F. , Dickman Martin B.
Published: June 2006

Exogenous proline can protect cells of
Saccharomyces cerevisiae
from oxidative stress. We altered intracellular proline levels by overexpressing the proline dehydrogenase gene (
PUT1
) of
S. cerevisiae
. Put1p performs the first enzymatic step of proline degradation in
S. cerevisiae
. Overexpression of Put1p results in low proline levels and hypersensitivity to oxidants, such as hydrogen peroxide and paraquat. A
put1
-disrupted yeast mutant deficient in Put1p activity has increased protection from oxidative stress and increased proline levels. Following a conditional life/death screen in yeast, we identified a tomato (
Lycopersicon esculentum
) gene encoding a QM-like protein (tQM) and found that stable expression of
tQM
in the Put1p-overexpressing strain conferred protection against oxidative damage from H
2
O
2
, paraquat, and heat. This protection was correlated with reactive oxygen species (ROS) reduction and increased proline accumulation. A yeast two-hybrid system assay was used to show that tQM physically interacts with Put1p in yeast, suggesting that tQM is directly involved in modulating proline levels. tQM also can rescue yeast from the lethality mediated by the mammalian proapoptotic protein Bax, through the inhibition of ROS generation. Our results suggest that tQM is a component of various stress response pathways and may function in proline-mediated stress tolerance in plants.
Overproduction of Wild-Type and Bioengineered Derivatives of the Lantibiotic Lacticin 3147
Authors: Cotter Paul D. , Draper Lorraine A. , Lawton Elaine M. , McAuliffe Olivia , Hill Colin , Ross R. Paul
Published: June 2006

Lacticin 3147 is a broad-spectrum two-peptide lantibiotic whose genetic determinants are located on two divergent operons on the lactococcal plasmid pMRC01. Here we introduce each of 14 subclones, containing different combinations of lacticin 3147 genes, into MG1363 (pMRC01) and determine that a number of them can facilitate overproduction of the lantibiotic. Based on these studies it is apparent that while the provision of additional copies of genes encoding the biosynthetic/production machinery and the regulator LtnR is a requirement for high-level overproduction, the presence of additional copies of the structural genes (i.e.,
ltnA1A2
) is not.
Potential of a 16S rRNA-Based Taxonomic Microarray for Analyzing the Rhizosphere Effects of Maize on
Agrobacterium
spp. and Bacterial Communities
Authors: Sanguin Hervé , Remenant Benoît , Dechesne Arnaud , Thioulouse Jean , Vogel Timothy M. , Nesme Xavier , Moënne-Loccoz Yvan , Grundmann Geneviève L.
Published: June 2006

Bacterial diversity is central to ecosystem sustainability and soil biological function, for which the role of roots is important. The high-throughput analysis potential of taxonomic microarray should match the breadth of bacterial diversity. Here, the power of this technology was evidenced through methodological verifications and analysis of maize rhizosphere effect based on a 16S rRNA-based microarray developed from the prototype of H. Sanguin et al. (Environ. Microbiol. 8:289-307, 2006). The current probe set was composed of 170 probes (41 new probes in this work) that targeted essentially the
Proteobacteria
. Cloning and sequencing of 16S rRNA amplicons were carried out on maize rhizosphere and bulk soil DNA. All tested clones that had a perfect match with corresponding probes were positive in the hybridization experiment. The hierarchically nested probes were reliable, but the level of taxonomic identification was variable, depending on the probe set specificity. The comparison of experimental and theoretical hybridizations revealed 0.91% false positives and 0.81% false negatives. The microarray detection threshold was estimated at 0.03% of a given DNA type based on DNA spiking experiments. A comparison of the maize rhizosphere and bulk soil hybridization results showed a significant rhizosphere effect, with a higher predominance of
Agrobacterium
spp. in the rhizosphere, as well as a lower prevalence of
Acidobacteria
,
Bacteroidetes
,
Verrucomicrobia
, and
Planctomycetes
, a new taxon of interest in soil. In addition, well-known taxonomic groups such as
Sphingomonas
spp.,
Rhizobiaceae
, and
Actinobacteria
were identified in both microbial habitats with strong hybridization signals. The taxonomic microarray developed in the present study was able to discriminate and characterize bacterial community composition in related biological samples, offering extensive possibilities for systematic exploration of bacterial diversity in ecosystems.
Microbial Diversity Associated with Odor Modification for Production of Fertilizers from Chicken Litter
Authors: Enticknap Julie J. , Nonogaki Hirofumi , Place Allen R. , Hill Russell T.
Published: June 2006

Litter from the chicken industry can present several environmental challenges, including offensive odors and runoff into waterways leading to eutrophication. An economically viable solution to the disposal of waste from chicken houses is treatment to produce a natural, granulated fertilizer that can be commercially marketed for garden and commercial use. Odor of the final product is important in consumer acceptance, and an earthy odor is desirable. By understanding and manipulating the microbial processes occurring during this process, it may be possible to modify the odors produced. Geosmin and related volatiles produced by soil actinomycetes are responsible for earthy odors, and actinomycetes are likely to be present in the composting manure. Bacterial communities at each stage of the process were analyzed by culturing studies and denaturing gradient gel electrophoresis (DGGE). The processing steps changed the culturable bacterial community, but the total community was shown by DGGE to be stable throughout the process. A local agricultural soil was analyzed in parallel as a potential source of geosmin-producing actinomycetes. This agricultural soil had higher microbial diversity than the compost at both the culturable and the molecular levels. Actinomycete bacteria were isolated and analyzed by AromaTrax, a gas chromatography-olfactometry system. This system enables the odor production of individual isolates to be monitored, allowing for rational selection of strains for augmentation experiments to improve the odor of the final fertilizer product.
Production of Xylitol from
d
-Xylose by a Xylitol Dehydrogenase Gene-Disrupted Mutant of
Candida tropicalis
Authors: Ko Byoung Sam , Kim Jinmi , Kim Jung Hoe
Published: June 2006

Xylitol dehydrogenase (XDH) is one of the key enzymes in
d
-xylose metabolism, catalyzing the oxidation of xylitol to
d
-xylulose. Two copies of the
XYL2
gene encoding XDH in the diploid yeast
Candida tropicalis
were sequentially disrupted using the Ura-blasting method. The
XYL2
-disrupted mutant, BSXDH-3, did not grow on a minimal medium containing
d
-xylose as a sole carbon source. An enzyme assay experiment indicated that BSXDH-3 lost apparently all XDH activity. Xylitol production by BSXDH-3 was evaluated using a xylitol fermentation medium with glucose as a cosubstrate. As glucose was found to be an insufficient cosubstrate, various carbon sources were screened for efficient cofactor regeneration, and glycerol was found to be the best cosubstrate. BSXDH-3 produced xylitol with a volumetric productivity of 3.23 g liter
−1
h
−1
, a specific productivity of 0.76 g g
−1
h
−1
, and a xylitol yield of 98%. This is the first report of gene disruption of
C. tropicalis
for enhancing the efficiency of xylitol production.
Molecular Epidemiology of Ceftiofur-Resistant
Escherichia coli
Isolates from Dairy Calves
Authors: Donaldson Sarah C. , Straley Beth A. , Hegde Narasimha V. , Sawant Ashish A. , DebRoy Chitrita , Jayarao Bhushan M.
Published: June 2006

Healthy calves (
n
= 96, 1 to 9 weeks old) from a dairy herd in central Pennsylvania were examined each month over a five-month period for fecal shedding of ceftiofur-resistant gram-negative bacteria. Ceftiofur-resistant
Escherichia coli
isolates (
n
= 122) were characterized by antimicrobial resistance (disk diffusion and MIC), serotype, pulsed-field gel electrophoresis subtypes, beta-lactamase genes, and virulence genes. Antibiotic disk diffusion assays showed that the isolates were resistant to ampicillin (100%), ceftiofur (100%), chloramphenicol (94%), florfenicol (93%), gentamicin (89%), spectinomycin (72%), tetracycline (98%), ticarcillin (99%), and ticarcillin-clavulanic acid (99%). All isolates were multidrug resistant and displayed elevated MICs. The
E. coli
isolates belonged to 42 serotypes, of which O8:H25 was the predominant serotype (49.2%). Pulsed-field gel electrophoresis classified the
E. coli
isolates into 27 profiles. Cluster analysis showed that 77 isolates (63.1%) belonged to one unique group. The prevalence of pathogenic
E. coli
was low (8%). A total of 117 ceftiofur-resistant
E. coli
isolates (96%) possessed the
bla
CMY2
gene. Based on phenotypic and genotypic characterization, the ceftiofur-resistant
E. coli
isolates belonged to 59 clonal types. There was no significant relationship between calf age and clonal type. The findings of this study revealed that healthy dairy calves were rapidly colonized by antibiotic-resistant strains of
E. coli
shortly after birth. The high prevalence of multidrug-resistant nonpathogenic
E. coli
in calves could be a significant source of resistance genes to other bacteria that share the same environment.