bluuebunny/crossref_metadata_2025 huggingface dataset:
Attitudes of Interior Designers toward CADD and CADD Education
Authors: Clemons Stephanie A. , McCullough Jane
Published: June 2008

Across the nation, institutions that teach interior design are adding computer–aided drafting and design (CADD) classes to their curriculum with the assumption that the courses will enhance their graduates’ marketability. In July 1987, a questionnaire was mailed to practicing interior designers to assess their attitudes toward CADD training for interior design students and toward CADD in general. By understanding the attitudes of practicing interior designers, teaching institutions can better determine if the need for CADD classes exists and what attitudes toward CADD the graduates will encounter when they enter the design field.

Responses from a nationwide random sample indicate that practicing interior designers have positive attitudes toward CADD and toward CADD training for students.
Glycolytic activation at the onset of contractions in isolated Xenopus laevis single myofibres
Authors: Walsh Brandon , Stary Creed M. , Howlett Richard A. , Kelley Kevin M. , Hogan Michael C.
Published: May 2008

Intracellular pH (pHi) was measured in isolated Xenopus laevis single myofibres at the onset of contractions, with and without glycolytic blockade, to investigate the time course of glycolytic activation. Single myofibres (n= 8; CON) were incubated in 2′,7′‐bis(carboxyethyl)‐5(6)‐carboxyfluorescein acetoyxmethyl ester (10 μm; for fluorescence measurement of pHi) and stimulated for 15 s at 0.67 Hz in anoxia in the absence (control condition; CON) and presence of a glycolytic inhibitor (1 mm iodoacetic acid; IAA). Intracellular pHi and tension were continuously recorded, and the differences in pHi between conditions were used to estimate the activation time of glycolysis. An immediate and steady increase in pHi (initial alkalosis) at the onset of contractions was similar between CON and IAA trials for the first 9 s of the contractile bout. However, from six contractions (∼10 s) throughout the remainder of the bout, IAA demonstrated a continued rise in pHi, in contrast to a progressive decrease in pHi in CON (P < 0.05). These results demonstrate, with high temporal resolution, that glycolysis is activated within six contractions (10 s at 0.67 Hz) in single Xenopus skeletal muscle fibres.
The effects of ageing and passive heating on cardiorespiratory and cerebrovascular responses to orthostatic stress in humans
Authors: Lucas R. A. I. , Cotter J. D. , Morrison S. , Ainslie P. N.
Published: May 2008

We tested the hypothesis that older adults, relative to younger adults, would be more prone to critical reductions in cerebral blood flow and oxygenation upon standing during passive heat stress. Six older (70 ± 4 years, mean ±s.d.) and six younger males (29 ± 4 years) were heated (oesophageal temperature raised 0.5°C) in a water‐perfused suit. Blood flow velocity in the middle cerebral artery (MCAv), cerebral oxygenation, mean arterial pressure (MAP) and end‐tidal partial pressure of carbon dioxide (
) were measured continuously before and during 3 min standing in each thermal state. At supine normothermic baseline, MCAv was 47% lower in older participants (P < 0.001), whilst MAP and cerebral oxygenation were similar between groups (P > 0.05). Heating lowered the supine MAP more in younger adults, and elevated heart rate only in this group. Upon initial standing in normothermia, older participants had a greater drop in MCAv (P < 0.05 versus young), a lesser drop in MAP (∼24 and ∼42% in older and younger participants, respectively), but slower recovery of MAP (27.3 ± 6.8 versus 18.6 ± 4.7 s, mean ±s.d., P= 0.004); heating did not exacerbate any postural responses in either age group. During the last minute of standing, MCAv and
were lower in older participants, though age differences were not evident in cerebral oxygenation (normothermic or heated). Thus, independent of heat stress, in addition to lower resting MCAv, there are further age‐related reductions in MCAv and slower corrections of MAP following standing. However, these asymptomatic changes seem to represent a physiologically acceptable insult which can be well tolerated in otherwise healthy older participants even during heat stress.
Hypotonicity reduces the activity of murine aquaporin‐2 promoter induced by dibutyryl cAMP
Authors: Saito Takako , Saito Tomoyuki , Kasono Keizo , Tamemoto Hiroyuki , Kawakami Masanobu , Sasaki Sei , Ishikawa San‐e
Published: May 2008

The present study was undertaken to determine whether hypotonicity regulates the aquaporin‐2 (AQP‐2) gene in vitro. The 5′‐flanking region of the AQP‐2 gene contains the tonicity‐response enhancer (TonE) promoter located between −570 and −560 bp, and another distinct hypertonicity‐responsive region between −6.1 and −4.3 kb of the AQP‐2 gene. The 5′‐flanking region of murine AQP‐2 gene up to −9.5 kb was cloned into a luciferase (Luc) reporter plasmid. The constructs, which have TonE and/or the hypertonicity‐responsive region, together with the murine AQP‐2 gene, were co‐transfected into murine IMCD3 cells. When the cells were co‐transfected with the construct containing more than 1.1 kb of the 5′‐flanking region of murine AQP‐2 gene (–9.5AQP2, −6.1AQP2 and −1.1AQP2) and the AQP‐2 gene, 24 h exposure to 5 μmol l−1 dibutyryl cAMP (DBcAMP) significantly increased the Luc activity by 2.3‐fold in the isotonic medium (300 mosmol kg−1). In the hypotonic medium (225 mosmol kg−1), basal activity was not altered, and the response of Luc activity to 24 h exposure to 5 μmol l−1DBcAMP was abolished. Similar findings were obtained in isosmotic, urea‐supplemented medium (estimated tonicity, 225 mosmol kg−1). The response of Luc activity to 5 μmol l−1 DBcAMP in the hypotonic medium was not affected in cells either transfected with 0.36 kb of the 5′‐flanking region of AQP‐2 or co‐transfected with −1.1AQP2 and a dominant‐negative TonE binding protein (pDNTonEBP). Pre‐incubation of cells with 1 μmol l−1 SP600125, an inhibitor of c‐Jun N‐terminal kinase (JNK), restored the response of Luc activity to 5 μmol l−1 DBcAMP under hypotonic conditions. These findings may indicate that hypotonicity reduces the cAMP‐induced AQP‐2 promoter activity mediated via TonE by activating JNK kinase.
Matching of blood flow to metabolic rate during recovery from moderate exercise in humans
Authors: Harper Allison J. , Ferreira Leonardo F. , Lutjemeier Barbara J. , Townsend Dana K. , Barstow Thomas J.
Published: May 2008

It is unclear whether measurement of limb or conduit artery blood flow during recovery from exercise provides an accurate representation of flow to the muscle capillaries where gas exchange occurs. To investigate this, we: (a) examined the kinetic responses of femoral artery blood flow (
), estimated muscle capillary blood flow (
) and estimated muscle oxygen uptake (
) following cessation of exercise; and (b) compared these responses to verify the adequacy of O2 delivery during recovery. Pulmonary
(
) was measured breath by breath,
was measured using Doppler ultrasonography, and deoxy‐haemoglobin/myoglobin (deoxy‐[Hb/Mb]) was estimated by near‐infrared spectroscopy over the rectus femoris in nine healthy subjects during a series of transitions from moderate knee‐extension exercise to rest. The time course of
was estimated by rearranging the Fick equation [i.e.
], using the primary component of
to represent
and deoxy‐[Hb/Mb] as a surrogate for arteriovenous O2 difference. There were no significant differences among the overall kinetics of
(τ, 31.4 ± 8.2 s),
[mean response time (MRT), 34.5 ± 20.4 s] and
(MRT, 31.7 ± 14.7 s). The
kinetics were also significantly correlated (P < 0.05) with those of both
and
. Both
and
appear to be coupled with
during recovery from moderate knee‐extension exercise, such that extraction falls (thus cellular energetic state is not further compromised) throughout recovery.
Response of Anaerobic Ammonium-Oxidizing Bacteria to Hydroxylamine
Authors: van der Star Wouter R. L. , van de Graaf Maarten J. , Kartal Boran , Picioreanu Cristian , Jetten Mike S. M. , van Loosdrecht Mark C. M.
Published: May 2008

Anaerobic ammonium oxidation is a recent addition to the microbial nitrogen cycle, and its metabolic pathway, including the production and conversion of its intermediate hydrazine, is not well understood. Therefore, the effect of hydroxylamine addition on the hydrazine metabolism of anaerobic ammonium-oxidizing (anammox) bacteria was studied both experimentally and by mathematical modeling. It was observed that hydroxylamine was disproportionated biologically in the absence of nitrite into dinitrogen gas and ammonium. Little hydrazine accumulated during this process; however, rapid hydrazine production was observed when nearly all hydroxylamine was consumed. A mechanistic model is proposed in which hydrazine is suggested to be continuously produced from ammonium and hydroxylamine (possibly via nitric oxide) and subsequently oxidized to N
2
. The electron acceptor for hydrazine oxidation is hydroxylamine, which is reduced to ammonium. A decrease in the hydroxylamine reduction rate, therefore, leads to a decrease in the hydrazine oxidation rate, resulting in the observed hydrazine accumulation. The proposed mechanism was verified by a mathematical model which could explain and predict most of the experimental data.
Rapid Ultrafiltration Concentration and Biosensor Detection of Enterococci from Large Volumes of Florida Recreational Water
Authors: Leskinen Stephaney D. , Lim Daniel V.
Published: May 2008

Monitoring recreational waters for fecal contamination by standard methodologies involves culturing indicator bacteria, such as fecal coliforms and enterococci. Delayed reporting of microbial water quality parameters increases the likelihood of public exposure to pathogens of fecal origin, making the development of rapid methods important for public health protection. A rapid assay for enterococci was developed using a combined ultrafiltration-biosensor procedure. Twelve 100-liter water samples were collected from upper Tampa Bay over a 9-month period. The samples were collected on site by dead-end hollow-fiber ultrafiltration. Postfiltration processing of the initial retentates included sonication and micrometer-level sieve passage to remove interfering particles. Centrifugation was utilized for secondary concentration. Grab samples were collected simultaneously with the ultrafiltered samples. Concentrations of enterococci in all grab and ultrafiltration samples were determined by the standard method (EPA method 1600) for calculation of recovery efficiencies and concentration factors. Levels of enterococci increased twofold in initial retentates and by 4 orders of magnitude in final retentates over ambient concentrations. An aliquot of each final retentate was adsorbed onto polystyrene waveguides for immunoassay analysis of enterococci with a microfluidic fiber optic biosensor, the Raptor. Enterococci were detected when concentrations in the ambient water exceeded the regulatory standard for a single sample (≥105 CFU/100 ml). The combined ultrafiltration-biosensor procedure required 2.5 h for detection compared to 24 for the standard method. This study demonstrated that enterococci can be detected rapidly using on-site ultrafiltration, secondary concentration, and biosensor analysis.
Reduction of Uranium(VI) to Uranium(IV) by Clostridia
Authors: Gao Weimin , Francis Arokiasamy J.
Published: May 2008

Several different species of clostridia reduced U(VI) to U(IV) to various degrees. The optimal pH for U(VI) reduction is 5 to 6 in most cases; a
Clostridium
sp. showed the highest rate at pH 4. Nitrate did not affect U(VI) reduction, indicating that this process in clostridia is nitrate independent.
Microencapsulation of Bacteriophage Felix O1 into Chitosan-Alginate Microspheres for Oral Delivery
Authors: Ma Yongsheng , Pacan Jennifer C. , Wang Qi , Xu Yongping , Huang Xiaoqing , Korenevsky Anton , Sabour Parviz M.
Published: May 2008

This paper reports the development of microencapsulated bacteriophage Felix O1 for oral delivery using a chitosan-alginate-CaCl2system. In vitro studies were used to determine the effects of simulated gastric fluid (SGF) and bile salts on the viability of free and encapsulated phage. Free phage Felix O1 was found to be extremely sensitive to acidic environments and was not detectable after a 5-min exposure to pHs below 3.7. In contrast, the number of microencapsulated phage decreased by 0.67 log units only, even at pH 2.4, for the same period of incubation. The viable count of microencapsulated phage decreased only 2.58 log units during a 1-h exposure to SGF with pepsin at pH 2.4. After 3 h of incubation in 1 and 2% bile solutions, the free phage count decreased by 1.29 and 1.67 log units, respectively, while the viability of encapsulated phage was fully maintained. Encapsulated phage was completely released from the microspheres upon exposure to simulated intestinal fluid (pH 6.8) within 6 h. The encapsulated phage in wet microspheres retained full viability when stored at 4°C for the duration of the testing period (6 weeks). With the use of trehalose as a stabilizing agent, the microencapsulated phage in dried form had a 12.6% survival rate after storage for 6 weeks. The current encapsulation technique enables a large proportion of bacteriophage Felix O1 to remain bioactive in a simulated gastrointestinal tract environment, which indicates that these microspheres may facilitate delivery of therapeutic phage to the gut.
Differential Interference with
Pythium ultimum
Sporangial Activation and Germination by
Enterobacter cloacae
in the Corn and Cucumber Spermospheres
Authors: Windstam Sofia , Nelson Eric B.
Published: May 2008

Differential protection of plants by
Enterobacter cloacae
was studied by investigating early sensing and response behavior of
Pythium ultimum
sporangia toward seeds in the presence or absence of
E. cloacae
. Ten percent of
P. ultimum
sporangia were activated within the first 30 min of exposure to cucumber seeds. In contrast, 44% of the sporangia were activated as early as 15 min after exposure to corn seeds with over 80% sporangial activation by 30 min. Germ tubes emerged from sporangia after 2.5 and 1.0 h in the cucumber and corn spermospheres, respectively. Seed application of the wild-type strain of
E. cloacae
(EcCT-501R3) reduced sporangial activation by 45% in the cucumber spermosphere, whereas no reduction was observed in the corn spermosphere. Fatty acid transport and degradation mutants of
E. cloacae
(strains EcL1 and Ec31, respectively) did not reduce sporangial activation in either of the spermospheres. Although wild-type or mutant strains of
E. cloacae
failed to reduce seed colonization incidence, pathogen biomass on cucumber seeds was reduced in the presence of
E. cloacae
strains EcCT-501R3 and Ec31 by 4 and 8 h after sowing, respectively. By 12 h, levels of
P. ultimum
on cucumber seeds treated with
E. cloacae
EcCT-501R3 did not differ from levels on noninoculated seeds. On corn seeds,
P. ultimum
biomass was not affected by the presence of any
E. cloacae
strain. When introduced after sporangial activation had occurred,
E. cloacae
failed to reduce
P. ultimum
biomass on cucumber seeds compared with that on nontreated seeds. Also, increasing numbers of sporangia used to inoculate seeds yielded increased pathogen biomass at each sampling time. This indicates a direct link between the level of seed-colonizing biomass of
P. ultimum
and the number of activated and germinated sporangia in the spermosphere, suggesting that
E. cloacae
suppresses
P. ultimum
seed infections by reducing sporangial activation and germination within the first 30 to 90 min after sowing.
Temporal Release of Fatty Acids and Sugars in the Spermosphere: Impacts on
Enterobacter cloacae
-Induced Biological Control
Authors: Windstam Sofia , Nelson Eric B.
Published: May 2008

The aim of this study was to determine the temporal release of fatty acids and sugars from corn and cucumber seeds during the early stages of seed germination in order to establish whether sugars found in exudate can prevent exudate fatty acid degradation by
Enterobacter cloacae
. Both saturated (long-chain saturated fatty acids [LCSFA]) and unsaturated (long-chain unsaturated fatty acids [LCUFA]) fatty acids were detected in corn and cucumber seed exudates within 15 min after seed sowing. LCSFA and LCUFA were released at a rate of 26.1 and 6.44 ng/min/seed by corn and cucumber seeds, respectively. The unsaturated portion of the total fatty acid pool from both plant species contained primarily oleic and linoleic acids, and these fatty acids were released at a combined rate of 6.6 and 0.67 ng/min/seed from corn and cucumber, respectively. In the absence of seed exudate sugars,
E. cloacae
degraded linoleic acid at rates of 29 to 39 ng/min, exceeding the rate of total fatty acid release from seeds. Sugars constituted a significant percentage of corn seed exudate, accounting for 41% of the total dry seed weight. Only 5% of cucumber seed exudate was comprised of sugars. Glucose, fructose, and sucrose were the most abundant sugars present in seed exudate from both plant species. Corn seeds released a total of 137 μg/seed of these three sugars within 30 min of sowing, whereas cucumber seeds released 0.83 μg/seed within the same time frame. Levels of glucose, fructose, and sucrose found in corn seed exudate (90 to 342 μg) reduced the rate of linoleic acid degradation by
E. cloacae
to 7.5 to 8.8 ng/min in the presence of either sugar, leaving sufficient concentrations of linoleic acid to activate
Pythium ultimum
sporangia Our results demonstrate that elevated levels of sugars in the corn spermosphere can prevent the degradation of LCUFA by
E. cloacae
, leading to its failure to suppress
P. ultimum
sporangial activation, germination, and subsequent disease development.
ThehmsHFRSOperon ofXenorhabdus nematophilaIs Required for Biofilm Attachment toCaenorhabditis elegans
Authors: Drace Kevin , Darby Creg
Published: May 2008

The bacteriumXenorhabdus nematophilais an insect pathogen and an obligate symbiont of the nematodeSteinernema carpocapsae. X. nematophilamakes a biofilm that adheres to the head of the model nematodeCaenorhabditis elegans, a capabilityX. nematophilashares with the biofilms made byYersinia pestisandYersinia pseudotuberculosis. As inYersiniaspp., theX. nematophilabiofilm requires a 4-gene operon,hmsHFRS. Also like itsYersiniacounterparts, theX. nematophilabiofilm is bound by the lectin wheat germ agglutinin, suggesting that β-linkedN-acetyl-d-glucosamine orN-acetylneuraminic acid is a component of the extracellular matrix.C. elegansmutants with aberrant surfaces that do not permitYersiniabiofilm attachment also are resistant toX. nematophilabiofilms. AnX. nematophila hmsHmutant that failed to make biofilms onC. eleganshad no detectable defect in symbiotic association withS. carpocapsae, nor was virulence reduced against the insectManduca sexta.
Short Peptide Induces an “Uncultivable” Microorganism To Grow In Vitro
Authors: Nichols D. , Lewis K. , Orjala J. , Mo S. , Ortenberg R. , O'Connor P. , Zhao C. , Vouros P. , Kaeberlein T. , Epstein S. S.
Published: May 2008

Microorganisms comprise the bulk of biodiversity, but only a small fraction of this diversity grows on artificial media. This phenomenon was noticed almost a century ago, repeatedly confirmed, and termed the “great plate count anomaly.” Advances in microbial cultivation improved microbial recovery but failed to explain why most microbial species do not grow in vitro. Here we show that at least some of such species can form domesticated variants capable of growth on artificial media. We also present evidence that small signaling molecules, such as short peptides, may be essential factors in initiating growth of nongrowing cells. We identified one 5-amino-acid peptide, LQPEV, that at 3.5 nM induces the otherwise “uncultivable” strain
Psychrobacter
sp. strain MSC33 to grow on standard media. This demonstrates that the restriction preventing microbial in vitro growth may be different from those offered to date to explain the “great plate count anomaly,” such as deficiencies in nutrient composition and concentrations in standard media, medium toxicity, and inappropriate incubation time. Growth induction of MSC33 illustrates that some microorganisms do not grow in vitro because they are removed from their native communities and the signals produced therein. “Uncultivable” species represent the largest source of unexplored biodiversity, and provide remarkable opportunities for both basic and applied research. Access to cultures of some of these species should be possible through identification of the signaling compounds necessary for growth, their addition to standard medium formulations, and eventual domestication.
Two-Log Increase in Sensitivity for Detection of Norovirus in Complex Samples by Concentration with Porcine Gastric Mucin Conjugated to Magnetic Beads
Authors: Tian Peng , Engelbrektson Anna , Mandrell Robert
Published: May 2008

Human histo-blood group antigens (HBGA) have been identified previously as candidate receptors for human norovirus (NOR). Type A, type H1, and Lewis HBGA in humans have been identified as major HBGA for NOR binding. We have found that pig stomach (gastric) mucin (PGM) contains blood group A, H1, and Lewis b HBGA and binds to multiple strains of NOR more broadly than do specific antibodies to NOR. Both genogroup I (GGI) and GGII NOR strains were recovered by PGM-conjugated magnetic beads. A fecal sample containing GGII NOR was detected at a dilution of 1:1,000,000 by the standard RNA extraction procedure, whereas NOR in a 1:100,000,000 dilution could be concentrated by PGM-conjugated magnetic beads and NOR in spiked food samples (e.g., oyster extract, strawberry, raspberry, and lettuce) was captured by PGM, thus minimizing the reverse transcription-PCR inhibitors in food and increasing sensitivity.
Gamma Irradiation Can Be Used To Inactivate
Bacillus anthracis
Spores without Compromising the Sensitivity of Diagnostic Assays
Authors: Dauphin Leslie A. , Newton Bruce R. , Rasmussen Max V. , Meyer Richard F. , Bowen Michael D.
Published: May 2008

The use of
Bacillus anthracis
as a biological weapon in 2001 heightened awareness of the need for validated methods for the inactivation of
B. anthracis
spores. This study determined the gamma irradiation dose for inactivating virulent
B. anthracis
spores in suspension and its effects on real-time PCR and antigen detection assays. Strains representing eight genetic groups of
B. anthracis
were exposed to gamma radiation, and it was found that subjecting spores at a concentration of 10
7
CFU/ml to a dose of 2.5 × 10
6
rads resulted in a 6-log-unit reduction of spore viability. TaqMan real-time PCR analysis of untreated versus irradiated Ames strain (K1694) spores showed that treatment significantly enhanced the detection of
B. anthracis
chromosomal DNA targets but had no significant effect on the ability to detect targets on the pXO1 and pXO2 plasmids of
B. anthracis
. When analyzed by an enzyme-linked immunosorbent assay (ELISA), irradiation affected the detection of
B. anthracis
spores in a direct ELISA but had no effect on the limit of detection in a sandwich ELISA. The results of this study showed that gamma irradiation-inactivated spores can be tested by real-time PCR or sandwich ELISA without decreasing the sensitivity of either type of assay. Furthermore, the results suggest that clinical and public health laboratories which test specimens for
B. anthracis
could potentially incorporate gamma irradiation into sample processing protocols without compromising the sensitivity of the
B. anthracis
assays.
In Vitro Study of the Effect of Cationic Biocides on Bacterial Population Dynamics and Susceptibility
Authors: Moore Louise E. , Ledder Ruth G. , Gilbert Peter , McBain Andrew J.
Published: May 2008

Cationic biocides (CBs) are widely used in domestic and public hygiene and to control biofouling and microbial contamination in industry. The increased use of biocides has led to concern regarding possible reductions in biocide effectiveness. Domestic drain microcosms were stabilized for 5 months and then exposed to polyhexamethylene biguanide (PHMB) at 0.1, 0.2, and 0.4g liter
−1
over 6 months and characterized throughout by differential culture, together with eubacterial-specific PCR-denaturing gradient gel electrophoresis. Additionally, MICs and minimal bactericidal concentrations (MBCs) for bacteria previously isolated from a domestic drain (
n
= 18) and the human skin (
n
= 13) were determined before, during, and after escalating, sublethal exposure (14 passages) to two quaternary ammonium compounds (QAC1 and QAC2), the bisbiguanide chlorhexidine (CHX), and PHMB. Exposure of the drain microcosm to PHMB did not decrease the total viable count although significant (
P
< 0.01) decreases in recovery were observed for the gram-positive cocci with associated clonal expansion of pseudomonads (from ca. 0.1% of the population to ca. 10%). This clonal expansion was also manifested as elevations in bacteria that could grow in the presence of PHMB, CHX, and QAC1. Decreases in susceptibility (greater than twofold) occurred for 10/31 of the test bacteria for QAC1, 14/31 for QAC2, 10/31 for CHX, and 7/31 for PHMB. Exposure of microcosms to PHMB targeted gram-positive species and caused the clonal expansion of pseudomonads. In terms of prolonged-sublethal passage on CBs, exposure to all the biocides tested resulted in susceptibility decreases for a proportion of test bacteria, but refractory clones were not generated.
Role of the Lysozyme Inhibitor Ivy in Growth or Survival ofEscherichia coliandPseudomonas aeruginosaBacteria in Hen Egg White and in Human Saliva and Breast Milk
Authors: Deckers Daphne , Vanlint Dietrich , Callewaert Lien , Aertsen Abram , Michiels Chris W.
Published: May 2008

Ivy is a lysozyme inhibitor that protectsEscherichia coliagainst lysozyme-mediated cell wall hydrolysis when the outer membrane is permeabilized by mutation or by chemical or physical stress. In the current work, we have investigated whether Ivy is necessary for the survival or growth ofE. coliMG1655 andPseudomonas aeruginosaPAO1 in hen egg white and in human saliva and breast milk, which are naturally rich in lysozyme and in membrane-permeabilizing components. Wild-typeE. coliwas able to grow in saliva and breast milk but showed partial inactivation in egg white. The knockout of Ivy did not affect growth in breast milk but slightly increased sensitivity to egg white and caused hypersensitivity to saliva, resulting in the complete inactivation of 104CFU ml−1of bacteria within less than 5 hours. The depletion of lysozyme from saliva completely restored the ability of theivymutant to grow like the parental strain.P. aeruginosa, in contrast, showed growth in all three substrates, which was not affected by the knockout of Ivy production. These results indicate that lysozyme inhibitors like Ivy promote bacterial survival or growth in particular lysozyme-rich secretions and suggest that they may promote the bacterial colonization of specific niches in the animal host.
Development of an In Vitro Activity Assay as an Alternative to the Mouse Bioassay for
Clostridium botulinum
Neurotoxin Type A
Authors: Rasooly Reuven , Do Paula M.
Published: May 2008

Currently, the only accepted assay with which to detect active
Clostridium botulinum
neurotoxin is an in vivo mouse bioassay. The mouse bioassay is sensitive and robust and does not require specialized equipment. However, the mouse bioassay is slow and not practical in many settings, and it results in the death of animals. Here, we describe an in vitro cleavage assay for SNAP-25 (synaptosome-associated proteins of 25 kDa) for measuring the toxin activity with the same sensitivity as that of the mouse bioassay. Moreover, this assay is far more rapid, can be automated and adapted to many laboratory settings, and has the potential to be used for toxin typing. The assay has two main steps. The first step consists of immunoseparation and concentration of the toxin, using immunomagnetic beads with monoclonal antibodies directed against the 100-kDa heavy chain subunit, and the second step consists of a cleavage assay targeting the SNAP-25 peptide of the toxin, labeled with fluorescent dyes and detected as a fluorescence resonance energy transfer assay. Our results suggest that the sensitivity of this assay is 10 pg/ml, which is similar to the sensitivity of the mouse bioassay, and this test can detect the activity of the toxin in carrot juice and beef. These results suggest that the assay has a potential use as an alternative to the mouse bioassay for analysis of
C. botulinum
type A neurotoxin.
Evaluation of Major Types of Shiga Toxin 2e-Producing
Escherichia coli
Bacteria Present in Food, Pigs, and the Environment as Potential Pathogens for Humans
Authors: Beutin Lothar , Krüger Ulrike , Krause Gladys , Miko Angelika , Martin Annett , Strauch Eckhard
Published: May 2008

Shiga toxin 2e (Stx2e)-producing strains from food (
n
= 36), slaughtered pigs (
n
= 25), the environment (
n
= 21), diseased pigs (
n
= 19), and humans (
n
= 9) were investigated for production of Stx2e by enzyme-linked immunosorbent assay, for virulence markers by PCR, and for their serotypes to evaluate their role as potential human pathogens. Stx2e production was low in 64% of all 110 strains. Stx2e production was inducible by mitomycin C but differed considerably between strains. Analysis by nucleotide sequencing and transcription of
stx
2e
genes in high- and low-Stx2e-producing strains showed that toxin production correlated with transcription rates of
stx
2e
genes. DNA sequences specific for the
int
,
Q
,
dam
, and
S
genes of the
stx
2e
bacteriophage P27 were found in 109 strains, indicating cryptic P27-like prophages, although 102 of these were not complete for all genes tested. Genes encoding intimin (
eae
), enterohemorrhagic
Escherichia coli
hemolysin (
ehx
), or other
stx
1
or
stx
2
variants were not found, whereas genes for heat-stable enterotoxins STI, STII, or EAST1 were present in 54.5% of the strains. Seven major serotypes that were associated with diseased pigs (O138:H14, O139:H1, and O141:H4) or with slaughter pigs, food, and the environment (O8:H4, O8:H9, O100:H30, and O101:H9) accounted for 60% of all Stx2e strains. The human Stx2e isolates did not belong to these major serotypes of Stx2e strains, and high production of Stx2e in human strains was not related to diarrheal disease. The results from this study and other studies do not point to Stx2e as a pathogenicity factor for diarrhea and hemolytic uremic syndrome in humans.
Presence of a Family of Plasmids (29 to 65 Kilobases) with a 26-Kilobase Common Region in Different Strains of the Sulfur-Oxidizing BacteriumAcidithiobacillus caldus
Authors: van Zyl Leonard J. , Deane Shelly M. , Louw Lilly-Ann , Rawlings Douglas E.
Published: May 2008

Three large cryptic plasmids from different isolates ofAcidithiobacillus calduswere rescued by using an in vitro transposition system that delivers a kanamycin-selectable marker and anEscherichia coliplasmid origin of replication. The largest of the plasmids, the 65-kb plasmid pTcM1, was isolated from a South AfricanA. caldusstrain, MNG. This plasmid was sequenced and compared to that of pTcF1 (39 kb, from strain “f,” South Africa) and pC-SH12 (29 kb, from strain C-SH12, Australia). With the exception of a 2.7-kb insertion sequence, pC-SH12 appears to represent the DNA common to all three plasmids and includes a number of accessory genes plus the plasmid “backbone” containing the replication region. The two larger plasmids carry, in addition, a number of insertion sequences of the ISL3 family and a composite transposon related to the Tn21subfamily containing a highly mosaic region within the borders of the inverted repeats. Genes coding for arsenic resistance, plasmid mobilization, plasmid stability, and a putative restriction-modification system occur within these mosaic regions.
Esterase Autodisplay: Enzyme Engineering and Whole-Cell Activity Determination in Microplates with pH Sensors
Authors: Schultheiss Eva , Weiss Svenja , Winterer Elisa , Maas Ruth , Heinzle Elmar , Jose Joachim
Published: May 2008

Among the GDSL family of serine esterases/lipases is a group of bacterial enzymes that posses C-terminal extensions involved in outer membrane anchoring or translocation. ApeE fromSalmonella entericaserovar Typhimurium, a member of this group, has been expressed inEscherichia coliand was resistant to protease digestion when the protease was added to whole cells, indicating a periplasmic localization. The five consensus blocks conserved within all GDSL esterases were identified in ApeE by multiple sequence alignment and separated from the C-terminal extension. The DNA sequence spanning the four invariant residues Ser, Gly, Asn, and His, and hence representing the catalytic domains of ApeE, was amplified by PCR and fused in frame to the transport domains of the autodisplay system. The resulting artificial esterase, called EsjA, was overexpressed in the cell envelope ofE. coliand was shown to be active by the use of α-naphthyl acetate (α-NA) as a substrate in an in-gel activity stain after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Surface exposure of EsjA was indicated by its accessibility to protease added to whole cells. The esterase activity of whole cells displaying EsjA was determined by a pH agar assay and by the use of microplates with integrated pH-dependent optical sensors. α-NA, α-naphthyl butyrate, and α-naphthyl caproate were used as substrates, and it turned out that the substrate preferences of artificial EsjA were altered in comparison to original ApeE. Our results indicate that autodisplay of esterase in combination with pH sensor microplates can provide a new platform technology for the screening of tailor-made hydrolase activities.
Listeria monocytogenes
Biofilm-Associated Protein (BapL) May Contribute to Surface Attachment of
L. monocytogenes
but Is Absent from Many Field Isolates
Authors: Jordan Suzanne J. , Perni Stefano , Glenn Sarah , Fernandes Isabel , Barbosa Manuela , Sol Manuela , Tenreiro Rogerio P. , Chambel Lelia , Barata Belarmino , Zilhao Isabel , Aldsworth Timothy G. , Adriao Andreia , Faleiro M. Leonor , Shama Gilbert , Andrew Peter W.
Published: May 2008

Listeria monocytogenes
is a food-borne pathogen capable of adhering to a range of surfaces utilized within the food industry, including stainless steel. The factors required for the attachment of this ubiquitous organism to abiotic surfaces are still relatively unknown. In silico analysis of the
L. monocytogenes
EGD genome identified a putative cell wall-anchored protein (Lmo0435 [BapL]), which had similarity to proteins involved in biofilm formation by staphylococci. An insertion mutation was constructed in
L. monocytogenes
to determine the influence of this protein on attachment to abiotic surfaces. The results show that the protein may contribute to the surface adherence of strains that possess BapL, but it is not an essential requirement for all
L. monocytogenes
strains. Several BapL-negative field isolates demonstrated an ability to adhere to abiotic surfaces equivalent to that of BapL-positive strains. BapL is not required for the virulence of
L. monocytogenes
in mice.
Microarray-Based Characterization of Microbial Community Functional Structure and Heterogeneity in Marine Sediments from the Gulf of Mexico
Authors: Wu Liyou , Kellogg Laurie , Devol Allan H. , Tiedje James M. , Zhou Jizhong
Published: May 2008

Marine sediments of coastal margins are important sites of carbon sequestration and nitrogen cycling. To determine the metabolic potential and structure of marine sediment microbial communities, two cores were collected each from the two stations (GMT at a depth of 200 m and GMS at 800 m) in the Gulf of Mexico, and six subsamples representing different depths were analyzed from each of these two cores using functional gene arrays containing ∼2,000 probes targeting genes involved in carbon fixation; organic carbon degradation; contaminant degradation; metal resistance; and nitrogen, sulfur, and phosphorous cycling. The geochemistry was highly variable for the sediments based on both site and depth. A total of 930 (47.1%) probes belonging to various functional gene categories showed significant hybridization with at least 1 of the 12 samples. The overall functional gene diversity of the samples from shallow depths was in general lower than those from deep depths at both stations. Also high microbial heterogeneity existed in these marine sediments. In general, the microbial community structure was more similar when the samples were spatially closer. The number of unique genes at GMT increased with depth, from 1.7% at 0.75 cm to 18.9% at 25 cm. The same trend occurred at GMS, from 1.2% at 0.25 cm to 15.2% at 16 cm. In addition, a broad diversity of geochemically important metabolic functional genes related to carbon degradation, nitrification, denitrification, nitrogen fixation, sulfur reduction, phosphorus utilization, contaminant degradation, and metal resistance were observed, implying that marine sediments could play important roles in biogeochemical cycling of carbon, nitrogen, phosphorus, sulfate, and various metals. Finally, the Mantel test revealed significant positive correlations between various specific functional genes and functional processes, and canonical correspondence analysis suggested that sediment depth, PO
4
3−
, NH
4
+
, Mn(II), porosity, and Si(OH)
4
might play major roles in shaping the microbial community structure in the marine sediments.
A PCR-Based Method for Monitoring
Legionella pneumophila
in Water Samples Detects Viable but Noncultivable Legionellae That Can Recover Their Cultivability
Authors: Dusserre Eric , Ginevra Christophe , Hallier-Soulier Sylvie , Vandenesch François , Festoc Gabriel , Etienne Jerome , Jarraud Sophie , Molmeret Maëlle
Published: May 2008

Legionella pneumophila
is the causative agent of Legionnaires' disease. This bacterium is ubiquitous in aqueous environments and uses amoebae as an intracellular replicative niche. Real-time PCR has been developed for rapid detection of
Legionella
DNA in water samples. In addition to culturable bacteria, this method may also detect dead and viable but noncultivable (VBNC) legionellae. In order to understand the significance of positive PCR results in this setting, we prepared water samples containing known concentrations of
L. pneumophila
and analyzed them comparatively by means of conventional culture, real-time PCR, viability labeling, and immunodetection (solid-phase cytometry). We also examined the influence of chlorination on the results of the four methods. The different techniques yielded similar results for nonchlorinated water samples but not for chlorinated samples. After treatment for 24 h with 0.5 and 1 ppm chlorine, all cultures were negative, PCR and immunodetection showed about 10
6
genome units and bacteria/ml, and total-viable-count (TVC) labeling detected 10
5
and 10
2
metabolically active bacteria/ml, respectively. Thus, PCR also detected bacteria that were VBNC. The recoverability of VBNC forms was confirmed by 5 days of coculture with
Acanthamoeba polyphaga
. Therefore, some TVC-positive bacteria were potentially infective. These data show that
L. pneumophila
PCR detects not only culturable bacteria but also VBNC forms and dead bacterial DNA at low chlorine concentrations.
Genome-Wide Gene Expression Patterns and Growth Requirements Suggest thatPelobacter carbinolicusReduces Fe(III) Indirectly via Sulfide Production
Authors: Haveman Shelley A. , DiDonato Raymond J. , Villanueva Laura , Shelobolina Evgenya S. , Postier Bradley L. , Xu Bo , Liu Anna , Lovley Derek R.
Published: May 2008

AlthoughPelobacterspecies are closely related toGeobacterspecies, recent studies suggested thatPelobacter carbinolicusmay reduce Fe(III) via a different mechanism because it lacks the outer-surfacec-type cytochromes that are required for Fe(III) reduction byGeobacter sulfurreducens. Investigation into the mechanisms for Fe(III) reduction demonstrated thatP. carbinolicushad growth yields on both soluble and insoluble Fe(III) consistent with those of other Fe(III)-reducing bacteria. Comparison of whole-genome transcript levels during growth on Fe(III) versus fermentative growth demonstrated that the greatest apparent change in gene expression was an increase in transcript levels for four contiguous genes. These genes encode two putative periplasmic thioredoxins; a putative outer-membrane transport protein; and a putative NAD(FAD)-dependent dehydrogenase with homology to disulfide oxidoreductases in the N terminus, rhodanese (sulfurtransferase) in the center, and uncharacterized conserved proteins in the C terminus. UnlikeG. sulfurreducens, transcript levels for cytochrome genes did not increase inP. carbinolicusduring growth on Fe(III).P. carbinolicuscould use sulfate as the sole source of sulfur during fermentative growth, but required elemental sulfur or sulfide for growth on Fe(III). The increased expression of genes potentially involved in sulfur reduction, coupled with the requirement for sulfur or sulfide during growth on Fe(III), suggests thatP. carbinolicusreduces Fe(III) via an indirect mechanism in which (i) elemental sulfur is reduced to sulfide and (ii) the sulfide reduces Fe(III) with the regeneration of elemental sulfur. This contrasts with the direct reduction of Fe(III) that has been proposed forGeobacterspecies.
Leishmania
Adaptor Protein-1 Subunits Are Required for Normal Lysosome Traffic, Flagellum Biogenesis, Lipid Homeostasis, and Adaptation to Temperatures Encountered in the Mammalian Host
Authors: Vince James E. , Tull Dedreia L. , Spurck Timothy , Derby Merran C. , McFadden Geoffrey I. , Gleeson Paul A. , Gokool Suzanne , McConville Malcolm J.
Published: May 2008

The adaptor protein-1 (AP-1) complex is involved in membrane transport between the Golgi apparatus and endosomes. In the protozoan parasite
Leishmania mexicana mexicana
, the AP-1 μ1 and σ1 subunits are not required for growth at 27°C but are essential for infectivity in the mammalian host. In this study, we have investigated the function of these AP-1 subunits in order to understand the molecular basis for this loss of virulence. The μ1 and σ1 subunits were localized to late Golgi and endosome membranes of the major parasite stages. Parasite mutants lacking either AP-1 subunit lacked obvious defects in Golgi structure, endocytosis, or exocytic transport. However, these mutants displayed reduced rates of endosome-to-lysosome transport and accumulated fragmented, sterol-rich lysosomes. Defects in flagellum biogenesis were also evident in nondividing promastigote stages, and this phenotype was exacerbated by inhibitors of sterol and sphingolipid biosynthesis. Furthermore, both AP-1 mutants were hypersensitive to elevated temperature and perturbations in membrane lipid composition. The pleiotropic requirements for AP-1 in membrane trafficking and temperature stress responses explain the loss of virulence of these mutants in the mammalian host.
SSD1
Is Integral to Host Defense Peptide Resistance in
Candida albicans
Authors: Gank Kimberly D. , Yeaman Michael R. , Kojima Satoshi , Yount Nannette Y. , Park Hyunsook , Edwards John E. , Filler Scott G. , Fu Yue
Published: May 2008

Candida albicans
is usually a harmless human commensal. Because inflammatory responses are not normally induced by colonization, antimicrobial peptides are likely integral to first-line host defense against invasive candidiasis. Thus,
C. albicans
must have mechanisms to tolerate or circumvent molecular effectors of innate immunity and thereby colonize human tissues. Prior studies demonstrated that an antimicrobial peptide-resistant strain of
C. albicans
, 36082
R
, is hypervirulent in animal models versus its susceptible counterpart (36082
S
). The current study aimed to identify a genetic basis for antimicrobial peptide resistance in
C. albicans
. Screening of a
C. albicans
genomic library identified
SSD1
as capable of conferring peptide resistance to a susceptible surrogate,
Saccharomyces cerevisiae
. Sequencing confirmed that the predicted translation products of 36082
S
and 36082
R
SSD1
genes were identical. However, Northern analyses corroborated that
SSD1
is expressed at higher levels in 36082
R
than in 36082
S
. In isogenic backgrounds,
ssd1
Δ/
ssd1
Δ null mutants were significantly more susceptible to antimicrobial peptides than parental strains but had equivalent susceptibilities to nonpeptide stressors. Moreover,
SSD1
complementation of
ssd1
Δ/
ssd1
Δ mutants restored parental antimicrobial peptide resistance phenotypes, and overexpression of
SSD1
conferred enhanced peptide resistance. Consistent with these in vitro findings,
ssd1
null mutants were significantly less virulent in a murine model of disseminated candidiasis than were their parental or complemented strains. Collectively, these results indicate that
SSD1
is integral to
C. albicans
resistance to host defense peptides, a phenotype that appears to enhance the virulence of this organism in vivo.
Competitive and Cooperative Effects in Quorum-Sensing-Regulated Galactoglucan Biosynthesis in
Sinorhizobium meliloti
Authors: McIntosh Matthew , Krol Elizaveta , Becker Anke
Published: May 2008

The symbiotic nitrogen-fixing bacterium
Sinorhizobium meliloti
possesses the Sin quorum-sensing system based on
N
-acyl homoserine lactones (AHLs) as signal molecules. The Sin system consists of SinI, the AHL synthase, and SinR, the LuxR-type regulator. This system regulates the expression of a multitude of
S. meliloti
genes through ExpR, another LuxR-type regulator. Analysis of the activity of the
sinI
promoter showed that the expression of
sinI
is dependent on
sinR
and enhanced by a combination of
expR
and Sin AHLs. The characterization of the ExpR binding site upstream of
sinI
and the identification of binding sites upstream of the galactoglucan biosynthesis genes
wgaA
(
expA1
) and
wgeA
(
expE1
) allowed the definition of a consensus sequence for these binding sites. Based on this consensus, two additional ExpR binding sites in the promoter regions of
exoI
and
exsH
, two genes related to the production of succinoglycan, were found. The specific binding of ExpR to the
wgaA
and
wgeA
promoters was enhanced in the presence of oxo-C
14
-HL. Positive regulation of the galactoglucan biosynthesis genes by ExpR was shown to be dependent on WggR (ExpG) and influenced by MucR, both of which are previously characterized regulators of these genes. Based on these results, a reworked model of the Sin-ExpR quorum-sensing regulation scheme of galactoglucan production in
S. meliloti
is suggested.