bluuebunny/crossref_metadata_2025 huggingface dataset:
Polycystic Ovaries and Polycystic Ovary Syndrome in Epilepsy: Evidence for Neurogonadal Disease
Authors: Harden Cynthia L.
Published: July 2005

Polycystic ovary syndrome (PCOS) is a mysterious reproductive disorder that results in subfertility. The underlying causes are not known, and even the definition is a moving target. Women with epilepsy have features of PCOS at a higher than expected rate, and polycystic ovaries (PCO) also are present at high rates in this population. Valproate is associated with weight gain and increased androgen levels, two features of PCOS. This review proposes that epilepsy, with its known adverse effects on luteinizing hormone pulsatility, could be a cause of PCOS and that valproate could be an imitator, if not also a cause of the syndrome.
A Thermochromic Dispersive Electrode Can Measure the Underlying Skin Temperature and Prevent Burns During Radiofrequency Ablation
Authors: THIAGALINGAM ARAVINDA , POULIOPOULOS JIM , BARRY MICHAEL ANTHONY (TONY) , SALISBURY ELIZABETH , PATHMANATHAN NIRMALA , BOYD ANITA , ROSS DAVID L. , KOVOOR PRAMESH
Published: July 2005

Introduction: Burns at the dispersive electrode are serious complications of diathermy and radiofrequency (RF) ablation procedures. We aimed to create a new methodology to reduce the incidence of dispersive electrode related skin burns. We hypothesized that a dispersive electrode incorporating a thermochromic liquid crystal (TLC) layer could accurately measure underlying skin temperatures and help prevent burns.

Methods and Results: The TLC electrode was compared with a standard dispersive electrode in 12 male sheep. RF current was delivered with the dispersive electrode fully applied or partially detached to simulate different clinical scenarios. The temperature of the TLC layer, calculated from the hue (color) every 15 seconds, was compared with fluoroptic skin temperature probes. TLC electrodes with a temperature range of 45–58°C were used in six sheep to assess the correlation of TLC temperature distribution with skin temperature and burns. TLC electrodes with a temperature range of 40–50°C were used in another 6 sheep to simulate clinical conditions in which the ablation was stopped if the TLC temperature was >42°C.

The TLC measured temperatures correlated well with fluoroptic probes at the skin surface (r = 0.94 ± 0.05, mean of the absolute difference in temperature difference 0.9 ± 0.58°C). Ablations with partial application of standard dispersive electrodes consistently caused skin burns. There were no burns under the TLC electrode when ablations were ceased for temperatures >42°C.

Conclusions: TLC‐equipped dispersive electrodes were able to accurately measure skin temperature under the electrode. This technology is likely to prevent dispersive electrode related burns.
Paradoxical Atrial Undersensing Due to Inappropriate Atrial Noise Reversion of Atrial Fibrillation in Dual‐Chamber Pacemakers
Authors: KOLB CHRISTOF , HALBFAß PHILIPP , ZRENNER BERNHARD , SCHMITT CLAUS
Published: July 2005

Background: Paradoxical atrial undersensing at high atrial sensing levels was described as false atrial noise reversion of dual‐chamber pacemakers during atrial fibrillation in a sheep model. It is unknown whether this phenomenon occurs in humans.

Methods: In total, 71 patients with implanted dual‐chamber pacemakers and atrial fibrillation were tested for the occurrence of paradoxical atrial undersensing. After determination of the sensing threshold of atrial fibrillation (30 seconds of continuous mode switch), the atrial sensing level was stepwise increased. If, after correct mode switch behavior at insensitive levels, loss of mode switch occurred at higher sensing levels and if the pacing mode was consistent with atrial noise reversion, paradoxical atrial undersensing was assumed.

Results: Paradoxical atrial undersensing could be provoked in 9 of 71 (13%) patients at a median sensing level of 0.4 (range 0.15–2.0) mV. Six different pacemaker models of five different manufacturers were affected. The occurrence of paradoxical atrial undersensing was significantly associated with the sensing threshold of atrial fibrillation (2.7 ± 1.5 mV for patients with paradoxical undersensing compared to 1.6 ± 1.3 mV for those without, P = 0.02). Decreasing the atrial sensing level avoided paradoxical undersensing in 8 of 9 patients while maintaining an adequate safety margin for the detection of atrial fibrillation.

Conclusion: Paradoxical atrial undersensing is inherent to all current dual‐chamber pacemakers. The incidence is about 13% when using very high atrial sensing levels. Inappropriate atrial noise reversion can be resolved in most of the cases by decreasing atrial sensing levels and knowledge of this phenomenon is important to avoid unwarranted atrial lead revisions.
On Geodetic Distance Computations in Spatial Modeling
Authors: Banerjee Sudipto
Published: July 2005

Summary Statisticians analyzing spatial data often need to detect and model associations based upon distances on the Earth's surface. Accurate computation of distances are sought for exploratory and interpretation purposes, as well as for developing numerically stable estimation algorithms. When the data come from locations on the spherical Earth, application of Euclidean or planar metrics for computing distances is not straightforward. Yet, planar metrics are desirable because of their easier interpretability, easy availability in software packages, and well‐established theoretical properties. While distance computations are indispensable in spatial modeling, their importance and impact upon statistical estimation and prediction have gone largely unaddressed. This article explores the different options in using planar metrics and investigates their impact upon spatial modeling.
Variable Selection for Marginal Longitudinal Generalized Linear Models
Authors: Cantoni Eva , Flemming Joanna Mills , Ronchetti Elvezio
Published: July 2005

SummaryVariable selection is an essential part of any statistical analysis and yet has been somewhat neglected in the context of longitudinal data analysis. In this article, we propose a generalized version of Mallows'sCp(GCp) suitable for use with both parametric and nonparametric models.GCpprovides an estimate of a measure of model's adequacy for prediction. We examine its performance with popular marginal longitudinal models (fitted using GEE) and contrast results with what is typically done in practice: variable selection based on Wald‐type or score‐type tests. An application to real data further demonstrates the merits of our approach while at the same time emphasizing some important robust features inherent toGCp.
Exact Two‐Sample Inference with Missing Data
Authors: Cheung Ying Kuen
Published: July 2005

Summary When comparing follow‐up measurements from two independent populations, missing records may arise due to censoring by events whose occurrence is associated with baseline covariates. In these situations, inferences based only on the completely followed observations may be biased if the follow‐up measurements and the covariates are correlated. This article describes exact inference for a class of modified U‐statistics under covariate‐dependent dropouts. The method involves weighing each permutation according to the retention probabilities, and thus requires estimation of the missing data mechanism. The proposed procedure is nonparametric in that no distributional assumption is necessary for the outcome variables and the missingness patterns. Monte Carlo approximation by the Gibbs sampler is proposed, and is shown to be fast and accurate via simulation. The method is illustrated in two small data sets for which asymptotic inferential procedures may not be appropriate.
Estimation of Breeding Values Using Selected Pedigree Records
Authors: Morton Richard , Howarth Jordan M.
Published: July 2005

SummaryFish bred in tanks or ponds cannot be easily tagged individually. The parentage of any individual may be determined by DNA fingerprinting, but is sufficiently expensive that large numbers cannot be so fingerprinted. The measurement of the objective trait can be made on a much larger sample relatively cheaply. This article deals with experimental designs for selecting individuals to be fingerprinted and for the estimation of the individual and family breeding values. The general setup provides estimates for both genetic effects regarded as fixed or random and for fixed effects due to known regressors. The family effects can be well estimated when even very small numbers are fingerprinted, provided that they are the individuals with the most extreme phenotypes.
Likelihood Methods for Treatment Noncompliance and Subsequent Nonresponse in Randomized Trials
Authors: O'Malley A. James , Normand Sharon‐Lise T.
Published: July 2005

SummaryWhile several new methods that account for noncompliance or missing data in randomized trials have been proposed, the dual effects of noncompliance and nonresponse are rarely dealt with simultaneously. We construct a maximum likelihood estimator (MLE) of the causal effect of treatment assignment for a two‐armed randomized trial assuming all‐or‐none treatment noncompliance and allowing for subsequent nonresponse. The EM algorithm is used for parameter estimation. Our likelihood procedure relies on a latent compliance state covariate that describes the behavior of a subject under all possible treatment assignments and characterizes the missing data mechanism as inFrangakis and Rubin(1999,Biometrika86,365–379). Using simulated data, we show that the MLE for normal outcomes compares favorably to the method‐of‐moments (MOM) and the standard intention‐to‐treat (ITT) estimators under (1) both normal and nonnormal data, and (2) departures from the latent ignorability and compound exclusion restriction assumptions. We illustrate methods using data from a trial to compare the efficacy of two antipsychotics for adults with refractory schizophrenia.
Bayesian Cure Rate Frailty Models with Application to a Root Canal Therapy Study
Authors: Yin Guosheng
Published: July 2005

SummaryDue to natural or artificial clustering, multivariate survival data often arise in biomedical studies, for example, a dental study involving multiple teeth from each subject. A certain proportion of subjects in the population who are not expected to experience the event of interest are considered to be “cured” or insusceptible. To model correlated or clustered failure time data incorporating a surviving fraction, we propose two forms of cure rate frailty models. One model naturally introduces frailty based on biological considerations while the other is motivated from the Cox proportional hazards frailty model. We formulate the likelihood functions based on piecewise constant hazards and derive the full conditional distributions for Gibbs sampling in the Bayesian paradigm. As opposed to the Cox frailty model, the proposed methods demonstrate great potential in modeling multivariate survival data with a cure fraction. We illustrate the cure rate frailty models with a root canal therapy data set.
Selling Foreign Investment Short
Authors: Overholt William H.
Published: July 2005

Yasheng Huang is a thoughtful, articulate observer of China's political
economy, who has gained international respect. Many high-level conferences
benefit from his insights, and this reviewer is indebted on a variety of
issues.

Selling China is essentially two books. One is a
very impressive, very carefully documented review of the negative
consequences for the Chinese economy of laws and policies that advantage
state enterprises at the expense of private enterprises – particularly of a
financial system that allocates financial resources overwhelmingly to
relatively inefficient state enterprises, and proportionately disadvantages
private enterprises. As Huang details, the current Chinese system allocates
advantages and disadvantages to firms according to a political pecking order
rather than by credit quality. China's markets are highly fragmented and
locally protected. China's banks allocate capital disproportionately to
state enterprises. Its stock and bond markets are largely restricted by
policy, although not by law, to state enterprises. Implicit government
guarantees of loans to the biggest state enterprises (SOEs), together with
weak private-sector accounting standards, make it irrational for the banks
to lend to private firms rather than to big SOEs.
The Risk of Partner Violence Among Low‐Income Hispanic Subgroups
Authors: Frias Sonia M. , Angel Ronald J.
Published: July 2005

Women with few social resources are at elevated risk of partner abuse. Certain evidence suggests that African American and Hispanic women, who are overrepresented in the lower socioeconomic strata, are at particularly high risk. We compare women's risk of partner violence, defined as moderate and severe, among 2,400 low‐income African American, Hispanic, and non‐Hispanic Whites from “Welfare, Children and Families: A Three City Study” and find that these groups differ in their risk of degrees of violence. Specific nation‐of‐origin Hispanic subgroups also manifest important differences in their violence risk profiles. We argue that a better understanding of victimization requires more detailed ethnic categorization and a more refined understanding of the meaning of domestic violence for different groups.
The Influence of Union Transitions on White Adults’ Attitudes Toward Cohabitation
Authors: Cunningham Mick , Thornton Arland
Published: July 2005

Data from the Intergenerational Panel Study of Parents and Children are used to identify the influences of adult union transitions on changes in attitudes toward cohabitation among a sample of 794 young adults. The analysis examines the extent to which attitudes about cohabitation change as a result of entry into and exit from cohabitation and marriage. A dynamic interpretation of union transitions is formulated, and results demonstrate that entry into a first cohabitation and divorce after direct entry into marriage are associated with increasingly positive attitudes toward cohabitation between the ages of 18 and 31. Some evidence suggests that direct entry into stable marriage leads young adults to view cohabitation less favorably.
MICROBIOLOGY AND NUTRITIONAL COMPOSITION OF AN EDIBLE LARVA (BUNAEA ALCINOE STOLL) OF THE NIGER DELTA
Authors: AMADI E.N. , OGBALU O.K. , BARIMALAA I.S. , PIUS M.
Published: July 2005

The microbial load and nutritional composition of the Bunaea alcinoe larvae were investigated. The bacterial population of the skin and intestinal content were 8.16 × 106and 5.70 × 105 cfu/g while the fungal populations were 2.07 × 106and 1.32 × 106 cfu/g, respectively. Twenty bacterial isolates belonging to the genera Staphylococcus, Bacillus, Micrococcus and Acinetobacter were isolated. Staphylococcus aureus occurred on both the skin and intestinal content. The nutritional composition of the larvae revealed high protein (55%) followed by fat (25%). Total available carbohydrate, moisture and ash were 4, 8 and 8%, respectively. The public health implication of the presence of S. aureus is highlighted. B. alcinoe larva is a rich source of protein and can be recommended in the diet of communities where protein is scarce or expensive.
EFFECT OF MODIFIED ATMOSPHERE PACKAGING AND TEMPERATURE ON THE SHELF LIFE OF SLICED PASTIRMA PRODUCED FROM FROZEN/THAWED MEAT
Authors: AKSU MUHAMMET İRFAN , KAYA MÜKERREM , OCKERMAN HERBERT W.
Published: July 2005

Pastirma is a dry‐cured meat product and is categorized as an intermediate moisture food. Nonsliced pastirma can be stored without refrigeration. Pastirma is consumed sliced without cooking. For preservation of quality characteristics, sliced pastirma needs to be packaged and stored at fairly low temperatures (<10C). In this study, pastirma was made from frozen M. Longissimus dorsi muscle stored at−18C for 240 days and then thawed at 10C for 24 h. Pastirma produced from frozen/thawed meat was sliced. The sliced pastirma samples were stored in modified atmosphere packaging (MAP) (50% N2 + 50% CO2) at 4C and 10C for 150 days and thiobarbutiric acid reactive substances, free fatty acids, nonprotein nitrogen, pH, L*, a* and b* values, total aerobic and lactic acid bacteria, Micrococcus/Staphylococcus, yeast‐mold, and Enterobacteriaceae counts in sliced product were determined during storage (0, 30, 60, 90, 120 and 150 days). Results showed that sliced pastirma samples made from frozen/thawed meat and MAP can be stored at 4C and 10C for 150 days.
TRANSPORT AND STUNNING AFFECT QUALITY OF ARCTIC CHAR FILLETS
Authors: JITTINANDANA S. , KENNEY P.B. , MAZIK P.M. , DANLEY M. , NELSON C.D. , KISER R.A. , HANKINS J.A.
Published: July 2005

Arctic char transport was conducted using AQUI‐S, gas carbon dioxide (CO

2

), ice slurry or water for 5.5 h followed by CO

2

or by percussive stunning. The treatment without transport represented the control. At harvest and at 24‐h postmortem, transport in an ice slurry followed by percussive stunning resulted in the highest muscle pH; CO

2

stunning reduced muscle pH regardless of transport treatment. At 24‐h postmortem, the control treatment resulted in the firmest cooked products, while other transport conditions did not affect texture. Stunning with CO

2

produced a firmer texture compared with percussive stunning for cooked products derived from 24‐h and 4‐day postmortem refrigerated fillets. The transport treatment and the stunning method did not affect the muscle pH after a 4‐day storage at 3C.
INFLUENCE OF DIETARY MANGANESE SOURCE AND SUPPLEMENTATION LEVEL ON PORK QUALITY DURING RETAIL DISPLAY
Authors: APPLE JASON K. , ROBERTS W. JOSHUA , MAXWELL CHARLES V. , BOGER CHARLES B. , FRIESEN KIM G. , RAKES LILLY K. , FAKLER TIM M.
Published: July 2005

Pork loins were used to test the effects of dietary manganese source and supplementation level on pork quality traits during retail display. Chops from pigs fed 350 ppm Mn from Availa‐Mn received higher (P < 0.05) Japanese color scores, and, after 4 days of retail display, were less discolored than chops from pigs fed the control diet or diets containing manganese sulfate (MnSO4) (diet × display; P = 0.092). Chops from pigs fed 350 ppm Mn from MnSO4lost less moisture after 2 days of display (diet × display; P = 0.058), and tended to have lower (P < 0.10) thiobarbituric acid reactive substances values than chops from pigs fed the control diets or diets supplemented with 700 ppm Mn from Availa‐Mn. The results indicate that feeding an additional 350 ppm Mn may enhance pork quality; however, the dietary inclusion of 700 ppm Mn, regardless of source, had no beneficial effects on pork color or lipid oxidation.
TU‐C‐J‐6B‐06: Dynamic Contrast Enhanced CT to Improve Localization of the GTV Within the Prostate
Authors: Jeukens CRLPN , van den Berg CAT , Donker R , Vulpen M , van Leeuwen MS , Lagendijk JJW , van der Heide UA
Published: July 2005

Purpose: To achieve a higher sensitivity and specificity in the localization of the GTV within the prostate using quantitative perfusion maps obtained from dynamic contrast enhanced CT (DCE‐CT) imaging. Method and Materials: DCE‐CT tracks the passage of a bolus of contrast agent through the prostate in time. With a 40 slice CT scanner we scan a large volume of the prostate with a high temporal (1.5 s) and spatial (0.5×0.5×5 mm) resolution. Moreover, because of the linearity between CT signal enhancement and contrast concentration increase, quantitative analysis is greatly facilitated. We calculate quantitative perfusion maps of the complete prostate by using the tissue homogeneity model, which determines the response of a voxel to the measured input bolus. Furthermore, we have performed an extensive signal‐to‐noise analysis to determine the optimum between voxel size and flow accuracy. We have included 11 patients in our study with biopsy proven prostate cancer (stage T3). The CT exam was performed prior to external beam radiotherapy. Results: For all 11 patients we have successfully calculated perfusion maps with an accuracy of 10% and a resolution of 1.5×1.5×5 mm. We find the average perfusion of the prostate to be 15 ml/100g/min, while the elevated regions have average values of 60–70 ml/100g/min. These hotspots, which are identified as the GTV, can be delineated in three dimensions based on the flow maps and are found to correlate well with the clinically known GTV. Conclusion: Using DCE‐CT we can determine three‐dimensional quantitative perfusion maps of the complete prostate. Elevated flow regions correlate well with the clinically known GTV, and may be of value to improve delineation of the GTV within the prostate.
Supernova Olivine from Cometary Dust
Authors: Messenger Scott , Keller Lindsay P. , Lauretta Dante S.
Published: July 2005

An interplanetary dust particle contains a submicrometer crystalline silicate aggregate of probable supernova origin. The grain has a pronounced enrichment in
18
O/
16
O (13 times the solar value) and depletions in
17
O/
16
O (one-third solar) and
29
Si/
28
Si (<0.8 times solar), indicative of formation from a type II supernova. The aggregate contains olivine (forsterite 83) grains <100 nanometers in size, with microstructures that are consistent with minimal thermal alteration. This unusually iron-rich olivine grain could have formed by equilibrium condensation from cooling supernova ejecta if several different nucleosynthetic zones mixed in the proper proportions. The supernova grain is also partially encased in nitrogen-15-rich organic matter that likely formed in a presolar cold molecular cloud.
Ecosystem Collapse in Pleistocene Australia and a Human Role in Megafaunal Extinction
Authors: Miller Gifford H. , Fogel Marilyn L. , Magee John W. , Gagan Michael K. , Clarke Simon J. , Johnson Beverly J.
Published: July 2005

Most of Australia's largest mammals became extinct 50,000 to 45,000 years ago, shortly after humans colonized the continent. Without exceptional climate change at that time, a human cause is inferred, but a mechanism remains elusive. A 140,000-year record of dietary δ
13
C documents a permanent reduction in food sources available to the Australian emu, beginning about the time of human colonization; a change replicated at three widely separated sites and in the marsupial wombat. We speculate that human firing of landscapes rapidly converted a drought-adapted mosaic of trees, shrubs, and nutritious grasslands to the modern fire-adapted desert scrub. Animals that could adapt survived; those that could not, became extinct.
Premelting at Defects Within Bulk Colloidal Crystals
Authors: Alsayed A. M. , Islam M. F. , Zhang J. , Collings P. J. , Yodh A. G.
Published: July 2005

Premelting is the localized loss of crystalline order at surfaces and defects at temperatures below the bulk melting transition. It can be thought of as the nucleation of the melting process. Premelting has been observed at the surfaces of crystals but not within. We report observations of premelting at grain boundaries and dislocations within bulk colloidal crystals using real-time video microscopy. The crystals are equilibrium close-packed, three-dimensional colloidal structures made from thermally responsive microgel spheres. Particle tracking reveals increased disorder in crystalline regions bordering defects, the amount of which depends on the type of defect, distance from the defect, and particle volume fraction. Our observations suggest that interfacial free energy is the crucial parameter for premelting in colloidal and atomic-scale crystals.
Tunable Supercurrent Through Semiconductor Nanowires
Authors: Doh Yong-Joo , van Dam Jorden A. , Roest Aarnoud L. , Bakkers Erik P. A. M. , Kouwenhoven Leo P. , De Franceschi Silvano
Published: July 2005

Nanoscale superconductor/semiconductor hybrid devices are assembled from indium arsenide semiconductor nanowires individually contacted by aluminum-based superconductor electrodes. Below 1 kelvin, the high transparency of the contacts gives rise to proximity-induced superconductivity. The nanowires form superconducting weak links operating as mesoscopic Josephson junctions with electrically tunable coupling. The supercurrent can be switched on/off by a gate voltage acting on the electron density in the nanowire. A variation in gate voltage induces universal fluctuations in the normal-state conductance, which are clearly correlated to critical current fluctuations. The alternating-current Josephson effect gives rise to Shapiro steps in the voltage-current characteristic under microwave irradiation.
Highly Pathogenic H5N1 Influenza Virus Infection in Migratory Birds
Authors: Liu J. , Xiao H. , Lei F. , Zhu Q. , Qin K. , Zhang X.-w. , Zhang X.-l. , Zhao D. , Wang G. , Feng Y. , Ma J. , Liu W. , Wang J. , Gao G. F.
Published: July 2005

H5N1 avian influenza virus (AIV) has emerged as a pathogenic entity for a variety of species, including humans, in recent years. Here we report an outbreak among migratory birds on Lake Qinghaihu, China, in May and June 2005, in which more than a thousand birds were affected. Pancreatic necrosis and abnormal neurological symptoms were the major clinical features. Sequencing of the complete genomes of four H5N1 AIV strains revealed them to be reassortants related to a peregrine falcon isolate from Hong Kong and to have known highly pathogenic characteristics. Experimental animal infections reproduced typical highly pathogenic AIV infection symptoms and pathology.
OusB, a Broad-Specificity ABC-Type Transporter fromErwinia chrysanthemi, Mediates Uptake of Glycine Betaine and Choline with a High Affinity
Authors: Choquet Gwénaëlle , Jehan Nathalie , Pissavin Christine , Blanco Carlos , Jebbar Mohamed
Published: July 2005

The ability ofErwinia chrysanthemito cope with environments of elevated osmolality is due in part to the transport and accumulation of osmoprotectants. In this study we have identified a high-affinity glycine betaine and choline transport system inE. chrysanthemi. By using a pool of Tn5-B21ousAmutants, we isolated a mutant that could grow in the presence of a toxic analogue of glycine betaine (benzyl-glycine betaine) at high osmolalities. This mutant was impaired in its ability to transport all effective osmoprotectants inE. chrysanthemi. The DNA sequence of the regions flanking the transposon insertion site revealed three chromosomal genes (ousVWX) that encode components of an ABC-type transporter (OusB): OusV (ATPase), OusW (permease), and OusX (periplasmic binding protein). The OusB components showed a significant degree of sequence identity to components of ProU fromSalmonella entericaserovar Typhimurium andEscherichia coli. OusB was found to restore the uptake of glycine betaine and choline through functional complementation of anE. colimutant defective in both ProU and ProP osmoprotectant uptake systems. Competition experiments demonstrated that choline, dimethylsulfoniacetate, dimethylsulfoniopropionate, and ectoine were effective competitors for OusB-mediated betaine transport but that carnitine, pipecolate, and proline were not effective. In addition, the analysis of single and double mutants showed that OusA and OusB were the only osmoprotectant transporters operating inE. chrysanthemi.
Combined Effect of High-Pressure Treatments and Bacteriocin-Producing Lactic Acid Bacteria on Inactivation of
Escherichia coli
O157:H7 in Raw-Milk Cheese
Authors: Rodriguez Eva , Arques Juan L. , Nuñez Manuel , Gaya Pilar , Medina Margarita
Published: July 2005

The effect of high-pressure (HP) treatments combined with bacteriocins of lactic acid bacteria (LAB) produced in situ on the survival of
Escherichia coli
O157:H7 in cheese was investigated. Cheeses were manufactured from raw milk inoculated with
E. coli
O157:H7 at approximately 10
5
CFU/ml. Seven different bacteriocin-producing LAB were added at approximately 10
6
CFU/ml as adjuncts to the starter. Cheeses were pressurized on day 2 or 50 at 300 MPa for 10 min or 500 MPa for 5 min, at 10°C in both cases. After 60 days,
E. coli
O157:H7 counts in cheeses manufactured without bacteriocin-producing LAB and not pressurized were 5.1 log CFU/g. A higher inactivation of
E. coli
O157:H7 was achieved in cheeses without bacteriocin-producing LAB when 300 MPa was applied on day 50 (3.8-log-unit reduction) than if applied on day 2 (1.3-log-unit reduction). Application of 500 MPa eliminated
E. coli
O157:H7 in 60-day-old cheeses. Cheeses made with bacteriocin-producing LAB and not pressurized showed a slight reduction of the pathogen. Pressurization at 300 MPa on day 2 and addition of lacticin 481-, nisin A-, bacteriocin TAB 57-, or enterocin AS-48-producing LAB were synergistic and reduced
E. coli
O157:H7 counts to levels below 2 log units in 60-day-old cheeses. Pressurization at 300 MPa on day 50 and addition of nisin A-, bacteriocin TAB 57-, enterocin I-, or enterocin AS-48-producing LAB completely inactivated
E. coli
O157:H7 in 60-day-old cheeses. The application of reduced pressures combined with bacteriocin-producing LAB is a feasible procedure to improve cheese safety.
Stable Isotope Fractionation of Tetrachloroethene during Reductive Dechlorination by
Sulfurospirillum multivorans
and
Desulfitobacterium
sp. Strain PCE-S and Abiotic Reactions with Cyanocobalamin
Authors: Nijenhuis Ivonne , Andert Janet , Beck Kirsten , Kästner Matthias , Diekert Gabriele , Richnow Hans-Hermann
Published: July 2005

Carbon stable isotope fractionation of tetrachloroethene (PCE) during reductive dechlorination by whole cells and crude extracts of
Sulfurospirillum multivorans
and
Desulfitobacterium
sp. strain PCE-S and the abiotic reaction with cyanocobalamin (vitamin B
12
) was studied. Fractionation was largest during the reaction with cyanocobalamin with αC = 1.0132. Stable isotope fractionation was lower but still in a similar order of magnitude for
Desulfitobacterium
sp. PCE-S (αC = 1.0052 to 1.0098). The isotope fractionation of PCE during dehalogenation by
S. multivorans
was lower by 1 order of magnitude (αC = 1.00042 to 1.0017). Additionally, an increase in isotope fractionation was observed with a decrease in cell integrity for both strains. For
Desulfitobacterium
sp. strain PCE-S, the carbon stable isotope fractionation factors were 1.0052 and 1.0089 for growing cells and crude extracts, respectively. For
S. multivorans
, αC values were 1.00042, 1.00097, and 1.0017 for growing cells, crude extracts, and the purified PCE reductive dehalogenase, respectively. For the field application of stable isotope fractionation, care is needed as fractionation may vary by more than an order of magnitude depending on the bacteria present, responsible for degradation.
Enhancement of the Efficiency of Secretion of Heterologous Lipase in
Escherichia coli
by Directed Evolution of the ABC Transporter System
Authors: Eom Gyeong Tae , Song Jae Kwang , Ahn Jung Hoon , Seo Yeon Soo , Rhee Joon Shick
Published: July 2005

The ABC transporter (TliDEF) from
Pseudomonas fluorescens
SIK W1, which mediated the secretion of a thermostable lipase (TliA) into the extracellular space in
Escherichia coli
, was engineered using directed evolution (error-prone PCR) to improve its secretion efficiency. TliD mutants with increased secretion efficiency were identified by coexpressing the mutated
tliD
library with the wild-type
tliA
lipase in
E. coli
and by screening the library with a tributyrin-emulsified indicator plate assay and a microtiter plate-based assay. Four selected mutants from one round of error-prone PCR mutagenesis, T6, T8, T24, and T35, showed 3.2-, 2.6-, 2.9-, and 3.0-fold increases in the level of secretion of TliA lipase, respectively, but had almost the same level of expression of TliD in the membrane as the strain with the wild-type TliDEF transporter. These results indicated that the improved secretion of TliA lipase was mediated by the transporter mutations. Each mutant had a single amino acid change in the predicted cytoplasmic regions in the membrane domain of TliD, implying that the corresponding region of TliD was important for the improved and successful secretion of the target protein. We therefore concluded that the efficiency of secretion of a heterologous protein in
E. coli
can be enhanced by in vitro engineering of the ABC transporter.
Role of Porins in Sensitivity of
Escherichia coli
to Antibacterial Activity of the Lactoperoxidase Enzyme System
Authors: De Spiegeleer Philipp , Sermon Jan , Vanoirbeek Kristof , Aertsen Abram , Michiels Chris W.
Published: July 2005

Lactoperoxidase is an enzyme that contributes to the antimicrobial defense in secretory fluids and that has attracted interest as a potential biopreservative for foods and other perishable products. Its antimicrobial activity is based on the formation of hypothiocyanate (OSCN
−
) from thiocyanate (SCN
−
), using H
2
O
2
as an oxidant. To gain insight into the antibacterial mode of action of the lactoperoxidase enzyme system, we generated random transposon insertion mutations in
Escherichia coli
MG1655 and screened the resultant mutants for an altered tolerance of bacteriostatic concentrations of this enzyme system. Out of the ca. 5,000 mutants screened, 4 showed significantly increased tolerance, and 2 of these had an insertion, one in the
waaQ
gene and one in the
waaO
gene, whose products are involved in the synthesis of the core oligosaccharide moiety of lipopolysaccharides. Besides producing truncated lipopolysaccharides and displaying hypersensitivity to novobiocin and sodium dodecyl sulfate (SDS), these mutants were also shown by urea-SDS-polyacrylamide gel electrophoresis analysis to have reduced amounts of porins in their outer membranes. Moreover, they showed a reduced degradation of
p
-nitrophenyl phosphate and an increased resistance to ampicillin, two indications of a decrease in outer membrane permeability for small hydrophilic solutes. Additionally,
ompC
and
ompF
knockout mutants displayed levels of tolerance to the lactoperoxidase system similar to those displayed by the
waa
mutants. These results suggest that mutations which reduce the porin-mediated outer membrane permeability for small hydrophilic molecules lead to increased tolerance to the lactoperoxidase enzyme system because of a reduced uptake of OSCN
−
.
Development of a Simple and Rapid Fluorogenic Procedure for Identification of
Vibrionaceae
Family Members
Authors: Richards Gary P. , Watson Michael A. , Parveen Salina
Published: July 2005

We describe a simple colony overlay procedure for peptidases (COPP) for the rapid fluorogenic detection and quantification of
Vibrionaceae
from seawater, shellfish, sewage, and clinical samples. The assay detects phosphoglucose isomerase with a lysyl aminopeptidase activity that is produced by
Vibrionaceae
family members. Overnight cultures are overlaid for 10 min with membranes containing a synthetic substrate, and the membranes are examined for fluorescent foci under UV illumination. Fluorescent foci were produced by all the
Vibrionaceae
tested, including
Vibrio
spp.,
Aeromonas
spp., and
Plesiomonas
spp. Fluorescence was not produced by non-
Vibrionaceae
pathogens.
Vibrio cholerae
strains O1, O139, O22, and O155 were strongly positive. Seawater and oysters were assayed, and 87 of 93 (93.5%) of the positive isolates were identified biochemically as
Vibrionaceae
, principally
Vibrio vulnificus
,
Vibrio parahaemolyticus
,
Aeromonas hydrophila
,
Photobacterium damselae
, and
Shewanella putrefaciens
. None of 50 nonfluorescent isolates were
Vibrionaceae
. No
Vibrionaceae
were detected in soil, and only
A. hydrophila
was detected in sewage. The COPP technique may be particularly valuable in environmental and food-testing laboratories and for monitoring water quality in the aquaculture industry.
Identification and Characterization of a SecondlexAGene ofXanthomonas axonopodisPathovar citri
Authors: Yang Mei-Kwei , Su Shu-Ray , Sung Vin-Long
Published: July 2005

We previously identified and characterized alexAgene fromXanthomonas axonopodispv. citri. For this study, we cloned and expressed alexAhomologue fromX. axonopodispv. citri. This gene was designatedlexA2, and the previously identifiedlexAgene was renamedlexA1. The coding region oflexA2is 606 bp long and shares 59% nucleotide sequence identity withlexA1. Analyses of the deduced amino acid sequence revealed that LexA2 has structures that are characteristic of LexA proteins, including a helix-turn-helix DNA binding domain and conserved amino acid residues required for the autocleavage of LexA. ThelexA2mutant, which was constructed by gene replacement, was 4 orders of magnitude more resistant to the DNA-damaging agent mitomycin C at 0.1 μg/ml and 1 order of magnitude more resistant to another DNA-damaging agent, methylmethane sulfonate at 30 μg/ml, than the wild type. AlexA1 lexA2double mutant had the same degree of susceptibility to mitomycin C as thelexA1orlexA2single mutant but was 1 order of magnitude more resistant to methylmethane sulfonate at 30 μg/ml than thelexA1orlexA2single mutant. These results suggest that LexA1 and LexA2 play different roles in regulating the production of methyltransferases that are required for repairing DNA damage caused by methylmethane sulfonate. A mitomycin C treatment also caused LexA2 to undergo autocleavage, as seen with LexA1. The results of electrophoresis mobility shift assays revealed that LexA2 does not bind thelexA1promoter. It binds to both thelexA2andrecApromoters. However, neither LexA2 nor LexA1 appears to regulaterecAexpression, aslexA1,lexA2, andlexA1 lexA2mutants did not become constitutive forrecAtranscription and RecA production. These results suggest thatrecAexpression inX. axonopodispv. citri is regulated by mechanisms that have yet to be identified.
Algal Viruses with Distinct Intraspecies Host Specificities Include Identical Intein Elements
Authors: Nagasaki Keizo , Shirai Yoko , Tomaru Yuji , Nishida Kensho , Pietrokovski Shmuel
Published: July 2005

Heterosigma akashiwo
virus (HaV) is a large double-stranded DNA virus infecting the single-cell bloom-forming raphidophyte (golden brown alga)
H. akashiwo
. A molecular phylogenetic sequence analysis of HaV DNA polymerase showed that it forms a sister group with
Phycodnaviridae
algal viruses. All 10 examined HaV strains, which had distinct intraspecies host specificities, included an intein (protein intron) in their DNA polymerase genes. The 232-amino-acid inteins differed from each other by no more than a single nucleotide change. All inteins were present at the same conserved position, coding for an active-site motif, which also includes inteins in mimivirus (a very large double-stranded DNA virus of amoebae) and in several archaeal DNA polymerase genes. The HaV intein is closely related to the mimivirus intein, and both are apparently monophyletic to the archaeal inteins. These observations suggest the occurrence of horizontal transfers of inteins between viruses of different families and between archaea and viruses and reveal that viruses might be reservoirs and intermediates in horizontal transmissions of inteins. The homing endonuclease domain of the HaV intein alleles is mostly deleted. The mechanism keeping their sequences basically identical in HaV strains specific for different hosts is yet unknown. One possibility is that rapid and local changes in the HaV genome change its host specificity. This is the first report of inteins found in viruses infecting eukaryotic algae.
Metabolic Engineering of Anthocyanin Biosynthesis in
Escherichia coli
Authors: Yan Yajun , Chemler Joseph , Huang Lixuan , Martens Stefan , Koffas Mattheos A. G.
Published: July 2005

Anthocyanins are red, purple, or blue plant pigments that belong to the family of polyphenolic compounds collectively called flavonoids. Their demonstrated antioxidant properties and economic importance to the dye, fruit, and cut-flower industries have driven intensive research into their metabolic biosynthetic pathways. In order to produce stable, glycosylated anthocyanins from colorless flavanones such as naringenin and eriodictyol, a four-step metabolic pathway was constructed that contained plant genes from heterologous origins: flavanone 3β-hydroxylase from
Malus domestica
, dihydroflavonol 4-reductase from
Anthurium andraeanum
, anthocyanidin synthase (ANS) also from
M. domestica
, and UDP-glucose:flavonoid 3-
O
-glucosyltransferase from
Petunia hybrida
. Using two rounds of PCR, each one of the four genes was first placed under the control of the
trc
promoter and its own bacterial ribosome-binding site and then cloned sequentially into vector pK184.
Escherichia coli
cells containing the recombinant plant pathway were able to take up either naringenin or eriodictyol and convert it to the corresponding glycosylated anthocyanin, pelargonidin 3-
O
-glucoside or cyanidin 3-
O
-glucoside. The produced anthocyanins were present at low concentrations, while most of the metabolites detected corresponded to their dihydroflavonol precursors, as well as the corresponding flavonols. The presence of side product flavonols is at least partly due to an alternate reaction catalyzed by ANS. This is the first time plant-specific anthocyanins have been produced from a microorganism and opens up the possibility of further production improvement by protein and pathway engineering.
Effect of 26-Oxygenosterols from
Ganoderma lucidum
and Their Activity as Cholesterol Synthesis Inhibitors
Authors: Hajjaj Hassan , Macé Catherine , Roberts Matthew , Niederberger Peter , Fay Laurent B.
Published: July 2005

Ganoderma lucidum
is a medicinal fungus belonging to the Polyporaceae family which has long been known in Japan as Reishi and has been used extensively in traditional Chinese medicine. We report the isolation and identification of the 26-oxygenosterols ganoderol A, ganoderol B, ganoderal A, and ganoderic acid Y and their biological effects on cholesterol synthesis in a human hepatic cell line in vitro. We also investigated the site of inhibition in the cholesterol synthesis pathway. We found that these oxygenated sterols from
G. lucidum
inhibited cholesterol biosynthesis via conversion of acetate or mevalonate as a precursor of cholesterol. By incorporation of 24,25-dihydro-[24,25-
3
H
2
]lanosterol and [3-
3
H]lathosterol in the presence of ganoderol A, we determined that the point of inhibition of cholesterol synthesis is between lanosterol and lathosterol. These results demonstrate that the lanosterol 14α-demethylase, which converts 24,25-dihydrolanosterol to cholesterol, can be inhibited by the 26-oxygenosterols from
G. lucidum
. These 26-oxygenosterols could lead to novel therapeutic agents that lower blood cholesterol.
Potential Regrowth and Recolonization of Salmonellae and Indicators in Biosolids and Biosolid-Amended Soil
Authors: Zaleski Kathleen J. , Josephson Karen L. , Gerba Charles P. , Pepper Ian L.
Published: July 2005

This study evaluated the potential for conversion of Class B to Class A biosolids with respect to salmonellae and fecal coliforms during solar drying in concrete lined drying beds. Anaerobically (8% solids) and aerobically (2% solids) digested Class B biosolids were pumped into field-scale drying beds, and microbial populations and environmental conditions were monitored. Numbers of fecal coliforms and salmonellae decreased as temperature and rate of desiccation increased. After 3 to 4 weeks, Class A requirements were achieved in both biosolids for the pathogens and the indicators. However, following rainfall events, significant increase in numbers was observed for both fecal coliforms and salmonellae. In laboratory studies, regrowth of fecal coliforms was observed in both biosolids and biosolid-amended soil, but the regrowth of salmonellae observed in the concrete-lined drying beds did not occur. These laboratory studies demonstrated that pathogens decreased in numbers when soil was amended with biosolids. Based on serotyping, the increased numbers of salmonellae seen in the concrete lined drying beds following rainfall events was most likely due to recolonization due to contamination from fecal matter introduced by animals and not from regrowth of salmonellae indigenous to biosolids. Overall, we conclude that the use of concrete-lined beds created a situation in which moisture added as rainfall accumulated in the beds, promoting the growth of fecal coliforms and salmonellae added from external sources.
AmyA, an α-Amylase with β-Cyclodextrin-Forming Activity, and AmyB from the Thermoalkaliphilic OrganismAnaerobranca gottschalkii: Two α-Amylases Adapted to Their Different Cellular Localizations
Authors: Ballschmiter Meike , Armbrecht Martin , Ivanova Krasimira , Antranikian Garabed , Liebl Wolfgang
Published: July 2005

Two α-amylase genes from the thermophilic alkaliphileAnaerobranca gottschalkiiwere cloned, and the corresponding enzymes, AmyA and AmyB, were investigated after purification of the recombinant proteins. Based on their amino acid sequences, AmyA is proposed to be a lipoprotein with extracellular localization and thus is exposed to the alkaline milieu, while AmyB apparently represents a cytoplasmic enzyme. The amino acid sequences of both enzymes bear high similarity to those of GHF13 proteins. The different cellular localizations of AmyA and AmyB are reflected in their physicochemical properties. The alkaline pH optimum (pH 8), as well as the broad pH range, of AmyA activity (more than 50% activity between pH 6 and pH 9.5) mirrors the conditions that are encountered by an extracellular enzyme exposed to the medium ofA. gottschalkii, which grows between pH 6 and pH 10.5. AmyB, on the other hand, has a narrow pH range with a slightly acidic pH optimum at 6 to 6.5, which is presumably close to the pH in the cytoplasm. Also, the intracellular AmyB is less tolerant of high temperatures than the extracellular AmyA. While AmyA has a half-life of 48 h at 70°C, AmyB has a half-life of only about 10 min at that temperature, perhaps due to the lack of stabilizing constituents of the cytoplasm. AmyA and AmyB were very similar with respect to their substrate specificity profiles, clearly preferring amylose over amylopectin, pullulan, and glycogen. Both enzymes also hydrolyzed α-, β-, and γ-cyclodextrin. Very interestingly, AmyA, but not AmyB, displayed high transglycosylation activity on maltooligosaccharides and also had significant β-cyclodextrin glycosyltransferase (CGTase) activity. CGTase activity has not been reported for typical α-amylases before. The mechanism of cyclodextrin formation by AmyA is unknown.
Suppression of Damping-Off Disease in Host Plants by the Rhizoplane Bacterium
Lysobacter
sp. Strain SB-K88 Is Linked to Plant Colonization and Antibiosis against Soilborne Peronosporomycetes
Authors: Islam Md. Tofazzal , Hashidoko Yasuyuki , Deora Abhinandan , Ito Toshiaki , Tahara Satoshi
Published: July 2005

We previously demonstrated that xanthobaccin A from the rhizoplane bacterium
Lysobacter
sp. strain SB-K88 suppresses damping-off disease caused by
Pythium
sp. in sugar beet. In this study we focused on modes of
Lysobacter
sp. strain SB-K88 root colonization and antibiosis of the bacterium against
Aphanomyces cochlioides
, a pathogen of damping-off disease. Scanning electron microscopic analysis of 2-week-old sugar beet seedlings from seeds previously inoculated with SB-K88 revealed dense colonization on the root surfaces and a characteristic perpendicular pattern of
Lysobacter
colonization possibly generated via development of polar, brush-like fimbriae. In colonized regions a semitransparent film apparently enveloping the root and microcolonies were observed on the root surface. This
Lysobacter
strain also efficiently colonized the roots of several plants, including spinach, tomato,
Arabidopsis thaliana
, and
Amaranthus gangeticus
. Plants grown from both sugar beet and spinach seeds that were previously treated with
Lysobacter
sp. strain SB-K88 displayed significant resistance to the damping-off disease triggered by
A. cochlioides
. Interestingly, zoospores of
A. cochlioides
became immotile within 1 min after exposure to a SB-K88 cell suspension, a cell-free supernatant of SB-K88, or pure xanthobaccin A (MIC, 0.01 μg/ml). In all cases, lysis followed within 30 min in the presence of the inhibiting factor(s). Our data indicate that
Lysobacter
sp. strain SB-K88 has a direct inhibitory effect on
A. cochlioides
, suppressing damping-off disease. Furthermore, this inhibitory effect of
Lysobacter
sp. strain SB-K88 is likely due to a combination of antibiosis and characteristic biofilm formation at the rhizoplane of the host plant.