bluuebunny/crossref_metadata_2025 huggingface dataset:
1141249460 A novel three‐dimensional in vitro system of trophoblast–endothelium cell interaction
Authors: Aldo P , Krikun G , Lockwood CJ , Mor G
Published: May 2006

Introduction:  Preeclampsia and IUGR have been linked to improper trophoblast migration that impairs maternal spiral artery transformation minimizing vessel dilation and therefore restricting blood flow to the developing fetus. Endothelial cells are thought to play an essential role in directing trophoblast migration and transformation, although the mechanism by which this occurs is not fully understood. We have developed a novel in vitro system that evaluates endothelial‐trophoblast interaction and signaling in a three‐dimensional environment.

Methods:  An immortalized human endometrial endothelial cell line (HEEC) (Krikun et al., 2004) and first trimester trophoblast cell lines HTR‐8 and Swan.71 were used. Endothelial transformation into vessel‐like structures occurred in Matrigel Basement Membrane Matrix. Cell membrane linker dye and OpenLab Image Analysis software were used to monitor trophoblast migration and transformation. Cytokine and chemokine production was determined using a multiplex bead assay.

Results:  Upon differentiation in matrigel, endothelial cells undergo a shift in cytokine production characterized by an increased secretion of chemokines. First trimester trophoblast cells migrate toward the differentiated endothelium, and reach it within 4–8 hr. Complete replacement of the endothelium is seen by 48 hr. The transformed tubes maintain structure and remain functional for several weeks allowing for further co‐culture studies.

Conclusion:  We report the characterization of a novel three‐dimensional in vitro system of trophoblast–endothelium cell interaction. We found significant changes in the phenotype (cytokine expression and LPS responses) of endothelial cells upon differentiation in matrigel as compared to the monolayer culture. These changes may be necessary for the endothelium to direct trophoblast migration. Furthermore, this model represents an ideal co‐culture system to evaluate the role of additional cell types, e.g. immune cells, involved in the process of spiral artery transformation.
1141635485 Genetic stress (IL‐10 deficiency) and toxicant (PCB)‐induced disruption of pregnancy
Authors: Tewari N , Murphy SP , Sharma S
Published: May 2006

Several epidemiologic studies have provided evidence that a significant number of pregnant women living within the World Trade Center disaster area experienced a high rate of low birth weight and premature deliveries. Our hypothesis is that these women were predisposed to pregnancy disruption effects of toxicants detected in the dust of the disaster area. We sought to examine the effects of PCB on pregnancy outcome in an IL‐10‐/‐ mouse model. IL‐10‐/‐ or congenic wild type mice of 6–8 weeks age were given i.p injections of Aroclor 1254 (PCB) at doses of 50 or 500 μg/mouse in 100 μL corn oil on gestational day (gd) 1 and 7 or an equivalent (100 μL) amount of corn oil. Mice were sacrificed on gd 13 and uteri and spleen were excised and collected. Uterine mononuclear cells (UMC) and splenic mononuclear cells (SMC) were isolated. These cell populations were subjected to phenotypic and functional characterization. A portion of uterine tissue was either snap frozen for placental PCB contents or for histology. No doses of Aroclor 1254 affected the pregnancy outcome in wild type mice. However, IL‐10‐/‐ mice experienced fetal resorption when injected with high dose (500 μg/mouse) of Aroclor 1254. Local immune dysregulation and placental anomalies including apoptosis and immunocyte infiltration are currently being investigated. Our results strongly suggest that IL‐10 deficiency and PCB‐mediated immunotoxicity lead to adverse pregnancy outcomes.

Acknowledgement:  Supported by NIEHS Superfund Basic Research Program Award P42ES13660.
1141642587 Ras‐activators in trophoblastic cell lines
Authors: Enkelmann A , Poehlmann TG , Roediger J , Sedlmayr P , Rubio I , Markert UR
Published: May 2006

Background:  Numerous cytokines influence functions and behaviour of trophoblast cells. The use of different signalling pathways is decisive for different functions, such as migration, proliferation and invasion. Epithelial growth factor (EGF), leukemia inhibitory factor (LIF) and interleukin‐6 (IL‐6) are major inducers. MAP Kinase (MAPK) pathways, which are related to invasiveness in many malignant cells, are expected to be involved.

Material and Methods:  The trophoblastic cell line AC1‐M59 and the choriocarcinoma cell line Jeg‐3 were stimulated with EGF, LIF or IL‐6. After 2–30 min, cells were lyzed and activated (GTP‐bound) ras was pulled down by using small modular GTPase‐binding domains derived from c‐Raf and sepharose micro‐beads. The ras‐containing pellet was analyzed by electrophoresis followed by Western blotting and detected with various anti‐ras antibodies. The remaining lysate was analyzed for phospho‐erk1/2.

Results:  In AC1‐M59 cells, EGF induced a strong and immediate ras and erk1/2 activation as visible at all analyzed time points. Effects of LIF were much lower and started 15 min after stimulation. IL‐6 did not induce detectable effects. Jeg‐3 cells displayed a high spontaneous ras activation which was not further increased after stimulation.

Discussion:  In trophoblastic cells, EGF activates signalling molecules of the MAPK pathways as similarly described for other cells. LIF is known to use the Jak/STAT pathway, which may be responsible for a slower ras activation than induced by EGF. The high spontaneous ras activation in Jeg‐3 cells may be related to their malign characteristics.

Conclusion:  EGF is a powerful activator of ras in trophoblastic cell lines, which may be a key signalling molecule for invasion of trophoblast cells.
1141651467 Role of calpain in human sperm
Authors: Ozaki Y , Goto S , Nozawa K , Nakanishi T , Sugiura M
Published: May 2006

We evaluated the role of calpain in human sperm for fertilization. Semen collected manually from healthy donors with informed consent was liquefied and following percoll gradient centrifugation. After exposure to different concentration of progesterone (P) or under hypoxic condition (H), the samples were used for immunostaining, SDS‐PAGE and western blot analysis. The increase of calcium ion concentration in the sperm was observed by fluorescent microscope (ARGUS‐50CA) system using Fura 2‐AM. The role of calpain for fertilization was speculated from the results of Hamster penetration test, Hamilton Motility Analyzer, Triple satin method and Acrobeads test using calpain inhibitors. Immunostaining in human sperm was observed using antibodies against the micro‐calpain in the types of whole acrosome, equatorial segment, head segment, neck segment or neck and tail segment. Most of these demonstrated acrosome type staining with anti‐pro micro‐calpain antibody. Western blot analysis revealed P or H treatment to cause a concentration or time‐dependent activation of micro‐calpain. In addition, calpain inhibitors significantly reduced the acrosome reaction and penetration. The results suggest that micro‐calpain in human sperm plays an important role for the fertilization.
Short‐term and long‐term outcomes of patients with malignant large bowel obstruction
Authors: Ng Wing‐Chi , Lee Janet Fung‐Yee , Ng Simon Siu‐Man , Yiu Raymond Ying‐Chang , Leung Ka‐Lau
Published: May 2006

Objective:  The aim of the present study was to review our experience in the surgical management of patients with obstructing colorectal cancers over an 11‐year period, 1987–1997.

Patients and methods:  Retrospective review of case records of 275 patients (male: 177; female 98) who had undergone emergency surgery for obstructing colorectal cancers was performed. Tumours proximal to splenic flexure were defined as proximal tumours while those at or below the splenic flexure were defined as distal tumours.

Results:  The obstruction was caused by proximal tumours in 88 (32%) patients. The resection rate and the primary anastomotic rate were higher for proximal tumours compared with distal tumours (95.5%vs 85.6%, P = 0.014; 92%vs 30.5%, P < 0.001). For distal tumours, stoma rate was found to be influenced by the following factors: preoperative albumin level, duration of observation after admission, operating surgeons’ years of experience, bowel perforation and site of the obstructing tumour. Multivariate analysis disclosed that surgeons’ experience was the only independent factor predicting stoma formation. The in‐hospital mortality and the anastomotic leakage rates were 15.3% and 5.6%, respectively. Tumour stage was the only prognostic factor affecting the disease‐free survival after curative resection. The 5‐year disease‐free survival rates for Dukes’ B and C disease were 66% and 37.2%, respectively.

Conclusions:  Tumour stage was a significant prognostic factor for patients with obstructing colorectal cancers. Emergency surgery for distal tumours should preferentially be performed by more experienced surgeons in order to achieve a higher anastomotic rate.
Cryosurgery for prostate cancer: How I do it
Authors: Ho Sammy Shing‐Chee
Published: May 2006

Background:  Cryosurgery for prostate cancer is the ablation of the cancer by inducing a lethal freezing temperature (−40°C) to kill the cancer cells. This is now one of the optional curative treatments for localized prostate cancer patients.

Methods:  A third generation cryotherapy machine was used. An ultrathin 17 Fr cryoneedle was used to puncture the prostate gland transperineally under real‐time transrectal ultrasound guidance. Cryogen gas, using argon, was used for active freezing and helium gas was used for active thawing. Thermoprobes were used to ensure the lethal temperature was reached. A urethral warmer device was necessary to reduce urethral damage. Foley catheterization was necessary for 2–3 days postoperatively.

Results:  Postoperative prostate specific antigen (PSA) level was monitored 3 monthly. Biochemical success (PSA < 0.4) can be achieved in most cases.

Conclusion:  This new cryoablation of prostate cancer can provide a minimally invasive option for localized prostate cancer patients.
Perineal radical prostatectomy
Authors: Mah Ida Soo‐Fan
Published: May 2006

Perineal radical prostatectomy (PRP) for carcinoma of the prostate was a popular operation before the introduction of retropubic prostatectomy in the 1940s. Recently, there has been renewed interest in PRP because patients are seeking less invasive treatment. With the modern modified technique, the PRP produces results comparable to the open retropubic prostatectomy. However, it is less surgically invasive with a short hospital stay. Compared with the laparoscopic technique, it is technically less demanding and has a much shorter learning curve.
Steady and Unsteady Radial Forces for a Centrifugal Pump With Impeller to Tongue Gap Variation
Authors: González José , Parrondo Jorge , Santolaria Carlos , Blanco Eduardo
Published: May 2006

Experimental and numerical studies are presented on the steady and unsteady radial forces produced in a single volute vaneless centrifugal pump. Experimentally, the unsteady pressure distributions were obtained using fast response pressure transducers. These measurements were compared with equivalent numerical results from a URANS calculation, using the commercial code FLUENT. Two impellers with different outlet diameters were tested for the same volute, with radial gaps between the blade and tongue of 10.0% and 15.8% of the impeller radius, for the bigger and smaller impeller diameters, respectively. Very often, pump manufacturers apply the similarity laws to this situation, but the measured specific speeds in this case were found to be slightly different. The steady radial forces for the two impellers were calculated from both the measured average pressure field and the model over a wide range of flow rates in order to fully characterize the pump behavior. Again, a deviation from the expected values applying the similarity laws was found. The data from the pressure fluctuation measurements were processed to obtain the dynamic forces at the blade passing frequency, also over a wide range of flow rates. Afterwards, these results were used to check the predictions from the numerical simulations. For some flow rates, the bigger diameter produced higher radial forces, but this was not to be a general rule for all the operating points. This paper describes the work carried out and summarizes the experimental and the numerical results, for both radial gaps. The steady and unsteady forces at the blade passing frequency were calculated by radial integration of the pressure distributions on the shroud side of the pump volute. For the unsteady forces, the numerical model allowed a separate analysis of the terms due to the pressure pulsations and terms related to the momentum exchange in the impeller. In this way, the whole operating range of the pump was studied and analyzed to account for the static and dynamic flow effects. The unsteady forces are very important when designing the pump shaft as they can produce a fatigue collapse if they are not kept under a proper working value.
The Electrochemical Performance of Anode-Supported SOFCs with LSM-Type Cathodes Produced by Alternative Processing Routes
Authors: Mertens J. , Haanappel V. A. C. , Tropartz C. , Herzhof W. , Buchkremer H. P.
Published: May 2006

The electrochemical performance of La0.65Sr0.3MnO3-type (LSM) anode-supported single cells, produced by alternative production processes, has been investigated at intermediate temperatures. In particular, three different variations of the production route were investigated in more detail: (1) the use of nonground LSM powder for the cathode current collector layer, (2) the use of noncalcined and nonground YSZ powder for the cathode functional layer, and (3) the use of tape casting versus warm pressing as the production process for anode substrates. Results from electrochemical measurements performed between 700 and 900°C with H2 (3vol%H2O) as fuel gas and air as the oxidant showed that performance increased with increasing grain size of the outer cathode current collector layer: the highest performance was achieved with nonground LSM powder. Furthermore, performance was not adversely influenced by the use of noncalcined and nonground YSZ for the cathode functional layer. Also the use of anode substrates with a thickness of about 0.7mm and produced by tape casting, instead of those with a thickness of about 1.5mm and applied by warm pressing, did not detrimentally affect the electrochemical performance of this type of SOFC.
Effect of Various Electrolyte Compositions on the NiO Degradation in Molten Carbonates
Authors: Scaccia Silvera , Frangini Stefano
Published: May 2006

In the present paper we report the NiO solubility in a variety of alkali carbonate compositions in the presence of MgO, CaCO3, BaCO3, and La2O3 additions under different conditions of gas composition and temperature. Two types of binary eutectic melts were chosen, namely, the standard electrolyte (62-38) (Li-K)2CO3 and (52–48)% (Li-Na)2CO3 mixtures. The ternary eutectic melt chosen was (43.5-31.5-25.0)mol%(Li-Na-K)2CO3. The observed reduction of NiO solubility induced by the additives can be explained in terms of a higher basic character of all the modified electrolytes, although the NiO dissolution still follows an acidic dissolution mechanism in all the conditions under study.
Effect of Channel Aspect Ratio on the Flow Performance of a Spiral-Channel Viscous Micropump
Authors: Kilani M. I. , Al-Salaymeh A. , Al-Halhouli A. T.
Published: May 2006

The paper investigates the effect of channel aspect ratio on the flow performance of a newly introduced spiral-channel viscous micropump. An approximate 2D analytical solution for the flow field, which ignores channel curvature but accounts for a finite wall height, is first developed at the lubrication limit. A number of 3D models for spiral pumps with different aspect ratios are then built and analyzed using the finite volume method. Numerical and analytical results are in good agreement and tend to support one another. The results are compared with an approximate 2D analytical solution developed for infinite aspect ratio, which neglects the effect of side walls, and assumes uniform velocity distribution across the channel width. The error in this approximation was found to exceed 5% for aspect ratios less than 10. Pressure and drag shape factors were introduced in the present work to express the effect of the pressure difference and boundary velocity on the flow rate at various aspect ratios for both moving and stationary walls. Also, it has been found numerically that the flow rate varies linearly with both the pressure difference and boundary velocity, which supports the validity of the linear lubrication model employed.
Optimization of current modulation function for proton spread‐out Bragg peak fields
Authors: Lu Hsiao‐Ming , Kooy Hanne
Published: May 2006

Proton treatments with spread‐out Bragg peak (SOBP) fields often use a rotating modulation wheel of varying thickness to modulate the pristine Bragg peak in depth and intensity. The technique of modulating also the beam current independently over the wheel rotation provides an additional control over the intensities of the pulled‐back Bragg peaks. As a result, a single wheel can be used over a large range of energies and SOBP parameters and field‐specific wheels are no longer necessary. An essential task in commissioning a particular treatment depth is the determination of this current modulation function. We have developed a method for the optimization of the current modulation function. The basic idea is to treat the entire beam nozzle, housing the various beam scattering and modulating components, as a whole and to characterize its effect as a transformation from a modulating beam current to a depth‐dose distribution. While this transformation is difficult to calculate theoretically due to the complex scattering paths in the nozzle and the phantom, it can, however, be determined by time‐resolved dose measurements. Using this transformation, we can calculate SOBP depth‐dose distributions for any current modulation function and optimize it by a simple numerical optimization. We have applied the new method to a number of proton beams with satisfactory results.
Dosimetric and radiobiological impact of dose fractionation on respiratory motion induced IMRT delivery errors: A volumetric dose measurement study
Authors: Duan Jun , Shen Sui , Fiveash John B. , Popple Richard A. , Brezovich Ivan A.
Published: May 2006

Respiratory motion can introduce substantial dose errors during IMRT delivery. These errors are difficult to predict because of the nonsynchronous interplay between radiation beams and tissues. The present study investigates the impact of dose fractionation on respiratory motion induced dosimetric errors during IMRT delivery and their radiobiological implications by using measured 3D dose. We focused on IMRT delivery with dynamic multileaf collimation (DMLC‐IMRT). IMRT plans using several beam arrangements were optimized for and delivered to a polystyrene phantom containing a simulated target and critical organs. The phantom was set in linear sinusoidal motion at a frequency of . The amplitude of the motion was in the longitudinal direction and in the lateral direction. Absolute doses were measured with a ionization chamber while dose distributions were measured with transverse films spaced apart. Measurements were performed for varying number of fractions with motion, with respiratory‐gated motion, and without motion. A tumor control probability (TCP) model for an inhomogeneously irradiated tumor was used to calculate and compare TCPs for the measurements and the treatment plans. Equivalent uniform doses (EUD) were also computed. For individual fields, point measurements using an ionization chamber showed substantial dose deviations ( to ) for the moving phantom as compared to the stationary phantom. However, much smaller deviations ( to ) were observed for the composite dose of all fields. The dose distributions and DVHs of stationary and gated deliveries were in good agreement with those of treatment plans, while those of the nongated moving phantom showed substantial differences. Compared to the stationaryphantom, the largest differences observed for the minimum and maximum target doses were and , respectively. Due to their random nature, these dose errors tended to average out over fractionated treatments. The results of five‐fraction measurements showed significantly improved agreement between the moving and stationary phantom. The changes in TCP were less than 4.3% for a single fraction, and less than 2.3% for two or more fractions. Variation of average EUD per fraction was small ( for a fraction size of ), even when the DVHs were noticeably different from that of the stationary tumor. In conclusion, IMRT treatment of sites affected by respiratory motion can introduce significant dose errors in individual field doses; however, these errors tend to cancel out between fields and average out over dose fractionation. 3D dose distributions, DVHs, TCPs, and EUDs for stationary and moving cases showed good agreement after two or more fractions, suggesting that tumors affected by respiration motion may be treated using IMRT without significant dosimetric and biological consequences.
The Embryonic Vertebrate Heart Tube Is a Dynamic Suction Pump
Authors: Forouhar Arian S. , Liebling Michael , Hickerson Anna , Nasiraei-Moghaddam Abbas , Tsai Huai-Jen , Hove Jay R. , Fraser Scott E. , Dickinson Mary E. , Gharib Morteza
Published: May 2006

The embryonic vertebrate heart begins pumping blood long before the development of discernable chambers and valves. At these early stages, the heart tube has been described as a peristaltic pump. Recent advances in confocal laser scanning microscopy and four-dimensional visualization have warranted another look at early cardiac structure and function. We examined the movement of cells in the embryonic zebrafish heart tube and the flow of blood through the heart and obtained results that contradict peristalsis as a pumping mechanism in the embryonic heart. We propose a more likely explanation of early cardiac dynamics in which the pumping action results from suction due to elastic wave propagation in the heart tube.
Conjunctive Representation of Position, Direction, and Velocity in Entorhinal Cortex
Authors: Sargolini Francesca , Fyhn Marianne , Hafting Torkel , McNaughton Bruce L. , Witter Menno P. , Moser May-Britt , Moser Edvard I.
Published: May 2006

Grid cells in the medial entorhinal cortex (MEC) are part of an environment-independent spatial coordinate system. To determine how information about location, direction, and distance is integrated in the grid-cell network, we recorded from each principal cell layer of MEC in rats that explored two-dimensional environments. Whereas layer II was predominated by grid cells, grid cells colocalized with head-direction cells and conjunctive grid × head-direction cells in the deeper layers. All cell types were modulated by running speed. The conjunction of positional, directional, and translational information in a single MEC cell type may enable grid coordinates to be updated during self-motion–based navigation.
Bacillus anthracis
Multiplication, Persistence, and Genetic Exchange in the Rhizosphere of Grass Plants
Authors: Saile Elke , Koehler Theresa M.
Published: May 2006

Bacillus anthracis
, the causative agent of anthrax, is known for its rapid proliferation and dissemination in mammalian hosts. In contrast, little information exists regarding the lifestyle of this important pathogen outside of the host. Considering that
Bacillus
species, including close relatives of
B. anthracis
, are saprophytic soil organisms, we investigated the capacity of
B. anthracis
spores to germinate in the rhizosphere and to establish populations of vegetative cells that could support horizontal gene transfer in the soil. Using a simple grass plant-soil model system, we show that
B. anthracis
strains germinate on and around roots, growing in characteristic long filaments. From 2 to 4 days postinoculation, approximately one-half of the
B. anthracis
CFU recovered from soil containing grass seedlings arose from heat-sensitive organisms, while
B. anthracis
CFU retrieved from soil without plants consisted of primarily heat-resistant spores. Coinoculation of the plant-soil system with spores of a fertile
B. anthracis
strain carrying the tetracycline resistance plasmid pBC16 and a selectable
B. anthracis
recipient strain resulted in transfer of pBC16 from the donor to the recipient as early as 3 days postinoculation. Our findings demonstrate that
B. anthracis
can survive as a saprophyte outside of the host. The data suggest that horizontal gene transfer in the rhizosphere of grass plants may play a role in the evolution of the
Bacillus cereus
group species.
Isolation and Characterization of Acid-Tolerant, Thermophilic Bacteria for Effective Fermentation of Biomass-Derived Sugars to Lactic Acid
Authors: Patel Milind A. , Ou Mark S. , Harbrucker Roberta , Aldrich Henry C. , Buszko Marian L. , Ingram Lonnie O. , Shanmugam K. T.
Published: May 2006

Biomass-derived sugars, such as glucose, xylose, and other minor sugars, can be readily fermented to fuel ethanol and commodity chemicals by the appropriate microbes. Due to the differences in the optimum conditions for the activity of the fungal cellulases that are required for depolymerization of cellulose to fermentable sugars and the growth and fermentation characteristics of the current industrial microbes, simultaneous saccharification and fermentation (SSF) of cellulose is envisioned at conditions that are not optimal for the fungal cellulase activity, leading to a higher-than-required cost of cellulase in SSF. We have isolated bacterial strains that grew and fermented both glucose and xylose, major components of cellulose and hemicellulose, respectively, to
l
(+)-lactic acid at 50°C and pH 5.0, conditions that are also optimal for fungal cellulase activity. Xylose was metabolized by these new isolates through the pentose-phosphate pathway. As expected for the metabolism of xylose by the pentose-phosphate pathway, [
13
C]lactate accounted for more than 90% of the total
13
C-labeled products from [
13
C]xylose. Based on fatty acid profile and 16S rRNA sequence, these isolates cluster with
Bacillus coagulans
, although the
B. coagulans
type strain, ATCC 7050, failed to utilize xylose as a carbon source. These new
B. coagulans
isolates have the potential to reduce the cost of SSF by minimizing the amount of fungal cellulases, a significant cost component in the use of biomass as a renewable resource, for the production of fuels and chemicals.
Characterization of Shiga Toxin Gene (
stx
)-Positive and Intimin Gene (
eae
)-Positive
Escherichia coli
Isolates from Wastewater of Slaughterhouses in France
Authors: Loukiadis Estelle , Kérourédan Monique , Beutin Lothar , Oswald Eric , Brugère Hubert
Published: May 2006

Wastewater samples from 12 slaughterhouses located in different regions in France were tested for the presence of
stx
-positive and
eae
-positive
Escherichia coli
isolates, and characteristics of the isolates obtained were determined. A total of 224 wastewater samples were collected in wastewater treatment plants at different stages of wastewater processing. Altogether, 5,001
E. coli
isolates were obtained by colony counting and screened for the presence of
stx
and
eae
genes by multiplex PCR.
stx
-positive and
eae
-positive
E. coli
isolates were detected in 25% of the samples collected; they were found in 13% and 3% of the samples obtained from treated effluent and sludge, respectively, suggesting that they could be spread into the environment. Screening of the samples collected by immunomagnetic separation allowed us to isolate 31 additional
E. coli
serogroup O157 isolates. Four of these isolates harbored
stx
and
eae
genes. All
stx
-positive and
eae
-positive
E. coli
isolates were analyzed for
eae
and
stx
genetic variants, as well as for additional virulence factors and serotypes. Our results suggest that the majority of the
stx
- and
eae
-positive
E. coli
isolates from wastewater have low virulence for humans. However, the diversity of the enterohemorrhagic
E. coli
-associated virulence factors in the strains indicates that the environment may play an important role in the emergence of new pathogenic enterohemorrhagic
E. coli
strains.
Quantitative Microbial Risk Assessment Models for Consumption of Raw Vegetables Irrigated with Reclaimed Water
Authors: Hamilton Andrew J. , Stagnitti Frank , Premier Robert , Boland Anne-Maree , Hale Glenn
Published: May 2006

Quantitative microbial risk assessment models for estimating the annual risk of enteric virus infection associated with consuming raw vegetables that have been overhead irrigated with nondisinfected secondary treated reclaimed water were constructed. We ran models for several different scenarios of crop type, viral concentration in effluent, and time since last irrigation event. The mean annual risk of infection was always less for cucumber than for broccoli, cabbage, or lettuce. Across the various crops, effluent qualities, and viral decay rates considered, the annual risk of infection ranged from 10
−3
to 10
−1
when reclaimed-water irrigation ceased 1 day before harvest and from 10
−9
to 10
−3
when it ceased 2 weeks before harvest. Two previously published decay coefficients were used to describe the die-off of viruses in the environment. For all combinations of crop type and effluent quality, application of the more aggressive decay coefficient led to annual risks of infection that satisfied the commonly propounded benchmark of ≤10
−4
, i.e., one infection or less per 10,000 people per year, providing that 14 days had elapsed since irrigation with reclaimed water. Conversely, this benchmark was not attained for any combination of crop and water quality when this withholding period was 1 day. The lower decay rate conferred markedly less protection, with broccoli and cucumber being the only crops satisfying the 10
−4
standard for all water qualities after a 14-day withholding period. Sensitivity analyses on the models revealed that in nearly all cases, variation in the amount of produce consumed had the most significant effect on the total uncertainty surrounding the estimate of annual infection risk. The models presented cover what would generally be considered to be worst-case scenarios: overhead irrigation and consumption of vegetables raw. Practices such as subsurface, furrow, or drip irrigation and postharvest washing/disinfection and food preparation could substantially lower risks and need to be considered in future models, particularly for developed nations where these extra risk reduction measures are more common.
Environmental Whole-Genome Amplification To Access Microbial Populations in Contaminated Sediments
Authors: Abulencia Carl B. , Wyborski Denise L. , Garcia Joseph A. , Podar Mircea , Chen Wenqiong , Chang Sherman H. , Chang Hwai W. , Watson David , Brodie Eoin L. , Hazen Terry C. , Keller Martin
Published: May 2006

Low-biomass samples from nitrate and heavy metal contaminated soils yield DNA amounts that have limited use for direct, native analysis and screening. Multiple displacement amplification (MDA) using φ29 DNA polymerase was used to amplify whole genomes from environmental, contaminated, subsurface sediments. By first amplifying the genomic DNA (gDNA), biodiversity analysis and gDNA library construction of microbes found in contaminated soils were made possible. The MDA method was validated by analyzing amplified genome coverage from approximately five
Escherichia coli
cells, resulting in 99.2% genome coverage. The method was further validated by confirming overall representative species coverage and also an amplification bias when amplifying from a mix of eight known bacterial strains. We extracted DNA from samples with extremely low cell densities from a U.S. Department of Energy contaminated site. After amplification, small-subunit rRNA analysis revealed relatively even distribution of species across several major phyla. Clone libraries were constructed from the amplified gDNA, and a small subset of clones was used for shotgun sequencing. BLAST analysis of the library clone sequences showed that 64.9% of the sequences had significant similarities to known proteins, and “clusters of orthologous groups” (COG) analysis revealed that more than half of the sequences from each library contained sequence similarity to known proteins. The libraries can be readily screened for native genes or any target of interest. Whole-genome amplification of metagenomic DNA from very minute microbial sources, while introducing an amplification bias, will allow access to genomic information that was not previously accessible.
Short-Term Metabolome Dynamics and Carbon, Electron, and ATP Balances in Chemostat-Grown
Saccharomyces cerevisiae
CEN.PK 113-7D following a Glucose Pulse
Authors: Wu Liang , van Dam Jan , Schipper Dick , Kresnowati M. T. A. Penia , Proell Angela M. , Ras Cor , van Winden Wouter A. , van Gulik Walter M. , Heijnen Joseph J.
Published: May 2006

The in vivo kinetics in
Saccharomyces cerevisiae
CEN.PK 113-7D was evaluated during a 300-second transient period after applying a glucose pulse to an aerobic, carbon-limited chemostat culture. We quantified the responses of extracellular metabolites, intracellular intermediates in primary metabolism, intracellular free amino acids, and in vivo rates of O
2
uptake and CO
2
evolution. With these measurements, dynamic carbon, electron, and ATP balances were set up to identify major carbon, electron, and energy sinks during the postpulse period. There were three distinct metabolic phases during this time. In phase I (0 to 50 seconds after the pulse), the carbon/electron balances closed up to 85%. The accumulation of glycolytic and storage compounds accounted for 60% of the consumed glucose, caused an energy depletion, and may have led to a temporary decrease in the anabolic flux. In phase II (50 to 150 seconds), the fermentative metabolism gradually became the most important carbon/electron sink. In phase III (150 to 300 seconds), 29% of the carbon uptake was not identified in the measurements, and the ATP balance had a large surplus. These results indicate an increase in the anabolic flux, which is consistent with macroscopic balances of extracellular fluxes and the observed increase in CO
2
evolution associated with nonfermentative metabolism. The identified metabolic processes involving major carbon, electron, and energy sinks must be taken into account in in vivo kinetic models based on short-term dynamic metabolome responses.
Genetic Population Structure of the Soil BacteriumMyxococcusxanthusat the Centimeter Scale
Authors: Vos Michiel , Velicer Gregory J.
Published: May 2006

Myxococcus xanthusis a gram-negative soil bacterium best known for its remarkable life history of social swarming, social predation, and multicellular fruiting body formation. Very little is known about genetic diversity within this species or how social strategies might vary among neighboring strains at small spatial scales. To investigate the small-scale population structure ofM. xanthus, 78 clones were isolated from a patch of soil (16 by 16 cm) in Tübingen, Germany. Among these isolates, 21 genotypes could be distinguished from a concatemer of three gene fragments:csgA(developmental C signal),fibA(extracellular matrix-associated zinc metalloprotease), andpilA(the pilin subunit of type IV pili). Accumulation curves showed that most of the diversity present at this scale was sampled. ThepilAgene contains both conserved and highly variable regions, and two frequency-distribution tests provide evidence for balancing selection on this gene. The functional domains in thecsgAgene were found to be conserved. Three instances of lateral gene transfer could be inferred from a comparison of individual gene phylogenies, but no evidence was found for linkage equilibrium, supporting the view thatM. xanthusevolution is largely clonal. This study shows thatM. xanthusis surrounded by a variety of distinct conspecifics in its natural soil habitat at a spatial scale at which encounters among genotypes are likely.
Overflow Metabolism in
Escherichia coli
during Steady-State Growth: Transcriptional Regulation and Effect of the Redox Ratio
Authors: Vemuri G. N. , Altman E. , Sangurdekar D. P. , Khodursky A. B. , Eiteman M. A.
Published: May 2006

Overflow metabolism in the form of aerobic acetate excretion by
Escherichia coli
is an important physiological characteristic of this common industrial microorganism. Although acetate formation occurs under conditions of high glucose consumption, the genetic mechanisms that trigger this phenomenon are not clearly understood. We report on the role of the NADH/NAD ratio (redox ratio) in overflow metabolism. We modulated the redox ratio in
E. coli
through the expression of
Streptococcus pneumoniae
(water-forming) NADH oxidase. Using steady-state chemostat cultures, we demonstrated a strong correlation between acetate formation and this redox ratio. We furthermore completed genome-wide transcription analyses of a control
E. coli
strain and an
E. coli
strain overexpressing NADH oxidase. The transcription results showed that in the control strain, several genes involved in the tricarboxylic acid (TCA) cycle and respiration were repressed as the glucose consumption rate increased. Moreover, the relative repression of these genes was alleviated by expression of NADH oxidase and the resulting reduced redox ratio. Analysis of a promoter binding site upstream of the genes which correlated with redox ratio revealed a degenerate sequence with strong homology with the binding site for ArcA. Deletion of
arcA
resulted in acetate reduction and increased the biomass yield due to the increased capacities of the TCA cycle and respiration. Acetate formation was completely eliminated by reducing the redox ratio through expression of NADH oxidase in the
arcA
mutant, even at a very high glucose consumption rate. The results provide a basis for studying new regulatory mechanisms prevalent at reduced NADH/NAD ratios, as well as for designing more efficient bioprocesses.
Extracellular Production of Neoculin, a Sweet-Tasting Heterodimeric Protein with Taste-Modifying Activity, by
Aspergillus oryzae
Authors: Nakajima Ken-ichiro , Asakura Tomiko , Maruyama Jun-ichi , Morita Yuji , Oike Hideaki , Shimizu-Ibuka Akiko , Misaka Takumi , Sorimachi Hiroyuki , Arai Soichi , Kitamoto Katsuhiko , Abe Keiko
Published: May 2006

Neoculin (NCL), a protein with sweetness approximately 500-fold that of sugar, can be utilized as a nonglycemic sweetener. It also has taste-modifying activity to convert sourness to sweetness. NCL is a heterodimer composed of an
N
-glycosylated acidic subunit (NAS) and a basic subunit (NBS), which are conjugated by disulfide bonds. For the production of recombinant NCL (rNCL) by
Aspergillus oryzae
, α-amylase with a KEX2 cleavage site, -K-R-, was fused upstream of each of NAS and NBS and the resulting fusion proteins were simultaneously expressed. For accurate and efficient cleavage of the fusion construct by KEX2-like protease, a triglycine motif was inserted after the KEX2 cleavage site. As NBS showed lower production efficiency than did NAS, a larger amount of the NBS expression plasmid than of NAS expression plasmid was introduced during cotransformation, resulting in successful production of rNCL in the culture medium. Moreover, to obtain a higher production yield of rNCL, the active form of
hacA
cDNA encoding a transcription factor that induces an unfolded protein response was cloned and expressed constitutively. This resulted in a 1.5-fold increase in the level of rNCL production (2.0 mg/liter). rNCL was purified by chromatography, and its NAS was found to be
N
-glycosylated as expected. The original sweetness and taste-modifying activity of rNCL were comparable to those of native NCL when confirmed by calcium imaging with human embryonic kidney cells expressing the human sweet taste receptor and by sensory tests.
High-Throughput Transposon Mutagenesis of
Corynebacterium glutamicum
and Construction of a Single-Gene Disruptant Mutant Library
Authors: Suzuki Nobuaki , Okai Naoko , Nonaka Hiroshi , Tsuge Yota , Inui Masayuki , Yukawa Hideaki
Published: May 2006

A simple and high-throughput transposon-mediated mutagenesis system employing two different types of transposons in combination with direct genomic DNA amplification and thermal asymmetric interlaced PCR (TAIL-PCR) was developed. Each of the two minitransposons based on IS
31831
(ISL3 family) and Tn
5
(IS4 family) was integrated into the
Corynebacterium glutamicum
R genome. By using BLAST and Perl, transposon insertion locations were automatically identified based on the sequences of TAIL-PCR products of mutant cells. Insertion locations of 18,000 mutants were analyzed, and a comprehensive insertion library covering nearly 80% of the 2,990 open reading frames of
C. glutamicum
R was generated. Eight thousand of the mutants, exhibiting disruption in 2,330 genes, survived on complex medium under normal laboratory conditions, indicating that the genes were not essential for cell survival. Of the 2,330 genes, 30 exhibited high similarity to essential genes of
Escherichia coli
or
Bacillus subtilis
. This approach could be useful in furthering genetic understanding of cellular life and facilitating the functional analysis of microorganisms.
Salmonella
Serovars in the Herpetofauna of Indiana County, Pennsylvania
Authors: Chambers David L. , Hulse Arthur C.
Published: May 2006

Herpetofaunal
Salmonella enterica
serovars have not been fully examined in any U.S. region. Thirty-three
Salmonella
serovars were isolated from 156 samples from 34 species, all within Indiana County, Pennsylvania. Results suggest that herpetofaunas could potentially pose a threat to humans. Further understanding of
Salmonella
in herpetofaunas may prevent future human cases.
Effect of Biofilm Growth on Expression of Surface Proteins of
Actinomyces naeslundii
Genospecies 2
Authors: Paddick James S. , Brailsford Susan R. , Rao Susmitha , Soares Renata F. , Kidd Edwina A. M. , Beighton David , Homer Karen A.
Published: May 2006

The predominant surface proteins of biofilm and planktonic
Actinomyces naeslundii
, a primary colonizer of the tooth surface, were examined. Seventy-nine proteins (the products of 52 genes) were identified in biofilm cells, and 30 of these, including adhesins, chaperones, and stress-response proteins, were significantly up-regulated relative to planktonic cells.
Viable Bacteria Present within Oral Squamous Cell Carcinoma Tissue
Authors: Hooper Samuel J. , Crean St John , Lewis Michael A. O. , Spratt David A. , Wade William G. , Wilson Melanie J.
Published: May 2006

Despite increasing interest in the possible relationships between bacteria and the different stages of cancer development, the association of bacteria with cancer of the oral cavity has yet to be adequately examined. With that in mind, the primary objective of this study was to identify any bacterial species within oral squamous cell carcinoma tissue using a standard microbiological culture approach. At the time of surgery, a 1-cm3portion of tissue was harvested from deep within the tumor mass using a fresh blade for each cut. Whenever possible, “superficial” portions from the mucosa overlying the tumor and nontumorous control specimens from at least 5 cm away from the primary tumor site were also obtained. Surface contamination was eliminated by immersion in Betadine and washing with phosphate-buffered saline. Each specimen was aseptically macerated and cultured on nonselective media under both aerobic and anaerobic conditions. Isolates were identified by 16S rRNA gene sequencing. Twenty deep-tissue specimens, 19 with corresponding superficial tissues and 12 with control tissues, were successfully processed. A diversity of bacterial taxa were isolated and identified, including several putatively novel species. Most isolates were found to be saccharolytic and acid-tolerant species. Notably, some species were isolated only from either the tumorous or nontumorous tissue type, indicating a degree of restriction. Successful surface decontamination of the specimens indicates that the bacteria detected were from within the tissue. A diversity of bacterial groups have been isolated from within oral squamous cell carcinoma tissue. The significance of these bacteria within the tumor warrants further study.
Human Papillomavirus Type 16 Integration in Cervical Carcinoma In Situ and in Invasive Cervical Cancer
Authors: Arias-Pulido Hugo , Peyton Cheri L. , Joste Nancy E. , Vargas Hernan , Wheeler Cosette M.
Published: May 2006

Integration of human papillomavirus type 16 (HPV-16) into the host DNA has been proposed as a potential marker of cervical neoplastic progression. In this study, a quantitative real-time PCR (qRT-PCR) was used to examine the physical status of HPV-16 in 126 cervical carcinoma in situ and 92 invasive cervical cancers. Based on criteria applied to results from this qRT-PCR assay, HPV-16 was characterized in carcinoma in situ cases as episomal (61.9%), mixed (i.e., episomal and integrated; 29.4%), and integrated (8.7%) forms. In invasive cervical cancer samples, HPV-16 was similarly characterized as episomal (39.1%), mixed (45.7%), and integrated (15.2%) forms. The difference in the frequency of integrated or episomal status estimated for carcinoma in situ and invasive cervical cancer cases was statistically significant (
P
= 0.003). Extensive mapping analysis of HPV-16 E1 and E2 genes in 37 selected tumors demonstrated deletions in both E1 and E2 genes with the maximum number of losses (78.4%) observed within the HPV-16 E2 hinge region. Specifically, deletions within the E2 hinge region were detected most often between nucleotides (nt) 3243 and 3539. The capacity to detect low-frequency HPV-16 integration events was highly limited due to the common presence and abundance of HPV episomal forms. HPV-16 E2 expressed from intact episomes may act in trans to regulate integrated genome expression of E6 and E7.
Comparative Evaluation of the Performance of the Abbott Real-Time Human Immunodeficiency Virus Type 1 (HIV-1) Assay for Measurement of HIV-1 Plasma Viral Load following Automated Specimen Preparation
Authors: Garcia-Diaz A. , Clewley G. S. , Booth C. L. , Labett W. , McAllister N. , Geretti A. M.
Published: May 2006

The performance of the new Abbott real-time human immunodeficiency virus type 1 (HIV-1) assay for HIV-1 RNA load determination in plasma was compared to that of the Abbott LCx HIV-1 RNA quantitative assay following automated RNA isolation by the Abbott m1000 extractor. The measured viral loads of 89 clinical specimens differed by mean 0.19 log
10
copies/ml (95% confidence interval, 0.12 to 0.26 log
10
copies/ml). Although the difference in viral load determinations was positively skewed in favor of the LCx assay, it did not reach statistical significance (
P
= 0.42). Results were linearly associated (
R
2
= 0.94) and strongly correlated (
R
= 0.96). Good performance was observed with HIV-1 subtypes other than B and circulating recombinant forms, although results obtained with two subtype G specimens and one H specimen showed a more substantial difference.
Rapid Detection of
Brucella
spp. in Blood Cultures by Fluorescence In Situ Hybridization
Authors: Wellinghausen Nele , Nöckler Karsten , Sigge Anja , Bartel Melanie , Essig Andreas , Poppert Sven
Published: May 2006

Brucellosis is a severe systemic disease in humans. We describe a new 16S rRNA-based fluorescence in situ hybridization assay that facilitates rapid and specific detection of all human pathogenic species of
Brucella
and that can be applied directly to positive blood cultures.