bluuebunny/crossref_metadata_2025 huggingface dataset:
Assessment of out‐of‐field absorbed dose and equivalent dose in proton fields
Authors: Clasie Ben , Wroe Andrew , Kooy Hanne , Depauw Nicolas , Flanz Jay , Paganetti Harald , Rosenfeld Anatoly
Published: December 2009

Purpose

In proton therapy, as in other forms of radiation therapy, scattered and secondary particles produce undesired dose outside the target volume that may increase the risk of radiation‐induced secondary cancer and interact with electronic devices in the treatment room. The authors implement a Monte Carlo model of this dose deposited outside passively scattered fields and compare it to measurements, determine the out‐of‐field equivalent dose, and estimate the change in the dose if the same target volumes were treated with an active beam scanning technique.

Methods

Measurements are done with a thimble ionization chamber and the Wellhofer MatriXX detector inside a Lucite phantom with field configurations based on the treatment of prostate cancer and medulloblastoma. The authors use aGEANT4 Monte Carlo simulation, demonstrated to agree well with measurements inside the primary field, to simulate fields delivered in the measurements. The partial contributions to the dose are separated in the simulation by particle type and origin.

Results

The agreement between experiment and simulation in the out‐of‐field absorbed dose is within 30% at 10–20 cm from the field edge and 90% of the data agrees within 2 standard deviations. In passive scattering, the neutron contribution to the total dose dominates in the region downstream of the Bragg peak (65%–80% due to internally produced neutrons) and inside the phantom at distances more than 10–15 cm from the field edge. The equivalent doses using 10 for the neutron weighting factor at the entrance to the phantom and at 20 cm from the field edge are 2.2 and 2.6 mSv/Gy for the prostate cancer and cranial medulloblastoma fields, respectively. The equivalent dose at 15–20 cm from the field edge decreases with depth in passive scattering and increases with depth in active scanning. Therefore, active scanning has smaller out‐of‐field equivalent dose by factors of 30–45 in the entrance region and this factor decreases with depth.

Conclusions

The dose deposited immediately downstream of the primary field, in these cases, is dominated by internally produced neutrons; therefore, scattered and scanned fields may have similar risk of second cancer in this region. The authors confirm that there is a reduction in the out‐of‐field dose in active scanning but the effect decreases with depth.GEANT4 is suitable for simulating the dose deposited outside the primary field. The agreement with measurements is comparable to or better than the agreement reported for other implementations of Monte Carlo models. Depending on the position, the absorbed dose outside the primary field is dominated by contributions from primary protons that may or may not have scattered in the brass collimating devices. This is noteworthy as the quality factor of the low LET protons is well known and the relative dose risk in this region can thus be assessed accurately.
Inclusion of the dose from kilovoltage cone beam CT in the radiation therapy treatment plans
Authors: Alaei Parham , Ding George , Guan Huaiqun
Published: December 2009

Purpose

Cone beam CT is increasingly being used for daily patient positioning verification during radiation therapy treatments. The daily use of CBCT could lead to accumulated patient doses higher than the older technique of weekly portal imaging. There have been several studies focusing on measurement or calculation of the patient dose from CBCT recently.

Methods

This study investigates the feasibility of configuring a kV x‐ray source in a commercial treatment planning system to calculate the dose to patient resulting from an IGRT procedure. The method proposed in this article can be used to calculate dose from CBCT imaging procedure and include that in the patient treatment plans.

Results

The kilovoltage beam generated by the CBCT imager has been modeled using the planning system. The modeled profiles agree with the measured ones to within 5%. The modeled beam was used to calculate dose to phantom in the pelvic region and the calculations were compared to TLD measurements. The agreement between calculated and measured doses ranges from 0% to 19% in soft tissue with larger variations observed near and within the bone.

Conclusions

The modeling of the beam produces reasonable results and the dose calculation comparisons indicate the potential for computing kilovoltage CBCT doses using a treatment planning system. Further improvements in the dose calculation algorithm are necessary, especially for dose calculations in and near the bone.
Computed tomography dose measurements with radiochromic films and a flatbed scanner
Authors: Rampado O. , Garelli E. , Ropolo R.
Published: December 2009

Gafchromic® XR‐QA films were developed for patient dosimetry in diagnostic radiology. A possible application of these films is the measurement of doses in computed tomography. In this study a method to evaluate the CTDI using Gafchromic XR‐QA film and a flatbed scanner was developed and tested. Film samples were cut to dimensions of in order to have an integration area similar to that of a pencil ionization chamber, with the possibility of changing the integration length. Prior to exposing these films to a computed tomography beam, the angular dependence of the film dose response was investigated by exposing film strips to a static x‐ray beam at different angles in the range 0°–180°. A difference of 49% was found between the response with the axis beam parallel to the film surface (90°) and with the axis beam perpendicular (0° and 180°). Integrating over a 360° exposure like the one in computed tomography, a difference of less than 2% was estimated, which is comparable with the measurement error obtainable with XR‐QA film. A calibration with a CT beam in the scout mode was performed and film strips were then exposed to single axial scans and to helical scans both in air and in phantoms. Two different types of flatbed scanners were used to read the film samples, a Microtek ScanMaker 9800XL scanner and an Epson Expression 10000 XL scanner, and the accuracy of the results were compared. For beam collimations above 10 mm differences between CTDI measured by film and CTDI measured by ionization chamber below 9% were found for the Epson scanner, with an average estimated error at level of 5%. For the Microtek scanner and for the same film samples, differences below 11% with an average error at level of 8% were founded. The uncertainty of the measured CTDI was provided by the method for each measurement, and it was shown that about the 95% of the differences between the CTDI measurements with radiochromic films and with the ionization chamber were below the estimated uncertainty, for both scanners. After an accurate calibration procedure and the consideration of the uncertainty associated with the measurement, Gafchromic® XR‐QA films can be used to evaluate the CTDI.
Performance characterization of a MVCT scanner using multislice thick, segmented cadmium tungstate‐photodiode detectors
Authors: Kirvan P. F. , Monajemi T. T. , Fallone B. G. , Rathee S.
Published: December 2009

Purpose

Megavoltage computed tomography (MVCT) and megavoltage cone beam computed tomography (MVCBCT) can be used for visualizing anatomical structures prior to radiation therapy treatments to assist in patient setup and target localization. These systems are less susceptible to metal artifacts and provide better CT number linearity than conventional CT scanners. However, their contrast is limited by the properties of the megavoltage photons and the low detective quantum efficiency (DQE) of flat panel detector systems currently available. By using higher DQE, thick, segmented cadmium tungstate detectors, the authors can improve the low contrast detectability of a MVCT system. This in turn would permit greater soft tissue visualization for a given radiation dose, allowing MVCT to be used in more clinical situations.

Methods

This article describes the evaluation of our prototype system that uses thick, segmented detectors. In order to create images using a dose that would be acceptable for day to day patient imaging, the authors evaluated their system using the low intensity bremsstrahlung component of a 6 MeV electron beam. The system was evaluated for its uniformity, high contrast resolution, low contrast detectability, signal to noise ratio, contrast to noise ratio, and CT number linearity.

Results

The prototype system was found to have a high contrast spatial resolution of about 5 line pairs per cm, and to be able to visualize a 15 mm 1.5% contrast target with 2 cGy of radiation dose delivered. vs radiation dose and mean pixel value vs electron density curves were linear.

Conclusions

This prototype system shows a large improvement in low contrast detectability over current MVCBCT systems.
PPARγ Activation: A Potential Treatment For Pulmonary Hypertension
Authors: Hansmann Georg , Zamanian Roham T.
Published: December 2009

Pharmacological activation of the transcription factor PPARγ affects the expression of key genes involved in the development of pulmonary hypertension (PH), subsequently has a variety of beneficial effects in the vasculature, and hence might be a powerful treatment of PH.
Measurement and Clinical Monitoring of Human Lymphocyte Clonality by Massively Parallel V-D-J Pyrosequencing
Authors: Boyd Scott D. , Marshall Eleanor L. , Merker Jason D. , Maniar Jay M. , Zhang Lyndon N. , Sahaf Bita , Jones Carol D. , Simen Birgitte B. , Hanczaruk Bozena , Nguyen Khoa D. , Nadeau Kari C. , Egholm Michael , Miklos David B. , Zehnder James L. , Fire Andrew Z.
Published: December 2009

Massively parallel sequencing of rearranged immune receptor genes permits detection and tracking of specific immune cell populations in normal and pathological contexts.
Antimicrobial Susceptibilities of Multidrug-Resistant
Campylobacter jejuni
and
C. coli
Strains:
In Vitro
Activities of 20 Antimicrobial Agents
Authors: Lehtopolku Mirva , Nakari Ulla-Maija , Kotilainen Pirkko , Huovinen Pentti , Siitonen Anja , Hakanen Antti J.
Published: December 2009

There is a paucity of information regarding antimicrobial agents that are suitable to treat severe infections caused by multidrug-resistant
Campylobacter
spp. Our aim was to identify agents that are potentially effective against multiresistant
Campylobacter
strains. The
in vitro
activities of 20 antimicrobial agents against 238
Campylobacter
strains were analyzed by determining MICs by the agar plate dilution method or the Etest. These strains were selected from 1,808
Campylobacter
isolates collected from Finnish patients between 2003 and 2005 and screened for macrolide susceptibility by using the disk diffusion test. The 238 strains consisted of 183 strains with erythromycin inhibition zone diameters of ≤23 mm and 55 strains with inhibition zone diameters of >23 mm. Of the 238
Campylobacter
strains, 19 were resistant to erythromycin by MIC determinations (MIC ≥ 16 μg/ml). Given that the resistant strains were identified among the collection of 1,808 isolates, the frequency of erythromycin resistance was 1.1%. All erythromycin-resistant strains were multidrug resistant, with 18 (94.7%) of them being resistant to ciprofloxacin (MIC ≥ 4 μg/ml). The percentages of resistance to tetracycline and amoxicillin-clavulanic acid (co-amoxiclav) were 73.7% and 31.6%, respectively. All macrolide-resistant strains were susceptible to imipenem, meropenem, and tigecycline. Ten (52.6%) multiresistant strains were identified as being
Campylobacter jejuni
strains, and 9 (47.4%) were identified as being
C. coli
strains. These data demonstrate that the incidence of macrolide resistance was low but that the macrolide-resistant
Campylobacter
strains were uniformly multidrug resistant. In addition to the carbapenems, tigecycline was also highly effective against these multidrug-resistant
Campylobacter
strains
in vitro
. Its efficacy for the treatment of human campylobacteriosis should be evaluated in clinical trials.
Inhibition of
Porphyromonas gingivalis
Biofilm by Oxantel
Authors: Dashper Stuart , Ang Ching-Seng , Liu Sze Wei , Paolini Rita , Veith Paul , Reynolds Eric
Published: December 2009

Porphyromonas gingivalis
is a major pathogen of chronic periodontitis and exists in a biofilm on the surface of the tooth root. Oxantel, a cholinergic anthelmintic and fumarate reductase inhibitor, significantly inhibited biofilm formation by
P. gingivalis
and disrupted established biofilms at concentrations below its MIC against planktonic cells. Oxantel was more effective against
P. gingivalis
in biofilm than metronidazole, a commonly used antibiotic for periodontitis.
Clinical Pharmacokinetics of Oral Controlled-Release 5-Fluorocytosine
Authors: Pai Manjunath P. , Bruce Hollie , Felton Linda A.
Published: December 2009

5-Fluorocytosine (5FC) is an oral antifungal that is currently used in combination with amphotericin B to treat
Cryptococcus neoformans
meningoencephalitis. The oral dosing of 5FC could be optimized by the use of a controlled-release (CR) formulation. The objective of the current study was to develop two prototype 5FC-CR formulations and evaluate the single-dose (1,500-mg) serum pharmacokinetic profiles of those formulations relative to the profile of the commercially available, immediate-release 5FC product (Ancobon) by the use of a phase 1, open-label, randomized, three-phase, crossover pharmacokinetic study design. Hydroxypropyl methylcellulose was utilized as the rate-controlling matrix to compound the 5FC-CR tablets. The two prototype 5FC-CR formulations demonstrated 80% release at 13.0 and 18.4 h, respectively, whereas the immediate-release product demonstrated 80% release at 0.28 h, as determined
in vitro
by the United States Pharmacopeia apparatus 2 dissolution method. Five subjects completed all three phases of the study without any adverse events. The mean maximum concentration, the area under the curve from time zero to 24 h, and the area under the curve from time zero to infinity were approximately 50% lower (
P
< 0.01) with the 5FC-CR formulations than with the immediate-release 5FC product. However, no statistically significant differences in the minimum concentrations at 24 h were noted between the formulations. The gastric absorption profile of 5FC-CR was well predicted by
in vitro
dissolution. Future exploration of a gastroretentive 5FC-CR formulation could overcome the marked lack of bioequivalence observed in the present study.
Clinical Pharmacodynamics of Cefepime in Patients Infected with
Pseudomonas aeruginosa
Authors: Crandon Jared L. , Bulik Catharine C. , Kuti Joseph L. , Nicolau David P.
Published: December 2009

We evaluated cefepime exposures in patients infected with
Pseudomonas aeruginosa
to identify the pharmacodynamic relationship predictive of microbiological response. Patients with non-urinary tract
P. aeruginosa
infections and treated with cefepime were included. Free cefepime exposures were estimated by using a validated population pharmacokinetic model.
P. aeruginosa
MICs were determined by Etest and pharmacodynamic indices (the percentage of the dosing interval that the free drug concentration remains above the MIC of the infecting organism [
f
T
> MIC], the ratio of the minimum concentration of free drug to the MIC [
f
C
min
/MIC], and the ratio of the area under the concentration-time curve for free drug to the MIC [
f
AUC/MIC]) were calculated for each patient. Classification and regression tree analysis was used to partition the pharmacodynamic parameters for prediction of the microbiological response. Monte Carlo simulation was utilized to determine the optimal dosing regimens needed to achieve the pharmacodynamic target. Fifty-six patients with pneumonia (66.1%), skin and skin structure infections (SSSIs) (25%), and bacteremia (8.9%) were included. Twenty-four (42.9%) patients failed cefepime therapy. The MICs ranged from 0.75 to 96 μg/ml, resulting in median
f
T
> MIC,
f
C
m
in
/MIC, and
f
AUC/MIC exposures of 100% (range, 0.8 to 100%), 4.3 (range, 0.1 to 27.3), and 206.2 (range, 4.2 to 1,028.7), respectively. Microbiological failure was associated with an
f
T
> MIC of ≤60% (77.8% failed cefepime therapy when
f
T
> MIC was ≤60%, whereas 36.2% failed cefepime therapy when
f
T
> MIC was >60%;
P
= 0.013). A similar
f
T
> MIC target of ≤63.9% (
P
= 0.009) was identified when skin and skin structure infections were excluded. While controlling for the SSSI source (odds ratio [OR], 0.18 [95% confidence interval, 0.03 to 1.19];
P
= 0.07) and combination therapy (OR, 2.15 [95% confidence interval, 0.59 to 7.88];
P
= 0.25), patients with
f
T
> MIC values of ≤60% were 8.1 times (95% confidence interval, 1.2 to 55.6 times) more likely to experience a poor microbiological response. Cefepime doses of at least 2 g every 8 h are required to achieve this target against CLSI-defined susceptible
P. aeruginosa
organisms in patients with normal renal function. In patients with non-urinary tract infections caused by
P. aeruginosa
, achievement of cefepime exposures of >60%
f
T
> MIC will minimize the possibility of a poor microbiological response.
Rates and Mechanisms of Resistance Development inMycobacterium tuberculosisto a Novel Diarylquinoline ATP Synthase Inhibitor
Authors: Huitric E. , Verhasselt P. , Koul A. , Andries K. , Hoffner S. , Andersson D. I.
Published: December 2009

R207910 (also known as TMC207) is an investigational drug currently in clinical studies for the treatment of multidrug-resistant (MDR) tuberculosis. It has a high degree of antimycobacterial activity and is equally effective against drug-susceptible and MDRMycobacterium tuberculosisisolates. In the present study, we characterized the development of resistance to R207910in vitro. Ninety-seven independent R207910-resistant mutants were selected from seven different clinical isolates ofM. tuberculosis(three drug-susceptible and four MDR isolates) at 10×, 30×, and 100× the MIC. At a concentration of 0.3 mg/liter (10× the MIC), the mutation rates ranged from 4.7 × 10−7to 8.9 × 10−9mutations per cell per division, and at 1.0 mg/liter (30× the MIC) the mutation rate ranged from 3.9 × 10−8to 2.4 × 10−9. No resistant mutants were obtained at 3 mg/liter (100× the MIC). The level of resistance ranged from 0.12 to 3.84 mg/liter for the mutants identified; these concentrations represent 4- to 128-fold increases in the MICs. For 53 of the resistant mutants, theatpEgene, which encodes a transmembrane and oligomeric C subunit of the ATP synthase and which was previously shown to be involved in resistance, was sequenced. For 15/53 mutants, five different point mutations resulting in five different amino acid substitutions were identified in theatpEgene. For 38/53 mutants, noatpEmutations were found and sequencing of the complete F0 ATP synthase operon (atpB,atpE, andatpFgenes) and the F1 ATP synthase operon (atpH,atpA,atpG,atpD, andatpCgenes) from three mutants revealed no mutations, indicating other, alternative resistance mechanisms. Competition assays showed no measurable reduction in the fitness of the mutants compared to that of the isogenic wild types.
Genes Involved in Yellow Pigmentation of
Cronobacter sakazakii
ES5 and Influence of Pigmentation on Persistence and Growth under Environmental Stress
Authors: Johler Sophia , Stephan Roger , Hartmann Isabel , Kuehner Kirsten A. , Lehner Angelika
Published: December 2009

Cronobacter
spp. are opportunistic food-borne pathogens that are responsible for rare but highly fatal cases of meningitis and necrotizing enterocolitis in neonates. While the operon responsible for yellow pigmentation in
Cronobacter sakazakii
strain ES5 was described recently, the involvement of additional genes in pigment expression and the influence of pigmentation on the fitness of
Cronobacter
spp. have not been investigated. Thus, the aim of this study was to identify further genes involved in pigment expression in
Cronobacter sakazakii
ES5 and to assess the influence of pigmentation on growth and persistence under conditions of environmental stress. A knockout library was created using random transposon mutagenesis. The screening of 9,500 mutants for decreased pigment production identified 30 colorless mutants. The mapping of transposon insertion sites revealed insertions in not only the carotenoid operon but also in various other genes involved in signal transduction, inorganic ions, and energy metabolism. To determine the effect of pigmentation on fitness, colorless mutants (Δ
crtE
, Δ
crtX
, and Δ
crtY
) were compared to the yellow wild type using growth and inactivation experiments, a macrophage assay, and a phenotype array. Among other findings, the colorless mutants grew at significantly increased rates under osmotic stress compared to that of the yellow wild type while showing increased susceptibility to desiccation. Moreover, Δ
crtE
and Δ
crtY
exhibited increased sensitivity to UVB irradiation.
Establishment of Cyanophycin Biosynthesis in
Pichia pastoris
and Optimization by Use of Engineered Cyanophycin Synthetases
Authors: Steinle Anna , Witthoff Sabrina , Krause Jens P. , Steinbüchel Alexander
Published: December 2009

Two strains of the methylotrophic yeast
Pichia pastoris
were used to establish cyanophycin (multi-
l
-arginyl-poly-
l
-aspartic acid [CGP]) synthesis and to explore the applicability of this industrially widely used microorganism for the production of this polyamide. Therefore, the CGP synthetase gene from the cyanobacterium
Synechocystis
sp. strain PCC 6308 (
cphA
6308
) was expressed under the control of the alcohol oxidase 1 promoter, yielding CGP contents of up to 10.4% (wt/wt), with the main fraction consisting of the soluble form of the polymer. To increase the polymer contents and to obtain further insights into the structural or catalytic properties of the enzyme, site-directed mutagenesis was applied to
cphA
6308
and the mutated gene products were analyzed after expression in
P. pastoris
and
Escherichia coli
, respectively. CphA
6308
Δ1, which was truncated by one amino acid at the C terminus; point mutated CphA
6308
C595S; and the combined double-mutant CphA
6308
Δ1C595S protein were purified. They exhibited up to 2.5-fold higher enzyme activities of 4.95 U/mg, 3.20 U/mg, and 4.17 U/mg, respectively, than wild-type CphA
6308
(2.01 U/mg). On the other hand, CphA proteins truncated by two (CphA
6308
Δ2) or three (CphA
6308
Δ3) amino acids at the C terminus showed similar or reduced CphA enzyme activity in comparison to CphA
6308
. In flask experiments, a maximum of 14.3% (wt/wt) CGP was detected after the expression of CphA
6308
Δ1 in
P. pastoris
. For stabilization of the expression plasmid, the
his4
gene from
Saccharomyces cerevisiae
was cloned into the expression vector used and the constructs were transferred to histidine auxotrophic
P. pastoris
strain GS115. Parallel fermentations at a one-to-one scale revealed 26°C and 6.0 as the optimal temperature and pH, respectively, for CGP synthesis. After optimization of fermentation parameters, medium composition, and the length of the cultivation period, CGP contents could be increased from 3.2 to 13.0% (wt/wt) in cells of
P. pastoris
GS115 expressing CphA
6308
and up to even 23.3% (wt/wt) in cells of
P. pastoris
GS115 expressing CphA
6308
Δ1.
Inducible Expression of Transmembrane Proteins on Bacterial Magnetic Particles in
Magnetospirillum magneticum
AMB-1
Authors: Yoshino Tomoko , Shimojo Akiko , Maeda Yoshiaki , Matsunaga Tadashi
Published: December 2009

Bacterial magnetic particles (BacMPs) produced by the magnetotactic bacterium
Magnetospirillum magneticum
AMB-1 are used for a variety of biomedical applications. In particular, the lipid bilayer surrounding BacMPs has been reported to be amenable to the insertion of recombinant transmembrane proteins; however, the display of transmembrane proteins in BacMP membranes remains a technical challenge due to the cytotoxic effects of the proteins when they are overexpressed in bacterial cells. In this study, a tetracycline-inducible expression system was developed to display transmembrane proteins on BacMPs. The expression and localization of the target proteins were confirmed using luciferase and green fluorescent protein as reporter proteins. Gene expression was suppressed in the absence of anhydrotetracycline, and the level of protein expression could be controlled by modulating the concentration of the inducer molecule. This system was implemented to obtain the expression of the tetraspanin CD81. The truncated form of CD81 including the ligand binding site was successfully displayed at the surface of BacMPs by using Mms13 as an anchor protein and was shown to bind the hepatitis C virus envelope protein E2. These results suggest that the tetracycline-inducible expression system described here will be a useful tool for the expression and display of transmembrane proteins in the membranes of BacMPs.
Identity, Diversity, and Molecular Phylogeny of the Endophytic Mycobiota in the Roots of Rare Wild Rice (
Oryza granulate
) from a Nature Reserve in Yunnan, China
Authors: Yuan Zhi-lin , Zhang Chu-long , Lin Fu-cheng , Kubicek Christian P.
Published: December 2009

Rice (
Oryza sativa
L.) is, on a global scale, one of the most important food crops. Although endophytic fungi and bacteria associated with rice have been investigated, little is known about the endophytic fungi of wild rice (
Oryza granulate
) in China. Here we studied the root endophytic mycobiota residing in roots of
O. granulate
by the use of an integrated approach consisting of microscopy, cultivation, ecological indices, and direct PCR. Microscopy confirmed the ubiquitousness of dark septate endophytes (DSEs) and sclerotium-like structures in root tissues. Isolations from 204 root segments from 15 wild rice plants yielded 58 isolates, for which 31 internal transcribed spacer (ITS)-based genotypes were recorded. The best BLAST match indicated that 34.5% of all taxa encountered may represent hitherto undescribed species. Most of the fungi were isolated with a very low frequency. Calculation of ecological indices and estimation of taxon accumulation curves indicated a high diversity of fungal species. A culture-independent approach was also performed to analyze the endophytic fungal community. Three individual clone libraries were constructed. Using a threshold of 90% similarity, 35 potentially different sequences (phylotypes) were found among 186 positive clones. Phylogenetic analysis showed that frequently detected clones were classified as Basidiomycota, and 60.2% of total analyzed clones were affiliated with unknown taxa.
Exophiala
,
Cladophialophora
,
Harpophora
,
Periconia macrospinosa
, and the
Ceratobasidium
/
Rhizoctonia
complex may act as potential DSE groups. A comparison of the fungal communities characterized by the two approaches demonstrated distinctive fungal groups, and only a few taxa overlapped. Our findings indicate a complex and rich endophytic fungal consortium in wild rice roots, thus offering a potential bioresource for establishing a novel model of plant-fungal mutualistic interactions.
Abundance of Ammonia-Oxidizing
Archaea
and
Bacteria
along an Estuarine Salinity Gradient in Relation to Potential Nitrification Rates
Authors: Bernhard Anne E. , Landry Zachary C. , Blevins Alison , de la Torre José R. , Giblin Anne E. , Stahl David A.
Published: December 2009

Abundance of ammonia-oxidizing
Archaea
(AOA) was found to be always greater than that of ammonia-oxidizing
Bacteria
along an estuarine salinity gradient, and AOA abundance was highest at intermediate salinity. However, AOA abundance did not correlate with potential nitrification rates. This lack of correlation may be due to methodological limitations or alternative energy sources.
Bibersteinia trehalosiInhibits the Growth ofMannheimia haemolyticaby a Proximity-Dependent Mechanism
Authors: Dassanayake Rohana P. , Call Douglas R. , Sawant Ashish A. , Casavant N. Carol , Weiser Glen C. , Knowles Donald P. , Srikumaran Subramaniam
Published: December 2009

Mannheimia(Pasteurella)haemolyticais the only pathogen that consistently causes severe bronchopneumonia and rapid death of bighorn sheep (BHS;Ovis canadensis) under experimental conditions. Paradoxically,Bibersteinia(Pasteurella)trehalosiandPasteurella multocidahave been isolated from BHS pneumonic lungs much more frequently thanM. haemolytica. These observations suggest that there may be an interaction between these bacteria, and we hypothesized thatB. trehalosiovergrows or otherwise inhibits the growth ofM. haemolytica. Growth curves (monoculture) demonstrated thatB. trehalosihas a shorter doubling time (∼10 min versus ∼27 min) and consistently achieves 3-log higher cell density (CFU/ml) compared toM. haemolytica. During cocultureM. haemolyticagrowth was inhibited whenB. trehalosientered stationary phase (6 h) resulting in a final cell density forM. haemolyticathat was 6 to 9 logs lower than expected with growth in the absence ofB. trehalosi. Coculture supernatant failed to inhibitM. haemolyticagrowth on agar or in broth, indicating no obvious involvement of lytic phages, bacteriocins, or quorum-sensing systems. This observation was confirmed by limited growth inhibition ofM. haemolyticawhen both pathogens were cultured in the same media but separated by a filter (0.4-μm pore size) that limited contact between the two bacterial populations. There was significant growth inhibition ofM. haemolyticawhen the populations were separated by membranes with a pore size of 8 μm that allowed free contact. These observations demonstrate thatB. trehalosican both outgrow and inhibitM. haemolyticagrowth with the latter related to a proximity- or contact-dependent mechanism.
Reliable Detection of Dead Microbial Cells by Using Fluorescent Hydrazides
Authors: Saint-Ruf Claude , Cordier Corinne , Mégret Jérome , Matic Ivan
Published: December 2009

We have developed a new method for accurate quantification of dead microbial cells. This technique employs the simultaneous use of fluorescent hydrazides and nucleic acid dyes. Fluorescent hydrazides allow detection of cells that cannot be detected with currently used high-affinity nucleic acid dyes. This is particularly important for nongrowing bacterial populations and for multicellular communities containing physiologically heterogeneous cell populations, such as colonies and biofilms.
Global Transcriptional, Physiological, and Metabolite Analyses of the Responses of
Desulfovibrio vulgaris
Hildenborough to Salt Adaptation
Authors: He Zhili , Zhou Aifen , Baidoo Edward , He Qiang , Joachimiak Marcin P. , Benke Peter , Phan Richard , Mukhopadhyay Aindrila , Hemme Christopher L. , Huang Katherine , Alm Eric J. , Fields Matthew W. , Wall Judy , Stahl David , Hazen Terry C. , Keasling Jay D. , Arkin Adam P. , Zhou Jizhong
Published: December 2009

The response of
Desulfovibrio vulgaris
Hildenborough to salt adaptation (long-term NaCl exposure) was examined by performing physiological, global transcriptional, and metabolite analyses. Salt adaptation was reflected by increased expression of genes involved in amino acid biosynthesis and transport, electron transfer, hydrogen oxidation, and general stress responses (e.g., heat shock proteins, phage shock proteins, and oxidative stress response proteins). The expression of genes involved in carbon metabolism, cell growth, and phage structures was decreased. Transcriptome profiles of
D. vulgaris
responses to salt adaptation were compared with transcriptome profiles of
D. vulgaris
responses to salt shock (short-term NaCl exposure). Metabolite assays showed that glutamate and alanine accumulated under salt adaptation conditions, suggesting that these amino acids may be used as osmoprotectants in
D. vulgaris
. Addition of amino acids (glutamate, alanine, and tryptophan) or yeast extract to the growth medium relieved salt-related growth inhibition. A conceptual model that links the observed results to currently available knowledge is proposed to increase our understanding of the mechanisms of
D. vulgaris
adaptation to elevated NaCl levels.
Effect of Growth Phase Feeding Strategies on Succinate Production by Metabolically Engineered
Escherichia coli
Authors: Jiang Min , Liu Shu-wen , Ma Jiang-feng , Chen Ke-quan , Yu Li , Yue Fang-fang , Xu Bing , Wei Ping
Published: December 2009

Aerobic growth conditions significantly influenced anaerobic succinate production in two-stage fermentation by
Escherichia coli
AFP111 with knockouts in
rpoS
,
pflAB
,
ldhA
, and
ptsG
genes. At a low cell growth rate limited by glucose, enzymes involved in the reductive arm of the tricarboxylic acid cycle and the glyoxylate shunt showed elevated activities, providing AFP111 with intracellular redox balance and increased succinic acid yield and productivity.
Identification of the Main Promoter Directing Cereulide Biosynthesis in Emetic
Bacillus cereus
and Its Application for Real-Time Monitoring of
ces
Gene Expression in Foods
Authors: Dommel Monica K. , Frenzel Elrike , Strasser Bernd , Blöchinger Claudia , Scherer Siegfried , Ehling-Schulz Monika
Published: December 2009

Cereulide, the emetic
Bacillus cereus
toxin, is synthesized by cereulide synthetase via a nonribosomal peptide synthetase (NRPS) mechanism. Previous studies focused on the identification, structural organization, and biochemical characterization of the
ces
gene locus encoding cereulide synthetase; however, detailed information about the transcriptional organization of the
ces
genes was lacking. The present study shows that the
cesPTABCD
genes are transcribed as a 23-kb polycistronic transcript, while
cesH
, encoding a putative hydrolase, is transcribed from its own promoter. Transcription initiation was mapped by primer extension and rapid amplification of cDNA ends (RACE). Deletion analysis of promoter elements revealed a main promoter located upstream of the
cesP
coding sequence, encoding a 4′-phosphopantetheinyl transferase. This promoter drives transcription of
cesPTABCD
. In addition, intracistronic promoter regions in proximity to the translational start sites of
cesB
and
cesT
were identified but were only weakly active under the chosen assay conditions. The identified main promoter was amplified from the emetic reference strain
B. cereus
F4810/72 and fused to luciferase genes in order to study promoter activity in complex environments and to establish a biomonitoring system to assess cereulide production in different types of foods.
ces
promoter activity was strongly influenced by the food matrix and varied by 5 orders of magnitude. The amount of cereulide toxin extracted from spiked foods correlated well with the bioluminescence data, thus illustrating the potential of the established reporter system for monitoring of
ces
gene expression in complex matrices.
Indigenous and Environmental Modulation of Frequencies of Mutation in
Lactobacillus plantarum
Authors: Machielsen Ronnie , van Alen-Boerrigter Ingrid J. , Koole Lucy A. , Bongers Roger S. , Kleerebezem Michiel , Van Hylckama Vlieg Johan E. T.
Published: December 2009

Reliability of microbial (starter) strains in terms of quality, functional properties, growth performance, and robustness is essential for industrial applications. In an industrial fermentation process, the bacterium should be able to successfully withstand various adverse conditions during processing, such as acid, osmotic, temperature, and oxidative stresses. Besides the evolved defense mechanisms, stress-induced mutations participate in adaptive evolution for survival under stress conditions. However, this may lead to accumulation of mutant strains, which may be accompanied by loss of desired functional properties. Defining the effects of specific fermentation or processing conditions on the mutation frequency is an important step toward preventing loss of genome integrity and maintaining the productivity of industrial strains. Therefore, a set of
Lactobacillus plantarum
mutator reporter strains suitable for qualitative and quantitative analysis of low-frequency mutation events was developed. The mutation reporter system constructed was validated by using chemical mutagenesis (
N
-methyl-
N
′-nitro-
N
-nitrosoguanidine) and by controlled expression of endogenous candidate mutator genes (e.g., a truncated derivative of the
L. plantarum hexA
gene). Growth at different temperatures, under low-pH conditions, at high salt concentrations, or under starvation conditions did not have a significant effect on the mutation frequency. However, incubation with sublethal levels of hydrogen peroxide resulted in a 100-fold increase in the mutation frequency compared to the background mutation frequency. Importantly, when cells of
L. plantarum
were adapted to 42°C prior to treatment with sublethal levels of hydrogen peroxide, there was a 10-fold increase in survival after peroxide treatment, and there was a concomitant 50-fold decrease in the mutation frequency. These results show that specific environmental conditions encountered by bacteria may significantly influence the genetic stability of strains, while protection against mutagenic conditions may be obtained by pretreatment of cultures with other, nonmutagenic stress conditions.
The Lactococcin G Immunity Protein Recognizes Specific Regions in Both Peptides Constituting the Two-Peptide Bacteriocin Lactococcin G
Authors: Oppegård Camilla , Emanuelsen Linda , Thorbek Lisbeth , Fimland Gunnar , Nissen-Meyer Jon
Published: December 2009

Lactococcin G and enterocin 1071 are two homologous two-peptide bacteriocins. Expression vectors containing the gene encoding the putative lactococcin G immunity protein (
lagC
) or the gene encoding the enterocin 1071 immunity protein (
entI
) were constructed and introduced into strains sensitive to one or both of the bacteriocins. Strains that were sensitive to lactococcin G became immune to lactococcin G when expressing the putative lactococcin G immunity protein, indicating that the
lagC
gene in fact encodes a protein involved in lactococcin G immunity. To determine which peptide or parts of the peptide(s) of each bacteriocin that are recognized by the cognate immunity protein, combinations of wild-type peptides and hybrid peptides from the two bacteriocins were assayed against strains expressing either of the two immunity proteins. The lactococcin G immunity protein rendered the enterococcus strain but not the lactococcus strains resistant to enterocin 1071, indicating that the functionality of the immunity protein depends on a cellular component. Moreover, regions important for recognition by the immunity protein were identified in both peptides (Lcn-α and Lcn-β) constituting lactococcin G. These regions include the N-terminal end of Lcn-α (residues 1 to 13) and the C-terminal part of Lcn-β (residues 14 to 24). According to a previously proposed structural model of lactococcin G, these regions will be positioned adjacent to each other in the transmembrane helix-helix structure, and the model thus accommodates the present results.
Development of a Common Oligonucleotide Reference Standard for Microarray Data Normalization and Comparison across Different Microbial Communities
Authors: Liang Yuting , He Zhili , Wu Liyou , Deng Ye , Li Guanghe , Zhou Jizhong
Published: December 2009

High-density functional gene arrays have become a powerful tool for environmental microbial detection and characterization. However, microarray data normalization and comparison for this type of microarray remain a challenge in environmental microbiology studies because some commonly used normalization methods (e.g., genomic DNA) for the study of pure cultures are not applicable. In this study, we developed a common oligonucleotide reference standard (CORS) method to address this problem. A unique 50-mer reference oligonucleotide probe was selected to co-spot with gene probes for each array feature. The complementary sequence was synthesized and labeled for use as the reference target, which was then spiked and cohybridized with each sample. The signal intensity of this reference target was used for microarray data normalization and comparison. The optimal amount or concentration were determined to be ca. 0.5 to 2.5% of a gene probe for the reference probe and ca. 0.25 to 1.25 fmol/μl for the reference target based on our evaluation with a pilot array. The CORS method was then compared to dye swap and genomic DNA normalization methods using the
Desulfovibrio vulgaris
whole-genome microarray, and significant linear correlations were observed. This method was then applied to a functional gene array to analyze soil microbial communities, and the results demonstrated that the variation of signal intensities among replicates based on the CORS method was significantly lower than the total intensity normalization method. The developed CORS provides a useful approach for microarray data normalization and comparison for studies of complex microbial communities.
Aspergillus fumigatus Survival in Alkaline and Extreme Zinc-Limiting Environments Relies on the Induction of a Zinc Homeostasis System Encoded by the
zrfC
and
aspf2
Genes
Authors: Amich Jorge , Vicentefranqueira Rocío , Leal Fernando , Calera José Antonio
Published: December 2009

Aspergillus fumigatus
has three zinc transporter-encoding genes whose expression is regulated by both pH and the environmental concentration of zinc. We have previously reported that the
zrfA
and
zrfB
genes of
A. fumigatus
are transcribed at higher levels and are required for fungal growth under acidic zinc-limiting conditions whereas they are dispensable for growth in neutral or alkaline zinc-limiting media. Here we report that the transporter of the zinc uptake system that functions in
A. fumigatus
growing in neutral or alkaline environments is encoded by
zrfC
. The transcription of
zrfC
occurs divergently with respect to the adjacent
aspf2
gene, which encodes an immunodominant antigen secreted by
A. fumigatus
. The two genes—
zrfC
and
aspf2
—are required to different extents for fungal growth in alkaline and extreme zinc-limiting media. Indeed, these environmental conditions induce the simultaneous transcription of both genes mediated by the transcriptional regulators ZafA and PacC. ZafA upregulates the expression of
zrfC
and
aspf2
under zinc-limiting conditions regardless of the ambient pH, whereas PacC represses the expression of these genes under acidic growth conditions. Interestingly, the mode of action of PacC for
zrfC-aspf2
transcription contrasts with the more widely accepted model for PacC function, according to which under alkaline growth conditions PacC would activate the transcription of alkaline-expressed genes but would repress the transcription of acid-expressed genes. In sum, this report provides a good framework for investigating several important aspects of the biology of species of
Aspergillus
, including the repression of alkaline genes by PacC at acidic pH and the interrelationship that must exist between tissue pH, metal availability in the host tissue, and fungal virulence.
Immunization with a Combination of Three Pneumococcal Proteins Confers Additive and Broad Protection against
Streptococcus pneumoniae
Infections in Mice
Authors: Wu Kaifeng , Zhang Xuemei , Shi Jing , Li Nan , Li Dairong , Luo Miao , Cao Ju , Yin Nanlin , Wang Hong , Xu Wenchun , He Yujuan , Yin Yibing
Published: December 2009

Pneumococcal polysaccharide-based vaccines are effective in preventing pneumococcus infection; however, some drawbacks preclude their widespread use in developing and undeveloped countries. Here, we evaluated the protective effects of ATP-dependent caseinolytic protease (ClpP), pneumolysin mutant (ΔA146 Ply), putative lipoate-protein ligase (Lpl), or combinations thereof against pneumococcal infections in mice. Vaccinated mice were intraperitoneally and/or intranasally challenged with different pneumococcal strains. In intraperitoneal challenge models with pneumococcal strain D39 (serotype 2), the most striking protection was obtained with the combination of the three antigens. Similarly, with the intranasal challenge models, (i) additive clearance of bacteria in lungs was observed for the combination of the three antigens and (ii) a combination vaccine conferred complete protection against intranasal infections of three of the four most common pneumococcal strains (serotypes 14, 19F, and 23F) and 80% protection for pneumococcal strain 6B. Even so, immunity to this combination could confer protection against pneumococcal infection with a mixture of four serotypes. Our results showed that the combination vaccine was as effective as the currently used vaccines (PCV7 and PPV23). These results indicate that system immunization with the combination of pneumococcal antigens could provide an additive and broad protection against
Streptococcus pneumoniae
in pneumonia and sepsis infection models.
Assessment of Live Candidate Vaccines for Paratuberculosis in Animal Models and Macrophages
Authors: Scandurra Gabriella M. , de Lisle Geoffrey W. , Cavaignac Sonia M. , Young May , Kawakami R. Pamela , Collins Desmond M.
Published: December 2009

Mycobacterium avium
subsp.
paratuberculosis
(basonym
M. paratuberculosis
) is the causative agent of paratuberculosis, a chronic enteritis of ruminants. To control the considerable economic effect that paratuberculosis has on the livestock industry, a vaccine that induces protection with minimal side effects is required. We employed transposon mutagenesis and allelic exchange to develop three potential vaccine candidates, which were then tested for virulence with macrophages, mice, and goats. All three models identified the WAg906 mutant as being the most attenuated, but some differences in the levels of attenuation were evident among the models when testing the other strains. In a preliminary mouse vaccine experiment, limited protection was induced by WAg915, as evidenced by a reduced bacterial load in spleens and livers 12 weeks following intraperitoneal challenge with
M. paratuberculosis
K10. While we found macrophages and murine models to be rapid and cost-effective alternatives for the initial screening of
M. paratuberculosis
mutants for attenuation, it appears necessary to do the definitive assessment of attenuation with a ruminant model.
Increase in NF-κB Binding Affinity of the Variant C Allele of the Toll-Like Receptor 9 −1237T/C Polymorphism Is Associated with
Helicobacter pylori
-Induced Gastric Disease
Authors: Ng Mike Tsz Hin , van't Hof Rob , Crockett Julie C. , Hope Mairi E. , Berry Susan , Thomson John , McLean Mairi H. , McColl Kenneth E. L. , El-Omar Emad M. , Hold Georgina L.
Published: December 2009

Colonization of the gastric mucosa by
Helicobacter pylori
can lead to serious clinical outcomes, including gastric cancer. Toll-like receptors (TLRs) play an important role in the host response to
H. pylori
through the recognition of pathogen-associated molecular patterns. TLR9, in particular, is partly responsible for initiating bacterial induced immunity by binding unmethylated CpG-DNA, which is abundant in bacteria. A well-documented single nucleotide polymorphism (SNP) within the
TLR9
promoter (
TLR9
−1237T/C), is associated with a variety of inflammatory disorders, including allergic asthma, inflammatory bowel disease, and atopy. Analysis of the
TLR9
promoter gene sequence has shown that carriage of the variant “C” allele at position −1237 creates a potential NF-κB binding site that would theoretically increase the transcriptional activity of the gene. In this study, we report that the
TLR9
−1237 C allele was significantly associated with the development of
H. pylori
-induced premalignant gastric changes. Functional analysis of the SNP, supporting the data generated from the genetic association study, showed that carriage of the C allele increased
TLR9
transcriptional activity driven mainly by activation of NF-κB. Collectively, these findings confirm that the
TLR9
−1237T/C polymorphism is a risk factor for the development of
H. pylori
-induced premalignant gastric changes and provide a plausible mechanistic explanation.