Assessing the validity and sensitivity of microbial processes within a hydrodynamic model.

Eutrophication due to excess anthropogenic nutrients in waterways is a significant issue worldwide. The pressure-stressor-response of a waterway to excessive nutrient loading is reliant on numerous physical and biological factors, including hydrodynamics and microbial processing. While substantial progress has been made towards simulating these mechanisms there are limited multi-disciplinary studies that relate the physical hydrodynamics of a site with the ecological response from linked laboratory and field studies. This paper presents the development of a coupled hydrodynamic and aquatic ecosystem response model, expanded to include an integrated microbial loop, that allows the explicit representation of heterotrophic bacteria growth and dissolved organic nutrient mineralisation. A unique long-term water quality dataset at an estuary in south-eastern Australia was used to validate and assess the model's sensitivity to complex biophysical processes driving the observed water quality variability. Results indicate that explicit time-varying bacterial mineralisation rates provide a substantially improved understanding of the broader aquatic ecosystem response than assigned fixed bulk rate parameter values, which are typically derived from non-local literature. Implementation of a microbial loop at the study site indicated that the model is sensitive to the boundary conditions, in particular catchment loads, with both net transport rates and the net growth rates of heterotrophic bacteria demonstrating different responses. Under average flow conditions, a smaller net transport and reduced nutrient availability has a pronounced effect of lowering net growth rates through the applied limitation factors. During high flow conditions, freshwater inflows increased net transport and nutrient loads, which resulted in higher net growth rates. Further, temporal variability in water temperature had a compounding effect on the model's response sensitivity. This approach has broader application in other riverine systems subject to eutrophication, and in interrogating linkages in hydrodynamic and microbial mediated processes (e.g., productivity). Future studies are recommended to better understand the sensitivity of aquatic ecosystem response models to microbial net growth rate kinetics at different temperatures and from top-down predation (e.g., zooplankton grazing).

Impact of macromolecular crowding on RNA/spermine complex coacervation and oligonucleotide compartmentalization.

We report the effect of neutral macromolecular crowders poly(ethylene glycol) (PEG) (8 kDa) and Ficoll (70 kDa) on liquid-liquid phase separation in a polyuridylic acid (polyU)/spermine complex coacervate system. The addition of PEG decreased both the amount of spermine required for phase separation and the coacervation temperature (TC). We interpret these effects on phase behavior as arising due to excluded volume and preferential interactions on both the secondary structure/condensation of spermine-associated polyU molecules and on the association of soluble polyU/spermine polyelectrolyte complexes to form coacervate droplets. Examination of coacervates formed in the presence of fluorescently-labeled PEG or Ficoll crowders indicated that Ficoll is accumulated while PEG is excluded from the coacervate phase, which provides further insight into the differences in phase behavior. Crowding agents impact distribution of a biomolecular solute: partitioning of a fluorescently-labeled U15 RNA oligomer into the polyU/spermine coacervates was increased approximately two-fold by 20 wt% Ficoll 70 kDa and by more than two orders of magnitude by 20 wt% PEG 8 kDa. The volume of the coacervate phase decreased in the presence of crowder relative to a dilute buffer solution. These findings indicate that potential impacts of macromolecular crowding on phase behavior and solute partitioning should be considered in model systems for intracellular membraneless organelles.

Pulsed-light inactivation of pathogenic and spoilage bacteria on cheese surface.

Cheese products are susceptible to postprocessing cross-contamination by bacterial surface contamination during slicing, handling, or packaging, which can lead to food safety issues and significant losses due to spoilage. This study examined the effectiveness of pulsed-light (PL) treatment on the inactivation of the spoilage microorganism Pseudomonas fluorescens, the nonenterohemorrhagic Escherichia coli ATCC 25922 (nonpathogenic surrogate of Escherichia coli O157:H7), and Listeria innocua (nonpathogenic surrogate of Listeria monocytogenes) on cheese surface. The effects of inoculum level and cheese surface topography and the presence of clear polyethylene packaging were evaluated in a full factorial experimental design. The challenge microorganisms were grown to early stationary phase and subsequently diluted to reach initial inoculum levels of either 5 or 7 log cfu/slice. White Cheddar and process cheeses were cut into 2.5×5 cm slices, which were spot-inoculated with 100 µL of bacterial suspension. Inoculated cheese samples were exposed to PL doses of 1.02 to 12.29 J/cm(2). Recovered survivors were enumerated by standard plate counting or the most probable number technique, as appropriate. The PL treatments were performed in triplicate and data were analyzed using a general linear model. Listeria innocua was the least sensitive to PL treatment, with a maximum inactivation level of 3.37±0.2 log, followed by P. fluorescens, with a maximum inactivation of 3.74±0.8 log. Escherichia coli was the most sensitive to PL, with a maximum reduction of 5.41±0.1 log. All PL inactivation curves were nonlinear, and inactivation reached a plateau after 3 pulses (3.07 J/cm(2)). The PL treatments through UV-transparent packaging and without packaging consistently resulted in similar inactivation levels. This study demonstrates that PL has strong potential for decontamination of the cheese surface.

Inactivation of Escherichia coli in milk and concentrated milk using pulsed-light treatment.

Pulsed light (PL) treatment has been viewed as an alternative to thermal treatments for the inactivation of pathogenic and spoilage microorganisms in recent years. The objectives of this study were to quantify the effectiveness of PL on inactivating Escherichia coli in cow milk and to evaluate the effect of total solids and fat content on inactivation. Samples of reconstituted milk with variable total solids levels (9.8, 25, and 45%) and commercial cow milk with different fat contents (skim milk, 2% fat, and whole milk) were inoculated with nonpathogenic E. coli ATCC 25922 at a concentration of 10(7)cfu/mL. One milliliter of the inoculated sample was placed in a thin layer in a glass chamber and exposed to PL doses of up to 14.9 J/cm(2), both in static mode and turbulent mode. Survivors were quantified using standard plate counting. All experiments were performed in triplicate. Pulsed light treatment of the concentrated milks of 25 and 45% solids content resulted in reductions of less than 1 log, even in turbulent mode, whereas for the milk with 9.8% solids content, reduction levels of 2.5 log cfu were obtained after treatment with 8.4 J/cm(2) in turbulent mode. In the skim milk, a 3.4 log cfu reduction at 14.9 J/cm(2) was obtained and a plateau of the inactivation curve typical of PL treatment was not achieved. Under the same conditions, both 2% and whole milk attained inactivation levels greater than 2.5 log cfu. These data indicate that PL is effective for the inactivation of E. coli in milk, but has limited effectiveness for microbial inactivation in concentrated milk, due to the absorption of light by the milk solids and shielding of the bacteria in the concentrated substrates. Milk fat also diminishes the effectiveness of PL to some extent, due to light-scattering effects.

Using radiotracer techniques for coastal hydrodynamic model evaluation.

A three-dimensional (3D) water circulation and contaminant transport model of Manila Bay has been developed with the aim of better understanding the formation and movement of harmful algal blooms. Radiotracer techniques were used to evaluate the model by recording the dispersion of a tracer at depths of 2 and 15 m near the injection point. The selected tracer was 99mTc eluted from a molybdenum/technetium medical generator. The rationale for the choice of the tracer and the location of the injection is discussed. At 2 m the transport was dominated by the prevailing winds, and at 15 m by tidally induced currents. The development of the hydrodynamic model and its experimental evaluation were iterative processes. The experimental study confirmed the need for full 3D modelling of Manila Bay; quantified the impact of the prevailing wind field on contaminant dispersion near the injection point; and allowed the calculation of transverse dispersivity to guide the selection of parameter values used in the overall model.

Comparison of MRI pulse sequences in defining prostate volume after permanent implantation.

PURPOSE: To determine the relative value of three MRI pulse sequences in defining the prostate volume after permanent implantation. METHODS AND MATERIALS: A total of 45 patients who received a permanent 125I implant were studied. Two weeks after implantation, an axial CT scan (2 mm thickness) and T1-weighted, T1-weighted fat saturation, and T2-weighted axial MRI (3-mm) studies were obtained. The prostate volumes were compared with the initial ultrasound planning volumes, and subsequently the CT, T1-weighted, and T1-weighted fat saturation MRI volumes were compared with the T2-weighted volumes. Discrepancies in volume were evaluated by visual inspection of the registered axial images and the registration of axial volumes on the sagittal T2-weighted volumes. In a limited set of patients, pre- and postimplant CT and T2-weighted MRI studies were available for comparison to determine whether prostate volume changes after implant were dependent on the imaging modality. RESULTS: T1-weighted and T1-weighted fat saturation MRI and CT prostate volumes were consistently larger than the T2-weighted MRI prostate volumes, with a volume on average 1.33 (SD 0.24) times the T2-weighted volume. This discrepancy was due to the superiority of T2-weighted MRI for prostate definition at the following critical interfaces: membranous urethra, apex, and anterior base-bladder and posterior base-seminal vesicle interfaces. The differences in prostate definition in the anterior base region suggest that the commonly reported underdose may be due to overestimation of the prostate in this region by CT. The consistent difference in volumes suggests that the degree of swelling observed after implantation is in part a function of the imaging modality. In patients with pre- and postimplant CT and T2-weighted MRI images, swelling on the T2-weighted images was 1.1 times baseline and on CT was 1.3 times baseline, confirming the imaging modality dependence of prostate swelling. CONCLUSION: Postimplant T2-weighted MRI images provided superior prostate definition in all critical regions of the prostate compared with CT and the other MRI sequences tested. In addition to defining an optimal technique, these findings call two prior observations into question. Under dosing at the anterior base region may be overestimated because of poor definition of the prostate-bladder muscle interface. The swelling observed after implantation was lower on T2-weighted images as well, suggesting that a fraction of postimplant swelling is a function of the imaging modality. These findings have implications for preimplant planning and postimplant evaluation. As implant planning techniques become more conformal, and registration methods become more efficient, T2-weighted MRI after implantation will improve the accuracy of postimplant dosimetry.

Compensation of x-ray beam penumbra in conformal radiotherapy.

In radiotherapy, the gross tumor volume is surrounded by a clinically defined margin to allow for the presence of undetected malignant cells. Additional margins are added to accommodate positioning uncertainties and organ motion, creating a planning target volume, or PTV. Finally, a margin is included in the beam apertures surrounding the PTV to account for the dose fall-off at the beam edges (i.e., the "penumbra"). For higher energy beams and for low density tissues adjacent to the PTV, the beam aperture margin should be increased to account for the increased range of scattered photons and electrons. However, increased margins also increase the volume of normal tissue irradiated. In this work, the beam aperture margin is reduced by using filters and multileaf collimator (MLC) techniques to create compensating rinds of increased beam intensity. These compensation techniques were evaluated for 6 and 18 MV x rays by calculating penumbral widths as a function of the increased beam intensity in the rind, the rind width, and tissue density. Dose calculations were performed using a 3D superposition algorithm, which includes an extrafocal source model. Calculations were validated experimentally with film dosimetry. Results show the distance between the 95%-50% isodose lines is reduced from 11 mm to 4 mm for 6 MV x rays in the lung phantom, when the beam intensity is increased by 20% in a 10 mm wide rind. At 18 MV, this distance is reduced from 16 mm to 6 mm with a 20% increase in rind intensity, but a 15 mm wide rind is required. In all cases, penumbra compensation did not result in any appreciable increase in scatter dose outside the field boundaries. These results suggest that penumbra compensation is a practical means of controlling the beam aperture margin.

Monitor unit settings for intensity modulated beams delivered using a step-and-shoot approach.

Two linear accelerators have been commissioned for delivering IMRT treatments using a step-and-shoot approach. To assess beam startup stability for 6 and 18 MV x-ray beams, dose delivered per monitor unit (MU), beam flatness, and beam symmetry were measured as a function of the total number of MU delivered at a clinical dose rate of 400 MU per minute. Relative to a 100 MU exposure, the dose delivered per MU by both linear accelerators was found to be within +/-2% for exposures larger than 4 MU. Beam flatness and symmetry also met accepted quality assurance standards for a minimum exposure of 4 MU. We have found that the performance of the two machines under study is well suited to the delivery of step-and-shoot IMRT. A system of dose calculation has also been commissioned for applying head scatter corrections to fields as small as 1x1 cm2. The accuracy and precision of the relative output calculations in water was validated for small fields and fields offset from the axis of collimator rotation. For both 6 and 18 MV x-ray beams, the dose per MU calculated in a water phantom agrees with measured data to within 1% on average, with a maximum deviation of 2.5%. The largest output factor discrepancies were seen when the actual radiation field size deviated from the set field size. The measured output in water can vary by as much 16% for 1x1 cm2 fields, when the measured field size deviates from the set field size by 2 mm. For a 1 mm deviation, this discrepancy was reduced to 8%. Steps should be taken to ensure collimator precision is tightly controlled when using such small fields. If this is not possible, very small fields should not contribute to a significant portion of the treatment, or uncertainties in the collimator position may effect the accuracy of the dose delivered.

Reveal for Salmonella test system.

The Reveal for Salmonella (RSS) test system is a presumptive qualitative test that detects the presence of Salmonella organisms in foods within 21 h total testing time, allowing the user to release negative products 24 h earlier than when using other rapid test kits. Foods are enriched with a proprietary resuscitation medium called Revive and then selectively enriched with either Selenite Cystine or Rappaport-Vassiliadis selective media. The enriched culture is used to inoculate the RSS detection device, which initiates a lateral flow through a reagent zone containing anti-Salmonella antibodies conjugated to colloidal gold particles that capture antigens present in the culture. The antigen-antibody complex migrates farther and is captured by an additional anti-Salmonella antibody, causing the colloidal gold to precipitate and form a visual line, indicating a positive result. A procedural control line also will form regardless of the presence of Salmonella organisms to indicate the test is working properly. Existing AOAC Official Methods for Salmonella organisms require a 48 h enrichment before testing. Hence, a food product has to be held before release, adding extra cost to the company and the consumer. The RSS test system was evaluated by quantitative spiking studies. Although AOAC encourages inclusion of naturally contaminated foods, almost all microbiological AOAC validation studies have been performed with artificially contaminated foods for absolute control over the study. The RSS test system is designed to test many food types for Salmonella organisms and has a limit of detection of 5-10 colony-forming units (cfu)/25 g with a false-negative rate of < 1% and a false-positive rate of < 5.0%. It showed an 81% overall agreement with the traditional procedure of the U.S. Department of Agriculture's Food Safety Inspection Service.

Analysis of the DNA content distribution of micronuclei using flow sorting and fluorescent in situ hybridization with a centromeric DNA probe.

The DNA content distributions of micronuclei induced in mouse 3T3 cells by ionizing radiation and chemicals was measured by flow cytometry. For a quantitative understanding of these distributions, micronuclei with increasing DNA contents were sorted and analysed for the presence of centromeric signals using fluorescent in situ hybridization (FISH) with a mouse centromeric gamma satellite probe. Radiation-induced micronuclei were found to be produced mainly by chromosome fragments, whereas micronuclei induced by the tear gas chlorobenzylidene malonitrile (CS) were found to be produced mainly by whole chromatids. In contrast, micronuclei induced by vinblastine (VBL) were, according to the shape of their DNA content distributions, produced mainly by whole chromosomes and by combinations of two or more whole chromosomes. With increasing DNA content, micronuclei induced by ionizing radiation also contained one or more whole chromosomes, whereas micronuclei induced by CS or VBL were found to contain several whole chromatids or chromosomes respectively. Computerized random breakage of chromosomes and random combination of chromosome fragments, whole chromatids and whole chromosomes were used according to the FISH results to simulate the measured DNA content distributions of micronuclei. A good agreement was obtained between measured and simulated distributions of micronuclei as well as between results of the measured frequency of micronuclei showing centromeric signals as a function of their DNA content and those predicted by the simulations. These results demonstrate the usefulness of flow cytometry and sorting combined with the FISH technique and computer simulations for producing a more detailed analysis of mechanisms of micronucleus induction.

Fumonisins Veratox. A new rapid quantitative ELISA for determination of fumonisin in food and feed.

Polyclonal antibodies against fumonisin B1 were produced by immunizing sheep with fumonisin B1-keyhole limpet hemocyanin as an immunogen. A quantitative competitive enzyme-linked immunosorbent assay was developed whereby free fumonisins or sample extract containing fumonisins and enzyme-labelled fumonisin competed for binding to the solid phase-bound antibodies. The color intensity of wells, formed by substrate reaction with the enzyme, was inversely related to FB1 concentration. Detection limits for the assay were 0.1 ng/mL fumonisin B1 and concentrations of fumonisins B1, B2, and B3 required for 50% binding inhibition were 5.5, 23 and 18 ng/mL, respectively. For food and feed analyses, samples were extracted with 70% methanol and dilution of the extracts were used directly for ELISA. ELISA results were compared to HPLC analyses by a reference laboratory and the correlation (r value) between ELISA and HPLC was 0.967. The assay may be used to quantitate fumonisins in food and feed within 30 minutes.

Evaluation of the micronucleus test in vitro using Chinese hamster cells: results of four chemicals weakly positive in the in vivo micronucleus test.

A rapid and simple procedure for the micronucleus test (MNT) in vitro using Chinese hamster ovary (CHO) cells was established in our laboratory. The assay is intended to quickly screen chromosomal aberrations in vitro within the framework of industrial genotoxicity studies. To test the sensitivity of the assay in the experiments described here, four substances, classified as noncarcinogens but reported as weak inducers of micronuclei (MN) in bone-marrow cells of mice, were evaluated in the MNT in vitro. Of the four compounds, ascorbic acid, phenol, and 2,6-diaminotoluene proved to be genotoxic in the MNT in vitro. Titanium dioxide, which could not be dissolved in the culture medium, did not induce MN. The MNT in vitro proved to be quick and relatively simple and to yield highly reproducible results when testing the four chemicals.

Enzyme-linked immunosorbent assay for detection of zearalenone in corn, wheat, and pig feed: collaborative study.

A direct competitive enzyme-linked immunosorbent assay (ELISA) screening method for zearalenone in corn, wheat, and feed at 500 ng/g was evaluated by 23 collaborators (22 laboratories) in an international collaborative study. Eighteen samples of spiked or naturally contaminated corn, wheat, and pig feed were prepared by the sponsoring laboratory and sent for testing with complete test kits to participating collaborators in Canada, Italy, Sweden, The Netherlands, and the United States. Test samples were extracted with methanol-water solution (70 + 30) by shaking on a wrist-action shaker for 3 min. A portion of the extract was mixed with an equal volume of zearalenone-enzyme conjugate, and the mixture was incubated with zearalenone-specific monoclonal antibodies coated onto microtiter wells. All test samples were assayed in duplicate. One of 52 (2%) blanks was reported positive. Thirty-nine of the 52 (75%) samples that were spiked at 500 ng/g were reported as positive. Forty-nine of the 51 (96%) samples with concentrations at or above 1000 ng/g were reported as positive. The overall incidence of false negatives was 6.0% and the incidence of false positives was 22.7% by the ELISA method. Only one (3.4%) false negative was reported for samples containing > or = 800 ng/g. In the spectrophotometric method, 8 collaborators determined approximate levels of zearalenone in test samples from standard curves constructed from spiked extracts (0-3000 ng/g of each commodity tested). This method gave and overall incidence of false negatives of 5.7% and false positives of 17.8%. Average relative standard deviations, RSDr (repeatability) and RSDR (reproducibility), were 11.6 and 25.1% for spiked samples and 11.7 and 33.1% for naturally contaminated samples, respectively. Standard curves were constructed with each set of samples assayed. Comparison of absorbance values from these standard curves indicate the performance of reagents and antibody used in the assay. The ELISA method has been adopted first action by AOAC INTERNATIONAL as a screening method for zearalenone at > or = 800 ng/g in corn, wheat, and pig feed.

Diagnostic value of C-reactive protein in acute appendicitis.

Serum C-reactive protein was measured in 56 patients hospitalized with a suspected diagnosis of acute appendicitis. Based on these determinations, four groups of patients were defined: Group A = 26 patients with acute appendicitis who had a C-reactive protein level higher than 2.5 mg/dl. Group B = 4 patients with a C-reactive protein level lower than 2.5 mg/dl who, after surgery based on a presumed diagnosis of acute appendicitis, were found to have a normal appendix. Group C = 22 patients with nonspecific abdominal pain, 18 (72 percent) of whom had an elevated C-reactive protein level, although in only 4 (7.1 percent) were these levels higher than 2.5 percent mg/dl. Group D = 4 patients who had diseases other than acute appendicitis. It is concluded that an increase in C-reactive protein levels to more than 2.5 mg/dl is not a definite indicator of acute appendicitis. However, if the C-reactive protein level in blood drawn 12 hours after the onset of symptoms is less than 2.5 mg/dl, acute appendicitis can be excluded.

PCR cloning of a repeated DNA fragment from Chinese hamster ovary (CHO) cell X chromosomes and mapping by fluorescence in situ hybridization.

Hamster chromosome-specific DNA sequences were amplified by primer directed DNA amplification using mixed base oligonucleotides in an arbitrarily primed polymerase chain reaction (AP-PCR) protocol. The template DNA was comprised of approximately 3000 chinese hamster ovary cell (CHO) chromosomes enriched by flow sorting from a human x hamster hybrid cell line. Labeling of the PCR product pool and fluorescence in situ hybridization (FISH) demonstrated preferential binding to the distal long arm of the CHO X chromosome. The PCR products were cloned, labeled by PCR and hybridized to metaphase spreads. Clones containing highly reiterated DNA were identified by FISH and sequenced. Here, we present the sequence and chromosomal location of one of the repeat clones that maps close to the secondary constriction on the long arm of the CHO X chromosome, pCAT2066-24.