Effect of Proximity to a Cattle Feedlot on Escherichia coli O157:H7 Contamination of Leafy Greens and Evaluation of the Potential for Airborne Transmission

The impact of proximity to a beef cattle feedlot on Escherichia coli O157:H7 contamination of leafy greens was examined. In each of 2 years, leafy greens were planted in nine plots located 60, 120, and 180 m from a cattle feedlot (3 plots at each distance). Leafy greens (270) and feedlot manure samples (100) were collected six different times from June to September in each year. Both E. coli O157:H7 and total E. coli bacteria were recovered from leafy greens at all plot distances. E. coli O157:H7 was recovered from 3.5% of leafy green samples per plot at 60 m, which was higher (P < 0.05) than the 1.8% of positive samples per plot at 180 m, indicating a decrease in contamination as distance from the feedlot was increased. Although E. coli O157:H7 was not recovered from air samples at any distance, total E. coli was recovered from air samples at the feedlot edge and all plot distances, indicating that airborne transport of the pathogen can occur. Results suggest that risk for airborne transport of E. coli O157:H7 from cattle production is increased when cattle pen surfaces are very dry and when this situation is combined with cattle management or cattle behaviors that generate airborne dust. Current leafy green field distance guidelines of 120 m (400 feet) may not be adequate to limit the transmission of E. coli O157:H7 to produce crops planted near concentrated animal feeding operations. Additional research is needed to determine safe set-back distances between cattle feedlots and crop production that will reduce fresh produce contamination.     

Determination of proton flux and conductance at pH 6.8 through single FO sectors from Escherichia coli

We have developed a mathematical model in concert with an assay that allows us to calculate proton (H+) flux and conductance through a single FO of the F1FO ATP synthase. Lipid vesicles reconstituted with just a few functional FO from Escherichia coli were loaded with 250 mM K+ and suspended in a low K+ solution. The pH of the weakly buffered external solution was recorded during sequential treatment with the potassium ionophore valinomycin, the protonophore carbonyl cyanide 3-chlorophenylhydrazone, and HCl. From these pH traces and separate determinations of vesicle size and lipid concentration we calculate the proton conductance through a single FO sector. This methodology is sensitive enough to detect small (15%) conductance changes. We find that wild-type FO has a proton flux of 3100 +/- 500 H+/s/FO at a transmembrane potential of 106 mV (25 degrees C and pH 6.8). This corresponds to a proton conductance of 4.4 fS.     

Differential detection of phospholipid fluidity, order, and spacing by fluorescence spectroscopy of bis-pyrene, prodan, nystatin, and merocyanine 540

The properties of liquid-ordered, solid-ordered, and liquid-disordered phases were investigated by steady-state fluorescence spectroscopy in liposomes composed of mixtures of dipalmitoylphosphatidylcholine and cholesterol (0-40 mol %) as a function of temperature (24-51 degrees C). The fluorescent probes used (bis-pyrene, nystatin, prodan, and merocyanine) were chosen because they differ in the location they occupy in the membrane and in the types of properties they sense. Comparison of phase diagrams with contour plots of the fluorescence data suggested that bis-pyrene is sensitive primarily to lipid order. In contrast, nystatin fluorescence intensity responded to changes in lipid fluidity. The shape of the prodan emission spectrum detected both liquid-solid and order-disorder transitions in the phase diagram. Merocyanine's behavior was more complex. First, it was more sensitive than any of the other probes to the membrane pretransition that occurs in the absence of cholesterol. Second, regardless of whether emission intensity, anisotropy, or spectral shape was observed, the probe appeared to distinguish two types of liquid-ordered phases, one with tightly packed lipids and one in which the apparent spacing among lipids was increased. The prodan data supported these results by displaying modest versions of these two observations. Together, the results identify eight regions within the phase diagram of distinguishable combinations of these physical properties. As an example of how this combined analysis can be applied to biological membranes, human erythrocytes were treated similarly. Temperature variation at constant cholesterol content revealed three of the eight combinations identified in our analysis of liposomes.     

Multiplexed expression and screening for recombinant protein production in mammalian cells

Background  

A variety of approaches to understanding protein structure and function require production of recombinant protein. Mammalian based expression systems have advantages over bacterial systems for certain classes of protein but can be slower and more laborious. Thus the availability of a simple system for production and rapid screening of constructs or conditions for mammalian expression would be of great benefit. To this end we have coupled an efficient recombinant protein production system based on transient transfection in HEK-293 EBNA1 (HEK-293E) suspension cells with a dot blot method allowing pre-screening of proteins expressed in cells in a high throughput manner.     

Synthesis and characterization of peptide grafted porous polymer microstructures

A scanning laser system has been used to generate three-dimensional trimethylolpropane trimethacrylate (TRIM) cross-linked poly(2-hydroxylethyl methacrylate) polymer microstructures through azo-bis(isobutyro)nitrile (AIBN) photopolymerization using a 20 x 0.5 NA microscope objective and 365 nm laser excitation. Macropores are observed to form without the use of porogens in regions of highest light flux. This is attributed to phase separation, which results from differences in monomer reactivity and miscibility. The microstructures were aminated and then protected with the photolabile protective group 6-nitroveratryloxycarbonyl (NVOC). This made it possible to selectively modify the microstructures with the same scanning laser system that was used to fabricate them, resulting in peptide grafted three-dimensional porous microstructures. On the basis of the absorbance of the dibenzofulvene-piperidine, these structures have an amine site density of approximately 0.1 nmol/feature. MALDI-TOF MS was used to characterize peptide photografted microstructures. N-Tris(2,4,6-trimethoxyphenyl)phosphonium (TMPP) labeling of the peptides greatly enhanced detection and allowed post-source decay sequencing of the peptides from the microstructures. The techniques described could be used to generate three-dimensional peptide grafted porous scaffolds for tissue engineering applications.     

Protein Dielectric Environment Modulates the Electron-Transfer Pathway in Photosynthetic Reaction Centers

The replacement of tyrosine by aspartic acid at position M210 in the photosynthetic reaction center of Rhodobacter sphaeroides results in the generation of a fast charge recombination pathway that is not observed in the wild-type. Apparently, the initially formed charge-separated state (cation of the special pair, P, and anion of the A-side bacteriopheophytin, H_A) can decay rapidly via recombination through the neighboring bacteriochlorophyll (B_A) soon after formation. The charge-separated state then relaxes over tens of picoseconds and recombination slows to the hundreds-of-picoseconds or nanosecond timescale. This dielectric relaxation results in a time-dependent blue shift of B_A^– absorption, which can be monitored using transient absorbance measurements. Protein dynamics also appear to modulate the electron transfer between H_A and the next electron carrier, Q_A (a ubiquinone). The kinetics of this reaction are complex in the mutant, requiring two kinetic terms, and the spectra associated with the two terms are distinct; a red shift of the H_A ground-state bleaching is observed between the shorter and longer H_A-to-Q_A electron-transfer phases. The kinetics appears to be pH-independent, suggesting a negligible contribution of static heterogeneity originating from protonation/deprotonation in the ground state. A dynamic model based on the energy levels of the two early charge-separated states, P⁺B_A^– and P⁺H_A^–, has been developed in which the energetics of these states is modulated by fast protein dielectric relaxations and this in turn alters both the kinetic complexity of the reaction and the reaction pathway.     

A new brace treatment similar for adolescent scoliosis and kyphosis based on restoration of thoracolumbar lordosis. Radiological and subjective clinical results after at least one year of treatment

Study design

A prospective treatment study with a new brace was conducted Objective. To evaluate radiological and subjective clinical results after one year conservative brace treatment with pressure onto lordosis at the thoracolumbar joint in children with scoliosis and kyphosis.

Summary of background data

Conservative brace treatment of adolescent scoliosis is not proven to be effective in terms of lasting correction. Conservative treatment in kyphotic deformities may lead to satisfactory correction. None of the brace or casting techniques is based on sagittal forces only applied at the thoracolumbar spine (TLI= thoracolumbar lordotic intervention). Previously we showed in patients with scoliosis after forced lordosis at the thoracolumbar spine a radiological instantaneous reduction in both coronal curves of double major scoliosis.

Methods

A consecutive series of 91 children with adolescent scoliosis and kyphosis were treated with a modified symmetric 30 degrees Boston brace to ensure only forced lordosis at the thoracolumbar spine. Scoliosis was defined with a Cobb angle of at least one of the curves [greater than or equal to] 25 degrees and kyphosis with or without a curve <25 degrees in the coronal plane. Standing radiographs were made i) at start, ii) in brace at beginning and iii) after one year treatment without brace.

Results

Before treatment start ??in brace?? radiographs showed a strong reduction of the Cobb angles in different curves in kyphosis and scoliosis groups (sagittal n = 5 all p < 0.001, pelvic obliquity p < 0.001). After one year of brace treatment in scoliosis and kyphosis group the measurements on radiographs made without brace revealed an improvement in 3 Cobb angles each.

Conclusion

Conservative treatment using thoracolumbar lordotic intervention in scoliotic and kyphotic deformities in adolescence demonstrates a marked improvement after one year also in clinical and postural criteria. An effect not obtained with current brace techniques.