Polyplex-induced cytosolic nuclease activation leads to differential transgene expression.

Cytosolic nucleases have been proposed to play an important role in limiting the effectiveness of polyplex-based gene delivery agents. In order to explore the effect of cell membrane disruption on nuclease activation, nuclease activity upon polyplex uptake and localization, and nuclease activity upon gene expression, we employed an oligonucleotide molecular beacon (MB). The MB was incorporated as an integral part of the polymer/DNA polyplex, and two-color flow cytometry experiments were performed to explore the relationship of MB cleavage with propidium iodide (PI) uptake, protein expression, and polyplex uptake. In addition, confocal fluorescence microcopy was performed to examine both polyplex and cleaved MB localization. The impact of cell membrane disruption was also probed using whole-cell patch clamp measurement of the plasma membrane's electrical conductance. Differential activation of cytosolic nuclease was observed with substantial activity for B-PEI and G5 PAMAM dendrimer (G5), less cleavage for jetPEI, and little activity for L-PEI. jetPEI and L-PEI exhibited substantially greater transgene expression, consistent with the lower amounts of MB oligonucleotide cleavage observed. Cytosolic nuclease activity, although dependent on the choice of polymer employed, was not related to the degree of cell plasma membrane disruption that occurred as measured by PI uptake or whole-cell patch clamp.

A chemometric method for correcting Fourier transform infrared spectra of biomaterials for interference from water in KBr discs.

Mid-infrared (mid-IR) analysis of solid biomaterials by the familiar KBr disc technique is all too often frustrated by water interference in the very important protein (amide) and carbohydrate (hydroxyl) regions of their spectra. A method was therefore devised that overcomes the difficulty and corrects IR spectra of solid biomaterials in KBr discs by mathematically eliminating the interference that arises from water molecules absorbed and bound in the KBr crystal lattice. The derivation of a linear system of chemometric equations that solves the water interference problem in a rigorous objective way is presented. Infrared spectra that result after correction by the method can be used reliably for quantitative analysis as well as structure identification. A major advantage is realized in quantitative analysis as the technique permits cryogenic pulverization of the biomaterial in KBr to prevent change in chemical structure and minimize the particle size to closely approach the solid solution condition required by the Beer-Lambert law. Extensive pulverization, which produces large water absorption bands that overlap and obscure the amide and hydroxyl regions of interest, is no longer problematic. The method is illustrated by removing strong water interference to extract the spectra of corn starch and gluten in pressed KBr discs. Results of the new method are compared with attenuated total reflection (ATR) spectra of gluten corrected using the conventional advanced ATR correction algorithm.

Starch graft poly(methyl acrylate) loose-fill foam: preparation, properties and degradation.

Starch graft poly(methyl acrylate) (S-g-PMA) was prepared by ceric ion initiation of methyl acrylate in an aqueous corn starch slurry (prime starch) which maximized the accessibility of the starch for graft polymerization. A new ceric ion reaction sequence was established as starch-initiator-methyl acrylate followed by addition of a small amount of ceric ion solution when the graft polymerization was almost complete to quench the reaction. As a result of this improved procedure, no unreacted methyl acrylate monomer remained, and thus, essentially no ungrafted poly(methyl acrylate) homopolymer was formed in the final grafted product. Quantities of the high purity S-g-PMA so prepared in pilot scale were converted to resin pellets and loose-fill foam by single screw and twin screw extrusion. The use of prime starch significantly improved the physical properties of the final loose-fill foam, in comparison to foam produced from regular dry corn starch. The S-g-PMA loose-fill foam had compressive strength and resiliency comparable to expanded polystyrene but higher bulk density. The S-g-PMA loose-fill foam also had better moisture and water resistance than other competitive starch-based materials. Studies indicated that the starch portion in S-g-PMA loose-fill foam biodegraded rapidly, whereas poly(methyl acrylate) remained relatively stable under natural environmental conditions.

Analysis of ethanol-glucose mixtures by two microbial sensors: application of chemometrics and artificial neural networks for data processing.

Although biosensors based on whole microbial cells have many advantages in terms of convenience, cost and durability, a major limitation of these sensors is often their inability to distinguish between different substrates of interest. This paper demonstrates that it is possible to use sensors entirely based upon whole microbial cells to selectively measure ethanol and glucose in mixtures. Amperometric sensors were constructed using immobilized cells of either Gluconobacter oxydans or Pichia methanolica. The bacterial cells of G. oxydans were sensitive to both substrates, while the yeast cells of P. methanolica oxidized only ethanol. Using chemometric principles of polynomial approximation, data from both of these sensors were processed to provide accurate estimates of glucose and ethanol over a concentration range of 1.0-8.0 mM (coefficients of determination, R(2)=0.99 for ethanol and 0.98 for glucose). When data were processed using an artificial neural network, glucose and ethanol were accurately estimated over a range of 1.0-10.0 mM (R(2)=0.99 for both substrates). The described methodology extends the sphere of utility for microbial sensors.

Toxigenic strains of Fusarium moniliforme and Fusarium proliferatum isolated from dairy cattle feed produce fumonisins, moniliformin and a new C21H38N2O6 metabolite phytotoxic to Lemna minor L.

Corn samples suspected of causing refusal-to-eat syndrome in dairy cattle were examined mycologically. Fusarium moniliforme (14 isolates) and F. proliferatum (12 isolates) were the predominant fungi present. These isolates were tested for mycotoxin production on rice at 25 degrees C. Each strain of F. moniliforme produced fumonisin B1 (FB1: 378-15,600 ppm) and fumonisin B2 (FB2: 2-1050 ppm). Each strain of F. proliferatum produced moniliformin (45-16,000 ppm), FB1 (27-6140 ppm), and FB2 (5-1550 ppm). In addition, a new Fusarium metabolite of molecular composition C21H38N2O6 was produced by 10 of the F. moniliforme isolates and 7 of the F. proliferatum isolates. The metabolite's 1H- and 13C-NMR, HRFAB/MS and IR spectra indicate an alpha amino acid. It is toxic to Lemna minor L. duckweed (LD50 100 micrograms/mL).

Transient infrared spectroscopy for detection of toxigenic fungi in corn: potential for on-line evaluation.

An urgent need for rapid sensors to detect contamination of food grains by toxigenic fungi such as Aspergillus flavus prompted research and development of Fourier transform infrared photoacoustic spectroscopy (FTIR-PAS) as a highly sensitive probe for fungi growing on the surfaces of individual corn kernels. However, the photoacoustic technique has limited potential for screening bulk corn because currently available photoacoustic detectors can accommodate only a single intact kernel at a time. Transient infrared spectroscopy (TIRS), on the other hand, is a promising new technique that can acquire analytically useful infrared spectra from a moving mass of solid materials. Therefore, the potential of TIRS for on-line, noncontact detection of A. flavus contamination in a moving bed of corn kernels was explored. Early test results based on visual inspection of TIRS spectral differences predict an 85% or 95% success rate in distinguishing healthy corn from grain infected with A. flavus. Four unique infrared spectral features which identified infected corn in FTIR-PAS were also found to be diagnostic in TIRS. Although the technology is still in its infancy, the preliminary results indicate that TIRS is a potentially effective screening method for bulk quantities of corn grain.

Degradation of starch-poly(beta-hydroxybutyrate-co-beta-hydroxyvalerate) bioplastic in tropical coastal waters.

Extruded bioplastic was prepared from cornstarch or poly(beta-hydroxybutyrate-co-beta-hydroxyvalerate) (PHBV) or blends of cornstarch and PHBV. The blended formulations contained 30 or 50% starch in the presence or absence of polyethylene oxide (PEO), which enhances adherence of starch granules to PHBV. Degradation of these formulations was monitored for 1 year at four stations in coastal water southwest of Puerto Rico. Two stations were within a mangrove stand. The other two were offshore; one of these stations was on a shallow shoulder of a reef, and the other was at a location in deeper water. Microbial enumeration at the four stations revealed considerable flux in the populations over the course of the year. However, in general, the overall population densities were 1 order of magnitude less at the deeper-water station than at the other stations. Starch degraders were 10- to 50-fold more prevalent than PHBV degraders at all of the stations. Accordingly, degradation of the bioplastic, as determined by weight loss and deterioration of tensile properties, correlated with the amount of starch present (100% starch >50% starch > 30% starch > 100% PHBV). Incorporation of PEO into blends slightly retarded the rate of degradation. The rate of loss of starch from the 100% starch samples was about 2%/day, while the rate of loss of PHBV from the 100% PHBV samples was about 0.1%/day. Biphasic weight loss was observed for the starch-PHBV blends at all of the stations. A predictive mathematical model for loss of individual polymers from a 30% starch-70% PHBV formulation was developed and experimentally validated. The model showed that PHBV degradation was delayed 50 days until more than 80% of the starch was consumed and predicted that starch and PHBV in the blend had half-lives of 19 and 158 days, respectively. Consistent with the relatively low microbial populations, bioplastic degradation at the deeper-water station exhibited an initial lag period, after which degradation rates comparable to the degradation rates at the other stations were observed. Presumably, significant biodegradation occurred only after colonization of the plastic, a parameter that was dependent on the resident microbial populations. Therefore, it can be reasonably inferred that extended degradation lags would occur in open ocean water where microbes are sparse.

Detection of ethanol in a two-component glucose/ethanol mixture using a nonselective microbial sensor and a glucose enzyme electrode.

Chemometric theory was applied to a microbial sensor for determinations of ethanol in the presence of glucose. Microbial sensors, consisting of Gluconobacter oxydans cells immobilized on Clark-type amperometric oxygen electrodes, exhibited good sensitivity but low selectivity toward ethanol and glucose. An Eksan-G commercial glucose analyzer was used as a second sensor for multivariate calibration and analyses. Microbial sensors exhibited nearly complete additivity for total glucose plus ethanol concentrations from 0.0 to 0.6 mM. Within this linear range, chemometric analyses provided estimates of ethanol concentration with measurement errors of less than 8%. Multivariate calibration thus is a promising approach to enhance the usefulness of microbial sensors.

Neural network pattern recognition of photoacoustic FTIR spectra and knowledge-based techniques for detection of mycotoxigenic fungi in food grains.

Fourier transform infrared photoacoustic spectroscopy (FTIR-PAS), a highly sensitive probe of the surfaces of solid substrates, is used to detect toxigenic fungal contamination in corn. Kernels of corn infected with mycotoxigenic fungi, such as Aspergillus flavus, display FTIR-PAS spectra that differ significantly form spectra of uninfected kernels. Photoacoustic infrared spectral features were identified, and an artificial neural network was trained to distinguish contaminated form uncontaminated corn by pattern recognition. Work is in progress to integrate epidemiological information about cereal crop fungal disease into the pattern recognition program to produce a more knowledge-based, and hence more reliable and specific, technique. A model of a hierarchically organized expert system is proposed, using epidemiological factors such as corn variety, plant stress and susceptibility to infection, geographic location, weather, insect vectors, and handling and storage conditions, in addition to the analytical data, to predict Al. flavus and other kinds of toxigenic fungal contamination that might be present in food grains.

Identification of Fourier transform infrared photoacoustic spectral features for detection of Aspergillus flavus infection in corn.

Aspergillus flavus and other pathogenic fungi display typical infrared spectra which differ significantly from spectra of substrate materials such as corn. On this basis, specific spectral features have been identified which permit detection of fungal infection on the surface of corn kernels by photoacoustic infrared spectroscopy. In a blind study, ten corn kernels showing bright greenish yellow fluorescence (BGYF) in the germ or endosperm and ten BGYF-negative kernels were correctly classified as infected or not infected by Fourier transform infrared photoacoustic spectroscopy. Earlier studies have shown that BGYF-positive kernels contain the bulk of the aflatoxin contaminating grain at harvest. Ten major spectral features, identified by visual inspection of the photoacoustic spectra of A. flavus mycelium grown in culture versus uninfected corn, were interpreted and assigned by theoretical comparisons of the relative chemical compositions of fungi and corn. The spectral features can be built into either empirical or knowledge-based computer models (expert systems) for automatic infrared detection and segregation of grains or kernels containing aflatoxin from the food and feed supply.

Multivariate FTIR analysis of substrates for protein, polysaccharide, lipid and microbe content: potential for solid-state fermentations.

Components of fermentation processes such as protein, polysaccharide and lipid, as well as microbes, such as fungi grown on solid substrates, are difficult to measure in situ. The potential of Fourier Transform Infrared (FTIR) analysis of solid-state fermentations from mid-infrared absorption spectra has been investigated. The problem under consideration was to build a calibration model containing no irrelevant information to enable a multivariate mathematical approach for prediction of component concentrations. Methods for solid sample preparation and preprocessing of FTIR data were developed to assure Beer-Lambert law compliance and produce a well-conditioned multivariate system. The model was tested using composite samples of zein protein, corn starch and azolectin lipid, and corn samples containing known levels of fungal contamination. Preliminary concentration estimates were remarkably close to the correct values, with less than 5% standard error of prediction for all components measured.

Some chemical and physical properties of extracellular polysaccharides produced by Butyrivibrio fibrisolvens strains.

Most strains of Butyrivibrio fibrisolvens are known to produce extracellular polysaccharides (EPs). However, the rheological and functional properties of these EPs have not been determined. Initially, 26 strains of Butyrivibrio were screened for EP yield and apparent viscosities of cell-free supernatants. Yields ranged from less than 1.0 to 16.3 mg per 100 mg of glucose added to the culture. Viscosities ranged from 0.71 to 5.44 mPa.s. Five strains (CF2d, CF3, CF3a, CE51, and H10b) were chosen for further screening. The apparent viscosity of the EP from each of these strains decreased by only 50 to 60% when the shear rate was increased from 20 to 1,000 s-1. Strain CE51 produced the EP having the highest solution viscosity. A detailed comparison of shear dependency of the EP from strain CF3 with xanthan gum showed that this EP was less shear sensitive than xanthan gum and, at a shear rate of 1,000 s-1, more viscous. EPs from strains CF3 and H10b were soluble over a wide range of pH (1 to 13) in 80% (vol/vol) ethanol-water or in 1% (wt/vol) salt solutions. The pH of 1% EP solutions was between 4.5 and 5.5. Addition of acid increased solution viscosities, whereas addition of base decreased viscosity. EPs from strains CF3, CE51, and H10b displayed qualitatively similar infrared spectra. Calcium and sodium were the most abundant minerals in the three EPs. The amounts of magnesium, calcium, and iron varied considerably among the EPs, but the potassium contents remained relatively constant.

Measurement of protein biomass by Fourier transform infrared-photoacoustic spectroscopy.

A relatively new analytical technique, Fourier transform infrared-photoacoustic spectroscopy (FTIR-PAS), provides spectra of bacteria, fungi, and other microorganisms in solid states not suitable for conventional absorption spectroscopy. In this paper the feasibility of quantitative measurement of protein biomass on solid substrates by FTIR-PAS is examined and discussed. By measuring photoacoustic absorption bands from amide groups in the protein of microorganisms, the increase in biomass that occurs during growth was monitored directly and accurately. Incorporation of polyacrylonitrile into the sample as an internal standard was shown to be a convenient method for improving both the reliability and the range of detection by photoacoustic spectroscopy. Results of FTIR-PAS measurements of known quantities of microbial mass in simulated growth experiments suggest that the technique may be especially suitable for assays of microorganisms used in solid-state biosyntheses of drugs, hormones, and other biological agents.