Bradley J. Hernlem

Dihaploid stocks of switchgrass isolated by a screening approach.

Manipulation of ploidy in switchgrass has potential to accelerate inbred production and to provide insight about genome structure through either sequencing or cytogenetic approaches. We have identified two dihaploid individuals isolated from among the progeny of a controlled cross between two individuals of the cultivars Alamo and Kanlow. The dihaploid lines were initially distinguished from the parental lines by their reduced heterozygosity and were subsequently confirmed through estimation of C values by flow cytometry and chromosome counts of metaphase root tip squash preparations. These plants are functionally sterile, with floral bracts that remain closed and inviable pollen. They can be easily distinguished from tetraploid individuals by their reduced stature, smaller epidermal cell size, and lower number of chloroplasts per guard cell. Aberrant meiosis in these individuals is evidenced by a lack of regular pairing at diakinesis and metaphase I and suggests that the non-homologous genomes are distinct from one another. The reduced genome size of these dihaploids will facilitate basic genome studies and genetic analyses that are impossible or problematic in polyploid accessions.

Development of monoclonal antibodies specific for Ricinus agglutinins

Abstract Ricin is a highly toxic, dichain ribosome-inactivating protein present in the seeds of Ricinus communis (castor), grown principally as a source of high quality industrial lubricant and as an ornamental. Because of its presence in industrial byproducts and its documented use for intentional poisoning, there is a need for analytical methodology to quantify ricin in both castor extracts and food matrices. We developed a panel of monoclonal antibodies to ricin, with most having strong cross-reactivity with RCA-1, a homologous but less toxic castor agglutinin. Some of the IgM-producing hybridomas appeared to produce a second, IgG isotype and were further analysed by fluorescence-activated cell sorting. The antibodies were effective in various ELISA formats, many with IC50s in the range of 0.1–10 ng/mL and minimal matrix effects in skim milk. Assay specificity can be adjusted for analytical needs by varying the combination of antibodies in a sandwich ELISA format.

Low Levels of Aflatoxin B1, Ricin, and Milk Enhance Recombinant Protein Production in Mammalian Cells

Gene expression in transduced mammalian cells correlates with virus titer, but high doses of vector for gene therapy leads to toxicity in humans and in animals. Changing the optimal tissue culture medium by adding low levels of environmental stressors, such as 1 µM of the fungal toxin aflatoxin B1 (AFB1), 1 ng of the castor bean protein toxin ricin, or 1% reconstituted milk, enhances transcription and increases production of proteins in transduced mammalian cells as demonstrated by production of the following three recombinant proteins: firefly luciferase, β-galactosidase, and green fluorescent protein (GFP). Higher concentrations of the stress-producing substances damage the cells beyond recovery, resulting in inhibited gene expression and cell death. We also evaluated the effect of the stressor substances on the enhanced infectivity of virus. The presented findings extend methods for large-scale transient recombinant protein production in mammalian cells and suggest that it may be possible to reduce the cytotoxicity of the adenovirus by reducing the virus titer without adversely affecting gene expression levels.

Non-Linear Relationships between Aflatoxin B1 Levels and the Biological Response of Monkey Kidney Vero Cells

Aflatoxin-producing fungi contaminate food and feed during pre-harvest, storage and processing periods. Once consumed, aflatoxins (AFs) accumulate in tissues, causing illnesses in animals and humans. Most human exposure to AF seems to be a result of consumption of contaminated plant and animal products. The policy of blending and dilution of grain containing higher levels of aflatoxins with uncontaminated grains for use in animal feed implicitly assumes that the deleterious effects of low levels of the toxins are linearly correlated to concentration. This assumption may not be justified, since it involves extrapolation of these nontoxic levels in feed, which are not of further concern. To develop a better understanding of the significance of low dose effects, in the present study, we developed quantitative methods for the detection of biologically active aflatoxin B1 (AFB1) in Vero cells by two independent assays: the green fluorescent protein (GFP) assay, as a measure of protein synthesis by the cells, and the microculture tetrazolium (MTT) assay, as a measure of cell viability. The results demonstrate a non-linear dose-response relationship at the cellular level. AFB1 at low concentrations has an opposite biological effect to higher doses that inhibit protein synthesis. Additional studies showed that heat does not affect the stability of AFB1 in milk and that the Vero cell model can be used to determine the presence of bioactive AFB1 in spiked beef, lamb and turkey meat. The implication of the results for the cumulative effects of low amounts of AFB1 in numerous foods is discussed.

Microwave Heating Inactivates Shiga Toxin (Stx2) in Reconstituted Fat-Free Milk and Adversely Affects the Nutritional Value of Cell Culture Medium

Microwave exposure is a convenient and widely used method for defrosting, heating, and cooking numerous foods. Microwave cooking is also reported to kill pathogenic microorganisms that often contaminate food. In this study, we tested whether microwaves would inactivate the toxicity of Shiga toxin 2 (Stx2) added to 5% reconstituted fat-free milk administered to monkey kidney Vero cells. Heating of milk spiked with Stx2 in a microwave oven using a 10% duty cycle (cycle period of 30 s) for a total of 165 kJ energy or thermal heating (pasteurization), widely used to kill pathogenic bacteria, did not destroy the biological effect of the toxin in the Vero cells. However, conventional heating of milk to 95 °C for 5 min or at an increased microwave energy of 198 kJ reduced the Stx2 activity. Gel electrophoresis showed that exposure of the protein toxin to high-energy microwaves resulted in the degradation of its original structure. In addition, two independent assays showed that exposure of the cell culture medium to microwave energy of 198 kJ completely destroyed the nutritional value of the culture medium used to grow the Vero cells, possibly by damaging susceptible essential nutrients present in the medium. These observations suggest that microwave heating has the potential to destroy the Shiga toxin in liquid food.

TNF as Biomarker for Rapid Quantification of Active Staphylococcus Enterotoxin A in Food

Staphylococcus aureus is a major bacterial pathogen which causes clinical infections and food poisoning. This bacterium produces a group of twenty-one enterotoxins (SEs). These enterotoxins have two separate but related biological activities. They cause gastroenteritis and function as superantigens that activate large numbers of T cells. The current method for detection of enterotoxins activity is an in vivo monkey or kitten bioassay; however, this method is not practical to test on a large number of samples. Several immunological assays have been developed however, but these assays cannot distinguish between active toxin which causes food poisoning and inactive toxin, which can bind antibody, but shows no toxicity. The current study demonstrates that short term ex vivo exposure of primary naïve CD4+ T-cells or splenocytes to SEA induces differential expression and secretion of tumor necrosis factor (TNF) protein. We used immunomagnetic beads coated with anti-SEA antibody to specifically isolate SEA from food. After the eluted toxin was added to the cells SEA biological activity was measured by quantifying TNF protein expression or secretion.

Plant Compounds Enhance the Assay Sensitivity for Detection of Active Bacillus cereus Toxin

Bacillus cereus is an important food pathogen, producing emetic and diarrheal syndromes, the latter mediated by enterotoxins. The ability to sensitively trace and identify this active toxin is important for food safety. This study evaluated a nonradioactive, sensitive, in vitro cell-based assay, based on B. cereus toxin inhibition of green fluorescent protein (GFP) synthesis in transduced monkey kidney Vero cells, combined with plant extracts or plant compounds that reduce viable count of B. cereus in food. The assay exhibited a dose dependent GFP inhibition response with ~25% inhibition at 50 ng/mL toxin evaluated in culture media or soy milk, rice milk or infant formula, products associated with food poisonings outbreak. The plant extracts of green tea or bitter almond and the plant compounds epicatechin or carvacrol were found to amplify the assay response to ~90% inhibition at the 50 ng/mL toxin concentration greatly increasing the sensitivity of this assay. Additional studies showed that the test formulations also inhibited the growth of the B. cereus bacteria, likely through cell membrane disruption. The results suggest that the improved highly sensitive assay for the toxin and the rapid inactivation of the pathogen producing the toxin have the potential to enhance food safety.

Sensitive, Rapid, Quantitative and in Vitro Method for the Detection of Biologically Active Staphylococcal Enterotoxin Type E.

Staphylococcus aureus is a major bacterial cause of clinical infections and foodborne illnesses through its production of a group of enterotoxins (SEs) which cause gastroenteritis and also function as superantigens to massively activate T cells. In the present study, we tested Staphylococcal enterotoxin type E (SEE), which was detected in 17 of the 38 suspected staphylococcal food poisoning incidents in a British study and was the causative agent in outbreaks in France, UK and USA. The current method for detection of enterotoxin activity is an in vivo monkey or kitten bioassay; however, this expensive procedure has low sensitivity and poor reproducibility, requires many animals, is impractical to test on a large number of samples, and raises ethical concerns with regard to the use of experimental animals. The purpose of this study is to develop rapid sensitive and quantitative bioassays for detection of active SEE. We apply a genetically engineered T cell-line expressing the luciferase reporter gene under the regulation of nuclear factor of activated T-cells response element (NFAT-RE), combined with a Raji B-cell line that presents the SEE-MHC (major histocompatibility complex) class II to the engineered T cell line. Exposure of the above mixed culture to SEE induces differential expression of the luciferase gene and bioluminescence is read out in a dose dependent manner over a 6-log range. The limit of detection of biologically active SEE is 1 fg/mL which is 109 times more sensitive than the monkey and kitten bioassay.

Rapid Cell-Based Assay for Detection and Quantification of Active Staphylococcal Enterotoxin Type D

Food poisoning by Staphylococcus aureus is a result of ingestion of Staphylococcal enterotoxins (SEs) produced by this bacterium and is a major source of foodborne illness. Staphylococcal enterotoxin D (SED) is one of the predominant enterotoxins recovered in Staphylococcal food poisoning incidences, including a recent outbreak in Guam affecting 300 children. Current immunology methods for SED detection cannot distinguish between the biologically active form of the toxin, which poses a threat, from the inactive form, which poses no threat. In vivo bioassays that measure emetic activity in kitten and monkeys have been used, but these methods rely upon expensive procedures using live animals and raising ethical concerns. A rapid (5 h) quantitative bioluminescence assay, using a genetically engineered T-cell Jurkat cell line expressing luciferase under regulation of nuclear factor of activated T cells response elements, in combination with the lymphoblastoid B-cell line Raji for antigen presentation, was developed. In this assay, the detection limit of biologically active SED is 100 ng/mL, which is 10 times more sensitive than the splenocyte proliferation assay, and 105 times more sensitive than monkey or kitten bioassay. Pasteurization or repeated freeze-thaw cycles had no effect on SED activity, but reduction in SED activity was shown with heat treatment at 100°C for 5 min. It was also shown that milk exhibits a protective effect on SED. This bioluminescence assay may also be used to rapidly evaluate antibodies to SED for potential therapeutic application as a measurement of neutralizing biological effects of SED.

Generation of Octaploid Switchgrass by Seedling Treatment with Mitotic Inhibitors

Switchgrass (Panicum virgatum L.) exists as multiple cytotypes with octaploid (8x) and tetraploid (4x) populations occupying distinct, overlapping ranges. These cytotypes tend to show differences in adaptation, yield potential, and other characters, but the specific result of whole-genome duplication is not clear and 8x and 4x switchgrass populations are reproductively isolated with limited genetic exchange. To create new opportunities for population improvement and to study the effects of whole genome duplication on switchgrass, seedling treatment of the tetraploid cultivar Liberty with microtubule inhibitors was used to generate an octaploid population. Resulting octaploids, tetraploids, and cytochimeras were resolved by intercrossing octaploid sectors to produce a population of 19 octaploid families. Fertility of octaploid sectors was significantly reduced relative to tetraploid sectors and caryopsis size significantly increased. Cell size was significantly increased which resulted in quantitative changes to leaf anatomy. During seedling and early vegetative growth stages, no differences in vigor or tillering ability were seen. This technique resulted in efficient genome doubling and was simple to perform. However, aneuploids were also identified with both larger and smaller than expected genome sizes.