Lack of Detectable Allergenicity in Genetically Modified Maize Containing “Cry” Proteins as Compared to Native Maize Based on In Silico & In Vitro Analysis

BackgroundGenetically modified, (GM) crops with potential allergens must be evaluated for safety and endogenous IgE binding pattern compared to native variety, prior to market release.ObjectiveTo compare endogenous IgE binding proteins of three GM maize seeds containing Cry 1Ab,1Ac,1C transgenic proteins with non GM maize.MethodsAn integrated approach of in silico & in vitro methods was employed. Cry proteins were tested for presence of allergen sequence by FASTA in allergen databases. Biochemical assays for maize extracts were performed. Specific IgE (sIgE) and Immunoblot using food sensitized patients sera (n = 39) to non GM and GM maize antigens was performed.ResultsIn silico approaches, confirmed for non sequence similarity of stated transgenic proteins in allergen databases. An insignificant (p> 0.05) variation in protein content between GM and non GM maize was observed. Simulated Gastric Fluid (SGF) revealed reduced number of stable protein fractions in GM then non GM maize which might be due to shift of constituent protein expression. Specific IgE values from patients showed insignificant difference in non GM and GM maize extracts. Five maize sensitized cases, recognized same 7 protein fractions of 88-28 kD as IgE bindng in both GM and non-GM maize, signifying absence of variation. Four of the reported IgE binding proteins were also found to be stable by SGF.ConclusionCry proteins did not indicate any significant similarity of >35% in allergen databases. Immunoassays also did not identify appreciable differences in endogenous IgE binding in GM and non GM maize.     

Whey Protein/Polysaccharide-Stabilized Oil Powders for Topical Application—Release and Transdermal Delivery of Salicylic Acid from Oil Powders Compared to Redispersed Powders

Oil-in-water (o/w) emulsions are commonly converted into solid-like powders in order to improve their physical and chemical stabilities. The aim of this study was to investigate whether whey protein/polysaccharide-stabilized o/w emulsions could be converted into stable oil powders by means of freeze-drying. Moreover, during this study, the effects of pH and polymer type on release and trans(dermal) delivery of salicylic acid, a model drug, from these oil powders were investigated and compared to those of the respective template emulsions and redispersed oil powders. Physical characterization of the various formulations was performed, such as droplet size analysis and oil leakage, and relationships drawn with regards to release and trans(dermal) delivery. The experimental outcomes revealed that the oil powders could be redispersed in water without changing the release characteristics of salicylic acid. pH and polymer type affected the release of salicylic acid from the oil powders, template emulsions, and redispersed powders similarly. Contrary, the transdermal delivery from the oil powders and from their respective redispersed oil powders was differently affected by pH and polymer type. It was hypothesized that the release had been influenced by the electrostatic interactions between salicylic acid and emulsifiers, whereas the transdermal performance could have been determined by the particle or aggregate sizes of the formulations.KEY WORDS: carrageenan, chitosan, oil powders, release, salicylic acid, topical delivery, whey proteins     

Induction of “Low Dose” Tolerance to a Bacterial Somatic Antigen in Neonatal Mice

THE concept of “low dose” tolerance rests on the observation that specific immunological unresponsiveness can be induced by extremely small doses of antigen, sometimes in quantities too small to provoke antibody formation as such^1–3. The initial observations were made with soluble serum proteins, which are generally not considered “strong” antigens^4,5. Low dose tolerance can be induced also to the highly immunogenic protein antigen derived from Salmonella flagella, as assessed at the level of serum antibody^6,7. Furthermore, recent studies at the level of antibody forming cells indicate that immunological tolerance to complex antigens such as sheep erythrocytes and bacterial extracts, as well as to serum proteins or chemical haptens, can be induced in neonatal or adult animals^9–14. In this regard, relatively large doses of antigen are reportedly necessary to induce tolerance in adult rodents to a bacterial antigen such as Escherichia coli lipopolysaccharide (LPS). For example, 10–15 mg of the LPS is necessary to induce tolerance, as measured by an indirect haemolytic plaque assay with antigen coated sheep erythrocytes^15,16. Lower doses of LPS reportedly induce only immunity.     

Reusable Solid-Phase Supports for Oligonucleotide Synthesis Using Hydroquinone-O,O'-diacetic Acid (“Q-Linker”)

Abstract

Reusable solid-phase supports for large scale oligonucleotide synthesis have been prepared by converting amino derivatized supports into hydroxyl supports. Rapid nucleo side attachment, via a Q-linker arm, was automatically performed on the DNA synthesizer using HBTU and DMAP as the coupling reagents. All steps were suitable for automation and ～ 1.5 h was required to prepare the supports for reuse. Up to twelve consecutive syntheses of a 20-mer phosphorothioate were performed on a synthesis column.     

What is Meant by “95% of Species”? An Argument for the Inclusion of Rapid Tolerance Testing

It is increasingly common for water quality guidelines and risk assessments to consider the proportion of species at risk from a particular toxicant, based on the species sensitivity distribution (SSD) for that toxicant. There is a premise that the sensitivity data from species included in the SSD are sufficient to predict the effect on species for which there are no data. We discuss and review assumptions that follow this premise and find that for most toxicant SSDs include too few species, and that component species are biased toward particular taxonomic groups, common species and species from North America and western Europe. Consequently, protecting a given percentage, for example, 95%, of species in an SSD will likely protect more or less than 95% of species in nature, by an unknown amount. For the assumptions of SSDs to be better met, there is a need for tolerance data on more species, from more taxonomic and other groups, including rare species and those from widespread localities. In order to achieve this, we argue for the inclusion of rapid tests, which we define as toxicity tests designed to require less effort to conduct, relative to traditional tests, so sensitivity can be quickly and approximately determine in many species. Their use will allow for more species, more representative of natural communities, to be tested and therefore allow the construction of less biased SSDs and thus more accurate guidelines and assessments of risk.     

Response to “Critical Assessment of the Evidence for Striped Nanoparticles”

Stirling et al., (10.1371/journal.pone.0108482) presented an analysis on some of our publications on the formation of stripe-like domains on mixed-ligand coated gold nanoparticles. The authors shed doubts on some of our results however no valid argument is provided against what we have shown since our first publication: scanning tunneling microscopy (STM) images of striped nanoparticles show stripe-like domains that are independent of imaging parameters and in particular of imaging speed. We have consistently ruled out the presence of artifacts by comparing sets of images acquired at different tip speeds, finding invariance of the stipe-like domains. Stirling and co-workers incorrectly analyzed this key control, using a different microscope and imaging conditions that do not compare to ours. We show here data proving that our approach is rigorous. Furthermore, we never solely relied on image analysis to draw our conclusions; we have always used the chemical nature of the particles to assess the veracity of our images. Stirling et al. do not provide any justification for the spacing of the features that we find on nanoparticles: ~1 nm for mixed ligand particles and ~ 0.5 nm for homoligand particles. Hence our two central arguments remain unmodified: independence from imaging parameters and dependence on ligand shell chemical composition. The paper report observations on our STM images; none is a sufficient condition to prove that our images are artifacts. We thoroughly addressed issues related to STM artifacts throughout our microscopy work. Stirling et al. provide guidelines for what they consider good STM images of nanoparticles, such images are indeed present in our literature. They conclude that the evidences we provided to date are insufficient, this is a departure from one of the authors’ previous article which concluded that our images were composed of artifacts. Given that four independent laboratories have reproduced our measurements and that no scientifically rigorous argument is presented to invalidate our STM images, and also given that Stirling et al. do not contest the quality of our recent STM images, we re-affirm that specific binary mixture of ligands spontaneously form features in their ligand shell that we describe as stripe-like domains ~1 nm in width.