Polyethylene glycol (PEG)-coated nanopharmaceuticals can cause mild to severe hypersensitivity reactions (HSRs), which can occasionally be life threatening or even lethal. The phenomenon represents an unsolved immune barrier to the use of these drugs, yet its mechanism is poorly understood. This study showed that a single i.v. injection in pigs of a low dose of PEGylated liposomes (Doxebo) induced a massive rise of anti-PEG IgM in blood, peaking at days 7−9 and declining over 6 weeks. Bolus injections of PEG-liposomes during seroconversion resulted in anaphylactoid shock (pseudo-anaphylaxis) within 2−3 min, although similar treatments of naı ̈ve animals led to only mild hemodynamic disturbance. Parallel measurement of pulmonary arterial pressure (PAP) and sC5b-9 in blood, taken as measures of HSR and complement activation, respectively, showed a concordant rise of the two variables within 3 min and a decline within 15 min, suggesting a causal relationship between complement activation and pulmonary hypertension. We also observed a rapid decline of anti-PEG IgM in the blood within minutes, increased binding of PEGylated liposomes to IgM + B cells in the spleen of immunized animals compared to control, and increased C3 conversion by PEGylated liposomes in the serum of immunized pigs. These observations taken together suggest rapid binding of anti-PEG IgM to PEGylated liposomes, leading to complement activation via the classical pathway, entailing anaphylactoid shock and accelerated blood clearance of liposome−IgM complexes. These data suggest that complement activation plays a causal role in severe HSRs to PEGylated nanomedicines and that pigs can be used as a hazard identification model to assess the risk of HSRs in preclinical safety studies.

In winter wheat (Triticum spp.) and barley (Hordeum vulgare) varieties, long exposures to nonfreezing cold temperatures accelerate flowering time (vernalization) and improve freezing tolerance (cold acclimation). However, when plants initiate their reproductive development, freezing tolerance decreases, suggesting a connection between the two processes. To better understand this connection, we used two diploid wheat (Triticum monococcum) mutants, maintained vegetative phase (mvp), that carry deletions encompassing VRN-1, the major vernalization gene in temperate cereals. Homozygous mvp/mvp plants never flower, whereas plants carrying at least one functional VRN-1 copy (Mvp/-) exhibit normal flowering and high transcript levels of VRN-1 under long days. The Mvp/- plants showed reduced freezing tolerance and reduced transcript levels of several cold-induced C-REPEAT BINDING FACTOR transcription factors and COLD REGULATED genes (COR) relative to the mvp/mvp plants. Diploid wheat accessions with mutations in the VRN-1 promoter, resulting in high transcript levels under both long and short days, showed a significant down-regulation of COR14b under long days but not under short days. Taken together, these studies suggest that VRN-1 is required for the initiation of the regulatory cascade that down-regulates the cold acclimation pathway but that additional genes regulated by long days are required for the down-regulation of the COR genes. In addition, our results show that allelic variation in VRN-1 is sufficient to determine differences in freezing tolerance, suggesting that quantitative trait loci for freezing tolerance previously mapped on this chromosome region are likely a pleiotropic effect of VRN-1 rather than the effect of a separate closely linked locus (FROST RESISTANCE-1), as proposed in early freezing tolerance studies.

The enhancement of winter hardiness is one of the most important tasks facing breeders of winter cereals. For this reason, the examination of those regulatory genes involved in the cold acclimation processes is of central importance. The aim of the present work was the functional analysis of two wheat CBF transcription factors, namely TaCBF14 and TaCBF15, shown by previous experiments to play a role in the development of frost tolerance. These genes were isolated from winter wheat and then transformed into spring barley, after which the effect of the transgenes on low temperature stress tolerance was examined. Two different types of frost tests were applied; plants were hardened at low temperature before freezing, or plants were subjected to frost without a hardening period. The analysis showed that TaCBF14 and TaCBF15 transgenes improve the frost tolerance to such an extent that the transgenic lines were able to survive freezing temperatures several degrees lower than that which proved lethal for the wild-type spring barley. After freezing, lower ion leakage was measured in transgenic leaves, showing that these plants were less damaged by the frost. Additionally, a higher Fv/Fm parameter was determined, indicating that photosystem II worked more efficiently in the transgenics. Gene expression studies showed that HvCOR14b, HvDHN5, and HvDHN8 genes were up-regulated by TaCBF14 and TaCBF15. Beyond that, transgenic lines exhibited moderate retarded development, slower growth, and minor late flowering compared with the wild type, with enhanced transcript level of the gibberellin catabolic HvGA2ox5 gene.

Cold hardening is necessary to achieve the genetically determined maximum freezing tolerance and to reduce yield losses in winter cereals. The aim of the present study was to determine a set of genes with an important role in this process, by comparing of chromosome 5A substitution lines with different levels of freezing tolerance, since chromosome 5A is a major regulator of this trait. During 21 days of treatment at 2 degrees C, 303 genes were up-regulated, while 222 were down-regulated at most sampling points, and 156 at around half of them (out of the 10,297 unigenes studied). The freezing-tolerant substitution line exhibited 1.5 times as many differentially expressed genes than the sensitive one. The transcription of 78 genes (39 up-regulated) proved to be chromosome 5A-dependent. These genes encoded proteins involved in transcriptional regulation, defence processes and carbohydrate metabolism. Three of the chromosome 5A-related genes, coding for a cold-responsive, a Ca-binding and an embryo and meristem-related protein, were genetically mapped and characterized in further detail. The present experimental system was appropriate for the selection of chromosome 5A-related genes involved in short- and long-term cold acclimation in wheat. By modifying the expression of these genes it may be possible to improve freezing tolerance.

No abstract

Cold acclimation is necessary for winter wheat (Triticum aestivum L.) to achieve its genetically determined maximum freezing tolerance, and cold also fulfils the vernalisation requirement. Chromosome 5A is a major regulator of these traits. The aim of the present study was to discover whether changes in the half-cell redox potential of the glutathione/glutathione disulphide (GSH/GSSG) and ascorbate/dehydroascorbate (AA/DHA) couples induced by cold acclimation are related to freezing tolerance and vernalisation requirement in a specific genetic system including chromosome 5A substitution lines. The amounts of H₂O₂ and AA, and the AA/DHA ratio showed a rapid and transient increase in the crown of all genotypes during the first week of acclimation, followed by a gradual increase during the subsequent 2 weeks. The amount of GSH and its ratio compared to GSSG quickly decreased during the first day, while later these parameters showed a continuous slow increase. The H₂O₂, AA and GSH concentrations, AA/DHA and GSH/GSSG ratios and the half-cell reduction potential of the GSH/GSSG couple were correlated with the level of freezing tolerance after 22 days at 2 °C; hence these parameters may have an important role in the acclimation process. In contrast to H₂O₂ and the non-enzymatic antioxidants, the lipid peroxide concentration and activity of the four antioxidant enzymes exhibited a transient increase during the first week, with no significant difference between genotypes. None of the parameters studied showed any relationship with the vegetative/generative transition state monitored as apex morphology and vernalisation gene expression.

The effect of wheat chromosome 5A on free amino acid accumulation induced by osmotic stress was compared in chromosome 5A substitution lines with different freezing tolerance. Treatment with 15% polyethylene glycol (PEG) resulted in greater total free amino acid content even after 3 days compared to the controls. The ratio of amino acids belonging to various amino acid families differed after 3-week treatment in the control and PEG-treated plants only in the case of the freezing-sensitive substitution line. There was a transient increase with a maximum after 3 days in the amounts of several amino acids, after which their concentrations exhibited a more gradual increase. During the first days of osmotic stress the Glu, Gln, Asp, Asn, Thr, Ser, Leu, and His concentrations were greater in the tolerant substitution line than in the sensitive one, while the opposite relationship was observed at the end of the PEG treatment. The coordinated changes in the levels of individual amino acids indicated that they are involved in both the short- and long-term responses to osmotic stress. The alterations differed in the two chromosome 5A substitution lines, depending on the stress tolerance of the chromosome donor genotype.

Phytoremediation is a plant based, cost effective technology to detoxify or stabilize contaminated soils. Fast growing, high biomass, perennial plants may be used not only in phytoremediation but also in energy production. Szarvasi-1 energy grass (Elymus elongatus subsp. ponticus cv. Szarvasi-1), a good candidate for this combined application, was grown in nutrient solution in order to assess its Cd, Cu, Ni, Pb and Zn accumulation and tolerance. Its shoot metal accumulation showed the order Pb