In vitro evaluation of archaeosome vehicles for transdermal vaccine delivery

Abstract

Archaeosomes composed of archaeal total polar lipids (TPL) or semi-synthetic analog vesicles have been used as vaccine adjuvants and delivery systems in animal models for many years. Typically administered by intramuscular or subcutaneous injections, archaeosomes can induce robust, long-lasting humoral and cell-mediated immune responses against entrapped antigens and provide protection in murine models of infectious disease and cancer. Herein, we evaluated various archaeosomes for transdermal delivery, since this route may help eliminate needle-stick injuries and needle re-use, and therefore increase patient compliance. Archaeosomes composed of TPL from different archaea (Halobacterium salinarum, Methanobrevibacter smithii, Haloferax volcanii) and various semi-synthetic glycolipid combinations were evaluated for their ability to diffuse across the skin barrier using an ex vivo pig skin model and the results were compared to conventional synthetic ester liposomes. Physicochemical characteristics were determined for selected formulations including vesicle size, size distribution, zeta potential, fluidity, antigen (ovalbumin) incorporation efficiency and release. Archaeosomes, in particular those composed of M. smithii TPL or the synthetic glycolipid sulfated S-lactosylarchaeol (SLA) mixed with uncharged glycolipid lactosyl archaeol (LA), appeared to be effective carriers for ovalbumin, achieving much better antigen distribution and vesicle accumulation in the skin epidermis than conventional liposomes. The enhanced skin permeation of archaeosomes may be attributed to their chemical structure and physicochemical properties such as particle size, surface charge, stability, and fluidity of their lipid bilayer.     

RNAi-Mediated Silencing of the Flavanone 3-Hydroxylase Gene and Its Effect on Flavonoid Biosynthesis in Strawberry Fruit

Anthocyanin biosynthesis requires the activities of several enzymes in vivo. Flavanone 3-hydroxylase (F3H) converts flavanone into dihydroflavanol at an early step in the anthocyanin biosynthesis pathway. In this study we constructed an RNAi gene-silencing vector that encodes a hairpin F3H RNA. Agrobacterium strain GV3101 harboring the F3H RNAi vector was injected into strawberry fruits which were still attached to the plants 14 days after pollination. The phenotype was observed 10 days postinjection, and fruits were tested by RT-PCR and northern blot assays. The results showed that the F3H gene was downregulated by approximately 70 % in the agroinfiltrated fruits compared with the control. HPLC–MS analysis showed that anthocyanin content was greatly reduced, flavonol was also decreased, and the levels of p-coumaroyl glucoside and p-coumaroyl-1-acetate were markedly increased. We conclude that the precursors were shunted to the phenylpropanoid pathway, and that F3H is one of the key enzymes required for the biosynthesis of flavonoids in strawberry fruit. According to our results, reducing gene function via RNA interference is a rapid, simple, and effective way to identify gene function in strawberry fruit.     

MAIGO2 is involved in abscisic acid‐mediated response to abiotic stresses and Golgi‐to‐ER retrograde transport

The central role of multisubunit tethering complexes in intracellular trafficking has been established in yeast and mammalian systems. However, little is known about their roles in the stress responses and the early secretory pathway in Arabidopsis. In this study, Maigo2 (MAG2), which is equivalent to the yeast Tip20p and mammalian Rad50‐interacting protein, is found to be required for the responses to salt stress, osmotic stress and abscisic acid in seed germination and vegetative growth, and MAG2‐like (MAG2L) is partially redundant with MAG2 in response to environmental stresses. MAG2 strongly interacts with the central region of ZW10, and both proteins are important as plant endoplasmic reticulum (ER)‐stress regulators. ER morphology and vacuolar protein trafficking are unaffected in the mag2, mag2l and zw10 mutants, and the secretory marker to the apoplast is correctly transported in mag2 plants, which indicate that MAG2 functions as a complex with ZW10, and is potentially involved in Golgi‐to‐ER retrograde trafficking. Therefore, a new role for ER–Golgi membrane trafficking in abiotic‐stress and ER‐stress responses is discovered.     

Field performance of STG06L-35-061, a new genetic resource developed from crosses between weed-suppressive indica rice and commercial southern U.S. long-grains

Aims

Weed control in rice is challenging, particularly in light of increased resistance to herbicides in weed populations including Echinochloa crus-galli (L.) Beauv. Indica rice cultivars can produce high yields and suppress barnyardgrass, but have not been commercially acceptable in the U.S. due to inferior agronomic traits and grain quality. Our objectives were to combine high yield and weed-suppressive characteristics from indica cultivars with commercially acceptable grain quality and plant types from long-grain cultivars grown in the southern U.S.

Methods

Crosses between indica and commercial tropical japonica (cv. Katy, and cv. Drew) rice were evaluated for weed suppression and agronomic traits in a breeding program.

Results

In some tests, the selection STG06L-35-061 was nearly as weed suppressive as PI 312777, the suppressive parent, and more suppressive than its tropical japonica parents. Its main crop yield is commercially acceptable, and intermediate between PI 312777 and Katy. Its milling quality and cooking quality are similar to long-grain commercial cultivars, and it has resistance to rice blast disease. Marker analyses identified introgressions from the indica parents on chromosomes 1 and 3 of STG06L-35-061 that require further analysis as possible sources of weed suppressive traits.

Conclusions

STG06L-35-061 might be suitable for organic rice or reduced input conventional systems.     

AtSec20 is involved in osmotic stress tolerance and AtSec20 mutation unaffects the integrity of intracellular organelles and the anterograde biosynthetic trafficking

The soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) play essential roles in intracellular trafficking. However, few experimental data have clarified their roles in the stress responses and the early secretary pathway in Arabidopsis. The AtSec20 gene encodes a protein that is homologous to yeast Sec20p and mammalian BNIP1, which are involved in the Golgi-to-ER retrograde trafficking in yeast and mammalian cells. In this study, AtSec20 is found to be required for the responses to salt stress, osmotic stress and gibberellin (GA) during seed germination and early seedling establishment. Mutation of AtSec20 unaffects the morphology of intracellular organelles, such as endoplasmic reticulum (ER), trans-Golgi network, and peroxisome, and vacuolar protein trafficking is normal in sec20 mutants. Collectively, these results imply that the AtSec20 is involved in abiotic stress tolerance, potentially via roles in retrograde vesicle fusion process in Arabidopsis.     

Arabidopsis CROLIN1, a Novel Plant Actin-binding Protein,Functions in Cross-linking and Stabilizing Actin Filaments

Higher order actin filament structures are necessary for cytoplasmic streaming, organelle movement, and other physiological processes. However, the mechanism by which the higher order cytoskeleton is formed in plants remains unknown. In this study, we identified a novel actin-cross-linking protein family (named CROLIN) that is well conserved only in the plant kingdom. There are six isovariants of CROLIN in the Arabidopsis genome, with CROLIN1 specifically expressed in pollen. In vitro biochemical analyses showed that CROLIN1 is a novel actin-cross-linking protein with binding and stabilizing activities. Remarkably, CROLIN1 can cross-link actin bundles into actin networks. CROLIN1 loss of function induces pollen germination and pollen tube growth hypersensitive to latrunculin B. All of these results demonstrate that CROLIN1 may play an important role in stabilizing and remodeling actin filaments by binding to and cross-linking actin filaments.     

Neil3 and NEIL1 DNA Glycosylases Remove Oxidative Damages from Quadruplex DNA and Exhibit Preferences for Lesions in the Telomeric Sequence Context

The telomeric DNA of vertebrates consists of d(TTAGGG)_n tandem repeats, which can form quadruplex DNA structures in vitro and likely in vivo. Despite the fact that the G-rich telomeric DNA is susceptible to oxidation, few biochemical studies of base excision repair in telomeric DNA and quadruplex structures have been done. Here, we show that telomeric DNA containing thymine glycol (Tg), 8-oxo-7,8-dihydroguanine (8-oxoG), guanidinohydantoin (Gh), or spiroiminodihydantoin (Sp) can form quadruplex DNA structures in vitro. We have tested the base excision activities of five mammalian DNA glycosylases (NEIL1, NEIL2, mNeil3, NTH1, and OGG1) on these lesion-containing quadruplex substrates and found that only mNeil3 had excision activity on Tg in quadruplex DNA and that the glycosylase exhibited a strong preference for Tg in the telomeric sequence context. Although Sp and Gh in quadruplex DNA were good substrates for mNeil3 and NEIL1, none of the glycosylases had activity on quadruplex DNA containing 8-oxoG. In addition, NEIL1 but not mNeil3 showed enhanced glycosylase activity on Gh in the telomeric sequence context. These data suggest that one role for Neil3 and NEIL1 is to repair DNA base damages in telomeres in vivo and that Neil3 and Neil1 may function in quadruplex-mediated cellular events, such as gene regulation via removal of damaged bases from quadruplex DNA.