Regenerative Drug Discovery Using Ear Pinna Punch Wound Model in Mice.

The ear pinna is a complex tissue consisting of the dermis, cartilage, muscles, vessels, and nerves. Ear pinna healing is a model of regeneration in mammals. In some mammals, including rabbits, punch wounds in the ear pinna close spontaneously; in common-use laboratory mice, they remain for life. Agents inducing ear pinna healing are potential regenerative drugs. We tested the effects of selected bioactive agents on 2 mm ear pinna wound closure in BALB/c mice. Our previous research demonstrated that a DNA methyltransferase inhibitor, zebularine, remarkably induced ear pinna regeneration. Although experiments with two other demethylating agents, RG108 and hydralazine, were unsuccessful, a histone deacetylase inhibitor, valproic acid, was another epigenetic agent found to increase ear hole closure. In addition, we identified a pro-regenerative activity of 4-ketoretinoic acid, a retinoic acid metabolite. Attempts to counteract the regenerative effects of the demethylating agent zebularine, with folates as methyl donors, failed. Surprisingly, a high dose of methionine, another methyl donor, promoted ear hole closure. Moreover, we showed that the regenerated areas of ear pinna were supplied with nerve fibre networks and blood vessels. The ear punch model proved helpful in testing the pro-regenerative activities of small-molecule compounds and observations of peripheral nerve regeneration.

Hybrid Bilayer PLA/Chitosan Nanofibrous Scaffolds Doped with ZnO, Fe3O4, and Au Nanoparticles with Bioactive Properties for Skin Tissue Engineering.

Burns affect almost half a million of Americans annually. In the case of full-thickness skin injuries, treatment requires a transplant. The development of bioactive materials that promote damaged tissue regeneration constitutes a great alternative to autografts. For this reason, special attention is focused on three-dimensional scaffolds that are non-toxic to skin cells and can mimic the extracellular matrix, which is mainly composed of nanofibrous proteins. Electrospinning, which enables the preparation of nanofibers, is a powerful tool in the field of biomaterials. In this work, novel hybrid poly (lactic acid)/chitosan biomaterials functionalized with three types of nanoparticles (NPs) were successfully developed. ZnO, Fe3O4, and Au NPs were investigated over their morphology by TEM method. The top layer was obtained from PLA nanofibers, while the bottom layer was prepared from acylated chitosan. The layers were studied over their morphology by the SEM method and their chemical structure by FT-IR. To verify their potential in burn wound treatment, the scaffolds' susceptibility to biodegradation as well as moisture permeability were calculated. Also, biomaterials conductivity was determined in terms of electrostimulation. Finally, cytotoxicity tests were carried out by XTT assay and morphology analysis using both fibroblasts cell line and primary cells. The hybrid nanofibrous scaffolds displayed a great potential in tissue engineering.

Normoergic NO-dependent changes, triggered by a SAR inducer in potato, create more potent defense responses to Phytophthora infestans.

In our experimental approach we examined how potato leaves exposed to a chemical agent might induce nitric oxide (NO) dependent biochemical modifications for future mobilization of an effective resistance to Phytophthora infestans. After potato leaf treatment with one of the following SAR inducers, i.e. ß-aminobutyric acid (BABA), 2,6-dichloroisonicotinic acid (INA) or Laminarin, we observed enhanced NO generation concomitant with biochemical changes related to a slight superoxide anion (O2(-)) and hydrogen peroxide (H2O2) accumulation dependent on minimal NADPH oxidase and peroxidase activities, respectively. These rather normoergic changes, linked to the NO message, were mediated by the temporary down-regulation of S-nitrosoglutathione reductase (GSNOR). In turn, after challenge inoculation signal amplification promoted potato resistance manifested in the up-regulation of GSNOR activity tuned with the depletion of the SNO pool, which was observed by our team earlier (Floryszak-Wieczorek et al., 2012). Moreover, hyperergic defense responses related to an early and rapid O2(-)and H2O2 overproduction together with a temporary increase in NADPH oxidase and peroxidase activities were noted. BABA treatment was the most effective against P. infestans resulting in the enhanced activity of ß-1,3-glucanase and callose deposition. Our results indicate that NO-mediated biochemical modifications might play an important role in creating more potent defense responses of potato to a subsequent P. infestans attack.

The proteome response of potato leaves to priming agents and S-nitrosoglutathione.

The primed mobilization for more potent defense responses to subsequent stress has been shown for many plant species, but there is a growing need to identify reliable molecular markers for this unique phenomenon. In the present study a proteomic approach was used to screen similarities in protein abundance in leaves of primed potato (Solanum tuberosum L.) treated with four well-known inducers of plant resistance, i.e. ß-aminobutyric acid (BABA), γ-aminobutyric acid (GABA), Laminarin and 2,6-dichloroisonicotinic acid (INA), respectively. Moreover, to gain insight into the importance of nitric oxide (NO) in primed protein accumulation the potato leaves were supplied by S-nitrosoglutathione (GSNO), as an NO donor. The comparative analysis, using two-dimensional electrophoresis and mass spectrometry, revealed that among 25 proteins accumulated specifically after BABA, GABA, INA and Laminarin treatments, 13 proteins were accumulated also in response to GSNO. Additionally, overlapping proteomic changes between BABA-primed and GSNO-treated leaves showed 5 protein spots absent in the proteome maps obtained in response to the other priming agents. The identified 18 proteins belonged, in most cases, to functional categories of primary metabolism. The selected proteins including three redox-regulated enzymes, i.e. glyceraldehyde 3-phosphate dehydrogenase, carbonic anhydrase, and fructose-bisphosphate aldolase, were discussed in relation to the plant defence responses. Taken together, the overlapping effects in the protein profiles obtained between priming agents, GSNO and cPTIO treatments provide insight indicating that the primed potato exhibits unique changes in the primary metabolism, associated with selective protein modification via NO.

Nitric oxide-mediated stress imprint in potato as an effect of exposure to a priming agent.

We investigated how potato exposed to a chemical agent could activate nitric oxide (NO)-dependent events facilitating more potent defense responses to a subsequent pathogen attack. Obtained data revealed that all applied inducers, i.e., ß-aminobutyric acid (BABA), γ-aminobutyric acid (GABA), laminarin, or 2,6-dichloroisonicotinic acid (INA), were active stimuli in potentiating NO synthesis in the primed potato. It is assumed, for the mechanism proposed in this paper, that priming involves reversible S-nitrosylated protein (S-nitrosothiols [SNO]) storage as one of the short-term stress imprint components, apart from epigenetic changes sensitized by NO. Based on BABA- and GABA-induced events, it should be stated that a rise in NO generation and coding the NO message in SNO storage at a relatively low threshold together with histone H2B upregulation might create short-term imprint activation, facilitating acquisition of a competence to react faster after challenge inoculation. Laminarin elicited strong NO upregulation with an enhanced SNO pool-altered biochemical imprint in the form of less effective local recall, nevertheless being fully protective in distal responses against P. infestans. In turn, INA showed the most intensified NO generation and abundant formation of SNO, both after the inducer treatment and challenge inoculation abolishing potato resistance against the pathogen. Our results indicate, for the first time, that a precise control of synthesized NO in cooperation with reversible SNO storage and epigenetic modifications might play an important role in integrating and coordinating defense potato responses in the priming phenomenon.