Repression of barley cathepsins, HvPap-19 and HvPap-1, differentially alters grain composition and delays germination.

During barley germination, cysteine proteases are essential in the mobilization of storage compounds providing peptides and amino acids to sustain embryo growth until photosynthesis is completely established. Knockdown barley plants, generated by artificial miRNA, for the cathepsins B- and F-like HvPap-19 and HvPap-1 genes, respectively, showed less cysteine protease activities and consequently lower protein degradation. The functional redundancy between proteases triggered an enzymatic compensation associated with an increase in serine protease activities in both knockdown lines, which was not sufficient to maintain germination rates and behaviour. Concomitantly, these transgenic lines showed alterations in the accumulation of protein and carbohydrates in the grain. While the total amount of protein increased in both transgenic lines, the starch content decreased in HvPap-1 knockdown lines and the sucrose concentration was reduced in silenced HvPap-19 grains. Consequently, phenotypes of HvPap-1 and HvPap-19 artificial miRNA lines showed a delay in the grain germination process. These data demonstrate the potential of exploring the properties of barley proteases for selective modification and use in brewing or in the livestock feeding industry.

Evaluation of annealed titanium oxide nanotubes on titanium: From surface characterization to in vivo assays.

The entire route from anodic oxidation and surface characterization, including in vitro experiments and finally in vivo osseointegration assays were performed with the aim to evaluate nanotubular and crystalline annealed titanium oxides as a suitable surface for grade 2 titanium permanent implants. Polished titanium (T0) was compared with anodized surfaces obtained in acidic media with fluoride, leading to an ordered nanotubular structure of titanium oxide on the metal surface, characterized by tube diameter of 89 ± 24 nm (Tnts). Samples were thermally treated in air (TntsTT) to increase the anatase crystalline phase on nanotubes, with minor alteration of the structure. Corrosion tests were performed to evaluate the electrochemical response after 1, 14, and 28 days of immersion in simulated body fluid. Based on the in vitro results, heat-treated titanium nanotubes (TntsTT) were selected as a promissory candidate to continue with the osseointegration in vivo assays. The in vivo results showed no major improvement in the osseointegration process when compared with untreated Ti after 30 days of implantation and there also was a lower increase in the development of new osseous tissue.

Effect of anodized zirconium implants on early osseointegration process in adult rats: a histological and histomorphometric study.

Since surface plays a key role in bioactivity, the response of the host to the biomaterial will determine the success or failure of the prosthesis. The purpose of this study is to make an exhaustive analysis of the histological and histochemical characteristics of new bone tissue around Zr implants anodized at 60 V (Zr60) supported by histomorphometric methods in a rat model. Fibrous tissue was observed around the control implants (Zr0) and osteoblasts were identified on the trabeculae close to the implantation site that showed typical cytological characteristics of active secretory cells, regardless of the surface condition. The histomorphometrical analysis revealed a significant increase in cancellous bone volume, trabecular thickness and in trabecular number together with a decrease in trabecular separation facing Zr60. TRAP staining showed that there was a relative increase in the number of osteoclasts for Zr60. In addition, a larger number of osteoclast with a greater number of nuclei were detected in the tibiae for Zr60. This research demonstrated that the new bone microarchitecture in contact with Zr60 is able to improve the early stages of the osseointegration process and consequently the primary stability of implants which is a crucial factor to reduce recovery time for patients.

Repression of drought-induced cysteine-protease genes alters barley leaf structure and responses to abiotic and biotic stresses.

To survive under water deficiency, plants alter gene expression patterns, make structural and physiological adjustments, and optimize the use of water. Rapid degradation and turnover of proteins is required for effective nutrient recycling. Here, we examined the transcriptional responses of the C1A cysteine protease family to drought in barley and found that four genes were up-regulated in stressed plants. Knock-down lines for the protease-encoding genes HvPap-1 and HvPap-19 showed unexpected changes in leaf cuticle thickness and stomatal pore area. The efficiency of photosystem II and the total amount of proteins were almost unaltered in stressed transgenic plants while both parameters decreased in stressed wild-type plants. Although the patterns of proteolytic activities in the knock-down lines did not change, the amino acid accumulation increased in response to drought, concomitant with a higher ABA content. Whilst jasmonic acid (JA) and JA-Ile concentrations increased in stressed leaves of the wild-type and the HvPap-1 knock-down lines, their levels were lower in the HvPap-19 knock-down lines, suggesting the involvement of a specific hormone interaction in the process. Our data indicate that the changes in leaf cuticle thickness and stomatal pore area had advantageous effects on leaf defense against fungal infection and mite feeding mediated by Magnaporthe oryzae and Tetranychus urticae, respectively.

Silencing barley cystatins HvCPI-2 and HvCPI-4 specifically modifies leaf responses to drought stress.

Protein breakdown and mobilization are some of the major metabolic features associated with abiotic stresses, essential for nutrient recycling and plant survival. Genetic manipulation of protease and/or protease inhibitors may contribute to modulate proteolytic processes and plant responses. The expression analysis of the whole cystatin family, inhibitors of C1A cysteine proteases, after water deprivation in barley leaves highlighted the involvement of Icy-2 and Icy-4 cystatin genes. Artificial microRNA lines independently silencing the two drought-induced cystatins were generated to assess their function in planta. Phenotype alterations at the final stages of the plant life cycle are represented by the stay-green phenotype of silenced cystatin 2 lines. Besides, the enhanced tolerance to drought and differential responses to water deprivation at the initial growing stages are observed. The mutual compensating expression of Icy-2 and Icy-4 genes in the silencing lines pointed to their cooperative role. Proteolytic patterns by silencing these cystatins were concomitant with modifications in the expression of potential target proteases, in particular, HvPap-1, HvPap-12, and HvPap-16 C1A proteases. Metabolomics analysis lines also revealed specific modifications in the accumulation of several metabolites. These findings support the use of plants with altered proteolytic regulation in crop improvement in the face of climate change.

In vitro and in vivo characterization of anodised zirconium as a potential material for biomedical applications.

In vitro studies offer the insights for the understanding of the mechanisms at the tissue-implant interface that will provide an effective functioning in vivo. The good biocompatibility of zirconium makes a good candidate for biomedical applications and the attractive in vivo performance is mainly due to the presence of a protective oxide layer. The aim of this study is to evaluate by in vitro and in vivo approach, the influence of surface modification achieved by anodisation at 30 and 60V on zirconium implants on the first steps of the osseointegration process. In this study cell attachment, proliferation and morphology of mouse myoblast C2C12-GFP and in mouse osteoprogenitor MC3T3-E1 cells was evaluated. Also, together with the immune system response, osteoclast differentiation and morphology with RAW 264.7 murine cell line were analysed. It was found that anodisation treatment at 60V enhanced cell spreading and the osteoblastic and osteoclastic cells morphology, showing a strong dependence on the surface characteristics. In vivo tests were performed in a rat femur osteotomy model. Dynamical and static histological and histomorphometric analyses were developed 15 and 30days after surgery. Newly formed bone around Zr60V implants showed a continuous newly compact and homogeneous bone just 15 after surgery, as judged by the enhanced thickness and mineralization rate. The results indicate that anodising treatment at 60V could be an effective improvement in the osseointegration of zirconium by stimulating adhesion, proliferation, morphology, new bone thickness and bone mineral apposition, making zirconium an emerging candidate material for biomedical applications.

Effect of anodization on the surface characteristics and electrochemical behaviour of zirconium in artificial saliva.

The paper is focused on elaboration of ZrO2 films on pure zirconium via anodizing in phosphoric acid with and without fluoride at constant potentials of 30 V and 60 V. The structure and composition of the films were investigated using scanning electronic microscopy, Raman spectroscopy and X-ray photoelectron spectroscopy. The composition of the oxides formed at both potentials can be identified as monoclinic ZrO2. In addition to Zr and O, the layers formed in phosphoric acid contain phosphorus originating from the phosphoric acid. When the phosphoric acid solution contains NaF, fluorine is also incorporated into the oxide layer. The oxides formed at a higher voltage have greater roughness than those formed at 30 V. Anodized samples exhibit smaller current densities during anodic polarization compared to the as-received zirconium covered with native oxide.

Structure and dielectric properties of electrochemically grown ZrO2 films.

The dielectric properties of electrochemically grown zirconium oxide films by anodisation of zirconium in 1.0 mol dm-3 phosphoric acid solution were investigated in a 3 to 30 V potential range with a view to inducing surface modifications for eventual use in biomedical and electronic applications. The oxide films grown at different potentials were characterised by Atomic Force Microscopy, X-ray photoelectron and Raman spectroscopies; the latter demonstrated the incorporation of phosphate ions into the passive films. Flat band potentials calculated from the Mott-Shottky analysis of the oxides semiconducting properties confirm the bilayer structure of the films. The oxide dielectric permittivity was evaluated from impedance spectroscopy measurements and the film oxide model proposed gave values independent of the oxide growth potential.

A comparative study of zirconium and titanium implants in rat: osseointegration and bone material quality.

Permanent metal implants are widely used in human medical treatments and orthopedics, for example as hip joint replacements. They are commonly made of titanium alloys and beyond the optimization of this established material, it is also essential to explore alternative implant materials in view of improved osseointegration. The aim of our study was to characterize the implant performance of zirconium in comparison to titanium implants. Zirconium implants have been characterized in a previous study concerning material properties and surface characteristics in vitro, such as oxide layer thickness and surface roughness. In the present study, we compare bone material quality around zirconium and titanium implants in terms of osseointegration and therefore characterized bone material properties in a rat model using a multi-method approach. We used light and electron microscopy, micro Raman spectroscopy, micro X-ray fluorescence and X-ray scattering techniques to investigate the osseointegration in terms of compositional and structural properties of the newly formed bone. Regarding the mineralization level, the mineral composition, and the alignment and order of the mineral particles, our results show that the maturity of the newly formed bone after 8 weeks of implantation is already very high. In conclusion, the bone material quality obtained for zirconium implants is at least as good as for titanium. It seems that the zirconium implants can be a good candidate for using as permanent metal prosthesis for orthopedic treatments.

Can anodised zirconium implants stimulate bone formation? Preliminary study in rat model.

The mechanical properties and good biocompatibility of zirconium and some of its alloys make these materials good candidates for biomedical applications. The attractive in vivo performance of zirconium is mainly due to the presence of a protective oxide layer. In this preliminary study, the surface of pure zirconium modified by anodisation in acidic media at low potentials to enhance its barrier protection given by the oxides and osseointegration. Bare, commercially pure zirconium cylinders were compared to samples anodised at 30 V through electrochemical tests and scanning electron microscopy (SEM). For both conditions, in vivo tests were performed in a rat tibial osteotomy model. The histological features and fluorochrome-labelling changes of newly bone formed around the implants were evaluated on the non-decalcified sections 63 days after surgery. Electrochemical tests and SEM images show that the anodisation treatment increases the barrier effect over the material and the in vivo tests show continuous newly formed bone around the implant with a different amount of osteocytes in their lacunae depending on the region. There was no significant change in bone thickness around either kind of implant but the anodised samples had a significantly higher mineral apposition, suggesting that the anodisation treatment stimulates and assists the osseointegration process. We conclude that anodisation treatment at 30 V can stimulate the implant fixation in a rat model, making zirconium a strong candidate material for permanent implants.