Francisco J. L. Aragão

Advancing dental implant surface technology--from micron- to nanotopography.

Current trends in clinical dental implant therapy include use of endosseous dental implant surfaces embellished with nanoscale topographies. The goal of this review is to consider the role of nanoscale topographic modification of titanium substrates for the purpose of improving osseointegration. Nanotechnology offers engineers and biologists new ways of interacting with relevant biological processes. Moreover, nanotechnology has provided means of understanding and achieving cell specific functions. The various techniques that can impart nanoscale topographic features to titanium endosseous implants are described. Existing data supporting the role of nanotopography suggest that critical steps in osseointegration can be modulated by nanoscale modification of the implant surface. Important distinctions between nanoscale and micron-scale modification of the implant surface are presently considered. The advantages and disadvantages of nanoscale modification of the dental implant surface are discussed. Finally, available data concerning the current dental implant surfaces that utilize nanotopography in clinical dentistry are described. Nanoscale modification of titanium endosseous implant surfaces can alter cellular and tissue responses that may benefit osseointegration and dental implant therapy.

RNAi-Mediated Resistance to Bean golden mosaic virus in Genetically Engineered Common Bean (Phaseolus vulgaris)

Bean golden mosaic virus (BGMV) is transmitted by the whitefly Bemisia tabaci in a persistent, circulative manner, causing the golden mosaic of common bean (Phaseolus vulgaris L.). The characteristic symptoms are yellow-green mosaic of leaves, stunted growth, or distorted pods. The disease is the largest constraint to bean production in Latin America and causes severe yield losses (40 to 100%). Here, we explored the concept of using an RNA interference construct to silence the sequence region of the AC1 viral gene and generate highly resistant transgenic common bean plants. Eighteen transgenic common bean lines were obtained with an intron-hairpin construction to induce post-transcriptional gene silencing against the AC1 gene. One line (named 5.1) presented high resistance (approximately 93% of the plants were free of symptoms) upon inoculation at high pressure (more than 300 viruliferous whiteflies per plant during the whole plant life cycle) and at a very early stage of plant development. Transgene-specific small interfering RNAs were detected in both inoculated and non-inoculated transgenic plants. A semiquantitative polymerase chain reaction analysis revealed the presence of viral DNA in transgenic plants exposed to viruliferous whiteflies for a period of 6 days. However, when insects were removed, no virus DNA could be detected after an additional period of 6 days.

The effects of implant surface nanoscale features on osteoblast- specific gene expression

This study investigated the influence of nanoscale implant surface features on osteoblast differentiation. Titanium disks (20.0 x 1.0 mm) with different nanoscale materials were prepared using sol-gel-derived coatings and characterized by scanning electron microscopy, atomic force microscopy and analyzed by X-ray Photoelectron Spectrometer. Human Mesenchymal Stem Cells (hMSCs) were cultured on the disks for 3-28 days. The levels of ALP, BSP, Runx2, OCN, OPG, and OSX mRNA and a panel of 76 genes related to osteogenesis were evaluated. Topographical and chemical evaluation confirmed nanoscale features present on the coated surfaces only. Bone-specific mRNAs were increased on surfaces with superimposed nanoscale features compared to Machined (M) and Acid etched (Ac). At day 14, OSX mRNA levels were increased by 2-, 3.5-, 4- and 3-fold for Anatase (An), Rutile (Ru), Alumina (Al), and Zirconia (Zr), respectively. OSX expression levels for M and Ac approximated baseline levels. At days 14 and 28 the BSP relative mRNA expression was significantly up-regulated for all surfaces with nanoscale coated features (up to 45-fold increase for Al). The PCR array showed an up-regulation on Al coated implants when compared to M. An improved response of cells adhered to nanostructured-coated implant surfaces was represented by increased OSX and BSP expressions. Furthermore, nanostructured surfaces produced using aluminum oxide significantly enhanced the hMSC gene expression representative of osteoblast differentiation. Nanoscale features on Ti implant substrates may improve the osseointegration response by altering adherent cell response.

Inheritance of foreign genes in transgenic bean (Phaseolus vulgaris L.) co-transformed via particle bombardment

Exploiting the biolistic process we have generated stable transgenic bean (Phaseolus vulgaris L.) plants with unlinked and linked foreign genes. Co-transformation was conducted using plasmid constructions containing a fusion of the gus and neo genes, which were co-introduced with the methionine-rich 2S albumin gene isolated from the Brazil nut and the antisense sequence of AC1, AC2, AC3 and BC1 genes from the bean golden mosaic geminivirus. The results revealed a co-transformation frequency ranging from 40% to 50% when using unlinked genes and 100% for linked genes. The introduced foreign genes were inherited in a Mendelian fashion in most of the transgenic bean lines. PCR and Southern blot hybridization confirmed the integration of the foreign genes in the plant genome.

Selection of transgenic meristematic cells utilizing a herbicidal molecule results in the recovery of fertile transgenic soybean [Glycine max (L.) Merril] plants at a high frequency

Abstract Imazapyr is a herbicidal molecule that concentrates in the apical meristematic region of the plant. Its mechanism of action is the inhibition of the enzymatic activity of acetohydroxyacid synthase, which catalyses the initial step in the biosynthesis of isoleucine, leucine and valine. The selectable marker gene, ahas, was previously isolated from Arabidopsis thaliana and contains a mutation at position 653 bp. Combining the use of imazapyr, the ahas gene and a multiple shooting induction protocol has allowed us to develop a novel system to select transgenic meristematic cells after the physical introduction of foreign genes. In this study, we describe a protocol to obtain a high frequency of fertile transgenic soybean plants that is variety-independent.

Rnai-mediated silencing of the myo-inositol-1-phosphate synthase gene (GmMIPS1) in transgenic soybean inhibited seed development and reduced phytate content

Inositol plays a role in membrane trafficking and signaling in addition to regulating cellular metabolism and controlling growth. In plants, the myo-inositol-1-phosphate is synthesized from glucose 6-phosphate in a reaction catalyzed by the enzyme myo-inositol-1-phosphate synthase (EC 5.5.1.4). Inositol can be converted into phytic acid (phytate), the most abundant form of phosphate in seeds. The path to phytate has been suggested to proceed via the sequential phosphorylation of inositol phosphates, and/or in part via phosphatidylinositol phosphate. Soybean [Glycine max (L.) Merrill] lines were produced using interfering RNA (RNAi) construct in order to silence the myo-inositol-1-phosphate (GmMIPS1) gene. We have observed an absence of seed development in lines in which the presence of GmMIPS1 transcripts was not detected. In addition, a drastic reduction of phytate (InsP6) content was achieved in transgenic lines (up to 94.5%). Our results demonstrated an important correlation between GmMIPS1 gene expression and seed development.

The ER luminal binding protein (BiP) mediates an increase in drought tolerance in soybean and delays drought-induced leaf senescence in soybean and tobacco.

The ER-resident molecular chaperone BiP (binding protein) was overexpressed in soybean. When plants growing in soil were exposed to drought (by reducing or completely withholding watering) the wild-type lines showed a large decrease in leaf water potential and leaf wilting, but the leaves in the transgenic lines did not wilt and exhibited only a small decrease in water potential. During exposure to drought the stomata of the transgenic lines did not close as much as in the wild type, and the rates of photosynthesis and transpiration became less inhibited than in the wild type. These parameters of drought resistance in the BiP overexpressing lines were not associated with a higher level of the osmolytes proline, sucrose, and glucose. It was also not associated with the typical drought-induced increase in root dry weight. Rather, at the end of the drought period, the BiP overexpressing lines had a lower level of the osmolytes and root weight than the wild type. The mRNA abundance of several typical drought-induced genes [NAC2, a seed maturation protein (SMP), a glutathione-S-transferase (GST), antiquitin, and protein disulphide isomerase 3 (PDI-3)] increased in the drought-stressed wild-type plants. Compared with the wild type, the increase in mRNA abundance of these genes was less (in some genes much less) in the BiP overexpressing lines that were exposed to drought. The effect of drought on leaf senescence was investigated in soybean and tobacco. It had previously been reported that tobacco BiP overexpression or repression reduced or accentuated the effects of drought. BiP overexpressing tobacco and soybean showed delayed leaf senescence during drought. BiP antisense tobacco plants, conversely, showed advanced leaf senescence. It is concluded that BiP overexpression confers resistance to drought, through an as yet unknown mechanism that is related to ER functioning. The delay in leaf senescence by BiP overexpression might relate to the absence of the response to drought.

High-efficiency transformation by biolistics of soybean, common bean and cotton transgenic plants

This protocol describes a method for high-frequency recovery of transgenic soybean, bean and cotton plants, by combining resistance to the herbicide imazapyr as a selectable marker, multiple shoot induction from embryonic axes of mature seeds and biolistics techniques. This protocol involves the following stages: plasmid design, preparation of soybean, common bean and cotton apical meristems for bombardment, microparticle-coated DNA bombardment of apical meristems and in vitro culture and selection of transgenic plants. The average frequencies (the total number of fertile transgenic plants divided by the total number of bombarded embryonic axes) of producing germline transgenic soybean and bean and cotton plants using this protocol are 9, 2.7 and 0.55%, respectively. This protocol is suitable for studies of gene function as well as the production of transgenic cultivars carrying different traits for breeding programs. This protocol can be completed in 7–10 months.

First transgenic geminivirus-resistant plant in the field

volume 27 number 12 december 2009 nature biotechnology unsatisfactory, and commercial cultivars are susceptible to early, moderate or severe infection2,4. After the first demonstration of pathogenderived resistance (PDR) in the pioneering work from Roger Beachy ́s lab describing coat protein–mediated resistance to tobacco mosaic virus5, several strategies have been used to genetically engineer tolerance or immunity to viruses in transgenic plants. These strategies are based on two broad classes: protein-mediated resistance and RNA silencing–mediated resistance. Now that we better understand the mechanisms of RNA interference (RNAi) and its biological functions, it is possible to look back on initial experiments from a new perspective. It is now known that plants naturally process viral RNAs to generate small sequences of a pathogen’s genetic material that can be specifically used against that pathogen through the RNA-induced silencing complex. An RNA-silencing (posttranscriptional gene silencing) mechanism was recognized as being responsible for resistance to RNA viruses. This mechanism depends on the formation of doublestranded RNA (dsRNA) whose antisense strand is complementary to the transcript of a targeted gene. These discoveries led to the introduction of constructs to produce intracellular generation of small interfering RNA (siRNA)-like species in transgenic plants, inducing targeted gene silencing and virus resistance. This is an important tool in generating plants resistant to a broad range of viruses6. However, not all viral genes used in transgenic constructs render plants resistant to infection. The use of inverted repeat constructs, resulting in dsRNA transcripts, is the most efficient means of generating transformed lines showing effective gene knockdown or virus resistance7. The most likely reason for this is that dsRNAs are fed directly into the silencing pathway at the level of the RNaseIII-like enzyme Dicer, and therefore they are not reliant on the action of plant-encoded RNA-dependent RNA polymerase proteins. Nevertheless, most examples of RNAi-mediated virus resistance pertain to RNA plant viruses. Indeed, attempts to obtain robust PDR to geminiviruses have not been as successful as those against RNA viruses, and development of geminivirus-resistant plants is considered a major challenge8. Because of the social and economic importance of common bean as a source of protein in the diet of over a billion people worldwide, we have been attempting since should be done remains undone.” If it is not clearly stated with meaning that weeds are a major constraint on the quality of life of most women in the developing world, then what should be done remains undone, and gender issues have not been adequately addressed with biotechnology.

The combination of micron and nanotopography by H2SO4/H2O2 treatment and its effects on osteoblast-specific gene expression of hMSCs

H(2)SO(4)/H(2)O(2) treatment of titanium implants imparts nanofeatures to the surface and alters the osteoblast response. The aim of this study was to evaluate the effect of H(2)SO(4)/H(2)O(2) treatment of commercially pure Titanium (cpTi) surfaces on gene expression of human mesenchymal stem cells (hMSCs) differentiated into osteoblasts. Commercially pure grade IV titanium disks (20.0 mm x 1.0 mm) were polished or polished and subsequently treated by grit blasting or grit-blasting/acid etching with an H(2)SO(4)/H(2)O(2) solution. The surfaces were divided into three groups: smooth (S), grit-blasted (Gb), and nanostructured: grit-blasted/acid etched (Nano). Surfaces were examined by scanning electron microscopy and atomic force microscopy. HMSCs were grown on the disks. The data points analyzed were at 3, 7, 14, and 28 days. Real-time PCR was used to measure the mRNA levels of ALP, BSP, Runx2, OCN, OPN, and OSX. The housekeeping gene GAPDH was used as a control. Descriptive statistics were calculated using Microsoft Excel. T-test was performed for comparison of mRNA levels when compared with S surfaces (p < 0.05). All osteoblast-specific genes were regulated in surface-dependent patterns and most of them were upregulated on the Nano surfaces. Runx2 and OSX mRNAs were more than threefold upregulated at days 14 and 28 on Nano. Higher levels for ALP (38-fold), BSP (76-fold), and OCN (3-fold) were also observed on the Nano surfaces. A grit-blasted surface imparted with nanofeatures by H(2)SO(4)/H(2)O(2) treatment affected adherent cell bone-specific gene expression. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res, 2010.