Structural basis of nanobody recognition of grapevine fanleaf virus and of virus resistance loss.

Grapevine fanleaf virus (GFLV) is a picorna-like plant virus transmitted by nematodes that affects vineyards worldwide. Nanobody (Nb)-mediated resistance against GFLV has been created recently, and shown to be highly effective in plants, including grapevine, but the underlying mechanism is unknown. Here we present the high-resolution cryo electron microscopy structure of the GFLV-Nb23 complex, which provides the basis for molecular recognition by the Nb. The structure reveals a composite binding site bridging over three domains of one capsid protein (CP) monomer. The structure provides a precise mapping of the Nb23 epitope on the GFLV capsid in which the antigen loop is accommodated through an induced-fit mechanism. Moreover, we uncover and characterize several resistance-breaking GFLV isolates with amino acids mapping within this epitope, including C-terminal extensions of the CP, which would sterically interfere with Nb binding. Escape variants with such extended CP fail to be transmitted by nematodes linking Nb-mediated resistance to vector transmission. Together, these data provide insights into the molecular mechanism of Nb23-mediated recognition of GFLV and of virus resistance loss.

Deciphering the transcriptional regulation and spatiotemporal distribution of immunity response in barley to Pyrenophora graminea fungal invasion.

BACKGROUND: Barley leaf stripe disease, caused by the fungus Pyrenophora graminea (Pg), is a worldwide crop disease that results in significant loss of barley yield. The purpose of the present work was to use transcriptomic profiling to highlight barley genes and metabolic pathways affected or altered in response to Pg infection and consequently elucidate their involvement and contribution in resistance to leaf stripe. RESULTS: Our study examined and compared the transcriptomes of two barley genotypes using an established differential display reverse-transcription polymerase chain reaction (DDRT-PCR) strategy at 14 and 20 days post-inoculation (dpi). A total of 54 significantly modulated expressed sequence tags (ESTs) were identified. The analysis of gene expression changes during the course of infection with Pg suggested the involvement of 15 upregulated genes during the immunity response. By using network-based analyses, we could establish a significant correlation between genes expressed in response to Pg invasion. Microscopic analysis and quantitative PCR (qPCR) profiling of callose synthase and cellulose synthases revealed a direct involvement of cell wall reinforcement and callose deposition in the Pg-resistant phenotype. CONCLUSIONS: We have identified a number of candidate genes possibly involved in the host-pathogen interactions between barley and Pg fungus, 15 of which are specifically expressed in Pg-resistant plants. Collectively, our results suggest that the resistance to leaf stripe in barley proceeds through callose deposition and different oxidation processes.

Induction of G1-phase cell cycle arrest and apoptosis pathway in MDA-MB-231 human breast cancer cells by sulfated polysaccharide extracted from Laurencia papillosa.

BACKGROUND: Marine algae consumption is linked to law cancer incidences in countries that traditionally consume marine products. Hence, Phytochemicals are considered as potential chemo-preventive and chemotherapeutic agents against cancer. We investigated the effects of the algal sulfated polysaccharide extract (ASPE) from the red marine alga L. papillosa on MDA-MB-231 human breast cancer cell line. METHODS: Flow cytometry analysis was performed to study the cell viability, cell cycle arrest and apoptosis. Changes in the expression of certain genes associated with cell cycle regulation was conducted by PCR real time analyses. Further investigations on apoptotic molecules was performed by ROS measurement and protein profiling. RESULTS: ASPE at low doses (10 µg/ml), inhibited cell proliferation, and arrested proliferating MDA-MB-231 cells at G1-phase. However, higher doses (50 µg/ml), triggered apoptosis in those cells. The low dose of ASPE also caused up-regulation of Cip1/p21 and Kip1/p27 and down-regulation of cyclins D1, D2, and E1 transcripts and their related cyclin dependent kinases: Cdk2, Cdk4, and Cdk6. The higher doses of ASPE initiated a dose-dependent apoptotic death in MDA-MB-231 by induction of Bax transcripts, inhibition of Bcl-2 and cleavage of Caspase-3 protein. Over-generation of reactive oxygen species (ROS) were also observed in MDA-MB-231 treated cells. CONCLUSIONS: These findings indicated that ASPE induces G1-phase arrest and apoptosis in MDA-MB-231 cells. ASPE may serve as a potential therapeutic agent for breast cancer.

NtRING1, putative RING-finger E3 ligase protein, is a positive regulator of the early stages of elicitin-induced HR in tobacco.

KEY MESSAGE: NtRING1 is a RING-finger protein with a putative E3 ligase activity. NtRING1 regulates HR establishment against different pathogens. Loss-/gain-of-function of NtRING1 altered early stages of HR phenotype establishment. Plant defence responses against pathogens often involve the restriction of pathogens by inducing a hypersensitive response (HR). cDNA clones DD11-39, DD38-11 and DD34-26 were previously obtained from a differential screen aimed at characterising tobacco genes with an elicitin-induced HR-specific pattern of expression. Our precedent observations suggested that DD11-39, DD38-11 and DD34-26 might play roles in the HR establishment. Only for DD11-39 a full-length cDNA sequence was obtained and the corresponding protein encoded for a type-HC RING-finger/putative E3 ligase protein which we termed NtRING1. The expression of NtRING1 was upregulated upon HR induction by elicitin, Ralstonia solanacearum, or tobacco mosaic virus (TMV) in tobacco. Silencing of NtRING1 remarkably delayed the establishment of elicitin-induced HR in tobacco as well as the expression of different early induction genes in tissues undergoing HR. Accordingly, transient overexpression of NtRING1 accelerated the HR launching upon elicitin treatment. Taking together, our data suggests that NtRING1 plays a functional role in the early establishment of HR.

Camelid nanobodies with high affinity for broad bean mottle virus: a possible promising tool to immunomodulate plant resistance against viruses.

Worldwide, plant viral infections decrease seriously the crop production yield, boosting the demand to develop new strategies to control viral diseases. One of these strategies to prevent viral infections, based on the immunomodulation faces many problems related to the ectopic expression of specific antibodies in planta. Camelid nanobodies, expressed in plants, may offer a solution as they are an attractive tool to bind efficiently to viral epitopes, cryptic or not accessible to conventional antibodies. Here, we report a novel, generic approach that might lead to virus resistance based on the expression of camelid specific nanobodies against Broad bean mottle virus (BBMV). Eight nanobodies, recognizing BBMV with high specificity and affinity, were retrieved after phage display from a large 'immune' library constructed from an immunized Arabic camel. By an in vitro assay we demonstrate how three nanobodies attenuate the BBMV spreading in inoculated Vicia faba plants. Furthermore, the in planta transient expression of these three selected nanobodies confirms their virus neutralizing capacity. In conclusion, this report supports that plant resistance against viral infections can be achieved by the in vivo expression of camelid nanobodies.

Acceleration of Alveolar Ridge Augmentation Using a Low Dose of Recombinant Human Bone Morphogenetic Protein-2 Loaded on a Resorbable Bioactive Ceramic.

PURPOSE: The aim of the present study was to evaluate the effect of a porous silica-calcium phosphate composite (SCPC50) loaded with and without recombinant human bone morphogenetic protein-2 (rhBMP-2) on alveolar ridge augmentation in saddle-type defects. MATERIALS AND METHODS: Micro-granules of SCPC50 resorbable bioactive ceramic were coated with rhBMP-2 10 mg and then implanted into a saddle-type defect (12 × 7 mm) in a dog mandible and covered with a collagen membrane. Control groups included defects grafted with SCPC50 granules without rhBMP-2 and un-grafted defects. Bone healing was evaluated at 8 and 16 weeks using histologic and histomorphometric techniques. The increase in bone height and total defect fill were assessed for each specimen using the ImageJ 1.46 program. The release kinetics of rhBMP-2 was determined in vitro. The height of the bone in the grafted defects and the total defect fill were statistically analyzed. RESULTS: SCPC50 enhanced alveolar ridge augmentation as indicated by the increased vertical bone height, bone surface area, and bone volume after 16 weeks. SCPC50-rhBMP-2 provided a sustained release profile of a low effective dose (BMP-2 4.6 ± 1.34 pg/mL per hour) during the 1- to 21-day period. The slow rate of release of rhBMP-2 from SCPC50 accelerated synchronized complete bone regeneration and graft material resorption in 8 weeks. Successful rapid reconstruction of the alveolar ridge by SCPC50 and SCPC50-rhBMP-2 occurred without any adverse excessive bone formation, inflammation, or fluid-filled voids. CONCLUSIONS: Results of this study suggest that SCPC50 is an effective graft material to preserve the alveolar ridge after tooth extraction. Coating SCPC50-rhBMP-2 further accelerated bone regeneration and a considerable increase in vertical bone height. These findings make SCPC50 the primary choice as a carrier for rhBMP-2. SCPC50-rhBMP-2 can serve as an alternative to autologous bone grafting.

Rat animal model for preclinical testing of microparticle urethral bulking agents.

OBJECTIVES: To develop an economic, practical and readily available animal model for preclinical testing of urethral bulking therapies, as well as to establish feasible experimental methods that allow for complete analysis of hard microparticle bulking agents. METHODS: Alumina ceramic beads suspended in hyaluronic acid were injected into the proximal urethra of 15 female rats under an operating microscope. We assessed overall lower urinary tract function, bulking material intraurethral integrity and local host tissue response over time. Microphotographs were taken during injection and again 6 months postoperatively, before urethral harvest. Urinary flow rate and voiding frequency were assessed before and after injection. At 6 months, the urethra was removed and embedded in resin. Hard tissue sections were cut using a sawing microtome, and processed for histological analysis using scanning electron microscopy, light microscopy and immunohistochemistry. RESULTS: Microphotographs of the urethra showed complete volume retention of the bulking agent at 6 months. There was no significant difference between average urinary frequency and mean urinary flow rate at 1 and 3 months postinjection as compared with baseline. Scanning electron microscopy proved suitable for evaluation of microparticle size and integrity, as well as local tissue remodeling. Light microscopy and immunohistochemistry allowed for evaluation of an inflammatory host tissue reaction to the bulking agent. CONCLUSIONS: The microsurgical injection technique, in vivo physiology and novel hard tissue processing for histology, described in the present study, will allow for future comprehensive preclinical testing of urethral bulking therapy agents containing microparticles made of a hard material.

Alveolar ridge preservation using resorbable bioactive ceramic composite: a histological study.

PURPOSE: The purpose of this study was to histologically evaluate newly generated vital bone using porous granules of bioactive and resorbable silica-calcium phosphate nanocomposite (SCPC) in extraction sockets. MATERIAL AND METHODS: Six patients with a non-restorable maxillary central incisor requiring extraction followed by implant placement participated in the study. Extraction sockets were grafted with granules of SCPC. After 6 months, a bone core sample was retrieved from the center of the healed socket for histologic analysis, and dental implants were placed. Alveolar bone width was clinically assessed immediately after tooth extraction and 6 months after bone grafting, at the time of implant placement. Alveolar bone height was radiographically assessed immediately after tooth extraction and 6 months after extraction. RESULTS: Histomorphometric analyses of sockets grafted with SCPC for 6 months revealed 46.8% +/- 14% new vital bone and 2.5% +/- 1.5% graft material remnants. In these sockets, the mean bone height resorption over the 6-month period of healing was 1.6 mm +/- 1.5 mm. The mean bone width resorption of 2 mm +/- 0.7 mm was found at the bone crest. CONCLUSION: The results of this study suggest that SCPC graft material reduces the amount of change in alveolar ridge dimensions after tooth extraction and facilitates the regeneration of new vital bone.

Bioactive Carbonate Apatite Cement with Enhanced Compressive Strength via Incorporation of Silica Calcium Phosphate Composites and Calcium Hydroxide.

Carbonate apatite (CO3Ap) is a bioceramic material with excellent properties for bone and dentin regeneration. To enhance its mechanical strength and bioactivity, silica calcium phosphate composites (Si-CaP) and calcium hydroxide (Ca(OH)2) were added to CO3Ap cement. The aim of this study was to investigate the effect of Si-CaP and Ca(OH)2 on the mechanical properties in terms of the compressive strength and biological characteristics of CO3Ap cement, specifically the formation of an apatite layer and the exchange of Ca, P, and Si elements. Five groups were prepared by mixing CO3Ap powder consisting of dicalcium phosphate anhydrous and vaterite powder added by varying ratios of Si-CaP and Ca(OH)2 and 0.2 mol/L Na2HPO4 as a liquid. All groups underwent compressive strength testing, and the group with the highest strength was evaluated for bioactivity by soaking it in simulated body fluid (SBF) for one, seven, 14, and 21 days. The group that added 3% Si-CaP and 7% Ca(OH)2 had the highest compressive strength among the groups. SEM analysis revealed the formation of needle-like apatite crystals from the first day of SBF soaking, and EDS analysis indicated an increase in Ca, P, and Si elements. XRD and FTIR analyses confirmed the presence of apatite. This combination of additives improved the compressive strength and showed the good bioactivity performance of CO3Ap cement, making it a potential biomaterial for bone and dental engineering applications.

Inhibition of osteoclast activities by SCPC bioceramic promotes osteoblast-mediated graft resorption and osteogenic differentiation.

Maximizing vital bone in a grafted site is dependent on a number of factors. These include resorption or turnover of the graft material, stimulation of bone formation pathway without a need for biological molecules added to the site and inhibition of cellular activities that compromise the mineralization of new bone matrix. In the present study, the dissolution profile of silica-calcium phosphate composite (SCPC) in physiological solution was measured and the data were fed to (ANN-NARX) prediction model to predict the time required for complete dissolution. The inductively coupled plasma-optical emission spectrometer ionic composition analysis of the culture medium incubated for 3 days with SCPC showed 57% decrease in Ca concentration and a significant increase in the concentration of Si (13.5 ± 1.8 µg/ml), P (249.4 ± 22 µg/ml), and Na (9.3 ± 0.52 µg/ml). In conjunction with the release of Si, P, and Na ions, the bone resorptive activity of osteoclasts was inhibited as indicated by the significant decrease in multinucleated tartrate resistant acidic phosphate stained cells and the volume of resorption pits on bone slices. In contrast, addition of SCPC to hBMSC cultured in conventional medium promoted higher Runt-related transcription factor 2 (p < .05), osteocalcin (p < .01), and bone sialo protein (p < .01) than that expressed by control cells grown in the absence of SCPC. The predicted dissolution time of 200 mg of porous SCPC particles in 10 ml phosphate buffered saline is 6.9 months. An important byproduct of the dissolution is inhibition of osteoclastic activity and promotion of osteoblastic differentiation and hence bone formation.

Effect of processing parameters on the microstructure and mechanical behavior of silica-calcium phosphate nanocomposite.

Silica-calcium phosphate nanocomposite (SCPC) is a bioactive ceramic characterized by superior bone regenerative capacity and resorbability when compared to traditional bioactive ceramics. The aim of the present study is to evaluate the effect of processing parameters on the microstructure and mechanical properties of SCPC. Cylinders were prepared by pressing the ceramic powder at 200, 300 or 400 MPa and sintering at 900, 1000 or 1100 degrees C for 3 h, respectively. XRD results indicate that the crystalline structure of the material is made of beta-NaCaPO(4) and alpha-cristobalite solid solutions. The increase in sintering temperature results in an increase in the grain size and the formation of a melting phase that coats the grains. TEM analyses reveal that the melting phase is amorphous and rich in silicon. The mechanical properties of SCPC cylinders are dependent on the content of the melting phase and the microstructure of the material. The ranges of compressive strength and modulus of elasticity of the SCPC are 62-204 MPa and 6-14 GPa, respectively, which are comparable to those of cortical bone. The results suggest that the interaction between crystalline and amorphous phases modulated the mechanical behavior of SCPC. It is possible to engineer the mechanical properties of SCPC by controlling the processing parameters to synthesize various fixation devices for orthopedic and cranio-maxillofacial applications.

A ceramic-based anticancer drug delivery system to treat breast cancer.

Drug delivery systems offer the advantage of sustained targeted release with minimal side effect. In the present study, the therapeutic efficacy of a porous silica-calcium phosphate nanocomposite (SCPC) as a new delivery system for 5-Fluorouracil (5-FU) was evaluated in vitro and in vivo. In vitro studies showed that two formulations; SCPC50/5-FU and SCPC75/5-FU hybrids were very cytotoxic for 4T1 mammary tumor cells. In contrast, control SCPCs without drug did not show any measurable toxic effect. Release kinetics studies showed that SCPC75/5-FU hybrid provided a burst release of 5-FU in the first 24 h followed by a sustained release of a therapeutic dose (30.7 microg/day) of the drug for up to 32 days. Moreover, subcutaneous implantation of SCPC75/5-FU hybrid disk in an immunocompetent murine model of breast cancer stopped 4T1 tumor growth. Blood analyses showed comparable concentrations of Ca, P and Si in animals implanted with or without SCPC75 disks. These results strongly suggest that SCPC/5-FU hybrids can provide an effective treatment for solid tumors with minimal side effects.

Synthesis and characterization of porous bioactive SiC tissue engineering scaffold.

Silicon carbide (SiC) is an inert material with excellent biocompatibility properties. A major issue that limits its use as a medical device is the difficult processing technique that requires hot pressing at a temperature (>2,000o C) and pressure (1,000-2,000 atm). In the present study, we developed a protocol to synthesize a porous SiC scaffold by pressing the powder at 50 MPa and heating at 900o C/2 hr. The surface of SiC was chemically modified by NaOH to facilitate sintering and induce bioactivity. Porous discs with 51.51 ± 3.17% porosity and interconnected pores in the size range from 1 to 1,000 µm were prepared using 40% PEG. The average compressive strength and Young's modulus of the scaffolds were 1.94 ± 0.70 and 169.2 ± 0.08 MPa, respectively. FTIR analysis confirmed the formation of biomimetic hydroxyapatite layer after 2 hr of immersion in simulated body fluid. The Ca/P ratio was dependent on the concentration of the silanol groups created on the material surface. Increasing the atomic % of silicon on the SiC surface from 33.27 ± 9.53% to 45.13 ± 4.74% resulted in a 76% increase in the osteocalcin expression by MC3T3-E1 cells seeded on the material after 7 days. The cells colonized the entire thickness of the template and filled the pores with mineralized extracellular matrix after 14 days. Taken all together, the porous SiC scaffolds can serve as a bone graft for tissue reconstruction and cell delivery in trauma surgery.

Tissue Engineering: What is New?

Soft and hard tissue engineering has expanded the frontiers of oral/maxillofacial augmentation. Soft tissue grafting enhancements include improving flap prevascularization and using stem cells and other cells to create not only the graft, but also the vascularization and soft tissue scaffolding for the graft. Hard tissue grafts have been enhanced by osteoinductive factors, such as bone morphogenic proteins, that have allowed the elimination of harvesting autogenous bone and thus decrease the need for other surgical sites. Advancements in bone graft scaffolds have developed via seeding with stem cells and improvement of the silica/calcium/phosphate composite to improve graft characteristics and healing.

Isolation, Structural characterization, and antiproliferative activity of phycocolloids from the red seaweed Laurencia papillosa on MCF-7 human breast cancer cells.

Hydrocolloids from seaweeds (phycocolloids) have interesting functional properties like antiproliferative activity. Marine algae consumptions are linked to law cancer incidences in countries that traditionally consume marine products. In this study, we have investigated water-soluble sulfated polysaccharides isolated from the red seaweed Laurencia papillosa and determined their chemical characteristics and biological activities on the human breast cancer cell line MCF-7. Total polysaccharides were extracted and fractionated from L. papillosa and characterized using FTIR-ATR and NMR spectrometry. In addition, their approximate molar mass was determined by GPC method. The chemical characterization of purified polysaccharides reveals the presence of sulfated polysaccharides differentially dispersed in the algal cell wall. They are the three types of carrageenan, kappa, iota and lambda carrageenans, named LP-W1, -W2 and -W3 respectively. Biological effects and cytotoxicity of the identified of the three sulfated polysaccharide fractions were evaluated in MCF-7 cell line. Our results showed a significant inhibition of MCF-7 cell viability by dose-dependent manner for cells exposed to LP-W2 and LP-W3 polysaccharides for 24h. The mechanistic of LP fractions-mediated apoptosis in MCF-7 cells was demonstrated. The biological effects of L. papillosa SPs indicate that it may be a promising candidate for breast cancer prevention and therapy.

Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein.

Double-stranded RNA (dsRNA) plays essential functions in many biological processes, including the activation of innate immune responses and RNA interference. dsRNA also represents the genetic entity of some viruses and is a hallmark of infections by positive-sense single-stranded RNA viruses. Methods for detecting dsRNA rely essentially on immunological approaches and their use is often limited to in vitro applications, although recent developments have allowed the visualization of dsRNA in vivo. Here, we report the sensitive and rapid detection of long dsRNA both in vitro and in vivo using the dsRNA binding domain of the B2 protein from Flock house virus. In vitro, we adapted the system for the detection of dsRNA either enzymatically by northwestern blotting or by direct fluorescence labeling on fixed samples. In vivo, we produced stable transgenic Nicotiana benthamiana lines allowing the visualization of dsRNA by fluorescence microscopy. Using these techniques, we were able to discriminate healthy and positive-sense single-stranded RNA virus-infected material in plants and insect cells. In N. benthamiana, our system proved to be very potent for the spatio-temporal visualization of replicative RNA intermediates of a broad range of positive-sense RNA viruses, including high- vs. low-copy number viruses.

Calcium release and physical properties of modified carbonate apatite cement as pulp capping agent in dental application.

BACKGROUND: Carbonate apatite (CO3Ap) and silica-calcium phosphate composite (SCPC) are bone substitutes with good prospect for dental application. SCPC creates a hydroxyapatite surface layer and stimulate bone cell function while, CO3Ap induce apatite crystal formation with good adaptation providing good seal between cement and the bone. Together, these materials will add favorable properties as a pulp capping material to stimulate mineral barrier and maintain pulp vitality. The aim of this study is to investigate modification of CO3Ap cement combined with SCPC, later term as CO3Ap-SCPC cement (CAS) in means of its chemical (Calcium release) and physical properties (setting time, DTS and pH value). METHODS: The study consist of three groups; group 1 (100% calcium hydroxide, group 2 CO3Ap (60% DCPA: 40% vaterite, and group 3 CAS (60% DCPA: 20% vaterite: 20% SCPC. Distilled water was employed as a solution for group 1, and 0.2 mol/L Na3PO4 used for group 2 and group 3.Samples were evaluated with respect to important properties for pulp capping application such as pH, setting time, mechanical strength and calcium release evaluation. RESULTS: The fastest setting time was in CO3Ap cement group without SCPC, while the addition of 20% SCPC slightly increase the pH value but did not improved the cement mechanical strength, however, the mechanical strength of both CO3Ap groups were significantly higher than calcium hydroxide. All three groups released calcium ions and had alkaline pH. Highest pH level, as well as calcium released level, was in the control group. CONCLUSION: The CAS cement had good mechanical and acceptable chemical properties for pulp capping application compared to calcium hydroxide as a gold standard. However, improvements and in vivo studies are to be carried out with the further development of this material.

Dissolution kinetics of a Si-rich nanocomposite and its effect on osteoblast gene expression.

Silica-calcium phosphate nanocomposite (SCPC) has recently been proposed as a novel resorbable, bioactive, and mechanically compatible template for bone reconstruction. The effect of the physicochemical properties on the surface reactivity and dissolution kinetics of SCPC immersed in simulated body fluid (SBF) was investigated and compared to that of bioactive glass (BG). Moreover, the stimulatory effect on osteoblast gene expression of SCPC was determined using quantitative real-time polymerase chain reaction (qRT-PCR), and compared to that of hydroxyapatite (HA-200). Mercury porosimetry revealed that surface areas of SCPC particles containing 10 (SCPC10), 30 (SCPC30), and 50 (SCPC50) wt % Si-content were 14-, 18-, and 32-times higher than that of BG. Inductively coupled plasma analysis showed that after 192 h of immersion, Si-rich SCPC50 exhibited controlled bulk-dissolution and released 43.1 ppm Si, which was sixfold higher than that released from BG (7.7 ppm). Moreover, SCPC50 showed a rapid Ca-uptake from SBF and developed a surface apatite layer after only 2 h, whereas a similar layer was detected on BG after 8 days of immersion under the same experimental conditions. qRT-PCR revealed that osteopontin and osteocalcin mRNA expression by osteoblast-like cells attached to Si-rich SCPC50 was significantly higher than that on HA-200 or polystyrene after 2 days in culture. This suggested a role of dissolved Si in stimulating the differentiation and mineralization of osteoblast precursor cells. The favorable physiochemical and bioactivity properties of Si-rich SCPC nanocomposite indicate that SCPC can have wide applications as a synthetic bone graft for cell delivery applications in tissue engineering.

Enhancement of osteoblast gene expression by mechanically compatible porous Si-rich nanocomposite.

Synthesis of a porous bioactive ceramic implant for load bearing applications is a challenging task in maxillofacial and orthopedic surgeries. A novel bioactive resorbable silica-calcium phosphate nanocomposite (SCPC) has recently been introduced as a potential bone graft. In the present study, we employed SCPC to develop a resorbable porous scaffold and analyzed the effects of composition and porosity on the mechanical properties. The ranges of compressive strength and modulus of elasticity of SCPC containing 32-56% porosity were 1.5-50 MPa and 0.14-2.1 GPa, respectively, which matched the corresponding values for trabecular bone. The compressive strength of dense SCPC was dependent on the Si content and acquired values (93-285 MPa) comparable to that of cortical bone. The superior mechanical properties of SCPC are attributed to the intricate interactions at the boundaries of the nanograins and to the homogenous distribution of hierarchical pore-structure throughout the material volume. X-ray computed tomography and mercury porosimetry analyses revealed high interconnectivity of the pores in the size range 3 nm to 650 microm. Quantitative real-time PCR analyses showed that neonatal rat calvarial osteoblasts attached to Si-rich SCPC expressed 5- and 26-fold higher osteocalcin mRNA levels compared to cells attached to ProOsteon hydroxyapatite disks and tissue culture polystyrene plates respectively, after four days in culture. Results of the present study strongly suggest that porous, bioactive resorbable SCPCs can serve as tissue engineering scaffolds for cell delivery to treat load-bearing bone defects in orthopedic and maxillofacial surgeries.

NtLRP1, a tobacco leucine-rich repeat gene with a possible role as a modulator of the hypersensitive response.

Plant defense responses against pathogens often involve the restriction of the pathogen to its site of penetration achieved through the combined effects of the hypersensitive response (HR) and its tightly connected localized acquired resistance (LAR). The tobacco DD9-3 expressed sequence tag was previously isolated from a screen designed to isolate genes induced early during the HR, thus potentially involved in the induction/regulation of the HR or LAR. Translation of the open reading frame of DD9-3 revealed a leucine-rich repeat (LRR) domain highly homologous with the receptor domain of a receptor kinase, suggesting a potential function in signaling pathways. The full-length cDNA was cloned. It encodes a small (232 amino acids) LRR protein, designated Nicotiana tabacum leucine-rich protein 1 (NtLRP1), containing a signal peptide, four leucine zipper repeats, five LRR repeats, and a C-terminal domain rich in proline. NtLRP1 expression is induced early during the HR initiated by elicitins, Ralstonia solanacearum, or Tobacco mosaic virus. NtLRP1 coupled with the green fluorescent protein localizes to the endoplasmic reticulum (ER). Loss-of-function through virus-induced gene silencing or through RNA interference did not modify the elicitin-induced HR or LAR. Gain-of-function experiments through transient Agrobacterium tumefaciens-mediated NtLRP1 expression in tobacco leaves caused the suppression of the HR induced by 2 nM elicitin and delayed the HR when the elicitin was applied at higher concentrations. The results suggest that NtLRP1 acts as a modulator of the HR and that retention in the ER is essential for its function.