Biological effects of silk fibroin 3D scaffolds on stem cells from human exfoliated deciduous teeth (SHEDs).

The aim is to investigate in vitro biological effects of silk fibroin 3D scaffolds on stem cells from human exfoliated deciduous teeth (SHEDs) in terms of proliferation, morphological appearance, cell viability, and expression of mesenchymal stem cell markers. Silk fibroin 3D scaffolding materials may represent promising suitable scaffolds for their application in regenerative endodontic therapy approaches. SHEDs were cultured in silk fibroin 3D scaffolds. Then, cell numbers were counted and the Alamar blue colorimetric assay was used to analyse cell proliferation after 24, 48, 72, and 168 h of culture. The morphological features of SHEDs cultured on silk fibroin scaffolds were evaluated by scanning electron microscopy (SEM). Finally, cell viability and the expression of mesenchymal stem cell markers were analysed by flow cytometry. One-way analysis of variance (ANOVA) followed by a Bonferroni post-test was performed (P < 0.05). At 24 and 48 h of culture, SHED proliferation on scaffolds was modest compared to the control although still significant (p < 0.05). However, cell proliferation progressively increased from 72 to 168 h compared with the control (p < 0.001; p < 0.01). In addition, flow cytometry analysis showed that the culture of SHEDs on silk fibroin scaffolds did not significantly alter the level of expression of the mesenchymal markers CD73, CD90, or CD105 up to 168 h; in addition, cell viability in silk fibroin was similar to than obtained in plastic. Moreover, SEM studies revealed a suitable degree of proliferation, cell spreading, and attachment, especially after 168 h of culture. The findings from the current study suggest that silk fibroin 3D scaffolds had a favourable effect on the biological responses of SHEDs. Further in vivo investigations are required to confirm these results.

Effects of composite films of silk fibroin and graphene oxide on the proliferation, cell viability and mesenchymal phenotype of periodontal ligament stem cells.

In regenerative dentistry, stem cell-based therapy often requires a scaffold to deliver cells and/or growth factors to the injured site. Graphene oxide (GO) and silk fibroin (SF) are promising biomaterials for tissue engineering as they are both non toxic and promote cell proliferation. On the other hand, periodontal ligament stem cells (PDLSCs) are mesenchymal stem cells readily accessible with a promising use in cell therapy. The purpose of this study was to investigate the effects of composite films of GO, SF and GO combined with fibroin in the mesenchymal phenotype, viability, adhesion and proliferation rate of PDLSCs. PDLSCs obtained from healthy extracted teeth were cultured on GO, SF or combination of GO and SF films up to 10 days. Adhesion level of PDSCs on the different biomaterials were evaluated after 12 h of culture, whereas proliferation rate of cells was assessed using the MTT assay. Level of apoptosis was determined using Annexin-V and 7-AAD and mesenchymal markers expression of PDLSCs were analyzed by flow cytometry. At day 7 of culture, MTT experiments showed a high rate of proliferation of PDLSCs growing on GO films compared to the other tested biomaterials, although it was slightly lower than in plastic (control). However PDLSCs growing in fibroin or GO plus fibroin films showed a discrete proliferation. Importantly, at day 10 of culture it was observed a significant increase in PDLSCs proliferation rate in GO films compared to plastic (P < 0.05), as well as in GO plus fibroin compared to fibroin alone (P < 0.001). Flow cytometry analysis showed that culture of PDLSCs in fibroin, GO or GO plus fibroin films did not significantly alter the level of expression of the mesenchymal markers CD73, CD90 or CD105 up to 168 h, being the cell viability in GO even better than obtained in plastic. Our findings suggest that the combination of human dental stem cells/fibroin/GO based-bioengineered constructs have strong potential for their therapeutic use in regenerative dentistry.

Construction of a BmNPV polyhedrin-plus Bac-to-Bac baculovirus expression system for application in silkworm, Bombyx mori.

The baculovirus expression vector system is one of the most powerful and versatile eukaryotic expression systems available. However, as the recombinant baculovirus is usually generated by replacing the foreign gene into the polyhedrin locus, the resulting polyhedrin-negative virus is less infectious to the host larvae when administered via oral ingestion. This limits the large-scale production of the recombinant protein, as the host larvae can only be inoculated through dorsal injection, which is a laborious task. In this paper, we describe a new Bombyx mori nucleopolyhedrovirus polyhedrin-plus Bac-to-Bac baculovirus expression system for application in silkworm, B. mori. In this system, the foreign gene and the polyhedrin are co-expressed, and polyhedra are produced as in the wild-type virus, and thus the recombinant baculovirus can be used directly via oral infection. It effectively improves the efficiency of the baculovirus expression system and also widens the application of baculovirus in other fields, such as the development of new biological insecticides.

Electrochemical Exfoliation of Graphite in Aqueous Sodium Halide Electrolytes toward Low Oxygen Content Graphene for Energy and Environmental Applications.

Graphene and graphene-based materials have shown great promise in many technological applications, but their large-scale production and processing by simple and cost-effective means still constitute significant issues in the path of their widespread implementation. Here, we investigate a straightforward method for the preparation of a ready-to-use and low oxygen content graphene material that is based on electrochemical (anodic) delamination of graphite in aqueous medium with sodium halides as the electrolyte. Contrary to previous conflicting reports on the ability of halide anions to act as efficient exfoliating electrolytes in electrochemical graphene exfoliation, we show that proper choice of both graphite electrode (e.g., graphite foil) and sodium halide concentration readily leads to the generation of large quantities of single-/few-layer graphene nanosheets possessing a degree of oxidation (O/C ratio down to ∼0.06) lower than that typical of anodically exfoliated graphenes obtained with commonly used electrolytes. The halide anions are thought to play a role in mitigating the oxidation of the graphene lattice during exfoliation, which is also discussed and rationalized. The as-exfoliated graphene materials exhibited a three-dimensional morphology that was suitable for their practical use without the need to resort to any kind of postproduction processing. When tested as dye adsorbents, they outperformed many previously reported graphene-based materials (e.g., they adsorbed ∼920 mg g-1 for methyl orange) and were useful sorbents for oils and nonpolar organic solvents. Supercapacitor cells assembled directly from the as-exfoliated products delivered energy and power density values (up to 15.3 Wh kg-1 and 3220 W kg-1, respectively) competitive with those of many other graphene-based devices but with the additional advantage of extreme simplicity of preparation.

Effect of aqueous and particulate silk fibroin in a rat model of experimental colitis.

Silk fibroin (SF) has anti-inflammatory properties and promotes wound healing. Moreover, SF particles act as carriers of active drugs against intestinal inflammation due to their capacity to deliver the compound to the damaged colonic tissue. The present work assesses the effect of SF in the trinitrobenzenesulfonic acid model of rat colitis that resembles human intestinal inflammation. SF (8mg/kg) was administered in aqueous solution orally and in two particulate formats by intrarectal route, following two technologies: spray drying to make microparticles and desolvation in organic solvent to produce nanoparticles. SF treatments ameliorated the colonic damage, reduced neutrophil infiltration and improved the compromised oxidative status of the colon. They also reduced the gene expression of pro-inflammatory cytokines like IL-1ß and the anti-inflammatory cytokine IL-10. Moreover, they improved the intestinal wall integrity by increasing the gene expression of some of its markers (villin, trefoil factor-3 and mucins), thus accelerating the healing. The immunomodulatory properties of SF particles were also tested in vitro in macrophages: they activated the immune response in basal conditions without increasing it after a pro-inflammatory insult. In conclusion, SF particles could be useful as carriers to deliver active drugs to the damaged intestinal colon with additional anti-inflammatory and healing properties.

Investigating the Dispersion Behavior in Solvents, Biocompatibility, and Use as Support for Highly Efficient Metal Catalysts of Exfoliated Graphitic Carbon Nitride.

The liquid-phase exfoliation of graphitic carbon nitride (g-C3N4) to afford colloidal dispersions of two-dimensional flakes constitutes an attractive route to facilitate the processing and implementation of this novel material toward different technological applications, but quantitative knowledge about its dispersibility in solvents is lacking. Here, we investigate the dispersion behavior of exfoliated g-C3N4 in a wide range of solvents and evaluate the obtained results on the basis of solvent surface energy and Hildebrand/Hansen solubility parameters. Estimates of the three Hansen parameters for exfoliated g-C3N4 from the experimentally derived data yielded δD ≈ 17.8 MPa(1/2), δP ≈ 10.8 MPa(1/2), and δH ≈ 15.4 MPa(1/2). The relatively high δH value suggested that, contrary to the case of other two-dimensional materials (e.g., graphene or transition metal dichalcogenides), hydrogen-bonding plays a substantial role in the efficient interaction, and thus dispersibility, of exfoliated g-C3N4 with solvents. Such an outcome was attributed to a high density of primary and/or secondary amines in the material, the presence of which was associated with incomplete condensation of the structure. Furthermore, cell proliferation tests carried out on thin films of exfoliated g-C3N4 using murine fibroblasts suggested that this material is highly biocompatible and noncytotoxic. Finally, the exfoliated g-C3N4 flakes were used as supports in the synthesis of Pd nanoparticles, and the resulting hybrids exhibited an exceptional catalytic activity in the reduction of nitroarenes.

Spread of Tomato yellow leaf curl virus Sar from the Mediterranean Basin: Presence in the Canary Islands and Morocco.

Severe outbreaks of tomato yellow leaf curl disease occurred during summer and autumn 1999 in tomato (Lycopersicon esculentum Mill.) crops in the Vecindario Region of Gran Canaria (Canary Islands, Spain) and Agadir (southwestern Atlantic coast of Morocco). Symptoms of the disease included upward curling of leaflet margins, reduction of leaflet area, and yellowing of young leaves, as well as stunting and flower abortion. High populations of whiteflies, Bemisia tabaci Gen., were present on tomatoes in Agadir, and analysis of adult individuals by random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) identified them as the biotype Q. Samples were collected from symptomatic tomato plants: 5 plants from Gran Canaria and 22 from three areas in Agadir, (7 from Agadir/1, 12 from Agadir/2, and 3 from Agadir/3) in the Koudya Region. Samples were analyzed for Tomato yellow leaf curl virus (TYLCV) Sar or Is (genus Begomovirus, family Geminiviridae) infection by squash blot hybridization under high stringency conditions with digoxigenin-labeled DNA probes specific to TYLCV-Sar or -Is, as described previously (1,3). The TYLCV-Sar probe hybridized to the five samples from Gran Canaria, and the TYLCV-Is probe hybridized to the 22 samples from Agadir. The TYLCV-Sar probe also hybridized to the three samples from Agadir/3. Primer pairs MA-14/MA-15 and MA-30/MA-31, designed for specific amplification of the intergenic region (IR) of TYLCV-Sar or -Is reported from Spain, respectively (1), were used in PCR to amplify one sample each from Gran Canaria, Agadir/1, and Agadir/3. A fragment of the expected size was obtained from the samples from Gran Canaria and Agadir/3 using MA14/MA15 (342 bp) and from the two samples from Agadir using MA30/MA31 (357 bp). PCR products were directly sequenced (GenBank Accession nos. AF215819 to AF215822). The nucleotide sequences of the IR fragments amplified from the Gran Canaria and Agadir/3 sample using MA-14/MA-15 indicated their closest relationship (99.0 and 96.7% identity, respectively) was to the corresponding region of a TYLCV-Sar isolate reported from Spain (GenBank Accession no. L27708). The nucleotide sequences of the IR fragments amplified from the Agadir/1 and Agadir/3 samples using MA-30/MA-31 indicated their closest relationship (98.1% identity) was to the corresponding region of the TYLCV-Is isolate reported from Spain (GenBank Accession no. AF071228). Based on the hybridization and sequence data, we conclude that the symptomatic plants from Gran Canaria were infected by TYLCV-Sar, those from Agadir/1 and Agadir/2 were infected by TYLCV-Is, and those from Agadir/3 had mixed infections with TYLCV-Is and TYLCV-Sar. The presence of TYLCV-Is in Morocco has been described recently (2). However, this is the first report of TYLCV-Sar in the Canary Islands and Morocco and extends its geographic range beyond the Iberian Peninsula and Italy. References: (1) J. Navas-Castillo et al. Plant Dis. 83:29, 1999. (2) M. Peterschmitt et al. Plant Dis. 83:1074, 1999. (3) S. Sánchez-Campos et al. Phytopathology 89:1038, 1999.

Reproductive Fitness and Random Amplified Polymorphic DNA Variation among Isolates of Pratylenchus vulnus.

The reproductive fitness of seven isolates of Pratylenchus vulnus from different geographical areas and hosts was assessed in monoxenic cultures (carrot), and greenhouse cultures (plum, sour orange, and quince). The genetic makeup of the different isolates was compared by Random Amplified Polymorphic DNA (RAPD-PCR). The apple (PvAP-S) and apricot (PvAT-F) isolates reproduced less in monoxenic cultures than the rose (PvRO-S) and walnut (PvWA-A and PvWA-U) isolates. On plum, the rose isolate (PvRO-S) reproduced better than the apple (PvAP-S) and walnut isolate from the United States (PvWA-U). On sour orange, the apple (PvAP-S), unknown origin (PvU-UK), and walnut isolate from Argentina (PvWA-A) multiplied well, whereas the walnut isolate from the United States (PvWA-U), apricot (PvAT-F), and rose (PvRO-S) did not. On quince, the apple (PvAP-S) and walnut (PvWA-U) isolates showed a higher reproduction than the one from unknown origin (PvU-UK). RAPD-PCR patterns among the seven P. vulnus isolates were similar, although high intraspecific varibility was detected. Very few bands of P. neglectus were shared by any population of P. vulnus. A high degree of similarity was found among the patterns corresponding to the rose (PvRO-S), apple (PvAP-S), walnut from the United States (PvWA-U), and unknown origin (PvUK-U) isolates. The apricot isolate (PvAT-F) was the most dissimilar among the seven isolates. No correlation could be established between the genetic variation of P. vulnus detected by RAPD-PCR and reproductive fitness. Results demonstrate high genetic varibility between geographically separated populations of P. vulnus.

Comparative inhibitory activity of the stilbenes resveratrol and oxyresveratrol on African swine fever virus replication.

Stilbenols are polyphenolic phytoalexins produced by plants in response to biotic or abiotic stress. These compounds have received much attention because of their significant biological effects. One of these is their antiviral action, which has previously been documented for two members of this class, namely resveratrol and oxyresveratrol. Here we tested the antiviral effect of these two compounds on African swine fever virus, the only member of the newly created family Asfarviridae and a serious limitation to porcine production worldwide. Our results show a potent, dose-dependent antiviral effect of resveratrol and oxyresveratrol in vitro. Interestingly, this antiviral activity was found for these synthetic compounds and also for oxyresveratrol extracted from new natural sources (mulberry twigs). The antiviral effect of these two drugs was demonstrated at concentrations that do not induce cytotoxicity in cultured cells. Moreover, these antivirals achieved a 98-100% reduction in viral titers. Both compounds allowed early protein synthesis but inhibited viral DNA replication, late viral protein synthesis and viral factory formation.

Efficient production of canine interferon-alpha in silkworm Bombyx mori by use of a BmNPV/Bac-to-Bac expression system.

We exploited the silkworm Bombyx mori for the production of recombinant canine interferon-alpha (CaIFN-alpha). The recombinant baculovirus harboring canine interferon gene was rapidly generated by the BmNPV/Bac-to-Bac system that was recently developed. In B. mori-derived cell lines, the expression of the recombinant protein reached maximal levels around 72-96 h post-infection. For the isolation of the expressed recombinant protein from B. mori larvae, the whole bodies of the infected larvae were homogenized, and the expressed protein was purified by affinity chromatography. Based on the fact that the recombinant CaIFN-alpha showed two bands on the sodium dodecyl sulfate polyacrylamide gel electrophoresis pattern, the expressed protein was thought to be glycosylated. The rCaIFN-alpha yield was about 528 microg per larva, showing that the expression in silkworm was successful. Furthermore, the recombinant protein was proven to be able to inhibit the infection of Madin-Darby canine kidney cells by the vesicular stomatitis virus, indicating that it is biologically active in vitro. The method established in this study provides an efficient way to produce a large amount of CaIFN-alpha and paves the way for further utilization of this protein as a therapeutic agent or vaccine adjuvant in dogs.

Multiple origins of cultivated grapevine (Vitis vinifera L. ssp. sativa) based on chloroplast DNA polymorphisms.

The domestication of the Eurasian grape (Vitis vinifera ssp. sativa) from its wild ancestor (Vitis vinifera ssp. sylvestris) has long been claimed to have occurred in Transcaucasia where its greatest genetic diversity is found and where very early archaeological evidence, including grape pips and artefacts of a 'wine culture', have been excavated. Whether from Transcaucasia or the nearby Taurus or Zagros Mountains, it is hypothesized that this wine culture spread southwards and eventually westwards around the Mediterranean basin, together with the transplantation of cultivated grape cuttings. However, the existence of morphological differentiation between cultivars from eastern and western ends of the modern distribution of the Eurasian grape suggests the existence of different genetic contribution from local sylvestris populations or multilocal selection and domestication of sylvestris genotypes. To tackle this issue, we analysed chlorotype variation and distribution in 1201 samples of sylvestris and sativa genotypes from the whole area of the species' distribution and studied their genetic relationships. The results suggest the existence of at least two important origins for the cultivated germplasm, one in the Near East and another in the western Mediterranean region, the latter of which gave rise to many of the current Western European cultivars. Indeed, over 70% of the Iberian Peninsula cultivars display chlorotypes that are only compatible with their having derived from western sylvestris populations.

Genetic structure of Atriplex halimus populations in the Mediterranean Basin.

BACKGROUND AND AIMS: The saltbush Atriplex halimus is a chenopodiaceous plant well adapted to dry saline habitats and widely distributed in the Mediterranean Basin. A study was carried out to analyse the genetic diversity of A. halimus at the level of the Mediterranean Basin. METHODS: To assess the intra- and interpopulational variation of A. halimus a total of 51 populations and six plants per populations was analysed with the RAPD-PCR technique. For the study of the phylogeny of the populations, 21 samples of A. halimus and seven samples of other species of Atriplex were analysed by the sequencing of the ITS (internal transcribed spacer) region of the ribosomal DNA. KEY RESULTS: The AMOVA analysis of the RAPD results showed that populations were divided into two discrete genetic groups, as the variation among groups accounted for 54.36 % of the total variance of the collection. At the same time, the intrapopulational diversity was high, as 301 out of 306 plants analysed constituted an individual RAPD haplotype. The sequencing of the ITS region also showed a significant separation of the two genetic groups, with a genetic distance of 0.023 nucleotide substitutions per site. Using A. breweri, A. canescens, A. glauca and A. prostrata as outgroups in the phylogenetic analysis, A. breweri and A. canescens are the species closest to A. halimus from this group, while A. prostrata is the most distant. CONCLUSIONS: The present work indicates that two genetic groups of A. halimus can be distinguished after analysing the genetic diversity of 51 populations from ten countries in the Mediterranean Basin.

Genetic diversity of Iberian populations of Bemisia tabaci (Hemiptera: Aleyrodidae) based on random amplified polymorphic DNA-polymerase chain reaction.

The genetic structure of six Iberian populations of the whitefly Bemisia tabaci, two of them biotype Q, one biotype B, and the other three a mixture of both, has been studied using random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). A total of 336 individuals was completely discriminated by means of 234 scored bands. Separate analyses of molecular variance of haploid males and diploid females using the pairwise number of differences between haplotypes showed that biotypes contribute significantly more to the observed variability than populations within biotypes. On average, gene flow between two biotypes of the same population is lower than between populations of identical biotypes. On the basis of these results and the nondetection under natural conditions of a single hybrid, we consider that both biotypes are genetically isolated under the ecological conditions prevailing in the south Iberian Peninsula. All populations of biotype Q presented similar values of intrapopulational diversity, which were higher than the values shown by populations of biotype B.

Genetic relationships among biotypes of Bemisia tabaci (Hemiptera: Aleyrodidae) based on AFLP analysis.

Genetic similarities between 13 samples belonging to nine reference biotypes and two field populations of Bemisia tabaci (Gennadius), one field population of B. medinae Gómez-Menor and another of B. afer Priesner & Hosny, were evaluated using amplified fragment length polymorphism (AFLP) markers. The results indicate that B. tabaci biotypes can be grouped together with a minimum similarity coefficient of 0.32 and separated from the two other species with a similarity coefficient of 0.07. Bemisia tabaci biotypes were grouped in four clusters which comprised: (i) Near East and Indian subcontinent biotypes; (ii) B and Q biotypes plus a Nigerian population from cowpea; (iii) New World A biotype; and (iv) S biotype and a Nigerian population from cassava. These results were consistent with a previous grouping of biotypes based on RAPD-PCR analysis. The AFLP assay allowed the scoring of a total of 354 polymorphic bands in two reaction events with the use of two primer combinations.

Complete sequence of the Pepino mosaic virus RNA genome.

We have determined the complete nucleotide sequence (Accession No. AF484251) of the Pepino mosaic virus (PepMV) RNA genome. PepMV is the etiological agent of a new disease which affects tomato crops in Europe and North America. The PepMV genome consists of one single stranded positive sense RNA 6410 nt long that contains five open reading frames (ORFs). ORF 1 is the putative RNA dependent RNA polymerase (RdRp), as it has the characteristic methyltransferase, NTP-binding and polymerase motifs. ORF 2 to 4 form the PepMV triple gene block. ORF 5 codes for the capsid protein. Two short untranslated regions flank the coding regions and there is a poly(A) tail at the 3'end of the genomic RNA. Thus, the genome organization of PepMV is that of a typical member of the genus Potexvirus. The nucleotide sequence obtained shares an overall 99% identity with the genomic RNA of a PepMV isolate from UK which has been partially sequenced. Protein coded by ORF4 is the least conserved between both isolates (95% amino acid identity), whereas proteins coded by ORF3 and ORF5 are identical.