Chemical, structural and cytotoxicity characterisation of experimental fluoride-doped calcium phosphates as promising remineralising materials for dental applications.

OBJECTIVES: This study aimed at evaluating the cytotoxicity, chemical and structural properties of experimental fluoride-doped calcium-phosphates as potential remineralising materials for dental applications. METHODS: Experimental calcium phosphates were formulated using ß-tricalcium phosphate, monocalcium phosphate monohydrate, calcium hydroxide, and different concentrations of calcium/sodium fluoride salts [(5 wt%: VSG5F), (10 wt%: VSG10F), (20 wt%: VSG20F)]. A fluoride-free calcium phosphate (VSG) was used as control. Each tested material was immersed in simulated body fluid (SBF), (24 h, 15 and 30 days) to assess their ability to crystallise into apatite-like. Cumulative fluoride release was assayed up to 45 days. Moreover, each powder was placed into a medium containing human dental pulp stem cells (200 mg/mL) and their cytotoxicity was analysed using the 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) assay (24 h, 48 h and 72 h incubation). These latter results were statistically analysed by ANOVA and Tukey's test (α = 0.05). RESULTS: All the experimental VSG-F materials produced fluoride-containing apatite-like crystals after SBF immersion. VSG20F presented prolonged release of fluoride ions into the storage media (45d). VSG, VSG10F and VSG20F showed a significant cytotoxicity at dilution of 1:1, while at 1:5, only VSG and VSG20F demonstrated a reduction in cell viability. At lower dilutions (1:10, 1:50 and 1:100) all specimens showed no significant toxicity to hDPSCs, but an increase in cell proliferation. SIGNIFICANCE: The experimental fluoride-doped calcium-phosphates are biocompatible and possess a clear ability to evoke fluoride-containing apatite-like crystallisation. Hence, they may be promising remineralising materials for dental applications.

Magnetic solid phase extraction as a nonchromatographic method for the quantification of ultratrace inorganic arsenic in rice by inductively coupled plasma-optical emission spectrometry (ICP OES).

An alternative analytical method was developed for the quantification of inorganic arsenic (iAs) in rice by ICP OES. Iron nanoparticles modified with an organophosphorus compound were used as the solid phase for MSPE of iAs from the plant matrix. The MSPE procedure was performed using 4 mL of a buffer solution with pH 4.0, 20 mg of the nanomaterial, and a 15-min extraction time. The total As (tAs) by ICP OES was also quantified using the same MSPE procedure after solubilization of the samples by a block digester. The accuracy of tAs and iAs quantification was verified using CRM NIST 1568b (97 % and 101 % recovery, respectively). The precision (RSD < 15 %) and LOD and LOQ (1.08 and 3.70 µg kg-1, respectively) of the proposed method were satisfactory. The rice samples had tAs contents between 0.090 and 0.295 mg kg-1 and iAs mass fractions between 0.055 and 0.109 mg kg-1.

Non-chromatographic arsenic speciation analyses in wild shrimp (Farfantepenaeus brasiliensis) using functionalized magnetic iron-nanoparticles.

A new iron-magnetic nanomaterial functionalized with organophosphorus compound was used as solid-phase for arsenic speciation analysis in seafood samples by ICP-MS. The procedure was optimized using chemometric tools and the variables pH = 4.0, 15 min extraction time, and 20 mg of mass of material were obtained as the optimum point. The inorganic arsenic (iAs) extracted using nanoparticles presented concentrations between 20 and 100 µg kg-1 in the evaluated samples. The method was validated for accuracy using CRMs DOLT-5 and DORM-4. It was possible to reuse the same magnetic nanomaterial for 6 successive cycles, and we obtained a detection limit of 16.4 ng kg-1. The proposed method is suitable for the use of inorganic speciation of As, presenting good accuracy, precision, relatively low cost, and acquittance to green chemistry principles.

A new heterofunctional support for enzyme immobilization: PEI functionalized Fe3O4 MNPs activated with divinyl sulfone. Application in the immobilization of lipase from Thermomyces lanuginosus.

Lipase from Thermomyces lanuginosus (TLL) was immobilized onto a novel heterofunctional support, divinyl sulfone (DVS) superparamagnetic nanoparticles (SPMNs) functionalized with polyethyleneimine (PEI). Particle size and zeta potential measurements, elemental analysis, X-ray powder diffraction, magnetic measurements, and infrared spectroscopy analysis were used to characterize the TLL preparations. At pH 10, it was possible to achieve 100 % of immobilization yield in 1 h. The immobilization pH gives TLL preparations with different stabilities; indeed the TLL preparation immobilized at pH 5.0 was the most stable during the thermal inactivation at all pH values. For the hydrolysis of racemic methyl mandelate, the nanobiocatalysts immobilized at pH 5.0 and blocked with ethylenediamine (EDA) and ethanolamine (ETA) obtained good enantioselectivities (68 % and 72 %, respectively) with high catalytic activities in the reaction medium at pH 7.0. The operational stability of the systems was evaluated in the esterification reaction of benzyl alcohol, obtaining up to 61 % conversion after the seventh reaction cycle. These results show that SPMN@PEI-DVS support is a robust strategy for the easy and rapid recovery of the nanobiocatalyst by applying a magnetic field, showing great potential for industrial applications.

Ethyl Butyrate Synthesis Catalyzed by Lipases A and B from Candida antarctica Immobilized onto Magnetic Nanoparticles. Improvement of Biocatalysts' Performance under Ultrasonic Irradiation.

The synthesis of ethyl butyrate catalyzed by lipases A (CALA) or B (CALB) from Candida antarctica immobilized onto magnetic nanoparticles (MNP), CALA-MNP and CALB-MNP, respectively, is hereby reported. MNPs were prepared by co-precipitation, functionalized with 3-aminopropyltriethoxysilane, activated with glutaraldehyde, and then used as support to immobilize either CALA or CALB (immobilization yield: 100 ± 1.2% and 57.6 ± 3.8%; biocatalysts activities: 198.3 ± 2.7 Up-NPB/g and 52.9 ± 1.7 Up-NPB/g for CALA-MNP and CALB-MNP, respectively). X-ray diffraction and Raman spectroscopy analysis indicated the production of a magnetic nanomaterial with a diameter of 13.0 nm, whereas Fourier-transform infrared spectroscopy indicated functionalization, activation and enzyme immobilization. To determine the optimum conditions for the synthesis, a four-variable Central Composite Design (CCD) (biocatalyst content, molar ratio, temperature and time) was performed. Under optimized conditions (1:1, 45 °C and 6 h), it was possible to achieve 99.2 ± 0.3% of conversion for CALA-MNP (10 mg) and 97.5 ± 0.8% for CALB-MNP (12.5 mg), which retained approximately 80% of their activity after 10 consecutive cycles of esterification. Under ultrasonic irradiation, similar conversions were achieved but at 4 h of incubation, demonstrating the efficiency of ultrasound technology in the enzymatic synthesis of esters.

Further stabilization of lipase from Pseudomonas fluorescens immobilized on octyl coated nanoparticles via chemical modification with bifunctional agents.

The lipase from Pseudomonas fluorescens (PFL) was adsorbed on superparamagnetic NiZnFe2O4 octyl-nanoparticles via interfacial activation, producing the biocatalyst OCTYL-NANO-PFL. In order to further improve the stability of the immobilized lipase, the immobilized enzyme biocatalyst was chemically modified with different concentrations of diverse bifunctional molecules (glutaraldehyde (GA), divinylsulfone (DVS) or p-benzoquinone (BQ)). The concentrations of bifunctional agents were varied (0.5, 1, 2.5 and 5% (v/v for GA and DVS and w/v for BQ)). The results showed a greatly improved stability after chemical modification with all bifunctional molecules, mainly with 5% (v/v) GA or 1% (v/v) DVS. The biocatalysts OCTYL-NANO-PFL-GA 5% and -DVS 1% were about 60 folds more stable at pH 7 than the unmodified preparation and, at pH 5, >200 folds for 5% GA modified enzyme. The most stable BQ treated biocatalysts, OCTYL-NANO-PFL-BQ 0.5%, was about 8.3 more stable than OCTYL-NANO-PFL at pH 7, while was 20 fold more stable at pH 9.

Time-resolved molecular dynamics of single and double hydrogen migration in ethanol.

Being the lightest, most mobile atom that exists, hydrogen plays an important role in the chemistry of hydrocarbons, proteins and peptides and most biomolecules. Hydrogen can undergo transfer, exchange and migration processes, having considerable impact on the chemical behavior of these molecules. Although much has been learned about reaction dynamics involving one hydrogen atom, less is known about those processes where two or more hydrogen atoms participate. Here we show that single and double hydrogen migrations occurring in ethanol cations and dications take place within a few hundred fs to ps, using a 3D imaging and laser pump-probe technique. For double hydrogen migration, the hydrogens are not correlated, with the second hydrogen migration promoting the breakup of the C-O bond. The probability of double hydrogen migration is quite significant, suggesting that double hydrogen migration plays a more important role than generally assumed. The conclusions are supported by state-of-the-art molecular dynamics calculations.

Comparison of the immobilization of lipase from Pseudomonas fluorescens on divinylsulfone or p-benzoquinone activated support.

NiZnFe2O4 superparamagnetic nanoparticles were coated with silica by impregnation with tetraethoxysilane (TEOS) and further activated with divinylsulfone (DVS) and p-benzoquinone (BQ) for covalent immobilization lipase from Pseudomonas fluorescens (PFL), producing the biocatalysts TEOS-NANO-DVS-PFL and TEOS-NANO-BQ-PFL. The optimal conditions for enzyme immobilization were found to be pH 7 and 0.1 M of both activating reagents. PFL was also immobilized on TEOS nanoparticles without any activation as a reference (TEOS-NANO-PFL). Results indicated that TEOS could be released from the nanoparticles at alkaline pH value. Optimal TEOS-NANO-PFL exhibited a recovered activity of 55% and a t1/2(60°C) of just over 150 min; while TEOS-NANO-DVS-PFL showed 82% of activity recovered and t1/2(60°C) of 225 min; being the TEOS-NANO-BQ-PFL the biocatalyst offering the best results (89% of recovered activity and a half-life over 1440 min), the maximum enzyme load was ≈300 U/g.

First Report of Tomato torrado virus Infecting Tomato in Italy.

In 2009 and 2010, approximately 2% of plants had disease symptoms, including initial leaflet chlorosis that later developed into necrotic spots and general necroses along the leaflet. Fruit production on affected plants was substantially reduced and necroses were also present. Total RNA was extracted from five symptomatic plant samples using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and analyzed by reverse transcription (RT)-PCR with specific primer pair: TR2F (5' GAAGGACGAAGAGCGACTG 3'), and TR2R (5' AAGGTAGGTATGCGTTTGC 3') (1). The primers amplified a 575-bp fragment within the coat protein Vp23 of Tomato torrado virus (ToTV). No RT-PCR products were observed when water or asymptomatic tomato plants were used as controls. The RT-PCR products were purified and directly sequenced in both directions. Pair-wise similarity analysis confirmed the presence of ToTV with 99% similarity to isolate PRI-ToTV0301 (GenBank Accession No. DQ388880) and 98% similarity to isolate Kra (Accession No. EU652402). A representative sequence was deposited with GenBank (Accession No. GU903899). To further confirm the presence of ToTV, dsRNA analysis was conducted on all five symptomatic plants and one healthy tomato plant (2). Electrophoresis of dsRNA showed two bands of approximately 5,400 and 7,800 nucleotides long, typical of ToTV in all samples, while a third band between the other two (approximately 6,400 nt) was detected. Serological testing using double-antibody sandwich-ELISA was also conducted on the five symptomatic and 25 additional plants from the same greenhouse that displayed typical Pepino mosaic virus (PepMV) symptoms only. Antibodies used for serological testing screened for the presence of PepMV, Tomato spotted wilt virus, Cucumber mosaic virus, and Tomato mosaic virus (Loewe Biochemica, Sauerlach, Germany). These tests detected PepMV in all samples with disease symptoms typical of PepMV, and in three of the five samples with the newly described symptoms. To our knowledge, this is the first report of ToTV in Italy, and in some plants, co-infection with PepMV was likely. All ToTV-infected tomato plants in the greenhouse were destroyed. References: (1) H. Pospieszny et al. Plant Dis. 91:1364, 2007. (2) J. Sambrook et al. Molecular Cloning. A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory Press, Woodbury, NY, 1989.

The First Citrus tristeza virus Outbreak Found in a Relevant Citrus Producing Area of Sicily, Italy.

In the course of a survey to select superior old citrus lines in the area of Siracusa (Sicily, Italy), trees in several blocks of Fortune (Citrus reticulata Blanco), Nova (C. reticulata Blanco), Satsuma (C. unshiu (Macfad.) mandarins Marc.), and Marsh grapefruit (C. paradisi Macfad.) propagated on sour orange (C. aurantium L.) rootstock showed stunting, decline, dieback, and small-sized fruits. Stunting was particularly evident in grapefruit. Declined plants consistently showed pin-holing in the cambial face of sour orange bark below the bud union line, which is often associated with Citrus tristeza virus (CTV) infection. Young shoots from 600 Fortune, 300 Nova, 400 Satsuma, and 20 Marsh grapefruit plants showing decline were analyzed by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) (Loewe Phytodiagnostica Biochemica, Sauerlach, Germany) and by immunoprinting-ELISA (Agritest Srl Valenzano-Bari-Italy) using CTV specific polyclonal antibodies. All decline tree samples reacted positively with both techniques while healthy greenhouse controls were negative. Total RNA was extracted from 50 of those plants, 25 Fortune and 15 Nova mandarins, 5 Satsuma, and 5 Marsh grapefruit (Qiagen RNeasy Plant minikit, Qiagen S.P.A., Milan, Italy), and tested in reverse transcription-polymerase chain reaction (RT-PCR) using specific primers for genes p20 (forward 5'-CGA GCT TAC TTT AGT GTT A-3' from CTV T36 genomic position 17767-17786 and reverse 5'-TAA TGT CAA ACT GAC CGC from CTV T36 position 18269-18286) and p23 (forward 5'-ACT AAC TTT AAT TCG AAC A-3' from CTV T36 position 18347-18286 and reverse 5'-AAC TTA TTC CGT CCA CTT C-3' from CTV T36 position 19026-19044) (2). In all cases, DNA fragments of the expected size were amplified. Equivalent samples from CTV-free greenhouse control plants did not react in ELISA and yielded no DNA after amplification with the same primers. When the history of the plants in the affected blocks was traced, it was found that all Fortune, Nova, satsuma and Marsh grapefruit trees had been propagated from budwood illegally imported from Spain 10 years before, suggesting the possibility that the imported buds were infected with CTV. The estimated number of infected plants in the area of Siracusa is approximately 10,000, and some evidence suggests that the virus might be spreading in the area (work in progress). Only scattered CTV-infected trees had been detected in Italy previously (1). To our knowledge, this is the first report of an important CTV outbreak in Italy. Additional surveys are being conducted to get a more accurate estimation of the CTV incidence, to determine if the virus is being dispersed by aphid vectors, and to biologically and molecularly characterize the virus strains present in the affected area. Presently, there are approximately 100,000 ha of citrus in Sicily, mostly grown on decline susceptible sour orange rootstock. The presence and potential spread of CTV is a major threat for this citrus industry. References: (1) M. Davino and G. Terranova. Frutticoltura 61:18, 1999. (2) A. Sambade et al. Plant Pathol. 51:257, 2002.

First Report of Tomato chlorosis virus in Italy.

During winter 2000-2001, an unusual disease of tomato was observed in some greenhouses in Sardinia, Sicily, and Apulia, in southern Italy. Plants were chlorotic and reduced in size, expanded leaves showed interveinal yellowing, and older leaves developed interveinal reddish-bronze necrosis and downward rolling. The symptoms resembled those recently reported from Portugal (1) as induced by Tomato chlorosis virus (ToCV) (family Closteroviridae, genus Crinivirus), a whitefly-transmitted virus new to Europe. Symptomatic leaf tissues were extracted and analyzed by reverse transcription-polymerase chain reaction as described by Louro et al.(1). The 439-bp ToCV-specific DNA fragment was amplified in samples collected from 6 of 14 greenhouses in Sardinia, 2 of 5 greenhouses in Sicily, and 1 of 1 greenhouse in Apulia. The sequence of the fragment obtained from a Sicilian isolate (GenBank Accession No. AY048854) showed more than 99% identity to ToCV isolates (Accession Nos. AF024630 and AF234029) from the United States and Portugal, respectively. Infestations of Trialeurodes vaporariorum and Bemisia tabaci have been reported in autumn. To our knowledge, this is the first report of ToCV in Italy. Although we found the virus in three regions of the country, its distribution is likely to be wider, since the symptoms can be mistaken for those of a physiological disorder or of Tomato infectious chlorosis virus, another crinivirus infecting tomato. Reference: (1) Louro et al. Eur. J. Plant Pathol. 106:589, 2000.

Delineation of the roles of amino acids involved in the catalytic functions of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase.

The roles of particular amino acids in substrate and coenzyme binding and catalysis of glucose-6-phosphate dehydrogenase of Leuconostoc mesenteroides have been investigated by site-directed mutagenesis, kinetic analysis, and determination of binding constants. The enzyme from this species has functional dual NADP(+)/NAD(+) specificity. Previous investigations in our laboratories determined the three-dimensional structure. Kinetic studies showed an ordered mechanism for the NADP-linked reaction while the NAD-linked reaction is random. His-240 was identified as the catalytic base, and Arg-46 was identified as important for NADP(+) but not NAD(+) binding. Mutations have been selected on the basis of the three-dimensional structure. Kinetic studies of 14 mutant enzymes are reported and kinetic mechanisms are reported for 5 mutant enzymes. Fourteen substrate or coenzyme dissociation constants have been measured for 11 mutant enzymes. Roles of particular residues are inferred from k(cat), K(m), k(cat)/K(m), K(d), and changes in kinetic mechanism. Results for enzymes K182R, K182Q, K343R, and K343Q establish Lys-182 and Lys-343 as important in binding substrate both to free enzyme and during catalysis. Studies of mutant enzymes Y415F and Y179F showed no significant contribution for Tyr-415 to substrate binding and only a small contribution for Tyr-179. Changes in kinetics for T14A, Q47E, and R46A enzymes implicate these residues, to differing extents, in coenzyme binding and discrimination between NADP(+) and NAD(+). By the same measure, Lys-343 is also involved in defining coenzyme specificity. Decrease in k(cat) and k(cat)/K(m) for the D374Q mutant enzyme defines the way Asp-374, unique to L. mesenteroides G6PD, modulates stabilization of the enzyme during catalysis by its interaction with Lys-182. The greatly reduced k(cat) values of enzymes P149V and P149G indicate the importance of the cis conformation of Pro-149 in accessing the correct transition state.

First Report of Root Rot Caused by Phytophthora cinnamomi on Avocado in Italy.

Root rot caused by Phytophthora cinnamomi Rands is generally recognized to be the most important disease of avocado (Persea americana Miller) wherever this tropical fruit tree is grown (3). The disease was first found in Italy in the spring of 1998. Eight-year-old trees, with symptoms ranging from initial to severe, were observed in an experimental field near Rocca di Caprileone, in Sicily. Few trees showed symptoms of both root rot and collar rot. Infected trees were of 13 commercial cultivars. Trees were grafted on two different rootstocks: Hass seedlings and G6 seedlings. G6 is a selection reported to have some field resistance to P. cinnamomi infections (2). However, no correlation was observed between symptom severity and rootstock. P. cinnamomi was isolated on BNPRAH selective medium (4) from trunk bark, feeder roots, and rhizosphere soil of diseased trees, and from roots of symptomless trees. The isolates, identified primarily on the basis of morphological and cultural characteristics, formed rosaceous colonies on potato dextrose agar (PDA) and on corn meal agar (CMA) coralloid-type mycelium, with abundant hyphal swellings, which were typically spherical and in clusters. Chlamydospores were either terminal or intercalary, and often occurred in characteristic grapelike clusters. Sporangia, which were produced in saline solution (1), were broadly ellipsoidal or ovoid, persistent, non-papillate and proliferous. The identification was confirmed by the electrophoresis of mycelial proteins on polyacrylamide slab gel. The electrophoretic patterns of total soluble proteins and eight isozymes (AKP [alkaline phosphatase], EST [esterase], FUM [fumarase], GLC [NAD-glucose dehydrogenase], G6PD [glucose-6-phosphate dehydrogenase], LDH [lactate dehydrogenase], MDH [malate dehydrogenase], and SOD [superoxide dismutase]) of the isolates from avocado were identical to those of two strains of P. cinnamomi, used as reference (isolate 70473 from International Mycological Institute, U.K., and an isolate from myrtle from the Institute of Plant Pathology, University of Catania, Italy). Conversely, the electrophoretic phenotype of the P. cinnamomi isolates from avocado was clearly distinct from those of reference strains of eight other species included in Waterhouse's taxonomic group VI. Pairings with isolates of a known mating type of P. cinnamomi, P. cryptogea, and P. drechsleri revealed that all the isolates from avocado were A2 mating type. It is possible that P. cinnamomi had been introduced into the experimental field on infected symptomless nursery trees. In Italy, root rot caused by P. cinnamomi could have a significant impact on commercial avocado plantings extending over about 20 ha. Moreover, this polyphagous pathogen may be a threat to other crops as well as to forest trees. References: (1) D. W. Chen and G. A. Zentmyer. Mycologia 62:397, 1970. (2) M. D. Coffey. Plant Dis. 71:1046, 1987. (3) D. C. Erwin and O. K. Ribeiro. 1996. Phytophthora Diseases Worldwide. American Phytopathological Society, St. Paul, MN. (4) H. Masago et al. Phytopathology 67:425, 1977.