Biofilm formation on bone grafts and bone graft substitutes: comparison of different materials by a standard in vitro test and microcalorimetry.

We analyzed the initial adhesion and biofilm formation of Staphylococcus aureus (ATCC 29213) and S. epidermidis RP62A (ATCC 35984) on various bone grafts and bone graft substitutes under standardized in vitro conditions. In parallel, microcalorimetry was evaluated as a real-time microbiological assay in the investigation of biofilm formation and material science research. The materials beta-tricalcium phosphate (beta-TCP), processed human spongiosa (Tutoplast) and poly(methyl methacrylate) (PMMA) were investigated and compared with polyethylene (PE). Bacterial counts (log(10) cfu per sample) were highest on beta-TCP (S. aureus 7.67 +/- 0.17; S. epidermidis 8.14 +/- 0.05) while bacterial density (log(10) cfu per surface) was highest on PMMA (S. aureus 6.12 +/- 0.2, S. epidermidis 7.65 +/- 0.13). Detection time for S. aureus biofilms was shorter for the porous materials (beta-TCP and processed human spongiosa, p < 0.001) compared to the smooth materials (PMMA and PE), with no differences between beta-TCP and processed human spongiosa (p > 0.05) or PMMA and PE (p > 0.05). In contrast, for S. epidermidis biofilms the detection time was different (p < 0.001) between all materials except between processed human spongiosa and PE (p > 0.05). The quantitative analysis by quantitative culture after washing and sonication of the material demonstrated the importance of monitoring factors like specific surface or porosity of the test materials. Isothermal microcalorimetry proved to be a suitable tool for an accurate, non-invasive and real-time microbiological assay, allowing the detection of bacterial biomass without removing the biofilm from the surface.

Bacterial biofilms and capsular contracture in patients with breast implants.

BACKGROUND: It has been hypothesized that bacterial biofilms on breast implants may cause chronic inflammation leading to capsular contracture. The association between bacterial biofilms of removed implants and capsular contracture was investigated. METHODS: Breast implants explanted between 2006 and 2010 at five participating centres for plastic and reconstructive surgery were investigated by sonication. Bacterial cultures derived from sonication were correlated with patient, surgical and implant characteristics, and the degree of capsular contracture. RESULTS: The study included 121 breast implants from 84 patients, of which 119 originated from women and two from men undergoing gender reassignment. Some 50 breast prostheses were implanted for reconstruction, 48 for aesthetic reasons and 23 implants were used as temporary expander devices. The median indwelling time was 4·0 (range 0·1-32) years for permanent implants and 3 (range 1-6) months for temporary devices. Excluding nine implants with clinical signs of infection, sonication cultures were positive in 40 (45 per cent) of 89 permanent implants and in 12 (52 per cent) of 23 temporary devices. Analysis of permanent implants showed that a positive bacterial culture after sonication correlated with the degree of capsular contracture: Baker I, two of 11 implants; Baker II, two of ten; Baker III, nine of 23; and Baker IV, 27 of 45 (P < 0·001). The most frequent organisms were Propionibacterium acnes (25 implants) and coagulase-negative staphylococci (21). CONCLUSION: Sonication cultures correlated with the degree of capsular contracture, indicating the potential causative role of bacterial biofilms in the pathogenesis of capsular contracture. Registration number: NCT01138891 (http://www.clinicaltrials.gov).

Corynebacterium bovis shoulder prosthetic joint infection: the first reported case.

We report the first case of Corynebacterium bovis shoulder prosthetic joint infection. The organism was isolated from intraoperative tissue culture and from the removed prosthesis using sonication. A 2-stage exchange and 3 months of antibiotic therapy were performed. C. bovis may cause implant-associated infections, which can manifest as low-grade infection.

Infection after total ankle replacement : A 5-year retrospective analysis of 92 implanted ankle prostheses (2004-2008).

Objectives: Total ankle replacement (TAR) is increasingly used for treatment of primary or posttraumatic arthritis of the ankle joint, if joint movement is intended to be preserved. Data on characteristics and treatment of ankle prosthetic joint infection (PJI) is limited and no validated therapeutic algorithm exist. Therefore, we analyzed all infections, which occurred in a cohort of implanted ankle prostheses during a 5-year-period.Methods: Between 06/2004 and 12/2008, all patients with an implanted ankle prosthesis at our institution were retrospectively reviewed. All patients were operated by the same surgical team. Ankle PJI was defined as visible purulence, acute inflammation on histopathology, sinus tract, or microbial growth in periprosthetic tissue or sonication fluid of the removed prosthesis. The surgery on the infected ankle prosthesis and the follow-up were performed by the surgical team, who implanted the prosthesis. A specialized septic team consisting of an orthopaedic surgeon and infectious diseases consultant were included in the treatment.Results: During the study period, 92 total ankle prostheses were implanted in 90 patients (mean age 61 years, range 28-80 years). 78 patients had posttraumatic arthritis, 11 rheumatoid arthritis and 3 other degenerative disorder. Ankle PJI occurred in 3 of 92 TAR (3.3%), occurring 1, 2 and 24 months after implantation; the causative organisms were Enterobacter cloacae, Streptococcus pyogenes and Staphylococcus epidermidis, respectively. The ankle prosthesis was removed in all infected patients, including debridement of the surrounding tissue was debrided and insertion of an antibiotic loaded spacer. Provisional arthrodesis was performed by external fixation in two patients and by plaster cast in one. A definitive ankle arthrodesis with a retrograde nail was performed 6 to 8 weeks after prosthesis removal. One patient needed a flap coverage. All 3 patients received intravenous antibiotic treatment for 2 weeks, followed by oral antibiotics for 4-6 weeks. At follow-up visit up to 18 months after start of treatment, all patients were without clinical or laboratory signs of infection.Conclusions: The infection incidence after TAR was 3.3%, which is slightly higher than reported after hip (<1%) or knee arthroplasty (<2%). A two-step approach consisting of removal of the infected prosthesis, combined with local and systemic antibiotic treatment, followed by definitive ankle arthrodesis shows good results. Larger patient cohort and longer follow-up evaluation is needed to define the optimal treatment approach for ankle PJI.

In vivo gene transfer into the ocular ciliary muscle mediated by ultrasound and microbubbles.

This study aimed to assess application of ultrasound (US) combined with microbubbles (MB) to transfect the ciliary muscle of rat eyes. Reporter DNA plasmids encoding for Gaussia luciferase, β-galactosidase or the green fluorescent protein (GFP), alone or mixed with 50% Artison MB, were injected into the ciliary muscle, with or without US exposure (US set at 1 MHz, 2 W/cm(2), 50% duty cycle for 2 min). Luciferase activity was measured in ocular fluids at 7 and 30 days after sonoporation. At 1 week, the US+MB treatment showed a significant increase in luminescence compared with control eyes, injected with plasmid only, with or without MB (×2.6), and, reporter proteins were localized in the ciliary muscle by histochemical analysis. At 1 month, a significant decrease in luciferase activity was observed in all groups. A rise in lens and ciliary muscle temperature was measured during the procedure but did not result in any observable or microscopic damages at 1 and 8 days. The feasibility to transfer gene into the ciliary muscle by US and MB suggests that sonoporation may allow intraocular production of proteins for the treatment of inflammatory, angiogenic and/or degenerative retinal diseases.

Differentiated chemical reactivity of nanoparticles toward DTT

RATIONALE: Induction of oxidative stress and impairment of the antioxidant defense are considered important biological responses following nanoparticle (NP) exposure. The acellular in vitro dithiothreitol (DTT) assay is proposed to measure the oxidative potential of NP. In addition, DTT can be considered as a model compound of sulfur containing antioxidants. The objective of this work is to evaluate the surface reactivity in solution of a NP panel toward DTT. METHOD: The NP panel was composed of four carbonaceous particles, six types of metal oxides and silver with primary size ranged from 7 to 300 nm. Suspensions were prepared in surfactant solution with 30 min sonication. DTT was used as reductant to evaluate the oxidative properties of the different NP. The determination of the NP ability to catalyze electron transfer from DTT to oxygen was carried out as described in Sauvain et al., Nanotoxicology, 2008, 2:3, 121−129. RESULTS: All the carbonaceous NP catalyzed the oxidation of DTT by oxygen following the mass based order: carbon black > diesel exhaust particle > nanotubes > fullerene. A contrasting reactivity was observed for the metallic NP. Except for nickel oxide and metallic silver, which reacted similarly to the carbonaceous NP, all other metal oxides hindered the oxidation of DTT by oxygen, with ZnO being the most effective one. CONCLUSIONS : DTT was stabilized against oxidation in the presence of metal oxide NP in the solution. This suggests that different chemical interactions take place compared with carbonaceous NP. To explain these differences, we hypothesize that DTT could form complexes with the metal oxide surface (or dissolved metal ions), rendering it less susceptible to oxidation. By analogy, such a process could be thought to apply in biological systems with sulfur−containing antioxidants, reducing their buffer capacity. Such NP could thus contribute to oxidative stress by an alternative mechanism.

Detecting the oxidative reactivity of nanoparticles: a new protocol for reducing artifacts

Understanding the oxidative reactivity of nanoparticles (NPs; <100 nm) could substantially contribute to explaining their toxicity. We attempted to refine the use of 2′7-dichlorodihydrofluorescein (DCFH) to characterize NP generation of reactive oxygen species (ROS). Several fluorescent probes have been applied to testing oxidative reactivity, but despite DCFH being one of the most popular for the detection of ROS, when it has been applied to NPs there have been an unexplainably wide variability in results. Without a uniform methodology, validating even robust results is impossible. This study, therefore, identified sources of conflicting results and investigated ways of reducing occurrence of artificial results. Existing techniques were tested and combined (using their most desirable features) to form a more reliable method for the measurement of NP reactivity in aqueous dispersions. We also investigated suitable sample ranges necessary to determine generation of ROS. Specifically, ultrafiltration and time-resolved scan absorbance spectra were used to study possible optical interference when using high sample concentrations. Robust results were achieved at a 5 µM DCFH working solution with 0.5 unit/mL horseradish peroxidase (HRP) dissolved in ethanol. Sonication in DCFH-HRP working solution provided more stable data with a relatively clean background. Optimal particle concentration depends on the type of NP and in general was in the µg/mL range. Major reasons for previously reported conflicting results due to interference were different experimental approaches and NP sample concentrations. The protocol presented here could form the basis of a standardized method for applying DCFH to detect generation of ROS by NPs.

Epidemiology and new developments in the diagnosis of prosthetic joint infection.

Although prosthetic joint infection (PJI) is a rare event after arthroplasty, it represents a significant complication that is associated with high morbidity, need for complex treatment, and substantial healthcare costs. An accurate and rapid diagnosis of PJI is crucial for treatment success. Current diagnostic methods in PJI are insufficient with 10-30% false-negative cultures. Consequently, there is a need for research and development into new methods aimed at improving diagnostic accuracy and speed of detection. In this article, we review available conventional diagnostic methods for the diagnosis of PJI (laboratory markers, histopathology, synovial fluid and periprosthetic tissue cultures), new diagnostic methods (sonication of implants, specific and multiplex PCR, mass spectrometry) and innovative techniques under development (new laboratory markers, microcalorimetry, electrical method, reverse transcription [RT]-PCR, fluorescence in situ hybridization [FISH], biofilm microscopy, microarray identification, and serological tests). The results of highly sensitive diagnostic techniques with unknown specificity should be interpreted with caution. The organism identified by a new method may represent a real pathogen that was unrecognized by conventional diagnostic methods or contamination during specimen sampling, transportation, or processing. For accurate interpretation, additional studies are needed, which would evaluate the long-term outcome (usually >2 years) with or without antimicrobial treatment. It is expected that new rapid, accurate, and fully automatic diagnostic tests will be developed soon.

Shikonin-loaded antibody-armed nanoparticles for targeted therapy of ovarian cancer.

Conventional chemotherapy of ovarian cancer often fails because of initiation of drug resistance and/or side effects and trace of untouched remaining cancerous cells. This highlights an urgent need for advanced targeted therapies for effective remediation of the disease using a cytotoxic agent with immunomodulatory effects, such as shikonin (SHK). Based on preliminary experiments, we found SHK to be profoundly toxic in ovarian epithelial cancer cells (OVCAR-5 and ID8 cells) as well as in normal ovarian IOSE-398 cells, endothelial MS1 cells, and lymphocytes. To limit its cytotoxic impact solely to tumor cells within the tumor microenvironment (TME), we aimed to engineer SHK as polymeric nanoparticles (NPs) with targeting moiety toward tumor microvasculature. To this end, using single/double emulsion solvent evaporation/diffusion technique with sonication, we formulated biodegradable NPs of poly(lactic-co-glycolic acid) (PLGA) loaded with SHK. The surface of NPs was further decorated with solubilizing agent polyethylene glycol (PEG) and tumor endothelial marker 1 (TEM1)/endosialin-targeting antibody (Ab) through carbodiimide/N-hydroxysuccinimide chemistry. Having characterized the physicochemical and morphological properties of NPs, we studied their drug-release profiles using various kinetic models. The biological impact of NPs was also evaluated in tumor-associated endothelial MS1 cells, primary lymphocytes, and epithelial ovarian cancer OVCAR-5 cells. Based on particle size analysis and electron microscopy, the engineered NPs showed a smooth spherical shape with size range of 120 to 250 nm and zeta potential value of -30 to -40 mV. Drug entrapment efficiency was ~80%-90%, which was reduced to ~50%-60% upon surface decoration with PEG and Ab. The liberation of SHK from NPs showed a sustained-release profile that was best fitted with Wagner log-probability model. Fluorescence microscopy and flow cytometry analysis showed active interaction of Ab-armed NPs with TEM1-positive MS1 cells, but not with TEM1-negative MS1 cells. While exposure of the PEGylated NPs for 2 hours was not toxic to lymphocytes, long-term exposure of the Ab-armed and PEGylated NPs was significantly toxic to TEM1-positive MS1 cells and OVCAR-5 cells. Based on these findings, we propose SHK-loaded Ab-armed PEGylated PLGA NPs as a novel nanomedicine for targeted therapy of solid tumors.

Intrinsic ROS-production capacity of nanoparticles

Engineered nanomaterials (ENMs) exhibit special physicochemical properties and thus are finding their way into an increasing number of industries, enabling products with improved properties. Their increased use brings a greater likelihood of exposure to the nanoparticles (NPs) that could be released during the life cycle of nano-abled products. The field of nanotoxicology has emerged as a consequence of the development of these novel materials, and it has gained ever more attention due to the urgent need to gather information on exposure to them and to understand the potential hazards they engender. However, current studies on nanotoxicity tend to focus on pristine ENMs, and they use these toxicity results to generalize risk assessments on human exposure to NPs. ENMs released into the environment can interact with their surroundings, change characteristics and exhibit toxicity effects distinct from those of pristine ENMs. Furthermore, NPs' large surface areas provide extra-large potential interfaces, thus promoting more significant interactions between NPs and other co-existing species. In such processes, other species can attach to a NP's surface and modify its surface functionality, in addition to the toxicity in normally exhibits. One particular occupational health scenario involves NPs and low-volatile organic compounds (LVOC), a common type of pollutant existing around many potential sources of NPs. LVOC can coat a NP's surface and then dominate its toxicity. One important mechanism in nanotoxicology is the creation of reactive oxygen species (ROS) on a NP's surface; LVOC can modify the production of these ROS. In summary, nanotoxicity research should not be limited to the toxicity of pristine NPs, nor use their toxicity to evaluate the health effects of exposure to environmental NPs. Instead, the interactions which NPs have with other environmental species should also be considered and researched. The potential health effects of exposure to NPs should be derived from these real world NPs with characteristics modified by the environment and their distinct toxicity. Failure to suitably address toxicity results could lead to an inappropriate treatment of nano- release, affect the environment and public health and put a blemish on the development of sustainable nanotechnologies as a whole. The main objective of this thesis is to demonstrate a process for coating NP surfaces with LVOC using a well-controlled laboratory design and, with regard to these NPs' capacity to generate ROS, explore the consequences of changing particle toxicity. The dynamic coating system developed yielded stable and replicable coating performance, simulating an important realistic scenario. Clear changes in the size distribution of airborne NPs were observed using a scanning mobility particle sizer, were confirmed using both liquid nanotracking analyses and transmission electron microscopy (TEM) imaging, and were verified thanks to the LVOC coating. Coating thicknesses corresponded to the amount of coating material used and were controlled using the parameters of the LVOC generator. The capacity of pristine silver NPs (Ag NPs) to generate ROS was reduced when they were given a passive coating of inert paraffin: this coating blocked the reactive zones on the particle surfaces. In contrast, a coating of active reduced-anthraquinone contributed to redox reactions and generated ROS itself, despite the fact that ROS generation due to oxidation by Ag NPs themselves was quenched. Further objectives of this thesis included development of ROS methodology and the analysis of ROS case studies. Since the capacity of NPs to create ROS is an important effect in nanotoxicity, we attempted to refine and standardize the use of 2'7-dichlorodihydrofluorescin (DCFH) as a chemical tailored for the characterization of NPs' capacity for ROS generation. Previous studies had reported a wide variety of results, which were due to a number of insufficiently well controlled factors. We therefore cross-compared chemicals and concentrations, explored ways of dispersing NP samples in liquid solutions, identified sources of contradictions in the literature and investigated ways of reducing artificial results. The most robust results were obtained by sonicating an optimal sample of NPs in a DCFH-HRP solution made of 5,M DCFH and 0.5 unit/ml horseradish peroxidase (HRP). Our findings explained how the major reasons for previously conflicting results were the different experimental approaches used and the potential artifacts appearing when using high sample concentrations. Applying our advanced DCFH protocol with other physicochemical characterizations and biological analyses, we conducted several case studies, characterizing aerosols and NP samples. Exposure to aged brake wear dust engenders a risk of potential deleterious health effects in occupational scenarios. We performed microscopy and elemental analyses, as well as ROS measurements, with acellular and cellular DCFH assays. TEM images revealed samples to be heterogeneous mixtures with few particles in the nano-scale. Metallic and non-metallic elements were identified, primarily iron, carbon and oxygen. Moderate amounts of ROS were detected in the cell-free fluorescent tests; however, exposed cells were not dramatically activated. In addition to their highly aged state due to oxidation, the reason aged brake wear samples caused less oxidative stress than fresh brake wear samples may be because of their larger size and thus smaller relative reactive surface area. Other case studies involving welding fumes and differently charged NPs confirmed the performance of our DCFH assay and found ROS generation linked to varying characteristics, especially the surface functionality of the samples. Les nanomatériaux manufacturés (ENM) présentent des propriétés physico-chimiques particulières et ont donc trouvés des applications dans un nombre croissant de secteurs, permettant de réaliser des produits ayant des propriétés améliorées. Leur utilisation accrue engendre un plus grand risque pour les êtres humains d'être exposés à des nanoparticules (NP) qui sont libérées au long de leur cycle de vie. En conséquence, la nanotoxicologie a émergé et gagné de plus en plus d'attention dû à la nécessité de recueillir les renseignements nécessaires sur l'exposition et les risques associés à ces nouveaux matériaux. Cependant, les études actuelles sur la nanotoxicité ont tendance à se concentrer sur les ENM et utiliser ces résultats toxicologiques pour généraliser l'évaluation des risques sur l'exposition humaine aux NP. Les ENM libérés dans l'environnement peuvent interagir avec l'environnement, changeant leurs caractéristiques, et montrer des effets de toxicité distincts par rapport aux ENM originaux. Par ailleurs, la grande surface des NP fournit une grande interface avec l'extérieur, favorisant les interactions entre les NP et les autres espèces présentes. Dans ce processus, d'autres espèces peuvent s'attacher à la surface des NP et modifier leur fonctionnalité de surface ainsi que leur toxicité. Un scénario d'exposition professionnel particulier implique à la fois des NP et des composés organiques peu volatils (LVOC), un type commun de polluant associé à de nombreuses sources de NP. Les LVOC peuvent se déposer sur la surface des NP et donc dominer la toxicité globale de la particule. Un mécanisme important en nanotoxicologie est la création d'espèces réactives d'oxygène (ROS) sur la surface des particules, et les LVOC peuvent modifier cette production de ROS. En résumé, la recherche en nanotoxicité ne devrait pas être limitée à la toxicité des ENM originaux, ni utiliser leur toxicité pour évaluer les effets sur la santé de l'exposition aux NP de l'environnement; mais les interactions que les NP ont avec d'autres espèces environnementales doivent être envisagées et étudiées. Les effets possibles sur la santé de l'exposition aux NP devraient être dérivés de ces NP aux caractéristiques modifiées et à la toxicité distincte. L'utilisation de résultats de toxicité inappropriés peut conduire à une mauvaise prise en charge de l'exposition aux NP, de détériorer l'environnement et la santé publique et d'entraver le développement durable des industries de la nanotechnologie dans leur ensemble. L'objectif principal de cette thèse est de démontrer le processus de déposition des LVOC sur la surface des NP en utilisant un environnement de laboratoire bien contrôlé et d'explorer les conséquences du changement de toxicité des particules sur leur capacité à générer des ROS. Le système de déposition dynamique développé a abouti à des performances de revêtement stables et reproductibles, en simulant des scénarios réalistes importants. Des changements clairs dans la distribution de taille des NP en suspension ont été observés par spectrométrie de mobilité électrique des particules, confirmé à la fois par la méthode dite liquid nanotracking analysis et par microscopie électronique à transmission (MET), et a été vérifié comme provenant du revêtement par LVOC. La correspondance entre l'épaisseur de revêtement et la quantité de matériau de revêtement disponible a été démontré et a pu être contrôlé par les paramètres du générateur de LVOC. La génération de ROS dû aux NP d'argent (Ag NP) a été diminuée par un revêtement passif de paraffine inerte bloquant les zones réactives à la surface des particules. Au contraire, le revêtement actif d'anthraquinone réduit a contribué aux réactions redox et a généré des ROS, même lorsque la production de ROS par oxydation des Ag NP avec l'oxygène a été désactivé. Les objectifs associés comprennent le développement de la méthodologie et des études de cas spécifique aux ROS. Etant donné que la capacité des NP à générer des ROS contribue grandement à la nanotoxicité, nous avons tenté de définir un standard pour l'utilisation de 27- dichlorodihydrofluorescine (DCFH) adapté pour caractériser la génération de ROS par les NP. Des etudes antérieures ont rapporté une grande variété de résultats différents, ce qui était dû à un contrôle insuffisant des plusieurs facteurs. Nous avons donc comparé les produits chimiques et les concentrations utilisés, exploré les moyens de dispersion des échantillons HP en solution liquide, investigué les sources de conflits identifiées dans les littératures et étudié les moyens de réduire les résultats artificiels. De très bon résultats ont été obtenus par sonication d'une quantité optimale d'échantillons de NP en solution dans du DCFH-HRP, fait de 5 nM de DCFH et de 0,5 unité/ml de Peroxydase de raifort (HRP). Notre étude a démontré que les principales raisons causant les conflits entre les études précédemment conduites dans la littérature étaient dues aux différentes approches expérimentales et à des artefacts potentiels dus à des concentrations élevées de NP dans les échantillons. Utilisant notre protocole DCFH avancé avec d'autres caractérisations physico-chimiques et analyses biologiques, nous avons mené plusieurs études de cas, caractérisant les échantillons d'aérosols et les NP. La vielle poussière de frein en particulier présente un risque élevé d'exposition dans les scénarios professionnels, avec des effets potentiels néfastes sur la santé. Nous avons effectué des analyses d'éléments et de microscopie ainsi que la mesure de ROS avec DCFH cellulaire et acellulaire. Les résultats de MET ont révélé que les échantillons se présentent sous la forme de mélanges de particules hétérogènes, desquels une faible proportion se trouve dans l'échelle nano. Des éléments métalliques et non métalliques ont été identifiés, principalement du fer, du carbone et de l'oxygène. Une quantité modérée de ROS a été détectée dans le test fluorescent acellulaire; cependant les cellules exposées n'ont pas été très fortement activées. La raison pour laquelle les échantillons de vielle poussière de frein causent un stress oxydatif inférieur par rapport à la poussière de frein nouvelle peut-être à cause de leur plus grande taille engendrant une surface réactive proportionnellement plus petite, ainsi que leur état d'oxydation avancé diminuant la réactivité. D'autres études de cas sur les fumées de soudage et sur des NP différemment chargées ont confirmé la performance de notre test DCFH et ont trouvé que la génération de ROS est liée à certaines caractéristiques, notamment la fonctionnalité de surface des échantillons.

Geração sonoquímica de oxidantes em solução aquosa saturada de tetracloreto de carbono

In this work the CCl4 degradation in aqueous solution by sonication with 40 kHz commercial ultrasonic bath was investigated. Sonochemical degradation of CCl4 occur by the cleavage of C-Cl bond into the cavitation bubbles. Oxidation reactions and the pH decreasing in the bulk solution during sonication were attributed to chlorine radicals produced by CCl4 sonolysis, leading to increase the chloride concentration. The formation of oxidizing agents was evaluated employing I- and Fe2+ ion solutions, converted to I2 and HIO, and Fe3+, respectively. The amount of chloride and hydronium ions produced after 3 min of irradiation was 11.52 and 12.19 mmol, respectively, suggesting that the same reaction was involved to produce these ions. Fe2+ oxidation and the pH variation were monitored to estimate chlorine radical formation rate in the presence (0.107 mumol s-1) and absence (0.092 mumol s-1) of metallic ion during the first minute of sonication.

Floculação de Chlorella sp. produzida em concentrado de dessalinização e estudo de método de extração de lipídeos intracelulares

Chlorella sp. was used to assess algal lipid production with concentrated desalination. In order to investigate the action of the flocculating agent calcium chloride and pH, a Box-Behnken Design and a Central Composite Design (CCD) were carried out. Also, Soxhlet and Supercritical Fluid Extraction (SFE), with and without sonication lipid extraction methods, were examined. The optimal flocculation conditions were pH 10.0 and 2.0 g/L of calcium chloride concentration. The highest lipid content of Chlorella sp. was obtained using the Soxhlet extraction method. The most abundant fatty acid extracted by Soxhlet and SFE, with and without sonication, was palmitic acid, whose proportions were 57.4%, 35.3% and 25.5%, respectively.

Polyclonal antibodies to the coat protein of Apple stem grooving virus expressed in Escherichia coli: production and use in immunodiagnosis

The coat protein gene of Apple stem grooving virus (ASGV) was amplified by RT-PCR, cloned, sequenced and subcloned in the expression vector pMal-c2. This plasmid was used to transform Escherichia coli BL21c+ competent cells. The ASGV coat protein (cp) was expressed as a fusion protein containing a fragment of E. coli maltose binding protein (MBP). Bacterial cells were disrupted by sonication and the ASGVcp/MBP fusion protein was purified by amylose resin affinity chromatography. Polyclonal antibodies from rabbits immunized with the fusion protein gave specific reactions to ASGV from infected apple (Malus domestica) cv. Fuji Irradiada and Chenopodium quinoa at dilutions of up to 1:1,000 and 1:2,000, respectively, in plate trapped ELISA. The ASGVcp/MBP fusion protein reacted to a commercial antiserum against ASGV in immunoblotting assay. The IgG against ASGVcp/MBP performed favorably in specificity and sensitivity to the virus. This method represents an additional tool for the efficient ASGV-indexing of apple propagative and mother stock materials, and for use in support of biological and molecular techniques.

Determination of patulin in apple juice by HPLC using a simple and fast sample preparation method

The goal of this work was to develop a simple and rapid preparation method for patulin analysis in apple juice without previous clean-up. This method combined sonication and liquid extraction techniques and was used for determination of patulin in 37 commercial apple juices available on the market in the South of Brazil. The method performance characteristics were determined using a sample obtained in a local market fortified at five concentration levels of patulin and done in triplicates. The coefficient of variation for repeatability at the fortification level of 20.70µg.L-1 was 3.53 % and the recovery 94.63 %, respectively. The correlation coefficient was 0.9996 and agrees with the requirements for a linear analytical method value. The detection limit was 0.21µg.L-1 and the quantification limit 0.70 µg.L-1. Only three of the analyzed samples were upper the allowed level of 50.00 µg.L-1 recommended for the World Health Organization.

The Combined treatment of UV Light and Titanium Dioxide (TiO2) in Grease Filtration Technique

The aim of this thesis was to identify the best grease removal technique with the application of low power of UV light to TiO2 coated grease filters. The treatment with various power series of ozone generating and ozone free lamps to normal grease filters and TiO2 coated grease filters were examined and the obtained results are compared to each other in this paper. The effect of ozone reaction was observed and compared with the effect of TiO2. The experiments were solely based on the photo oxidation and photo catalytic oxidation reactions. TiO2 is a green catalyst used in the photocatalytic reaction. Sunflower oil was used for grease production and tetracholoroethylene as a solvent. Grease samples were collected from the ventilation duct connected to the cooking hood system. Sample extraction was done in ultrasonic bath with the principle of sonication. The sample analysis was done by FTIR machine. The result determining the concentration of grease was the quantification of saturated C-H bonds in the chosen peak group of the spectrum. A very low power of UVC light functions perfectly with the Titanium dioxide. The experimental results have shown the combined treatment of titanium dioxide and UV light is an effective method in grease removal process. The photocatalytic reaction with titanium dioxide is better than photo oxidation reaction with ozone treatment. Photocatalytic reaction is environmentally friendly, energy efficient and economical.