The effect of biomass immobilization support material and bed porosity on hydrogen production in an upflow anaerobic packed-bed bioreactor

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Exenatide and sitagliptin decrease interleukin 1β, matrix metalloproteinase 9, and nitric oxide synthase 2 gene expression but does not reduce alveolar bone loss in rats with periodontitis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Immediate tooth replantation in rats: effect of systemic antibiotic therapy with amoxicillin and tetracycline

The aim of this study was to evaluate the influence of systemic administration of antibiotics (amoxicillin and tetracycline) at the different phases of the repair process (7, 15, 30 days) in immediate rat tooth replantation. Ninety rats had their incisors extracted and stored in saline for 5 min. Next, the teeth were replanted, and the animals were assigned to three groups according to the antibiotic administered by oral gavage: control group, amoxycillin group, and tetracycline group. Euthanasia was performed at 7, 15, and 30 days after replantation. Regardless of the evaluation period, the connective tissue underlying the epithelial attachment and the periodontal ligament showed statistically significant difference relative to the acute inflammatory infiltrate, which was more intense in the control group followed by the tetracycline group. These results point to the fact that systemic antibiotic therapy (SAT) in immediate tooth replantation is beneficial to pulpal and periodontal ligament repair and that amoxycillin is an excellent option. There is a lack of randomized studies assessing how the use of systemic antibiotics could influence tooth healing after immediate replantation.

Evaluation of rhamnolipid and surfactin to reduce the adhesion and remove biofilms of individual and mixed cultures of food pathogenic bacteria

Biofilms represent a great concern for food industry, since they can be a source of persistent contamination leading to food spoilage and to the transmission of diseases. To avoid the adhesion of bacteria and the formation of biofilms, an alternative is the pre-conditioning of surfaces using biosurfactants, microbial compounds that can modify the physicochemical properties of surfaces changing bacterial interactions and consequently adhesion. Different concentrations of the biosurfactants, surfactin from Bacillus subtilis and rhamnolipids from Pseudomonas aeruginosa, were evaluated to reduce the adhesion and to disrupt biofilms of food-borne pathogenic bacteria. Individual cultures and mixed cultures of Staphylococcus aureus, Listeria monocytogenes and Salmonella Enteritidis were studied using polystyrene as the model surface. The pre-conditioning with surfactin 0.25% reduced by 42.0% the adhesion of L monocytogenes and S. Enteritidis, whereas the treatment using rhamnolipids 1.0% reduced by 57.8% adhesion of L monocytogenes and by 67.8% adhesion of S. aureus to polystyrene.Biosurfactants were less effective to avoid adhesion of mixed cultures of the bacteria when compared with individual cultures. After 2 h contact with surfactin at 0.1% concentration, the pre-formed biofilms of S. aureus were reduced by 63.7%, L. monocytogenesby 95.9%, S. Enteritidis by 35.5% and the mixed culture biofilm by 58.5%. The rhamnolipids at 0.25% concentration removed 58.5% the biofilm of S. aureus, 26.5% of L monocytogenes, 23.0% of S. Enteritidis and 24.0% the mixed culture after 2 h contact. In general, the increase in concentration of biosurfactants and in the time of contact decreased biofilm removal percentage. These results suggest that surfactin and rhamnolipids can be explored to control the attachment and to disrupt biofilms of individual and mixed cultures of the food-borne pathogens. (C) 2011 Elsevier Ltd. All rights reserved.

A model to explain spermatophore implantation in cephalopods (Mollusca: Cephalopoda) and a discussion on its evolutionary origins and significance

Male coleoid cephalopods produce spermatophores that can attach autonomously on the female's body during a complex process of evagination called the spermatophoric reaction, during which the ejaculatory apparatus and spiral filament of the spermatophore are everted and exposed to the external milieu. In some deepwater cephalopods, the reaction leads to the intradermal implantation of the spermatophore, a hitherto enigmatic phenomenon. The present study builds upon several lines of evidence to propose that spermatophore implantation is probably achieved through the combination of (1) an evaginating-tube mechanism performed by the everting ejaculatory apparatus and (2) the anchorage provided by the spiral filament's stellate particles. The proposed theoretical model assumes that, as it is exposed to the external milieu, each whorl of the spiral filament anchors to the surrounding tissue by means of its sharp stellate particles. As the ejaculatory apparatus tip continues evaginating, it grows in diameter and stretches lengthwise, enlarging the diameter of the whorl and propelling it, consequently tearing and pushing the anchored tissue outward and backward, and opening space for the next whorl to attach. After the ejaculatory apparatus has been everted and has perforated tissue, the cement body is extruded, possibly aiding in final attachment, and the sperm mass comes to lie inside the female tissue, encompassed by the everted ejaculatory apparatus tube. It is proposed that this unique, efficient spermatophore attachment mechanism possibly evolved in intimate relationship with the adoption of an active mode of life by coleoids. The possible roles of predation pressure and sperm competition in the evolution of this mechanism are also discussed. (c) 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105, 711726.

Oral immunization with attenuated Salmonella vaccine expressing Escherichia coli O157:H7 intimin gamma triggers both systemic and mucosal humoral immunity in mice

Human infections with EHEC such as O157:H7 have been a great concern for worldwide food-industry surveillance. This pathogen is commonly associated with bloody diarrhea that can evolve to the life-threatening hemolytic uremic syndrome. Animals are the natural reservoir where this pathogen remains asymptomatically, in steps of ingestion and colonization of the bowel. The bacterium is shed in the feces, contaminating the surroundings, including water and food that are directed for human consumption. A major player in this colonization process is intimin, an outer membrane adhesion molecule encoded by the E. coli attachment and effacement (eae) gene that has been shown to be essential for intimate bacterial attachment to eukaryotic host cells. In an attempt to reduce the colonization of animal reservoirs with EHEC O157:H7, we designed a vaccine model to induce an immune response against intimin gamma. The model is based on its recombinant expression in attenuated Salmonella, used as a suitable vaccine vector because of its recognized ability to deliver recombinant antigens and to elicit all forms of immunity: mucosal, systemic, and humoral responses. To test this model, mice were orally immunized with a S. enterica serovar Typhimurium strain carrying the pYA3137eaeA vector, and challenged with E. coli O157:H7. Here we show that immunization induced the production of high levels of specific IgG and IgA antibodies and promoted reduction in the fecal shedding of EHEC after challenge. The live recombinant vaccine reported herein may contribute to the efforts of reducing animal intestinal mucosa colonization.

Hybrid Scaffolds Built From PET and Collagen as a Model For Vascular Graft Architecture

A hybrid material with excellent mechanical and biological properties is produced by electrospinning a co-solution of PET and collagen. The fibers are mapped using SEM, confocal Raman microscopy and collagenase digestion assays. Fibers of different compositions and morphologies are intermingled within the same membrane, resulting in a heterogeneous scaffold. The collagen distribution and exposure are found to depend on the PET/collagen ratio. The materials are chemically and mechanically characterized and biologically tested with fibroblasts (3T3-L1) and a HUVEC culture in vitro. All of the hybrid scaffolds show better cell attachment and proliferation than PET. These materials are potential candidates to be used as vascular grafts.

Periodontitis and Gingivitis in Inflammatory Bowel Disease

BACKGROUND: The oral cavity is frequently affected in patients with inflammatory bowel disease (IBD), especially in patients with Crohn's disease (CD). Periodontitis is thought to influence systemic autoimmune or inflammatory diseases. We aimed to analyze the relationship of periodontitis and gingivitis markers with specific disease characteristics in patients with IBD and to compare these data with healthy controls. METHODS: In a prospective 8-month study, systematic oral examinations were performed in 113 patients with IBD, including 69 patients with CD and 44 patients with ulcerative colitis. For all patients, a structured personal history was taken. One hundred thirteen healthy volunteers served as a control group. Oral examination focussed on established oral health markers for periodontitis (bleeding on probing, loss of attachment, and periodontal pocket depth) and gingivitis (papilla bleeding index). Additionally, visible oral lesions were documented. RESULTS: Both gingivitis and periodontitis markers were higher in patients with IBD than in healthy control. In univariate analysis and logistic regression analysis, perianal disease was a risk factor for periodontitis. Nonsmoking decreased the risk of having periodontitis. No clear association was found between clinical activity and periodontitis in IBD. In only the CD subgroup, high clinical activity (Harvey-Bradshaw index > 10) was associated with 1 periodontitis marker, the loss of attachment at sites of maximal periodontal pocket depth. Oral lesions besides periodontitis and gingivitis were not common, but nevertheless observed in about 10% of patients with IBD. CONCLUSIONS: IBD, and especially perianal disease in CD, is associated with periodontitis. Optimal therapeutic strategies should probably focus on treating both local oral and systemic inflammation.

In vitro characterization of a synthetic calcium phosphate bone graft on periodontal ligament cell and osteoblast behavior and its combination with an enamel matrix derivative.

OBJECTIVES Recent studies suggest that a combination of enamel matrix derivative (EMD) with grafting material may improve periodontal wound healing/regeneration. Newly developed calcium phosphate (CaP) ceramics have been demonstrated a viable synthetic replacement option for bone grafting filler materials. AIMS This study aims to test the ability for EMD to adsorb to the surface of CaP particles and to determine the effect of EMD on downstream cellular pathways such as adhesion, proliferation, and differentiation of primary human osteoblasts and periodontal ligament (PDL) cells. MATERIALS AND METHODS EMD was adsorbed onto CaP particles and analyzed for protein adsorption patterns via scanning electron microscopy and high-resolution immunocytochemistry with an anti-EMD antibody. Cell attachment and cell proliferation were quantified using CellTiter 96 One Solution Cell Assay (MTS). Cell differentiation was analyzed using real-time PCR for genes encoding Runx2, alkaline phosphatase, osteocalcin, and collagen1α1, and mineralization was assessed using alizarin red staining. RESULTS Analysis of cell attachment revealed significantly higher number of cells attached to EMD-adsorbed CaP particles when compared to control and blood-adsorbed samples. EMD also significantly increased cell proliferation at 3 and 5 days post-seeding. Moreover, there were significantly higher mRNA levels of osteoblast differentiation markers including collagen1α1, alkaline phosphatase, and osteocalcin in osteoblasts and PDL cells cultured on EMD-adsorbed CaP particles at various time points. CONCLUSION The present study suggests that the addition of EMD to CaP grafting particles may influence periodontal regeneration by stimulating PDL cell and osteoblast attachment, proliferation, and differentiation. Future in vivo and clinical studies are required to confirm these findings. CLINICAL RELEVANCE The combination of EMD and CaP may represent an option for regenerative periodontal therapy in advanced intrabony defects.

In vitro-activity of oily calcium hydroxide suspension on microorganisms as well as on human alveolar osteoblasts and periodontal ligament fibroblasts.

BACKGROUND Findings from animal and human studies have indicated that an oily calcium hydroxide suspension (OCHS) may improve early wound healing in the treatment of periodontitis. Calcium hydroxide as the main component is well known for its antimicrobial activity, however at present the effect of OCHS on the influence of periodontal wound healing/regeneration is still very limited. The purpose of this in vitro study was to investigate the effect of OCHS on periodontopathogenic bacteria as well as on the attachment and proliferation of osteoblasts and periodontal ligament fibroblasts. METHODS Human alveolar osteoblasts (HAO) and periodontal ligament (PDL) fibroblasts were cultured on 3 concentrations of OCHS (2.5, 5 and 7.5 mg). Adhesion and proliferation were counted up to 48 h and mineralization was assayed after 1 and 2 weeks. Furthermore potential growth inhibitory activity on microorganisms associated with periodontal disease (e.g. Porphyromonas gingivalis, Tannerella forsythia, Aggregatibacter actinomycetemcomitans) as well as the influence of periodontopathogens and OCHS on the HAO and PDL fibroblasts counts were determined. RESULTS More than a 2-fold increase in adherent HAO cells was observed at 4 h following application of OCHS when compared to the control group (p = 0.007 for 2.5 mg). Proliferation of HAO cells at 48 h was stimulated by moderate concentrations (2.5 mg; 5 mg) of OCHS (each p < 0.001), whereas a high concentration (7.5 mg) of OCHS was inhibitory (p = 0.009). Mineralization was observed only for HAO cells treated with OCHS. OCHS did not exert any positive effect on attachment or proliferation of PDL fibroblasts. Although OCHS did not have an antibacterial effect, it did positively influence attachment and proliferation of HAO cells and PDL fibroblasts in the presence of periodontopathogens. CONCLUSIONS The present data suggests that OCHS promotes osteoblast attachment, proliferation and mineralization in a concentration-dependent manner and results are maintained in the presence of periodontal pathogens.

Brand Authenticity: An Integrative Framework and Measurement Scale

Although brand authenticity is gaining increasing interest in consumer behavior research and managerial practice, literature on its measurement and contribution to branding theory is still limited. This article develops an integrative framework of the concept of brand authenticity and reports the development and validation of a scale measuring consumers' perceived brand authenticity (PBA). A multi-phase scale development process resulted in a 15-item PBA scale measuring four dimensions: credibility, integrity, symbolism, and continuity. This scale is reliable across different brands and cultural contexts. We find that brand authenticity perceptions are influenced by indexical, existential, and iconic cues, whereby some of the latters' influence is moderated by consumers' level of marketing skepticism. Results also suggest that PBA increases emotional brand attachment and word-of-mouth, and that it drives brand choice likelihood through self-congruence for consumers high in self-authenticity.

Temporal sequence of hard and soft tissue healing around titanium dental implants.

The objective of the present review was to summarize the evidence available on the temporal sequence of hard and soft tissue healing around titanium dental implants in animal models and in humans. A search was undertaken to find animal and human studies reporting on the temporal dynamics of hard and soft tissue integration of titanium dental implants. Moreover, the influence of implant surface roughness and chemistry on the molecular mechanisms associated with osseointegration was also investigated. The findings indicated that the integration of titanium dental implants into hard and soft tissue represents the result of a complex cascade of biological events initiated by the surgical intervention. Implant placement into alveolar bone induces a cascade of healing events starting with clot formation and continuing with the maturation of bone in contact with the implant surface. From a genetic point of view, osseointegration is associated with a decrease in inflammation and an increase in osteogenesis-, angiogenesis- and neurogenesis-associated gene expression during the early stages of wound healing. The attachment and maturation of the soft tissue complex (i.e. epithelium and connective tissue) to implants becomes established 6-8 weeks following surgery. Based on the findings of the present review it can be concluded that improved understanding of the mechanisms associated with osseointegration will provide leads and targets for strategies aimed at enhancing the clinical performance of titanium dental implants.

Biological and protective properties of immune sera directed to the influenza virus neuraminidase

The envelope of influenza A viruses contains two large antigens, hemagglutinin (HA) and neuraminidase (NA). Conventional influenza virus vaccines induce neutralizing antibodies that are predominantly directed to the HA globular head, a domain that is subject to extensive antigenic drift. Antibodies directed to NA are induced at much lower levels, probably as a consequence of the immunodominance of the HA antigen. Although antibodies to NA may affect virus release by inhibiting the sialidase function of the glycoprotein, the antigen has been largely neglected in past vaccine design. In this study, we characterized the protective properties of monospecific immune sera that were generated by vaccination with recombinant RNA replicon particles encoding NA. These immune sera inhibited hemagglutination in an NA subtype-specific and HA subtype-independent manner and interfered with infection of MDCK cells. In addition, they inhibited the sialidase activities of various influenza viruses of the same and even different NA subtypes. With this, the anti-NA immune sera inhibited the spread of H5N1 highly pathogenic avian influenza virus and HA/NA-pseudotyped viruses in MDCK cells in a concentration-dependent manner. When chickens were immunized with NA recombinant replicon particles and subsequently infected with low-pathogenic avian influenza virus, inflammatory serum markers were significantly reduced and virus shedding was limited or eliminated. These findings suggest that NA antibodies can inhibit virus dissemination by interfering with both virus attachment and egress. Our results underline the potential of high-quality NA antibodies for controlling influenza virus replication and place emphasis on NA as a vaccine antigen. IMPORTANCE The neuraminidase of influenza A viruses is a sialidase that acts as a receptor-destroying enzyme facilitating the release of progeny virus from infected cells. Here, we demonstrate that monospecific anti-NA immune sera inhibited not only sialidase activity, but also influenza virus hemagglutination and infection of MDCK cells, suggesting that NA antibodies can interfere with virus attachment. Inhibition of both processes, virus release and virus binding, may explain why NA antibodies efficiently blocked virus dissemination in vitro and in vivo. Anti-NA immune sera showed broader reactivity than anti-HA sera in hemagglutination inhibition tests and demonstrated cross-subtype activity in sialidase inhibition tests. These remarkable features of NA antibodies highlight the importance of the NA antigen for the development of next-generation influenza virus vaccines.

NADPH-CYTOCHROME P-450 REDUCTASE: EVIDENCE FOR TWO SEPARATE STRUCTURAL DOMAINS AND ISOLATION AND CHARACTERIZATION OF THE MEMBRANE ASSOCIATED SEGMENT

Homogenous detergent-solubilized NADPH-Cytochrome P-450 reductase was incorporated into microsomes and liposomes. This binding occurred spontaneously at temperatures between 4(DEGREES) and 37(DEGREES) and appeared to involve hydrophobic forces as the binding was not disrupted by 0.5 M sodium chloride. This exogenously-added reductase was active catalytically towards native cytochrome P-450, suggesting an association with the microsomal membrane similar to endogenous reductase. Homogeneous detergent-solubilized reductase was disaggregated by Renex-690 micelles, confirming the presence of a hydrophobic combining region on the enzyme. In contrast to these results, steapsin protease-solubilized reductase was incapable of microsomal attachment and did not interact with Renex-690 micelles. Detergent-solubilized reductase (76,500 daltons) was converted into a form with the electrophoretic mobility of steapsin protease-solubilized reductase (68,000 daltons) and a 12,500 dalton peptide (as determined by polyacrylamide-SDS gel electrophoresis) when the liposomal-incorporated enzyme was incubated with steapsin protease. The 68,000 dalton fragment thus obtained had properties identical with steapsin protease-solubilized reductase, i.e. it was catalytically active towards cytochrome c but inactive towards cytochrome P-450 and did not bind liposomes. The 12,500 dalton fragment remained associated with the liposomes when the digest was fractionated by gel filtration, suggesting that this is the segment of the enzyme which is embedded in the phospholipid bilayer. Thus, detergent-solubilized reductase appears to contain a soluble catalytic domain and a separate and separable membrane-binding domain. This latter domain is required for attaching the enzyme to the membrane and also to facilitate the catalytic interaction between the reductase and its native electron acceptor, cytochrome P-450. The membrane-binding segment of the reductase was isolated by preparative gel electrophoresis in SDS following its generation by proteolytic treatment of liposome-incorporated reductase. The peptide has a molecular weight of 6,400 as determined by gel filtration in 8 M guanidine hydrochloride and has an amino acid composition which is not especially hydrophobic. Following removal of SDS and dialysis out of 6 M urea, the membrane-binding peptide was unable to inhibit the activity of a reconstituted system containing purified reductase and cytochrome P-450. Moreover, when reductase and cytochrome P-450 were added to liposomes which contained the membrane-binding peptide, it was determined that mixed function oxidase activity was reconstituted as effectively as when vesicles without the membrane-binding peptide were used. Thus, the membrane-binding peptide was ineffective as an inhibitor of mixed function oxidase activity, suggesting perhaps that it facilitates catalysis by anchoring the catalytic domain of the reductase proximal to cytochrome P-450 (i.e. in the same mixed micelle) rather than through a specific interaction with cytochrome P-450. ^

Microtubule dynamics and glutathione metabolism in phagocytizing human polymorphonuclear leukocytes.

Glutathione oxidants such as tertiary butyl hydroperoxide were shown previously to prevent microtubule assembly and cause breakdown of preassembled cytoplasmic microtubules in human polymorphonuclear leukocytes. The objectives of the present study were to determine the temporal relationship between the attachment and ingestion of phagocytic particles and the assembly of microtubules, and simultaneously to quantify the levels of reduced glutathione and products of its oxidation as potential physiological regulators of assembly. Polymorphonuclear leukocytes from human peripheral blood were induced to phagocytize opsonized zymosan at 30 degrees C. Microtubule assembly was assessed in the electron microscope by direct counts of microtubules in thin sections through centrioles. Acid extracts were assayed for reduced glutathione (GSH) and oxidized glutathione (GSSG), by the sensitive enzymatic procedure of Tietze. Washed protein pellets were assayed for free sulfhydryl groups and for mixed protein disulfides with glutathione (protein-SSG) after borohydride splitting of the disulfide bond. Resting cells have few assembled microtubules. Phagocytosis induces a cycle of rapid assembly followed by disassembly. Assembly is initiated by particle contact and is maximal by 3 min of phagocytosis. Disassembly after 5-9 min of phagocytosis is preceded by a slow rise in GSSG and coincides with a rapid rise in protein-SSG. Protein-SSG also increases under conditions in which butyl hydroperoxide inhibits the assembly of microtubules that normally follows binding of concanavalin A to leukocyte cell surface receptors. No evidence for direct involvement of GSH in the induction of assembly was obtained. The formation of protein-SSG, however, emerges as a possible regulatory mechanism for the inhibition of microtubule assembly and induction of their disassembly.