Evaluation Of Bacterial Diversity Recovered From Petroleum Samples Using Different Physical Matrices

Unraveling the microbial diversity and its complexity in petroleum reservoir environments has been a challenge throughout the years. Despite the techniques developed in order to improve methodologies involving DNA extraction from crude oil, microbial enrichments using different culture conditions can be applied as a way to increase the recovery of DNA from environments with low cellular density for further microbiological analyses. This work aimed at the evaluation of different matrices (arenite, shale and polyurethane foam) as support materials for microbial growth and biofilm formation in enrichments using a biodegraded petroleum sample as inoculum in sulfate reducing condition. Subsequent microbial diversity characterization was carried out using Scanning Electronic Microscopy (SEM), Denaturing Gradient Gel Electrophoresis (DGGE) and 16S rRNA gene libraries in order to compare the microbial biomass yield, DNA recovery efficiency and diversity among the enrichments. The DNA from microbial communities in petroleum enrichments was purified according to a protocol established in this work and used for 16S rRNA amplification with bacterial generic primers. The PCR products were cloned, and positive clones were screened by Amplified Ribosomal DNA Restriction Analysis (ARDRA). Sequencing and phylogenetic analyses revealed that the bacterial community was mostly represented by members of the genera Petrotoga, Bacillus, Pseudomonas, Geobacillus and Rahnella. The use of different support materials in the enrichments yielded an increase in microbial biomass and biofilm formation, indicating that these materials may be employed for efficient biomass recovery from petroleum reservoir samples. Nonetheless, the most diverse microbiota were recovered from the biodegraded petroleum sample using polyurethane foam cubes as support material. (C) 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. This is an open access article under the CC BY-NC-ND license47371272

Crystalline TiO2 coating on titanium surface : eletrochemical stability and biological properties

Orientador: Valentim Adelino Ricardo BarãoTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de PiracicabaResumo: Os implantes dentários são comumente usados na reabilitação protética e o processo de osseointegração é mandatório para o sucesso e longevidade do tratamento. A osseointegracão envolve processos organizados, dos quais fazem parte proteínas, citocinas inflamatórias, células e a precipitação de hidroxiapatita. Todos esses processos podem ser influenciados pelas propriedades da superfície do implante. O titânio (Ti) e suas ligas são os materiais mais utilizados devido às suas adequadas propriedades mecânicas e biocompatibilidade. Tais propriedades são devidas à formação natural de uma camada instável de dióxido de titânio (TiO2) na superfície, protetora ao processo de corrosão. Além disso, a bioatividade do Ti pode ser reduzida de acordo com o envelhecimento natural da superfície. Assim, foram propostos dois estudos in vitro: a incorporação de um filme de TiO2 estável e cristalino, a fim de melhorar as propriedades físico-químicas do Ti (1), seguido da fotofuncionalização por luz ultravioleta (UV), capaz de renovar a superfície (2). No primeiro estudo, filmes de TiO2 pulverizados foram formados na superfície de Ti, com diferentes fases cristalinas (anatase, rutilo e fase mista). Os filmes foram avaliados quanto ao comportamento eletroquímico, propriedades de superfície, adsorção de proteína e precipitação da hidroxiapatita. Para isso, as superfícies foram caracterizadas quanto a composição química, topografia, fase cristalina e energia livre de superfície. Testes eletroquímicos foram conduzidos usando solução de fluido corpóreo (SFC). A adsorção de albumina foi medida pelo método do ácido bicinconínico. A precipitação da hidroxiapatita foi avaliada após 28 dias de imersão em SBF. Em geral, as fases cristalinas de TiO2 apresentaram melhor comportamento eletroquímico, especialmente a mista, que apresentou a maior resistência à polarização (p<0,05). O rutilo apresentou maior diminuição da densidade de corrente e taxa de corrosão, enquanto a fase mista apresentou comportamento passivo mais estável (p<0,05). A fase mista aumentou a adsorção de albumina (p<0,05). A morfologia da hidroxiapatita foi dependente da fase cristalina, sendo mais evidente no grupo misto. A combinação das fases anatase e rutilo nos filmes de TiO2 parece ser mais adequada para a aplicação em implantes, devido à maior proteção contra corrosão, maior adsorção de proteína e bioatividade. Assim, no segundo estudo, apenas a fase mista do TiO2 foi usada para testar sua influência e da fotofuncionalização (meio de ativação da superfície) nas alterações físico-químicas de superfície e comportamento celular. As superfícies foram analisadas quanto à morfologia, topografia, composição química, fase cristalina e molhabilidade. Células pré-osteoblásticas (MC3T3E1) foram utilizadas para avaliar a morfologia, adesão, viabilidade, mineralização e expressão de citocinas (IFN-?, TNF-?, IL-4, IL-6 e IL-17). A fotofuncionalização aumentou a molhabilidade das superfícies tratadas com TiO2 e não tratadas (p<0,05). O TiO2 fotofuncionalizado apresentou maior adesão e viabilidade celular nos dias 2 e 4 (p<0,05). A fotofuncionalização potencializou essa viabilidade e induziu maior mineralização (dia 14) para ambas as superfícies (p<0,05). Em geral, as proteínas avaliadas foram levemente afetadas pelos tratamentos UV ou TiO2. Assim, a incorporação de TiO2 misto na superfície do Ti, seguida da fotofuncionalização por UV é promissora para aplicação em implantes dentáriosAbstract: Dental implants are commonly used in prosthetic rehabilitation, and the osseointegration is mandatory for the success and longevity of the treatment. The osseointegration process involves organized mechanisms, in which proteins, inflammatory cytokines, cells and hydroxyapatite orchestrate such process. These mechanisms can be governed by the surface properties of an implant material. Titanium (Ti) and its alloys are the most used material due to their adequate mechanical properties and bioactivity. Such properties are due to the unstable titanium dioxide oxide (TiO2) naturally formed on its surface, which is protective to the corrosion process. Furthermore, the bioactivity of Ti can be reduced according to the natural aging of the surface. Thus, two in vitro studies were proposed: an incorporation of a stable and crystalline TiO2 film to improve the physical-chemical properties of Ti (1), followed by an ultraviolet light (UV) photofunctionalization, which is able to renew the surface (2). In the first study, sputtered TiO2 films were grown on titanium (Ti) surface, with different crystalline phases (anatase, rutile and mixed phase). Films were evaluated regarding their electrochemical behavior, surface properties, protein adsorption and hydroxyapatite precipitation. The surfaces were characterized as chemical composition, topography, crystalline phase and surface free energy (SFE). Electrochemical tests were conducted using simulated body fluid (SBF). Albumin adsorption was measured by bicinchoninic acid method. Hydroxyapatite (HA) precipitation was evaluated after 28 days of immersion in SBF. In general, TiO2 crystalline phases showed improved electrochemical behavior, specially mixed TiO2 that showed the highest polarization resistance (p<0.05). Rutile phase exhibited a greater influence on decreasing the current density and corrosion rate, while the mixed phase displayed a more stable passive behavior (p<0.05). Regarding protein interaction, mixed phase increased the albumin adsorption (p<0.05). The morphology of HA was dependent to the crystalline phase, being more evident in the mixed group. The combination of anatase and rutile structures to generate TiO2 films seems to be more suitable for biomedical implants application as greater corrosion protection, higher protein adsorption and bioactivity were accounted. Therefore, in the second study, we only used mixed TiO2 phase to verify its influence and the photofunctionalization on the physical-chemical surface alterations and cell behavior. Surfaces were analyzed in terms of morphology, topography, chemical composition, crystalline phase and wettability. Pre-osteoblastic cells (MC3T3E1) were used to assess cell morphology and adhesion, viability, mineralization and cytokine expression (IFN-?, TNF-?, IL-4, IL-6 and IL-17). Photofunctionalization increased the wettability of both surface conditions (p<0.05). TiO2-treated samples featured normal cell morphology and spreading, and greater cell viability at 2 and 4 days (p<0.05). UV potentiated such viability and induced higher mineralization (day 14) for both surfaces (p<0.05). In general, assessed proteins were found slightly affected by either UV or TiO2 treatments. Thus, mixed TiO2 incorporation on Ti surface followed by a UV photofunctionalization seems to be promising to be applied on dental implantsMestradoPrótese DentalDoutora em Clínica Odontológica001CAPE

Screening of Marine-derived Fungi Isolated from the sponge Didemnun ligulum for Biodegradation of Pentachlorophenol

Contamination by pesticides employed in agriculture has caused serious environmental harm. Pentachlorophenol (PCP) is a phenolic organochlorine compound and a dangerous pollutant which was banned from Brazil since 1985; however, there are still many contaminated areas. This pesticide is a serious problem because it has high toxicity and persistence at the environment due to its resistance to biotic and abiotic degradation. The use of microorganisms as degrading agents is considered an efficient method to reduce the adverse effects of environmental contaminants. It is noteworthy that fungi from marine environment are adapted to extreme conditions, including high chlorine concentrations, and can produce unique enzymes with interesting properties. Therefore, marine-derived fungi have an excellent enzymatic potential for the biotransformation of xenobiotics such as organochlorine pesticides. In this work, fifteen fungi strains isolated from a marine invertebrate, the ascidian Didemnun ligulum, were evaluated according to their ability to grow in solid culture media (3% malt extract agar) in the presence of different concentrations (10, 25, 30, 40, and 50 mg L–1) of PCP. Among the tested strains, nine could grow in at least one concentration, and Trichoderma harzianum CBMAI 1677 showed optimal growth at the higher evaluated concentration (50 mg L–1), showing toxicity resistance and suggesting its potential for biodegradation of PCP. In a later work, it was observed that T. harzianum CBMAI 1677 was able to degrade PCP. These results confirmed the efficiency of marine-derived fungi to biodegrade persistent compounds and could enable the development of bioremediation methodologies using these microorganism

Evaluation of bacterial diversity recovered from petroleum samples using different physical matrices

Unraveling the microbial diversity and its complexity in petroleum reservoir environments has been a challenge throughout the years. Despite the techniques developed in order to improve methodologies involving DNA extraction from crude oil, microbial enrichments using different culture conditions can be applied as a way to increase the recovery of DNA from environments with low cellular density for further microbiological analyses. This work aimed at the evaluation of different matrices (arenite, shale and polyurethane foam) as support materials for microbial growth and biofilm formation in enrichments using a biodegraded petroleum sample as inoculum in sulfate reducing condition. Subsequent microbial diversity characterization was carried out using Scanning Electronic Microscopy (SEM), Denaturing Gradient Gel Electrophoresis (DGGE) and 16S rRNA gene libraries in order to compare the microbial biomass yield, DNA recovery efficiency and diversity among the enrichments. The DNA from microbial communities in petroleum enrichments was purified according to a protocol established in this work and used for 16S rRNA amplification with bacterial generic primers. The PCR products were cloned, and positive clones were screened by Amplified Ribosomal DNA Restriction Analysis (ARDRA). Sequencing and phylogenetic analyses revealed that the bacterial community was mostly represented by members of the genera Petrotoga, Bacillus, Pseudomonas, Geobacillus and Rahnella. The use of different support materials in the enrichments yielded an increase in microbial biomass and biofilm formation, indicating that these materials may be employed for efficient biomass recovery from petroleum reservoir samples. Nonetheless, the most diverse microbiota were recovered from the biodegraded petroleum sample using polyurethane foam cubes as support material.47371272

PERFIL NUTRICIONAL DE IDOSOS HOSPITALIZADOS

This study aimed to know the nutritional profile of elderly patients hospitalized in a public hospital. A retrospective survey of 250 nutritional assessment sheets of elderly people admitted to the hospital service was carried out, where anthropometric data, nutritional diagnosis and laboratory tests were collected. Prevalence of low weight and lean mass depletion was detected among the oldest old, as well as a significant correlation between the levels of albumin and creatine with the muscular depletion measured by the calf circumference. Regarding food intake, it was observed that 14% ingested a lower amount than prescribed, and the long-lived elderly people needed more food intervention. It is concluded that the incidence of nutritional risk and malnutrition among hospitalized elderly people is higher among long-lived elderly people, and that in this group there is a negative correlation between serum albumin levels with BMI and CP.Este estudo teve como objetivo conhecer o perfil nutricional de pacientes idosos hospitalizados em um hospital público. Realizou-se pesquisa retrospectiva de 250 fichas de avaliação nutricional de idosos admitidos no serviço hospitalar, onde foram coletados dados antropométricos, diagnóstico nutricional e exames laboratoriais. Foi detectado prevalência de baixo peso e depleção de massa magra entre os idosos longevos, além de uma correlação significativa entre os níveis de albumina e creatina com a depleção muscular medida pela circunferência da panturrilha. Em relação a ingesta alimentar, observou-se que 14% ingeriram um valor menor do que o prescrito, sendo que os idosos longevos necessitaram de maior intervenção alimentar. Conclui-se que a incidência de risco nutricional e desnutrição entre idosos hospitalizados é maior entre os idosos longevos, e que neste grupo há uma correlação negativa entre os níveis séricos de albumina com IMC e CP

Cellulolytic and proteolytic ability of bacteria isolated from gastrointestinal tract and composting of a hippopotamus

The bioprospection for cellulase and protease producers is a promise strategy for the discovery of potential biocatalysts for use in hydrolysis of lignocellulosic materials as well as proteic residues. These enzymes can increment and turn viable the production of second generation ethanol from different and alternative sources. In this context, the goal of this study was the investigation of cellulolytic and proteolytic abilities of bacteria isolated from the gastrointestinal tract of a hippopotamus as well as from its composting process. It is important to highlight that hippopotamus gastrointestinal samples were a non-typical sources of efficient hydrolytic bacteria with potential for application in biotechnological industries, like biofuel production. Looking for this, a total of 159 bacteria were isolated, which were submitted to qualitative and quantitative enzymatic assays. Proteolytic analyzes were conducted through the evaluation of fluorescent probes. Qualitative assays for cellulolytic abilities revealed 70 positive hits. After quantitative analyzes, 44 % of these positive hits were selected, but five (5) strains showed cellulolytic activity up to 11,8 FPU/mL. Regarding to proteolytic activities, six (6) strains showed activity above 10 %, which overpassed results described in the literature. Molecular analyzes based on the identification of 16S rDNA, revealed that all the selected bacterial isolates were affiliated to Bacillus genus. In summary, these results strongly indicate that the isolated bacteria from a hippopotamus can be a potential source of interesting biocatalysts with cellulolytic and proteolytic activities, with relevance for industrial applications.Brazilian research agency (FAPESP)Brazilian research agency (CAPES)Brazilian research agency (CNPq)Univ Fed Sao Paulo, Dept Biol Sci, Rua Sao Nicolau 210, BR-09913030 Sao Paulo, BrazilSao Paulo Zoo Pk Fdn, Av Miguel Estefano 4241, BR-04301905 Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Dept Biophys, Rua 3 Maio,100, BR-04044020 Sao Paulo, SP, BrazilFed Univ Latin Amer Integrat, Latin Amer Inst Life Sci & Nat, Av Tarquinio Joslin Santos 1000, BR-85870901 Foz Do Iguacu, Parana, BrazilUniv Fed Sao Paulo, Dept Biol Sci, Rua Sao Nicolau 210, BR-09913030 Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Biophys, Rua 3 Maio,100, BR-04044020 Sao Paulo, SP, BrazilFAPESP: 2010/51992-5CNPq: 475166/2013-2Web of Scienc

Estudo comparativo da expressão do colágeno na parede cólica de pacientes operados por doença diverticular do sigmóide

Objective: This study aimed to investigate the expression of collagen in the colon wall of patients with diverticular disease of the sigmoid. Method: For this study, samples of sigmoid colon of 15 patients named Disease Group, seven men and eight women, aged 37-57 years underwent surgery for the treatment of diverticulitis were selected. For the Control Group, patients’ specimens undergoing emergency surgery for trauma sigmoid were used and they didn’t have associated diseases. The surgeries were performed in hospitals in the city of Jundiaí. The histological study of surgical specimens were performed by staining with hematoxylin - eosin (HE) and picrosirius histochemical method for collagen quantification. Results: The collagen areas expressed in the colon wall (F), glandular tissue (E) and Total Area of Disease Group are significantly higher than areas of the Control Group (p=0.003, p=0.026 and p=0.010) respectively. The comparison of the proportions of the collagen areas (Proportion F) and muscle tissue (Proportion M) in the two groups was statistically significant (p=0.044 and p=0.026) respectively. The other variables investigated, area of muscle tissue (M) and proportion (E), showed no significant differences. Conclusion: In this study, the collagen expression in the colon wall of the patients operated for sigmoid diverticular disease was higher than in patients without disease.Objetivo: O presente trabalho teve como objetivo estudar a expressão do colágeno na parede cólica de pacientes com doença diverticular do sigmoide. Método: Para a realização deste estudo, foram selecionadas amostras de colo sigmoide de 15 pacientes (Grupo Doente), sete homens e oito mulheres, com faixa etária entre 37 a 57 anos de idade, dos quais 15 foram submetidos à cirurgia para o tratamento de diverticulite. As cirurgias foram realizadas em hospitais na cidade de Jundiaí, São Paulo. Para o Grupo Controle foram utilizados espécimes de pacientes submetidos à cirurgia de urgência por traumatismo de sigmoide e que não apresentavam doenças associadas. A avaliação histológica dos espécimes cirúrgicos foi realizada pela coloração da hematoxilina-eosina (HE) e pelo método histoquímico do picrosirius para a quantificação do colágeno. Resultados: As áreas expressas de colágeno na parede cólica (F), no tecido glandular (E), e na Área Total dos pacientes do Grupo Doente são significantemente maiores do que nas áreas do Grupo Controle (p=0,003, p=0,026 e p=0,010, respectivamente). A comparação das proporções da área de colágeno (Proporção F) e do tecido muscular (Proporção M) nos dois grupos foi estatisticamente significante (p=0,044 e p=0,026, respectivamente). As demais variáveis investigadas, área de tecido muscular (M) e proporção (E), não apresentaram diferenças significantes. Conclusão: Neste estudo, a expressão do colágeno na parede cólica dos pacientes operados por doença diverticular do sigmoide foi maior do que nos pacientes sem doença.Dados abertos - Sucupira - Teses e dissertações (2013 a 2016

Disponibilização de fluoreto na saliva após o uso de dentifrícios contendo NAF ou MFP

Dentifrícios fluoretados são reconhecidos pela sua eficácia na prevenção de cárie, a qual é baseada no efeito de F solúvel (na forma iônica e MFP) disponível na formulação. Nos dentifrícios a base de fluoreto de sódio (NaF), todo o F está na forma iônica, enquanto nas formulações a base de monofluorofosfato (Na2FPO3, MFP), o F é ligado covalentemente ao fosfato e requer a hidrólise enzimática para liberar F iônico. Neste estudo, foi avaliado F solúvel (iônico e na forma de MFP) disponível na saliva após a escovação com dentifrícios brasileiros mais vendidos, formulados a base de NaF ou Na2FPO3 (contendo, respectivamente, SiO2 e CaCO3 como abrasivos). Em um estudo in vivo, cruzado e cego, 12 voluntários escovaram seus dentes por 1 minuto, com 1 g dos dentifrícios: a. dentifrício não fluoretado, b. dentifrício a base de Na2FPO3/CaCO3 (1361.1 ± 28.7 ppm F solúvel, Sorriso Dentes Brancos®) ou c. dentifrício a base de NaF/SiO2 (1422.1 ± 6.21 ppm F solúvel, Colgate Total 12®). Saliva não estimulada foi coletada antes e 5, 15, 30, 45 e 60 minutos após a escovação. As concentrações de F solúvel total (FST) e F iônico (FI) foram dosadas por um eletrodo íon-seletivo. A concentração de FST foi similar após o uso das duas formulações de dentifrícios fluoretados. O percentual de F iônico após o uso de dentifrício a base de MFP/CaCO3 variou de 30,8% a 84,4% durante o período de estudo. Os resultados sugerem que as formulações comumente utilizadas no Brasil são capazes de aumentar a concentração de F na saliva para interferir com o processo de cárie dental.Fluoride (F) toothpastes are well recognized for their effectiveness in caries prevention, which is based on the effect of soluble F (ionic and as MFP ion) available from the formulation. In sodium fluoride (NaF) toothpastes, all F is ionic, whereas in sodium monofluorphospohate (Na2FPO3, MFP) toothpastes, F is covalently bound to phosphate and requires enzymatic hydrolysis to release F ion. In this study, we evaluated soluble F (ionic and as MFP ion) availability in saliva following toothbrushing with top-selling Brazilian toothpastes with formulations based on NaF or Na2FPO3 (containing, respectively, SiO2 and CaCO3 as abrasives). In an in vivo, crossover and blind design, 12 volunteers brushed their teeth for 1 min with 1 g of the toothpastes: a. non-fluoridated toothpaste, b. Na2FPO3/CaCO3 toothpaste (1361.1 ± 28.7 ppm soluble F, Sorriso Dentes Brancos®) or c. NaF/SiO2 toothpaste (1422.1 ± 6.21 ppm soluble F, Colgate Total 12®). Unstimulated saliva was collected before brushing and 5, 15, 30, 45 and 60 min following toothbrushing. Total soluble F (TSF) and ionic F concentrations were measured by means of ion-selective electrode. The concentration of TSF was similar after the use of two formulations of fluoride dentifrices. The percentage of ionic F after the used of the MFP/CaCO3 toothpaste ranged from 30.8% to 84.4% during the study period. The results suggest that formulations commonly used in Brazilian dentifrices are able to increase the F concentration in saliva to interfere with the dental caries process