Lactobacillus crispatus represses vaginolysin expression by BV associated Gardnerella vaginalis and reduces cell cytotoxicity

Using a chemically-defined medium simulating genital tract secretions, we have shown that pre-adhering Lactobacillus crispatus to Hela epithelial cells reduced cytotoxicity caused by Gardnerella vaginalis. This effect was associated to the expression of vaginolysin and was specific to L. crispatus interference, as other vaginal facultative anaerobes had no protective effect.This work was supported by Portuguese National Funds (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684). JC, and MER acknowledge the financial support of individual Grants SFRH/BD/93963/2013, and SFRH/BPD/95401/2013 respectively. NC is an Investigador FCT.info:eu-repo/semantics/publishedVersio

Simulated Vaginal Fluid: Candida resistant strains biofilm characterization and vapor phase of essential oil effect

Introduction : Vulvovaginal candidiasis is a disease that affects millions of women worldwide. Oral formulations, topical creams or ointments are the conventional dosage forms, with an increase in drug administration through vaginal via. The use of simulated biological fluids (e.g. vaginal fluid) in the evaluation of antifungal therapies may better mimic the real biological environments and therefore provide a better understanding of the behavior of the antifungal. Methods : The main objective of this work was to compare planktonic growth and biofilm formation of Candida species, on common growth medium, Sabouraud Dextrose Broth (SDB) and on vaginal simulation conditions, Simulated Vaginal Fluid (SVF), through the optical density determination, colony-forming units and scanning electron microscopy. In addition, under the same conditions this study also evaluated the ability of vapor phase of oregano and white thyme essential oils (VP-EOs), potential alternative treatment, to inhibit biofilm formation and to destroy mature biofilms of vaginal isolates, through the colony-forming units determination. Results : Candida isolates maintained the same biofilm formation capacity and morphology in both media (SVF and SDB). Furthermore, the results obtained in this work related with VP-EOs effect agree with results acquired, previously, with SDB. This means that the effect of VP-EOs is not affected by the SVF medium, and that this fluid allows the dissolution of the volatile and bioactive compounds. Conclusions : These results can predict the in vivo behaviour, suggesting a potential effective application of VP-EOs as prophylactic or therapeutic treatment for biofilm-related vulvovaginal candidiasis.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and grant ref 2020.05720.BD for Liliana Fernandes. Also, this study was supported by LABBELS — Associate Laboratory in Biotechnology, Bioengineering and Microelectromechanical Systems, LA/P/0029/2020 and Maria Elisa Rodrigues thanks FCT for funding through program DL 57/2016—Norma transitóriainfo:eu-repo/semantics/publishedVersio

The effectiveness of voriconazole in therapy of Candida glabratas biofilms oral infections and its influence on the matrix composition and gene expression

Candida glabrata is one of most prevalente yeast in fungal infections, especially in immunocompromised patients. Its azole resistance results in a low therapeutic response, particularly when associated with biofilms. The main goal of this work was to study the effectiveness of voriconazole (Vcz) against C. glabrata biofilms oral pathologies, as esophageal or oropharyngeal candidiasis. Antifungal susceptibilities were determined in pre-formed 24-h-biofilms and ERG genes expression was determined by qRT-PCR. Protein quantification was performed using BCA Kit, carbohydrate was estimated according to the Dubois assay and b-1,3 glucans concentration were determined using Glucatell kit. Finally, ergosterol, Vcz, and fluconazole (Flu) concentrations within the bio-film matrices were determined by RP-HPLC. Results showed that C. glabrata biofilms were more susceptible to Vcz than to Flu and that ERG genes expression evidenced an overexpression of the three ERG genes in the presence of both azoles. The matrix content presented a remarked decrease in proteins and an increase in carbohydrates, namely b-1,3 glucans. Ergosterol was successfully detected and quantified in the biofilm matrices, with no differences in all the considered conditions. Vcz demonstrated better diffusion through the biofilms and better cell penetration capacities, than Flu, indicating that the structure of the drug molecule fully influences its dissemination through the biofilm matrices. This work showed that Vcz is notably more effective than Flu for the treatment of resistant C. glabrata oral biofilms, which demonstrates a clinical relevance in its future use for the treatment of oropharyngeal/esophageal candidiasis caused by this species.This work was supported by the Programa Operacional, Fatores de competitividade— COMPETE and by national funds through FCT—Fundação para a Ciência e a Tecnologia on the scope of the projects FCT [PTDC/SAU-MIC/119069/2010], [RECI/EBB-EBI/0179/2012], [PEst-OE/EQB/LA0023/2013] and Célia F. Rodrigues’ [SFRH/ BD/93078/2013] PhD Grant. The authors thank the Project ‘‘BioHealth—Biotechnology and Bioengineering approaches to improve health quality’’, Ref. NORTE-07-0124-FEDER000027, co-funded by the Programa Operacional Regional do Norte (ON.2—O Novo Norte), QREN, FEDER. The authors would like to acknowledge Pfizer, S.A. for the kindly donation of Voriconazole and Fluconazole.info:eu-repo/semantics/publishedVersio

Antimicrobial activity of phenolic extracts of Eucalyptus globulus and Juglans regia against dairy industry pathogens

Book of Abstracts of CEB Annual Meeting 2017[Excerpt] Bovine mastitis (BM) is the most expensive pathology for dairy industry and Staphylococcus aureus is amongst the most prevalent causative agents of this disease. Nowadays, it is known that S. aureus contaminated milk can enter the dairy production chain and be the origin of food contamination. Due to the poor efficacy of antibiotics and to the ability to form biofilms evidenced by this pathogen, BM has become increasingly difficult to control and to eradicate. Phenolic plant extracts are nowadays being evaluated since they are a rich source of bioactive molecules. Thus, in this work the antimicrobial activity of E. globulus and J. regia alone and in combination against S. aureus BM isolates was evaluated and compared with penicillin G (Pen G), an antibiotic commonly used in the treatment of this pathology. The evaluation of the cytotoxic potential of both extracts was also performed. [...]info:eu-repo/semantics/publishedVersio

Strategies for adaptation of mAb-producing CHO cells to serum-free medium

Large-scale production of biopharmaceuticals commonly requires the use of serum-free medium, for safety and cost reasons. However, serum is essential to most mammalian cells growth, and its removal implies a very time-consuming process for cell adaptation. Thus, the aim of the study was to evaluate different strategies for cell adaptation to serum-free medium. Three cell types were used to assess the impact of transfection on adaptation: one common CHO-K1 cell line and two CHO-K1 cells transfected with different technologies for antibody production. Cultures were started with a known cell concentration in Dulbecco’s Modified Eagle Medium supplemented with 10% serum. The effect of five combinations of supplements, that could support cells during adaptation, was tested. These supplements included insulin and trace elements (copper sulfate, zinc sulfate, sodium selenite, ammonium iron citrate, ferrous ammonium sulfate, ammonium metavanadate, nickel chloride and stannous chloride). A methodology of gradual adaptation was followed, consisting on sequential steps of serum reduction, after assuring good cell adaptation from the previous step. After reaching 0.625% serum, medium was gradually switched to the chemically defined serum-free EX-CELL CHO DHFR- medium. It was observed that supplements influence cell adaptation to serum-free medium. Indeed, the combinations containing the trace element ammonium iron citrate gave the worst results, with cell death at 2.5% serum. In contrast, the combination of ammonium metavanadate, nickel chloride and stannous chloride proved to be the most favorable to the three cell lines. Comparing the cells, it seems that the ability to produce antibody and the transfection methodology used does not have a great impact on adaptation. During the study, some procedure details were identified as particularly important and should be carefully considered in the process of cell adaptation to serum-free medium. These include the use of a higher initial cell concentration that will allow the survival of an increased number of cells during the process; avoiding harsh procedures to the cells such as centrifugation and the use of enzymes (i.e. trypsin), due to a higher cell sensibility during adaptation; and to give enough time for a full cell adaptation at each step

Candida albicans antimicrobial and antibiofilm activity of novel endodontic solvents

Background: Candida albicans is the most prevalent fungi isolated in endodontic infections. In this study, the ability of C. albicans biofilm to tolerate the novel solvent mixtures methyl ethyl ketone (MEK)/tetrachloroethylene (TCE) and MEK/orange oil (OOil) sequentially to the standard irrigation of sodium hypochlorite (NaOCl) and ethylenediaminetetraacetic (EDTA) was evaluated. Methods: Biofilm cell cultures of C. albicans SC 5314 were treated sequentially with NaOCl and EDTA and exposed to MEK/TCE or MEK/OOil. The effect of the antimicrobial treatment was evaluated using the disk diffusion method for planktonic culture, and the enumeration of colony-forming units (CFUs) and scanning electron microscope (SEM) for biofilm cell culture. Results: C. albicans mature biofilm (24 h) was significantly reduced in biomass and cell viability after solvent mixtures exposure, compared with the previous NaOCl and EDTA treatments. MEK/OOil combination caused a total reduction of biofilm, while with MEK/TCE, there was a 3-log (CFU/cm2) reduction compared with the sequence NaOCl and EDTA, and a 4-log (CFU/cm2) reduction compared with the control. Conclusions: The additional exposure of a preformed 24 h C. albicans biofilm to novel solvent mixtures MEK/TCE and MEK/OOil caused a positive antibiofilm impact, overcoming the performance of the conventional endodontic irrigating protocol.This article was supported by National Funds through FCT-Fundação para a Ciência e a Tecnologia, I.P., within CINTESIS, R&D Unit (reference UIDB/4255/2020). This work was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and grant ref 2020.05720.BD for Liliana Fernandes; BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020-Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

Preliminary evaluation of microcarrier culture for growth and monoclonal antibody production of CHO-K1 cells

Large-scale biopharmaceutical production commonly relies on suspension cell cultures that provide higher yields than adherent cultures. However, most mammalian cells grow adherently and therefore need to be adapted to suspended growth, which is not always simple or feasible. Microcarrier culture introduces new possibilities and makes achievable the practical high yield culture of anchorage-dependent cells in suspension systems. The aim of this study was to evaluate and optimize the use of microcarrier culture for the growth and antibody production of CHO-K1 cells. For this, the macroporous Cultispher microcarriers were used, and the initial cell adhesion to the microcarriers (occurring in the first 5-6 hours) and further cell proliferation were assessed. Cultures of antibody-producing CHO-K1 cells were performed in 50 ml vented conical tubes, and different conditions were tested: initial cell concentration (2x105 cells/ml and 4x105 cells/ml), microcarrier concentration (1 g/L and 2 g/L), type of rocking during the first 6 hours of adhesion (pulse or continuous) and rocking after initial adhesion (no rocking and 60 rpm). Cell concentration and viability in the microcarriers were assessed periodically (hourly for the adhesion phase, and daily after that). It was observed that an increase in the initial cell concentration does not enhance initial adhesion, possibly due to saturation of the microcarrier surface. For its turn, increasing microcarrier concentration, without further increasing initial cell concentration does not improve cell densities achieved in the culture. Concerning rocking, the most favorable type for the adhesion phase was pulse rocking and, after this, a continuous rocking provided an improved cell proliferation. In conclusion, microcarrier cultures proved to be a viable alternative to suspended cultures for the growth and antibody production of CHO-K1 cells

Community violence and alcohol abuse among adolescents: a sex comparison

OBJECTIVE: To verify whether there is an association between victimization and alcohol use among boys and girls in Porto Alegre, RS, Brazil. METHODS: This was a cross-sectional study of students from public schools, aged 10 to 19 years, from the fifth grade of primary education to the third grade of secondary education, selected by two-stage cluster sampling, defined by the socioeconomic characteristics of the neighborhood in which each school is located and by school class. Data collection was by means of a questionnaire which was completed anonymously and voluntarily. RESULTS: Fifty-four percent of the older adolescent boys (aged 14-19) drank alcohol, compared to 17% of the younger adolescent boys (10-13), as did 58% of the older adolescent girls and 19% of the younger adolescent girls. Fifty-seven percent of the students had suffered severe victimization, and 53% had suffered moderate victimization. When the samples were separated by sex, it was observed that boys and girls who drank alcohol reported 2.6 and 1.8 times more severe victimization respectively, while alcohol was associated with 3.1 and 2.5 times greater prevalence of moderate victimization, among boys and girls respectively. When episodes of drunkenness were analyzed, it was observed that adolescents got drunk more than pre-adolescents and that exposure to violence exhibited an increased association with drunkenness. Thirty-two percent of the boys (prevalence ratio, PR = 4.4; 95%CI 2.6-7.3) and 22% of the girls (PR = 2.2; 95%CI 1.2-4.1) who had been the victims of severe violence reported being drunk at least once. CONCLUSIONS: Adolescents of both sexes who consume more alcohol are at greater risk of suffering community violence.OBJETIVO: Verificar a existência de associação entre vitimização e uso de álcool entre meninos e meninas de Porto Alegre (RS). MÉTODOS: Foi realizado um estudo transversal. Participaram estudantes de escolas públicas com idade entre 10 e 19 anos, da 5ª série do ensino fundamental até a 3ª série do ensino médio, compondo amostra por conglomerados em dois estágios, definidas pelas características socioeconômicas do bairro da escola e pela turma. A coleta de dados foi realizada por questionário padronizado de forma anônima e voluntária. RESULTADOS: O álcool foi utilizado por 54% dos meninos adolescentes (14-19) e 17% dos adolescentes (10-13), por 58% das meninas adolescentes mais velhas e 19% das adolescentes mais novas. Cinqüenta e sete por cento dos estudantes sofreram vitimização severa, e 53% sofreram vitimização moderada. Quando as amostras separadas por sexos foram avaliadas, observou-se que meninos e meninas que faziam uso de álcool relataram 2,6 e 1,8, respectivamente, mais vitimização grave, ao passo que o uso de álcool entre meninos e meninas se associou a 3,1 e 2,5 mais prevalência de vitimização moderada, respectivamente. Para os episódios de embriaguez, observou-se que adolescentes se embriagaram mais que pré-adolescentes e que as exposições à violência mostraram associação aumentada para embriaguez. Um percentual de 32% de meninos (razão de prevalência, RP = 4,4; IC95% 2,6-7,3) e 22% de meninas (RP = 2,2; IC95% 1,2-4,1) vítimas de violência severa relatou embriaguez pelo menos uma vez. CONCLUSÕES: Adolescentes de ambos os sexos que consomem mais bebidas alcoólicas têm maior risco de sofrerem violência comunitária.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação Universidade Federal de Ciências da Saúde de Porto AlegreUniversidade Luterana do BrasilUniversidade Federal de São Paulo (UNIFESP)UFCSPAUNIFESP, EPMSciEL

Antisense locked nucleic acid gapmers to control Candida albicans filamentation

Whereas locked nucleic acid (LNA) has been extensively used to control gene expression, it has never been exploited to control Candida virulence genes. Thus, the main goal of this work was to compare the efficacy of five different LNA-based antisense oligonucleotides (ASO) with respect to the ability to control EFG1 gene expression, to modulate filamentation and to reduce C. albicans virulence. In vitro, all LNA-ASOs were able to significantly reduce C. albicans filamentation and to control EFG1 gene expression. Using the in vivo Galleria mellonella model, important differences among the five LNA-ASOs were revealed in terms of C. albicans virulence reduction. The inclusion of PS-linkage and palmitoyl-2-amino-LNA chemical modification in these five LNA gapmers proved to be the most promising combination, increasing the survival of G. mellonella by 40%. Our work confirms that LNA-ASOs are useful tools for research and therapeutic development in the candidiasis field.This study was supported by the Portuguese Foundation forScience and Technology (FCT) under the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European RegionalDevelopment Fund under the scope of Norte2020-ProgramaOperacional Regional do Norte and Daniela Eira Araújo [SFRH/BD/121417/2016] PhD grant. The authors also acknowledge theproject funding by the“02/SAICT/2017–Projetos de Investiga-ção Científica e Desenvolvimento Tecnológico (IC&DT)–POCI-01-0145-FEDER-028893”. VILLUM Fonden is acknowledgedfor funding the Biomolecular Nano-scale Engineering Center(BioNEC), a Villum center of excellence, grant numberVKR18333. Funding received by iBB-Institute for Bioengineer-ing and Biosciences from FCT (UID/BIO/04565/2020) andPrograma Operacional Regional de Lisboa 2020 (Project No.007317) is also acknowledged. We acknowledge Dr. LucíliaGoreti Pinto, Life and Health Sciences Research Institute(ICVS), School of Medicine, University of Minho, forprocessing and sectioningG. mellonellatissue samples.The authors declare no conflict of interest.info:eu-repo/semantics/publishedVersio