Combined Application of Antisense Oligomers to Control Transcription Factors of Candida albicans Biofilm Formation.

Antisense oligomers (ASOs) have been little exploited to control determinants of Candida albicans virulence. Biofilm formation is an important virulence factor of C. albicans, that is regulated by a complex network of transcription factors (such as EFG1, BRG1 and ROB1). Thus, the main goal of this work was to project ASOs, based on the 2'-OMethyl chemical modification, to target BRG1 and ROB1 mRNA and to validate its application either alone or in combination with the EFG1 mRNA target, to reduce C. albicans biofilm formation. The ability of ASOs to control gene expression was evaluate by qRT-PCR. The effect on biofilm formation was determined by the total biomass quantification, and simultaneously the carbohydrates and proteins reduction on extracellular matrix. It was verified that all the oligomers were able to reduce the levels of gene expression and the ability of C. albicans to form biofilms. Furthermore, the combined application of the cocktail of ASOs enhances the inhibition of C. albicans biofilm formation, minimizing biofilm thickness by reducing the quantity of matrix content (protein and carbohydrate). So, our work confirms that ASOs are useful tools for research and therapeutic development on the control of Candida species biofilm formation.

SPINNE: An app for human vertebral height estimation based on artificial neural networks.

The absence or poor preservation of vertebrae often prevent the application of the anatomical method for stature estimation. The main objective of this paper was to develop a web app based on artificial neural network (ANN) models to estimate the vertebral height of absent or poorly preserved vertebrae from other vertebrae and thus enable the application of anatomical methods. Artificial neural models were developed based on the vertebral height of vertebrae C2 to S1 of a sample composed of 56 adult male and 69 adult female individuals. The skeletons belong to the Identified Skeletal Collection of the University of Coimbra and the ages at death of these individuals ranged from 22 to 58 years old. Statistical analysis and algorithmic development were performed with the R language, R Core Team (2018). Intra- and inter-observer errors regarding the vertebral height were small for all vertebrae (<0.45 mm). Significant models to estimate vertebral height were obtained for both sexes and for the sex-pooled group, although none with an R2 higher than 0.48 and 0.34 for the C2 and the S1, respectively. The root mean square error (RMSE) regarding the predicted vertebral height and the observed vertebral height was almost always smaller than 1.0 mm while most R2 values were higher than 0.6 although models with worse performances were obtained for some vertebrae located at the ends of the vertebral column (C3, L4, and L5). The ANN models have clear potential to predict vertebral height. This mathematical approach may be used to enable the application of the anatomical method for stature estimation when some vertebrae are absent or poorly preserved. The application of the ANN models can be carried out by using the new web based app SPINNE.

Antimicrobial assessment of phage therapy using a porcine model of biofilm infection.

Antibiotic resistant bacterial communities persist in many types of wounds, chronic wounds in particular, in the form of biofilms. Biofilm formation is a major cause of severe infections and the main reason for a negative treatment outcome and slow healing progression. Chronic wounds are a silent epidemic essentially affecting people with co-morbid conditions such as diabetes and obesity and elderly persons particularly those with movement limitations. The development of complementary and alternative effective strategies to antibiotics for the treatment of chronic wounds is highly desired. Phage therapy constitutes a very promising approach in the control of topical microbial populations. In this work newly isolated phages were tested for their efficacy to control bacterial species that predominate in chronic wounds. Phage effectiveness was studied on 24-h old biofilms formed in polystyrene microplates and in porcine skin explants using two treatment approaches: individual phage and a cocktail of phages against four main pathogens commonly isolated from chronic wounds. The two models produced variations in the surface colonization ability, assessed by viable bacterial counts and microscopy visualization after using peptide nucleic acid (PNA) or locked nucleic acid probes (LNA) and 2'-O-methyl (2'-OMe) in fluorescence in situ hybridization (FISH), and in the phage-host interactions. Phages alone and combined caused greater reductions in the number of viable cells when biofilms had been formed on porcine skins and with greater variations detected at 4 h and 24 h of sampling. These results suggest that porcine skin models should be preferentially used to assess the use of phages and phage cocktails intended for topical use in order to understand the fate, throughout treatment time, of the population when dealing with biofilm-related infections.

Phage therapy as an alternative or complementary strategy to prevent and control biofilm-related infections.

The complex heterogeneous structure of biofilms confers to bacteria an important survival strategy. Biofilms are frequently involved in many chronic infections in consequence of their low susceptibility to antibiotics as well as resistance to host defences. The increasing need of novel and effective treatments to target these complex structures has led to a growing interest on bacteriophages (phages) as a strategy for biofilm control and prevention. Phages can be used alone, as a cocktail to broaden the spectra of activity, or in combination with other antimicrobials to improve their efficacy. Here, we summarize the studies involving the use of phages for the treatment or prevention of bacterial biofilms, highlighting the biofilm features that can be tackled with phages or combined therapy approaches.

The dental pulp stem cell niche based on aldehyde dehydrogenase 1 expression.

AIM: To detect cells expressing the stem cell marker ALDH1 (aldehyde dehydrogenase1) in the pulp of human permanent teeth and to investigate the expression of ALDH1 in isolated dental pulp cells. METHODOLOGY: Pulp tissue was collected and processed for immunohistochemistry to detect ALDH1-, STRO-1- and CD90-positive cells. In addition, cells were isolated and analysed by flow cytometry for ALDH1 activity and for the cell surface markers CD44, CD73, CD90, STRO-1 and CD45. Cells were also examined for multidifferentiation capacity. Within these cells, an ALDH1(+) cell subpopulation was selected and evaluated for multidifferentiation capacity. RESULTS: The immunohistochemistry analyses showed that ALDH1-, CD90- and STRO-1-positive cells were located mainly in the perivascular areas and nerve fibres of dental pulps. Cells on the fifth passage had high expression for CD44, CD73 and CD90, whereas moderate labelling was observed for STRO-1 and ALDH1 in flow cytometry analysis. On the same passages, cells were able to differentiate into osteogenic, adipogenic and chondrogenic lineages. The ALDH1(+) cell subpopulation also demonstrated multilineage differentiation ability. CONCLUSIONS: Dental pulp stem cells reside in the vicinity of blood vessels and nerve fibres, indicating the possible existence of more than one stem cell niche in dental pulps. Furthermore, ALDH1 was expressed by isolated dental pulp cells, which had mesenchymal stem cell characteristics. Thus, it can be suggested that ALDH1 may be used as a DPSC marker.

Association among XRCC1, XRCC3, and BLHX gene polymorphisms and chromosome instability in lymphocytes from patients with endometriosis and ovarian cancer.

Endometriosis is a complex disease that has both benign and malignant characteristics. It affects 5-10% of women of reproductive age. Studies have demonstrated the existence of common genetic changes in endometriosis and ovarian cancer, suggesting a possible association between these 2 diseases. However, the mechanisms that lead to the development of cancer from endometriosis remain unknown. In this study, we evaluated 3 groups of women: 72 patients with endometriosis, 70 with ovarian cancer, and 70 healthy individuals (controls). Repair (XRCC1 codons 194 and 399, XPD codons 312 and 751, and XRCC3 codon 241)- and metabolism (BLHX codon 443)-related gene polymorphisms were analyzed using the polymerase chain reaction-restriction fragment length polymorphism technique; the efficiency of DNA damage repair was analyzed in vitro in lymphocytes exposed to bleomycin. The logistic regression model was used to evaluate key associations. The results showed an increased average of chromosome breakage in bleomycin-treated lymphocytes from patients with endometriosis and ovarian cancer compared with healthy women. We also detected significant association between XRCC1, XRCC3, and BLHX polymorphisms and a high frequency of chromosomal damage. Women with endometriosis or ovarian cancer may have an altered mechanism of DNA repair, and these defects may be related to a higher incidence of ovarian cancer.