Ratiometric Imaging of Extracellular pH in Dental Biofilms

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites are not distributed evenly in dental biofilms. A complex interplay of sorption to and reaction with organic matter in the biofilm reduces the diffusion paths of solutes and creates steep gradients of reactive molecules, including organic acids, across the biofilm. Quantitative fluorescent microscopic methods, such as fluorescence life time imaging or pH ratiometry, can be employed to visualize pH in different microenvironments of dental biofilms. pH ratiometry exploits a pH-dependent shift in the fluorescent emission of pH-sensitive dyes. Calculation of the emission ratio at two different wavelengths allows determining local pH in microscopic images, irrespective of the concentration of the dye. Contrary to microelectrodes the technique allows monitoring both vertical and horizontal pH gradients in real-time without mechanically disturbing the biofilm. However, care must be taken to differentiate accurately between extra- and intracellular compartments of the biofilm. Here, the ratiometric dye, seminaphthorhodafluor-4F 5-(and-6) carboxylic acid (C-SNARF-4) is employed to monitor extracellular pH in in vivo grown dental biofilms of unknown species composition. Upon exposure to glucose the dye is up-concentrated inside all bacterial cells in the biofilms; it is thus used both as a universal bacterial stain and as a marker of extracellular pH. After confocal microscopic image acquisition, the bacterial biomass is removed from all pictures using digital image analysis software, which permits to exclusively calculate extracellular pH. pH ratiometry with the ratiometric dye is well-suited to study extracellular pH in thin biofilms of up to 75 µm thickness, but is limited to the pH range between 4.5 and 7.0.     

A Dual Tracer 18F-FCH/18F-FDG PET Imaging of an Orthotopic Brain Tumor Xenograft Model

Early diagnosis of low grade glioma has been a challenge to clinicians. Positron Emission Tomography (PET) using ¹⁸F-FDG as a radio-tracer has limited utility in this area because of the high background in normal brain tissue. Other radiotracers such as ¹⁸F-Fluorocholine (¹⁸F-FCH) could provide better contrast between tumor and normal brain tissue but with high incidence of false positives. In this study, the potential application of a dual tracer ¹⁸F-FCH/¹⁸F-FDG-PET is investigated in order to improve the sensitivity of PET imaging for low grade glioma diagnosis based on a mouse orthotopic xenograft model. BALB/c nude mice with and without orthotopic glioma xenografts from U87 MG-luc2 glioma cell line are used for the study. The animals are subjected to ¹⁸F-FCH and ¹⁸F-FDG PET imaging, and images acquired from two separate scans are superimposed for analysis. The ¹⁸F-FCH counts are subtracted from the merged images to identify the tumor. Micro-CT, bioluminescence imaging (BLI), histology and measurement of the tumor diameter are also conducted for comparison. Results show that there is a significant contrast in ¹⁸F-FCH uptake between tumor and normal brain tissue (2.65 ± 0.98), but with a high false positive rate of 28.6%. The difficulty of identifying the tumor by ¹⁸F-FDG only is also proved in this study. All the tumors can be detected based on the dual tracer technique of ¹⁸F-FCH/ ¹⁸F-FDG-PET imaging in this study, while the false-positive caused by ¹⁸F-FCH can be eliminated. Dual tracer ¹⁸F-FCH/¹⁸F-FDG PET imaging has the potential to improve the visualization of low grade glioma. ¹⁸F-FCH delineates tumor areas and the tumor can be identified by subtracting the ¹⁸F-FCH counts. The sensitivity was over 95%. Further studies are required to evaluate the possibility of applying this technique in clinical trials.     

Clinical Relevance of Telomere Status and Telomerase Activity in Colorectal Cancer

The role of telomeres and telomerase in colorectal cancer (CRC) is well established as the major driving force in generating chromosomal instability. However, their potential as prognostic markers remains unclear. We investigated the outcome implications of telomeres and telomerase in this tumour type. We considered telomere length (TL), ratio of telomere length in cancer to non-cancer tissue (T/N ratio), telomerase activity and TERT levels; their relation with clinical variables and their role as prognostic markers. We analyzed 132 CRCs and paired non-cancer tissues. Kaplan-Meier curves for disease-free survival were calculated for TL, T/N ratio, telomerase activity and TERT levels. Overall, tumours had shorter telomeres than non-tumour tissues (P < 0.001) and more than 80% of CRCs displayed telomerase activity. Telomere lengths of non-tumour tissues and CRCs were positively correlated (P < 0.001). Considering telomere status and clinical variables, the lowest degree of telomere shortening was shown by tumours located in the rectum (P = 0.021). Regarding prognosis studies, patients with tumours showing a mean TL < 6.35 Kb experienced a significantly better clinical evolution (P < 0.001) and none of them with the highest degree of tumour telomere shortening relapsed during the follow-up period (P = 0.043). The mean TL in CRCs emerged as an independent prognostic factor in the Cox analysis (P = 0.017). Telomerase-positive activity was identified as a marker that confers a trend toward a poor prognosis. In CRC, our results support the use of telomere status as an independent prognostic factor. Telomere status may contribute to explaining the different molecular identities of this tumour type.     

The formation of titan cells in Cryptococcus neoformans depends on the mouse strain and correlates with induction of Th2‐type responses

Cryptococcus neoformans is a pathogenic yeast that can form titan cells in the lungs, which are fungal cells of abnormal enlarged size. Little is known about the factors that trigger titan cells. In particular, it is not known how the host environment influences this transition. In this work, we describe the formation of titan cells in two mouse strains, CD1 and C57BL/6J. We found that the proportion of C. neoformans titan cells was significantly higher in C57BL/6J mice than in CD1. This higher proportion of titan cells was associated with a higher dissemination of the yeasts to the brain. Histology sections demonstrated eosinophilia in infected animals, although it was significantly lower in the CD1 mice which presented infiltration of lymphocytes. Both mouse strains presented infiltration of granulocytes, but the amount of eosinophils was higher in C57BL/6J. CD1 mice showed a significant accumulation of IFN‐γ, TNF‐α and IL17, while C57BL/BL mice had an increase in the anti‐inflammatory cytokine IL‐4. IgM antibodies to the polysaccharide capsule and total IgE were more abundant in the sera from C57BL/6J, confirming that these animals present a Th2‐type response. We conclude that titan cell formation in C. neoformans depends, not only on microbe factors, but also on the host environment.     

Platelet proteomics applied to the search for novel antiplatelet therapeutic targets

Introduction: Unwanted platelet activation is associated with numerous diseases, mainly thrombosis-related. In this context, proteomics has emerged as a novel tool with potential for drug target discovery and to scrutinize the effects of antiplatelet drugs.

Areas covered: The present review presents the main findings of platelet proteomic studies to date in the context of drug target discovery and perspectives for the future ahead. It includes data and evidences obtained from literature searches on PubMed as well as commentaries derived from the authors’ experience and opinions.

Expert commentary: Platelet proteomics applied to drug target discovery is a young field. Recent studies have shown promising data, especially in the context of coronary artery disease. However, challenges remain such as establishing definitive guidelines for blood collection and platelet isolation, essential to guarantee data reproducibility. Recent advances in quantitative platelet proteomics should lead to novel studies with higher clinical impact in the near future.     

Nanoencapsulation of Rose-Hip Oil Prevents Oil Oxidation and Allows Obtainment of Gel and Film Topical Formulations

The rose-hip oil holds skin regenerating properties with applications in the dermatological and cosmetic area. Its nanoencapsulation might favor the oil stability and its incorporation into hydrophilic formulations, besides increasing the contact with the skin and prolonging its effect. The aim of the present investigation was to develop suitable rose-hip-oil-loaded nanocapsules, to verify the nanocapsule effect on the UV-induced oxidation of the oil and to obtain topical formulations by the incorporation of the nanocapsules into chitosan gel and film. The rose-hip oil (500 or 600 μL), polymer (Eudragit RS100®, 100 or 200 mg), and acetone (50 or 100 mL) contents were separately varied aiming to obtain an adequate size distribution. The results led to a combination of the factors acetone and oil. The developed formulation showed average diameter of 158?±?6 nm with low polydispersity, pH of 5.8?±?0.9, zeta potential of +9.8?±?1.5 mV, rose-hip oil content of 54?±?1 μL/mL and tendency to reversible creaming. No differences were observed in the nanocapsules properties after storage. The nanoencapsulation of rose-hip oil decreased the UVA and UVC oxidation of the oil. The chitosan gel and film containing rose-hip-oil-loaded nanocapsules showed suitable properties for cutaneous use. In conclusion, it was possible to successfully obtain rose-hip-oil-loaded nanocapsules and to confirm the nanocapsules effect in protecting the oil from the UV rays. The chitosan gel and film were considered interesting alternatives for incorporating the nanoencapsulated rose-hip oil, combining the advantages of the nanoparticles to the advantages of chitosan.