ContactJ: Characterization of lipid droplet-mitochondrial contacts using fluorescence microscopy and image analysis.

Lipid droplets (LDs) are the major lipid storage organelles of eukaryotic cells and together with mitochondria key regulators of cell bioenergetics. LDs communicate with mitochondria and other organelles forming "metabolic synapse" contacts to ensure that lipid supply occurs where and when necessary. Although transmission electron microscopy analysis allows an accurate and precise analysis of contacts, the characterization of a large number of cells and conditions can become a long-term process. In order to extend contact analysis to hundreds of cells and multiple conditions, we have combined confocal fluorescence microscopy with advanced image analysis methods. In this work, we have developed the ImageJ macro script ContactJ, a novel and straight image analysis method to identify and quantify contacts between LD and mitochondria in fluorescence microscopy images allowing the automatic analysis. This image analysis workflow combines colocalization and skeletonization methods, enabling the quantification of LD-mitochondria contacts together with a complete characterization of organelles and cellular parameters. The correlation and normalization of these parameters contribute to the complex description of cell behavior under different experimental energetic states. ContactJ is available here: https://github.com/UB-BioMedMicroscopy/ContactJ/tree/1.0.

Impact of locking solutions on conditioning biofilm formation in tunnelled haemodialysis catheters and inflammatory response activation.

INTRODUCTION: The surface of tunnelled cuffed catheters provides an optimal environment for the development of biofilms, which have recently been described as conditioning films because of the presence of adherent biological materials. These biofilms are associated with infection and thrombosis and potentially increase patients' inflammatory response. These complications could be reduced by the use of locking solutions. OBJECTIVE: To analyse biofilm formation, using confocal and electron microscopy, in tunnelled cuffed catheters locked with three different solutions and to determine the relationship between these solutions and inflammatory response. STUDY DESIGN: This prospective study included 35 haemodialysis patients with tunnelled cuffed catheter removal for non-infection-related reasons. The participants were divided into three groups according to the lock solution used: (1) heparin 1: 5000 IU; (2) citrate 4%; and (3) taurolidine 1.35%, citrate 4% and heparin 500 IU (taurolock); in the latter group, 25,000 IU taurolidine-urokinase was used in the last weekly session. All tunnelled cuffed catheters were cultured, and the inner surface was evaluated with confocal and electron microscopy. The inflammatory profile of included patients was determined at tunnelled cuffed catheter removal. RESULTS: There were no differences in clinical or demographic variables between the three subgroups. Biofilm thickness was lower in the taurolidine group than in the citrate 4% and heparin groups (28.85 ± 6.86 vs 49.99 ± 16.56 vs 56.2 ± 15.67 µm, respectively; p < 0.001), as was biofilm volume (1.01 ±1.18 vs 3.7 ± 2.15 vs 5.55 ±2.44, µm3, respectively; p < 0.001). The mean interleukin-6 value was 39%, which was 50% lower than in the citrate and heparin groups, but without significance differences. CONCLUSION: Our results show that biofilms were found in all tunnelled cuffed catheters, but the thickness and volume were significantly lower in tunnelled cuffed catheters locked with taurolidine solution. Therefore, the type of locking solution used in tunnelled cuffed catheters should maintain tunnelled cuffed catheter sterility and prevent catheter-related bloodstream infections. No significant difference was observed in the inflammatory profile according to the type of locking solution.

Bicosomes: bicelles in dilute systems.

Bicelles are discoidal phospholipid nanostructures at high lipid concentrations. Under dilute conditions, bicelles become larger and adopt a variety of morphologies. This work proposes a strategy to preserve the discoidal morphology of bicelles in environments with high water content. Bicelles were formed in concentrated conditions and subsequently encapsulated in liposomes. Later dilution of these new structures, called bicosomes, demonstrated that lipid vesicles were able to isolate and protect bicelles entrapped inside them from the medium. Characterization of systems before and after dilution by dynamic light-scattering spectroscopy and cryo-transmission electron microscopy showed that free bicelles changed in size and morphology, whereas encapsulated bicelles remained unaltered by the effect of dilution. Free and entrapped bicelles (containing the paramagnetic contrast agent gadodiamide) were injected into rat brain lateral ventricles. Coronal and sagittal visualization was performed by magnetic resonance imaging. Whereas rats injected with free bicelles did not survive the surgery, those injected with bicosomes did, and a hyperintensity effect due to gadodiamide was observed in the cerebrospinal fluid. These results indicate that bicosomes are a good means of preserving the morphology of bicelles under dilution conditions.

Spontaneous fibrillation of the native neuropeptide hormone Somatostatin-14.

Natural Somatostatin-14 is a small cyclic neuropeptide hormone with broad inhibitory effects on endocrine secretions. Here we show that natural Somatostatin-14 spontaneously self-assembles in water and in 150 mM NaCl into liquid crystalline nanofibrils, which follow characteristic structural features of amyloid fibrils. These non-covalent highly stable structures are based on the Somatostatin native backbone conformation and are formed under non-denaturing conditions. Our results support the hypothesis that self-assembly into amyloid fibrils is a generic property of the polypeptide chain under appropriate conditions. Given recent advances on the mechanisms of biological storage and sorting modes of peptide/protein hormones into secretory granules, we propose that Somatostatin-14 fibrillation could be relevant to the regulated secretion pathway of this neuropeptide hormone. Such a hypothesis is consistent with the emerging concept of the existence of non-disease related but functional amyloids.

alphavbeta3 integrin expression and pinopod formation in normal and out-of-phase endometria of fertile and infertile women.

BACKGROUND: There is scanty and contradictory information regarding the comparison of traditional histological dating criteria of the endometrium with the expression of the new markers of endometrial receptivity such as alphavbeta3 integrin and pinopods. Also, definite data with respect to the potential correlation existing between these different new markers in defining the putative window of implantation are lacking. METHODS: The temporal relationship between alphavbeta3 integrin expression and pinopod formation in normal and out-of-phase endometrial biopsies from normal healthy women (n = 12) and infertile patients (n = 36) was investigated. Two endometrial biopsies (postovulatory day +7 to +8 and 4 days later) were performed during a single menstrual cycle in each subject. Estradiol and progesterone serum concentrations were quantified on the same days as endometrial sampling. RESULTS: No statistically significant difference regarding alphavbeta3 integrin expression, pinopod formation, and hormone concentrations was found between fertile controls and infertile patients irrespective of endometria being in-phase or out-of-phase. Although a coordinate high level of expression of alphavbeta3 integrin and pinopod on postovulatory days 7-8 was observed, there was an evident lack of temporal co-expression of these markers over the luteal phase in the endometrial samples investigated. CONCLUSIONS: There is a clear dissociation in the temporal expression of the most cited markers postulated to frame the window of implantation. The functional significance (if any) of these new markers remains to be established.