There is broad interest in the question of fluid-fluid phase coexistence in membranes, in particular, whether evidence for liquid-disordered (l(d))-liquid-ordered (l(o)) two-phase regions or membrane "rafts" can be found in natural membranes. In model membrane systems, such phase behavior is observed, and we have used deuterium nuclear magnetic resonance spectroscopy to map the phase boundaries of ternary mixtures containing 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), chain-perdeuterated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC-d(62)), and cholesterol. For both this ternary model system and the binary DPPC-d(62)/cholesterol system, we present clear evidence for l(d)-l(o) two-phase coexistence. We have selected sample compositions to focus on this region of fluid-fluid phase coexistence and to determine its temperature and composition ranges. The deuterium nuclear magnetic resonance spectra for compositions near the l(d)-l(o) phase boundary at high cholesterol concentrations show evidence of exchange broadening or critical fluctuations in composition, similar to that reported by Vist and Davis. There appears to be a line of critical compositions ranging from 48 degrees C for a DOPC/DPPC-d(62)/cholesterol composition of 0:75:25, to approximately -8 degrees C for the composition 57:14:29. At temperatures below this two-phase region, there is a region of three-phase coexistence (l(d)-l(o)-gel). These results are collected and presented in terms of a partial ternary phase diagram that is consistent with previously reported results of Vist and Davis.

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Direct visualization of raft-like l(o) (liquid-ordered) domains in model systems and cells using microscopic techniques requires fluorescence probes with known partitioning preference for one of the phases present. However, fluorescent probes may display dissimilar partitioning preferences in different lipid systems and can also affect the phase behaviour of the host lipid bilayer. Therefore a detailed understanding of the behaviour of fluorescent probes in defined lipid bilayer systems with known phase behaviour is essential before they can be used for identifying domain phase states. Using giant unilamellar vesicles composed of the ternary lipid mixture DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine)/DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine)/cholesterol, for which the phase behaviour is known, we examined nine commonly used fluorescent probes using confocal fluorescence microscopy. The partitioning preference of each probe was assigned either on the basis of quantification of the domain area fractions or by using a well-characterized l(d) (liquid-disordered)-phase marker. Fluorescent probes were examined both individually and using dual or triple labelling approaches. Most of the probes partitioned individually into the l(d) phase, whereas only NAP (naphtho[2,3-a]pyrene) and NBD-DPPE [1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl] preferred the l(o) phase. We found that Rh-DPPE (Lissamine rhodamine B-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine) increased the miscibility transition temperature, T(mix). Interestingly, the partitioning of DiIC18 (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) was influenced by Bodipy-PC [2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexa-decanoyl-sn-glycero-3-phosphocholine]. The specific use of each of the fluorescent probes is determined by its photostability, partitioning preference, ability to detect lipid phase separations and induced change in T(mix). We demonstrate the importance of testing a specific fluorescent probe in a given model membrane system, rather than assuming that it labels a particular lipid phase.

The microbiota isolated from the urine of bladder carcinoma patients exhibits significantly increased compositional abundance of some bacterial genera compared to the urine of healthy patients. Our aim was to compare the microbiota composition of cancerous tissues and urine samples collected from the same set of patients in order to improve the accuracy of diagnostic measures. Tissue samples were collected from patients during cancer tissue removal by transurethral resection. In parallel, urine samples were obtained by transurethral resectoscopy from the same patients. The V3–V4 region of the bacterial 16S rRNA gene was sequenced and analyzed using the Kraken pipeline. In the case of four patients, duplicate microbiota analysis from distant parts of the cancerous tissues was highly reproducible, and independent of the site of tissue collection of any given patient. Akkermansia, Bacteroides, Clostridium sensu stricto, Enterobacter and Klebsiella, as “five suspect genera”, were over-represented in tissue samples compared to the urine. To our knowledge, this is the first study comparing urinary and bladder mucosa-associated microbiota profiles in bladder cancer patients. More accurate characterization of changes in microbiota composition during bladder cancer progression could provide new opportunities in the development of appropriate screening or monitoring methods.