Investigation of the Membrane Fluidity Regulation of Fatty Acid Intracellular Distribution by Fluorescence Lifetime Imaging of Novel Polarity Sensitive Fluorescent Derivatives.

Free fatty acids are essential structural components of the cell, and their intracellular distribution and effects on membrane organelles have crucial roles in regulating the metabolism, development, and cell cycle of most cell types. Here we engineered novel fluorescent, polarity-sensitive fatty acid derivatives, with the fatty acid aliphatic chain of increasing length (from 12 to 18 carbons). As in the laurdan probe, the lipophilic acyl tail is connected to the environmentally sensitive dimethylaminonaphthalene moiety. The fluorescence lifetime imaging analysis allowed us to monitor the intracellular distribution of the free fatty acids within the cell, and to simultaneously examine how the fluidity and the microviscosity of the membrane environment influence their localization. Each of these probes can thus be used to investigate the membrane fluidity regulation of the correspondent fatty acid intracellular distribution. We observed that, in PC-12 cells, fluorescent sensitive fatty acid derivatives with increased chain length compartmentalize more preferentially in the fluid regions, characterized by a low microviscosity. Moreover, fatty acid derivatives with the longest chain compartmentalize in lipid droplets and lysosomes with characteristic lifetimes, thus making these probes a promising tool for monitoring lipophagy and related events.

Effect of liver fibrosis on survival in patients with intrahepatic cholangiocarcinoma: a SEER population-based study.

BACKGROUND: Intrahepatic cholangiocarcinoma (iCCA) is a biliary tract malignancy with rising incidence in recent decades. While the causative role of cirrhosis in the development of iCCA is well established, the role of cirrhosis as a prognostic factor in iCCA is debatable. MATERIALS AND METHODS: The study population consisted of 512 patients diagnosed with iCCA between 2004-2016 collected from the Surveillance, Epidemiology and End Results (SEER) database. The impact of fibrosis on overall and cancer-specific survival 12, 36 and 60 months following diagnosis, was evaluated in the entire cohort and in sub-groups stratified according to treatment approach and the American Joint Committee on Cancer (AJCC) tumor stage using a Cox proportional-hazards model. RESULTS: After adjusting for age, sex, race, year of diagnosis, AJCC stage, and surgical treatment strategy, advanced fibrosis was associated with worse cancer-specific survival across follow up periods (HR 1.49 (1.13-1.96, p = 0.005); HR 1.44 (1.14-1.83, p = 0.002) and HR 1.45 (1.15-1.83, p = 0.002) for 12, 36 and 60 months, respectively). Similar effects were observed for overall survival. Among patients that underwent surgical resection, advanced fibrosis was associated with worse overall survival and cancer-specific survival across follow up periods. Fibrosis was associated with worse overall and cancer-specific survival in patients with a later stage (III-IV) at diagnosis but this effect was not demonstrated in early stages. CONCLUSIONS: Patients with iCCA and advanced liver fibrosis have an increased risk of both overall and cancer-specific mortality compared to patients with earlier stages of fibrosis.

Novel inhibitors of leukocyte transendothelial migration.

Leukocyte transendothelial migration is one of the most important step in launching an inflammatory immune response and chronic inflammation can lead to devastating diseases. Leukocyte migration inhibitors are considered as promising and potentially effective therapeutic agents to treat inflammatory and auto-immune disorders. In this study, based on previous trioxotetrahydropyrimidin based integrin inhibitors that suboptimally blocked leukocyte adhesion, twelve molecules with a modified scaffold were designed, synthesized, and tested in vitro for their capacity to block the transendothelial migration of immune cells. One of the molecules, namely, methyl 4-((2-(tert-butyl)-6-((2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene) methyl) phenoxy) methyl) benzoate, (compound 12), completely blocked leukocyte transendothelial migration, without any toxic effects on immune or endothelial cells (IC50 = 2.4 µM). In vivo, compound 12 exhibited significant therapeutic effects in inflammatory bowel disease (IBD)/Crohn's disease, multiple sclerosis, fatty liver disease, and rheumatoid arthritis models. A detailed acute and chronic toxicity profile of the lead compound in vivo did not reveal any toxic effects. Such a type of molecule might therefore provide a unique starting point for designing a novel class of leukocyte transmigration blocking agents with broad therapeutic applications in inflammatory and auto-immune pathologies.

Hagfish slime threads as a biomimetic model for high performance protein fibres.

Textile manufacturing is one of the largest industries in the world, and synthetic fibres represent two-thirds of the global textile market. Synthetic fibres are manufactured from petroleum-based feedstocks, which are becoming increasingly expensive as demand for finite petroleum reserves continues to rise. For the last three decades, spider silks have been held up as a model that could inspire the production of protein fibres exhibiting high performance and ecological sustainability, but unfortunately, artificial spider silks have yet to fulfil this promise. Previous work on the biomechanics of protein fibres from the slime of hagfishes suggests that these fibres might be a superior biomimetic model to spider silks. Based on the fact that the proteins within these 'slime threads' adopt conformations that are similar to those in spider silks when they are stretched, we hypothesized that draw processing of slime threads should yield fibres that are comparable to spider dragline silk in their mechanical performance. Here we show that draw-processed slime threads are indeed exceptionally strong and tough. We also show that post-drawing steps such as annealing, dehydration and covalent cross-linking can dramatically improve the long-term dimensional stability of the threads. The data presented here suggest that hagfish slime threads are a model that should be pursued in the quest to produce fibres that are ecologically sustainable and economically viable.

Hagfish slime ecomechanics: testing the gill-clogging hypothesis.

Hagfish are able to produce substantial amounts of slime when harassed, but the precise ecological function of the slime is unclear. One possibility is that the slime acts as a defence against gill-breathing predators, whose gills may become entangled with the slime's mixture of mucins and fibrous threads during an attack. We previously demonstrated that hagfish slime does not bind water tightly, but instead behaves like a fine sieve that slows water down via viscous entrainment. These properties are consistent with the gill-clogging hypothesis, which we tested here by quantifying the effects of hagfish slime on water flow through an artificial gill model and real fish gills. Our results indicate that the slime is capable of clogging gills and increasing the resistance that they present to the flow of water. We also characterized the behaviour of slime release from live hagfish and the effect of convective mixing on the formation of slime in vitro. Our observations show that exudate is locally released from the slime glands as a coherent jet and that hagfish do not appear to use their slime as a protective envelope. We found that convective mixing between the exudate and seawater is necessary for proper slime formation, but excessive mixing leads to the slime's collapse. We suggest that the loose binding of water by the slime may be an optimal solution to the problem of delivering an expanding jet of flow-inhibiting material to the gills of would-be predators.

Composition, morphology and mechanics of hagfish slime.

Hagfish slime consists of mucins and protein threads that are released from slime glands and mix with seawater to produce an ephemeral material with intriguing physical properties. We recently characterized the mechanics of the slime's fibrous component, and here we report the first mechanical properties of the mucin component and the slime as a whole. Our results suggest that hagfishes can produce remarkable quantities of the slime because it is almost three orders of magnitude more dilute than typical mucus secretions. Mechanical experiments using whole slime produced in vitro demonstrate that the slime threads dominate the slime's material properties and impart elasticity. Mucins impart viscosity at the strain rates tested and are important for rapid deployment of the slime. We also found that slime threads are tapered at both ends, which suggested to us that hagfish slime might best be modeled as a discontinuous fibre-reinforced composite. Our measurements demonstrate that the mucins are not capable of providing shear linkage between threads, but this is not necessary because the threads are long enough to span an entire slime mass. Our findings suggest that hagfish slime consists mainly of bulk seawater entrained between mucin-coated threads, and in this way functions more like a fine sieve than coherent mucus. These results are consistent with the hypothesis that the slime has evolved as a defense against gill-breathing predators.