Spontaneous nano-emulsification: Process optimization and modeling for the prediction of the nanoemulsion’s size and polydispersity

The aim of the present study was to optimize the size and polydispersity of a lipid nanoemulsion as a function of the oil (Labrafac® WL1349), surfactant (Kolliphor® HS 15) and cosurfactant (Span® 80) phase composition and temperature. The nanoemulsions were prepared using a low-energy self-emulsification method. The Z-average diameter and the polydispersity index (PDI) were modeled with mixture experiments. Nanoemulsions from 20 nm to 120 nm with PDI < 0.2 were obtained at the three different tested temperatures (30 °C, 50 °C and 90 °C). The nanoemulsion size was able to be controlled with the oil, surfactant and cosurfactant concentrations. Interestingly, the smallest PDIs were obtained at 30 °C, and the cosurfactant concentration was able to be adjusted to optimize the formulation and to obtain nanoemulsions in the 20–120 nm range with a PDI smaller than 0.14. These nanoemulsions have shown a good stability at 4 °C in storage conditions and at 37 °C in diluted conditions

A Dual Fluorescence–Spin Label Probe for Visualization and Quantification of Target Molecules in Tissue by Multiplexed FLIM–EPR Spectroscopy

Simultaneous visualization and concentration quantification of molecules in biological tissue is an important though challenging goal. The advantages of fluorescence lifetime imaging microscopy (FLIM) for visualization, and electron paramagnetic resonance (EPR) spectroscopy for quantification are complementary. Their combination in a multiplexed approach promises a successful but ambitious strategy because of spin label-mediated fluorescence quenching. Here, we solved this problem and present the molecular design of a dual label (DL) compound comprising a highly fluorescent dye together with an EPR spin probe, which also renders the fluorescence lifetime to be concentration sensitive. The DL can easily be coupled to the biomolecule of choice, enabling in vivo and in vitro applications. This novel approach paves the way for elegant studies ranging from fundamental biological investigations to preclinical drug research, as shown in proof-of-principle penetration experiments in human skin ex vivo

Meiotic kinetochores fragment into multiple lobes upon cohesin loss in aging eggs.

Chromosome segregation errors during female meiosis are a leading cause of pregnancy loss and human infertility. The segregation of chromosomes is driven by interactions between spindle microtubules and kinetochores. Kinetochores in mammalian oocytes are subjected to special challenges: they need to withstand microtubule pulling forces over multiple hours and are built on centromeric chromatin that in humans is decades old. In meiosis I, sister kinetochores are paired and oriented toward the same spindle pole. It is well established that they progressively separate from each other with advancing female age. However, whether aging also affects the internal architecture of centromeres and kinetochores is currently unclear. Here, we used super-resolution microscopy to study meiotic centromere and kinetochore organization in metaphase-II-arrested eggs from three mammalian species, including humans. We found that centromeric chromatin decompacts with advancing maternal age. Kinetochores built on decompacted centromeres frequently lost their integrity and fragmented into multiple lobes. Fragmentation extended across inner and outer kinetochore regions and affected over 30% of metaphase-II-arrested (MII) kinetochores in aged women and mice, making the lobular architecture a prominent feature of the female meiotic kinetochore. We demonstrate that a partial cohesin loss, as is known to occur in oocytes with advancing maternal age, is sufficient to trigger centromere decompaction and kinetochore fragmentation. Microtubule pulling forces further enhanced the fragmentation and shaped the arrangement of kinetochore lobes. Fragmented kinetochores were frequently abnormally attached to spindle microtubules, suggesting that kinetochore fragmentation could contribute to the maternal age effect in mammalian eggs

Mechanism of TAp73 inhibition by ΔNp63 and structural basis of p63/p73 hetero-tetramerization.

Members of the p53 tumor suppressor family are expressed as multiple isoforms. Isoforms having an N-terminal transactivation domain are transcriptionally active while those ones lacking this domain often inhibit the transcriptional activity of other family members. In squamous cell carcinomas, the high expression level of ΔNp63α inhibits the tumour suppressor function of TAp73β. This can in principle be due to blocking of the promotor or by direct interaction between both proteins. P63 and p73 can hetero oligomerize through their tetramerization domains and a hetero-oligomer consisting of two p63 and two p73 molecules is thermodyna mically more stable than both homo tetramers. Here we show that hetero tetramer complexes exist also in differentiating keratinocytes. Through structure determination of the hetero tetramer we reveal why this hetero tetramer is the thermodynamically prefer red species. Based on this structure we have created mutants that either enable the formation of only heterotetramers or only homotetramers, allowing to investigate the function of these heterotetramers. Using these tools we show that inhibition of TAp73β in squamous cell carcinomas is due to promotor squelching and not direct interaction

Mechanism of TAp73 inhibition by ΔNp63 and structural basis of p63/p73 hetero-tetramerization.

Members of the p53 tumor suppressor family are expressed as multiple isoforms. Isoforms having an N-terminal transactivation domain are transcriptionally active while those ones lacking this domain often inhibit the transcriptional activity of other family members. In squamous cell carcinomas, the high expression level of ΔNp63α inhibits the tumour suppressor function of TAp73β. This can in principle be due to blocking of the promotor or by direct interaction between both proteins. P63 and p73 can hetero oligomerize through their tetramerization domains and a hetero-oligomer consisting of two p63 and two p73 molecules is thermodyna mically more stable than both homo tetramers. Here we show that hetero tetramer complexes exist also in differentiating keratinocytes. Through structure determination of the hetero tetramer we reveal why this hetero tetramer is the thermodynamically prefer red species. Based on this structure we have created mutants that either enable the formation of only heterotetramers or only homotetramers, allowing to investigate the function of these heterotetramers. Using these tools we show that inhibition of TAp73β in squamous cell carcinomas is due to promotor squelching and not direct interaction