Water-miscible organic cosolvents enhance phosphatidylinositol-specific phospholipase C phosphotransferase as well as phosphodiesterase activity

AbstractPhosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis catalyzes the hydrolysis of phosphatidylinositol (PI) in a Ca2+-independent two-step mechanism: (i) an intramolecular phosphotransferase reaction to form inositol 1,2-(cyclic)-phosphate (cIP), followed by (ii) a cyclic phosphodiesterase activity that converts cIP to inositol 1-phosphate (I-1-P). Moderate amounts of water-miscible organic solvents have previously been shown to dramatically enhance the cyclic phosphodiesterase activity, that is, hydrolysis of cIP. Cosolvents [isopropanol (iPrOH), dimethylsufoxide (DMSO), and dimethylformamide (DMF)] also enhance the phosphotransferase activity of PI-PLC toward PI initially presented in vesicles, monomers, or micelles. Although these water-miscible organic cosolvents caused large changes in PI particle size and distribution (monitored with pyrene-labeled PI fluorescence, 31P NMR spectroscopy, gel filtration, and electron microscopy) that differed with the activating solvent, the change in PI substrate structure in different cosolvents was not correlated with the enhanced catalytic efficiency of PI-PLC toward its substrates. PI-PLC stability was decreased in water/organic cosolvent mixtures (e.g., the Tm for PI-PLC thermal denaturation decreased linearly with added iPrOH). However, the addition of myo-inositol, a water-soluble inhibitor of PI-PLC, helped stabilize the protein. At 30% iPrOH and 4 °C (well below the Tm for PI-PLC in the presence of iPrOH), cosolvent-induced changes in protein secondary structure were minimal. iPrOH and diheptanoylphosphatidylcholine, each of which activates PI-PLC for cIP hydrolysis, exhibited a synergistic effect for cIP hydrolysis that was not observed with PI as substrate. This behavior is consistent with a mechanism for cosolvent activation that involves changes in active site polarity along with small conformational changes involving the barrel rim tryptophan side chains that have little effect on protein secondary structure

Boosting and Taming Wave Breakup in Second Harmonic Generation

Modulation instability is a universal phenomenon that can be found in a wide variety of nonlinear systems where, in the presence of a noise seed, peaks of random intensities can be generated. Several dynamical systems admit exact solutions in the form of breathers or solitons on a finite background. The vast majority of soliton studies has been restricted so far to one-dimensional systems. In contrast, the occurrences of localized structures in fully spatiotemporal systems has been only sporadically explored. In this work, we experimentally study the conditions for the wave-breaking of spatially extended optical beams in the process of second harmonic generation. Whenever the pump energy of the picosecond-long fundamental beam reaches a critical level, the beam shape at the second harmonic in a KTP crystal breaks into small filaments. These filaments exhibit extreme local intensity peaks, and their statistical distribution can be modified by the input energy of the fundamental beam. Moreover, by analyzing similar wave-breaking dynamics in a PPLN crystal in the presence of a higher nonlinear quadratic response, we observe that the spatial beam breaking may even gradually vanish as the laser intensity grows larger, leading to a spatial reshaping into a smooth and wider beam, accompanied by a substantial broadening of its temporal spectrum

Solid polymer electrolytes from a fluorinated copolymer bearing cyclic carbonate pendant groups

A poly(vinylidene fluoride-co-(2-oxo-1,3-dioxolan-4-yl)methyl 2-(trifluoromethyl)acrylate) random copolymer, poly(VDF-co-MAF-cyCB), with a MAF-cyCB weight fraction of 59% was synthesized via free radical copolymerization of VDF and MAF-cyCB, which is a methacrylate bearing cyclocarbonate side-chain. This copolymer showed nano-structured morphology, where crystalline PVDF-rich domains co-existed with amorphous poly(VDF-co-MAF-cyCB) segments. Solid polymer electrolytes were further obtained by loading the poly(VDF-co-MAF-cyCB) copolymer with various amounts of LiClO4. The added lithium salt was dissolved in the poly(VDF-co-MAF-cyCB) amorphous phase, which allowed the formation of an ionic conducting phase exhibiting ionic conductivity values as high as 2 × 10−4 S cm−1 at room temperature for an optimum cyCB/Li+ molar ratio of 5. The addition of LiClO4 up to the optimum cyCB/Li+ molar ratio of 5 also increased the phase separation between the crystalline and amorphous phases, the mechanical properties of the material (up to 107 at 102 rad s−1) and the ionic conductivity (>10−3 S cm−1 at 80 °C). Furthermore, an electrochemical stability window from 1.4 to 4.9 V vs. Li/Li+ and relatively high values for the measured lithium ions transference numbers (0.68 at 40 °C) were observed, making the investigated system a promising candidate for next generation solid polymer electrolytes

High-sensitivity calcium biosensor on the mitochondrial surface reveals that IP3R channels participate in the reticular Ca2+ leak towards mitochondria

International audienceGenetically encoded biosensors based on fluorescent proteins (FPs) are widely used to monitor dynamics and sub-cellular spatial distribution of calcium ion (Ca2+) fluxes and their role in intracellular signaling pathways. The development of different mutations in the Ca2+-sensitive elements of the cameleon probes has allowed sensitive range of Ca2+ measurements in almost all cellular compartments. Region of the endoplasmic reticulum (ER) tethered to mitochondria, named as the mitochondrial-associated membranes (MAMs), has received an extended attention since the last 5 years. Indeed, as MAMs are essential for calcium homeostasis and mitochondrial function, molecular tools have been developed to assess quantitatively Ca2+ levels in the MAMs. However, sensitivity of the first generation Ca2+ biosensors on the surface of the outer-mitochondrial membrane (OMM) do not allow to measure μM or sub-μM changes in Ca2+ concentration which prevents to measure the native activity (unstimulated exogenously) of endogenous channels. In this study, we assembled a new ratiometric highly sensitive Ca2+ biosensor expressed on the surface of the outer-mitochondrial membrane (OMM). It allows the detection of smaller differences than the previous biosensor in or at proximity of the MAMs. Noteworthy, we demonstrated that IP3-receptors have an endogenous activity which participate to the Ca2+ leak channel on the surface of the OMM during hypoxia or when SERCA activity is blocked

Phospholipid bilayer surface configuration probed quantitatively by (31)P field-cycling NMR

(31)P relaxation of the diester phosphate of phospholipids in unilamellar vesicles has been studied from 0.004 to 11.7 T. Relaxation at very low fields, below 0.1 T, shows a rate increase that reflects a residual dipolar interaction with neighboring protons, probably dominated by the glycerol C3 protons. This interaction is not fully averaged by faster motion such as rotational diffusion perpendicular to the membrane surface. The remaining dipolar interaction, modulated by overall rotational diffusion of the vesicle and lateral diffusion of the lipid molecules, is responsible for the very low-field relaxation. These measurements yield a good estimate of the time-average angle between the membrane surface and the vector connecting the phosphorus to the glycerol C3 protons, based on the classic theory by Woessner [Woessner, D. E. (1962) J. Chem. Phys. 37, 647–654]. Dynamic information is also obtained. Implications for solid-state NMR and other studies are discussed

Interdisciplinary research in artificial intelligence: challenges and opportunities

The use of artificial intelligence (AI) in a variety of research fields is speeding up multiple digital revolutions, from shifting paradigms in healthcare, precision medicine and wearable sensing, to public services and education offered to the masses around the world, to future cities made optimally efficient by autonomous driving. When a revolution happens, the consequences are not obvious straight away, and to date, there is no uniformly adapted framework to guide AI research to ensure a sustainable societal transition. To answer this need, here we analyze three key challenges to interdisciplinary AI research, and deliver three broad conclusions: 1) future development of AI should not only impact other scientific domains but should also take inspiration and benefit from other fields of science, 2) AI research must be accompanied by decision explainability, dataset bias transparency as well as development of evaluation methodologies and creation of regulatory agencies to ensure responsibility, and 3) AI education should receive more attention, efforts and innovation from the educational and scientific communities. Our analysis is of interest not only to AI practitioners but also to other researchers and the general public as it offers ways to guide the emerging collaborations and interactions toward the most fruitful outcomes